WO2025241867A1 - Anti-b7-h4 antibody-drug conjugate and application thereof - Google Patents

Abstract

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

Description

Anti-B7-H4 antibody-drug conjugates and their applications

This application is based on and claims priority to CN application number 202410657156.0, filed on May 24, 2024, the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

This application relates to the field of pharmaceutical technology, specifically to anti-B7-H4 antibody-drug conjugates and their applications.

Background Technology

B7-H4 (B7 homolog 4), also known as V-set domain-containing T-cell activation inhibitor 1 (VTCN1), B7S1, or B7x, is a member of the B7 family of immunomodulatory proteins. It inhibits T-cell function and negatively regulates the T-cell immune response by acting on unknown receptors on T cells, playing a crucial role in tumor immune escape. While B7-H4 expression is relatively low in normal tissues, it is highly expressed in various solid tumors, such as breast cancer, ovarian cancer, endometrial cancer, and cholangiocarcinoma, and is associated with poor prognosis in many of these tumors.

Several B7-H4-targeted therapies are currently under development, including monoclonal antibodies, bispecific antibodies, CAR-T therapy, and antibody-drug conjugates (ADCs), but none have yet been approved for marketing. Among these, antibody-drug conjugates utilize specific antibodies against B7-H4 to deliver small-molecule toxins (payloads) specifically to tumor cells expressing the B7-H4 protein. While retaining the tumor-killing properties of the small-molecule toxins, they selectively reduce off-target toxicity, showing significant potential in improving the benefit-risk ratio of anti-tumor therapy and promising greater benefits for patients.

Summary of the Invention

The first aspect of the present invention provides an antibody-drug conjugate or a stereoisomer thereof of formula (I), a prodrug thereof, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.
Ab-(LD) _a
Formula (I)

in:

Ab represents an antibody or its antigen-binding fragment;

L is ^L1 - ^L2 - ^L3 - ^L4 , where ^L1 is connected to Ab and ^L4 is connected to D;

^L1 is selected from Ra ^is selected from hydrogen, _C1 - _C6 alkyl, _C1 - _C6 deuterated alkyl, _C3 - _C8 cycloalkyl, and _C3 - _C8 deuterated cycloalkyl;

^L2 is selected from -( _CH2 ) _m - _X1 - _X2- ( _CH2 ) _n- ( _CH2OCH2 ) _p- ( _{CH2 ) q} -C(=O)- and -( _C1 - _C4 )alkylene-C(=O)-, wherein,

X ₁ is selected from C1, _C3 - _C8 cycloalkyl, 3-8 membered heterocyclic, _C6 - _C10 aryl, and 5-9 membered heteroaryl.

X ₂ is selected from bond, -O-, -NH-, -C(=O)- and -C(=O)NH-,

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

_The -( _CH2 ) _m - _X1 - _X2- ( _CH2 ) _n- ( _CH2OCH2 ) _p- ( _CH2 ) _q -C(=O)- and -( _C1 - _C4 )alkylene-C(=O)- are optionally substituted by one or more groups selected from hydrogen, halogen, hydroxyl, amino, -( _C1 - _C4 )alkylene-hydroxyl and -O-( _C1 - _C4 )alkylene-hydroxyl;

^L3 is selected from amino acid residues and peptide residues consisting of 2-10 amino acid residues;

^L4 is The * terminal is connected to D;

D is

^R1 and ^R2 are each independently selected from hydrogen, deuterium, halogen, hydroxyl, amino, _C1 - _C6 alkyl, _C1 - _C6 deuterated alkyl, _C1 - _C6 haloalkyl, and _C3 - _C8 cycloalkyl, or,

^R1 and ^R2 together with the carbon atom they are attached to form _C3 - _C6 cycloalkyl or 3-6 membered heterocyclic groups;

^R3 and ^R4 are each independently selected from hydrogen, deuterium, halogen, _C1 - _C6 alkyl, _C1 - _C6 deuterated alkyl, and _C1 - _C6 haloalkyl, or,

^R3 and ^R4 together with the carbon atoms they are attached to form a 5-6 membered heterocyclic group, which may be optionally substituted by one or more groups selected from hydrogen, deuterium, halogen, hydroxyl, amino, _C1 - _C6 alkyl, _C1 - _C6 deuterated alkyl and _C1 - _C6 haloalkyl;

a can be any value between 1 and 10.

In some embodiments, ^Ra is selected from hydrogen, _C1 - _C4 alkyl, and _C1 - _C4 deuterated alkyl.

In some embodiments, ^Ra is selected from hydrogen, methyl, ethyl, isopropyl, deuterated methyl, deuterated ethyl, and deuterated isopropyl.

In some implementations, the N-terminus of ^L1 is connected to ^L2 .

In some implementations, ^L1 is selected from... The * end is connected to ^L2 .

In some implementations, ^L1 is selected from... The * end is connected to ^L2 .

In some embodiments, _X1 is selected from hydroxyl groups, 5-6 membered heterocyclic groups, and 5-6 membered heteroaryl groups.

In some embodiments, _X1 is selected from bonds, 5-6 membered heterocyclic groups and 5-6 membered heteroaryl groups, wherein the 5-6 membered heterocyclic group contains one or two heteroatoms selected from N and O, and the 5-6 membered heteroaryl group contains one, two or three heteroatoms selected from N and O.

In some embodiments, _X1 is selected from pyrrolidinyl, tetrahydrofuranyl, 1,3-dioxocyclopentyl, 1,3-dioxocyclohexyl, pyridyl, and pyrimidinyl.

In some embodiments, _X1 is selected from 1,3-dioxocyclopentyl, 1,3-dioxocyclohexyl, and pyridyl.

In some implementations, _X2 is selected from the bond and -C(=O)NH-.

In some implementations, _X2 is -C(=O)NH-.

In some implementations, m is 0, 1, 2, or 3.

In some implementations, m is 1 or 2.

In some implementations, n is 0, 1, 2, or 3.

In some implementations, n is 1.

In some implementations, p is 0, 3, 4, 5, 6, 7, 8, 9, or 10.

In some implementations, p is 4 or 8.

In some implementations, q is 0, 1, 2, or 3.

In some implementations, q is 1.

In some implementations, q is 0.

In some implementations, the ^L2 is selected from -( _CH2 ) _m - _X2- ( _CH2 ) _n- ( _{CH2OCH2 ) p-} ( _CH2 ) _q -C(=O)-.

In some embodiments, the ^L2 is selected from -( _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 ) - . The} -( _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 by one or more groups selected from hydrogen, hydroxyl, amino, -methylene-hydroxyl, -O-ethylene-hydroxyl.

In some embodiments, the ^L2 is selected from _{-CH2CH2 -} C( ₌ O)NH- _CH2- ( _CH2OCH2 ) ₈ - _CH2 -C(=O)-, -CH ₂ -C(=O)-, -CH ₂ CH ₂ -C(=O)-, -CH(CH ₃ )-C(=O)-, -CH(CH ₂ CH ₃ )-C(=O)-, -CH(CH ₃ )CH ₂ -C(=O)-, and -CH ₂ CH(CH ₃ )-C(=O)-, which -CH ₂ CH ₂ -C(=O)NH-CH ₂ -(CH ₂ OCH ₂ ) ₈ -CH ₂ -C(=O)-, _-CH2 -C(＝O)-, _-CH2CH2- C(＝O)-, _-CH ( _CH3 )-C(＝O)-, -CH( _CH2CH3 )-C(＝O)-, -CH( _CH3 ) _CH2 -C(＝O)-, and _-CH2CH ( _CH3 )-C(＝O)- may be optionally substituted by one _or more groups selected from H, OH, _NH2 , _CH2OH , and -O- _{CH2CH2OH .}

_In some embodiments, the ^L2 is selected from -CH( _CH2OH )-C(＝O)-, -CH( _CH2OCH2CH2OH )-C(＝ _O )-,

In some implementations, ^L2 is -CH( _CH2OH )-C(=O)-.

In some implementations, the C (=O) terminal of ^L2 is connected to ^L3 .

In some implementations, ^L2 is

In some implementations, ^L2 is The * end is connected to ^L3 .

In some embodiments, ^L3 is a peptide residue consisting of 2-4 (preferably 4) amino acid residues, wherein the amino acids are selected from glycine, phenylalanine, valine, alanine, lysine, citrulline, serine, glutamic acid, and aspartic acid. In some embodiments, the amino acids are selected from glycine and phenylalanine.

In some embodiments, ^L3 is glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) (SEQ ID NO:12).

In some implementations, the C (=O) terminal of ^L3 is connected to ^L4 .

In some implementations, ^L3 is

In some implementations, ^L3 is The * end is connected to ^L4 .

In some embodiments, ^R1 and ^R2 are each independently selected from hydrogen, deuterium, _C1 - _C4 alkyl, _C1 - _C4 deuterated alkyl, _C1 - _C4 haloalkyl and _C3 - _C6 cycloalkyl.

In some embodiments, ^R1 and ^R2 are each independently selected from hydrogen, deuterium, methyl, deuterated methyl, halomethyl and cyclopropyl.

In some implementations, ^R1 and ^R2 are each independently hydrogen.

In some embodiments, ^R1 and ^R2 together with the carbon atom they are attached to form cyclopropyl, cyclobutyl, oxacyclobutyl, or azircyclobutyl.

In some embodiments, ^R1 and ^R2 together with the carbon atom they are attached to form cyclopropyl and cyclobutyl groups.

In some embodiments, ^R3 and ^R4 are each independently selected from hydrogen, deuterium, halogen, _C1 - _C4 alkyl, _C1 - _C4 deuterated alkyl, and _C1 - _C4 haloalkyl.

In some embodiments, ^R3 and ^R4 are each independently selected from hydrogen, deuterium, fluorine, chlorine, methyl, deuterated methyl and halomethyl.

In some embodiments, ^R3 is a methyl group.

In some implementations, ^R4 is fluorine.

In some embodiments, ^R3 and ^R4 together with the carbon atom they are attached to form a 5-6 heterocyclic group, the 5-6 heterocyclic group containing one or two heteroatoms selected from O, and the 5-6 heterocyclic group optionally being substituted by one or more groups selected from hydrogen, deuterium, halogens and _C1 - _C4 alkyl groups.

In some embodiments, ^R3 and ^R4 together with the carbon atoms they are bonded to form The It may be optionally substituted with one, two or three groups selected from hydrogen, deuterium and fluorine.

