Anti-b7-h4 antibody-drug conjugate and medicinal use thereof

Description

"Antibody-drug conjugate anti-B7-H4 and its medicinal use" Separated from BR112021024406-9, filed on June 8, 2020. FIELD OF THE INVENTION

[001] The present invention belongs to the field of biomedicine. Specifically, the present invention relates to an antibody-drug conjugate and its medicinal use. BACKGROUND OF THE INVENTION

[002] Tumor immunotherapy is a sustainable long-term hotspot of research and development in the field of tumor therapy, and T-cell tumor immunotherapy is in a central position. Tumor evasion is a major obstacle to tumor immunotherapy. Most tumors express antigens that can be recognized by the host immune system to varying degrees. However, in many cases, inefficient activation of effector T cells triggers an insufficient immune response, and therefore tumor cells promote tumor growth through their inhibitory effects on the immune system. Tumor immunotherapy consists of making full use of and mobilizing killer T cells and / or other immune system cells in tumor patients to kill the tumor.

[003] Research on the CD28 receptor and its ligands has led to the characterization of molecules referred to as the B7 superfamily. Members of the B7 family are a class of immunoglobulins with immunoglobulin-like V (IgV) and immunoglobulin-like C (IgC) domains. Its members include co-stimulatory factors B7.1 (CD80) and B7.2 (CD86), inducible co-stimulatory factor ligand (ICOS-L / B7-H2), programmed death-1 ligand (PD-L1 / B7-H1), programmed death-2 ligand (PD-L2 / B7-DC), B7-H3 and B7-H4, and similar molecules.

[004] Human B7-H4 is a type I transmembrane protein consisting of 282 amino acids. Its encoding gene is located Petition 870260061229, dated 06 / 22 / 2026, page 9 / 414 2 / 118 located in the p11.1 region of chromosome 1 (Choi IH et al., J Immunol. November 1, 2003; 171(9): 4650-4). B7-H4 has a negative regulatory effect on the immune response of T cells. B7-H4 plays a broad inhibitory role in differentiation and development, cell cycle progression, and cytokine production of CD4+ and CD8+ T cells (Sica GL et al., Immunity. June 2003; 18(6): 849-61). No immune cell disorders or autoimmune phenomena were found in B7-H4 knockout mice (Zhu G et al., Blood. February 19, 2009; 113(8): 1759-67; Suh WK et al., Blood. Mol Cell Biol. September 2006; 26(17): 6403-11). Currently, the B7-H4 receptor and its signaling transduction pathway are still unclear.

[005] Recent studies have found that the B7-H4 protein is abundantly expressed in a variety of tumor tissues, allowing tumor cells to evade attack by the body's immune system. Targeting the B7-H4 molecule for tumor therapy provides a new method for tumor immunotherapy.

[006] It is now known that human B7-H4 is expressed in cancer cells, such as breast cancer, ovarian cancer, lung cancer, cervical cancer, kidney cancer, bladder cancer, and liver cancer. B7-H4 mRNA expression is found in the spleen, lung, thymus, liver, skeletal muscle, kidney, pancreas, testicle, and ovary.

[007] At the protein level, low levels of B7-H4 expression are found in tissues such as the breast (duct and lobule), fallopian tube epithelium, and endometrial gland. Related studies have also shown that B7-H4 is overexpressed in tumor-associated macrophages (TAMs) (Kryczek, I. et al., J. Exp. Med. 2006, 203(4): 871-881), and macrophages constitute an important component of the tumor microenvironment and can represent up to 50% of the tumor mass. Petition 870260061229, dated 06 / 22 / 2026, page 10 / 414 3 / 118

[008] One strategy for cancer treatment is to use antibodies as vehicles to deliver cytotoxic molecules to cancer cells and then kill the cancer cells through the dissociated small molecules. The drugs used in this strategy are called antibody-drug conjugates. Adcetris and Kadcyla are currently marketed antibody-drug conjugates. Currently, many multinational pharmaceutical companies are developing monoclonal antibodies against B7-H4 and / or their drug conjugates to enhance the patient's own immune system response to tumors and to achieve the goal of directly killing tumor cells. Related patents are, for example, WO2013025779, US20140322129 and similar. Anti-B7-H4 monoclonal antibodies from Medimmune, FivePrime and other companies are still in preclinical development; anti-B7-H4 antibody conjugates from Genentech are also in preclinical development. SUMMARY OF THE INVENTION

[009] The objective of the present invention is to provide an anti-B7-H4 antibody-drug conjugate that has high affinity, high selectivity, high endocytic efficiency, high anticancer activity, high stability, high safety, and low toxicity and side effects, obtained through the following technical solutions:

[0010] An antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, Ab-(L2-LrD)y(A) where: D is a cytotoxic drug; L1 and L2 are bonding units; y is a number from 1 to 20; Petition 870260061229, dated 06 / 22 / 2026, page 11 / 414 4 / 118 Ab is a B7-H4 antibody or antigen-binding fragment thereof, comprising a variable region of antibody light chain and a variable region of antibody heavy chain; The antibody heavy chain variable region comprises at least one HCR, as shown in the selected sequence from the group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11; the antibody light chain variable region comprises at least one LCDR, as shown in the selected sequence from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14.

