Anti-PSMA antibody-drug conjugate and use thereof

Abstract

The present application relates to an anti-PSMA antibody-drug conjugate and the use thereof. Specifically provided are an antibody-drug conjugate as shown in 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).

Description

Anti-psma antibody drug conjugates and uses thereof

[0001] This application is based on and claims priority to CN application No. 202410662943.4, 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-PSMA antibody drug conjugates and uses thereof. BACKGROUND

[0003] Prostate specific membrane antigen (PSMA), also known as glutamate carboxypeptidase II and folate hydrolase 1, is a type II transmembrane glycoprotein enzyme composed of an intracellular region, a transmembrane region and an extracellular region. PSMA is expressed in low amounts in other normal human tissues except for prostate secretory epithelium, and its expression is significantly up-regulated in prostate cancer (PCa), and the expression level is closely related to the severity of the disease; in addition, PSMA is also expressed in the neovasculature of other non-prostate cancer solid tumors, such as gastric cancer, pancreatic cancer, breast cancer, lung cancer, etc.

[0004] Currently, there are various PSMA targeted therapies under development, including monoclonal antibodies, bispecific antibodies, CAR-T, targeted radioligands, antibody-drug conjugates (ADC), etc., and only targeted radioligands have marketed drugs. Among them, antibody-drug conjugates target the tumor cells expressing PSMA protein with small molecule toxins (payloads) through PSMA specific antibodies, which selectively reduces the off-target toxic side effects of small molecule toxins while retaining the tumor killing properties of small molecule toxins, and has great potential in improving the risk-benefit ratio of anti-tumor therapy, and is 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 -L 3 -L 4wherein L 1 is attached to Ab, L 4 is attached to D;

[0009] L 1 is selected from R a is selected from hydrogen, C1-C6alkyl, C1-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 -(C1-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 -(C1-C4)alkylene-C(=O)- are optionally substituted with one or more groups selected from hydrogen, halogen, hydroxyl, amino, -(C1-C4)alkylene-hydroxyl, and -O-(C1-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 2 are each independently selected from hydrogen, deuterium, halogen, hydroxyl, amino, C1-C6alkyl, C1-C6deuteroalkyl, C1-C6haloalkyl, 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 3-6 membered heterocyclyl;

[0020] R 3 and R 4 are each independently selected from hydrogen, deuterium, halogen, C1-C6alkyl, C1-C6deuteroalkyl, and C1-C6haloalkyl, 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-C6deuteroalkyl, and C1-C6haloalkyl;

[0022] a is an arbitrary number between 1-10.

[0023] In some embodiments, the R a is selected from hydrogen, C1-C4alkyl, and C1-C4deuteroalkyl.

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

[0025] In some embodiments, the L 1 is attached to the N-terminus of L 2 .

[0026] In some embodiments, the L 1 is selected from where the *-terminus is attached to L 2 .

[0027] In some embodiments, the L 1 is selected from where the *-terminus is attached to L 2 .

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

[0029] In some embodiments, the X1is selected from 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.

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

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

[0032] In some embodiments, said X2is selected from the group consisting of 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 the group consisting of -(CH2) m -X2-(CH2) n -(CH2OCH2) p -(CH2) q -C(=0)-.

[0044] In some embodiments, said L 2 is selected from the group consisting of -(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 H, OH, NH2, CH2OH and -O-CH2CH2OH.

[0045] In some embodiments, the L 2 selected from -CH2CH2-C(=O)NH-CH2-(CH2OCH2)8-CH2C(=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-CH2C(=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 -O-CH2CH2OH.

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

[0047] In some embodiments, the L 2 is -CH(CH2OH)-C(=0)-.

[0048] In some embodiments, the L 2 is -CH(CH2OH)-C(=0)-. 3

[0049] In some embodiments, the L 2 is -CH(CH2OH)-C(=0)-.

[0050] In some embodiments, the L 2 is -CH(CH2OH)-C(=0)-. 3

[0051] In some embodiments, the L 3 is 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. In some embodiments, the amino acids are selected from the group consisting of glycine and phenylalanine.

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

[0053] In some embodiments, the L 3 is -CH(CH2OH)-C(=0)-. 4

[0054] In some embodiments, the L 3 is -CH(CH2OH)-C(=0)-.

[0055] In some embodiments, the L 3 is -CH(CH2OH)-C(=0)-. 4

[0056] In some embodiments, the R 1 and R 2 are each independently selected from the group consisting of hydrogen, deuterium, C1-C4 alkyl, C1-C4 deuterated alkyl, C1-C4 halogenated alkyl, and C3-C6 cycloalkyl.

[0057] In some embodiments, the R 1 and R 2 are each independently selected from the group consisting of hydrogen, deuterium, methyl, deuterated methyl, halogenated methyl, 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 independently selected from:

[0070] In some embodiments, the Ab is an anti-PSMA 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 the sequence of CDR1 contained in the VH of SEQ ID NO: 12, or a sequence of CDR1 contained in the VH of SEQ ID NO: 12 having one or several (for example, 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of CDR1 contained in the VH of SEQ ID NO: 12;

[0074] (ii) a VH CDR2 that has the sequence of CDR2 contained in the VH of SEQ ID NO: 12, or a sequence of CDR2 contained in the VH of SEQ ID NO: 12 having one or several (for example, 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of CDR2 contained in the VH of SEQ ID NO: 12; and

[0075] (iii) a VH CDR3 that has the sequence of CDR3 contained in the VH of SEQ ID NO: 12, or a sequence of CDR3 contained in the VH of SEQ ID NO: 12 having one or several (for example, 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of CDR3 contained in the VH of SEQ ID NO: 12;

[0076] and / or

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

[0078] (iv) a VL CDR1 that has the sequence of CDR1 contained in the VL of SEQ ID NO: 13, or a sequence of CDR1 contained in the VL of SEQ ID NO: 13 having one or several (for example, 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of CDR1 contained in the VL of SEQ ID NO: 13;

[0079] (v) a VL CDR2 that has the sequence of CDR2 contained in the VL of SEQ ID NO: 13, or a sequence of CDR2 contained in the VL of SEQ ID NO: 13 having one or several (for example, 1 or 2) amino acid substitutions, deletions, or additions compared to the sequence of CDR2 contained in the VL of SEQ ID NO: 13; and

[0080] (vi) a VL CDR3 having the sequence of CDR3 contained in the VL as set forth in SEQ ID NO: 13, 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.

[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 sequence of CDR1, CDR2, and CDR3 contained in the VH as set forth in SEQ ID NO: 12; and / or

[0086] (b) the sequence of CDR1, CDR2, and CDR3 contained in the VL as set forth in SEQ ID NO: 13.