In some implementations, D is

In some implementations, each L is independently selected from:

In some implementations, each LD is independently selected from:

In some embodiments, the Ab is an anti-B7-H4 antibody or its antigen-binding fragment.

In some embodiments, the antibody or its antigen-binding fragment comprises:

(a) The following three heavy chain variable region (VH) complementarity-determining regions (CDRs):

(i) VH CDR1, having the sequence of CDR1 contained in VH as shown in SEQ ID NO:7 or SEQ ID NO:8, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of CDR1 contained in VH.

(ii) VH CDR2, having the sequence of CDR2 contained in VH as shown in SEQ ID NO:7 or SEQ ID NO:8, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the sequence of CDR2 contained in VH; and

(iii) VH CDR3, having the CDR3 sequence contained in VH as shown in SEQ ID NO:7 or SEQ ID NO:8, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the CDR3 sequence contained in VH;

and/or

(b) The following three light chain variable region (VL) CDRs:

(iv) VL CDR1, having the sequence of CDR1 contained in VL as shown in SEQ ID NO:9, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of CDR1 contained in VL.

(v) VL CDR2, having the sequence of CDR2 contained in VL as shown in SEQ ID NO:9, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of one or two amino acids) compared to the sequence of CDR2 contained in VL; and

(vi) VL CDR3, having the sequence of CDR3 contained in VL as shown in SEQ ID NO:9, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the sequence of CDR3 contained in VL.

In some implementations, the permutation described in any one of (i)-(vi) is a conservative permutation.

In some implementations, 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.

In some implementations, 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.

In some embodiments, the antibody or its antigen-binding fragment comprises:

(a) The sequences of CDR1, CDR2 and CDR3 contained in VH as shown in SEQ ID NO:7 or SEQ ID NO:8; and/or

(b) The CDR1, CDR2 and CDR3 sequences contained in the VL as shown in SEQ ID NO:9.

In some embodiments, the antibody or its antigen-binding fragment comprises:

(a) The following three heavy chain variable region (VH) CDRs:

(i) VH CDR1, which consists of the following sequence: SEQ ID NO:1, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to SEQ ID NO:1.

(ii) VH CDR2, which consists of the following sequence: SEQ ID NO:2, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to SEQ ID NO:2, and

(iii) VH CDR3, which consists of the following sequence: SEQ ID NO:3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to SEQ ID NO:3;

and/or

(b) The following three light chain variable region (VL) CDRs:

(iv) VL CDR1, which consists of the following sequence: SEQ ID NO:4, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to SEQ ID NO:4.

(v) VL CDR2, which consists of the following sequence: SEQ ID NO:5, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to SEQ ID NO:5, and

(vi) VL CDR3, which consists of the following sequence: SEQ ID NO:6, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to SEQ ID NO:6.

In some implementations, the permutation described in any one of (i)-(vi) is a conservative permutation.

In some embodiments, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:1; VH CDR2 as shown in SEQ ID NO:2; and VH CDR3 as shown in SEQ ID NO:3; and/or, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:4; VL CDR2 as shown in SEQ ID NO:5; and VL CDR3 as shown in SEQ ID NO:6.

In some embodiments, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:1; VH CDR2 as shown in SEQ ID NO:2; and VH CDR3 as shown in SEQ ID NO:3; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:4; VL CDR2 as shown in SEQ ID NO:5; and VL CDR3 as shown in SEQ ID NO:6.

In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment is shown in SEQ ID NO:9.

In some embodiments, the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:10, and the light chain amino acid sequence of the antibody is shown in SEQ ID NO:11.

In some embodiments, the antibody or its antigen-binding fragment is linked to L via its thiol group.

In some implementations, each 'a' is independently any value between 1 and 8.

In some implementations, each 'a' is independently any value between 3 and 8.

In some implementations, each 'a' is independently any value between 3 and 6, such as 3-4, 4-5, 5-6, or, for example, 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. For example, about 3.0, about 3.2, about 3.4, about 4.8, about 5.0, about 5.2, about 5.4.

In some embodiments, the antibody-drug conjugate represented by formula (I) is selected from:

in,

The definitions of Ab and a are as described in any implementation scheme;

In MH-ADC2, MH-ADC4, MH-ADC6, and MH-ADC8, Ab is connected to the e bit and/or the f bit.

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

In some implementations, in MH-ADC2, MH-ADC4, MH-ADC6, and MH-ADC8, a thiol group on the Ab is attached to the e-position.

In some implementations, in MH-ADC2, MH-ADC4, MH-ADC6, and MH-ADC8, a thiol group on the Ab is attached to the f position.

In some embodiments, in MH-ADC2, MH-ADC4, MH-ADC6, and MH-ADC8, one or more thiol groups on the Ab are connected to the e-site of one or more linkers, and another or more thiol groups are connected to the f-site of another or more linkers. In some embodiments, in MH-ADC2, one or more thiol groups on the Ab are connected to the e-site of one or more linkers, and another or more thiol groups are connected to the f-site of another or more linkers.

In some implementations, in MH-ADC2, MH-ADC4, MH-ADC6, and MH-ADC8, one or more thiol groups on the Ab are attached to the e-position, and another or more thiol groups are attached to the f-position.

In some implementations, in MH-ADC2, MH-ADC4, MH-ADC6, and MH-ADC8, the thiol group on the Ab is attached to the e-position.

In some implementations, in MH-ADC2, MH-ADC4, MH-ADC6, and MH-ADC8, the thiol group on the Ab is attached to the f position.

A second aspect of the invention provides a pharmaceutical composition comprising the antibody-drug conjugate or its stereoisomer as described in the first aspect of the invention, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, and optionally one or more pharmaceutical excipients.

A third aspect of the invention provides a composition comprising at least one antibody-drug conjugate or its stereoisomer as described in the first aspect of the invention, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate.

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

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

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

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

The fourth aspect of the present invention provides the use of the antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, or the pharmaceutical composition of the second aspect of the present invention, or the composition of the third aspect of the present invention, in the preparation of a drug acting on the B7-H4 target.

The fifth aspect of the present invention provides the use of the antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, as described in the first aspect of the present invention, or the pharmaceutical composition as described in the second aspect of the present invention, or the composition as described in the third aspect of the present invention, in the preparation of a medicament for treating and/or preventing diseases.

In some implementations, the disease is a disease related to B7-H4.

In some implementations, the disease is one associated with abnormal B7-H4 expression.

In some implementations, the disease is cancer or an autoimmune disease.

In some implementations, the disease is selected from breast cancer, ovarian cancer, endometrial cancer, and bile duct cancer.

Terminology Definition

In this application, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, to better understand this application, definitions and explanations of relevant terms are provided below.

In this application, the term “and/or” should be considered as a specific disclosure of each of two or more specified features or elements, having or not having the other features or elements. Therefore, the term “and/or” as used in phrases, such as “A and/or B” herein, is intended to include “A and B”, “A or B”, “A” (alone) and “B” (alone).

It should be understood that wherever the terms “include” or “contain” are used to describe aspects, similar aspects described using the terms “composed of” and/or “substantially composed of” are also provided.

When the stereoisomers of the compounds described in this application are specifically designated by chemical name as (R)- or (S)- isomers, they should be understood as having a predominant configuration of (R)- or (S)-, respectively. Any asymmetric carbon atom may exist in the (R)-, (S)-, or (R, S)- configurations, preferably in the (R)- or (S)- configuration.

In this application, the term "prodrug" refers to a derivative that can be hydrolyzed, oxidized, or otherwise reacted under biological conditions (in vitro or in vivo) to provide the compound of this application. Prodrugs become active compounds only after undergoing the reaction under biological conditions, or they do not have or only have low activity in their unreacted forms. Prodrugs can generally be prepared using known methods, such as those described in Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff, ed., 5th edition).

In this application, the term "pharmaceutically acceptable salt" means (i) a salt formed by an acidic functional group present in the compounds provided in this application and a suitable inorganic or organic cation (base), including but not limited to, alkali metal salts such as sodium, potassium, and lithium salts; alkaline earth metal salts such as calcium and magnesium salts; other metal salts such as aluminum, iron, zinc, copper, nickel, and cobalt salts; inorganic base salts such as ammonium salts; and organic base salts such as tert-octylamine, dibenzylamine, morpholine, glucosamine, phenylglycine alkyl ester, ethylenediamine, N-methylglucosamine, guanidine, diethylamine, triethylamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, chloroprocaine, procaine, diethanolamine, N-benzyl-phenylethylamine, piperazine, tetramethylamine, and tris(hydroxymethyl)aminomethane. (ii) Salts formed by the basic functional groups present in the compounds provided in this application and suitable inorganic or organic anions (acids), including but not limited to: hydrohalides, such as hydrofluoric acid, hydrochloride, hydrobromide, hydroiodide, etc.; inorganic acid salts, such as nitrates, perchlorates, sulfates, phosphates, etc.; lower alkyl sulfonates, such as methanesulfonates, trifluoromethanesulfonates, ethanesulfonates, etc.; aryl sulfonates, such as benzenesulfonates, p-benzenesulfonates, etc.; organic acid salts, such as acetates, malates, fumarates, succinates, citrates, tartrates, oxalates, maleates, etc.; amino acid salts, such as glycinates, trimethylglycinates, arginines, ornithines, glutamates, aspartates, etc.

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

In this application, the terms "solvent" and "solvent compound" are used interchangeably to refer to a compound existing in combination with a solvent molecule. This combination may include a stoichiometric amount of a solvent, such as a monohydrate or dihydrate, or may include any amount of water; similarly, methanol or ethanol may form an "alcohol," which may be stoichiometric or non-stoichiometric. The term "solvent compound" as used herein refers to a solid form, i.e., a compound in solution of a solvent that, while solvated, is not a solvate compound as used herein.

In this application, unless otherwise expressly indicated, the descriptive phrases “each…independently selected” and “…independently selected” used throughout this document are interchangeable and should be interpreted broadly. They can mean either that the specific options expressed by the same or different symbols in different groups do not affect each other, or that the specific options expressed by the same or different symbols in the same group do not affect each other.

In various parts of this application, the substituents of the compounds are described according to the type or range of groups. In particular, this application includes every independent secondary combination of the members of these types and ranges of groups.

In this application, the term "alkyl" refers to a straight-chain or branched monovalent saturated hydrocarbon group. For example, _C1 - _C6 alkyl refers to a group having 1 to 6 carbon atoms, such as 1, 2, 3, 4, 5, or 6 carbon atoms; _C1 - _C4 alkyl refers to a group 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, etc.