[0011] In one embodiment of the present invention, the antibody heavy chain variable region can also comprise at least one HCDR, as shown in the selected sequence from the group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, or, the antibody heavy chain variable region can also comprise at least one HCDR, as shown in the selected sequence from the group consisting of: SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45; The antibody light chain variable region comprises at least one LCDR, as shown in the selected sequence from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, Petition 870260061229, dated 06 / 22 / 2026, page 12 / 414 5 / 118 SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34.

[0012] In one embodiment of the present invention, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof as described above, wherein the anti-B7-H4 antibody or antigen-binding fragment thereof comprises a variable region of antibody heavy chain, wherein the variable region of antibody heavy chain comprises: HCDR1, as shown in SEQ ID NO:3, HCDR2, as shown in SEQ ID NO:4, and HCDR3, as shown in SEQ ID NO:5; or, HCDR1, as shown in SEQ ID NO:9, HCDR2, as shown in SEQ ID NO:10, and HCDR3, as shown in SEQ ID NO:11.

[0013] In one embodiment of the present invention, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof as described above, wherein the anti-B7-H4 antibody or antigen-binding fragment thereof comprises a variable region of antibody heavy chain, wherein the variable region of antibody heavy chain comprises: HCDR1, as shown in SEQ ID NO:23, HCDR2, as shown in SEQ ID NO:24 and HCDR3, as shown in SEQ ID NO:25; or, HCDR1, as shown in SEQ ID NO:29, HCDR2, as shown in SEQ ID NO:30, and HCDR3, as shown in SEQ ID NO:31.

[0014] In one embodiment of the present invention, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof as described above, wherein the anti Petition 870260061229, dated 06 / 22 / 2026, page 13 / 414 6 / 118 anti-B7-H4 body or antigen-binding fragment thereof comprises antibody heavy chain variable region, wherein the antibody heavy chain variable region comprises: HCDR1, as shown in SEQ ID NO:43, HCDR2, as shown in SEQ ID NO:44 and HCDR3, as shown in SEQ ID NO:25; or, HCDR1, as shown in SEQ ID NO:29, HCDR2, as shown in SEQ ID NO:45, and HCDR3, as shown in SEQ ID NO:31.

[0015] In one embodiment of the present invention, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof as described above, wherein the variable antibody heavy chain region of Ab comprises the CDRs of any one selected from the group consisting of the following (1) to (4): (1) HCDR1, as shown in SEQ ID NO:3, HCDR2, as shown in SEQ ID NO:4 and HCDR3, as shown in SEQ ID NO:5; (2) HCDR1, as shown in SEQ ID NO:9, HCDR2, as shown in SEQ ID NO:10 and HCDR3, as shown in SEQ ID NO:11; (3) HCDR1, as shown in SEQ ID NO:23, HCDR2, as shown in SEQ ID NO:24 and HCDR3, as shown in SEQ ID NO:25; or, (4) HCDR1, as shown in SEQ ID NO:29, HCDR2, as shown in SEQ ID NO:30 and HCDR3, as shown in SEQ ID NO:31.

[0016] The antibody heavy chain variable region of Ab may also comprise selected CDRs from the group consisting of Petition 870260061229, dated 06 / 22 / 2026, page 14 / 414 7 / 118 following (5) to (6): (5) HCDR1, as shown in SEQ ID NO:43, HCDR2, as shown in SEQ ID NO:44 and HCDR3, as shown in SEQ ID NO:25; or, (6) HCDR1, as shown in SEQ ID NO:29, HCDR2, as shown in SEQ ID NO:45 and HCDR3, as shown in SEQ ID NO:31.

[0017] In one embodiment of the present invention, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof as described above, wherein the anti-B7-H4 antibody or antigen-binding fragment thereof comprises a variable antibody light chain region, wherein the variable antibody light chain region comprises: LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, respectively; or LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively.

[0018] In some embodiment of the present invention, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof as described above, wherein the anti-B7-H4 antibody or antigen-binding fragment thereof comprises a variable antibody light chain region, wherein the variable antibody light chain region comprises: LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, respectively; or LCDR1, LCDR2 and LCDR3, as shown in SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34, respectively.

[0019] In one embodiment of the present invention, the antibody-drug conjugate of general formula (A) or a pharmaceutically produced salt Petition 870260061229, dated 06 / 22 / 2026, p. 15 / 414 8 / 118 acceptable or solvate of the same as described above, wherein the antibody heavy chain variable region of Ab comprises the CDRs of any one selected from the group consisting of the following (1) to (4): (1) LCDR1, as shown in SEQ ID NO:6, LCDR2, as shown in SEQ ID NO:7 and LCDR3, as shown in SEQ ID NO:8; (2) LCDR1, as shown in SEQ ID NO:12, LCDR2, as shown in SEQ ID NO:13 and LCDR3, as shown in SEQ ID NO:14; (3) LCDR1, as shown in SEQ ID NO:26, LCDR2, as shown in SEQ ID NO:27 and LCDR3, as shown in SEQ ID NO:28; or, (4) LCDR1, as shown in SEQ ID NO:32, LCDR2, as shown in SEQ ID NO:33 and LCDR3, as shown in SEQ ID NO:34.