[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 consisting of the sequence of SEQ ID NO: 6, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to SEQ ID NO: 6,

[0090] (ii) a VH CDR2 consisting of the sequence of SEQ ID NO: 7, or a sequence that has one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions compared to SEQ ID NO: 7, and

[0091] (iii) a VH CDR3 that consists of the sequence of SEQ ID NO:8, or a sequence with one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions as compared to SEQ ID NO:8;

[0092] and / or

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

[0094] (iv) a VL CDR1 that consists of the sequence of SEQ ID NO:9, or a sequence with one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions as compared to SEQ ID NO:9,

[0095] (v) a VL CDR2 that consists of the sequence of SEQ ID NO: 10, or a sequence with one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions as compared to SEQ ID NO: 10, and

[0096] (vi) a VL CDR3 that consists of the sequence of SEQ ID NO: 11, or a sequence with one or several (e.g., 1 or 2) amino acid substitutions, deletions, or additions as compared to SEQ ID NO: 11.

[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: 6; a VH CDR2 as set forth in SEQ ID NO: 7; and, a VH CDR3 as set forth in SEQ ID NO: 8; and / or, the VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 9; a VL CDR2 as set forth in SEQ ID NO: 10; and, a VL CDR3 as set forth in SEQ ID NO: 11.

[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: 6; a VH CDR2 as set forth in SEQ ID NO: 7; and, a VH CDR3 as set forth in SEQ ID NO: 8; and, the VL of the antibody or antigen-binding fragment thereof comprises a VL CDR1 as set forth in SEQ ID NO: 9; a VL CDR2 as set forth in SEQ ID NO: 10; and, a VL CDR3 as set forth in SEQ ID NO: 11.

[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: 12, 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: 13.

[0101] In some embodiments, the amino acid sequence of the heavy chain of the antibody is set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain of the antibody is set forth in SEQ ID NO: 2.

[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 3-7.5, such as 3-4.5, 4.5-6, 6-7.5, 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, about 5.7, about 5.9, about 6.1, about 6.3, about 6.5, about 6.7, about 6.9, about 7.1, about 7.3, about 7.5. Such as about 3.0, about 3.2, about 3.4, about 4.8, about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4, about 6.6, about 6.8, about 7.0, about 7.2, about 7.4.

[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 of the embodiments;

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

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

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

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

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

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

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

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

[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 PSMA 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 disease associated with PSMA.

[0126] In some embodiments, the disease is a disease associated with abnormal PSMA expression.

[0127] In some embodiments, the disease is cancer.

[0128] In some embodiments, the disease is selected from the group consisting of prostate cancer, gastric cancer, pancreatic cancer, breast cancer, and lung cancer.

[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 similar aspects described with the terms "consisting of and / or "consisting essentially of" are also contemplated.

[0133] Stereoisomers in the compounds described herein, when specifically designated as (R)- or (S)-isomers in the chemical name, are to be understood as predominantly in the (R)-isomer or (S)-isomer configuration, respectively. Any asymmetric carbon atom can be present 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 of a compound of the present application that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide the compound of the present application. Prodrugs are biologically active or inactive compounds that become active when they undergo a chemical reaction under biological conditions. They are inactive or have lower activity in the form in which they are not reacted. Prodrugs can 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 the members of the group(s) or range(s) recited.

[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 group includes 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 they 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: 4) amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS)4(SEQ ID NO: 5) 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, and the like. 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, ameliorate or eliminate the disease state or condition targeted. A subject is successfully "treated" if one or more signs and symptoms of the disease state or condition are observed and / or detectably reduced or ameliorated in the subject 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 will also be appreciated that treatment of the disease state or condition includes not only treatment of the disease state or condition in its entirety, but also includes treatment that does not achieve a total cure, but that achieves some biologically relevant or medically relevant outcome.

[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 attenuating 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 binding activity to tumor cell lines expressing PSMA, especially excellent binding activity to tumor cell lines with high expression of PSMA;

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

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

[0169] 4) The antibody-drug conjugate of the present application has 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.

[0173] Sequence information

[0174] The sequence information involved in the present application is shown in Table 1 below.

[0175] Table 1 DETAILED DESCRIPTION

[0176] The present application is further illustrated by the following description of specific embodiments, but this is not intended to be limiting of the present application. Based on the teachings of the present application, those skilled in the art will be able to make various modifications or improvements, without departing from the basic scope and spirit of the present application. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be obtained on the market.

[0177] In the following examples, the abbreviations of some drugs and their corresponding structural formulas are shown as follows:

[0178] Deruxtecan:

[0179] Among them, Deruxtecan (i.e. MC-GGFG-DXd) can be prepared according to CN104755494A example 58.

[0180] In the following examples, the AB-PG1-XG1-026 antibody used is a known antibody (see US20040033229A1), and the Fc part is mutated (L234A, L235A) to obtain the antibody AB-PG1-XG1-026 (LALA). The heavy chain sequence (SEQ ID NO: 1), light chain sequence (SEQ ID NO: 2), heavy chain variable region sequence (SEQ ID NO: 12), light chain variable region sequence (SEQ ID NO: 13), heavy chain CDR1 sequence (SEQ ID NO: 6), heavy chain CDR2 sequence (SEQ ID NO: 7), heavy chain CDR3 sequence (SEQ ID NO: 8), light chain CDR sequence (SEQ ID NO: 9), light chain CDR2 sequence (SEQ ID NO: 10) and light chain CDR3 sequence (SEQ ID NO: 11) of the AB-PG1-XG1-026 (LALA) antibody 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 also be used to identify the amino acid residues in the CDR region of the variable region.

[0181] Example 1. Preparation of linker-drug

[0182] Example 1.1

[0183] First step

[0184] Dissolve 1a (10.00 g, 95.20 mmol) in acetonitrile (100 mL), add tert-butyldimethylsilyl chloride (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, allow the reaction mixture to warm to room temperature and stir for 16 h. A solid precipitates, filter the reaction mixture directly and collect the filter cake to give 1b (11.50 g), yield: 55%.

[0185] MS-ESI calculated [M+H] = 220, found 220. +

[0186] Second step

[0187] Add 1b (5.55 g, 25.33 mmol), acetone (55.5 mL) and maleic anhydride (2.48 g, 25.33 mmol) sequentially to a reaction flask, stir the mixture at room temperature for 2 h after the addition is complete. Concentrate the reaction mixture directly to give a yellow oil (8.25 g). Dissolve the yellow oil in toluene (82 mL), add triethylamine (5.12 g, 50.66 mmol), and heat the mixture to 120 °C and reflux for 2 h. Concentrate the reaction mixture directly under reduced pressure to give a residue, purify the residue by column chromatography on silica gel (MeOH:DCM = 0-100%) to give 1c (1.53 g), yield: 33%.

