B7H4 antibody-drug conjugates and uses thereof

Abstract

The present invention provides an anti-B7H4 antibody-drug conjugate that specifically binds to B7H4 and a composition comprising the same, the structure of the anti-B7H4 antibody-drug conjugate being as shown in formula (I-1). The present invention further provides a method and use of the antibody-drug conjugate of the present invention. The anti-B7H4 antibody-drug conjugate of the present invention has better inhibitory activity against the in vitro proliferation of tumor cells and better tumor inhibitory effect in vivo. [Formula 1] JPEG2025510606000106.jpg66165

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from Chinese patent application No. 202210334522X, filed on March 30, 2022. This application incorporates the entire text of the above Chinese patent application by reference. The present invention provides an antibody-drug conjugate that specifically binds to B7H4 and a composition comprising the same. Methods and uses of the antibody-drug conjugate of the present invention are further provided. [Background technology]

[0002] Immune checkpoint inhibitors are the most common form of immunotherapy in oncology research. Immune checkpoint molecules are often highly expressed in the tumor microenvironment and allow tumors to evade immune system attacks by suppressing T cell activation and inducing T cell exhaustion. The B7 family and the TNF family are two major families of costimulatory molecules, and the B7 family currently has 10 molecules, namely CD80 (B7.1), CD86 (B7.2), B7H1 (PD-L1 / CD274), B7-DC (PD-L2 / CD273), B7H2 (ICOSL), B7H3 (CD276), B7H4 (B7S1 / B7x / Vtcn1), B7H5 (VISTA), B7H6 and B7H7 (HHLA2). Several members of the B7 family and their receptors have already been proven to be immune checkpoints, such as PD-L1 / PD1, CTLA4 and VISTA.

[0003] B7H4 is a relatively new B7 family member that is widely expressed in living cells at the mRNA level, but its protein level expression is very limited, with low levels of expression only in some ductal epithelial cells of the body, such as breast ducts and lobules, fallopian tube epithelium, endometrium, and other tissues. In contrast, B7H4 is highly expressed in various tumor tissues, such as breast cancer, especially triple-negative breast cancer, ovarian cancer, and endometrial cancer, in tumor cells. From the perspective of expression profile, B7H4 can be considered as a highly specific tumor-associated antigen. On the one hand, B7H4 is a novel immune checkpoint molecule, and in vitro, it has been demonstrated that B7H4 inhibits T cell proliferation, activation, and cytokine production by interacting with its unknown T cell surface receptor. Tumor cells suppress T cell activation by highly expressing B7H4 molecules and by suppressive macrophages that highly express B7H4 molecules in the tumor microenvironment, thereby achieving immune evasion. The B7H4 expression profile in tumors does not overlap with PD-L1. Antibody-targeted therapy against B7H4 and reactivation of the immune system by blocking the negative regulatory effects of B7H4 are promising avenues for treating tumors positive for B7H4 expression.

[0004] At present, many pharmaceutical companies are developing monoclonal antibodies, drug conjugates, or bispecific antibodies against B7-H4. The antibody-drug conjugates on the market include Adcetris and Kadcyla. At present, many multinational pharmaceutical companies are developing monoclonal antibodies or drug conjugates against B7-H4 to improve the immune system response to the patient's own tumor and achieve the goal of directly killing tumor cells. Related patents include W02013025779, US20140322129, etc. Anti-B7-H4 monoclonal antibodies from companies such as Medimmune and FivePrime are currently in preclinical development, and Genentech's anti-B7-H4 antibody-drug conjugate is also already in the preclinical development stage. Summary of the Invention [Problem to be solved by the invention]

[0005] The technical problem to be solved by the present invention is to provide an anti-B7H4 antibody-drug conjugate and its preparation method and use, in order to overcome the drawback of the limited number of anti-B7H4 antibody-drug conjugates in the prior art. Compared with the prior art, the anti-B7H4 antibody-drug conjugate of the present invention has one or more advantageous effects selected from the following group: (1) better inhibitory activity against in vitro proliferation of tumor cells; (2) better endocytosis; (3) better tumor inhibitory effect in vivo; (4) better affinity to human and monkey B7H4; (5) better targeting to B7H4; (6) better bystander killing effect; (7) better plasma stability; and (8) better safety. [Means for solving the problem]

[0006] The present invention solves the above technical problems mainly by the following technical means. In one aspect, the present application provides an anti-B7H4 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the structure of the anti-B7H4 antibody-drug conjugate is represented by formula (I): Ab-(LMD) p (I) During the ceremony, L and M are linker units, D is a cytotoxic drug, p represents the average number of bonds, and p is selected from an integer or decimal number of 1 to 10, preferably an integer or decimal number of 3 to 8; The Ab is an anti-B7H4 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0007] In one aspect, the present application provides an anti-B7H4 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the structure of the anti-B7H4 antibody-drug conjugate is represented by formula (I): Ab-(LMD) p (I) During the ceremony, L and M are linker units, -MD is a cytotoxic drug, p represents the average number of bonds, and p is selected from an integer or decimal number of 1 to 10, preferably an integer or decimal number of 3 to 8; The Ab is an anti-B7H4 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0008] In one aspect, the present application provides an anti-B7H4 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the structure of the anti-B7H4 antibody-drug conjugate is represented by formula (I-1): [ka] During the ceremony, M is -L 2 -L 1 -C(O)-, L 2 is -O- or -S-, L 1 is -(C(R 1a )(R 1b )) m -CH 2 -, C 3 -C 6 is a saturated cycloalkyl or a 3- to 6-membered saturated heterocyclyl, 3 -C 6The saturated cycloalkyl and the 3- to 6-membered saturated heterocyclyl each independently optionally have one or more R 2a is replaced by m is selected from 1, 2, 3, or 4; the heteroatoms in the 3- to 6-membered saturated heterocyclyl are selected from N, O, and S, and the number of heteroatoms is 1 to 3; R 1a are each independently hydrogen, halogen, hydroxy, amino, or C 1 -C 6 alkyl, 1 -C 6 The alkyl is optionally substituted with one or more R; R 1b and R 2a are each independently hydrogen, halogen, hydroxy, amino, or C 1 -C 6 alkyl, 1 -C 6 The alkyl is optionally substituted with one or more R; Each R is independently hydrogen or halogen; L is a linker unit, p represents the average number of bonds, and p is selected from an integer or decimal number of 1 to 10; Ab is an anti-B7H4 antibody or an antigen-binding fragment thereof. In some embodiments, the average bond number p is preferably any integer or decimal number from 3 to 8.

[0009] In certain preferred embodiments of the present invention, certain groups in the compounds represented by formula (I-1), (II-1), or (II-2) or pharma- ceutically acceptable salts thereof are defined below, and groups not mentioned are as described in any of the embodiments of this application (abbreviated as "in some embodiments").

[0010] In some embodiments, the anti-B7H4 antibody-drug conjugate of the present invention, its isomer, its pharma- ceutically acceptable salt, or mixtures thereof, wherein R 1aeach independently represents halogen, hydroxy, amino, or C 1 -C 6 alkyl, 1 -C 6 The alkyl is optionally substituted with one or more R, each of which is independently hydrogen or halogen.

[0011] In some embodiments, the anti-B7H4 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively.

[0012] In some embodiments, the anti-B7H4 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO:7, or at least 95%, 96%, 97%, 98% or 99% identity thereto, and a light chain variable region having an amino acid sequence set forth in SEQ ID NO:8, or at least 95%, 96%, 97%, 98% or 99% identity thereto. In some embodiments, the anti-B7H4 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:7, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:8.

[0013] In some embodiments, the anti-B7H4 antibody or antigen-binding fragment thereof of the present invention is a murine antibody or fragment thereof, a chimeric antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, or a fully human antibody or antigen-binding fragment thereof. In some embodiments, the anti-B7H4 antibody or antigen-binding fragment thereof of the invention is a humanized antibody or fragment thereof. In some embodiments, the anti-B7H4 antibody or antigen-binding fragment thereof of the present invention is a Fab, Fab', Fab'-SH, Fv, scFv, F(ab') 2 , an sdAb, a bispecific antibody or a linear antibody. In some embodiments, the anti-B7H4 antibodies of the invention are monoclonal antibodies.

[0014] In some embodiments, the antibody of the present invention is an IgG1 type antibody, an IgG2 type antibody, an IgG3 type antibody, or an IgG4 type antibody. In some embodiments, the antibody of the invention is an IgG1 type antibody. In some embodiments, the anti-B7H4 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain having an amino acid sequence set forth in SEQ ID NO:9, or at least 95%, 96%, 97%, 98% or 99% identity thereto, and a light chain having an amino acid sequence set forth in SEQ ID NO:10, or at least 95%, 96%, 97%, 98% or 99% identity thereto.

[0015] In some embodiments, the anti-B7H4 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:9, and a light chain having the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the anti-B7H4 antibody of the present invention is anti-B7H4 antibody DB1001. The amino acid sequence of DB1001 is as shown in the sequence listing. In some embodiments, in the anti-B7H4 antibody-drug conjugate of the present invention, its isomer, its pharma- ceutically acceptable salt, or a mixture thereof, L of M 2 The termini are attached to linker units L.

[0016] In some embodiments, the anti-B7H4 antibody-drug conjugate of the present invention, its isomer, its pharma- ceutically acceptable salt, or mixtures thereof, wherein L 1 is-(C(R 1a )(R 1b ))m -CH 2 - and R 1a is hydrogen, halogen and C 1 -C 6 alkyl; R 1b is hydrogen, halogen and C 1 -C 6 alkyl, e.g. R 1a is halogen and C 1 -C 6 alkyl; R 1b is hydrogen, halogen and C 1 -C 6 is selected from alkyl.

[0017] In some embodiments, the anti-B7H4 antibody-drug conjugate of the present invention, its isomer, its pharma- ceutically acceptable salt, or mixtures thereof, wherein L 1 is-(C(R 1a )(R 1b )) m -CH 2 - and R 1a is hydrogen or -CH 3 and R 1b is hydrogen and -CH 3 For example, R 1a Ha-CH 3 and R 1b is hydrogen and -CH 3 is selected from. In some embodiments, the anti-B7H4 antibody-drug conjugate of the present invention, its isomer, its pharma- ceutically acceptable salt, or mixtures thereof, wherein L 1 is -(C(R 1a )(R 1b )) m -CH 2 and m is 1 or 2.

[0018] In some embodiments, the anti-B7H4 antibody-drug conjugate of the present invention, its isomer, its pharma- ceutically acceptable salt, or mixtures thereof, wherein L 1 teeth, [ka] In the formula, the left side of the structural fragment is preferably selected from L 2 is bound to.

[0019] In some embodiments, the anti-B7H4 antibody-drug conjugate of the present invention, its isomer, its pharma- ceutically acceptable salt, or mixtures thereof, wherein L 1 is C 3 -C 6 is a saturated cycloalkyl or a 3- to 6-membered saturated heterocyclyl, 3 -C 6 The saturated cycloalkyl and the 3- to 6-membered saturated heterocyclyl each independently optionally have one or more R 2a is replaced by R 2a are each independently hydrogen, halogen, or C 1 -C 6 is selected from alkyl. In some embodiments, the anti-B7H4 antibody-drug conjugate of the present invention, its isomer, its pharma- ceutically acceptable salt, or mixtures thereof, wherein L 2 and -C(O)- is C 3 -C 6 It is attached to different atoms of a saturated cycloalkyl or a 3- to 6-membered saturated heterocyclyl.

[0020] In some embodiments, the anti-B7H4 antibody-drug conjugate of the present invention, its isomer, its pharma- ceutically acceptable salt, or mixtures thereof, wherein L 1 may optionally be one or more R 2a C replaced with 3 -C 6 is a saturated cycloalkyl, R 2a are each independently hydrogen, halogen, or C 1 -C 6 is selected from alkyl. In some embodiments, the anti-B7H4 antibody-drug conjugate of the present invention, its isomer, its pharma- ceutically acceptable salt, or mixtures thereof, wherein L 1 is C 3 -C 6It is a saturated cycloalkyl. For example, L 1 is cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, preferably cyclobutyl.

[0021] In some embodiments, the anti-B7H4 antibody-drug conjugate of the present invention, its isomer, its pharma- ceutically acceptable salt, or mixtures thereof, wherein L 1 may optionally contain one, two or three R 2a Replaced by [ka] and R 2a are each independently hydrogen, halogen, or C 1 -C 6 is selected from alkyl.

[0022] In some embodiments, the anti-B7H4 antibody-drug conjugate of the present invention, its isomer, its pharma- ceutically acceptable salt, or mixtures thereof, wherein L 1 teeth, [ka] is selected from.

[0023] In some embodiments, the anti-B7H4 antibody-drug conjugate, its isomer, pharma- ceutically acceptable salt, or mixture thereof of the present invention comprises a compound comprising the formula: M is -L 2 -L 1 -C(O)-, L 2 is -O-, L 1 is -(C(R 1a )(R 1b )) m -CH 2 - or C 3 -C 6 is a saturated cycloalkyl, 3 -C 6 A saturated cycloalkyl may optionally have one or more R 2a is replaced by m is selected from 1 or 2; R 1a are each independently hydrogen, halogen, or C 1 -C 6 alkyl, 1 -C 6 The alkyl is optionally substituted with one or more R; R 1b and R 2a are each independently hydrogen, halogen, or C 1 -C 6 alkyl, 1 -C 6 The alkyl is optionally substituted with one or more R; Each R is independently hydrogen or halogen.

[0024] In some embodiments, the anti-B7H4 antibody-drug conjugate, its isomer, pharma- ceutically acceptable salt, or mixture thereof of the present invention comprises a compound comprising the formula: M is -L 2 -L 1 -C(O)-, L 2 is -O-, L 1 is -(C(R 1a )(R 1b )) m -CH 2 - or alternatively 1, 2 or 3 R 2a Replaced with [ka] and m is selected from 1 or 2; R 1a each independently represents a halogen and C 1 -C 6 alkyl, 1 -C 6 The alkyl is optionally substituted with one or more R; R 1b and R 2a are each independently hydrogen, halogen, or C1 -C 6 alkyl, 1 -C 6 The alkyl is optionally substituted with one or more R; Each R is independently hydrogen or halogen. In some embodiments, the anti-B7H4 antibody-drug conjugate, its isomer, its pharma- ceutically acceptable salt, or mixture thereof of the present invention, wherein -M- is [ka] is selected from.

[0025] In some embodiments, the anti-B7H4 antibody-drug conjugate, its isomer, its pharma- ceutically acceptable salt, or mixture thereof of the present invention, wherein -M- is [ka] It is.

[0026] In some embodiments, the anti-B7H4 antibody-drug conjugate, its isomer, its pharma- ceutically acceptable salt, or mixture thereof of the present invention, wherein -M- is [ka] It is.

[0027] In some embodiments, the anti-B7H4 antibody-drug conjugate of the present invention further comprises a cytotoxic drug. [ka] teeth, [ka] The structure is selected from any one of the following:

[0028] In some embodiments, the anti-B7H4 antibody-drug conjugate, its isomer, its pharma- ceutically acceptable salt, or mixture thereof of the present invention is a compound represented by the formula: a -L b -L c - and Said-L a -teeth, [ka] and In the formula, W is -(C(R wa )(R wb )) wn - and Y is -(OCH 2 CH 2 ) yn -O yp - and Z is -(C(R za )(R zb )) zn and wn is 1, 2, 3 or 6; The 0 or 1 methylene unit of W is each independently -Cyr-, -N(R wx )C(O)-, -C(O)N(R wx )- or -C(O)-, yn is 0, 4 or 8, yp is 0 or 1, zn is 1, 2 or 3; Each methylene unit of Z is independently -Cyr-, -N(R zx )C(O)-, -C(O)N(R zx )- or -C(O)-; -Cyr- is a 3- to 10-membered saturated cycloalkyl, said -Cyr- being unsubstituted or independently containing 1 to 3 substituents R cx is replaced by In the formula, each R wa , R wb , R za , R zb , R wx , R zx , R cx are each independently hydrogen, halogen, or -OR r or Rr C selectively substituted with 1-6 is alkyl, In the formula, each R r are each independently hydrogen, halogen or C 1-6 is alkyl, Said-L b - is a peptide residue consisting of 2 to 7 amino acids, b The peptide residue of -L is a peptide residue consisting of an amino acid selected from the group consisting of phenylalanine, glycine, alanine, valine, citrulline, lysine, serine, glutamic acid, and aspartic acid, and is preferably b - represents a peptide residue consisting of 2 to 4 amino acids, b - is a peptide residue consisting of an amino acid selected from the group consisting of phenylalanine, glycine, alanine, valine, citrulline and lysine, Said-L c -teeth, [ka] and In the formula, R L1 , R L2 are each independently hydrogen, halogen, -OH and C 1-6 alkyl.

[0029] In some embodiments, the anti-B7H4 antibody-drug conjugate, its isomer, its pharma- ceutically acceptable salt, or mixture thereof of the present invention is a compound represented by the formula: a -L b -L c - and Said-L a -teeth, [ka] and In the formula, W is -(C(R wa )(R wb )) wn - and Y is -(OCH 2 CH 2) yn -O yp - and Z is -(C(R za )(R zb )) zn and wn is 1, 2, 3 or 6; The 0 or 1 methylene unit of W is each independently -Cyr-, -N(R wx )C(O)-, -C(O)N(R wx )- or -C(O)-, yn is 0, 4 or 8, yp is 0 or 1, zn is 1, 2 or 3; Each methylene unit of Z is independently -Cyr-, -N(R zx )C(O)-, -C(O)N(R zx )- or -C(O)-; -Cyr- is a 3- to 10-membered saturated cycloalkyl, said -Cyr- being unsubstituted or independently containing 1 to 3 substituents R cx is replaced by In the formula, each R wa , R wb , R za , R zb , R wx , R zx , R cx are each independently hydrogen, halogen, or -OR r or R r C selectively substituted with 1-6 is alkyl, In the formula, each R r are each independently hydrogen, halogen or C 1-6 is alkyl, Said-L b -teeth, [ka] is selected from the group consisting of Said-L c -teeth, [ka] and In the formula, R L1 , R L2 are each independently hydrogen, halogen, -OH and C 1-6 alkyl.

[0030] In some embodiments, the anti-B7H4 antibody-drug conjugate, its isomer, pharma- ceutically acceptable salt, or mixture thereof of the present invention is a conjugate of the formula: a -teeth, [ka] and preferably [ka] It is.

[0031] In some embodiments, the anti-B7H4 antibody-drug conjugate, its isomer, pharma- ceutically acceptable salt, or mixture thereof of the present invention is a conjugate of the formula: b -teeth, [ka] and preferably [ka] It is.

[0032] In some embodiments, the anti-B7H4 antibody-drug conjugate, its isomer, pharma- ceutically acceptable salt, or mixture thereof of the present invention is a conjugate of the formula: c -teeth, [ka] It is.

[0033] In some embodiments, in the anti-B7H4 antibody-drug conjugate of the present invention, its isomer, its pharma- ceutically acceptable salt, or a mixture thereof, the linker unit L is a The end binds to Ab, and Lc The end binds to M. In some embodiments, in the anti-B7H4 antibody-drug conjugate of the present invention, its isomer, its pharma- ceutically acceptable salt, or a mixture thereof, the linker unit L is a The end binds to Ab, and L c The end is bound to the linker unit M.

[0034] In some embodiments, the anti-B7H4 antibody-drug conjugate, its isomer, its pharma- ceutically acceptable salt, or mixture thereof of the present invention, wherein L is [ka] It is.