[0020] In one embodiment of the present invention, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof as described above, wherein the variable heavy chain and light chain regions of the anti-B7-H4 antibody or antigen-binding fragment thereof comprise: (1) HCDR1, as shown in SEQ ID NO:3, HCDR2, as shown in SEQ ID NO:4 and HCDR3, as shown in SEQ ID NO:5, respectively; and LCDR1, as shown in SEQ ID NO:6, LCDR2, as shown in SEQ ID NO:7 and LCDR3, as shown in SEQ ID NO:8; or, (2) HCDR1, as shown in SEQ ID NO:9, HCDR2, as shown in SEQ ID NO:10 and HCDR3, as shown in SEQ ID NO:11, respectively; and LCDR1, as shown in SEQ ID NO:12, LCDR2, as Petition 870260061229, dated 06 / 22 / 2026, page 16 / 414 9 / 118 shown in SEQ ID NO:13 and LCDR3, as shown in SEQ ID NO:14.

[0021] In one embodiment of the present invention, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof as described above, wherein the variable heavy chain and light chain regions of the anti-B7-H4 antibody or antigen-binding fragment thereof comprise: (3) HCDR1, as shown in SEQ ID NO:23, HCDR2, as shown in SEQ ID NO:24 and HCDR3, as shown in SEQ ID NO:25; and LCDR1, as shown in SEQ ID NO:26, LCDR2, as shown in SEQ ID NO:27 and LCDR3, as shown in SEQ ID NO:28; or, (4) HCDR1, as shown in SEQ ID NO:29, HCDR2, as shown in SEQ ID NO:30 and HCDR3, as shown in SEQ ID NO:31; and LCDR1, as shown in SEQ ID NO:32, LCDR2, as shown in SEQ ID NO:33, and LCDR3, as shown in SEQ ID NO:34.

[0022] In one embodiment of the present invention, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof as described above, wherein the variable heavy chain and light chain regions of the anti-B7-H4 antibody or antigen-binding fragment thereof comprise: (5) HCDR1, as shown in SEQ ID NO:43, HCDR2, as shown in SEQ ID NO:44 and HCDR3, as shown in SEQ ID NO:25, respectively; and LCDR1, as shown in SEQ ID NO:26, LCDR2, as shown in SEQ ID NO:27, and LCDR3, as shown in SEQ ID NO:28; or, Petition 870260061229, dated 06 / 22 / 2026, page 17 / 414 10 / 118 (6) HCDR1, as shown in SEQ ID NO:29, HCDR2, as shown in SEQ ID NO:45 and HCDR3, as shown in SEQ ID NO:31, respectively; and LCDR1, as shown in SEQ ID NO:32, LCDR2, as shown in SEQ ID NO:33, and LCDR3, as shown in SEQ ID NO:34.

[0023] In one embodiment of the present invention, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof as described above, wherein the antibody heavy chain variable region and the antibody light chain variable region of Ab comprise the CDRs of any one selected from the group consisting of the following (1) to (4): (1) HCDR1, as shown in SEQ ID NO:3, HCDR2, as shown in SEQ ID NO:4 and HCDR3, as shown in SEQ ID NO:5; and LCDR1, as shown in SEQ ID NO:6, LCDR2, as shown in SEQ ID NO:7 and LCDR3, as shown in SEQ ID NO:8; (2) HCDR1, as shown in SEQ ID NO:9, HCDR2, as shown in SEQ ID NO:10 and HCDR3, as shown in SEQ ID NO:11; and LCDR1, as shown in SEQ ID NO:12, LCDR2, as shown in SEQ ID NO:13 and LCDR3, as shown in SEQ ID NO:14; (3) HCDR1, as shown in SEQ ID NO:23, HCDR2, as shown in SEQ ID NO:24 and HCDR3, as shown in SEQ ID NO:25; and LCDR1, as shown in SEQ ID NO:26, LCDR2, as shown in SEQ ID NO:27 and LCDR3, as shown in SEQ ID NO:28; or, (4) HCDR1, as shown in SEQ ID NO:29, HCDR2, Petition 870260061229, dated 06 / 22 / 2026, page 18 / 414 11 / 118 as shown in SEQ ID NO:30 and HCDR3, as shown in SEQ ID NO:31; and LCDR1, as shown in SEQ ID NO:32, LCDR2, as shown in SEQ ID NO:33, and LCDR3, as shown in SEQ ID NO:34.

[0024] The antibody heavy chain variable region and antibody light chain variable region of Ab may also comprise CDRs selected from the group consisting of the following (5) or (6): (5) HCDR1, as shown in SEQ ID NO:43, HCDR2, as shown in SEQ ID NO:44 and HCDR3, as shown in SEQ ID NO:25; and LCDR1, as shown in SEQ ID NO:26, LCDR2, as shown in SEQ ID NO:27 and LCDR3, as shown in SEQ ID NO:28; (6) HCDR1, as shown in SEQ ID NO:29, HCDR2, as shown in SEQ ID NO:45 and HCDR3, as shown in SEQ ID NO:31; and LCDR1, as shown in SEQ ID NO:32, LCDR2, as shown in SEQ ID NO:33, and LCDR3, as shown in SEQ ID NO:34.

[0025] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof as described above, wherein Ab is a murine antibody or fragment thereof, a chimeric antibody or fragment thereof, a human antibody or fragment thereof, and a humanized antibody or fragment thereof.