In this application, the term "alkylene" refers to a straight-chain or branched divalent saturated hydrocarbon group, such as _C1 - _C4 alkylene, which refers to a group having 1 to 4 carbon atoms, such as 1, 2, 3, or 4 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene, ethylene, propylene, butylene, etc.

In this application, the term "cycloalkyl" refers to a monovalent saturated hydrocarbon group consisting of carbon atoms. For example, C3 _{- C8} cycloalkyl refers to a group consisting of 3-8 carbon atoms (e.g., 3, 4, 5, 6, 7, or 8). The _C3 - _C8 cycloalkyl includes _C3 - _C6 cycloalkyl, _C3 cycloalkyl, _C4 cycloalkyl, _C5 cycloalkyl, _C6 cycloalkyl, etc. The cycloalkyl group includes monocyclic, bicyclic, or polycyclic rings, including spirocyclic, fused, or bridged rings. Non-limiting examples include, but are not limited to, cyclobutyl, cyclopentyl, or cyclohexyl.

In this application, the term "heterocyclic group" refers to a saturated or partially unsaturated cyclic group composed of ring atoms, wherein 1, 2, 3, or 4 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 may optionally be oxidized; preferably, the carbon atom is optionally substituted with =O. For example, a 3-8 membered heterocyclic group refers to one composed of 3-8 (e.g., 3, 4, 5, 6, 7, or 8) ring atoms, and a 5-6 membered heterocyclic group refers to one composed of 5 or 6 ring atoms. The 3-8 membered heterocyclic groups include 3-7 membered heterocyclic groups, 4-7 membered heterocyclic groups, 5-6 membered heterocyclic groups, etc. Non-limiting examples include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, or morpholinyl groups.

In this application, the term "aryl" refers to an unsaturated carbocyclic group having a conjugated π-electron system, such as a _C6 - _C10 aryl group consisting of 6 to 10 carbon atoms. Non-limiting examples include, but are not limited to, phenyl and naphthyl groups.

In this application, the term "heteroaryl" refers to an unsaturated group consisting of ring atoms with a conjugated π-electron system, wherein 1, 2, 3, or 4 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 may optionally be oxidized. For example, 5-9-membered heteroaryls consist of 5 to 9 (e.g., 5, 6, 7, 8, or 9) ring atoms, including 5-6-membered heteroaryls, etc. The heteroaryls include monocyclic and polycyclic compounds, examples of which include, but are not limited to, imidazolyl, pyridinyl, quinolinyl, or isoquinolinyl compounds.

In this application, the term "halogen" refers to fluorine, chlorine, bromine, or iodine.

In this application, the term "halogenated" refers to a modified group being substituted with one or more halogens, such as 1, 2, 3, 4, 5, or 6 halogens. For example, "halogenated alkyl" refers to a group obtained by substituting one or more hydrogen atoms of any of the aforementioned alkyl groups (e.g., _C1 - _C6 alkyl, _C1 - _C4 alkyl, etc.) with a halogen, and non-limiting examples include, but are not limited to, _CF3 , _CHF2 , _{or CF2CF3} .

In this application, the term "deuterated" refers to a group whose modified group is substituted with one or more deuterium atoms, such as 1, 2, 3, 4, ₅ , or 6 deuterium atoms. For example, "deuterated alkyl" refers to a group in which one or more hydrogen atoms of any of the aforementioned alkyl groups (e.g., _C1 -C6 alkyl, _C1 - _C4 alkyl, etc.) are substituted with deuterium atoms, such as monodeuterated methyl, dideuterated methyl, trideuterated methyl, etc. For example, "deuterated cycloalkyl" refers to a group in which one or more hydrogen atoms of any of the aforementioned cycloalkyl groups (e.g., _C3 - _C8 cycloalkyl, _C3 - _C6 cycloalkyl, etc.) are substituted with deuterium atoms, such as monodeuterated cyclopropyl, dideuterated cyclopropyl, trideuterated cyclopropyl, etc.

In this application, the term "amino acid residue" refers to the incomplete amino acid structure remaining after the amino group of an amino acid loses a hydrogen atom and the carboxyl group loses a hydroxyl group.

In this application, the term "antibody" is used in its broadest sense, including intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, provided they possess the desired biological activity. In this application, "antibody" and "immunoglobulin" are used interchangeably.

In this application, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that the antibodies constituting the cluster are identical except for a small number of possible natural mutations. Monoclonal antibodies possess high specificity against a single determinant (epitope) of an antigen, while polyclonal antibodies, in contrast, contain different antibodies targeting different determinants (epitopes). Besides specificity, the advantage of monoclonal antibodies is that their synthesis is unaffected by contamination from other antibodies. The modifier "monoclonal" here indicates that the antibody is characterized by originating from a substantially homogeneous group of antibodies, and should not be construed as requiring special methods for preparation.

In some embodiments of this application, monoclonal antibodies further include chimeric antibodies, i.e., a portion of the heavy chain and/or light chain is identical or homologous to a certain type, class, or subclass of antibody, while the remainder is identical or homologous to another type, class, or subclass of antibody, provided they possess the desired biological activity (see, for example, US 4,816,567; and Morrison et al., 1984, PNAS, 81:6851-6855). Chimeric antibodies that can be used in this application include primatized antibodies, which comprise a variable region antigen-binding sequence from a non-human primate (e.g., ancient monkey, chimpanzee, etc.) and a human constant region sequence.

The monoclonal antibodies used in this application can be produced by many methods. For example, the monoclonal antibodies used in this application can be obtained by hybridoma methods using cells from many species, including mice, hamsters, rats, and humans (see, for example, Kohler et al., 1975, Nature, 256:495), or by recombinant DNA technology (see, for example, US 4,816,567), or isolated from phage antibody libraries (see, for example, Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, Journal of Molecular Biology, 222:581-597).

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

In this application, the term "antigen-binding fragment" of an antibody refers to a polypeptide containing 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 an antigen; it is also referred to as an "antigen-binding moiety." See also Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. In some cases, 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, diabody antibodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.

In this application, the term "Fd fragment" refers to an antibody fragment composed of VH and CH1 domains; the term "Fv fragment" refers to an antibody fragment composed of VL and VH domains of a single arm of an antibody; the term "dAb fragment" refers to an antibody fragment composed of a VH domain (Ward et al., Nature 341:544 546 (1989)); the term "Fab fragment" refers to an antibody fragment composed of VL, VH, CL and CH1 domains; the term "F(ab') ₂ fragment" refers to an antibody fragment containing two Fab fragments connected by a disulfide bridge on the hinge region; the term "Fab'fragment" refers to one of two Fab' fragments formed by breaking the disulfide bond on the hinge region of F(ab') ₂ by reducing the F(ab') ₂ fragment.

In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), where the VL and VH domains pair to form a monovalent molecule by enabling them to generate linkers for a single polypeptide chain (see, for example, Bird et al., Science 242:423 426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879 5883 (1988)). Such scFv molecules may have a general structure: _NH₂ -VL-linker-VH-COOH or _NH₂ -VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS (SEQ ID NO:13) amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 (SEQ ID NO:14) can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers that can be used in this 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.

In this application, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the essential properties of a protein/peptide containing an amino acid sequence. For example, a conservative substitution can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). 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 and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, 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).

Those skilled in the art will understand that in the antibody-drug conjugate of this application, the antibody is linked to the linker-payload via -S-, and -S- is not an externally attached thiol group, but rather a thiol group contained in the antibody itself after the antibody is reduced and the disulfide bond is broken.

In this application, the drug-to-antibody ratio (DAR) refers to the number of drug molecules conjugated to the antibody (e.g., 'a' in Formula I). The number of drug molecules contained in the antibody-drug conjugates described herein can be an integer or a decimal. Whether integer or decimal, it refers to the average number of drug molecules conjugated to each antibody. "a is any value between 1 and 10" means that 'a' can be any integer selected from 1 to 10 (inclusive) or any decimal selected from 1 to 10, such as 3.9 or 4.0. Furthermore, those skilled in the art will understand that even using the same preparation method, the DAR values of antibody-drug conjugates prepared in different batches may not be exactly the same; for example, they may fluctuate within a range not exceeding 0.5.

In this application, the term "about" is understood to mean within +/-10%, +/-9%, +/-8%, +/-7%, +/-6%, +/-5%, +/-4%, +/-3%, +/-2%, +/-1%, +/-0.5%, +/-0.4%, +/-0.3%, +/-0.2%, and +/-0.1% of the stated value. Unless otherwise apparent from the context, all numerical values provided herein are modified by the term "about".

In this application, pharmaceutical excipients refer to the excipients and additives used in the production of pharmaceuticals and the formulation of prescriptions. They are substances, other than the active ingredient, that have undergone reasonable safety assessments and are included in the pharmaceutical preparation. Besides acting as a formifier, carrier, and improving stability, pharmaceutical excipients also have important functions such as solubilization, co-solubilization, and sustained-release. They are important components that may affect the quality, safety, and efficacy of pharmaceuticals. Based on their origin, they can be classified as natural substances, semi-synthetic substances, and fully synthetic substances. Based on their function and use, pharmaceutical excipients can be classified as follows: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, antioxidants, chelating agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, filter aids, release inhibitors, etc. Based on their route of administration, they can be classified as oral, injection, mucosal, transdermal or local, nasal or oral inhalation, and ocular administration, etc. The same pharmaceutical excipient can be used in pharmaceutical preparations with different routes of administration and has different functions and uses.

In this application, the pharmaceutical composition can be formulated into various suitable dosage forms depending on the route of administration. Examples include tablets, capsules, granules, oral solutions, oral suspensions, oral emulsions, powders, tinctures, syrups, injections, suppositories, ointments, creams, pastes, ophthalmic preparations, pills, implants, aerosols, powder inhalers, and sprays. The pharmaceutical composition or suitable dosage form may contain 0.01 mg to 1000 mg of the antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate of this application.

In this application, the term "treatment" is used to alleviate, reduce, improve, or eliminate a targeted disease state or symptom. A subject is successfully "treated" if, in accordance with the methods described herein, a therapeutic amount of the ligand-conjugated drug or its racemic, enantiomer, diastereomer, pharmaceutically acceptable salt, or mixture of the foregoing forms is received, and one or more indications and symptoms exhibit an observable and/or detectable reduction or improvement. It should also be understood that treatment of the disease state or symptom includes not only complete treatment but also the achievement of some biological or medically relevant outcome without achieving complete treatment.