[0035] In some embodiments, in the anti-B7H4 antibody drug conjugate of the invention, the linker unit L is [ka] It is.

[0036] In some embodiments, the structure of the anti-B7H4 antibody-drug conjugate of the present invention is represented by formula (II-A): [ka] In the formula, p represents the average number of bonds, and p is an integer or decimal number of 1 to 10, and preferably an integer or decimal number of 3 to 8, and Ab, L, and M are each as defined in any embodiment of the present invention. In some embodiments, in the anti-B7H4 antibody-drug conjugate of the present invention, its isomer, pharma- ceutically acceptable salt thereof, or a mixture thereof, the structure of the anti-B7H4 antibody-drug conjugate is represented by formula (II-1) or (II-2): [ka] During the ceremony, p represents the average number of bonds, and p is selected from an integer or decimal number of 1 to 10, preferably an integer or decimal number of 3 to 8; Ab is an antibody or antigen-binding fragment thereof described in any embodiment herein, L 2 is -O- or -S-, preferably -O-; X 1 may optionally contain one, two or three R 2a C replaced with 3 -C 6 saturated cycloalkyl, preferably selected from one, two or three R 2a Replaced with [ka] and X 2 is -(C(R 1a )(R 1b )) m -CH 2 - selected from m is selected from 1 or 2; R 1a is hydrogen, halogen or C optionally substituted with one, two or three R 1 -C 6 alkyl, preferably halogen or C optionally substituted with one, two or three R 1 -C 6 is alkyl, R 1b or R 2a each independently represents hydrogen, halogen, or C optionally substituted with one, two, or three R 1 -C 6 It may be alkyl, Each R may independently be hydrogen or halogen.

[0037] In some embodiments, in the anti-B7H4 antibody-drug conjugate, its isomer, pharma- ceutically acceptable salt thereof, or mixtures thereof of the present invention, the anti-B7H4 antibody-drug conjugate is [ka]

[0038] [ka]

[0039] [ka]

[0040] [ka] is selected from the structural formula During the ceremony, p represents the average number of bonds, and p is selected from an integer or decimal number of 1 to 10, preferably an integer or decimal number of 3 to 8; Ab is an anti-B7H4 antibody or antigen-binding fragment thereof described in any embodiment herein; In yet another aspect, the present invention provides an anti-B7H4 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, the anti-B7H4 antibody-drug conjugate comprising:

[0041] [ka]

[0042] [ka]

[0043] [ka] is selected from the structural formula During the ceremony, p represents the average number of bonds, and p is selected from integers or decimals of 1 to 10, preferably integers or decimals of 3 to 8. The amino acid sequences of DB1001 are as shown in the sequence listing. The average number of bonds p is 3.89, or p is 5.4.

[0044] In some embodiments, the average bond number p of the present invention is selected from integers or decimals of 1 to 10. In some embodiments, the average bond number p of the present invention may be an integer or decimal number of 2 to 8. For example, the average bond number p may be an integer or decimal number of 3 to 8. For example, the average bond number p may be an integer or decimal number of 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.

[0045] In yet another aspect, the present invention provides a method for preparing an antibody-drug conjugate comprising the step of mixing said antibody dissolved in a buffer with said linker-cytotoxin dissolved in a solvent under the action of a reducing agent to obtain said antibody-drug conjugate. In yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-B7H4 antibody-drug conjugate described herein, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, and a pharma- ceutically acceptable carrier or excipient. In yet another aspect, the present invention provides the use of an anti-B7H4 antibody-drug conjugate, its isomer, a pharma- ceutically acceptable salt thereof, or a mixture or pharmaceutical composition thereof described herein in the preparation of a medicament for use in the treatment and / or prevention of a B7H4-mediated disease or disorder, preferably a B7H4-expressing positive cancer.

[0046] In yet another aspect, the present invention provides a method for treating and / or preventing a B7H4-mediated disease or disorder, comprising administering to a subject in need thereof an anti-B7H4 antibody drug conjugate, its isomer, a pharma- ceutically acceptable salt thereof, or a mixture or pharmaceutical composition thereof, as described herein, preferably, wherein said disease or disorder is a cancer with high expression of B7H4. In yet another aspect, the present invention provides an anti-B7H4 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture or pharmaceutical composition thereof, as described herein, for use in the treatment and / or prevention of a B7H4-mediated disease or disorder, preferably a B7H4-expressing positive cancer.

[0047] In some embodiments, the cancer of the present invention is selected from breast cancer, ovarian cancer, and endometrial cancer. In yet another aspect, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate as described herein or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition as described herein, and one or more additional therapeutic agents. In yet another aspect, the invention provides a kit comprising an antibody drug conjugate described herein, or a pharmaceutical composition described herein.

[0048] Definition of Terms The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, all of which are within the skill of the art. In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless expressly defined elsewhere in this specification, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. For definitions and terms in the art, those skilled in the art can refer to Current Protocols in Molecular Biology (Ausubel), among others. The abbreviations of amino acid residues are the standard three-letter and / or one-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. As used herein (including the claims), singular forms include their corresponding plural forms, unless the context clearly indicates otherwise.

[0049] The term "about" refers to a variation within a range of 0.5% to 10% above or below a specified numerical value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified numerical value.

[0050] The term "antibody" refers to any form of antibody having the desired biological activity. It is therefore used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies. The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each antibody comprising the population is identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single epitope. In contrast, conventional (polyclonal) antibody preparations generally contain a large number of antibodies against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and should not be construed as requiring production of the antibody by any particular method.

[0051] The term "full-length antibody" refers to an immunoglobulin molecule that, as it exists in nature, comprises four peptide chains, i.e., two heavy (H) chains (approximately 50-70 kDa for full length) and two light (L) chains (approximately 25 kDa for full length) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) and more conserved spaced regions, called framework regions (FRs). Each VH or VL region is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0052] The term "CDR" refers to the complementarity determining region in the variable sequence of an antibody. There are three CDRs in each of the heavy and light chain variable regions, designated HCDR1, HCDR2 and HCDR3, or LCDR1, LCDR2 and LCDR3 for each of the heavy and light chain variable regions. The precise amino acid sequence boundaries of the variable region CDRs of the antibody of the present invention can be determined by the methods of Chothia (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), based on the three-dimensional structure of the antibody and the topology of the CDR loops, Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and affinity propagation clustering using a large number of crystal structures. The CDRs of the antibodies of the present invention may be delimited by one of ordinary skill in the art according to any protocol in the art (e.g., different designation systems or combinations).

[0053] The term "antigen-binding fragment" of an antibody ("parent antibody") includes antibody fragments or derivatives, and generally includes at least one fragment of the antigen-binding or variable region (e.g., one or more CDRs) of the parent antibody that retains at least some of the binding specificity of the parent antibody. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules such as scFv, nanobodies formed from antibody fragments, and multispecific antibodies. When antigen-binding activity is expressed on a molar basis, a binding fragment or derivative generally retains at least 10% of its antigen-binding activity. Preferably, a binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen-binding affinity of the parent antibody. It is also contemplated that an antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functionally conservative variants" of an antibody) that do not significantly alter its biological activity.

[0054] The term "chimeric antibody" refers to an antibody having the variable domains of a first antibody and the constant domains of a second antibody, where the first and second antibodies are derived from different species. Typically, the variable domains are obtained from an antibody of rodent or other origin (the "parent antibody") and the constant domain sequences are obtained from an antibody of human origin, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject compared to the parent rodent antibody. The term "humanized antibody" refers to forms of antibodies that contain antibody sequences of human and non-human (e.g., mouse, rat) origin. Generally, a humanized antibody contains substantially all of at least one, and usually two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions being those of a human immunoglobulin sequence. A humanized antibody may optionally contain at least a portion of a human immunoglobulin constant region (Fc).

[0055] In this application, the term "halogen" generally refers to fluorine, chlorine, bromine, and iodine, and may be, for example, fluorine or chlorine. In this application, the term "alkyl group" generally refers to a residue derived by removing a hydrogen atom from an alkane. The alkyl group may be substituted or unsubstituted, substituted or unsubstituted. The term "alkyl group" generally refers to a saturated linear or branched aliphatic hydrocarbon group having a residue derived from removing hydrogen atoms from the same carbon atom or from two different carbon atoms of a parent alkane, which may be a linear or branched group containing 1 to 20 carbon atoms, e.g., a linear alkyl group containing 1 to 12 carbon atoms, e.g., 1 to 6 carbon atoms. Non-limiting examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, and the like. An alkyl group can be substituted or unsubstituted, substituted or unsubstituted, for example, when substituted, the substituents can be substituted at any available point of attachment, and the substituents can be independently substituted with one or more substituents selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo, for example, hydrogen, protium, deuterium, tritium, halogen, -NO 2 , -CN, -OH, -SH, -NH 2 , -C(O)H, -CO 2 H, -C(O)C(O)H, -C(O)CH 2 C(O)H, -S(O)H, -S(O) 2 H, -C(O)NH 2 , -SO 2 NH 2 , -OC(O)H, -N(H)SO 2 H or C 1-6 It may be aliphatic, for example when substituted with cycloalkyl it is cycloalkylalkyl.

[0056] In this application, the term "alkylene group" generally refers to a saturated linear or branched aliphatic hydrocarbon group having two residues derived by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane, which may be a linear or branched group containing 1 to 20 carbon atoms, for example, the term "methylene group" may refer to a residue derived by removing two hydrogen atoms from a group of one carbon atom. The methylene group may be substituted or unsubstituted, substituted or unsubstituted, for example, an alkylene group containing 1 to 12 carbon atoms, for example, 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include methylene (-CH 2 -), 1,1-ethylene (-CH(CH 3 )-), 1,2-ethylene (-CH 2 CH 2 )-, 1,1-propylene (-CH(CH 2 CH 3 )-), 1,2-propylene (-CH 2 CH(CH 3 )-), 1,3-propylene (-CH 2 CH 2 CH 2 -), 1,4-butylene (-CH 2 CH 2 CH 2 CH 2 -) and 1,5-butylene (-CH 2 CH 2 CH 2 CH 2 CH 2Alkylene groups may be substituted or unsubstituted, substituted or unsubstituted, for example, when substituted, the substituents may be substituted at any available point of attachment, the substituents may be independently substituted with one or more substituents selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo, for example, hydrogen, protium, deuterium, tritium, halogen, -NO 2 , -CN, -OH, -SH, -NH 2 , -C(O)H, -CO 2 H, -C(O)C(O)H, -C(O)CH 2 C(O)H, -S(O)H, -S(O) 2 H, -C(O)NH 2 , -SO 2 NH 2 , -OC(O)H, -N(H)SO 2 H or C 1-6 It may be aliphatic. The methylene or alkylene groups may be substituted or unsubstituted.

[0057] The term "alkoxy group" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), where alkyl or cycloalkyl are as defined herein. Non-limiting examples of alkoxy groups include methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. An alkoxy group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0058] In this application, the term "alkenyl group" generally refers to a straight-chain or branched hydrocarbon group containing one or more double bonds. Specific examples of alkenyl groups include allyl, homoallyl, vinyl, crotyl, butenyl, pentenyl, and hexenyl. Specific examples of C2-6 alkenyl groups having one or more double bonds include butadienyl, pentadienyl, hexadienyl, and hexatrienyl groups and their branched forms. The position of the unsaturated bond (double bond) may be anywhere on the carbon chain. The alkenyl group may be substituted or unsubstituted. In this application, the term "alkenylene group" generally refers to a group having a residue derived by removing two hydrogen atoms from a carbon atom of an alkene. Examples include allylene, vinylene, butenylene, pentenylene, hexenylene, etc. Alkenylene groups may be substituted or unsubstituted.

[0059] In this application, the term "alkynyl group" generally refers to unsaturated linear or branched alkynyl groups such as ethynyl, 1-propynyl, propargyl, butynyl, etc. Alkynyl groups can be substituted or unsubstituted. In this application, the term "alkynylene group" generally refers to a group having a residue derived by removing two hydrogen atoms from a carbon atom of an alkyne. Examples include ethynylene, propynylene, propynylene, butynylene, etc. Alkynylene groups may be substituted or unsubstituted.

[0060] In this application, the term "aryl group" generally means having a residue derived by removing a hydrogen atom from an aromatic ring. The term "aromatic ring" can refer to a 6-14 membered all carbon monocyclic or fused polycyclic ring (i.e., rings sharing adjacent pairs of carbon atoms) having a conjugated π electron system, which can be 6-10 membered, such as benzene and naphthalene. The aromatic ring can be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, where the ring attached to the parent structure is an aromatic ring. The aryl group can be substituted or unsubstituted, and if substituted, the substituents can be one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio. The aryl group can be substituted or unsubstituted.

[0061] In this application, the term "heteroaryl group" generally refers to a group having a residue derived by removing a hydrogen atom from a carbon atom of a heteroaromatic ring. The term "heteroaromatic ring" refers to a heteroaromatic system containing 1-4 heteroatoms, 5-14 ring atoms, where the heteroatoms may be selected from the group consisting of oxygen, sulfur, and nitrogen. The heteroaryl group may be 5-10 membered, 5-membered, or 6-membered, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl group may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, where the ring attached to the parent structure is a heteroaromatic ring. Heteroaryl groups can be optionally substituted or unsubstituted, and if substituted, the substituents can be one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio. Heteroaryl groups can be substituted or unsubstituted.

[0062] In this application, the term "cycloalkyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent, the cycloalkyl ring containing 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, preferably 3 to 10 carbon atoms, preferably 3 to 8 carbon atoms, more preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc., and polycyclic cycloalkyl groups include spiro, fused and bridged ring cycloalkyl groups. Cycloalkyl groups may be substituted or unsubstituted, and if substituted, the substituents may be substituted at any available point of attachment, and the substituents are preferably substituted with one or more substituents independently selected from the group consisting of hydrogen, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0063] In this application, the term "partially unsaturated" generally refers to a ring structure having at least one double or triple bond between ring molecules. The term "partially unsaturated" encompasses ring structures having multiple unsaturations, but is not intended to include aromatic or heteroaromatic rings as defined herein. The term "unsaturated" means that a portion has one or more degrees of unsaturation.

[0064] In this application, the term "heterocyclyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic light substituent containing 3 to 20 ring atoms, one or more of which are heteroatoms selected from nitrogen, oxygen or sulfur, and the remaining ring atoms are carbon. Preferably, it contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms, more preferably, it contains 3 to 8 ring atoms, of which 1 to 3 are heteroatoms, more preferably, it contains 3 to 6 ring atoms, of which 1 to 3 are heteroatoms, and most preferably, it contains 5 or 6 ring atoms, of which 1 to 3 are heteroatoms. Non-limiting examples of monocyclic heterocyclyls include pyrrolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl. Polycyclic heterocyclyls include spiro, fused and bridged heterocyclyls. The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, and the ring attached to the parent structure is a heterocyclyl. The heterocyclyl group may be substituted or unsubstituted, and if substituted, the substituents may be substituted at any available point of attachment, and the substituents are preferably substituted with one or more substituents independently selected from the group consisting of hydrogen atoms, halogen, alkyl, alkoxy, haloalkyl, hydroxy, hydroxyalkyl, cyano, amino, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl.

[0065] In this application, the term "ring atom" generally refers to an atom contained in a ring structure. For example, a ring atom may be a carbon atom on a benzene ring or a nitrogen atom on a pyridine ring. When a ring atom is bonded to a hydrogen atom, the ring atom may be substituted or unsubstituted. In this application, the term "independently" generally means that a variable applies to any situation, regardless of whether there are variables with the same or different definitions in the same compound. For example, the variable may refer to the type, number, or type of substituents in a compound, or the type of atoms in a compound. For example, if R occurs twice in a compound and R is defined as "independently carbon or nitrogen", then both R may be carbon, both R may be nitrogen, or one R may be carbon and the other R may be nitrogen.

[0066] In this application, the term "optionally" or "optionally" generally means that the event or circumstance described thereafter may, but need not, occur, and that the description includes cases in which the event or circumstance occurs and cases in which the event or circumstance does not occur. For example, "a heterocyclyl group optionally substituted with an alkyl group" means that an alkyl group may, but need not, be present, and can include cases in which the heterocyclyl group is substituted with an alkyl group and cases in which the heterocyclyl group is not substituted with an alkyl group.

[0067] In this application, the term "substituted" generally means that one or more hydrogen atoms in a group, for example up to 5, for example 1 to 3 hydrogen atoms, are replaced independently of one another with the corresponding number of substituents. Substituents are present only at their possible chemical positions, and a person skilled in the art can determine (through experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxy groups with free hydrogen may be unstable when attached to carbon atoms with unsaturated (e.g. olefinic) bonds.

[0068] In this application, the term "replaced" by zero or more (e.g., zero or one or more, zero or one, zero) methylene units generally refers to the fact that when the structure includes one or more methylene units, the one or more methylene units may be unreplaced or may be replaced with one or more groups other than methylene (e.g., -NHC(O)-, -C(O)NH-, -C(O)-, -OC(O)-, -C(O)O-, -NH-, -O-, -S-, -SO-, -SO2-, -PH-, -P(=O)H-, -NHSO2-, -SONH-, -C(=S)-, -C(=NH)-, -N=N-, -C=N-, -N=C-, or -C(=N2)-).

[0069] In the present application, the "bonding" of group X to group Y may generally be in any orientation, which generally means that when group X is used in a linker Y and group Z, two or more attachment sites of said group X may be optionally bonded to group Y or group Z. In the present application, the term "compound" generally refers to a substance having two or more different elements. For example, the compound of the present application may be an organic compound, for example, the compound of the present application may be a compound having a molecular weight of 500 or less, a compound having a molecular weight of 1000 or less, a compound having a molecular weight of 1000 or more, a compound having a molecular weight of 10,000 or more, or a compound having a molecular weight of 100,000 or more. In the present application, the compound may also refer to a compound linked by a chemical bond, for example, a compound in which one or more molecules having a molecular weight of 1000 or less are linked to a biopolymer by a chemical bond, and the biopolymer may be a glycan, a protein, a nucleic acid, a polypeptide, etc. For example, the compound of the present application may include a compound comprising a protein bound to one or more molecules having a molecular weight of 1000 or less, a compound comprising a protein bound to one or more molecules having a molecular weight of 10,000 or less, and a compound comprising a protein bound to one or more molecules having a molecular weight of 100,000 or less.

[0070] As used herein, terms such as "alkyl group," "alkenyl group," "cycloalkyl group," and the like are used in a variety of ways to indicate the number of atoms present in the group in a particular instance, as would be understood by one of ordinary skill in the art, e.g., C 1 -C 4 Alkyl, C 3 -C 7 Cycloalkoxy group, C 1 -C 4 The subscript number following the "C" indicates the number of carbon atoms present in the group. For example, C 3 Alkyl refers to alkyl having 3 carbon atoms (e.g., n-propyl, isopropyl), C 1-10 In the formula, the members of the group can have any number of carbon atoms falling within the range of 1-10.