[0026] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein Ab also comprises light chain structural region sequences and region Petition 870260061229, dated 06 / 22 / 2026, page 19 / 414 12 / 118 heavy chain structural, which are respectively derived from human germline light chain and heavy chain sequences or mutant sequence(s) thereof;

[0027] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein Ab also comprises constant heavy chain regions, wherein the constant heavy chain regions comprise those derived from human IgG1 or a variant thereof, IgG2 or a variant thereof, IgG3 or a variant thereof, or IgG4 or a variant thereof, preferably those derived from human IgG1, IgG2 or IgG4, more preferably the constant heavy chain region of IgG1 with ADCC toxicity highlighted after amino acid mutation, most preferably the constant heavy chain region as shown in SEQ ID NO: 54;

[0028] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein Ab also comprises a constant light chain region derived from human κ chain, λ chain or variant thereof, preferably derived from human κ chain, more preferably from the constant light chain region, as shown in SEQ ID NO:55.;

[0029] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein Ab comprises the variable light chain region with the following sequences: SEQ ID NO:16 or SEQ ID NO:18.

[0030] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein Ab comprises the variable light chain region with the following sequences: SEQ ID Petition 870260061229, dated 06 / 22 / 2026, p. 20 / 414 13 / 118 NO:16, SEQ ID NO:18, SEQ ID NO:36, SEQ ID NO:38, or variable light chain regions with at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% homology to SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:36, SEQ ID NO:38.

[0031] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein Ab comprises the variable heavy chain region with the following sequences: SEQ ID NO:15 or SEQ ID NO:17.

[0032] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein Ab comprises the variable heavy chain region with the following sequences: SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:35, SEQ ID NO:37, or variable heavy chain regions with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology to SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:35, SEQ ID NO:37.

[0033] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein the variable heavy chain region and the variable light chain region of Ab are any one selected from the group consisting of the following: (1) the variable heavy chain region, as shown in SEQ ID NO: 15 and the variable light chain region, as shown in SEQ ID NO: 16; (2) the variable heavy chain region, as shown in SEQ ID NO: 17 and the variable light chain region, as shown in SEQ ID NO: 18.

[0034] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt Petition 870260061229, dated 06 / 22 / 2026, page 21 / 414 14 / 118 or solvate thereof, as described above, wherein the variable heavy chain region and the variable light chain region of Ab are any one selected from the group consisting of the following: (1) the variable heavy chain region, as shown in SEQ ID NO: 15 and the variable light chain region, as shown in SEQ ID NO: 16; (2) the variable heavy chain region, as shown in SEQ ID NO: 17 and the variable light chain region, as shown in SEQ ID NO: 18; (3) the variable heavy chain region, as shown in SEQ ID NO: 35 and the variable light chain region, as shown in SEQ ID NO: 36; or, (4) the variable heavy chain region, as shown in SEQ ID NO: 37 and the variable light chain region, as shown in SEQ ID NO: 38.

[0035] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein Ab comprises a light chain with the following sequences: SEQ ID NO:20 or SEQ ID NO:22.

[0036] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein Ab comprises a light chain with the following sequences: SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:40, SEQ ID NO:42, or a full-length light chain with at least 80%, 85%, 90%, 95% or 99% homology to SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:40, SEQ ID NO:42.

[0037] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein Ab comprises Petition 870260061229, dated 06 / 22 / 2026, p. 22 / 414 15 / 118 the heavy chain with the following sequences: SEQ ID NO:19 or SEQ ID NO:21.

[0038] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein Ab comprises the heavy chain with the following sequences: SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:39, SEQ ID NO:41, or the full-length heavy chain with at least 80%, 85%, 90%, 95% or 99% homology to SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:39, SEQ ID NO:41.

[0039] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein Ab comprises: (1) the light chain with SEQ ID NO: 20 and the heavy chain with SEQ ID NO: 19; or, (2) the light chain with SEQ ID NO: 22 and the heavy chain with SEQ ID NO: 21.

[0040] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein Ab comprises: (1) the light chain with SEQ ID NO: 20 and the heavy chain with SEQ ID NO: 19; or, (2) the light chain with SEQ ID NO: 22 and the heavy chain with SEQ ID NO: 21; or, (3) the light chain with SEQ ID NO: 40 and the heavy chain with SEQ ID NO: 39; or, (4) the light chain with SEQ ID NO: 42 and the heavy chain with SEQ ID NO: 41.

[0041] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt Petition 870260061229, dated 06 / 22 / 2026, page 23 / 414 16 / 118 or solvate thereof, as described above, wherein the antigen-binding fragment of the anti-B7-H4 antibody is selected from the group consisting of Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (diabody), disulfide-linked stabilized V region (dsFv) and antigen-binding fragments of a peptide comprising CDRs.

[0042] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein the cytotoxic drug is selected from the group consisting of toxin, chemotherapeutic, antibiotic, radioisotope and nucleolytic enzyme; preferably a tubulin inhibitor or a DNA topoisomerase inhibitor that inhibits cell division; more preferably DM1, DM3, DM4, SN-38, MMAF or MMAE; also preferably, the tubulin inhibitor SN-38, MMAE or MMAF. Wherein the structure of MMAF and SN-38 is as shown in the following formula:

[0043] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein the cytotoxic drug is selected from camptothecin derivatives, preferably Exatecan, Petition 870260061229, dated 06 / 22 / 2026, page 24 / 414 17 / 118

[0044] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, which is a compound of general formula (I) or a pharmaceutically acceptable salt or solvate thereof, Ab--1-2 in which: Li and L2 are bonding units; y is a number selected from 1 to 8, preferably a number selected from 2 to 4; Ab is an anti-B7-H4 antibody or antigen-binding fragment thereof as defined above.