[0188] MS-ESI calculated [M-H] = 184, found 184. -

[0189] 1 H NMR (400 MHz, DMSO-d6) δ 7.00 (s, 2H), 6.05 (s, 1H), 4.30 (dd, J = 9.6, 5.6 Hz, 1H), 3.98 (dd, J = 10.8, 5.6 Hz, 1H), 3.84 (dd, J = 10.8, 10.8 Hz, 1H).

[0190] Third step

[0191] ​​To a solution of 1d (10.00 g, 13.59 mmol) in THF (450 mL) was added wet Pd / C (2 g, 20% w / w) and the mixture was stirred under hydrogen atmosphere for 66 h. The reaction solution was filtered, the filter cake was rinsed with a mixture of DCM / MeOH, and the combined filtrates were dried over anhydrous sodium sulfate, filtered, and concentrated to give the crude product. The crude product was purified by silica gel column chromatography (MeOH:DCM = 0-100%) to give 1e (6.10 g) in 69% yield.

[0192] MS-ESI calculated [M+H] = 668, found 668. +

[0193] Fourth Step

[0194] To a solution of 1f (2.70 g, 5.97 mmol) and trifluoroacetic acid (680 mg, 5.97 mmol) in N,N-dimethylformamide (54 mL) was added 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) under nitrogen atmosphere. The reaction was stirred at 0-10 °C for 2 h. To the reaction was added HCl (0.05 N, 54 mL) dropwise under ice bath. Solid was precipitated and the suspension was diluted with 2-methyltetrahydrofuran (100 mL). After stirring until dissolution, the solution was separated. The aqueous phase was extracted with 2-methyltetrahydrofuran (100 mL x 2), and the combined organic phases were washed with saturated brine (50 mL x 1), dried over anhydrous sodium sulfate, filtered, and 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 1g (6.00 g) in 84% yield. aq

[0195] MS-ESI calculated [M+H] = 1079, found 1079. +

[0196] Fifth Step

[0197] ​​​To a solution of 1 g (6.00 g, 5.56 mmol) in DCM / MeOH (120 mL / 12 mL) was added diethylamine (24 mL) dropwise under ice-bath cooling. After the addition was completed, the mixture was allowed to warm to room temperature and stirred for 4 h. The reaction mixture was directly concentrated to give a brown solid. Methyl tert-butyl ether was added to the solid, which was filtered and the filter cake was washed with methyl tert-butyl ether three times. The filtrate was concentrated to give a yellow solid. The solid was purified by preparative liquid chromatography to give 1h (150 mg). The remaining solid was purified by silica gel column chromatography (MeOH:DCM = 0-100%) to give 1h (800 mg). The combined yield was 59%.

[0198] MS-ESI calculated value [M+H] + = 857, found 857.

[0199] Sixth step

[0200] To a reaction flask were sequentially added 1c (65 mg, 0.234 mmol), 1h (0.20 g, 0.23 mmol), and N,N-dimethylformamide (4 mL). After the mixture was stirred to dissolve, it was placed in an ice bath, and 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.13 g, 0.35 mmol) and 2,4,6-trimethylpyridine (85 mg, 0.70 mmol) were added. The mixture was stirred at 0-5 °C for 1 h. The reaction mixture was adjusted to pH = 4-5 with HCl (0.5 N), and purified by preparative liquid chromatography to give compound 1 (70 mg). The yield was 14%. aq

[0201] MS-ESI calculated value [M+H] + = 1024, found 1024.

[0202] 1 ​H 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).

[0203] Example 1.2

[0204] first step

[0205] 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%.

[0206] MS-ESI calculated value [M+Na] + =1020, the actual measured value is 1020.

[0207] Step 2

[0208] 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 stirring was continued for 1 h. The reaction solution was adjusted to pH 6-7 with HCl (0.05 M) and purified by preparative liquid phase separation to obtain compound 2 (134 mg) with a yield of 49%. (aq) (0.05 M) to pH 6-7 and purified by preparative liquid phase separation to obtain compound 2 (134 mg) with a yield of 49%.

[0209] 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).

[0210] Example 1.3

[0211] First step

[0212] 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.

[0213] MS-ESI calculated [M+Na] = 236, found 236. +

[0214] Second Step

[0215] 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.

[0216] MS-ESI calculated [M+H] = 270, found 270. +

[0217] Third Step

[0218] 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)

[0219] MS-ESI calculated [M+H] = 260, found 260. + ​​​​

[0220] Step 4

[0221] 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%.

[0222] MS-ESI calculated value [M+H] + =339, the actual measured value is 339.

[0223] 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).

[0224] Step 5

[0225] 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%.

[0226] MS-ESI calculated value [M+H] + =475, the actual measured value is 475.

[0227] Step 6

[0228] Dissolve 3i (74 mg, 0.16 mmol) in N,N-dimethylformamide (1.30 mL), replace with nitrogen for 3 times, add 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (88 mg, 0.23 mmol) under ice-bath, after stirring for 10 min, add 1h (132 mg, 0.16 mmol), after stirring to dissolve, add 2,4,6-trimethylpyridine (54 mg, 0.45 mmol), continue to control the temperature at 0-10 °C for 2 h. Adjust the pH of the reaction solution to 6-7 with HCl (0.05 N), prepare liquid phase separation and purification to obtain compound 3 (71 mg) with a yield of 34%. (aq)

[0229] MS-ESI calculated value [M+H] + = 1313, measured value is 1313.

[0230] Example 1.4

[0231] Step 1

[0232] Under nitrogen atmosphere, dissolve 4a (5.00 g, 26.74 mmol) in N,N-dimethylformamide (50 mL), cool to 0-5 °C, then add NaH (1.28 g, 32.09 mmol), after stirring for 10 min, add tert-butyl bromoacetate (6.23 g, 32.09 mmol), keep the mixed system at 0-5 °C and stir for 2 h. Add water (200 mL) to the reaction solution, extract the aqueous phase with ethyl acetate (200 mL), wash the organic phase with saturated brine (200 mL x 2), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure to obtain 4b (4.60 g) with a yield of 43%.

[0233] MS-ESI calculated value [M+H] + = 302, 304, measured value is 302, 304.

[0234] 1 H NMR (400 MHz, CDCl3) δ 8.60 (d, J = 2.0 Hz, 1H), 7.84 (dd, J = 8.4, 2.4 Hz, 1H), 7.45 (d, J = 8.4 Hz, 1H), 4.69 (s, 2H), 4.10 (s, 2H), 1.49 (s, 9H).