[0071] One or more hydrogen atoms in the group, for example up to 5, for example 1 to 3 hydrogen atoms, are substituted independently of one another with the corresponding number of substituents. Substituents are present only at their possible chemical positions, and a person skilled in the art can determine (through experiment or theory) possible or impossible substitutions without undue effort. For example, amino or hydroxy groups with free hydrogen may be unstable when bonded to carbon atoms with unsaturated (e.g. olefinic) bonds. In the present application, the compounds or ligand-drug complexes of the present application include their tautomers, mesomers, racemates, enantiomers, and / or diastereomers. In the present application, the term "diastereomer" generally refers to stereoisomers that have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers may have different physical properties, such as melting points, boiling points, spectral properties, and reactivity. In the present application, the terms "tautomers" or "tautomeric forms" are used interchangeably and generally refer to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also called prototropic tautomers) include interconversions via proton transfer, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions via rearrangement of some of the bond electrons. As used herein, the term "mesomer" generally refers to an atom containing asymmetry within a molecule, but with symmetry such that the total optical rotation within the molecule is 0. The terms "racemate" or "racemic mixture" refer to a composition consisting of equimolar amounts of two enantiomeric substances.

[0072] As used herein, the term "isomer" of a compound or a ligand-drug complex generally includes tautomers, mesomers, racemates, enantiomers, and diastereomers of a compound. The term "ligand-drug conjugate" generally refers to a ligand linked to a biologically active cytotoxic drug via a stable linking unit. In this application, the "ligand-drug conjugate" may be an antibody drug conjugate (ADC), which may mean that a monoclonal antibody or antibody fragment is linked to a biologically active cytotoxic drug via a stable linking unit.

[0073] In this application, the term "ligand" generally refers to a macromolecular compound capable of recognizing and binding to an antigen or receptor associated with a target cell. The role of the ligand may be to present a drug to a target cell population bound to the ligand, including but not limited to protein hormones, lectins, growth factors, antibodies, or other molecules capable of binding to cells, receptors, and / or antigens. In this application, the ligand may be represented as Pc, where the ligand antigen forms a link with the linking unit via a heteroatom on the ligand. The ligand may be an antibody or an antigen-binding fragment thereof (Ab), which may be selected from a chimeric antibody, a humanized antibody, a fully human antibody, or a murine antibody.

[0074] The term "cytotoxic drug" generally refers to a toxic drug that may have a chemical molecule that is highly disruptive in tumor cells and their normal growth. A cytotoxic drug can kill tumor cells at a sufficiently high concentration. Said "cytotoxic drug" may be a toxin, such as a small molecule toxin or an enzymatically active toxin derived from bacteria, fungi, plants or animals, a radioisotope (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 or radioisotopes of Lu), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, and may be toxic drugs including, but not limited to, camptothecin derivatives, such as the camptothecin derivative exatecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]imidazo[1,2-b]quinoline-10,13(9H,15H)-dione). In some embodiments of the invention, the cytotoxic drug is also referred to as a cytotoxin.

[0075] The term "linker unit" or "linker structure" generally refers to a chemical structure fragment or bond that is attached to a ligand at one end and to a cytotoxic drug at the other end, and may be attached to a cytotoxic drug after another linker is attached. The direct or indirect bond to the ligand may be a direct bond to the ligand via a covalent bond in the group, or may be a bond to the ligand via a linker structure. For example, the linker structure may be a chemical structure fragment or bond that includes an acid-labile linker structure (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker structure, a photolabile linker structure, a dimethyl linker structure, or a disulfide-containing linker structure.

[0076] The term "selectively linked to another molecular moiety" generally means that the structure is not linked to any other chemical structure, or that the structure is linked (e.g., via a chemical bond or via a linker structure) to one or more other chemical structures that are different from the structure (e.g., a ligand described herein). The term "drug conjugation number" generally refers to the average number of cytotoxic drugs loaded on each ligand, and can be expressed as the ratio of the amount of cytotoxic drug to the amount of antibody, and the range of cytotoxic drug loading can be 0-12, e.g., 1-10 cytotoxic drugs can be linked to each ligand (Ab). In the present embodiment, the drug conjugation number is expressed as Na, and can be, for example, an average value of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The drug conjugation number of each ADC molecule after the coupling reaction can be identified using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assay, and HPLC characterization.

[0077] In this application, certain atoms of the compounds of this application may occur in more than one isotopic form. For example, hydrogen is represented as protium ( 1 H), deuterium ( 2 H) and tritium ( 3 H), and carbon can exist in three different isotopes ( 12 C. 13 C sum 14C) can occur naturally. Examples of isotopes that can be incorporated into the compounds of the present application include: 15 N, 18 O. 17 O. 18 F, 32 P, 33 P, 129 I, 131 I, 123 I, 124 I, 125 I, or similar isotopes. Thus, the compounds of the present application may be enriched in one or more of these isotopes relative to the natural abundance of these isotopes. As known to those skilled in the art, such isotopically enriched compounds may be used for a variety of purposes. For example, deuterium ( 2 Replacement with heavier isotopes such as deuterium (H) may offer certain therapeutic advantages that may be due to higher metabolic stability. 2 The natural abundance of hydrogen (H) is about 0.015%. Thus, in nature, there is one deuterium atom for every approximately 6500 hydrogen atoms. Thus, the deuterium-containing compounds of the present application have a deuterium abundance of greater than 0.015% at one or more positions (in some cases). Unless otherwise indicated, the structures described herein may also include compounds that differ only in the presence or absence of one or more isotopically enriched atoms. For example, any compound in which a hydrogen atom is replaced with deuterium or tritium, or a carbon atom is replaced with carbon-13 or carbon-14, but otherwise corresponds to the structure of the present application, is within the scope of the present application.

[0078] In this application, the term "pharmaceutical composition" generally refers to a mixture containing one or more compounds described herein or their physiologically / pharmaceutical acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically / pharmaceutical acceptable carriers and excipients. The pharmaceutical composition can facilitate administration to an organism and promote the absorption of the active ingredient, thereby exerting biological activity. The preparation of conventional pharmaceutical compositions can be referred to the Chinese Pharmacopoeia. The pharmaceutical composition may be in the form of a sterile injectable water or oil suspension for intramuscular and subcutaneous administration. The suspension may be formulated according to known techniques using suitable dispersing or wetting agents and suspending agents as mentioned above. The sterile injectable preparation may be a sterile injectable solution or suspension prepared in a non-toxic parenterally acceptable diluent or solvent, for example, a solution prepared in 1,3-butanediol. In addition, sterile non-volatile oils may be conveniently used as a solvent or suspending medium. For example, any blended non-volatile oils, including synthetic mono- or diglycerides, may be used. In addition, fatty acids such as oleic acid may also be used in the preparation of injectables.

[0079] As used herein, a "pharmaceutically acceptable salt" or a "pharmaceutically usable salt" generally refers to a salt of a compound or ligand-drug conjugate of the present application, or a salt of a compound described herein, which is safe and / or effective when used in a mammalian body and has the desired biological activity, and the antibody-antibody drug conjugate compound of the present application can form a salt with an acid, and non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0080] In this application, the term "pharmaceutical acceptable carrier" generally refers to a carrier or vehicle for administering therapeutic agents such as antibodies or polypeptides, genes, and other therapeutic agents. The term refers to any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition and can be administered without causing undue toxicity. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, and inactivated virus particles. Such carriers are well known to those skilled in the art. Pharmaceutically acceptable carriers in therapeutic compositions can include liquids such as water, saline, glycerol, and ethanol. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may also be present in these carriers.

[0081] As used herein, the terms "treatment" and "treating" generally refer to a method for obtaining beneficial or desired results, including, but not limited to, a therapeutic benefit. Therapeutic benefit includes, but is not limited to, eradicating, inhibiting, reducing, or ameliorating the underlying disease being treated. Additionally, therapeutic benefit may be achieved by eradicating, reducing, or ameliorating one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the patient, although the patient may still have the underlying disorder.

[0082] As used herein, the terms "prevention" and "preventing" generally refer to a method for obtaining a beneficial or desired result, including, but not limited to, a prophylactic benefit. For prophylactic benefit, the pharmaceutical composition may be administered to a patient who is at risk of developing a particular disease, or to a patient who has been reported to have one or more physiological symptoms of a disease, even if the disease has not yet been diagnosed.

[0083] As used herein, the term "subject" or "patient" generally refers to humans (i.e., male or female of any age group, e.g., pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young, middle-aged, or elderly)) and / or other primates (e.g., cynomolgus monkeys, rhesus monkeys), mammals, including commercially relevant mammals such as cows, pigs, horses, sheep, goats, cats, and / or dogs, and / or birds, including commercially relevant birds such as chickens, ducks, geese, quail, and / or turkeys.

[0084] The terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to an amount of the ligand-drug conjugate of the present invention, when administered to a cell, tissue, or subject, alone or in combination with other therapeutic agents, that is effective to prevent or ameliorate one or more symptoms of a disease or condition, or the progression of a disease or condition. A therapeutically effective dose also refers to an amount sufficient to improve symptoms, e.g., treat, cure, prevent or or ameliorate an associated medical condition, or increase the rate of treatment, cure, prevention or amelioration of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers to that ingredient alone. When administered in combination, a therapeutically effective dose refers to the total amount of active ingredients that induce a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent results in an increase of at least 10%, usually at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% of a diagnostic criterion or parameter.

[0085] The term "cancer" is used herein to refer to a group of cells that exhibit abnormally high levels of proliferation and growth. Cancers can be benign (also called benign tumors), precancerous, or malignant. Cancer cells can be solid cancer cells or leukemic cancer cells. The term "tumor" as used herein refers to one or more cells that comprise cancer. The term "tumor growth" is used herein to refer to the proliferation or growth of one or more cells that comprise cancer, resulting in a corresponding increase in the size or extent of the cancer.

[0086] Anti-B7H4 antibody The term "B7-H4" or "B7H4" refers to a member of the human B7 protein family, also known as CD276, which is a type I transmembrane protein with four Ig-like extracellular domains. B7-H4 is one of the immune checkpoint proteins expressed on the surface of antigen-presenting cells or cancer cells and has an inhibitory effect on the functional activation of T cells. The term "B7-H4" includes any variant or isoform of B7-H4 that is naturally expressed by cells. The antibodies of the present invention may cross-react with B7-H4 from non-human species. Alternatively, the antibodies may be specific for human B7-H4 and may not show cross-reactivity with other species. B7-H4 or any variant or isoform thereof may be isolated from cells or tissues that naturally express them or may be produced by recombinant techniques using techniques commonly used in the art and described herein. Preferably, the anti-B7-H4 antibody targets human B7-H4 with normal glycosylation patterns.

[0087] As used herein, the terms "anti-B7H4 antibody," "anti-B7H4," "B7H4 antibody," or "antibody that binds to B7H4" mean that the antibody is capable of binding to the B7H4 protein or a fragment thereof with sufficient affinity such that it can be used as a diagnostic and / or therapeutic agent in targeting B7H4. In some embodiments, the CDR sequences of an antibody used in a drug conjugate, composition, use or method of the invention comprise the CDR sequences derived from antibody PR008199 described in PCT / CN2021 / 102952. In some embodiments, the variable region sequences of an antibody used in a drug conjugate, composition or use of the invention comprise the variable region sequences derived from antibody PR008199 described in PCT / CN2021 / 102952. In some embodiments, the amino acid sequence of an antibody used in a drug conjugate, composition, use or method of the invention comprises the full length amino acid sequence derived from antibody PR008199 described in PCT / CN2021 / 102952.

[0088] The anti-B7H4 antibody DB1001 (PR008199) or its antigen-binding fragment according to the present invention is prepared with reference to the description in PCT / CN2021 / 102952. In some embodiments, the CDR sequence of the antibody used in the drug conjugate, composition, use or method of the present invention comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and LCDR1, LCDR2 and LCDR3 having the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively. In some embodiments, the variable region sequence of the antibody used in the drug conjugate, composition or use of the present invention comprises a heavy chain variable region having the amino acid sequence shown in SEQ ID NO:7, and a light chain variable region having the amino acid sequence shown in SEQ ID NO:8. In some embodiments, the antibody used in the drug conjugates, compositions, uses or methods of the invention comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:9, and a light chain having the amino acid sequence set forth in SEQ ID NO:10. In some embodiments, the antibody used in the drug conjugates, compositions, uses or methods of the invention is the anti-B7H4 antibody DB1001.

[0089] The amino acid sequences of the variable regions of anti-B7H4 antibody DB1001 are as follows, and the CDR regions were determined according to the IMGT numbering rules.

[0090] DB1001 heavy chain variable region: QVQLVESGGGVVQPGRSLRLSCAASGTFRSFGMHWVRQAPGKGLEWVAVISYDASNEYYADSVKGRFIISRDNSKDTLYLQMNSLRAGDTAVYYCAKGGALRWYFAYWGQGTLVTVSS(SEQ ID NO:7) [ka] [ka] [ka]

[0091] DB1001 light chain variable region: EIVMTQSPATLSVSPGERATLSCRASQSISSNLGWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYRSWPPLTFGGGTKVEIK(SEQ ID NO:8) [ka] [ka] [ka]

[0092] Heavy chain amino acid sequence of DB1001 QVQLVESGGGVVQPGRSLRLSCAASGTFRSFGMHWVRQAPGKGLEWVAVISYDASNEYYADSVKGRFIISRDNSKDTLYLQMNSLRAGDTAVYYCAKGGALRWYFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:9)

[0093] DB1001 light chain amino acid sequence EIVMTQSPATLSVSPGERATLSCRASQSISSNLGWYQQKPGQAPRLLIYGASTRATGIPA RFSGSGSGTEFTLTISSLQSEDFAVYYCQQYRSWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:10)

[0094] Any suitable method for producing antibodies can be used to produce the antibodies of the present invention. Any suitable form of B7H4 can be used as an immunogen (antigen) to produce antibodies. By way of example and not limitation, any B7H4 variant or fragment thereof can be used as an immunogen. In some embodiments, hybridoma cells producing mouse monoclonal anti-human B7H4 antibodies can be produced by methods known in the art. Antibodies from rodents (such as mice) can cause undesirable antibody immunogenicity when used in vivo as therapeutics, and repeated use can generate an immune response against the therapeutic antibody in humans, which at least results in a loss of therapeutic efficacy and, in severe cases, a potentially fatal allergic reaction. One method of reducing the immunogenicity of rodent antibodies involves the production of chimeric antibodies in which mouse variable regions are fused to human constant regions (Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-43). However, complete retention of the rodent variable regions in a chimeric antibody may still cause adverse immunogenicity in patients. Loop grafting of complementarity determining regions (CDRs) of rodent variable domains onto human frameworks (i.e., humanization) has been used to minimize rodent sequences (Jones et al. (1986) Nature 321:522; Verhoeyen et al. (1988) Science 239:1534).

[0095] In some embodiments, chimeric or humanized antibodies of the invention may be made based on the sequences of mouse monoclonal hybridoma antibodies produced. DNA encoding heavy and light immunoglobulin chains can be obtained from the mouse hybridoma of interest and engineered to contain non-mouse (e.g., human) immunoglobulin sequences using standard molecular biology techniques. In some embodiments, chimeric B7H4 antibodies according to the invention may be prepared using methods known in the art to operably link hybridoma-derived immunoglobulin heavy and light chain variable regions to human IgG constant regions (see, e.g., U.S. Patent No. 4,816,567 to Cabilly et al.) to obtain chimeric heavy and light chains. In some embodiments, the chimeric antibodies of the invention comprise a constant region that may be selected from any human IgG subtype, such as IgG1, IgG2, IgG3, IgG4, preferably IgG4.

[0096] In some embodiments, chimeric B7H4 antibodies of the present invention can be obtained from "mix and match" transfection of expressing cells with chimeric light chain and chimeric heavy chain expression plasmids, and the B7H4 binding of such "mixed and matched" antibodies can be tested using the binding assays described above and other conventional binding assays (such as ELISAs). Humanized antibodies of the invention can be made by inserting murine CDR regions into human germline framework regions using methods known in the art (see U.S. Patent Nos. 5,225,539 to Winter et al., and 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

[0097] In some embodiments, the amino acid changes include amino acid deletions, insertions or substitutions. In some embodiments, the anti-B7H4 antibodies or antigen-binding fragments thereof of the present invention include antibodies that are mutated by amino acid deletions, insertions or substitutions, but have amino acid sequences that are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the above antibodies (particularly in the CDR regions shown in the sequences above). In some embodiments, the antibodies of the present invention have no more than 1, 2, 3, 4 or 5 amino acid mutations by amino acid deletions, insertions or substitutions in the CDR regions when compared to the CDR regions shown in the particular sequences.

[0098] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., a substitution) at one or more amino acid positions. In some embodiments, it may be desirable to produce cysteine ​​engineered antibodies, such as "ThioMAbs," in which one or more residues of an antibody are substituted with a cysteine ​​residue.

[0099] In some embodiments, the antibodies provided herein can be further modified to contain other non-protein moieties that are known in the art and readily available. Moieties suitable for derivatization of antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0100] Drug Conjugates The present application provides an anti-B7H4 antibody-drug conjugate, its isomer, pharma-ceutically acceptable salt thereof, or a mixture thereof, which may have one or more effects selected from the following group: (1) having inhibitory activity against in vitro proliferation of tumor cells; (2) having target inhibition properties; (3) having plasma stability; (4) having tumor suppressing effect in vivo; (5) having bystander effect; (6) having anti-transporter transport ability; (7) having tumor targeting ability in vivo; and (8) having good in vivo safety.

[0101] The present application provides an anti-B7H4 antibody-drug conjugate, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, wherein the structure of the anti-B7H4 antibody-drug conjugate is represented by formula (I-1): [ka] During the ceremony, M is -L 2 -L 1 -C(O)-, L 2 is -O- or -S-, L 1 is -(C(R 1a )(R 1b )) m -CH 2 -, C 3 -C 6 is a saturated cycloalkyl or a 3- to 6-membered saturated heterocyclyl, 3 -C 6 The saturated cycloalkyl and the 3- to 6-membered saturated heterocyclyl each independently optionally have one or more R 2a is replaced by m is selected from 1, 2, 3 or 4; R 1a are each independently hydrogen, halogen, hydroxy, amino, or C 1 -C 6 alkyl, 1 -C 6 Alkyl is optionally substituted with one or more R, preferably halogen or C optionally substituted with one, two or three R. 1 -C 6 is alkyl, R 1b and R 2a are each independently hydrogen, halogen, hydroxy, amino, or C 1 -C 6 alkyl, 1 -C 6 The alkyl is optionally substituted with one or more R; Each R is independently hydrogen or halogen; L is a linker unit, p represents the average number of bonds, and p is selected from an integer or decimal number of 1 to 10, preferably an integer or decimal number of 3 to 8; Ab is an anti-B7H4 antibody or an antigen-binding fragment thereof.

[0102] In some embodiments, the anti-B7H4 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the light chain variable region comprising LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; preferably, the anti-B7H4 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:7 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:8; more preferably, the anti-B7H4 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:9 and a light chain having the amino acid sequence set forth in SEQ ID NO:10.

[0103] In some embodiments, in formulas (I-1), (II-1) and (II-2), L 1 is -(C(R 1a )(R 1b )) m -CH 2 - and R 1a is C optionally substituted with halogen, hydroxy, amino and R 1 -C 6 alkyl; R 1b is C optionally substituted with hydrogen, halogen, hydroxy, amino and R 1 -C 6 alkyl, m is selected from 1, 2, 3 or 4, and each R may independently be hydrogen or halogen, preferably R 1a is halogen and C 1 -C 6 alkyl; R 1b is hydrogen, halogen and C 1 -C 6 alkyl, m is selected from 1 or 2, preferably R 1a Ha-CH 3 and R1b is hydrogen and -CH 3 and m is selected from 1 or 2, preferably L 1 teeth, [ka] is selected from.