[0045] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, which is an antibody-drug conjugate of general formula (II) or a pharmaceutically acceptable salt or solvate thereof: Petition 870260061229, dated 06 / 22 / 2026, p. 25 / 414 18 / 118 where: Li and L2 are bonding units; y is a number selected from 1 to 8, preferably a number selected from 2 to 4; Ab is an anti-B7-H4 antibody or antigen-binding fragment thereof as defined above.

[0046] In a preferred embodiment, the antibody-drug conjugate of general formula (A) or a pharmaceutically acceptable salt or solvate thereof, as described above, which is an antibody-drug conjugate of general formula (III) or a pharmaceutically acceptable salt or solvate thereof, in which: L1 and L2 are bonding units; y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number selected from 4 to 8; or, y is preferably a number selected from 2 to 10, also preferably a number selected from 6 to 10, more preferably a number from 7 to 9, and most preferably an integer from 7, 8, 9; Ab is an anti-B7-H4 antibody or antigen-binding fragment thereof as defined above.

[0047] In a preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate of Petition 870260061229, dated 06 / 22 / 2026, page 26 / 414 19 / 118 same, as described above, where Li is as shown in the following general formula (B): (B) where: Mi is -CR1R2-; R1 and R2 are either the same or different, and are independently selected from the group consisting of hydrogen, alkyl, halogen, hydroxyl, and amino groups; N is an integer from 0 to 5, preferably 1, 2, or 3.

[0048] In a preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein L2 is as shown in the following general formula (C): (C) where: M2 is -CR4R5-; R3 is selected from the group consisting of hydrogen, halogen, hydroxyl, amino, alkyl, alkoxyl, and cycloalkyl atoms; R4 and R5 are either the same or different, and are independently selected from the group consisting of hydrogen, alkyl, halogen, hydroxyl, and amino groups; m is an integer from 0 to 5, preferably 1, 2, or 3.

[0049] In a preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein L2 is as shown in the figure. Petition 870260061229, dated 06 / 22 / 2026, page 27 / 414 20 / 118 following general formula (D): -K1-K2-K3-K4(D) s is an integer from 2 to 8, also preferably an integer from 4 to 8, most preferably an integer from 4 to 6; K2is -NR1(CH2CH2O)pCH2CH2C(O)-, -NR1(CH2CH2O)pCH2C(O)-, -S(CH2)pC(O)- or a single bond, p is an integer from 1 to 20, preferably 1 to 6; R1 is selected from the group consisting of hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkyl; K3 is a tetrapeptide residue, preferably the tetrapeptide residue is a peptide residue formed by amino acids selected from the group consisting of two or more of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamate and aspartate; more preferably the tetrapeptide residue GGFG; K4 is -NR2(CR3R4)t-, R2, R3 or R4 are, each independently, hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkyl, et is 1 or 2, preferably, as for L2, ,0 K1é oMs, s é 5; K2 is a link; K3 is the tetrapeptide residue GGFG; K4é -NR2(CR3R4)t-, R2, R3 or R4são, each one independent Petition 870260061229, dated 06 / 22 / 2026, p. 28 / 414 21 / 118 strongly, hydrogen, deuterium, hydroxyl, amino, C1-6 alkyl, halogen, C1-6 haloalkyl, C1-6 deuterated alkyl and C1-6 hydroxyalkyl, et is 1 or 2.

[0050] In a preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein the K1 terminus of the -L2- ligand unit is linked to Ab, and the K4 terminus is linked to L1.

[0051] In a preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein Li is -O-(CRaRb)m-CR5R6-C(O)-, -O-CR5R6-(CRaRb)m-, -O-CR5R6-, -NH-(CRaRb)m-CR5R6-C(O)- or -S-(CRaRb)m-CR5R6-C(O)-; Rae Rbsão, each independently selected from the group consisting of hydrogen, deuterium, halogen and alkyl; R is haloalkyl or cycloalkyl; R6 is selected from the group consisting of hydrogen, haloalkyl and cycloalkyl; or, R5e R6e the carbon atom to which they are attached, form a cycloalkyl; m is 0, 1, 2, 3 or 4.

[0052] Preferably, the L1 terminal is connected to the L2 linking unit.

[0053] In a preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein L1 is as shown in the following general formula (E): (AND), R5 is a haloalkyl or cycloalkyl group. R6 is selected from the group consisting of hydrogen, ha Petition 870260061229, dated 06 / 22 / 2026, p. 29 / 414 22 / 118 loalkyl and cycloalkyl, or, R5e R6e the carbon atom to which they are attached, form a cycloalkyl; preferably, R5 is selected from the group consisting of C1-6 haloalkyl and C3-6 cycloalkyl. R6 is selected from the group consisting of hydrogen, C1-6 haloalkyl and cycloalkyl, or, R5 and R6 and the carbon atom to which they are attached form a C3-6 cycloalkyl; m is an integer from 0 to 4; More preferably, general formula (E) is selected from the group consisting of the following substituents: \ ηρ. A? oeo ;