[0235] Step 2

[0236] ​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%.

[0237] MS-ESI calculated value [M+H] + = 403, measured value 403.

[0238] Third step

[0239] 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%.

[0240] MS-ESI calculated value [M+H] + = 239, measured value 239.

[0241] Fourth step

[0242] 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%.

[0243] MS-ESI calculated value [M+H] + = 319, measured value 319.

[0244] 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).

[0245] Fifth step

[0246] 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).

[0247] MS-ESI calculated value [M+H] + = 263, measured value 263.

[0248] Sixth step

[0249] 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%.

[0250] MS-ESI calculated value [M+H] + = 1101, measured value 1101.

[0251] 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).

[0252] Example 2. Preparation of antibody-drug conjugates

[0253] 1. Preparation of MH-ADC1

[0254] AB-PG1-XG1-026(LALA) antibody was dialyzed into 20 mM His / His-HCl pH 6.0-6.2 buffer to obtain an antibody intermediate. An appropriate amount of the antibody intermediate was taken, and 5 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 6.0-6.2 buffer was supplemented to make the final concentration of the antibody in the reaction system 10-20 mg / mL, the molar ratio of TCEP to the antibody (2.0-6.5):1.0, and the final concentration of DTPA 1 mM. After thorough mixing, the reduction reaction was carried out in a constant-temperature homogenizer at a temperature of 12-36°C for 1.0-3.5 hours. After the reduction was completed, DMSO and an appropriate amount of 10 mM linker-drug (compound 1) stock solution were added to the reaction system to make the volume of DMSO account for 10% of the total volume of the coupling reaction and the molar ratio of the linker-drug to the antibody 3.5-10.0. After thorough mixing, the coupling reaction was carried out in a constant-temperature homogenizer at a temperature of 3-16°C for 1-3 hours to obtain MH-ADC1, wherein Ab is AB-PG1-XG1-026(LALA) antibody. After the coupling was completed, the ADC sample was dialyzed into dialysis fluid (10 mM His / His-HCl, pH 6.0±0.2) for subsequent bulk preparation or storage at ≤-60°C.

[0255] It was detected that the relevant process parameters and DAR value detection data of products with different DAR values are shown in Table 2. The DAR value detection method is described in Example 3.

[0256] Table 2. Process parameters and DAR values of MH-ADC1 samples

[0257] Referring to MH-ADC1, compound 2, compound 3 and compound 4 were coupled respectively to prepare MH-ADC3, MH-ADC5 and MH-ADC7, wherein Ab is AB-PG1-XG1-026(LALA) antibody, and a is a number between 3.0 and 7.5.

[0258] 2. Preparation of MH-ADC2

[0259] AB-PG1-XG1-026(LALA) antibody was dialyzed into 20 mM His / His-HCl buffer to obtain an antibody intermediate. An appropriate amount of the antibody intermediate was taken, 5 mM tris (2-carboxyethyl) phosphine hydrochloride (TCEP) stock solution and 10 mM diethylene triamine pentaacetic acid (DTPA) stock solution were sequentially added, and 20 mM His / His-HCl 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 (2.0-6.5): 1, and the final concentration of DTPA 1 mM. After thorough mixing, the reduction reaction was carried out in a constant temperature mixer, the temperature was 12-22℃, and the reduction reaction time was 3.5 hours. After the reduction was completed, an appropriate amount of DMSO and 10 mM linker-drug (compound 1) stock solution were added to each reaction system, so that the volume fraction of DMSO in the total coupling reaction volume was 10%, and the molar ratio of the linker-drug to the antibody was (3.5-10.0): 1. After thorough mixing, the coupling reaction was carried out in a constant temperature mixer, the temperature was 3-16℃, and the coupling reaction time was 1-3 hours. After the coupling was completed, an appropriate amount of 0.15 M Tris buffer was added to the reaction system to adjust the pH of the reaction solution to 7.8-8.2, and the hydrolysis was carried out at 22-37℃ for 12-24 hours to obtain MH-ADC2, wherein Ab is AB-PG1-XG1-026(LALA) antibody, and Ab is connected to e and / or f of the linker through its thiol group, for example, one or more thiols on Ab are connected to e of one or more linkers, and one or more other thiols are connected to f of one or more other linkers, or, all thiols on Ab are connected to e of the linker, or, all thiols on Ab are connected to f of the linker. The ADC sample was dialyzed into dialysis buffer (20 mM His / His-HCl, pH 6.0±0.2) for subsequent bulk preparation, or stored at ≤-60℃.

[0260] It was detected that the relevant process parameters and DAR value detection data of products with different DAR values are shown in Table 3, and the DAR value detection method is described in Example 3.

[0261] Table 3. Process parameters and DAR values of MH-ADC2 samples

[0262] Referring to MH-ADC2, compounds 2, 3 and 4 were coupled to prepare MH-ADC4, MH-ADC6 and MH-ADC8, wherein Ab is AB-PG1-XG1-026(LALA) antibody, and a is a number between 3.0 and 7.5.

[0263] 3. Preparation of positive control ADC

[0264] Positive control ADC

[0265] AB-PG1-XG1-026(LALA) antibody was dialyzed into 20 mM His / His-HCl pH 6.0-6.2 buffer to obtain an antibody intermediate. An appropriate amount of the antibody intermediate was taken, and 5 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 6.0-6.2 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 2.5:1, and the final concentration of DTPA 1 mM. After thorough mixing, the reaction was carried out in a constant-temperature mixer at 4-8°C for 3.5 hours. After the reduction was completed, an appropriate amount of DMSO and 10 mM linker-drug (Deruxtecan) stock solution were added to the reaction system to make the DMSO account for 10% of the total volume of the coupling reaction and the molar ratio of the linker-drug to the antibody 4.8:1. After thorough mixing, the coupling reaction was carried out in a constant-temperature mixer at 4-10°C for 2 hours to obtain a positive control ADC, wherein Ab is AB-PG1-XG1-026(LALA) antibody. After the coupling was completed, the ADC sample was dialyzed into dialysis buffer (20 mM His / His-HCl, pH 6.0) using an ultrafiltration centrifuge tube, and was stored at ≤-60°C. The DAR value (HIC-HPLC) of the positive control ADC obtained was 3.96, and the detection method is described in Example 3.