[0104] In some embodiments, in formulas (I-1), (II-1) and (II-2), L 1 is C 3 -C 6 is a saturated cycloalkyl or a 3- to 6-membered saturated heterocyclyl, 3 -C 6 The saturated cycloalkyl and the 3- to 6-membered saturated heterocyclyl each independently optionally have one or more R 2a is replaced by R 2a are each independently hydrogen, halogen, hydroxy, amino, or C 1 -C 6 alkyl, preferably L 1 may optionally be one or more R 2a C replaced with 3 -C 6 is a saturated cycloalkyl, R 2a are each independently hydrogen, halogen, or C 1 -C 6 alkyl, preferably L 1 is C 3 -C 6 is a saturated cycloalkyl, preferably L 1 teeth, [ka] is selected from.

[0105] In some embodiments, in formulas (I-1), (II-1) and (II-2), L 1 teeth, [ka] is selected from.

[0106] In some embodiments, in formulas (I-1), (II-1) and (II-2), M is -L 2 -L 1 -C(O)-, L 2 is -O- or -S-, preferably -O-; L 1 is -(C(R 1a )(R 1b )) m -CH 2 - or C 3 -C 6 is a saturated cycloalkyl, 3 -C 6 A saturated cycloalkyl may optionally have one or more R 2a and preferably L 1 may optionally contain 1, 2 or 3 R 2a Replaced with [ka] and m is selected from 1 or 2; R 1a each independently represents a halogen and C 1 -C 6 alkyl, 1 -C 6 The alkyl is optionally substituted with one or more R; R 1b and R 2a are each independently hydrogen, halogen, or C 1 -C 6 alkyl, 1 -C 6 The alkyl is optionally substituted with one or more R, each of which is independently hydrogen or halogen.

[0107] In some preferred embodiments, in formulas (I-1), (II-1) and (II-2), M is [ka] is selected from.

[0108] In some particularly preferred embodiments, the anti-B7H4 antibody-drug conjugate, its isomer, pharma- ceutically acceptable salt, or mixture thereof of the present invention is a compound represented by the formula: [ka] It is.

[0109] In some embodiments, in the anti-B7H4 antibody-drug conjugate, its isomer, pharma- ceutically acceptable salt thereof, or mixtures thereof of the present invention, the structure of the anti-B7H4 antibody-drug conjugate is [ka] Represented by formula (II-1) or (II-2), During the ceremony, L 2 is -O- or -S-, preferably L 2 is -O-, X 1 may optionally contain one, two or three R 2a C replaced with 3 -C 6 saturated cycloalkyl, preferably selected from one, two or three R 2a Replaced with [ka] and X 2 is -(C(R 1a )(R 1b )) m -CH 2 - selected from m is selected from 1 or 2; R 1a is a C optionally substituted with halogen or one, two or three R 1 -C 6 is alkyl, R 1b or R 2aeach independently represents hydrogen, halogen, or C optionally substituted with one, two, or three R 1 -C 6 It may be alkyl, Each R may independently be hydrogen or halogen; p represents the average number of bonds, and n is an integer or decimal number selected from 1 to 10, preferably an integer or decimal number selected from 3 to 8; Ab is an anti-B7H4 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences set forth in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; preferably, the anti-B7H4 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO:7, and a light chain variable region having the amino acid sequence set forth in SEQ ID NO:8; more preferably, the anti-B7H4 antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:9, and a light chain having the amino acid sequence set forth in SEQ ID NO:10.

[0110] In some embodiments, the average bond number p of the present invention may be an integer or decimal number of 2 to 8. For example, the average bond number p may be an integer or decimal number of 3 to 8. For example, the average bond number p may be an integer or decimal number of 1 to 2, 2 to 3, 3 to 4, 4 to 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, or 9 to 10.

[0111] Pharmaceutical Compositions and Formulations In yet another aspect, the present invention provides a pharmaceutical composition comprising an anti-B7H4 antibody-drug conjugate described herein, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, and a pharma- ceutically acceptable carrier or excipient. As will be appreciated, the anti-B7H4 antibody drug conjugates provided herein, or their isomers, pharma- ceutically acceptable salts, or mixtures thereof, or pharmaceutical compositions thereof, may be administered in combination with suitable carriers, excipients, and other agents in the formulation to provide improved transfer, delivery, tolerability, and the like.

[0112] The term "pharmaceutical composition" refers to a formulation that allows for the presence of a biologically active effective form of the active ingredient contained therein and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. Pharmaceutical formulations containing the anti-B7H4 antibodies described herein can be prepared by mixing the anti-B7H4 antibody-drug conjugate of the present invention or a pharma- ceutical acceptable salt thereof having the desired purity with one or more optional pharmaceutical adjuvants (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. ed. (1980)), preferably in the form of an aqueous solution or a lyophilized formulation.

[0113] The pharmaceutical composition or formulation of the present invention may further comprise one or more other active ingredients as required for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In some embodiments, the additional active ingredients are chemotherapeutic agents, immune checkpoint inhibitors, growth inhibitors, antibiotics, or various known anti-tumor or anti-cancer agents, and the active ingredients are present in suitable combinations in amounts effective for the intended use. In some embodiments, the pharmaceutical composition of the present invention further comprises a composition of a polynucleotide encoding an anti-B7H4 antibody. In yet another aspect, the present invention provides a pharmaceutical composition comprising an antibody-drug conjugate as described herein or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition as described herein, and one or more additional therapeutic agents. In yet another aspect, there is provided a kit comprising an antibody drug conjugate as described herein, or an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof, or a pharmaceutical composition as described herein, preferably further comprising an administration device.

[0114] Medicinal Use In yet another aspect, the present invention provides the use of an antibody-drug conjugate as described herein, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof or a pharmaceutical composition as described herein, in the preparation of a medicament for use in the treatment and / or prevention of a B7H4-mediated disease or disorder, preferably a B7H4-expressing positive cancer. In yet another aspect, the present invention provides an antibody-drug conjugate as described herein, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof or a pharmaceutical composition as described herein, for use in the treatment and / or prevention of a B7H4-mediated disease or disorder, preferably a B7H4-expressing positive cancer.

[0115] In yet another aspect, the present invention provides a method for treating and / or preventing a B7H4 mediated disease or disorder, comprising administering to a subject in need thereof an antibody drug conjugate as described herein, an isomer thereof, a pharma- ceutically acceptable salt thereof, or a mixture thereof or a pharmaceutical composition as described herein, preferably, wherein said disease or disorder is a cancer with high expression of B7H4. In some embodiments, the cancer is selected from breast cancer, ovarian cancer, and endometrial cancer. In some embodiments, methods of administration of the invention include, but are not limited to, oral, intravenous, subcutaneous, intramuscular, intra-arterial, intra-articular (e.g., into an arthritic joint), via inhalation or aerosol delivery, or intratumoral administration. In some embodiments, the invention provides for the combined administration of a therapeutically effective amount of one or more therapies (e.g., therapeutic methods and / or other therapeutic agents) to a subject, which in some embodiments include surgical therapy and / or radiation therapy.

[0116] In some embodiments, the method or use provided by the present invention further comprises administering one or more therapies (e.g., therapeutic methods and / or other therapeutic agents) to an individual. The antibody-drug conjugate of the present invention or its pharma- ceutically acceptable salt thereof can be used alone or in combination with other therapeutic agents in the treatment. For example, it can be administered in combination with at least one other therapeutic agent.

[0117] The positive effects of the present invention are as follows: In the anti-B7H4 antibody-drug conjugate of the present invention, the anti-B7H4 antibody or its antigen-binding fragment is linked to a biologically active cytotoxic drug via a linker unit, and after the antibody-drug conjugate is transferred to a tumor cell, the linker is cleaved to release the cytotoxic drug HMD. For example, the antibody-drug conjugate DB1001-X1 of the present invention releases the low molecular weight compound P-II-3 after being transferred to a cell, and the antibody-drug conjugate DB1001-X2 of the present invention releases the low molecular weight compound P-III-30 after being transferred to a cell. The cytotoxic drug of the present invention has significantly enhanced proliferation inhibitory activity against NCI-N87 cells, JIMT-1 cells, Colo205 cells and MDA-MB-231 cells, and can exert excellent antitumor effects. The anti-B7H4 antibody drug conjugate of the present invention has good endocytosis towards B7H4 positive expressing MDA-MB-468 cells, which is superior to the reference ADC-1. The antibody-drug conjugate of the present application has significant growth inhibitory activity against B7H4-positive expressing cells MDA-MB-468, colorectal cancer HT29, human breast cell carcinoma MX-1, and endometrial cancer cell RL95-2.

[0118] The anti-B7H4 antibody drug conjugate of the present invention is able to block the inhibition of different donor-derived T cells by B7H4 positive cells and is superior to the reference ADC-1. The anti-B7H4 antibody-drug conjugate of the present invention has good stability in human, rat and monkey plasma in vitro. At the same time, due to the low plasma concentration and short half-life of the cytotoxic drug P-III-30, the systemic exposure of P-III-30 is low, which provides higher safety. The anti-B7H4 antibody-drug conjugate of the present invention has significant antitumor activity against MDA-MB-468 tumor-bearing mice, MX-1 breast cancer cell tumor-bearing mice, MCF-7 breast cancer cell tumor-bearing mice, RL95-2 endometrial cancer cell tumor-bearing mice, and OVCAR-3 ovarian cancer cell tumor-bearing mice. Therefore, the present invention has good prospects for application in the field of diseases associated with positive expression of B7H4 (eg cancer). [Brief description of the drawings]

[0119] [Figure 1] 1 shows efficacy evaluation of antibody-drug conjugates in MDA-MB-468 tumor-bearing mice. [Diagram 2] 1 shows species cross-reactivity of antibody drug conjugates in human, cynomolgus monkey, and mouse B7H4-transfected HEK293T cells. [Diagram 3] 1 shows the binding specificity of antibody-drug conjugates and cells overexpressing human B7 family proteins. [Figure 4] Figure 1 shows the integrated area of ​​fluorescent signal due to endocytosis of antibody drug conjugates in MDA-MB-468 cells at different time points. [Diagram 5] 1 shows the endocytosis efficiency of antibody-drug conjugates in MDA-MB-468 cells. [Figure 6] 1 shows the in vitro growth inhibition rate of antibody-drug conjugates against HT29-B7H4. [Figure 7] The in vitro growth inhibition efficiency of antibody-drug conjugates, monoclonal antibodies, and payloads (cytotoxic drugs) against tumor cells MX-1 is shown (3D cell culture method). [Figure 8] The in vitro growth inhibition efficiency of antibody-drug conjugates, monoclonal antibodies, and payloads (cytotoxic drugs) against tumor cells RL95-2 (3D cell culture method). [Figure 9] The in vitro growth inhibitory efficiency of antibody-drug conjugates, monoclonal antibodies, and payloads (cytotoxic drugs) against tumor cells JIMT-1 (3D cell culture method). [Figure 10]Activation of PBMC-derived T cells by antibody-drug conjugates. [Figure 11] Activation of PBMC-derived T cells by antibody-drug conjugates. [Figure 12] 1 shows the bystander killing effect of antibody drug conjugates on HT29-Luc cells incubated with HT29-B7H4. [Figure 13] Figure 1 shows payload (cytotoxic drug) release kinetics of antibody drug conjugates after 21 days of in vitro plasma incubation in human, monkey, and rat. [Figure 14] 1 shows efficacy evaluation of antibody-drug conjugates in mice bearing breast cancer MX-1 tumors. [Figure 15] 1 shows efficacy evaluation of antibody-drug conjugates in endometrial cancer RL95-2 tumor-bearing mice. [Figure 16] 1 shows efficacy evaluation of antibody-drug conjugates in ovarian cancer OVCAR-3 tumor-bearing mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0120] Sample measurement 1. ADC DAR value analysis method -HIC-HPLC (hydrophobic chromatography) High-performance liquid chromatograph: Waters e2965 high-performance liquid chromatography system. Chromatography column: MabPac® HIC-Butyl 5 μm 4.6 × 100 mm (Manufacturer: Thermo). Mobile phase A: 1.5 M (NH4) 2 SO 4 + 50mM K 2 HPO 4 (pH 7.0). Mobile phase B: 50mM K 2 HPO 4 (pH 7.0) / isopropanol (75:25 V / V).

[0121] Elution is carried out according to the following elution procedure. [Table 1] Detection conditions: the flow rate of the mobile phase was set to 1 ml / min, the detection wavelength was 280 nm, and the column temperature was 30°C.

[0122] 2. SEC Purity Analysis - SEC-HPLC (Size Exclusion Chromatography) High performance liquid chromatograph: 1260 Agilent Liquid Chromatograph. Chromatography column: Waters Xbridge BEH200 SEC (7.8×300 mm, 3.5 μm). Mobile phase: 50mM NaH 2 PO 4 + 200mM arginine (pH 6.80) + 10% isopropanol. [Table 2] Detection conditions: the flow rate of the mobile phase was set to 0.5 ml / min, the detection wavelength was 280 nm, and the column temperature was 30°C.

[0123] The present invention includes all combinations of the specific embodiments described. Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description provided below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are provided by way of illustration only, since various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. EXAMPLES

[0124] The following examples are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and should not be construed as limiting the scope thereof.

[0125] Example 1, Anti-B7H4 Antibody The antibody of the present invention was prepared with reference to PCT / CN2021 / 102952, in which the variable region amino acid sequences of anti-B7H4 antibody DB1001 (PR008199) are as follows, and the CDR regions were determined according to the Kabat numbering convention.

[0126] DB1001 heavy chain variable region: QVQLVESGGGVVQPGRSLRLSCAASGTFRSFGMHWVRQAPGKGLEWVAVISYDASNEYYADSVKGRFIISRDNSKDTLYLQMNSLRAGDTAVYYCAKGGALRWYFAYWGQGTLVTVSS(SEQ ID NO:7) [ka] [ka] [ka]

[0127] DB1001 light chain variable region: EIVMTQSPATLSVSPGERATLSCRASQSISSNLGWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYRSWPPLTFGGGTKVEIK(SEQ ID NO:8) [ka] [ka] [ka]

[0128] Based on the above sequence, primers are designed to construct VH / VK gene fragments by PCR to obtain the variable region. The antibody variable region is further subjected to homologous recombination with the constant region gene fragment to construct the complete antibody sequence DB1001. After transfection into CHO cells, antibody DB1001 is obtained according to the usual expression and purification methods.

[0129] Heavy chain amino acid sequence of DB1001 QVQLVESGGGVVQPGRSLRLSCAASGTFRSFGMHWVRQAPGKGLEWVAVISYDASNEYYADSVKGRFIISRDNSKDTLYLQMNSLRAGDTAVYYCAKGGALRWYFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:9)

[0130] DB1001 light chain amino acid sequence EIVMTQSPATLSVSPGERATLSCRASQSISSNLGWYQQKPGQAPRLLIYGASTRATGIPA RFSGSGSGTEFTLTISSLQSEDFAVYYCQQYRSWPPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:10)

[0131] Example 2. Preparation of anti-B7H4 antibody drug conjugates (ADCs) 2.1. Preparation of cytotoxic payload Preparation Example 1 [ka]

[0132] Step 1: To a solution of KI4 (900 mg, 1.69 mmol), HATU (691 mg, 1.88 mmol), and 3a (320 mg, 2.00 mmol) in DMF (18 mL) was added DIEA (500 mg, 3.87 mmol) at 0 °C under nitrogen atmosphere. Stir at 25 °C for 3 h. TLC (EA) showed the reaction of the raw materials was complete. The reaction solution was added dropwise to 320 mL of deionized water and filtered to give 850 mg of a gray solid, with a yield of 87%.

[0133] Step 2: 3b (100 mg, 0.174 mmol) in MeOH / DCM (1 / 1, 3 mL) with NaHCO 3 (42 mg, 0.50 mmol) solid was added and stirred at 25°C for 3 h, TLC (EA) showed the reaction was complete, the reaction was filtered, rotary dried at low temperature, slurried in aq. HCl (0.5 M, 10 mL), filtered, purified by prep-HPLC (0.1% TFA), and lyophilized to give 15 mg of a grey solid, 16% yield. MS m / z(ESI):534 [M+1]. H-NMR(400MHz,DMSO-D):8.45(d, 1H), 7.81(d, 1H), 7.32(s, 1H), 6.52(m, 1H), 5.58-5.56(m, 1H), 5.44(s, 2H), 5.14(dd, 2H), 3.96(m, 1H), 3.48(m, 1H), 3.19(m, 2H), 2.53-2.28(m, 3H), 2.48(s, 3H), 2.20-2.00(m, 4H), 1.95-1.80(m, 2H), 0.89(t, 3H).

[0134] Preparation Example 2 [ka]

[0135] Step 1 Under nitrogen atmosphere, DIEA (60.6 mg, 0.47 mmol) was added dropwise to a solution of KI4 (100 mg, 0.19 mmol), HATU (85.7 mg, 0.23 mmol), and 23a (21.5 mg, 0.21 mmol) in DMF (2 mL), and the mixture was allowed to react at 0 °C for 2 h after the addition was complete. LCMS showed that the reaction of the raw materials was complete. The reaction solution was added dropwise to 20 mL of water and stirred to precipitate a solid, which was then filtered to give 60.2 mg of a gray solid P-III-30, with a yield of 61%. MS-ESI: m / z 522.2 [M+H] + . 1 H NMR(400MHz,DMSO-d6) δ 8.42(d, J=8.7Hz, 1H), 7.79(d, J=11.0Hz, 1H), 7.30(s, 1H), 6.53(s, 1H), 5.62 - 5.53(m, 1H), 5.42(s, 2H), 5.30 - 5.16(m, 2H), 4.63(d, J=4.6Hz, 1H), 4.09 - 3.99(m, 1H), 3.22 - 3.11(m, 2H), 2.40(s, 3H), 2.28(dd, J=13.7, 7.2Hz, 1H), 2.22 - 2.08(m, 3H), 1.94 - 1.78(m, 2H), 1.08(d, J=6.1Hz, 3H), 0.87(t, J=7.3Hz, 3H).

[0136] Preparation Example 3 [ka]

[0137] Step 1 KI4 (50 mg, 0.094 mmol), 25a (11 mg, 0.094 mmol), and HATU (39 mg, 0.103 mmol) were added to DMF (3 mL), DIEA (30 mg, 0.235 mmol) was added after nitrogen gas replacement, and the reaction was allowed to proceed for 1.5 h at 25° C., after which LCMS showed the reaction was complete. The reaction was added dropwise to stirred water (50 mL), allowed to stand for 5 min after addition was complete, filtered, and the filter cake was lyophilized to give 20 mg of a grey solid 25 in 40% yield. MS-ESI: m / z 534.3 [M+H] + . 1HNMR(400MHz,DMSO-d6) δ 8.26(d, J=9.1Hz, 1H), 7.72(d, J=10.9Hz, 1H), 7.28(s, 1H), 6.51(s, 1H), 6.12(s, 1H), 5.59 - 5.50(m, 1H), 5.40(s, 2H), 5.15(d, J=18.8Hz, 1H), 4.98(d, J=19.0Hz, 1H), 3.28 - 3.16(m, 1H), 3.15 - 3.02(m, 1H), 2.74 - 2.52(m, 2H), 2.36(s, 3H), 2.24 - 2.04(m, 4H), 1.92 - 1.79(m, 4H), 0.86(t, J=7.3Hz, 3H).