[0054] In a more preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, as described above, wherein the -L2-L1- structure is as follows: K2 is a connection; K3 is the tetrapeptide residue GGFG; R5 is haloalkyl or C3-6 cycloalkyl; R6 is selected from the group consisting of hydrogen, haloalkyl and C3-6 cycloalkyl; Petition 870260061229, dated 06 / 22 / 2026, p. 30 / 414 23 / 118 or, R5e R6e the carbon atom to which they are attached, form a C3-6 cycloalkyl; R2, R3, or R4 are each independently either hydrogen or alkyl; s is an integer from 2 to 8; preferably, s is 4, 5, or 6; m is an integer from 0 to 4; Preferably, -L2-L1- is selected from the group consisting of the following structures: Petition 870260061229, dated 06 / 22 / 2026, p. 31 / 414 24 / 118

[0055] In a preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, as described above, which is the antibody-drug conjugate of general formula (IV) or a pharmaceutically acceptable salt or solvate thereof: in which: W is selected from the group consisting of C1-8 alkyl, C1-8 alkyl-cycloalkyl or linear heteroalkyl of 1 to 8 atoms, the heteroalkyl comprising 1 to 3 heteroatom(s) selected from the group consisting of N, O or S, wherein the C1-8 alkyl, cycloalkyl or linear heteroalkyl is, each independently, optionally also substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxyl and cycloalkyl; K2 is selected from the group consisting of Petition 870260061229, dated 06 / 22 / 2026, page 32 / 414 25 / 118 -NR1(CH2CH2O)piCH2CH2C(O)-, -NR1(CH2CH2O)piCH2C(O)-, -S(CH2)piC(O)- or linkage, R1 is selected from the group consisting of hydrogen atom, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl, and pi is an integer from 1 to 20; K3 is a peptide residue consisting of 2 to 7 amino acids; the amino acids may be substituted or unsubstituted. When substituted, substituents may be attached at any available attachment point, and the substituents are one or more independently selected from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxyl, and cycloalkyl; R2 is independently selected from the group consisting of hydrogen atom, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl; R3 and R4 are each independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl atoms; R5 is selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R6 is selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl atoms; or, R5e R6e the carbon atom to which they are attached, form a cycloalkyl or heterocyclyl; m is an integer from 0 to 4; y is 1 to 10 and y is a decimal or an integer, preferably y is a number from 2 to 10, more preferably y is a number from 4 to 10, also preferably a number from 6 to 9, and most preferably an integer of 7, 8 or 9; Ab is an anti-B7-H4 antibody or binding fragment to Petition 870260061229, dated 06 / 22 / 2026, p. 33 / 414 26 / 118 antigen of the same.

[0056] In a more preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, as described above, which is an antibody-drug conjugate of general formula (IA) or a pharmaceutically acceptable salt or solvate thereof: (IA) y is 1 to 10, y is a decimal or integer.

[0057] In a more preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, as described above, which is an antibody-drug conjugate of general formula (IB) or a pharmaceutically acceptable salt or solvate thereof: (IB) y is 1 to 10, y is a decimal or integer.

[0058] In a more preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, as described above, which is an antibody-drug conjugate of general formula (II-A) or a pharmaceutically acceptable salt or solvate thereof:

[0059] In a more preferred embodiment, combined anticor Petition 870260061229, dated 06 / 22 / 2026, p. 34 / 414 27 / 118 po-drug or a pharmaceutically acceptable salt or solvate thereof, as described above, which is an antibody-drug conjugate of general formula (II-B) or a pharmaceutically acceptable salt or solvate thereof:

[0060] In a more preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, as described above, which is an antibody-drug conjugate of general formula (IV-A) or a pharmaceutically acceptable salt or solvate thereof: where s is an integer from 2 to 8, R2a R6, mey are as defined in the general formula above (IV); Preferably, s is an integer from 4, 5, or 6, y is a number from 4 to 10, preferably a number from 6 to 9, and more preferably 7 or 8.

[0061] In a more preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, as described above, selected from the group consisting of the following compounds: Petition 870260061229, dated 06 / 22 / 2026, p. 35 / 414 28 / 118 Petition 870260061229, dated 06 / 22 / 2026, p. 36 / 414 29 / 118