[0266] 4. Preparation of isotype control ADC

[0267] Isotype control ADC

[0268] The Anti-HEL IgG1 LALA antibody (Biointron, B109802) was dialyzed into 20 mM His / His-HCl pH 6.0 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 diethylenetriaminepentaacetic acid (DTPA) stock solution were added in turn, and 20 mM His / His-HCl 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 2.0:1, and the final concentration of DTPA 1 mM. After mixing well, the reaction was carried out in a constant temperature mixer at 25°C for 2 hours. After reduction, an appropriate amount of 5 mM linker-drug (compound 1) stock solution was added to the reaction system to make the molar ratio of linker-drug to antibody 4.5:1. After mixing well, the coupling and hydrolysis reactions were carried out in a constant temperature mixer at 30°C for 18 hours to obtain the isotype control ADC, wherein Ab is Anti-HEL IgG1 LALA antibody, and the Ab is connected to the e and / or f positions of the linker through its thiol group, for example, one or more thiol groups on the Ab are connected to one or more e positions of the linker, and one or more or another thiol groups are connected to one or more or another f positions of the linker, or the thiol groups on the Ab are all connected to the e positions of the linker, or the thiol groups on the Ab are all connected to the f positions of the linker. The ADC sample was dialyzed into dialysis buffer (20 mM His / His-HCl, 10 mM Met, pH 5.2) and stored at ≤-60°C. The DAR value (HIC-HPLC) of the isotype control ADC obtained was 4.0, and the detection method is described in Example 3.

[0269] Example 3. Detection of the drug antibody coupling ratio of antibody-drug conjugates

[0270] The average drug-to-antibody ratio (DAR) of the ADC was detected by high performance liquid hydrophobic chromatography (HIC-HPLC). After the ADC sample was equilibrated to room temperature and mixed well, ultrapure water was used as the diluent, and the sample was diluted to a target concentration of 3 mg / mL according to the protein concentration. After mixing on a vortex mixer, the diluted sample solution was taken into an inner tube with a pipette, and the sample number was marked on the sample bottle. The high performance liquid chromatograph (Waters e2695) and hydrophobic chromatography column (TOSOH, TSKgel Butyl-NPR 4.6mm I.D.*3.5cm) were used to complete column equilibration, and then the sample was detected. The detection wavelength was 280nM, the column temperature was 30℃, the flow rate was 0.6mg / mL, and the sample amount was 50-100μg. The mobile phase A formula was 20mM phosphate buffer, 1.5M ammonium sulfate, pH 7.0; the mobile phase B formula was 20mM phosphate buffer, 25% isopropanol (v / v), pH 7.0. The gradient program was 0%-0% (0-2min), 0%-100% (2-17min), 100%-0% (17-17.1min), 0%-0% (17.1-23min). The liquid chromatogram was integrated, and the percentages of ADCs with 0, 2, 4, 6, and 8 drugs coupled (DAR0%, DAR2%, DAR4%, DAR6%, and DAR8%, respectively) were calculated according to the integration results. The average drug-to-antibody ratio was calculated using the following formula.

[0271] DAR value = (0 x DAR0% + 2 x DAR2% + 4 x DAR4% + 6 x DAR6% + 8 x DAR8%) / (DAR0% + DAR2% + DAR4% + DAR6% + DAR8%).

[0272] Example 4. Inhibition of tumor cell proliferation in vitro by antibody-drug conjugates

[0273] LNCAP cell test method: human tumor cells LNCAP (Cobioer, CBP60346) in the logarithmic growth phase were trypsinized, resuspended with fresh complete culture medium, counted, and adjusted to 4.5 x 10 4 cells / mL, 75μL / well, and the edge wells were supplemented with 200μL PBS. After the cell culture plates were placed in a 37℃, 5% CO2 incubator for 4-6 hours, 75μL of different concentrations of the ADC sample to be tested (final concentration 2500ng / mL-0.03ng / mL, 3.5-fold dilution) or buffer control was added to the corresponding wells of the cell culture plates, and the plates were incubated in the carbon dioxide incubator for 7 days. The test plates were removed, and 50μL of CellTiter-Glo® reagent was added to each well. After incubation at room temperature for 10 minutes, the luminescence intensity of each well was detected by a microplate reader. 2.0 Cell Viability Assay Kit (Nanjing Nvigan, DD1101-02), 100 μL / well, incubate at room temperature for 10-30 min, mix well and then detect luminescence readings using a multifunctional microplate reader (Molecular Devices, SpectraMax i3x). Use SoftMax Pro software to analyze the data, with "Concentration (ng / mL)" (the final concentration of the sample in the well) as the horizontal coordinate and "RLU" as the vertical coordinate, and use the four-parameter method for fitting and calculate the EC 50 value.

[0274] Stable PSMA-expressing 22RV1 cell test method: Take the PSMA-22RV1 cells (Cobioer, CBP74210) in the logarithmic growth phase, trypsinize, resuspend with fresh complete culture medium, count and adjust to 1×10 4 / mL, add to a white-walled bottomed 96-well cell culture plate, 100 μL / well. Place the cell culture plate in a 37°C, 5% CO2 incubator and incubate overnight. The next day, take one of the 96-well plates seeded with cells, add 100 μL / well of CellTiter Glo assay reagent and place for 30 minutes, read the values, defined as G0 data. Take another parallel plate, add 11.1 μL / well of 10× test ADC sample diluted by gradient (3-fold dilution for the first point, 8-fold dilution for the subsequent points), so that the highest 1× final concentration of the test sample in the 96-well cell plate is 150 μg / mL, a total of 9 concentration gradients, continue to place in the carbon dioxide incubator for 168 h, then equilibrate the test plate to room temperature, add 100 μL / well of CellTiter Glo assay reagent (Promega, G7558), and detect luminescence readings using a multifunctional microplate reader (Biotek, Synergy H1). Calculate the cell proliferation rate corresponding to each well, and use Graphpad Prism software to fit the curve and calculate the GI 50 value (GI 50 50) and IC 50 value.

[0275] Stable PSMA-expressing HEK293 cell test method: Take the PSMA-HEK293 cells (Genomeditech, GM-C26312) in the logarithmic growth phase, trypsinize, resuspend with fresh complete culture medium, count and adjust to 1.5×10 4 / mL, 100 μL / well. The cell culture plate was placed in a 37℃, 5% CO2 incubator for overnight culture. The next day, the cell culture plate was taken out, 100 μL of cell culture solution in each well was aspirated and discarded, and 100 μL / well of the gradient-diluted (starting concentration 150 μg / mL, 3-fold dilution for the first point, 8-fold dilution for the subsequent points) ADC sample to be tested was added, and the cell culture plate was continued to be placed in the carbon dioxide incubator for 120 h. After that, the cell culture plate was taken out, and 100 μL / well of the GMTiter TM luminescence method cell viability detection reagent (Genomeditech, GM-040504) 100 μL / well, and the luminescence reading value was detected by using a multifunctional enzyme label instrument (Molecular Devices, SpectraMax L). The cell survival rate corresponding to each well was calculated, and the Graphpad Prism software was used to fit the curve and calculate the IC 50 value of the compound.