In this application, the term "prevention" aims to avoid, reduce, prevent, or delay the onset of a disease or disease-related symptoms, provided that such disease or disease-related symptoms have not yet appeared before the administration of the relevant drug. "Prevention" does not necessarily require the complete prevention of the onset of a disease or disease-related symptoms. For example, reducing the risk of a subject developing a specific disease or disease-related symptoms after the administration of the relevant drug, or lessening the severity of subsequently occurring related symptoms, can be considered as "prevention" of the onset or development of the disease.

Beneficial effects

1) The antibody-drug conjugate of the present invention has a binding effect on tumor cell lines expressing B7H4, and has excellent binding activity, especially for tumor cell lines with high expression of B7H4.

2) The antibody-drug conjugate of the present invention exhibits endocytic activity in tumor cell lines expressing B7H4, especially in tumor cell lines with high B7H4 expression, where it has excellent endocytic activity.

3) The antibody-drug conjugate of the present invention has excellent inhibitory activity against tumor cell lines expressing B7H4, especially those overexpressing B7H4;

4) The antibody-drug conjugate of the present invention has target-dependent killing activity;

5) The antibody-drug conjugate of the present invention has excellent in vivo proliferation inhibition activity;

6) The antibody-drug conjugate of the present invention has excellent pharmacokinetic properties;

7) The antibody-drug conjugate of the present invention has good plasma stability.

Attached Figure Description

Figure 1. Binding curves of antibodies and antibody-drug conjugates with human breast cancer tumor cell line MX-1.

Figure 2. Endocytotic activity of antibodies and antibody-drug conjugates in human breast cancer tumor cell line MX-1.

Figure 3. Antitumor effect of MH-ADC2 in the MX-1 nude mouse subcutaneous xenograft model.

Figure 4. Results of total antibodies and ADCs in the serum of rats after a single intravenous administration of MH-ADC2.

Figure 5. Results of total antibodies and ADCs in serum after a single intravenous administration of MH-ADC2 to cynomolgus monkeys.

Sequence information

The sequence information involved in this application is shown in Table 1 below.

Table 1

Detailed Implementation

The following description of specific embodiments further illustrates this application, but it is not intended to limit the scope of the application. Those skilled in the art can make various modifications or improvements based on the teachings of this application without departing from its basic ideas and scope. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

In the following examples, the abbreviations of some drugs and their corresponding structural formulas are shown below:

Deruxtecan:

Deruxtecan (i.e., MC-GGFG-DXd) can be prepared according to Example 58 of CN104755494A.

In the following examples, the 2A7-H1 antibody used was obtained by humanizing the VH (SEQ ID NO:7) of the 2A7 antibody in patent WO2007067991A2 (with a one-amino acid mutation: V67F) and mutating the Fc (L234A, L235A). The heavy chain sequence (SEQ ID NO:10), light chain sequence (SEQ ID NO:11), heavy chain variable region sequence (SEQ ID NO:8), light chain variable region sequence (SEQ ID NO:9), heavy chain CDR1 sequence (SEQ ID NO:1), heavy chain CDR2 sequence (SEQ ID NO:2), heavy chain CDR3 sequence (SEQ ID NO:3), light chain CDR sequence (SEQ ID NO:4), light chain CDR2 sequence (SEQ ID NO:5), and light chain CDR3 sequence (SEQ ID NO:6) of the 2A7-H1 antibody are shown in Table 1. The CDR region sequence is defined using the Kabat numbering system, but any other well-known CDR region sequence determination method in the art can also be used to identify the amino acid residues in the CDR region within the variable region.

Example 1. Preparation of linker-drug

Example 1.1

first step

Dissolve 1a (10.00 g, 95.20 mmol) in acetonitrile (100 mL), add tert-butyldimethylchlorosilane (15.06 g, 99.92 mmol), cool the mixture to 0 °C, and add 1,8-diazobisspirocyclic [5.4.0]undec-7-ene (13.73 g, 90.21 mmol) dropwise. After the addition is complete, the reaction system is restored to room temperature and stirred for 16 h. A solid precipitates out. The reaction system is directly filtered, and the filter cake is collected to obtain 1b (11.50 g), yield: 55%.

The MS-ESI calculated value [M+H] ⁺ ＝220, and the measured value is 220.

Step 2

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

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

¹ H NMR (400MHz, DMSO-d ₆ ) δ7.00 (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).

Step 3

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

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

Step 4

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

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

Step 5

1 g (6.00 g, 5.56 mmol) was dissolved in a mixed solvent of DCM/MeOH (120 mL/12 mL). Diethylamine (24 mL) was added dropwise under ice bath conditions. After the addition was complete, the mixture was naturally heated to room temperature and stirred for 4 h. The reaction solution was directly concentrated to obtain a brown solid crude product. Methyl tert-butyl ether was added and the mixture was stirred. The mixture was filtered, and the filter cake was washed three times with methyl tert-butyl ether. The filtrate was concentrated to obtain a yellow solid crude product. 1 g of the crude product was purified by preparative liquid chromatography to obtain 1 h (150 mg). The remaining crude product was purified by silica gel column chromatography (MeOH:DCM = 0-100%) to obtain 1 h (800 mg). The combined yield was 59%.

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

Step 6

Add 1c (65 mg, 0.234 mmol), 1h (0.20 g, 0.23 mmol), and N,N-dimethylformamide (4 mL) sequentially to the reaction flask. After stirring to dissolve, place the flask in an ice bath. Add 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.13 g, 0.35 mmol) and 2,4,6-trimethylpyridine (85 mg, 0.70 mmol). Maintain the mixture at 0–5 °C and stir for 1 h. Adjust the pH of the reaction solution to 4–5 with HCl _aq (0.5 N) and purify using preparative liquid chromatography to obtain compound 1 (70 mg), yield: 14%.

The MS-ESI calculated value [M+H] ⁺ ＝1024, and the measured value is 1024.

^1H NMR (400MHz, DMSO-d6 ₎ )δ8.65(t,J＝6.8Hz,1H),8.60(d,J＝8.8Hz,1H),8.29(q,J＝5.6Hz,2H),8.09(d ,J＝8.0Hz,1H),7.99(t,J＝5.6Hz,1H),7.81(d,J＝11.2Hz,1H),7.80(s,1H),7.2 9-7.14(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),3.64-3.54(m,2 H),3.20-3.09(m,1H),3.00(dd,J＝14.0,4.4Hz,1H),2.73(dd,J＝13.6,9.6Hz, 1H), 2.39 (s, 3H), 2.26-2.17 (m, 2H), 1.94-1.83 (m, 2H), 0.86 (t, J = 7.2Hz, 3H).

Example 1.2

first step

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%.

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

Step 2

Compound 2c (85 mg, 0.19 mmol) was dissolved in N,N-dimethylformamide (1.70 mL), and trifluoroacetic acid (22 mg, 0.19 mmol) was added. The mixture was cooled to 0 °C, and then 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 sequentially. After the addition was complete, the reaction mixture was stirred at 0 °C–10 °C for 1 h. The reaction solution was adjusted to pH 6–7 with HCl _(aq) (0.05 M) and purified by preparative liquid chromatography to obtain compound 2 (134 mg), yield: 49%.

¹ H NMR (400MHz, DMSO-d ₆ )δ8.66(t,J＝6.4Hz,1H),8.60(d,J＝8.8Hz,1H),8.28(t,J＝6.0Hz,1H),8.15(t,J＝5.6Hz,1H),8.09(d,J＝8.0Hz,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＝16.8Hz,1H),5.70-5.62(m,1H),5.56-5.45(m,2H), 5.35(d,J＝18.8Hz,1H),4.68(d,J＝6.8Hz,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＝7.2Hz,3H).

Example 1.3

first step

3a (14.72 g, 150.16 mmol) was dissolved in acetone (118 mL), and 3b (20.00 g, 150.16 mmol) was added under ice bath conditions. The mixture was stirred for 5 min, and the reaction was monitored by TLC until complete. The reaction solution was concentrated to obtain a solid. The solid was dissolved in acetic anhydride (28 mL), and sodium acetate (24.63 g, 300.32 mmol) was added. The reaction system was heated to 90 °C and refluxed for 2 h. The reaction solution was filtered to remove insoluble solids, and the filter cake was washed with toluene. 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 3c (17.43 g), yield: 43%.

MS-ESI calculated value [M+Na] ⁺ ＝236, actual measured value is 236.

Step 2

3c (12.95 g, 60.74 mmol) and 3d (9.00 g, 60.74 mmol) were dissolved in toluene (180 mL), and p-toluenesulfonic acid (2.10 g, 12.15 mmol) was added. The mixture was heated to 90 °C and refluxed for 2 h. The reaction solution was cooled to room temperature and concentrated under reduced pressure to obtain the residue. The residue was dissolved in ethyl acetate, and the organic phase was washed successively with saturated sodium bicarbonate and saturated brine (200 mL x 1). The solution was 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 3e (5.53 g), yield: 33%.

MS-ESI calculated value [M+H] ⁺ ＝270, actual measured value is 270.

Step 3

3e (3.87 g, 14.37 mmol) was dissolved in THF (77.4 mL), and lithium hydroxide (1.37 g, 57.49 mmol) was dissolved in _H₂O (38.7 mL) and added to the above solution. The mixture was stirred at room temperature for 30 min. Ethyl acetate (15 mL) was added to the reaction solution, and the pH was adjusted to about 2 with HCl _(aq) (1N). The aqueous phase was extracted with ethyl acetate (38 mL x 3), and the organic phases were combined. The organic phase was dried over saturated brine (15 mL) and anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain crude product 3f (3.06 g).

The MS-ESI calculated value [M+H] ⁺ ＝260, and the measured value is 260.

Step 4

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 was 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) was added to the reaction solution, followed by the addition of HCl _(aq) (0.7 N, 140 mL). The mixture was stirred for 30 min. The aqueous phase was separated and extracted with dichloromethane (70 mL). The organic phases were combined and 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%.

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

¹ H NMR (400MHz, CDCl ₃ )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).

Step 5

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%.

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

Step 6

Compound 3 (74 mg, 0.16 mmol) was dissolved in N,N-dimethylformamide (1.30 mL), purged three times with nitrogen, and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (88 mg, 0.23 mmol) was added under ice bath conditions. After stirring for 10 min, 1 h (132 mg, 0.16 mmol) was added and stirred until dissolved. Then, 2,4,6-trimethylpyridine (54 mg, 0.45 mmol) was added, and the reaction was continued at 0℃–10℃ for 2 h. The pH of the reaction solution was adjusted to 6–7 with HCl _(aq) (0.05 N), and compound 3 (71 mg) was obtained by preparative liquid chromatography-purification, yield: 34%.