[0062] In a more preferred embodiment, the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, as described above, is selected from the group consisting of the following compounds: Petition 870260061229, dated 06 / 22 / 2026, p. 37 / 414 30 / 118 (Compound 4) (Compound 5) Petition 870260061229, dated 06 / 22 / 2026, p. 38 / 414 31 / 118 (Compound 8) Petition 870260061229, dated 06 / 22 / 2026, p. 39 / 414 32 / 118 hu2F8 (Compound 11) (Compound 12) Petition 870260061229, dated 06 / 22 / 2026, pp. 40 / 414 33 / 118 (Compound 16) Petition 870260061229, dated 06 / 22 / 2026, pp. 41 / 414 34 / 118 Petition 870260061229, dated 06 / 22 / 2026, p. 42 / 414 35 / 118 Petition 870260061229, dated 06 / 22 / 2026, pp. 43 / 414 36 / 118 Petition 870260061229, dated 06 / 22 / 2026, pp. 44 / 414 37 / 118 Petition 870260061229, dated 06 / 22 / 2026, p. 45 / 414 38 / 118 Petition 870260061229, dated 06 / 22 / 2026, pp. 46 / 414 39 / 118 Petition 870260061229, dated 06 / 22 / 2026, p. 47 / 414 40 / 118 Petition 870260061229, dated 06 / 22 / 2026, pp. 48 / 414 41 / 118 Petition 870260061229, dated 06 / 22 / 2026, page 49 / 414 42 / 118 Petition 870260061229, dated 06 / 22 / 2026, page 50 / 414 43 / 118 (Compound 53) (Compound 54) (Compound 55) (Compound 56) Petition 870260061229, dated 06 / 22 / 2026, page 51 / 414 44 / 118 (Compound 58) (Compound 60) Petition 870260061229, dated 06 / 22 / 2026, page 52 / 414 45 / 118 (Compound 61) — y (Compound 62) (Compound 63) (Compound 64) -1y Petition 870260061229, dated 06 / 22 / 2026, page 53 / 414 46 / 118 Petition 870260061229, dated 06 / 22 / 2026, page 54 / 414 47 / 118 Petition 870260061229, dated 06 / 22 / 2026, p. 55 / 414 48 / 118 Petition 870260061229, dated 06 / 22 / 2026, p. 56 / 414 49 / 118 Petition 870260061229, dated 06 / 22 / 2026, p. 57 / 414 50 / 118 Petition 870260061229, dated 06 / 22 / 2026, p. 58 / 414 51 / 118 Petition 870260061229, dated 06 / 22 / 2026, p. 59 / 414 52 / 118 Petition 870260061229, dated 06 / 22 / 2026, p. 60 / 414 53 / 118 Petition 870260061229, dated 06 / 22 / 2026, p. 61 / 414 54 / 118 Petition 870260061229, dated 06 / 22 / 2026, p. 62 / 414 55 / 118 Petition 870260061229, dated 06 / 22 / 2026, p. 63 / 414 56 / 118 Petition 870260061229, dated 06 / 22 / 2026, p. 64 / 414 57 / 118 Petition 870260061229, dated 06 / 22 / 2026, page 65 / 414 58 / 118 (Compound 117) (Compound 118) (Compound 119) (Compound 120) (Compound 121) (Compound 128) Petition 870260061229, dated 06 / 22 / 2026, page 66 / 414 59 / 118 (Compound 131) (Compound 134) (Compound 135) (Compound 136) Petition 870260061229, dated 06 / 22 / 2026, page 67 / 414 60 / 118 J y (Compound 140) (Compound 143) (Compound 144) Petition 870260061229, dated 06 / 22 / 2026, page 68 / 414 61 / 118 (Compound 145) hu2F7 oo (Compound 146) (Compound 149) (Compound 150) Diagram legend Compound = composite where y is a number selected from 1 to 10, preferably a number selected from 2 to 10, in addition, a number selected from 6 to 10, more preferably a number from 7 to 9, and most preferably an integer from 7, 8, 9; or, y is a number selected from 2 to 10, preferably a number from 4 to 8, more preferably a number from 6 to 8, also preferably a number from 7 to 8, and most preferably 8.

[0063] In a preferred embodiment, the present invention relates to the method for preparing the antibody-drug conjugate of general formula (IV) or a pharmaceutically acceptable salt or solvate of Petition 870260061229, dated 06 / 22 / 2026, p. 69 / 414 62 / 118 itself, which comprises the following step: after Ab is reduced, it is subjected to the coupling reaction with general formula (F) to obtain the compound with general formula (IV); in which: Ab is an anti-B7H4 antibody or antigen-binding fragment thereof; W, K2, K3, R2a R6, mey are as defined in the general formula (IV).

[0064] In a preferred embodiment, the general formula (F) is a compound of general formula (F-1): or a tautomer, mesomer, racemate, enantiomer, diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein K2, K3, R2a R6, are as defined above -L2-L1-.

[0065] In a preferred embodiment, the compound of general formula Petition 870260061229, dated 06 / 22 / 2026, page 70 / 414 63 / 118 (F) or general formula (F-1) is selected from the group consisting of: Petition 870260061229, dated 06 / 22 / 2026, page 71 / 414 64 / 118 Petition 870260061229, dated 06 / 22 / 2026, p. 72 / 414 65 / 118

[0066] In a preferred embodiment, the present invention relates to a pharmaceutical composition comprising the antibody-drug conjugate according to any of the present invention, or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable excipients, diluents or vehicles.

[0067] In a preferred embodiment, the present invention relates to the use of the antibody-drug conjugate of general formula (A), or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for the treatment of a disease related to human B7-H4, preferably a medicament for the treatment of a cancer with high B7-H4 expression.

[0068] In a more preferred embodiment, the cancer is selected from the group consisting of human brain astroblastoma, human pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft tissue sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe cell carcinoma of the kidney, clear cell carcinoma, colon cancer, colorectal cancer, proliferative round cell tumor of connective tissue, ependymoma, Ewing's sarcoma, extraosseous mucoid chondrosarcoma, fibrogenesis imperfecta of osseum, fibrous dysplasia of bone, gallbladder or cholangiocarcinoma, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, cancer renal, leukemia, liposarcoma / malignant lipomatous tumor,liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, cancer of, Petition 870260061229, dated 06 / 22 / 2026, page 73 / 414 66 / 118 pancreas, papillary thyroid cancer, parathyroid adenoma, pediatric cancer, peripheral schwannoma, pheocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, metastatic renal cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thymic cancer, metastatic thyroid cancer, and uterine cancer. DESCRIPTION OF THE DRAWINGS

[0069] Figure 1: In vivo efficacy of antibody-drug conjugates: hu2G6-MC-MMAF and hu2F7-MC-MMAF showed inhibitory and elimination effects on MX-1 xenograft tumors at doses of 1.5 mg / kg and 3 mg / kg.