[0138] Reference Example 1 [ka]

[0139] Step 1 KI4 (50 mg, 0.094 mmol), 26a (18 mg, 0.094 mmol), and HATU (39 mg, 0.103 mmol) were added to DMF (3 mL), and after nitrogen gas replacement, DIEA (48 mg, 0.376 mmol) was added. After 1.5 h at 25 °C, LCMS showed the reaction was complete. The reaction was added dropwise to stirred water (50 mL), left to stand for 5 min after addition was complete, filtered, and the filter cake was lyophilized to give 30 mg of a grey solid 26b in 52% yield. MS-ESI: m / z 607.4 [M+H] + .

[0140] Step 2 26b (30 mg, 0.049 mmol) was dissolved in DCM (2 mL), purged with nitrogen gas, cooled to 0°C, TFA (0.5 mL) was added, and the mixture was reacted at 0°C for 1.5 h, after which LCMS showed the reaction was complete. The reaction solution was rotovapped at low temperature, washed once with DCM, and then lyophilized with acetonitrile and water to give 20 mg of a yellow solid in 80% yield. MS-ESI: m / z 507.1 [M+H] + . 1HNMR(400MHz,DMSO-d6) δ 8.67(d, J=8.6Hz, 1H), 7.86 - 7.66(m, 4H), 7.32(s, 1H), 6.56(brs, 1H), 5.63 - 5.54(m, 1H), 5.43(s, 2H), 5.29(d, J=18.9Hz, 1H), 5.22(d, J=18.9Hz, 1H), 3.23 - 3.15(m, 2H), 3.13 - 3.03(m, 2H), 2.57 - 2.51(m, 2H), 2.43 - 2.38(m, 3H), 2.27 - 2.08(m, 2H), 1.94 - 1.78(m, 2H), 0.87(t, J=7.3Hz, 3H).

[0141] 2.2. Preparation of linker-payload Linker-Cytotoxin X1 [ka]

[0142] Step 1 27a (5.00 g, 43.0 mmol), NaHCO 3 Benzyl bromide (11.0 g, 64.6 mmol) was added dropwise to a solution of (10.9 g, 129 mmol) in DMF (50 mL) under a nitrogen atmosphere, and the mixture was reacted at 25° C. for 17 hours. TLC (PE / EA=2 / 1) showed the reaction was complete, and the reaction solution was added to 500 mL of water, extracted twice with EA (250 mL), separated, washed with saturated aqueous sodium chloride (500 mL), and then diluted with anhydrous Na 2 SO 4 The mixture was dried at 40° C., concentrated and passed through a column (PE:EA=3:2) to obtain 5.1 g of a colorless liquid, the yield is 57.1%.

[0143] Step 2 A solution of 27b (4.50 g, 21.8 mmol) in THF (10 mL) was added dropwise to a solution of KI2 (4.00 g, 10.9 mmol) and TsOH (800 mg, 4.65 mmol) in THF (30 mL) at 0 °C under nitrogen atmosphere, and the mixture was reacted at 25 °C for 2 h. TLC (PE / EA = 1 / 2) showed the reaction was complete. The reaction solution was added to 200 mL of water, extracted twice with EA (200 mL), and separated. The mixture was then washed with anhydrous NaCl. 2 SO 4 It was dried at 40° C., concentrated and passed through a column (PE / EA=3 / 2) to give 1.56 g of white solid, the yield is 26%.

[0144] Step 3 To a mixture of 27c (800 mg, 1.55 mmol) in EtOH (8 mL) and EA (8 mL) was added Pd / C (80 mg) under hydrogen atmosphere at 0° C. and stirred at 0° C. for 2.5 h. LCMS showed the reaction was complete. The reaction was filtered through diatomaceous earth, the filter cake was washed with EA (200 mL), concentrated, dissolved in THF (20 mL) and spun to dryness to give 600 mg of a white solid, 91% yield.

[0145] Step 4 To a solution of 27d (220 mg, 0.515 mmol), HY-13631A (250 mg, 0.47 mmol) and HATU (214 mg, 0.56 mmol) in DMF (6 mL) was added DIEA (152 mg, 1.18 mmol) under nitrogen atmosphere at 0 °C and reacted at 0 °C for 2 h. LCMS showed the reaction was complete. The reaction was added to an aqueous citric acid solution (pH = 4) (150 mL), filtered, the filter cake was washed with 175 mL of water, filtered, dried and dried on an oil pump to give 260 mg of brown solid, the yield is 66%.

[0146] Step 5 Diethylamine (8 mL) was added dropwise to a solution of 27e (260 mg, 0.309 mmol) in DCM (30 mL) under nitrogen atmosphere at 0°C, and the mixture was reacted at 0°C for 3 hours. LCMS showed that the reaction was complete. The reaction solution was added to a petroleum ether solution (600 mL) at 0°C, and a solid precipitated. After the solid was allowed to stand and adsorbed to the bottom of the bottle, the solution was poured out and dried with an oil pump to obtain 90 mg of a brown solid, with a yield of 47.1%.

[0147] Step 6 HATU (74 mg, 0.19 mmol) was added to a solution of 27f (90 mg, 0.13 mmol), KI-1 (92 mg, 0.19 mmol) and DIEA (50 mg, 0.39 mmol) in DMF (2.5 mL) under nitrogen atmosphere at 0° C., and the mixture was reacted at 0° C. for 2 h. LCMS showed that the reaction was essentially complete. At 0° C., the reaction solution was added to an aqueous citric acid solution (30 mL) with a pH of 4, and a flocculent solid was precipitated, which was filtered and separated through a separation plate (DCM / MecOH=10 / 1) to obtain 9.2 mg of a pale yellow solid X1, with a yield of 6%. MS m / z(ESI):1074 [M+1]. H-NMR(400MHz,MeOD):7.65(d, 1H), 7.62(s, 1H), 7.30-7.21(m, 5H), 6.79(s, 2H), 5.69-5.65(m, 1H), 5.57(d, 1H), 5.43-5.10(m, 3H), 4.70(d, 2H), 4.48-4.39(m, 2H), 4.10-4.05(m, 1H), 4.01-3.75(m, 5H),3.46(t, 2H), 3.22-3.15(m, 2H), 3.07-3.00(m, 1H), 2.75(m, 1H), 2.62(m, 1H), 2.45(s, 3H), 2.37-2.20(m, 6H), 2.10-2.02(m, 2H), 2.00-1.92(m, 2H) 1.68-1.57(m, 6H), 1.01(t, 3H).

[0148] Linker-Cytotoxin X2 [ka]

[0149] Step 1 34a (5 g, 48.0 mmol), K 2 CO 3 (19.9 g, 144.0 mmol) is dissolved in DMF (20 mL), benzyl bromide (12.3 g, 72.0 mmol) is added dropwise, and the mixture is reacted at 25°C for 17 hours. The reaction of the raw materials is detected to be complete by TLC (PE / EA=3 / 1). The reaction mixture is added to water (200 mL), extracted and separated with EA (250 mL), washed with saturated NaCl, and then washed with anhydrous Na 2 SO 4 After drying at 40° C., it was concentrated and passed through a column (PE:EA=2:1) ​​to obtain 8.7 g of colorless liquid 34b, the yield was 93%. MS-ESI: m / z 195.1 [M+H] + .

[0150] Step 2 Dissolve 34c (7.3 g, 19.8 mmol) and TsOH (1.46 g, 8.5 mmol) in THF (20 mL), protect with nitrogen, cool to 0 °C, add 43b (7.7 g, 39.6 mmol) in THF (10 mL) dropwise, and react at 0 °C for 2 h after the addition is complete. TLC (PE / EA = 2 / 1) shows that most of the raw materials have reacted. Pour the reaction mixture into 100 mL of water, extract with DCM (100 mL), separate, wash with saturated NaCl, and add anhydrous Na 2 SO 4 After drying at 40° C., it was passed through a column (PE / EA=1 / 1) to obtain 3.9 g of colorless viscous material 34d, the yield is 39%. MS-ESI: m / z 503.3 [M+H] + .

[0151] Step 3 To a mixture of 34d (1.9 g, 3.78 mmol) in EtOH (100 mL) and EA (100 mL), Pd / C (1 g, 10 wt.%) was added under hydrogen atmosphere at 0° C. and reacted at 0° C. for 3 h. TLC (PE / EA=2 / 1) showed the reaction was complete. The reaction was filtered through diatomaceous earth, the filter cake was washed with EA / EtOH (1:1, 100 mL×3), the filtrate was concentrated, dissolved in THF (50 mL×3), spun dry, and repeated three times to obtain 1 g of gray solid 34e, the yield was 64%. MS-ESI: m / z 435.2 [M+Na] + .

[0152] Step 4 DIEA (303 mg, 2.35 mmol) was added dropwise to a solution of 34e (426 mg, 1.03 mmol), KI4 (500 mg, 0.94 mmol) and HATU (429 mg, 1.13 mmol) in DMF (20 mL) at 0 °C under nitrogen atmosphere, and the mixture was reacted at 0 °C for 2 h after the completion of the addition. LCMS showed that the reaction was completed. The reaction solution was added dropwise to 300 mL of water, stirred, then left to stand for 5 min, filtered, and the filter cake was dissolved in a solution of DCM / MeOH (10:1, 100 mL), then dried by rotary evaporation, and the sample was stirred and purified by column chromatography (EA:MeOH=30:1) to obtain 600 mg of yellow solid 34f, with a yield of 77%. MS-ESI: m / z 830.3 [M+H] + .

[0153] Step 5 Diethylamine (5mL) was added dropwise to a solution of 34f (150mg, 0.18mmol) in DCM (5mL) under nitrogen atmosphere at 0℃, and the mixture was reacted at 0℃ for 2 hours. LCMS showed that the reaction was completed. Petroleum ether solution (100mL×6) was added to the reaction solution, and a solid precipitated. After the solid precipitated by standing, the solution was poured out and dried by oil pump to obtain 120mg of white powder 34g, LCMS showed that the product content was 70%, and the yield was 76%. MS-ESI: m / z 608.3 [M+H] + .

[0154] Step 6 A solution of 34g (60 mg, 0.099 mmol), 43h (51 mg, 0.108 mmol), and DIEA (32 mg, 0.25 mmol) in DMF (1 mL) was added to a solution of HATU (45 mg, 0.118 mmol) in DMF (1 mL) at 0 °C under nitrogen atmosphere and reacted at 0 °C for 2 h. LCMS showed that the reaction of the raw materials was complete. The reaction solution was directly passed through a reverse phase column with eluent ((MeCN / MeOH=1 / 1):H2O=60%:40%) and purified to obtain 14.8 mg of yellow solid X2, with a yield of 14%. MS-ESI: m / z 1062.4 [M+H] + . 1HNMR(400MHz, Methanol-d4) δ 7.69 - 7.61(m, 2H), 7.22 - 7.16(m, 2H), 7.16 - 7.09(m, 3H), 6.76(s, 2H), 5.70 - 5.64(m, 1H), 5.60(d, J=16.4Hz, 1H), 5.40 - 5.31(m, 2H), 5.26(d, J=19.0Hz, 1H), 4.65 - 4.50(m, 7H), 4.25 - 4.16(m, 1H), 3.87(d, J=16.7Hz, 1H), 3.83 - 3.76(m, 3H), 3.72(d, J=17.0Hz, 2H), 3.44(t, J=7.1Hz, 2H), 3.25 - 3.17(m, 2H), 3.10 - 3.02(m, 1H), 2.92 - 2.83(m, 1H), 2.45 - 2.39(m, 5H), 2.32 - 2.20(m, 5H), 1.97 - 1.89(m, 2H), 1.63 - 1.50(m, 4H), 1.34 - 1.20(m, 6H), 0.99(t, J=7.3Hz, 3H).

[0155] Linker-Cytotoxin X3 [ka]

[0156] Step 1 32a (2.00 g, 6.6 mmol), K 2 CO 3 Bromopropene (960 mg, 7.92 mmol) was added to MeCN (20 mL) (1.82 g, 13.2 mmol) and stirred at 20 °C for 5 h. TLC (PE / EA=1 / 2) showed the reaction was completed. The reaction solution was poured into water (100 mL), adjusted to pH 5, extracted with EA (100 mL) three times, dried over anhydrous sodium sulfate, rotary evaporated, and purified through column (PE / EA=2 / 1) to obtain 1.83 g of white solid 32b, the yield was 81%.

[0157] Step 2 A solution of 32b (1.38 g, 4.02 mmol) in DCM (10 mL) and TFA (10 mL) was added and stirred at 25° C. for 17 h. TLC (PE / EA=1 / 3) showed the reaction was complete. The reaction was spun down to give 0.91 g of yellow viscous material 32c in negligible yield.

[0158] Step 3 32c (910 mg, 4.87 mmol), NaHCO 3 (613 mg, 7.3 mmol) in DME / HO (20 mL / 10 mL) was added with 41d (1.92 g, 4.87 mmol) and stirred at 25 °C for 3 h. TLC (DCM / MeOH = 1 / 1) showed the reaction was completed. The reaction solution was poured into 100 mL of water, adjusted to pH 5 with aq. HCl (1 N), extracted twice with EA (150 mL), dried over anhydrous sodium sulfate, rotary evaporated and purified through column (DCM / MeOH = 20 / 1) to give 1.53 g of white solid 32e, the yield was 67%. MS-ESI: m / z 467.4 [M+H] + .

[0159] Step 4 A solution of 32f (3 g, 5.83 mmol) in MeOH (50 mL) was added to Pd / C (600 mg) and stirred under a hydrogen balloon at 25° C. for 5 h. TLC (EA) showed the reaction was complete. The reaction was filtered and spun to dryness to give 1.9 g of white solid 32f, 77% yield.

[0160] Step 5 To 32g (789 mg, 1.86 mmol), KI4 (900 mg, 1.69 mmol), triethylamine (342 mg, 3.38 mmol) in DMF (10 mL), add HATU (707 mg, 1.86 mmol) and stir at 0°C for 3.5 h. TLC (EA) indicates the reaction is complete. The reaction solution is then cooled to 50°C for 3.5 h. 2 Pour into 2H2O (80 mL), extract twice with EA (100 mL), dry over anhydrous sodium sulfate, rotovaporize and purify through column (EA) to give 1.186 g of white solid 32h, 83% yield. MS-ESI: m / z 842.3 [M+H] +.

[0161] Step 6 32h (1.186 g, 1.41 mmol) in DCM / diethylamine (20 mL, 20 / 1) was stirred at 25° C. for 17 h. TLC (DCM / MeOH=10 / 1) showed the reaction was complete. The reaction was poured into petroleum ether (200 mL) and filtered to give 768 mg of white solid 32i, 88% yield. MS-ESI: m / z 620.3 [M+H] + .

[0162] Step 7 HATU (414 mg, 1.09 mmol) was added to 32i (676 mg, 1.09 mmol), 32e (508 mg, 1.09 mmol), DIEA (423 mg, 3.27 mmol) in DMF (10 mL) and stirred at 20 °C for 17 h. TLC (PE / EA = 1 / 5) showed the reaction was complete. The reaction solution was poured into water (30 mL) and filtered, and the filter cake was purified through a column (DCM / MeOH = 50 / 1) to give 511 mg of white solid 32j, the yield is 44%. MS-ESI: m / z 1068.3 [M+H] + .

[0163] Step 8 A solution of 32j (482 mg, 0.451 mmol) in diethylamine / DCM (10 mL, 1 / 5) is stirred at 10° C. for 17 h. TLC (EA) shows the reaction is complete. The reaction is poured into PE (300 mL) and filtered to give 301 mg of white solid 32k in negligible yield.

[0164] Step 9 To 32k (301 mg, 0.356 mmol), Pd(PPh3)4 (82 mg, 0.071 mmol) in THF (5 mL) was added morpholine (93 mg, 1.07 mmol) and stirred at 25 °C for 5 h. LCMS showed the reaction was complete. The reaction was worked up to 108 mg of white solid 32l, 38% yield. MS-ESI: m / z 806.3 [M+H] + .

[0165] Step 10 Bromoacetyl bromide (27 mg, 0.134 mmol) was added to 32l (108 mg, 0.134 mmol), triethylamine (41 mg, 0.402 mmol) in THF (2 mL) and DMF (2 mL), and stirred at 0° C. for 1 h. TLC (DCM / MeOH=10 / 1) showed the reaction was complete. The reaction solution was directly purified to give 15 mg of white solid X3, with a yield of 12%. MS-ESI: m / z 926.3 [M+H] + . 1HNMR(400MHz,DMSO-d6) δ 12.11(s, 1H), 8.54 - 8.42(m, 3H), 8.27 - 8.16(m, 2H), 7.78(d, J=11.0Hz, 1H), 7.30(s, 1H), 6.53(s, 1H), 5.61 - 5.51(m, 1H), 5.42(s, 2H), 5.20 - 5.05(m, 2H), 4.56 - 4.42(m, 2H), 4.32 - 4.22(m, 1H), 3.96 - 3.87(m, 3H), 3.79(d, J=5.6Hz, 2H), 3.70(d, J=5.9Hz, 2H), 3.25 - 3.08(m, 2H), 2.61 - 2.53(m, 2H), 2.45 - 2.36(m, 4H), 2.36 - 2.22(m, 3H), 2.20 - 2.03(m, 4H), 1.99 - 1.68(m, 4H), 0.87(t, J=7.3Hz, 3H).

[0166] Linker-Cytotoxin X4 [ka]

[0167] Step 1 A solution of 33a (2.00 g, 2.58 mmol) in MeOH (20 mL) was added to Pd / C (400 mg, 10 wt.%) and stirred at 20 °C for 5 h. TLC (EA) showed the reaction was complete. The reaction was filtered and spun to dryness to give 1.3 g of white solid 33b, 74% yield.

[0168] Step 2 HATU (305 mg, 0.802 mmol) was added to 33b (0.55 g, 0.802 mmol), KI4 (427 mg, 0.802 mmol) and DIPEA (310 mg, 2.40 mmol) in DMF (5 mL) and stirred at 0 °C for 2 h. TLC (DCM / MeOH = 1 / 10) showed the reaction was complete. The reaction solution was poured into water (40 mL) and filtered to obtain the crude product, which was purified by column chromatography (DCM / MeOH = 20 / 1) to give 360 ​​mg of yellow solid 33c, with a yield of 41%.

[0169] Step 3 To 33c (360 mg, 0.326 mmol) in DCM (10 mL) was added diethylamine (2 mL). The mixture was stirred at 25° C. for 17 h. TLC (DCM / MeOH=5 / 1) showed the reaction was complete. The reaction was poured into PE (100 Ml) and filtered to give 205 mg of white solid 33d, 71% yield. MS-ESI: m / z 881.3 [M+H] + .

[0170] Step 4 To 33d (205 mg, 0.233 mmol) and triethylamine (118 mg, 1.17 mmol) in DMF (1 mL) and water (1 mL), add a solution of bromoacetyl bromide (94 mg, 0.446 mmol) in THF (2 mL), and stir at 0°C for 1 h. Directly prepare 15 mg of white solid X4 from the reaction solution, with a yield of 6%. MS-ESI: m / z 1001.2 [M+H] + . 1HNMR(400MHz,DMSO-d6) δ 8.57 - 8.50(m, 1H), 8.50 - 8.43(m, 2H), 8.35 - 8.29(m, 1H), 8.19 - 8.12(m, 2H), 7.80(d, J=10.8Hz, 1H), 7.27 - 7.14(m, 7H), 6.53(s, 1H), 5.59 - 5.51(m, 1H), 5.44 - 5.39(m, 2H), 5.20 - 5.07(m, 2H), 4.56 - 4.44(m, 3H), 3.92(s, 3H), 3.80 - 3.68(m, 5H), 3.41(s, 1H), 3.21 - 3.12(m, 2H), 2.83 - 2.74(m, 1H), 2.58 - 2.55(m, 3H), 2.39(s, 4H), 2.18 - 2.03(m, 4H), 1.93 - 1.78(m, 2H), 0.87(t, J=7.3Hz, 3H).