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

Example 1.4

first step

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

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

¹ H NMR (400MHz, CDCl ₃ ) δ8.60 (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).

Step 2

Under a 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), and cesium carbonate (13.53 g, 41.52 mmol) were dissolved in anhydrous toluene (50 mL). The mixture was heated to an internal temperature of 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), yield: 67%.

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

Step 3

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), discarding the organic phase. The pH of the aqueous phase was adjusted to 8-9 with ammonia, and then extracted with ethyl acetate (100 mL x 2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 4e (1.43 g), yield: 86%.

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

Step 4

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 evaporated to dryness to obtain 2.45 g of white solid intermediate. 2.45 g of white solid intermediate was added to a reaction flask, followed by acetic anhydride (5 mL) and sodium acetate (2.09 g, 15.36 mmol). 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 and filtered. The filtrate was concentrated under reduced pressure to obtain the residue. The residue was separated by silica gel column chromatography (EA: hexanes = 0-100%) to obtain 4f (1.97 g), yield: 80%.

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

1H NMR (400MHz, DMSO-d ₆ )δ8.51(d,J＝2.0Hz,1H),7.83(dd,J＝8.4,2.4Hz,1H),7.6(d,J＝8.4Hz,1H),7.24(s,2H),4.67(s,2H),4.15(s,2H),1.44(s,9H).

Step 5

4f (0.90 g, 2.83 mmol) was dissolved in dichloromethane (9 mL), and trifluoroacetic acid (1.8 mL) was added. The mixture was stirred at room temperature for 5 h. The reaction solution was directly evaporated to dryness, and dichloromethane (45 mL x 5) was concentrated with an oil pump until no obvious oily residue was found, yielding 4 g (0.93 g) of crude product.

The MS-ESI calculated value [M+H] ⁺ ＝263, and the measured value is 263.

Step 6

4 g (0.06 g), 1 h (0.20 g, 0.23 mmol), and N,N-dimethylformamide (4 mL) were added sequentially to the reaction flask. After stirring and dissolving, the mixture was placed in an ice bath. 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (0.13 g, 0.35 mmol) and 2,4,6-trimethylpyridine (0.26 g, 2.15 mmol) were added, and the mixture was stirred at 0–5 °C for 1 h. The reaction mixture was adjusted to pH 4–5 with HCl _(aq) (0.5 N) and then transferred to a preparative HPLC system with neutral pure water to prepare and isolate compound 4 (70 mg), yield: 27%.

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

^1H NMR (400MHz, DMSO-d6 ₎ )δ8.65(t,J＝6.8Hz,1H),8.60(d,J＝8.8Hz,1H),8.34-8.24(m,2H),8.09(d, J＝8.0Hz,1H),7.99(t,J＝5.6Hz,1H),7.81(d,J＝11.2Hz,1H),7.79(s,1H),7 .30-7.14(m,5H),7.04(s,2H),6.68(s,1H),5.91(d,J＝16.8Hz,1H),5.70-5 .61(m,1H),5.56-5.45(m,2H),5.36(d,J＝19.6Hz,1H),5.05(t,J＝6.0Hz,1H ),4.68(d,J＝6.4Hz,2H),4.59(dd,J＝9.2,6.0Hz,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＝8.8,4.4Hz,1H),2.74(dd,J＝13.6,9.6Hz,1H) ,2.39(s,3H),2.26-2.18(m,2H),1.94-1.82(m,2H),0.86(t,J=7.6Hz,3H).

Example 2. Preparation of antibody-drug conjugates

1. Preparation of MH-ADC1

MH-ADC1

The 2A7-H1 antibody was dialyzed into 20 mM His/His-HCl pH 6.0 ± 0.2 buffer to obtain the antibody intermediate. An appropriate amount of the antibody intermediate was taken and sequentially added to 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) stock solution and 10 mM diethylenetriaminepentaacetic acid (DTPA) stock solution, followed by the addition of 20 mM His/His-HCl pH 6.0 ± 0.2 buffer to achieve a final antibody concentration of 20 mg/mL, a TCEP to antibody molar ratio of (1.5–3.2):1.0, and a final DTPA concentration of 1 mM. After thorough mixing, the mixture was placed in a constant-temperature mixer for reduction reaction at 20–30 °C and 400 rpm for 1.0–3.0 hours. After reduction, an appropriate amount of 5 mM linker-drug (compound 1) stock solution was added to each reaction system, with a linker-drug to antibody molar ratio of 3.5–8.0. After thorough mixing, the mixture was placed in a constant-temperature mixer for coupling reaction at 20–30°C and 400 rpm for 0.5–2.0 hours to obtain MH-ADC1, in which the Ab is 2A7-H1 antibody. After coupling, the ADC sample was dialyzed into dialysis buffer (10 mM His/His-HCl, pH 6.0 ± 0.2) and stored at ≤-60°C or used for ADC stock preparation.

The relevant process parameters and DAR value test data for products with different DAR values are shown in Table 2 below. The DAR value test method is described in Example 3.

Table 2. MH-ADC1 Sample Preparation Process Parameters and DAR Values

Referring to MH-ADC1, compounds 2, 3, or 4 were conjugated to prepare MH-ADC3, MH-ADC5, and MH-ADC7, respectively, where Ab is the 2A7-H1 antibody and a is a value between 3 and 6.

2. Preparation of MH-ADC2

MH-ADC2

The 2A7-H1 antibody was dialyzed into 10 mM Tris pH 8.0 ± 0.2 buffer to obtain the antibody intermediate. An appropriate amount of the antibody intermediate was taken and sequentially added to 10 mM tris(2-carboxyethyl)phosphonic acid hydrochloride (TCEP) stock solution and 10 mM diethylenetriaminepentaacetic acid (DTPA) stock solution, followed by the addition of 10 mM Tris pH 8.0 ± 0.2 buffer to achieve a final antibody concentration of 20 mg/mL, a TCEP to antibody molar ratio of (1.5–3.5):1, and a final DTPA concentration of 1 mM. After thorough mixing, the mixture was placed in a constant-temperature mixer for reduction at 25°C and 400 rpm for 2 hours. After reduction, an appropriate amount of 5 mM linker-drug (compound 1) stock solution was added to each reaction system, with a linker-drug to antibody molar ratio of (3.0–8.0):1. After thorough mixing, the mixture is placed in a constant-temperature mixer for coupling reaction at 22–37°C and 400 rpm for 16–24 hours to obtain MH-ADC2. The Ab in this mixture is a 2A7-H1 antibody. The Ab is linked to the e- and/or f-positions of the linker via its thiol groups. For example, one or more thiol groups on the Ab are linked to one or more e-positions of the linker, another or more thiol groups are linked to another or more f-positions of the linker, or all thiol groups on the Ab are linked to the e-positions of the linker, or all thiol groups on the Ab are linked to the f-positions of the linker. After coupling, the ADC sample is dialyzed into dialysis buffer (20 mM His/His-HCl, pH 5.2 ± 0.2) and stored at ≤-60°C or used for ADC stock preparation.

The relevant process parameters and DAR value test data for products with different DAR values are shown in Table 3 below. The DAR value test method is described in Example 3.

Table 3. MH-ADC2 Sample Preparation Process Parameters and DAR Values

Referring to MH-ADC2, compounds 2, 3, or 4 were conjugated to prepare MH-ADC4, MH-ADC6, and MH-ADC8, respectively, where Ab is the 2A7-H1 antibody and a is a value between 3 and 6.

3. Preparation of positive control ADC

Positive control ADC

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

Example 3. Detection of Antibody-Drug Conjugate Ratio

High-performance liquid chromatography-hydrophobic chromatography (HIC-HPLC) was used to detect the average drug-to-antibody ratio (DAR) of ADCs. After equilibrating the ADC samples to room temperature and mixing, ultrapure water was used as the diluent to dilute to the target concentration of 5 mg/mL based on the protein concentration, and the mixture was vortexed for 20 seconds. The diluted sample solution was pipetted into an inner tube, and the sample number was marked on the vial. Using an Agilent HPLC system (Agilent, 1260 Bio) and a hydrophobic column (TOSOH, TSKgel Butyl-NPR column (2.5), 4.6 mm * 35 mm), the column was equilibrated before sample detection. The main acquisition method parameters are shown in Table 4. Mobile phase A consisted of 20 mM sodium phosphate, 1.5 M ammonium sulfate, pH 7.0; mobile phase B consisted of 20 mM sodium phosphate, 25% isopropanol (v/v), pH 7.0. Integrate the liquid phase spectrum and calculate the percentage of ADCs with no drug conjugation and those conjugated with 2, 4, 6, and 8 drugs based on the integration results (DAR 0%, DAR 2%, DAR 4%, DAR 6%, and DAR 8%, respectively). Calculate the average drug-antibody conjugation ratio using the following formula.

DAR value=(0×DAR0%+2×DAR2%+4×DAR4%+6×DAR6%+8×DAR8%)/(DAR0%+DAR2%+DAR4%+DAR6%+DAR8%).

Table 4. Parameters for Drug Antibody Conjugate Ratio Acquisition Method

Example 4. Detection of the binding activity of antibody-drug conjugates to human tumor cells

Flow cytometry was used to detect the in vitro binding activity of antibody-drug conjugates (ADCs) and naked antibodies to human tumor cells. Logarithmically growing human tumor cells MX-1 (Cobioer, catalog number CBP60640) were digested with trypsin, resuspended in flow cytometry buffer, counted, centrifuged, and resuspended in pre-chilled flow cytometry buffer to a concentration of 2 × ^10⁶ cells/mL. 50 μL of each well was added to a microplate. 50 μL of the ADC sample, naked antibody 2A7-H1, or buffer control (starting at 20 μg/mL, 4-fold dilution, for a total of 8 test concentrations) was added to the corresponding wells of the microplate. After incubation on ice in the dark for 30 min, the cells were centrifuged at 1000 rpm, the supernatant was discarded, and 200 μL of pre-chilled flow cytometry buffer was added to each well to resuspend the cells. The cells were then centrifuged again, and the supernatant was discarded. This washing process was repeated twice. The secondary antibody (PE Goat anti-Human IgG Fc Secondary Antibody, Invitrogen, catalog number 12-4998-82) was diluted 1:200 with flow cytometry buffer. 100 μL of the diluted secondary antibody was added to each well of a microplate and incubated on ice in the dark for 30 min. After centrifugation and discarding the supernatant, 200 μL of pre-chilled flow cytometry buffer was added to each well to resuspend and wash the cells. 100 μL of fixative (BD, catalog number 554655) was added to each well and the cells were fixed on ice in the dark for 10 min. After centrifugation at 1000 rpm for 5 min and discarding the supernatant, the cells were resuspended and washed twice with 200 μL of pre-chilled flow cytometry buffer. The cells were then resuspended with 100 μL of pre-chilled flow cytometry buffer, and the fluorescence intensity was detected using a flow cytometer (BECKMAN COULTER, CytoFLEX).