[0276] The experimental results are shown in Tables 4 and 5. The tested ADCs have excellent inhibitory activity on PSMA-expressing tumor cell lines and PSMA-overexpressing cell lines, and are significantly better than the positive control ADC and the isotype control ADC, which embodies the target-dependent killing effect. The isotype control ADC is a negative control, and the antigen of the antibody Anti-HEL IgG1 LALA (Biointron, B109802) is chicken egg lysozyme.

[0277] Table 4. In vitro proliferation inhibition activity of antibody-drug conjugates on human tumor cells

[0278] Table 5. In vitro proliferation inhibition activity of antibody-drug conjugates on PSMA-overexpressing cells

[0279] Example 5. Detection of bystander killing activity of antibody-drug conjugates

[0280] The stable overexpression of human PSMA HEK293 cell lines and control HEK293 cell lines 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, and the overexpression cell lines and control cell lines were inoculated into 96-well cell culture plates at a ratio of 1:1, with a total volume of 50 μL. At the same time, control wells of only overexpressing PSMA HEK293 cells and only HEK293 control cells were set. The ADC samples were gradient diluted, and the diluted ADC samples were added to the aforementioned wells inoculated with co-cultured cells or only overexpressing cell lines, control HEK293 cell lines according to the preset final concentration and well plate distribution, 50 μL, mixed well and placed in a 5% CO2, 37°C incubator for 120h. Take out the cell culture plate, add cell viability detection reagent 100ul / well, detect with multifunctional enzyme label instrument, for specific detection method information, see example 4.

[0281] 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 with negative target expression, but after mixing the target overexpression cells with the negative cells, the ADC also has a killing effect on the cells with negative target expression.

[0282] Example 6. In vivo pharmacodynamic detection of antibody-drug conjugate in human tumor cell mouse subcutaneous xenograft model

[0283] Take the human tumor cells in logarithmic growth phase, resuspend in serum-free culture medium after digestion and counting, inoculate subcutaneously in immunodeficient mice, 100 μL per animal, to establish a mouse subcutaneous xenograft model. After the tumor grows to a measurable range, measure the long diameter and short diameter of each tumor with a vernier caliper, and calculate the tumor volume according to the following formula: V=(a×b 2 ) / 2, where a represents the long diameter of the tumor, and b represents the short diameter of the tumor.

[0284] When the tumor volume reaches an average of 100mm 3 ~200mm 3 , randomly group according to tumor volume and mouse weight. Inject the tumor-bearing mice with solvent control or different doses of ADC through the tail vein, single dose or adjust the dose frequency according to the efficacy. Measure the long and short diameters of the tumor twice a week, and record the animal body weight. Calculate the tumor volume of each group, and calculate the tumor growth inhibition rate TGI according to the following formula: TGI=100%×[1-(TVt T -TV0 T ) / (TVt C -TV0 C)]. Wherein (TVt T TV0 represents the tumor volume of the dosing group on the day of measurement. T TV0 represents the tumor volume of the dosing group on the day of measurement. C TV0 represents the tumor volume of the dosing group on the day of measurement. C TV0 represents the tumor volume of the dosing group on the day of measurement.

[0285] In the LNCaP clone FGC mouse subcutaneous xenograft model endogenously expressing human PSMA, tumor-bearing NOD / SCID mice were injected with different doses of MH-ADC2-5# (6 mg / kg, 3 mg / kg and 1 mg / kg) via tail vein on D0 after grouping and D10 after the first administration, respectively, and all of them showed significant inhibition of tumor growth. During the experiment, no obvious abnormalities and weight loss were observed in the animals of each test group compared with the solvent control group (5% glucose injection), and the tolerance was good. By the end of the experiment (D21), the tumors of the 6 mg / kg dose group and part of the tumors of the 3 mg / kg and 1 mg / kg groups were reduced compared with D0, and the results are shown in Table 6.

[0286] Table 6. In vivo efficacy results of LNCaP clone FGC mouse subcutaneous xenograft model

[0287] The experimental results show that the tested ADC has a strong in vivo proliferation inhibition effect in the mouse subcutaneous xenograft model of human tumor cells, and has good safety.

[0288] Example 7. Pharmacokinetic study of antibody-drug conjugate

[0289] The 3-5 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 0.5 h, 1 h, 4 h, 8 h, 24 h, 48 h, 72 h, 96 h, 120 h, 168 h, 336 h, 504 h, 672 h after the start of administration, and the serum was separated and stored at -80℃±10℃ ultra-low temperature refrigerator for detection.

[0290] ADC detection: 100 μL / well of coating working solution (containing 1 μg / mL of recombinant human PSMA protein in PBS) was added to the enzyme-labeled plate, and after sealing with a sealing film, it was incubated at 2-8°C for 18-20 h. After washing with a plate washer, the residual liquid was wiped off on absorbent paper, 300 μL / well of blocking solution (5% skimmed milk powder) was added, and it was incubated at room temperature for 1.5-2.5 h. 3 μL / well of standard curve sample, blank control sample, QC sample and sample to be tested was added to the dilution plate, and then 297 μL of diluent (2% skimmed milk powder) was added to the corresponding dilution plate hole, and mixed at room temperature. The liquid in the 96-well enzyme-labeled plate was discarded, and the plate was washed and dried as described above, 100 μL / well of the above-processed sample was added according to the plate layout, and after sealing with a sealing film, it was incubated at room temperature for 55-65 min. After washing and drying again, 100 μL / well of detection working solution I (biotin-labeled anti-toxin antibody, 1:4000) was added, and after sealing with a sealing film, it was incubated at room temperature for 55-65 min. After washing and drying again, 100 μL / well of detection working solution II (SA-HRP, 1:200) was added, and after sealing with a sealing film, it was incubated at room temperature for 25-35 min. After washing and drying again, 100 μL / well of TMB color developing working solution was added, and after sealing with a sealing film, it was incubated at room temperature for 8-10 min. 100 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 absorbance value was read at 450-630 nm. The SoftMax Pro 7.0.3Gxp software of the enzyme-labeled instrument was used for regression calculation.