[0171] 2.3 Preparation of anti-B7H4 antibody-drug conjugate Preparation of antibody-drug conjugate ADC DB1001-X1 [ka] Prepare a reducing agent and a protecting agent using ultrapure water as follows: 2 mg / mL TCEP (Tris-2-carboxyethyl-phosphine, manufacturer: Thermo) aqueous solution and 100 mmol / L EDTA (disodium ethylenediaminetetraacetate, manufacturer: Sigma) aqueous solution.

[0172] 160mg of 17.99mg / mL DB1001 (PR08199) monoclonal antibody was placed in a 600mL centrifuge bottle, 30mM His-HAc was added, and the antibody concentration was diluted to 10mg / mL with a pH 5.5 buffer solution. 100mM EDTA aqueous solution was added according to 5% of the total volume of the reaction solution, and after shaking and mixing uniformly, 2mg / mL TCEP aqueous solution was added to reduce the antibody, and the molar ratio of TCEP to antibody was 2.3:1. After shaking and mixing uniformly, the mixture was placed in a cooled thermostatic mixer at 37℃ for 2h. According to the final concentration molar ratio of drug to antibody of 12:1, the DMA solution of the Linker-payload was added, and DMA was supplemented according to 10% of the total volume of the reaction solution, and after shaking and mixing uniformly, the mixture was placed in a cooled thermostatic mixer at 4℃ for 1h. The sample was placed in an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore) and the sample was replaced with a sample storage buffer. First, the sample was ultrafiltered three times with a buffer solution of pH 5.5 using 30 mM His-HAc containing 10% DMSO, and then ultrafiltered six times with a buffer solution of pH 5.5 using 30 mM His-HAc without DMSO, to obtain 125.6 mg of the antibody-drug conjugate DB1001-X1, with a concentration of 12.854 mg / mL and a yield of 78.5%. As a result of detection by HIC, the drug binding ratio (DAR) of the antibody-drug conjugate DB1001-X1 was 3.89, and the SEC purity was 100%.

[0173] Preparation of antibody-drug conjugate ADC DB1001-X2 [ka] A reducing agent and a protecting agent are prepared with ultrapure water as follows: 2 mg / mL TCEP (Tris-2-carboxyethyl-phosphine, manufacturer: Thermo) aqueous solution and 100 mmol / L EDTA (disodium ethylenediaminetetraacetate, manufacturer: Sigma) aqueous solution. Dissolve linker-payload X2 in dry DMA (N,N-Dimethylacetamide, manufacturer: China Pharmaceutical Group) to prepare a 10 mg / mL linker-payload DMA solution.

[0174] 160 mg of 17.99 mg / mL DB1001 (PR08199) monoclonal antibody is placed in a 600 mL centrifuge bottle, 30 mM His-HAc is added, and the antibody concentration is diluted to 10 mg / mL with pH 5.5 buffer. 100 mM EDTA aqueous solution is added according to 5% of the total volume of the reaction solution, and after shaking and mixing uniformly, 2 mg / mL TCEP aqueous solution is added to reduce the antibody, and the molar ratio of TCEP to antibody is 2.3:1. After shaking and mixing uniformly, the mixture is placed in a cooling type thermostatic mixer at 37 ° C. and reacted for 2 h. According to the final concentration molar ratio of drug to antibody of 12:1, the DMA solution of the Linker-payload is added, and DMA is supplemented according to 10% of the total volume of the reaction solution, and after shaking and mixing uniformly, the mixture is placed in a cooling type thermostatic mixer at 4 ° C. and reacted for 1 h. The sample was placed in an ultrafiltration tube (MWCO 30KD, manufacturer: Millipore) and the sample was replaced with a sample storage buffer. First, the sample was ultrafiltered three times with a buffer solution of pH 5.5 using 30 mM His-HAc containing 10% DMSO, and then ultrafiltered six times with a buffer solution of pH 5.5 using 30 mM His-HAc without DMSO, to obtain 128.3 mg of the antibody-drug conjugate DB1001-X2, with a concentration of 10.522 mg / mL and a yield of 80.18%. As detected by HIC, the drug-drug conjugate DB1001-X2 had a drug-drug binding ratio (DAR) of 5.4 and an SEC purity of 98.1%.

[0175] Preparation of reference ADC-1 Reference ADC-1 is prepared with reference to compound 34 (hu2F7-etitecan) of WO2020244657A1.

[0176] Example 3. In vitro proliferation inhibition test of small molecule compounds (cytotoxic drugs) 3.1. In vitro growth inhibition test of small molecule compounds (cytotoxic drugs) against tumor cells Purpose of the test Detect the inhibitory activity of drug compounds against the proliferation of NCI-N87, JIMT-1 and MBA-MB-231 tumor cells in vitro. After treating the cells with different concentrations of compounds in vitro and culturing for 6 days, the proliferation of the cells was detected using CTG (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, Cat. No.: G7558) reagent and the IC 50 Based on the value, the in vitro activity of the compound is evaluated.

[0177] 1. Cell culture: NCI-N87 / JIMT-1 / MBA-MB-231 are cultured in RPMI-1640 medium containing 10% FBS. 2. Cell preparation: Take NCI-N87 / JIMT-1 / MBA-MB-231 cells in logarithmic growth phase, wash once with PBS, add 2-3mL trypsin to digest for 2-3min, after the cells are completely digested, add 10-15mL cell culture medium to elute the digested cells, centrifuge at 1000rpm for 5min, discard the supernatant, then add 10-20mL cell culture medium to resuspend the cells and prepare a single cell suspension. 3. Cell plating: NCI-N87 / JIMT-1 / MBA-MB-231 single cell suspensions were homogenously mixed and cultured in cell culture medium to obtain a live cell density of 6x10 4 Adjust the cell density to 100 cells / mL, mix the density-adjusted cell suspension evenly, and add 50 μL / well to a 96-well cell culture plate. Incubate the culture plate in an incubator (37°C, 5% CO) for 18 hours. 2 ).

[0178] 4. Compound preparation: Dissolve the compounds in DMSO to prepare stock solutions with an initial concentration of 10 mM. The total eight concentrations of the small molecule compounds were 300, 100, 30, 10, 3, 1, 0.3, and 0.1 nM, respectively. 5. Sample addition: Add the prepared test samples of different concentrations to the culture plate, and prepare two replicate wells for each sample. Incubate the culture plate in an incubator for 6 days (37°C, 5% CO2 ). 6. Color development procedure: Take out a 96-well cell culture plate, add 50uL of CTG reagent to each well, and incubate at room temperature for 10 minutes. 7. Plate reading operation: Take out the 96-well cell culture plate, place it on the microplate reader, and measure the chemiluminescence with the microplate reader.

[0179] Data analysis: Data was processed and analyzed using Microsoft Excel and Graphpad Prism 5. [Table 3] Conclusion: According to the results in Table 1, the toxin of the drug conjugate of this application has obviously enhanced proliferation inhibitory activity against NCI-N87 cells, JIMT-1 and MDA-MB-231 cells.

[0180] 3.2. In vitro growth inhibition test of small molecule compounds (cytotoxic drugs) against tumor cells Purpose of the test Detect the inhibitory activity of drug compounds on the proliferation of NCI-N87 cells and Colo205 tumor cells in vitro. After treating the cells with different concentrations of compounds in vitro and culturing for 6 days, the cell proliferation was detected using CTG (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, Cat. No.: G7558) reagent and the IC 50 Based on the value, the in vitro activity of the compound is evaluated.

[0181] 1. Cell culture: NCI-N87 / Colo205 cells were cultured in RPMI-1640 medium containing 10% FBS. 2. Cell preparation: Take NCI-N87 / Colo205 cells in logarithmic growth phase, wash once with PBS, add 2-3mL trypsin to digest for 2-3min, after the cells are completely digested, add 10-15mL cell culture medium to elute the digested cells, centrifuge at 1000rpm for 5min, discard the supernatant, then add 10-20mL cell culture medium to resuspend the cells and prepare a single cell suspension. 3. Cell plating: NCI-N87 / Colo205 single cell suspensions were homogenously mixed and cultured in cell culture medium to a density of 6x10 live cells. 4 Adjust the cell density to 100 cells / mL, mix the density-adjusted cell suspension evenly, and add 50 μL / well to a 96-well cell culture plate. Incubate the culture plate in an incubator (37°C, 5% CO) for 18 hours. 2 ).

[0182] 4. Compound preparation: Dissolve the compounds in DMSO to prepare stock solutions with an initial concentration of 10 mM. The total eight concentrations of the small molecule compounds were 300, 100, 30, 10, 3, 1, 0.3, and 0.1 nM, respectively. 5. Sample addition: Add the prepared test samples of different concentrations to the culture plate, and prepare two replicate wells for each sample. Incubate the culture plate in an incubator for 6 days (37°C, 5% CO 2 ). 6. Color development procedure: Take out a 96-well cell culture plate, add 50uL of CTG reagent to each well, and incubate at room temperature for 10 minutes. 7. Plate reading operation: Take out the 96-well cell culture plate, place it on the microplate reader, and measure the chemiluminescence with the microplate reader.

[0183] Data analysis: Data was processed and analyzed using Microsoft Excel and Graphpad Prism 5. [Table 4] Conclusion: According to the results in Table 2, the toxin of the drug conjugate of this application has obviously enhanced proliferation inhibitory activity against NCI-N87 and Colo205 cells, and is superior to Reference Example 1.

[0184] Example 4. Cellular endocytosis activity of antibody-drug conjugates Purpose of the test The endocytosis effect of the present antibody-drug conjugate against the B7H4 target was detected in B7H4-expressing MDA-MB-468 cells. The cells were incubated with a fixed concentration of antibody-drug conjugate (ADC) and the endocytosis indicator reagent pHrodo, and the endocytosis ability of the antibody-drug conjugate was evaluated at different time points by observing the fluorescent signal generated by cellular pHrodo associated with the entry of the antibody-drug conjugate into the cells.

[0185] Experimental Method 1. Cell culture: MDA-MB-468 cells are cultured using Leibovitz's L-15 medium containing 10% FBS. 2. Cell preparation: Take MDA-MB-468 cells in logarithmic growth phase, wash once with PBS, then digest for 2-3 min, add 10-15 mL of cell culture medium after the cells are completely digested, elute the digested cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, add cell culture medium to resuspend the cells, prepare a single cell suspension, and adjust the viable cell density to 3x10 5 Adjust to cells / ml. 3. Cell plating: Add 50uL / well to a 96-well cell culture plate. Incubate the culture plate in an incubator (37°C, 5% CO2) for 48 hours. 2 ).

[0186] 4. Sample addition: The antibody-drug conjugate to be detected and Fab-pHrodo are incubated together to form a complex, the concentration is 120nM, and the concentration is prepared by 5-fold gradient dilution, and added to the cells at 50uL / well. There are a total of 8 concentrations, with two replicate wells for each concentration. 5. Cell culture: Incubate the culture plate in an incubator (37°C) for 48 hours. 6. Plate reading procedure: When the corresponding time point is reached, the 96-well cell culture plate is taken out, the cells are digested, and the cell number and fluorescence number are read by FACS.

[0187] [Table 5] Conclusion: Based on the results in Table 3, the antibody-drug conjugate of the present application has an endocytosis effect on MDA-MB-468 cells with high expression of B7H4.

[0188] Example 5: In vitro growth inhibition test of antibody-drug conjugates against tumor cells The CellTiter-Glo (registered trademark) chemiluminescent cell viability assay (i.e., CTG method) was used to evaluate the inhibitory effect of cell proliferation by incubation of B7H4-positive expressing cells MDA-MB-468 and B7H4-negative expressing cells MDA-MB-231 with anti-B7H4 ADCs DB1001-X1 and DB1001-X2 for 7 days.

[0189] Logarithmic growth phase cells were harvested and plated at a density of 2000 cells / well and incubated at 37°C, 5% CO 2 The cells were then cultured overnight in a 37°C incubator. On the next day, DB1001-X1 and DB1001-X2 were diluted 5-fold with complete medium to obtain 9 concentration gradients (starting with the highest concentration of 300 nM), which were then added to the cell culture plate at 50 μL / well, with complete medium as the blank control, and two replicate wells were set up and incubated in a 37°C incubator for 7 days. After the incubation was completed, the cell culture plate was taken out and equilibrated to room temperature, and then 50 μL of CTG detection reagent (Promega, Cat#: G7573) was added to each well, shaken to mix evenly, and then placed in a dark place for 10 minutes, after which the signal value was detected by a microplate reader. The S-shaped dose-response curve was plotted using a nonlinear regression model using GraphPad Prism software, and the IC 50Calculate the value. Formula for cell viability = (Lum 試験対象薬物 -Lum ブランク対照 ) / (Lum 溶媒ブランク対照 -Lum ブランク対照 )×100%.

[0190] The experimental results are shown in the table below. [Table 6] Experimental conclusion: According to the results in Table 4, the antibody-drug conjugates DB1001-X1 and DB1001-X2 of the present application have significantly enhanced proliferation inhibitory activity against B7H4 positive expressing MDA-MB-468.

[0191] Example 6: Efficacy evaluation of antibody-drug conjugates in MDA-MB-468 tumor-bearing mice To study the inhibitory effect of DB1001-X1 and DB1001-X2 on in vivo tumor formation, tumors were formed in mice using B7H4 positive expressing cells MDA-MB-468, and the antitumor effects of DB1001-X1 and DB1001-X2 were then evaluated. 1. Test substances and materials Blank control group (control group): saline DB1001-X1 (treatment group): 3mg / kg DB1001-X1 (treatment group): 10mg / kg DB1001-X2 (treatment group): 3mg / kg DB1001-X2 (treatment group): 10mg / kg 2. Preparation method: All samples are prepared by diluting with saline. 3. Test animals: 6-8 week-old female CB-17 SCID mice, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0192] 4. Test method: 10×10 6 MDA-MB-468 cells were inoculated subcutaneously into the right dorsal region of 6-8 week-old female NOD / SCID mice, and tumors were grown to approximately 172 mm 3When the tumor-bearing mice reached 10 days after the first dose, they were randomly assigned by StudyDirector™ and injected intravenously (iv) once with DB1001-X1 or DB1001-X2 at doses of 3 mg / kg and 10 mg / kg, respectively, starting on that day (day 0). Tumor volumes and body weights were measured twice weekly and the data were recorded. The vehicle control group or treatment group had 5 mice each. The tumor inhibition rate was calculated by measuring the tumor volume. The formula for calculating tumor volume is V=0.5 a × b 2 where a and b respectively indicate the long and short diameters of the tumor. The tumor-inhibiting effect of the compound is evaluated by T / C (%). The percentage value of T / C (%) is an index reflecting tumor growth inhibition, and T and C indicate the average tumor volume on a certain day in the administration group and the control group, respectively. The tumor growth inhibition rate is calculated by the following formula, TGI (%) = [1-(T i -T 0 ) / (V i -V 0 )] × 100, T i is the mean tumor volume of a given treatment group on a given day, and T 0 is the mean tumor volume at the start of treatment in this treatment group, and V i One day (T i is the mean tumor volume of the vehicle control group (on the same day as the first day), and V 0 is the average tumor volume at the start of administration in the solvent control group. Intergroup significance analysis between the test subject group and the vehicle group was performed using one-way ANOVA.

[0193] [Table 7]

[0194] [Table 8] The experimental results are shown in Figure 1 and Tables 5 and 6. Antibody drug conjugates DB1001-X1 and DB1001-X2 showed significantly enhanced dose-dependent tumor inhibitory activity after a single administration.

[0195] Example 7. Cross-species cross-reactivity of antibody-drug conjugates Purpose of the test Detect species cross-reactivity of antibody drug conjugate DB1001-X2 in human, cynomolgus monkey, and mouse B7H4-transfected HEK293T cells.

[0196] Experimental Method 1. All cell lines were o C, 5% CO 2 The cells are cultured in complete medium under the conditions described above. 2. Collect cells in logarithmic growth phase and detect cell viability by trypan blue exclusion to ensure that cell viability is above 90%. Centrifuge at 1000 r / min for 5 min, discard the supernatant, wash the cells once with PBS, and resuspend in FACS Buffer to prepare a single cell suspension at a cell density of 5 × 10 6 Adjust to cells / mL. 3. Add 50 μL of cell suspension to each 96-well plate, and dilute the working solution to obtain a total of eight concentrations from the highest concentration of 100 nM, which is then diluted 3-fold. Mix uniformly, then leave at 4°C and incubate for 40 min. 4. Wash the cells three times with FACS Buffer, 400 μL each time, and centrifuge at 1000 r / min for 5 min. Finally, resuspend the cells in 100 μL of FACS Buffer. 5. Add 2 μL of PE-labeled secondary antibody (PE anti-human IgG Fc antibody), mix evenly, and incubate in the dark at 4° C. for 40 minutes. 6. Wash the cells three times with FACS Buffer, 400 μL each time, and centrifuge at 1000 r / min for 5 min. Finally, resuspend the cells in 250 μL of FACS Buffer. 7. Detect the fluorescence value by flow cytometry.

[0197] result [Table 9] As shown in Table 7 and FIG. 2, DB1001-X2 has similar affinity for human and cynomolgus monkey B7-H4, but does not bind to mouse B7-H4. In the following examples, Isotype ADC refers to the following: the antibody is a negative control antibody, the linker-cytotoxin is X2, and is prepared with reference to DB1001-X2.

[0198] Example 8. Target specificity of antibody drug conjugates Purpose of the test The binding specificity of the antibody-drug conjugate DB1001-X2 to cells overexpressing human B7 family proteins was detected by flow cytometry. Experimental Method 1. Establish a HEK293T monoclonal cell line stably expressing human PD-L1 (B7-H1), PD-L2 (B7-DC), ICOSLG (B7-H2), CD276 (B7-H3), B7H4, VISTA (B7-H5), CD80 (B7-1), and CD86 (B7-2), and a CHO-K1 monoclonal cell line stably expressing human B7-H7 (HHLA2). 2. All cell lines were o C, 5% CO 2 The cells are cultured in complete medium under the conditions described above.

[0199] 3. Collect cells in logarithmic growth phase and detect cell viability by trypan blue exclusion to ensure that cell viability is above 90%. Centrifuge at 1000 r / min for 5 min, discard the supernatant, wash the cells once with PBS, and resuspend in FACS Buffer to prepare a single cell suspension at a cell density of 5 × 10 6 Adjust to cells / mL. 4. Add 50 μL of cell suspension to each 96-well plate, and make the highest concentration of the working solution to be tested 100 nM or 300 nM, totaling one concentration. Mix uniformly, then leave at 4°C and incubate for 40 min. 5. Wash the cells three times with FACS Buffer, 400 μL each time, centrifuge at 1000 r / min for 5 min, and finally resuspend the cells in 100 μL of FACS Buffer. 6. Add 2 μL of PE-labeled secondary antibody (PE anti-human IgG Fc antibody), mix evenly, and incubate in the dark at 4° C. for 40 minutes. 7. Wash the cells three times with FACS Buffer, 400 μL each time, centrifuge at 1000 r / min for 5 min, and finally resuspend the cells in 250 μL of FACS Buffer. 8. Detect the fluorescence value by flow cytometry.