The experimental results are shown in Figure 1. The tested ADCs showed strong binding activity to the tumor cell line MX-1, which highly expresses B7H4, and the binding activities of MH-ADC1-2# and MH-ADC2-3# were comparable to those of the naked antibody.

Example 5. Detection of endocytic activity of antibody-drug conjugates in human tumor cells

Logarithmic growth phase human tumor cells MX-1 were harvested, digested with trypsin, resuspended in flow cytometry buffer, counted, centrifuged, and resuspended in pre-chilled flow cytometry buffer to a concentration of 1× ^10⁶ cells/mL. The target ADC sample, naked antibody 2A7-H1 (final concentration 10 μg/mL), or flow cytometry buffer (blank control) was added to the cells. After incubation on ice in the dark for 30 min, the cells were centrifuged at 1000 rpm, the supernatant was discarded, and the cells were washed four times with pre-chilled cell culture medium to remove unbound antibodies or ADCs. After centrifugation and supernatant discarding, the cells were resuspended in pre-chilled cell culture medium, and 100 μL was added to each well of a microplate. The cells were incubated at 4℃ and 37℃ for 0 h, 1 h, 2 h, and 4 h, respectively. After centrifugation at 1000 rpm and supernatant discarding, 200 μL of pre-chilled flow cytometry buffer was added to each well to resuspend the cells, followed by centrifugation and supernatant discarding. The secondary antibody (PE Goat anti-Human IgG Fc Secondary Antibody, Invitrogen, catalog number 12-4998-82) was diluted 1:200 with flow cytometry buffer. 100 μL of the diluted secondary antibody was added to each well of a microplate and incubated on ice in the dark for 30 min. After centrifugation and discarding the supernatant, 200 μL of pre-chilled flow cytometry buffer was added to each well to resuspend and wash the cells. 100 μL of fixative (BD, catalog number 554655) was added to each well and the cells were fixed at 2–8 °C for 30 min. After centrifugation at 1000 rpm for 5 min and discarding the supernatant, the cells were resuspended and washed with 200 μL of pre-chilled flow cytometry buffer, and then centrifuged and the supernatant was discarded. The cells were resuspended with 100 μL of pre-chilled flow cytometry buffer, and the fluorescence intensity was detected using a flow cytometer (BECKMAN COULTER, CytoFLEX). The percentage of endocytosis in the test sample is represented by the ratio of the decrease in fluorescence intensity to the initial fluorescence intensity.

The experimental results are shown in Figure 2. The tested ADCs showed strong endocytic activity in the tumor cell line MX-1, which was highly expressed in B7H4, and the endocytic activity of MH-ADC1-2# and MH-ADC2-3# was comparable to that of the naked antibody.

Example 6. In vitro proliferation inhibition of tumor cells by antibody-drug conjugates

Logarithmic growth phase human tumor cells were digested, resuspended in fresh complete culture medium, and adjusted to an appropriate concentration. 50 μL/well was added to a 96-well cell culture plate. The cell culture plate was incubated overnight at 37°C in a 5% _CO2 incubator. The next day, 50 μL of different concentrations of the target ADC sample, small molecule DMSO solution, or buffer control was added to the corresponding wells of the cell culture plate. The plate was then incubated in a CO2 incubator for 7 days. After equilibration to room temperature, the luminescence values were detected using a CellTiter Glo assay kit (Promega, G7558) and a multi-functional microplate reader (Spark, Tecan). The cell inhibition rate was calculated using the following formula: Survival rate (%) = (RLU _ADC - RLU _blank ) / (RLU _buffer - RLU _blank ) × 100%. The drug efficacy inhibition rate curve was plotted using Prism Graphpad software, and the _IC50 value was calculated (see Tables 5 and 6).

Table 5. Antibody-drug conjugates' inhibitory activity against human tumor cell proliferation in vitro - Experiment 1

Table 6. Antibody-drug conjugates' inhibitory activity against human tumor cell proliferation in vitro - Experiment 2

The experimental results showed that the tested ADC had strong inhibitory activity against multiple tumor cell lines with different target expression levels and B7H4 overexpressing cell lines, and its inhibitory activity against multiple cell lines was significantly better than that of the positive control ADC.

Example 7. Detection of the bystander killing activity of antibody-drug conjugates

HEK293 cell lines stably overexpressing human B7H4 and control HEK293 cell lines in logarithmic growth phase were taken, digested with trypsin, neutralized with fresh culture medium (RPMI 1640 + 10% FBS), centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in RPMI 1640 + 10% FBS. After cell counting, the cell suspension was diluted according to the preset cell density, and the overexpressing cell lines and control cell lines were seeded 1:1 into 96-well cell culture plates with a total seeding volume of 50 μL. Simultaneously, control wells were set up with only HEK293 cells overexpressing B7H4 and only HEK293 control cells. ADC samples were serially diluted, and 50 μL of the diluted ADC sample was added to the wells seeded with co-cultured cells or only overexpressing cell lines and control HEK293 cell lines according to the preset final concentration and well distribution. After mixing, the wells were incubated in a 5% _CO2 incubator at 37°C for 120 h. Remove the cell culture plate, add 100 μL of cell viability assay reagent per well, and detect the cell viability using a multi-functional microplate reader.

The results showed that the ADC in this disclosure has a clear bystander killing effect. Within a certain incubation concentration range, the ADC does not kill control cells that are negative for target expression. However, when target overexpression cells are mixed with negative cells, the ADC also has a killing effect on target negative expression cells.

Example 8. In vivo efficacy detection of antibody-drug conjugate in a mouse subcutaneous xenograft model of human tumor cells

Human tumor cells in the logarithmic growth phase were digested, counted, and resuspended in serum-free culture medium. These cells were then subcutaneously inoculated into immunodeficient mice, with 100 μL inoculated into each animal, to establish a mouse subcutaneous xenograft model. Once the tumors had grown to a measurable size, the major and minor diameters of each tumor were measured using calipers, and the tumor volume was calculated using the following formula: V = (a × ^b² )/2, where a represents the major diameter and b represents the minor diameter.

Once the tumor volume reached an average ^of approximately 100-200 ^mm³ , mice were randomly grouped based on tumor volume and body weight. Tumor-bearing mice were administered either a single dose of the solvent control or different doses of the ADC via tail vein injection, or once every two weeks. The long and short diameters of the tumor were measured twice weekly, and animal body weight was recorded. Tumor volume for each group was statistically analyzed, and the tumor growth inhibition rate (TGI) was calculated using the following formula: TGI = 100% × [1 - ( _TVtT - _TV0T ) / ( _TVtC - _TV0C )]. Where ( _TVtT represents the tumor volume of the treated group on the day of measurement, _TV0T represents the tumor volume of the treated group at the time of grouping; _TVtC represents the tumor volume of the solvent control group on the day of measurement, and _TV0C represents the tumor volume of the solvent control group at the time of grouping).

In a nude mouse subcutaneous xenograft model of human breast cancer MX-1 cells, tumor-bearing nude mice were injected intravenously via tail vein with MH-ADC2-3# at doses of 0.5 mg/kg, 1 mg/kg, and 3 mg/kg (day of administration designated as D0). By the end of the experiment (D18), the tumors in the 3 mg/kg dose group had completely disappeared, while the 1 mg/kg and 0.5 mg/kg doses also significantly inhibited tumor growth. The tumor proliferation inhibition rates (TGI%) for the three dose groups were 105%, 87%, and 68%, respectively. The tumor growth curves are shown in Figure 3. During the experiment, no significant abnormalities or weight loss were observed in the test groups compared to the solvent control group (5% glucose injection), indicating good tolerance. The experimental results demonstrate that the tested ADC exhibits excellent in vivo proliferation inhibition in the MX-1 nude mouse subcutaneous xenograft model and has good safety.

Example 9. Pharmacokinetic Study of Antibody-Drug Conjugate

Different doses of the test ADC were injected into the tail vein of 6-8 week old SD rats at a volume of 5 mL/kg. Blood samples were collected and serum separated before administration (0 h) and at 5 min, 1 h, 4 h, 10 h, 24 h, 48 h, 72 h, 96 h, 168 h, 240 h, 336 h, 504 h, and 672 h after administration. The serum samples were then frozen at -70℃±10℃ for later testing.

Different doses of the test ADC were administered intravenously to cynomolgus monkeys aged 2–5 years (at a fixed interval of 0.5 h). Blood samples were collected at 0 h (before administration) and at 0.5 h, 1 h, 4 h, 10 h, 24 h, 48 h, 72 h, 96 h, 168 h, 240 h, 336 h, 504 h, and 672 h after administration, and serum was separated and stored at -70℃±10℃ for later testing.

ADC detection: Add 100 μL/well of coating working solution (1 μg/mL antitoxin antibody) to the microplate, seal with sealing film, and incubate at 2℃～8℃ for 16h～20h. Discard the liquid in the 96-well microplate, wash the plate 3 times with 300 μL/well washing buffer, and pat the 96-well plate dry on clean absorbent paper to remove residual liquid. Add 300 μL/well blocking buffer and incubate at room temperature for 1.5h～2h. Add 5 μL/well each of the standard curve sample, blank control sample, QC sample, and test sample to the dilution plate wells, then add 245 μL of analysis buffer to the corresponding dilution plate wells, cover with sealing film, and mix on a shaker at room temperature at no less than 500 rpm for no less than 10 minutes. Discard the liquid in the 96-well microplate, wash the plate 3 times with 300 μL/well washing buffer, and pat the 96-well plate dry on clean absorbent paper to remove residual liquid. Add 100 μL/well of the pre-treated sample according to the plate layout diagram. Seal the plate with a sealing film and incubate at room temperature (150 rpm–300 rpm) for 110–120 min. After washing, add 100 μL/well of detection working solution I (50 μg/mL biotin-labeled antigen). Seal the plate with a sealing film and incubate at room temperature (150 rpm–300 rpm) for 55–65 min. After washing and drying the plate again, add 100 μL/well of detection working solution II (SA-HRP, Jackson, catalog number 016-030-084, 1:10000). Seal the plate with a sealing film and incubate at room temperature (150 rpm–300 rpm) for 55–65 min. After washing and drying the plate again, add 100 μL/well of TMB colorimetric working solution. Seal the plate with a sealing film and incubate at room temperature (7–9 min). Add 50 μL/well of stop solution and gently tap to mix. Place the 96-well microplate into the microplate reader, use a detection wavelength of 450nm and a reference wavelength of 620nm for reading. Regression calculations were performed using the SoftMax Pro 7.0.3Gxp software included with the microplate reader.