[0291] Total anti-detection: 100 μL / well of coating working solution (containing 1 μg / mL of recombinant human PSMA protein in PBS) was added to the enzyme-labeled plate, and after sealing the plate with a sealing film, it was incubated at 2-8°C for 18-20 h. After washing the plate with a plate washer, the residual liquid was wiped off on the blotting paper, 300 μL / well of blocking solution (5% skimmed milk powder) was added, and it was incubated at room temperature for 1.5-2.5 h. 3 μL / well of standard curve sample, blank control sample, QC sample, etc. was added to the dilution plate hole, and then 297 μL of diluent (2% skimmed milk powder) was added to the corresponding dilution plate hole, and it was mixed under room temperature conditions. The liquid in the 96-well enzyme-labeled plate was discarded, and the plate was washed and wiped as described above, 100 μL / well of the previously treated sample was added according to the plate layout, and after sealing with a sealing film, it was incubated at room temperature for 55-65 min. After washing and wiping the plate as described above, 100 μL / well of detection working solution (Goat Anti-Human IgG1-Fc Secondary Antibody (HRP), abcam, item number ab97225, 1:40000) was added, and after sealing with a sealing film, it was incubated at room temperature for 55-65 min. After washing and wiping the plate again, 100 μL / well of TMB color developing working solution was added, and after sealing with a sealing film, it was incubated at room temperature in the dark for 8-10 min. 100 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 absorbance value was read at 450-630 nm. The SoftMax Pro 7.0.3Gxp software provided with the enzyme-labeled instrument was used for regression calculation.

[0292] Example 8. Mass spectrometry open ring confirmation of antibody-drug conjugates

[0293] RP-MS method was used for open ring confirmation of antibody-drug conjugates. The sample was diluted to 1 mg / mL with 50 mM ammonium formate (pH ~ 6.0), 50 μL of the diluted sample solution was taken, 1 μL of Rapi PNGaseF was added, and it was incubated at 37°C for 1 h, 3 μL of 0.2% formic acid aqueous solution was added, 0.5 M TCEP aqueous solution 2.5 μL was added, and it was incubated at room temperature for 30 min. After the pretreatment was completed, 1 μL of 10% FA was added, and RP-MS detection (Thermo QE) was performed. The instrument and key parameter information are shown in Table 7 below.

[0294] Table 7. Key parameter information table

[0295] The hydrolysis ring-opening of the ADC molecules was confirmed by comparing the molecular weight of each peak of the heavy chain and the light chain with the theoretical molecular weight after hydrolysis. The detection results of MH-ADC2-5# are shown in Table 8. It was confirmed by detection that the molecular weight detection results of each component were consistent with the theoretical values, and the ring opening was confirmed. The molecular weight of each peak of the heavy chain and the light chain of the remaining MH-ADC2 molecules in Example 2 and the MH-ADC4, MH-ADC6, and MH-ADC8 molecules were also consistent with the corresponding theoretical molecular weight after ring opening, and the ring opening was confirmed.

[0296] Table 8. Detection results of deglycosylation and reduction molecular weight (MH-ADC2-5#)

[0297] Note: L1+1OR represents one ring-opened small molecule coupled to the light chain; H3+3OR represents three ring-opened small molecules coupled to the heavy chain, and so on.

[0298] Example 9. Plasma stability study of antibody-drug conjugate

[0299] An appropriate amount of the 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 was purified by protein A chromatography or by antigen-coupled magnetic beads. The purified ADC sample was subjected to ultrafiltration centrifugal concentration, and the concentrated sample was detected, calculated for drug loading according to the method described in Example 3 or Example 8, and the plasma stability of the ADC was studied by the change in the DAR value after different incubation times. The experimental results showed that the ADC (e.g., MH-ADC2-5#) prepared in the application had good plasma stability, and the DAR value did not change significantly or changed little after different incubation times.

[0300] A number of embodiments have been described herein, but the description is illustrative only and is not intended to be limiting in any way whatsoever. It will be apparent to those of ordinary skill in the art that many embodiments and implementations other than those described herein are possible that are within the scope of the embodiments described herein. Although many possible combinations of features have been set forth herein, many other combinations are possible. Unless specifically set forth herein, any feature or element of any embodiment can be used in combination with any other feature or element of any other embodiment, or in combination with any other feature or element of the same embodiment.

Claims

1. 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, X2is selected from the group consisting of a bond, -0-, -NH-, -C(=0)-, and -C(=0)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 with 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.

2. 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.

3. 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, X1is selected from the group consisting of a bond, 5-6 membered heterocyclyl, and 5-6 membered heteroaryl, Preferably, X1is 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, X1is selected from the group consisting of a bond, pyrrolidinyl, tetrahydrofuranyl, 1,3-dioxolanyl, 1,3-dioxanyl, pyridinyl, and pyrimidinyl, Preferably, X1is selected from the group consisting of 1,3-dioxolanyl, 1,3-dioxanyl, and pyridinyl; X2is selected from the group consisting of a bond and -C(=0)NH-, Preferably, X2is -C(=0)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-CH2C(=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-CH2C(=O)-, -CH2-C(=0)-, -CH2CH2-C(=0)-, -CH(CH3)-C(=0)-, -CH(CH2CH3)-C(=0)-, -CH(CH3)CH2-C(=0)-, and -CH2CH(CH3)-C(=0)- 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.

4. 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: 3); Preferably, L 3 is Preferably, L 3 is wherein the * end is attached to L 4 is connected.

5. 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 optionally substituted with 1, 2, or 3 groups selected from hydrogen, deuterium, and fluorine; Preferably, D is 6. 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:

7. 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-PSMA 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): (i) a VH CDR1 that has the sequence of CDR1 contained in the VH of SEQ ID NO: 12, or a sequence of CDR1 contained in the VH of SEQ ID NO: 12 that comprises one or several (e.g. one or two) amino acid substitutions, deletions or additions when compared to the sequence of CDR1 contained in the VH of SEQ ID NO: 12; (ii) a VH CDR2 that has the sequence of CDR2 contained in the VH of SEQ ID NO: 12, or a sequence of CDR2 contained in the VH of SEQ ID NO: 12 that comprises one or several (e.g. one or two) amino acid substitutions, deletions or additions when compared to the sequence of CDR2 contained in the VH of SEQ ID NO: 12; and (iii) a VH CDR3 that has the sequence of CDR3 contained in the VH of SEQ ID NO: 12, or a sequence of CDR3 contained in the VH of SEQ ID NO: 12 that comprises one or several (e.g. one or two) amino acid substitutions, deletions or additions when compared to the sequence of CDR3 contained in the VH of SEQ ID NO: 12; and / or (b) the following 3 light chain variable region (VL) CDRs: (iv) a VL CDR1 that has the sequence of CDR1 contained in the VL of SEQ ID NO: 13, or a sequence of CDR1 contained in the VL of SEQ ID NO: 13 that comprises one or several (e.g. one or two) amino acid substitutions, deletions or additions when compared to the sequence of CDR1 contained in the VL of SEQ ID NO: 13; (v) a VL CDR2 that has the sequence of CDR2 contained in the VL of SEQ ID NO: 13, or a sequence of CDR2 contained in the VL of SEQ ID NO: 13 that comprises one or several (e.g. one or two) amino acid substitutions, deletions or additions when compared to the sequence of CDR2 contained in the VL of SEQ ID NO: 13; and (vi) a VL CDR3 that has the sequence of CDR3 contained in the VL of SEQ ID NO: 13, or a sequence of CDR3 contained in the VL of SEQ ID NO: 13 that comprises one or several (e.g. one or two) amino acid substitutions, deletions or additions when compared to the sequence of CDR3 contained in the VL of SEQ ID NO: 13; 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 CDR1, CDR2 and CDR3 contained in the VH of SEQ ID NO: 12; and / or (b) the sequence of CDR1, CDR2 and CDR3 contained in the VL of SEQ ID NO: 13; Preferably, the antibody or antigen binding fragment thereof comprises: (a) the following 3 heavy chain variable region (VH) CDRs: (i) a VH CDR1 that has the sequence of CDR1 contained in the VH of SEQ ID NO: 12, or a sequence of CDR1 contained in the VH of SEQ ID NO: 12 that comprises one or several (e.g. one or two) amino acid substitutions, deletions or additions when compared to the sequence of CDR1 contained in the VH of SEQ ID NO: 12; (ii) a VH CDR2 that has the sequence of CDR2 contained in the VH of SEQ ID NO: 12, or a sequence of CDR2 contained in the VH of SEQ ID NO: 12 that comprises one or several (e.g. one or two) amino acid substitutions, deletions or additions when compared to the sequence of CDR2 contained in the VH of SEQ ID NO: 12; and (iii) a VH CDR3 that has the sequence of CDR3 contained in the VH of SEQ ID NO: 12, or a sequence of CDR3 contained in the VH of SEQ ID NO: 12 that comprises one or several (e.g. one or two) amino acid substitutions, deletions or additions when compared to the sequence of CDR3 contained in the VH of SEQ ID NO: 12; (i) a VH CDR1 consisting of the sequence of SEQ ID NO:6, or a sequence with one or several (e.g. one or two) amino acid substitutions, deletions, or additions compared to SEQ ID NO:6, (ii) a VH CDR2 consisting of the sequence of SEQ ID NO:7, or a sequence with one or several (e.g. one or two) amino acid substitutions, deletions, or additions compared to SEQ ID NO:7, and (iii) a VH CDR3 consisting of the sequence of SEQ ID NO:8, or a sequence with one or several (e.g. one or two) amino acid substitutions, deletions, or additions compared to SEQ ID NO:8; and / or (b) the following 3 light chain variable region (VL) CDRs: (iv) a VL CDR1 consisting of the sequence of SEQ ID NO:9, or a sequence with one or several (e.g. one or two) amino acid substitutions, deletions, or additions compared to SEQ ID NO:9, (v) a VL CDR2 consisting of the sequence of SEQ ID NO: 10, or a sequence with one or several (e.g. one or two) amino acid substitutions, deletions, or additions compared to SEQ ID NO: 10, and (vi) a VL CDR3 consisting of the sequence of SEQ ID NO: 11, or a sequence with one or several (e.g. one or two) amino acid substitutions, deletions, or additions compared to SEQ ID NO: 11; 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 depicted in SEQ ID NO: 6; a VH CDR2 as depicted in SEQ ID NO: 7; and, a VH CDR3 as depicted in SEQ ID NO: 8; and / or, the VL of the antibody or antigen binding fragment thereof comprises a VL CDR1 as depicted in SEQ ID NO: 9; a VL CDR2 as depicted in SEQ ID NO: 10; and, a VL CDR3 as depicted in SEQ ID NO:

11. Preferably, the VH of the antibody or antigen binding fragment thereof comprises a VH CDR1 as depicted in SEQ ID NO: 6; a VH CDR2 as depicted in SEQ ID NO: 7; and, a VH CDR3 as depicted in SEQ ID NO: 8; and, the VL of the antibody or antigen binding fragment thereof comprises a VL CDR1 as depicted in SEQ ID NO: 9; a VL CDR2 as depicted in SEQ ID NO: 10; and, a VL CDR3 as depicted in SEQ ID NO:

11. 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: 12, 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:

13. Preferably, the amino acid sequence of the heavy chain of the antibody is as set forth in SEQ ID NO: 1, and the amino acid sequence of the light chain of the antibody is as set forth in SEQ ID NO:

2. Preferably, the antibody or antigen binding fragment thereof is linked to L via its thiol group.

8. 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 solvate of any of the foregoing, 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 7.5, such as between 3 and 4.5, between 4.5 and 6, between 6 and 7.5, 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, about 5.7, about 5.9, about 6.1, about 6.3, about 6.5, about 6.7, about 6.9, about 7.1, about 7.3, about 7.

5.

9. 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, and MH-ADC8, Ab is linked (e.g., via its thiol group) to both e and f, e.g., one or more thiol groups on Ab are linked to e and another or additional thiol groups on Ab are linked to f, or, all thiol groups on Ab are linked to e, or, all thiol groups on Ab are linked to f.

10. 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 solvate of any of the foregoing, and optionally one or more pharmaceutical excipients.

11. 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 solvate of any of the foregoing; Preferably, the composition comprises the antibody drug conjugate MH-ADC1 of claim 9, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a solvate of any of the foregoing, and the antibody drug conjugate MH-ADC2, or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a solvate of any of the foregoing. Preferably, the composition comprises the antibody drug conjugate MH-ADC3 as described 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 as described 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-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 as described 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-ADC8 or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

12. Use of the antibody drug conjugate or a stereoisomer thereof, a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 10 or the composition according to claim 11 for the manufacture of a medicament acting on the PSMA target, or for the manufacture of a medicament for the treatment and / or prevention of a disease, Preferably, the disease is a disease associated with PSMA; Preferably, the disease is a disease associated with abnormal expression of PSMA; Preferably, the disease is cancer; More preferably, the disease is selected from the group consisting of prostate cancer, gastric cancer, pancreatic cancer, breast cancer, and lung cancer. Preferably, the disease is a disease associated with PSMA; Preferably, the disease is a disease associated with abnormal expression of PSMA; Preferably, the disease is cancer; More preferably, the disease is selected from the group consisting of prostate cancer, gastric cancer, pancreatic cancer, breast cancer, and lung cancer.

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