[0200] Experimental Results As shown in Figure 3, DB1001-X2 bound to human B7-H4 but did not detectably bind to other human B7 family proteins B7-H1, B7-DC, B7-H2, B7-H3, B7-H5, B7-1, B7-2, and B7-H7. Conclusion of the experiment DB1001-X2 can specifically bind to human B7-H4 protein and does not cross-react with other B7 family proteins.

[0201] Example 9: Cellular endocytosis activity of antibody-drug conjugates Purpose of the test The endocytosis efficiency of the antibody-drug conjugate of the present application against the B7H4 target in MDA-MB-468 cells expressing B7H4 was tested in comparison with other antibody-drug conjugates against B7H4. The endocytosis ability of the antibody-drug conjugate was evaluated by incubating the cells with a fixed concentration of the antibody-drug conjugate and the endocytosis indicator Incucyte® Fabfluor-pH and continuously observing the changes in the fluorescent signal of live cells for 24 hours.

[0202] Experimental Method 1. Cell culture: MDA-MB-468 cells are cultured using Leibovitz's L-15 medium containing 10% FBS. 2. Cell preparation: Take MDA-MB-468 cells in logarithmic growth phase, wash once with PBS, then digest for 2-3 min, add 10-15 mL of cell culture medium after the cells are completely digested, elute the digested cells, centrifuge at 1000 rpm for 5 min, discard the supernatant, add cell culture medium to resuspend the cells, prepare a single cell suspension, and adjust the viable cell density to 1x10 5 Adjust to cells / mL. 3. Cell plating: Add 50uL / well to a 96-well cell culture plate. Incubate the culture plate overnight in an incubator (37℃, 5% CO 2 ). 4. Label the antigen-drug conjugate to be tested: Dilute the stock solution to be tested to 240 nM in 4x working solution (final concentration 60 nM) and Incucyte® Fabfluor-pH stock solution to 720 nM in 4x working solution (final concentration 180 nM), mix the two thoroughly and incubate at 37°C for 15 minutes in the dark. 5. Capture the analysis image: The labeled test working solution is transferred to the corresponding hole of the experimental plate, the experimental plate is transferred to the Incucyte live cell analysis device, the scanning photography process is set, and the image is acquired using the Incucyte live cell analysis system. The quantification was continued for 24 hours at 1 hour intervals. The analysis result was expressed as the total fluorescent area (μm 2 / image).

[0203] Experimental Results Figure 4 shows the integrated area of ​​fluorescent signal generated by endocytosis of the present antibody drug conjugate DB1001-X2 and the control antibody drug conjugate reference ADC-1 at different time points in MDA-MB-468 cells. Figure 5 shows a comparison of the endocytosis efficiency of the present antibody drug conjugate DB1001-X2 and the control antibody drug conjugate in MDA-MB-468 cells. [Table 10]

[0204] Conclusion of the experiment According to the results in Table 8, Figure 4 and Figure 5, the antibody-drug conjugate of the present application has an endocytosis effect in MDA-MB-468 cells expressing B7H4, which is superior to the reference ADC-1.

[0205] Example 10: In vitro growth inhibition test of antibody-drug conjugates against tumor cells (2D cell culture method) Purpose of the test The inhibitory effect of the present antibody drug conjugates against the B7H4 target on the in vitro proliferation of human colorectal cancer HT29 overexpressing B7H4 was tested using the CellTiter-Glo® chemiluminescent cell viability assay (i.e., CTG method) in comparison with other antibody drug conjugates against B7H4.

[0206] Experimental Method Cells in logarithmic growth phase were harvested and plated at a density of 15,000 cells / well and the plates were incubated for 37 min. o C, 5% CO 2 The cells were then placed in a 37°C incubator and cultured overnight. On the next day, the test object was diluted with complete medium to obtain a final concentration of 10 nM drug, and then added to the cell culture plate at 50 μL / well, with complete medium as the blank control, and three replicate wells were set up and incubated in a 37°C incubator for 7 days. After the incubation was completed, the cell culture plate was taken out and equilibrated to room temperature, and then 50 μL of CTG detection reagent (Promega, Cat#: G7573) was added to each well, shaken to mix evenly, and then placed in a dark place for 10 minutes, after which the signal value was detected and read by a microplate reader. The GraphPad Prism software was used to calculate the cell viability using the formula = (Lum 試験対象薬物 -Lum ブランク対照 ) / (Lum 溶媒ブランク対照 -Lum ブランク対照 )×100%.

[0207] Experimental Results [Table 11] Conclusion of the experiment According to the experimental results in Table 9 and Figure 6, the antibody-drug conjugate DB1001-X2 of the present application has significant growth inhibitory activity against human colorectal cancer cells HT29 overexpressing B7H4, and is superior to the reference ADC-1.

[0208] Example 11: In vitro growth inhibition test of antibody-drug conjugates against tumor cells (3D cell culture method) Purpose of the test The CellTiter-Glo® chemiluminescent cell viability assay (i.e., CTG method) was used to evaluate the growth inhibitory effects of anti-B7H4 ADC DB1001-X2 and anti-B7H4 mAb DB1001 against B7H4-high expressing human breast cancer MX-1, B7H4-low expressing endometrial cancer RL95-2, and B7H4-negative expressing breast cancer JIMT-1 under 3D culture conditions.

[0209] Experimental Method Cells in logarithmic growth phase were harvested and resuspended in cell culture medium to prepare a single cell suspension with a viable cell density of 1x10 5 Adjust the concentration of cells / mL, mix 3.5 mL of the cell suspension with 6.5 mL of 1% methylcellulose evenly, avoid air bubbles as much as possible, add 90 μL of the cell suspension to each well of a 96-well plate, and incubate at 37°C, 5% CO 2 The plate is then incubated overnight in a 37°C incubator. The next day, the test object is diluted with complete medium to obtain a 10x solution, and 10μL of the test object solution is added to each well, with the highest final concentration being 100nM, and 9 concentrations are diluted 3x, with complete medium as the blank control, and three replicate wells are set up and incubated in a 37°C incubator for 6 days. After the incubation is completed, the cell culture plate is taken out and equilibrated to room temperature, and then 100μL of CTG detection reagent (Promega, Cat#:G7573) is added to each well, and the plate is shaken to mix evenly and placed in the dark for 10 minutes. The cell plate is then left at room temperature for 30 minutes to stabilize the luminescence signal, and the Luminescence is read by EnVision. The GraphPad Prism software is used to plot the S-shaped dose-response curve using a nonlinear regression model, and the IC 50Calculate the value. Formula for cell viability = (Lum 試験対象薬物 -Lum ブランク対照 ) / (Lum 溶媒ブランク対照 -Lum ブランク対照 )×100%. Isotype ADC: the antibody is a negative control antibody, the linker-cytotoxin is X2, and is prepared with reference to DB1001-X2.

[0210] Experimental Results [Table 12] Conclusion of the experiment As shown in Table 10 and Figures 7, 8 and 9, DB1001-X2 had strong in vitro killing activity against both tumor cell lines with high and low expression of B7H4, but had no killing activity against cells that did not express B7H4, explaining that this killing activity was dependent on the expression of B7H4.

[0211] Example 12: T cell activation by antibody drug conjugates Purpose of the test The ability to block B7H4-mediated inhibition of T cells and activate IFN-gamma production by T cells was tested by comparing the present antibody drug conjugates against the B7H4 target with other antibody drug conjugates against B7H4. Experimental Method 1. Logarithmic growth phase 293T-OS8-human B7H4 cells (Kangyuan Bochuang Co., Ltd.) were harvested and centrifuged at 300 g for 5 minutes. 2. 1x10 cells 5 Resuspend at 1x10 cells / ml 4 Plate at a density of 100 μL / well and incubate overnight. 3. Using a Miltenyi T cell isolation kit (Miltenyi, CaT# 130-096-535), follow the instructions to isolate PBMCs from healthy donor 1 (Shanghai Miaoshun, Catalog No. PB100C-W, Lot No. A10Z983077) and healthy donor 2 (Shanghai Miaoshun, Catalog No. PB050C-W, Lot No. P122010104C) to obtain human primary T cells.

[0212] 4. Prepare test articles at 2x final concentration, with a final concentration of 0.02 nM. 5. Remove the culture medium from the experimental wells, and then add 2x10 human primary T cell lines from donor 1 and donor 2 to the experimental wells containing the 293T-OS8-humanB7H4 cell line. 5 / 100μL / well was added. 6. Then add 100 μL / well of test control antibody in duplicate wells. After culturing for 3 days, the supernatant was collected and the concentration of IFN-gamma was detected by ELISA. Isotype ADC: the antibody is a negative control antibody, the linker-cytotoxin is X2, and is prepared with reference to DB1001-X2.

[0213] Experimental Results [Table 13] Conclusion of the experiment As shown in Table 11, Figures 10 and 11, DB1001-X2 is able to block the inhibition of T cells from different donors by B7H4 positive cells, and is significantly superior to the reference ADC-1.

[0214] Example 13: Antibody Drug Conjugate Bystander Killing Purpose of the test The ability of small molecule drugs released after endocytosis to diffuse from B7H4-positive cells to nearby B7H4-negative cells and generate a bystander effect of cell killing thereagainst was detected by comparing the antibody-drug conjugates of the present application against the B7H4 target with other antibody-drug conjugates against B7H4. Unlabeled HT29 cells overexpressing B7H4 (HT29-B7H4) and HT29 cells transfected with luciferase but not expressing B7H4 (HT29-Luc2) were mixed in an appropriate ratio and incubated with the test subject for a certain period of time. After that, luciferase substrate was added, and the luciferase catalytic substrate produced by the live cells produced luciferin. The luciferin value was used to detect the luciferase content, i.e., reflect the number of live cells.

[0215] Experimental Method 1. Collect the cells in logarithmic growth phase, add 1640 medium containing 2% serum to resuspend the cells, adjust the cell numbers of HT29-B7H4 and HT29-Luc2, and add 45uL of cells to a 96-well plate, so that the ratio of the two cells is 3:1, i.e., the number of HT29-B7H4 cells is 11250 / well, the number of HT29-Luc2 cells is 3750 / well, and the number of HT29-Luc2 cells is 3750 / well. o C, 5% CO 2 Incubate the cells overnight in an incubator. 2. Prepare a 10x concentration of the test object solution, add 10μL of drug solution to each well of the cells in a 96-well plate, so that the final concentration of the test object is 10nM, and set up three replicate wells. Measure the luminescence value on day 0. 3. Incubate cells in a 96-well plate containing drugs at 37℃, 5% CO 2 The culture was continued for 7 days under the above conditions.

[0216] 4. After the incubation is completed, remove the cell culture plate and equilibrate it to room temperature. Then, add 50μL of CTG detection reagent (Promega, Cat#:G7573) to each well of HT29-B7H4 single incubation, shake to mix evenly, place in a dark place and let stand for 10 minutes, then detect with a microplate reader and read the signal value. 5. Add equal volumes of Bright-Glo solution to each well of HT29-Luc2 single incubation and co-incubation, and lyse the cells by shaking on an orbital shaker for 15 minutes. Then, read the luminescence value on a multi-function microplate reader. 5. Data analysis: Using GraphPad Prism software, the calculation formula for cell viability = (Lum 試験対象薬物 -Lum ブランク対照 ) / (Lum 溶媒ブランク対照 -Lum ブランク対照 )×100%.

[0217] Experimental Results [Table 14] Conclusion of the experiment As shown in Table 12 and FIG. 12, DB1001-X2 has a superior bystander killing effect on B7H4-negative cells incubated with B7H4-positive cells.

[0218] Example 14: Stability of antibody-drug conjugates in human, rat, and monkey plasma in vitro Purpose of the test The antibody-drug conjugates were incubated in vitro with human, mouse and monkey plasma for 21 days, samples were taken at different time points and the plasma stability of the antibody-drug conjugates was assessed by measuring the amount of small molecule toxin (cytotoxic drug) released into plasma using liquid chromatography and mass spectrometry.

[0219] Experimental Method DB1001-X2 was diluted to a final concentration of 150 μg / mL in human, rat or cynomolgus serum and incubated continuously at 37° C. for 21 days with intermediate sampling time points of TO, 2 h, 8 h, 1 day, 4 days, 7 days, 14 days, and 21 days. Plasma samples were precipitated with acetonitrile at a volume ratio of 1:1, centrifuged to remove the supernatant, and then analyzed by LC-MS / MS (liquid phase: Thermo Vanquish, triple quadrupole mass spectrometry: Thermo TSQ Quantis). The release rate of the antibody drug conjugate was calculated using the concentration of the cytotoxic drug P-III-30.

[0220] Experimental Results Referring to Table 13 and FIG. 13, 150 μg / mL of DB1001-X2 loaded (cytotoxic drug) P-III-30 showed less than 1% release in human, rat and monkey plasma after 21 days of incubation. [Table 15] Conclusion of the experiment DB1001-X2 has good stability in human, rat and monkey plasma in vitro.

[0221] Example 15: Pharmacokinetics of antibody conjugate drugs Purpose of the test DB1001-X2 was administered intravenously to cynomolgus monkeys twice, once every 3 weeks. The pharmacokinetic properties were studied to provide a reference for subsequent studies.

[0222] Experimental Method Four cynomolgus monkeys, half female and half male, weighing 2.2-3.6 kg at the time of group division, were divided into two groups, the DB1001-X2 low dose group and the DB1001-X2 high dose group, with the administration doses set at 30 and 80 mg / kg, respectively, once every three weeks for a total of two doses, and the administration volume was 5 mL / kg. Sampling time: Collect TK blood samples once before the first dose and 5 min, 0.5 h, 2 h, 4 h, 8 h, 24 h, 48 h, 72 h, 120 h, 168 h, 336 h, and 504 h after the end of the dose, and collect TK blood samples once each at 5 min, 0.5 h, 2 h, 4 h, 8 h, 24 h, and 48 h after the end of the final dose and before dissection. Sampling method: Collect approximately 0.5 mL of whole blood from the forelimb vein or other appropriate vein.

[0223] Blood sample processing: After collecting blood, place it into a blood collection tube labeled with EDTA·K2 as an anticoagulant. Gently invert the sample tube several times to ensure mixing, then immediately store in ice water. Within 1 hour of collection, centrifuge at 3800 rpm for 10 minutes at 4℃, aspirate and dispense 100μL of plasma per tube (2 tubes), temporarily store in dry ice, and transfer to an ultra-low temperature refrigerator (-60℃ or lower) within 4 hours. Record the time of whole blood collection and plasma collection in the test record. After all sample collection is complete, they are transported under dry ice conditions to the detection department. Sample Analysis: Plasma concentrations of total antibody and ADC drugs in serum are analyzed using established ELISA methods. Plasma concentrations of the cytotoxic drug P-III-30 in plasma are analyzed using established LC-MS / MS methods.

[0224] The experimental results are shown in Table 14. [Table 16]

[0225] Conclusion of the experiment According to the results in Table 14, no pharmacokinetic difference was observed between DB1001-X2 and the whole antibody, indicating that DB1001-X2 has good stability in plasma in vivo. Meanwhile, low plasma concentrations of P-III-30 were detected in cynomolgus monkeys at doses of 30 mg / kg and 80 mg / kg, which was attributed to the stability of DB1001-X2 and the short systemic half-life of P-III-30, indicating low systemic exposure of P-III-30 and higher safety.

[0226] Example 16: Efficacy evaluation of antibody-drug conjugates against mice bearing breast cancer cells MX-1 tumors with high expression of human B7H4 To examine the inhibitory effect of DB1001-X2 on in vivo tumor formation, we evaluated the antitumor effect of DB1001-X2 after in vivo formation of transplanted tumors in mice using B7H4-positive highly expressing human breast cancer cells MX-1. 1. Test substances and materials Blank control group (control group): saline DB1001-X2 (treatment group): 1mg / kg DB1001-X2 (treatment group): 3mg / kg 2. Preparation method: All samples are prepared by diluting with saline. 3. Test animals: 6-8 week-old female BALB / c nude mice purchased from Jishui Yaokang Biotechnology Co., Ltd.

[0227] 4. Test method: 5×10 6 MX-1 cells were inoculated subcutaneously into the right dorsal region of 6-8 week-old female BALB / c nude mice, and tumors were approximately 160.82 mm 3 When the tumor-bearing mice reached the age of 0, they were randomly grouped by StudyDirectorTM and injected with the test subjects by intravenous (iv) injection starting on that day (day 0), once every two weeks for a total of two injections, with DB1001-X2 at a dose of 1 mg / kg and 3 mg / kg, respectively, and ISO-ADC at a dose of 3 mg / kg. The study endpoint was the 27th day after grouping, and tumor volume and body weight were measured twice a week and the data were recorded. The control group or treatment group had 5 mice each. The tumor inhibition rate was calculated by measuring the tumor volume.

[0228] The formula for calculating tumor volume is V=0.5 a × b 2where a and b respectively indicate the long and short diameters of the tumor. The tumor inhibition effect of the compound is evaluated by T / C (%). The percentage value of T / C (%) is an index reflecting tumor growth inhibition, and T and C indicate the average tumor volume on a certain day in the administration group and the control group, respectively. The tumor growth inhibition rate is calculated by the following formula: TGI (%) = [1-(T i -T 0 ) / (V i -V 0 )] × 100, T i is the mean tumor volume of a given treatment group on a given day, and T 0 is the mean tumor volume at the start of treatment in this treatment group, and V i One day (T i is the mean tumor volume of the vehicle control group (on the same day as the first day), and V 0 is the mean tumor volume at the start of treatment in the vehicle control group. Comparison between two pairs of samples was performed using independent sample T-Test, and data was analyzed using SPSS, with P<0.05 indicating significant differences. The plotting software was GraphPad Prism.

[0229] [Table 17] According to the experimental results shown in FIG. 14 and Table 15, antibody-drug conjugate DB1001-X2 exhibited significant dose-dependent tumor-inhibitory activity after administration.

[0230] Example 17: Efficacy evaluation of antibody-drug conjugates against human B7H4-positive breast cancer cell MCF-7 tumor-bearing mice To compare the inhibitory effects of DB1001-X2 and similar competitors on in vivo tumor formation, the antitumor effect of DB1001-X2 was evaluated after in vivo transplantation of tumors in mice using B7H4-positive expressing human breast cancer cells MCF-7. 1. Test substances and materials Blank control group (control group): saline DB1001-X2 (treatment group): 3mg / kg 2. Preparation method: All samples are prepared by diluting with saline. 3. Test animals: 6-7 week-old female NCG mice, purchased from Jiangsu Jiaxing Pharmaceutical Biotechnology Co., Ltd.

[0231] 4. Test method: MCF-7 cells in the logarithmic growth phase (inoculation number N+12) were harvested, the medium was removed, and the cells were washed twice with PBS before inoculation (cell viability before and after tumor bearing was 96.03% and 94.81%, respectively). The inoculation amount was 1×10 7 cells / 75 μL / mouse (Matrigel was added at a 1:1 ratio). On the 19th day after inoculation, the average tumor volume was 107.44 mm 3 When tumor volume reached 1000 mg / kg, 50 mice were randomly divided into 10 groups of 5 mice each based on tumor volume. The day of grouping was defined as D0, and dosing began on D0, the day of grouping. Excess mice were euthanized. The test subjects were injected once at a dose of 3 mg / kg. Tumor volume and body weight were measured twice a week, and the data were recorded. The vehicle control group or treatment group had 5 mice in each group. The test endpoint time was 25 days after grouping, and the tumor inhibition rate was calculated by measuring the tumor volume.