Total antibody detection: Add 100 μL/well of coating working solution (recombinant human B7H4 protein solution, 0.5 μg/mL) to the microplate, seal with sealing film, and incubate at 2℃～8℃ for 16h～20h. Discard the liquid in the 96-well microplate, wash the plate 3 times with 300 μL/well washing buffer, and pat the 96-well plate dry on clean absorbent paper to remove residual liquid. Add 300 μL/well blocking buffer and incubate at room temperature for 1.5h～2.5h. Add 5 μL/well each of the standard curve sample, blank control sample, QC sample, and test sample to the dilution plate wells, then add 245 μL of analysis buffer to the corresponding dilution plate wells, cover with sealing film, and mix on a shaker at room temperature at no less than 500 rpm for no less than 10 minutes. Discard the liquid in the 96-well ELISA plate. Wash and dry the plate as described above. Add 100 μL/well of the pre-treated sample according to the plate layout diagram. Seal the plate with sealing film and incubate at 25°C and 300 rpm for 115–125 min. Wash and dry the plate again. Add 100 μL/well of the detection working solution (Mouse Anti-Human IgG1-Fc Secondary Antibody (HRP), Sino Biological, catalog number 10702-MM01T-H). Seal the plate with sealing film and incubate at 25°C and 300 rpm for 55–65 min. Wash and dry the plate again. Add 100 μL/well of TMB chromogenic working solution. Seal the plate with sealing film and incubate at 25°C for 8–12 min. Add 50 μL/well of stop solution and gently tap to mix. Place the 96-well ELISA plate in a microplate reader and read the plate at a detection wavelength of 450 nm and a reference wavelength of 620 nm. Regression calculations were performed using the SoftMax Pro 7.0.3Gxp software that comes with the microplate reader.

After a single intravenous injection of MH-ADC2-3# at doses of 2, 6, and 20 mg/kg into SD rats, there were no significant differences in serum ADC and total anti-antibody curves among the groups (see Figure 4). Serum ADC and total anti-antibody exposure levels in the animals generally increased with increasing dose, exhibiting linear pharmacokinetic characteristics.

After a single intravenous injection of MH-ADC2-3# at doses of 1, 3, and 10 mg/kg in cynomolgus monkeys, there were no significant differences in serum ADC and total anti-antibody curves among the groups (see Figure 5). The increase in serum ADC and total anti-antibody exposure levels in the animals was roughly proportional to the increase in dose, exhibiting linear pharmacokinetic characteristics.

Example 10. Antibody-Drug Conjugate Mass Spectrometry Analysis and Ring-Opening Confirmation

Ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-QFS-TOF-MS) was used to determine the intact molecular weight of the antibody-drug conjugate after N-sugar removal. The ring-opening status of the antibody-drug conjugate was confirmed by comparing the intact molecular weight after N-sugar removal with the theoretical molecular weight. 500 μg of sample was placed in an ultrafiltration centrifuge tube and centrifuged at 12,000 rpm for 10 min. 5 μL of N-glucosidase and 95 μL of water were added to the ultrafiltration tube containing the sample and mixed thoroughly (enzyme:sample = 1:100), resulting in a final concentration of approximately 5 mg/mL. The centrifuge tube containing the sample was incubated in a 37℃ water bath for 16-20 h. After removing the N-sugar, 95 μL of the sample was transferred to an inner tube, placed in a sample vial, and then placed in the instrument's sample tray. Instrument and key parameter information are shown in the table below.

Table 7. Key Parameter Information Table

The ring-opening status of the samples was confirmed by comparing the intact molecular weight of the de-N-sugar corresponding to each peak with the theoretical molecular weight. The detection results of MH-ADC2-3# are shown in Table 8. The detection results confirmed that the intact molecular weight of the de-N-sugar of each component was consistent with its theoretical value, confirming ring opening. In Example 2, the intact molecular weights of the de-N-sugar of each component of the remaining MH-ADC2 molecules, as well as MH-ADC4, MH-ADC6, and MH-ADC8 molecules, were also consistent with the corresponding theoretical molecular weights after ring opening, confirming ring opening.

Table 8. Results of molecular weight determination of N-glycan-free complete molecules (MH-ADC2-3#)

Example 11. Plasma stability study of antibody-drug conjugates

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. The sample was incubated at 37°C in a biochemical incubator for different time points, and then collected. The samples were purified by protein A chromatography or by antigen-conjugated magnetic beads. The purified ADC samples were concentrated by ultrafiltration and centrifugation, and then deglycosylated and analyzed according to the method described in Example 10. Based on molecular weight, the mass number of different numbers of small molecule drugs conjugated was analyzed. The average conjugation ratio (DAR) was calculated based on the percentage of each peak area and the number of conjugated drugs. The plasma stability of the ADC was studied by the change in DAR value after different incubation times. The experimental results showed that the ADC prepared in this application (e.g., MH-ADC2-3#) has good plasma stability. After different incubation times, the DAR value showed no significant change compared to 0 h, or the DAR value changed only slightly.

This document describes several embodiments, but these descriptions are exemplary and not limiting, and it will be apparent to those skilled in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein. Although many possible combinations of features are shown herein and discussed in detail, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature or element of any embodiment may be used in combination with, or substitute for, any feature or element of any other embodiment.

Claims (12)

The antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, as shown in formula (I),
Ab-(LD) _a
Formula (I) in: Ab represents an antibody or its antigen-binding fragment; L is ^L1 - ^L2 - ^L3 - ^L4 , where ^L1 is connected to Ab and ^L4 is connected to D; ^L1 is selected from Ra ^is selected from hydrogen, _C1 - _C6 alkyl, _C1 - _C6 deuterated alkyl, _C3 - _C8 cycloalkyl, and _C3 - _C8 deuterated cycloalkyl; ^L2 is selected from -( _CH2 ) _m - _X1 - _X2- ( _CH2 ) _n- ( _CH2OCH2 ) _p- ( _{CH2 ) q} -C(=O)- and -( _C1 - _C4 )alkylene-C(=O)-, wherein, X ₁ is selected from C1, _C3 - _C8 cycloalkyl, 3-8 membered heterocyclic, _C6 - _C10 aryl, and 5-9 membered heteroaryl. X ₂ is selected from bond, -O-, -NH-, -C(=O)- and -C(=O)NH-, m, n, p, and q are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. _The -( _CH2 ) _m - _X1 - _X2- ( _CH2 ) _n- ( _CH2OCH2 ) _p- ( _CH2 ) _q -C(=O)- and -( _C1 - _C4 )alkylene-C(=O)- are optionally substituted by one or more groups selected from hydrogen, halogen, hydroxyl, amino, -( _C1 - _C4 )alkylene-hydroxyl and -O-( _C1 - _C4 )alkylene-hydroxyl; ^L3 is selected from amino acid residues and peptide residues consisting of 2-10 amino acid residues; ^L4 is The * terminal is connected to D; D is ^R1 and ^R2 are each independently selected from hydrogen, deuterium, halogen, hydroxyl, amino, _C1 - _C6 alkyl, _C1 - _C6 deuterated alkyl, _C1 - _C6 haloalkyl, and _C3 - _C8 cycloalkyl, or, ^R1 and ^R2 together with the carbon atom they are attached to form _C3 - _C6 cycloalkyl or 3-6 membered heterocyclic groups; ^R3 and ^R4 are each independently selected from hydrogen, deuterium, halogen, _C1 - _C6 alkyl, _C1 - _C6 deuterated alkyl, and _C1 - _C6 haloalkyl, or, ^R3 and ^R4 together with the carbon atoms they are attached to form a 5-6 membered heterocyclic group, which may be optionally substituted by one or more groups selected from hydrogen, deuterium, halogen, hydroxyl, amino, _C1 - _C6 alkyl, _C1 - _C6 deuterated alkyl and _C1 - _C6 haloalkyl; a can be any value between 1 and 10. The antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate according to claim 1, wherein, ^Ra is selected from hydrogen, _C1 - _C4 alkyl, and _C1 - _C4 deuterated alkyl; Preferably, ^Ra is selected from hydrogen, methyl, ethyl, isopropyl, deuterated methyl, deuterated ethyl, and deuterated isopropyl; Preferably, ^L1 is selected from The * end is connected to ^L2 . The antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate according to claim 1 or 2, wherein, X ₁ is selected from 5-6 membered heterocyclic groups and 5-6 membered heteroaryl groups. Preferably, _X1 is selected from bonds, 5-6 membered heterocyclic groups, and 5-6 membered heteroaryl groups, wherein the 5-6 membered heterocyclic group contains one or two heteroatoms selected from N and O, and the 5-6 membered heteroaryl group contains one, two, or three heteroatoms selected from N and O. Preferably, _X1 is selected from alkyl, pyrrolidinyl, tetrahydrofuranyl, 1,3-dioxocyclopentyl, 1,3-dioxocyclohexyl, pyridyl, and pyrimidinyl. Preferably, _X1 is selected from 1,3-dioxocyclopentyl, 1,3-dioxocyclohexyl, and pyridyl; X ₂ is selected from the bond and -C(＝O)NH-, Preferably, _X2 is -C(=O)NH-; m can be 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 is selected from -(CH ₂ ) _m -X ₂ -(CH ₂ ) _n -(CH ₂ OCH ₂ ) _p -C(＝O)-, -(CH ₂ ) _m -X ₁ -X ₂ -(CH ₂ ) _n -(CH ₂ OCH ₂ ) _p -(CH ₂ ) _q -C(＝O)-, -X ₁ -(CH ₂ OCH ₂ ) _p -C(＝O)-, -(CH ₂ ) _n -(CH ₂ OCH ₂ ) _p -(CH ₂ ) _q -C(=O)- and -(C ₁ -C ₄ )alkylene-C(=O)-, said -(CH ₂ ) _m -X ₂ -(CH ₂ ) _n -(CH ₂ OCH ₂ ) _p -C(=O)-, -(CH ₂ ) _m -X ₁ -X ₂ -(CH ₂ ) _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 by one or more groups selected from hydrogen, hydroxyl, amino, -methylene-hydroxyl, -O-ethylene-hydroxyl; Preferably, ^L2 is selected from _{-CH2CH2 -} C(=O)NH- _CH2- ( _CH2OCH2 ) _{8- CH2} -C( ₌ O)-, -CH ₂ -C(=O)-, -CH ₂ CH ₂ -C(=O)-, -CH(CH ₃ )-C(=O)-, -CH(CH ₂ CH ₃ )-C(=O)-, -CH(CH ₃ )CH ₂ -C(=O)-, and -CH ₂ CH(CH ₃ )-C(=O)-, which -CH ₂ CH ₂ -C(=O)NH-CH ₂ -(CH ₂ OCH ₂ ) ₈ -CH ₂ -C(=O)-, _-CH2 -C(＝O)-, _-CH2CH2- C(＝O)-, _-CH ( _CH3 )-C(＝O)-, -CH( _CH2CH3 )-C(＝O)-, -CH( _CH3 ) _CH2 -C(＝O)- _, and _-CH2CH ( _CH3 )-C(＝O)- may be optionally substituted by one or more groups selected from H, OH, _NH2 , _CH2OH , and -O- _{CH2CH2OH ;} Preferably, ^L2 is _selected from -CH( _CH2OH )-C(＝O)-, -CH _{( CH2OCH2CH2OH} )-C(＝O)-, Preferably, ^L2 is -CH( _CH2OH )-C(＝O)-; Preferably, ^L2 is Preferably, ^L2 is The * end is connected to ^L3 . The antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate according to any one of claims 1-3, wherein, ^L3 is a peptide residue composed of 2-4 (preferably 4) amino acid residues, wherein the amino acid is selected from glycine, phenylalanine, valine, alanine, lysine, citrulline, serine, glutamic acid and aspartic acid, preferably, the amino acid is selected from glycine and phenylalanine. Preferably, ^L3 is glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly) (SEQ ID NO:12); Preferably, ^L3 is Preferably, ^L3 is The * end is connected to ^L4 . The antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate according to any one of claims 1-4, wherein, ^R1 and ^R2 are each independently selected from hydrogen, deuterium, _C1 - _C4 alkyl, _C1 - _C4 deuterated alkyl, _C1 - _C4 haloalkyl, and _C3 - _C6 cycloalkyl. Preferably, ^R1 and ^R2 are each independently selected from hydrogen, deuterium, methyl, deuterated methyl, halomethyl, and cyclopropyl. Preferably, ^R1 and ^R2 are each independently hydrogen; or, ^R1 and ^R2, together with the carbon atoms they are attached to, form cyclopropyl, cyclobutyl, oxocyclobutyl, and aziridine. Preferably, ^R1 and ^R2 together with the carbon atoms they are attached to form cyclopropyl and cyclobutyl groups; ^R3 and ^R4 are each independently selected from hydrogen, deuterium, halogen, _C1 - _C4 alkyl, _C1 - _C4 deuterated alkyl, and _C1 - _C4 haloalkyl. Preferably, ^R3 and ^R4 are each independently selected from hydrogen, deuterium, fluorine, chlorine, methyl, deuterated methyl, and halomethyl. Preferably, ^R3 is a methyl group. Preferably, ^R4 is fluorine; or, ^R3 and ^R4, together with the carbon atoms they are attached to, form a 5-6 heterocyclic group, which contains one or two heteroatoms selected from O. The 5-6 heterocyclic group may optionally be substituted with one or more groups selected from hydrogen, deuterium, halogens, and _C1 - _C4 alkyl groups. Preferably, ^R3 and ^R4 together with the carbon atoms they are bonded to form The It may be optionally substituted with one, two, or three groups selected from hydrogen, deuterium, and fluorine; Preferably, D is The antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate according to any one of claims 1-5, wherein each L is independently selected from:

Alternatively, each LD can be selected independently from:



The antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate according to any one of claims 1-6, wherein, Ab is an anti-B7-H4 antibody or its antigen-binding fragment; Preferably, the antibody or its antigen-binding fragment comprises: (a) The following three heavy chain variable region (VH) complementarity-determining regions (CDRs): (i) VH CDR1, having the sequence of CDR1 contained in VH as shown in SEQ ID NO:7 or SEQ ID NO:8, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of CDR1 contained in VH. (ii) VH CDR2, having the sequence of CDR2 contained in VH as shown in SEQ ID NO:7 or SEQ ID NO:8, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to the sequence of CDR2 contained in VH; and (iii) VH CDR3, having the CDR3 sequence contained in VH as shown in SEQ ID NO:7 or SEQ ID NO:8, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the CDR3 sequence contained in VH; and/or (b) The following three light chain variable region (VL) CDRs: (iv) VL CDR1, having the sequence of CDR1 contained in VL as shown in SEQ ID NO:9, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of CDR1 contained in VL. (v) VL CDR2, having the sequence of CDR2 contained in VL as shown in SEQ ID NO:9, or having a sequence with one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of one or two amino acids) compared to the sequence of CDR2 contained in VL; and (vi) VL CDR3, having the sequence of CDR3 contained in VL as shown in SEQ ID NO:9, or having a sequence with one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to the sequence of CDR3 contained in VL. Preferably, the substitution described in any one 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 its antigen-binding fragment comprises: (a) The sequences of CDR1, CDR2 and CDR3 contained in VH as shown in SEQ ID NO:7 or SEQ ID NO:8; and/or (b) The CDR1, CDR2 and CDR3 sequences contained in the VL shown in SEQ ID NO:9; Preferably, the antibody or its antigen-binding fragment comprises: (a) The following three heavy chain variable region (VH) CDRs: (i) VH CDR1, which consists of the following sequence: SEQ ID NO:1, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to SEQ ID NO:1. (ii) VH CDR2, which consists of the following sequence: SEQ ID NO:2, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to SEQ ID NO:2, and (iii) VH CDR3, which consists of the following sequence: SEQ ID NO:3, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to SEQ ID NO:3; and/or (b) The following three light chain variable region (VL) CDRs: (iv) VL CDR1, which consists of the following sequence: SEQ ID NO:4, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to SEQ ID NO:4. (v) VL CDR2, which consists of the following sequence: SEQ ID NO:5, or a sequence having one or more amino acid substitutions, deletions, or additions (e.g., substitutions, deletions, or additions of 1 or 2 amino acids) compared to SEQ ID NO:5, and (vi) VL CDR3, which consists of the following sequence: SEQ ID NO:6, or a sequence having one or more amino acid substitutions, deletions or additions (e.g., substitutions, deletions or additions of 1 or 2 amino acids) compared to SEQ ID NO:6; Preferably, the substitution described in any one of (i)-(vi) is a conservative substitution; Preferably, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:1; VH CDR2 as shown in SEQ ID NO:2; and VH CDR3 as shown in SEQ ID NO:3; and/or, the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:4; VL CDR2 as shown in SEQ ID NO:5; and VL CDR3 as shown in SEQ ID NO:6; Preferably, the VH of the antibody or its antigen-binding fragment comprises: VH CDR1 as shown in SEQ ID NO:1; VH CDR2 as shown in SEQ ID NO:2; and VH CDR3 as shown in SEQ ID NO:3; and the VL of the antibody or its antigen-binding fragment comprises: VL CDR1 as shown in SEQ ID NO:4; VL CDR2 as shown in SEQ ID NO:5; and VL CDR3 as shown in SEQ ID NO:6; Preferably, the amino acid sequence of the heavy chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region of the antibody or its antigen-binding fragment is as shown in SEQ ID NO:9; Preferably, the heavy chain amino acid sequence of the antibody is shown in SEQ ID NO:10, and the light chain amino acid sequence of the antibody is shown in SEQ ID NO:11; Preferably, the antibody or its antigen-binding fragment is linked to L via its thiol group. The antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate according to any one of claims 1-7, wherein, Each 'a' is an independent value between 1 and 8; Preferably, each 'a' is an independent value between 3 and 8; Preferably, each 'a' is an independent value between 3 and 6, such as 3-4, 4-5, 5-6, or for example, about 3.1, about 3.3, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.9, about 5.1, about 5.3, about 5.5. The antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate according to any one of claims 1-8, wherein the antibody-drug conjugate represented by formula (I) is selected from:


in, The definition of Ab is as described in claim 1 or 7, and the definition of a is as described in claim 1 or 8; In MH-ADC2, MH-ADC4, MH-ADC6, and MH-ADC8, an Ab (e.g., through its thiol group) is attached to the e-position and/or the f-position. For example, one or more thiol groups on the Ab are attached to the e-position, another or more thiol groups are attached to the f-position, or all thiol groups on the Ab are attached to the e-position, or all thiol groups on the Ab are attached to the f-position. A pharmaceutical composition comprising the antibody-drug conjugate or its stereoisomer as described in any one of claims 1-9, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, and optionally one or more pharmaceutical excipients. A composition comprising at least one antibody-drug conjugate or stereoisomer of any one of claims 1-9, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate; Preferably, the composition comprises the antibody-drug conjugate MH-ADC1 or its stereoisomer as described in claim 9, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, and the antibody-drug conjugate MH-ADC2 or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate. Preferably, the composition comprises the antibody-drug conjugate MH-ADC3 or its stereoisomer as described in claim 9, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, and the antibody-drug conjugate MH-ADC4 or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate. Preferably, the composition comprises the antibody-drug conjugate MH-ADC5 or its stereoisomer as described in claim 9, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, and the antibody-drug conjugate MH-ADC6 or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate. Preferably, the composition comprises the antibody-drug conjugate MH-ADC7 or its stereoisomer as described in claim 9, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, and the antibody-drug conjugate MH-ADC8 or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate. Use of the antibody-drug conjugate or its stereoisomer, its prodrug, its pharmaceutically acceptable salt or its pharmaceutically acceptable solvate, or the pharmaceutical composition of claim 10 or the composition of claim 11 in the preparation of a medicament targeting B7-H4, or in the preparation of a medicament for the treatment and/or prevention of disease. Preferably, the disease is a disease related to B7-H4; Preferably, the disease is one associated with abnormal B7-H4 expression; Preferably, the disease is cancer or an autoimmune disease; More preferably, the disease is selected from breast cancer, ovarian cancer, endometrial cancer, and bile duct cancer.