[0232] The formula for calculating tumor volume is V=0.5 a × b 2 where a and b respectively indicate the long and short diameters of the tumor. The tumor inhibition effect of the compound is evaluated by T / C (%). The percentage value of T / C (%) is an index reflecting tumor growth inhibition, and T and C indicate the average tumor volume on a certain day in the administration group and the control group, respectively. The tumor growth inhibition rate is calculated by the following formula: TGI (%) = [1-(T i -T 0 ) / (V i -V 0 )] × 100, T i is the mean tumor volume of a given treatment group on a given day, and T 0 is the mean tumor volume at the start of treatment in this treatment group, and V i One day (T i is the mean tumor volume of the vehicle control group (on the same day as the first day), and V 0 is the mean tumor volume at the start of treatment in the vehicle control group. Comparison between two pairs of samples was performed using independent sample T-Test, and data was analyzed using SPSS, with P<0.05 indicating significant differences. The plotting software was GraphPad Prism. Experimental Results: The antibody-drug conjugate DB1001-X2 of the present invention exhibited significant dose-dependent antitumor activity after a single administration.

[0233] Example 18: Efficacy evaluation of antibody-drug conjugates against mice bearing RL95-2 endometrial cancer cells with low expression of human B7H4 To examine the inhibitory effect of DB1001-X2 on in vivo tumor formation, we evaluated the antitumor effect of DB1001-X2 after in vivo transplantation of tumors into mice using B7H4-positive low-expressing human endometrial cancer cells, RL95-2. 1. Test substances and materials Blank control group (control group): Phosphate-buffered saline (DPBS) DB1001-X2 (treatment group): 1mg / kg DB1001-X2 (treatment group): 3mg / kg DB1001-X2 (treatment group): 10mg / kg 2. Preparation: All samples are prepared by dilution in phosphate buffered saline (DPBS). 3. Test animals: 6-8 week-old female BALB / c nude mice, purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd.

[0234] 4. Test method: Collect logarithmic growth phase RL95-2 cells and inject 5 x 10 cells into the right cervical dorsum of each mouse. 6RL95-2 cells were subcutaneously inoculated, the inoculation volume was 0.2 mL, and the cell suspension was PBS plus Matrigel (volume ratio 1:1). For the in vivo drug efficacy experiment, on the 20th day after cell inoculation, when the average tumor volume reached 134 mm3, the mice were randomly divided into groups of 5 mice according to the tumor volume and administered the drug. The day of grouping was defined as D0, and administration began on D0, the day of grouping. The test subject was injected once every 2 weeks, with a dose of 3 mg / kg, for a total of 2 injections, and the test endpoint was the 28th day after grouping. Tumor volume and body weight were measured twice a week, and the data were recorded.

[0235] The formula for calculating tumor volume is V=0.5 a × b 2 where a and b respectively indicate the long and short diameters of the tumor. The tumor inhibition effect of the compound is evaluated by T / C (%). The percentage value of T / C (%) is an index reflecting tumor growth inhibition, and T and C indicate the average tumor volume on a certain day in the administration group and the control group, respectively. The tumor growth inhibition rate is calculated by the following formula: TGI (%) = [1-(T i -T 0 ) / (V i -V 0 )] × 100, T i is the mean tumor volume of a given treatment group on a given day, and T 0 is the mean tumor volume at the start of treatment in this treatment group, and V i One day (T i is the mean tumor volume of the vehicle control group (on the same day as the first day), and V 0 is the mean tumor volume at the start of treatment in the vehicle control group. Statistical analysis. Differences between groups were evaluated by performing statistical analysis using GraphPad Prism software based on the data on day 28 after group division. Comparisons between three or more groups were analyzed using one-way ANOVA (Dunnett's multiple comparison test and Tukey's multiple comparison test), and p<0.05 was considered to be a significant difference.

[0236] [Table 18]

[0237] According to the experimental results shown in FIG. 15 and Table 16, the antibody-drug conjugate DB1001-X2: After administration, it showed significant dose-dependent tumor suppression activity. Example 19: Efficacy evaluation of antibody-drug conjugates against mice bearing ovarian cancer cells OVCAR-3 tumors with low expression of human B7H4 To examine the inhibitory effect of DB1001-X2 on in vivo tumor formation, we evaluated the antitumor effect of DB1001-X2 after tumor formation in mice in vivo using B7H4-positive low-expressing human ovarian cancer cells OVCAR-3. 1. Test substances and materials Blank control group (control group): saline DB1001-X2 (treatment group): 1mg / kg DB1001-X2 (treatment group): 3mg / kg DB1001-X2 (treatment group): 10mg / kg 2. Preparation method: All samples are prepared by diluting with saline. 3. Test animals: 6-8 week-old female BALB / c nude mice purchased from Jishui Yaokang Biotechnology Co., Ltd.

[0238] 4. Test method: 1×10 7 OVCAR-3 cells were inoculated subcutaneously into the right dorsal region of 6-8 week-old female BALB / c nude mice and resuspended in 1:1 PBS and Matrigel (0.2 ml / mouse), allowing tumors to grow to approximately 142.85 mm 3 When the tumor-bearing mice reached the age of 0, they were randomly grouped by StudyDirectorTM and injected with the test subjects by intravenous (iv) injection starting from that day (day 0), once every two weeks for a total of two injections, with DB1001-X2 at doses of 1 mg / kg, 3 mg / kg and 10 mg / kg, respectively. The study endpoint was the 27th day after grouping, and tumor volumes and body weights were measured twice a week and the data were recorded. The control group or treatment group had 5 mice each. The tumor inhibition rate was calculated by measuring the tumor volume.

[0239] The formula for calculating tumor volume is V=0.5 a × b 2 where a and b respectively indicate the long and short diameters of the tumor. The tumor inhibition effect of the compound is evaluated by T / C (%). The percentage value of T / C (%) is an index reflecting tumor growth inhibition, and T and C indicate the average tumor volume on a certain day in the administration group and the control group, respectively. The tumor growth inhibition rate is calculated by the following formula: TGI (%) = [1-(T i -T 0 ) / (V i -V 0 )] × 100, T i is the mean tumor volume of a given treatment group on a given day, and T 0 is the mean tumor volume at the start of treatment in this treatment group, and V i One day (T i is the mean tumor volume of the vehicle control group (on the same day as the first day), and V 0 is the mean tumor volume at the start of treatment in the vehicle control group. Comparison between two pairs of samples was performed using independent sample T-Test, and data was analyzed using SPSS, with P<0.05 indicating significant differences. The plotting software was GraphPad Prism.

[0240] [Table 19]

[0241] According to the experimental results shown in FIG. 16 and Table 17, antibody-drug conjugate DB1001-X2 exhibited significant dose-dependent tumor-inhibitory activity after administration.

[0242] Although the specific embodiments of the present invention have been described above, they are merely examples, and it is understood by those skilled in the art that many changes or modifications can be made to these embodiments without departing from the principle and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. An anti-B7H4 antibody-drug conjugate, its isomer, a pharmaceutically acceptable salt thereof, or a mixture thereof, wherein the structure of the anti-B7H4 antibody-drug conjugate is 【Chemistry 1】 It is expressed by equation (I-1), During the ceremony, M is -L 2 -L 1 -C(O)-, L 2 is -O- or -S-, L 1 is -(C(R 1a )(R 1b )) m -CH 2 -, C 3 -C 6 is a saturated cycloalkyl or a 3- to 6-membered saturated heterocyclyl, and the C 3 -C 6 saturated cycloalkyl and 3- to 6-membered saturated heterocyclyl are each independently optionally substituted with one or more R 2a s, m is selected from 1, 2, 3, or 4, and the heteroatoms in the 3-6 member saturated heterocycline are selected from N, O, and S, and the number of heteroatoms is 1 to 3. R 1a These are, independently, hydrogen, halogen, hydroxyl, amino, and C. 1 -C 6 Selected from alkyl, the C 1 -C 6 Alkyl is selectively substituted with one or more R groups. R 1b and R 2a These are, independently, hydrogen, halogen, hydroxyl, amino, and C. 1 -C 6 Selected from alkyl, the C 1 -C 6 Alkyl is selectively substituted with one or more R groups. R is independently either hydrogen or halogen. L is the linker unit, p represents the average number of connections, and p is selected from integers or decimals between 1 and 10. Ab is an anti-B7H4 antibody or its antigen-binding fragment, an anti-B7H4 antibody-drug conjugate, its isomer, a pharmaceutically acceptable salt thereof, or a mixture thereof.

2. The anti-B7H4 antibody drug conjugate according to claim 1, its isomers, pharmaceutically acceptable salts thereof, or mixtures thereof, wherein the anti-B7H4 antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and the light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6, respectively.

3. The anti-B7H4 antibody drug conjugate according to claim 1, the isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, wherein the anti-B7H4 antibody or its antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 7, or a heavy chain variable region having at least 95%, 96%, 97%, 98%, or 99% identity therewith, and the amino acid sequence shown in SEQ ID NO: 8, or a light chain variable region having at least 95%, 96%, 97%, 98%, or 99% identity therewith.

4. The anti-B7H4 antibody drug conjugate, isomer thereof, pharmaceutically acceptable salt thereof, or mixture thereof according to claim 1, wherein the anti-B7H4 antibody or antigen-binding fragment thereof is a mouse antibody or fragment thereof, a chimeric antibody or fragment thereof, a humanized antibody or fragment thereof.

5. The anti-B7H4 antibody or its antigen-binding fragment is Fab, Fab', Fab'-SH, Fv, scFv, F(ab') 2 An anti-B7H4 antibody-drug conjugate according to claim 1, selected from sdAb, a bispecific antibody, or a linear antibody, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof.

6. The anti-B7H4 antibody-drug conjugate according to claim 1, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, wherein the antibody is an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.

7. The anti-B7H4 antibody drug conjugate according to claim 1, comprising an anti-B7H4 antibody or its antigen-binding fragment, a heavy chain having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 9, or a light chain having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 10, or a light chain having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 10, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof.

8. The anti-B7H4 antibody-drug conjugate according to claim 1, its isomers, pharmaceutically acceptable salts thereof, or mixtures thereof, wherein p is an integer or decimal between 3 and 8.

9. L 1 is, -(C(R 1a ) (Caution 1b )) m -CH 2 - and R 1a These are hydrogen, halogens and C 1 -C 6 Selected from alkyl groups, R 1b These are hydrogen, halogens and C 1 -C 6 An anti-B7H4 antibody-drug conjugate according to claim 1, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, selected from alkyl groups.

10. L 1 is, -(C(R 1a ) (Caution 1b )) m -CH 2 - and R 1a ha-CH 3 And R 1b It is hydrogen and -CH 3 An anti-B7H4 antibody-drug conjugate according to claim 9, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, selected from among.

11. L 1 is, -(C(R 1a ) (Caution 1b )) m -CH 2 An anti-B7H4 antibody-drug conjugate according to claim 1, wherein m is 1 or 2, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof.

12. L 1 teeth, 【Chemistry 2】 An anti-B7H4 antibody-drug conjugate according to claim 1, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, selected from among.

13. L 1 C 3 -C 6 The C is a saturated cycloalkyl or a 3-6 member saturated heterocycline. 3 -C 6 Saturated cycloalkyls and 3- to 6-membered saturated heterocyclines each independently and selectively contain one or more R 2a Replaced with R 2a These are, independently, hydrogen, halogen, and C 1 -C 6 An anti-B7H4 antibody-drug conjugate according to claim 1, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, selected from alkyl groups.

14. L 1 This is a selective selection of one or more R 2a C replaced by 3 -C 6 It is a saturated cycloalkyl, and R 2a These are, independently, hydrogen, halogen, and C 1 -C 6 An anti-B7H4 antibody-drug conjugate according to claim 13, selected from alkyl groups, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof.

15. L 1 Selectively, one, two, or three R 2a Replaced with 【Transformation 3】 And R 2a These are, independently, hydrogen, halogen, and C 1 -C 6 An anti-B7H4 antibody-drug conjugate according to claim 14, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, selected from alkyl groups.

16. L 1 teeth, 【Chemistry 4】 An anti-B7H4 antibody-drug conjugate according to claim 1, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, selected from among.

17. M is -L 2 -L 1 -C(O)-, L 2 It is -O-, L 1 is, -(C(R 1a ) (Caution 1b )) m -CH 2 - or C 3 -C 6 It is a saturated cycloalkyl, and the C 3 -C 6 Saturated cycloalkyls selectively have one, two, or three R groups. 2a Replaced by, m is selected from 1 or 2. R 1a is, independently of each other, hydrogen, halogen and C 1 -C 6 alkyl selected from, said C 1 -C 6 alkyl is optionally substituted with one or more R, R 1b and R 2a each independently represents hydrogen, halogen, and C 1 -C 6 alkyl, and the C 1 -C 6 alkyl is optionally substituted with one or more R The anti-B7H4 antibody-drug conjugate according to claim 1, its isomers, pharmaceutically acceptable salts thereof, or mixtures thereof, wherein R is independently hydrogen or halogen.

18. M is -L 2 -L 1 -C(O)-, L 2 It is -O-, L 1 is, -(C(R 1a ) (Caution 1b )) m -CH 2 - Or selectively one, two, or three R 2a Replaced with 【Transformation 5】 And, m is selected from 1 or 2. R 1a These are, independently, halogen and C 1 -C 6 Selected from alkyl, the C 1 -C 6 Alkyl is selectively substituted with one or more R groups. R 1b and R 2a These are, independently, hydrogen, halogen, and C 1 -C 6 Selected from alkyl, the C 1 -C 6 Alkyl is selectively substituted with one or more R groups. The anti-B7H4 antibody-drug conjugate according to claim 17, its isomers, pharmaceutically acceptable salts thereof, or mixtures thereof, wherein R is independently hydrogen or halogen.

19. The aforementioned -M- is, 【Transformation 6】 An anti-B7H4 antibody-drug conjugate according to claim 1, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, selected from among.

20. The aforementioned L is -L a -L b -L c - and Said-L a -teeth, 【Transformation 7】 And, In the formula, W is -(C(R wa ) (Caution wb )) wn - and Y is - (OCH 2 CH 2 ) yn -O yp - and Z is -(C(R za ) (Caution zb )) zn And, wn is 1, 2, 3, or 6. The 0 or 1 methylene units of W are each independently -Cyr-, -N(R) wx )C(O)-, -C(O)N(R wx )-, or -C(O)-, yn is 0, 4, or 8, and yp is 0 or 1. Zn is 1, 2, or 3. Each methylene unit of Z is independently -Cyr-, -N(R) zx )C(O)-, -C(O)N(R zx )-, or -C(O)-, -Cyr- is a 3- to 10-membered saturated cycloalkyl group, and -Cyr- is either unsubstituted or independently has 1 to 3 substituents R cx It has been replaced with, In the formula, each R wa , R wb , R za , R zb , R wx , R zx , R cx These are, independently, hydrogen, halogen, and -OR r or R r C selectively substituted by 1-6 It is alkyl, In the formula, each R r These are, independently, hydrogen, halogen, or C 1-6 It is alkyl, Said-L b - indicates a peptide residue consisting of 2 to 4 amino acids, and the -L b The peptide residue is a peptide residue consisting of an amino acid selected from the group consisting of phenylalanine, glycine, alanine, valine, citrulline, and lysine. Said-L c -teeth, 【Transformation 8】 And, In the formula, R L1 , R L2 These are, independently, hydrogen, halogen, -OH, and C. 1-6 An anti-B7H4 antibody-drug conjugate according to claim 1, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, selected from the group consisting of alkyl groups.

21. Said-L a -teeth, 【Chemistry 9】 The anti-B7H4 antibody-drug conjugate according to claim 20, its isomers, pharmaceutically acceptable salts thereof, or mixtures thereof.

22. Said-L b -teeth, 【Chemistry 10】 Selected from the group consisting of, Preferably, the -L b -teeth, 【Chemistry 11】 The anti-B7H4 antibody-drug conjugate according to claim 20, its isomers, pharmaceutically acceptable salts thereof, or mixtures thereof.

23. Said-L c -teeth, 【Chemistry 12】 The anti-B7H4 antibody-drug conjugate according to claim 20, its isomers, pharmaceutically acceptable salts thereof, or mixtures thereof.

24. The aforementioned L is, 【Chemistry 13】 The anti-B7H4 antibody-drug conjugate according to claim 20, its isomers, pharmaceutically acceptable salts thereof, or mixtures thereof.

25. The structure of the anti-B7H4 antibody-drug conjugate is as follows: 【Chemistry 14】 It is expressed by equation (II-1) or (II-2), During the ceremony, p represents the average number of connections, and p is selected from an integer or decimal between 1 and 10, preferably from an integer or decimal between 3 and 8. Ab is as described in claim 1, L 2 is -O- or -S-, preferably -O-, X 1 This is a selective selection of one, two, or three R's. 2a C replaced by 3 -C 6 Selected from saturated cycloalkyl groups, preferably selectively comprising one, two, or three R groups. 2a Replaced with 【Chemistry 15】 And, X 2 is, -(C(R 1a ) (Caution 1b )) m -CH 2 - Selected from, m is selected from 1 or 2. R 1a C is selectively substituted with hydrogen, halogen, or one, two, or three R atoms. 1 -C 6 It is alkyl, R 1b or R 2a Each of these is independently a C atom selectively substituted with hydrogen, halogen, or one, two, or three R atoms. 1 -C 6 It is alkyl, The anti-B7H4 antibody-drug conjugate according to claim 1, its isomers, pharmaceutically acceptable salts thereof, or mixtures thereof, wherein R is independently hydrogen or halogen.

26. The aforementioned anti-B7H4 antibody-drug conjugate is 【Chemistry 16-1】 【Chemistry 16-2】 【Chemistry 16-3】 【Chemistry 16-4】 Selected from the structural formulas, During the ceremony, p is as described in claim 1, Ab is as described in claim 1, the anti-B7H4 antibody-drug conjugate according to claim 1, its isomers, pharmaceutically acceptable salts thereof, or mixtures thereof.

27. The aforementioned anti-B7H4 antibody-drug conjugate is 【Chemistry 17-1】 【Chemistry 17-2】 【Chemistry 17-3】 Selected from, During the ceremony, The anti-B7H4 antibody-drug conjugate, its isomers, pharmaceutically acceptable salts thereof, or mixtures thereof, wherein p represents the average number of bonds, and p is an integer or decimal between 1 and 10, preferably selected from an integer or decimal between 3 and 8.

28. A pharmaceutical composition comprising an anti-B7H4 antibody-drug conjugate according to any one of claims 1 to 27, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, and a pharmaceutically acceptable carrier or excipient.

29. The use of an anti-B7H4 antibody-drug conjugate, an isomer thereof, a pharmaceutically acceptable salt thereof, or a mixture thereof, in the preparation of a pharmaceutically acceptable drug for use in the treatment and / or prevention of a B7H4-mediated disease or disorder, preferably the disease or disorder being a B7H4-positive cancer, and more preferably the cancer being selected from breast cancer, ovarian cancer and endometrial cancer, according to any one of claims 1 to 27.

30. A pharmaceutical composition according to claim 28 for treating and / or preventing a B7H4-mediated disease or disorder, wherein the disease or disorder is a B7H4-positive expressing cancer, and more preferably the cancer is selected from breast cancer, ovarian cancer and endometrial cancer.

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