[0070] Figure 2: HPLC spectra of hu2F7-MC-MMAF with drug loadings of 2 and 4, respectively.

[0071] Figure 3: In vivo antitumor efficacy of hu2F7-MC-MMAF with drug loadings of 2 and 4, respectively.

[0072] Figure 4: In vivo antitumor efficacy of hu2F7-MC-MMAF with drug loadings of 2 and 4, respectively. The figure shows mouse tumors in each group on day 21. DETAILED DESCRIPTION OF THE INVENTION 1. Terms

[0073] For easier understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention pertains.

[0074] The three-letter codes and the one-letter codes of amino acids used in the present invention are described in J. Biol. Chem, 243, p3558 (1968). Petition 870260061229, dated 06 / 22 / 2026, p. 74 / 414 67 / 118

[0075] The term antibody described in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The composition and order of amino acids in the constant regions of the heavy chain of immunoglobulins are different, thus their antigenicity is also different. Accordingly, immunoglobulins can be classified into five types, also known as immunoglobulin isotypes, namely, IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are the μ chain, δ chain, γ chain, α chain, and ε chain, respectively. The same type of Ig can be classified into different subclasses according to the difference in their amino acid composition of the hinge region and the number and position of the heavy chain disulfide bonds. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4.The light chain is classified as either a κ chain or a λ chain according to the difference in the constant region. Each of the five types of Ig can have either a κ chain or a λ chain.

[0076] In the present invention, the variable region of the antibody light chain of the present invention may also comprise a constant region of the light chain, which comprises human or murine κ, λ chains or variants thereof.

[0077] In the present invention, the antibody heavy chain variable region of the present invention may also comprise the heavy chain constant region, comprising human or murine IgG1,2,3,4 or a variant thereof.

[0078] The sequence of approximately 110 amino acids next to The N-terminal of the antibody's heavy and light chains varies greatly and is the variable region (V region); the remaining amino acid sequence near the C-terminal is relatively stable and is the constant region (C region). The variable region comprises 3 hypervariable regions. Petition 870260061229, dated 06 / 22 / 2026, page 75 / 414 68 / 118 variable (HVR) and 4 structural regions (FR) with relatively conservative sequences. The 3 hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDR). Each variable light chain (VL) region and variable heavy chain (VH) region consists of 3 CDR regions and 4 FR regions, arranged from the amino terminal to the carboxyl terminal in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3. The number and position of CDR amino acid residues in the VL and VH regions of the antibody or antigen-binding fragment of the present invention meet the Kabat or Chothia numbering criteria and the Kabat or AbM definition criteria (http: / / bioinf.org.uk / abs / ).

[0079] The term antigen-presenting cell or APC is a cell that presents the foreign antigen complexed with MHC on its surface. T cells use T cell receptors (TCRs) to recognize such complexes. Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), monocytes, B lymphoblasts, and monocyte-derived dendritic cells (DCs). The term antigen presentation refers to the process by which APCs capture antigens and allow them to be recognized by T cells, for example, as a component of the MHC-I / MHC-II conjugate.

[0080] The term B7-H4 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 functional activation. Petition 870260061229, dated 06 / 22 / 2026, page 76 / 414 69 / 118 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 derived from non-human species. Alternatively, the antibodies may also be specific for human B7-H4 and may not cross-react with other species. B7-H4 or any variant or isoform thereof may be isolated from cells or tissues that naturally express it, or produced by recombination techniques using techniques commonly used in the art and those described herein. Preferably, the anti-B7-H4 antibody targets human B7-H4 with a normal glycosylation pattern.

[0081] The term recombinant human antibody includes human antibodies prepared, expressed, created or isolated by recombinant methods, and the techniques and methods involved are quite common in the art, for example (1) antibodies isolated from transgenic or transchromosomal animals (e.g., mice) expressing human immunoglobulin genes, or hybridomas prepared therefrom; (2) antibodies isolated from host cells transformed to express the antibodies, such as transfectionomas; (3) antibodies isolated from recombinant combinatorial human antibody libraries; as well as (4) antibodies prepared, expressed, created or isolated by splicing human immunoglobulin gene sequences to other DNA sequences and by other methods.These recombinant human antibodies comprise variable regions and constant regions, which use specific sequences of human germline immunoglobulins encoded by germline genes, but also comprise subsequent rearrangements and mutations, such as those that occur during antibody maturation.

[0082] The term murine antibody in the present invention is an Petition 870260061229, dated 06 / 22 / 2026, page 77 / 414 70 / 118 monoclonal antibody against human B7-H4 prepared according to the knowledge and skills in the art. During preparation, the test subject is injected with B7-H4 antigen, and then hybridomas expressing antibodies with the desired sequences or functional properties are isolated. In a preferred embodiment of the present invention, the murine B7-H4 antibody or antigen-binding fragment thereof may also comprise the constant light chain region of murine κ, λ chain or a variant thereof, or also comprise the constant heavy chain region of murine IgG1, IgG2, IgG3 or IgG4 or a variant thereof.

[0083] The term human antibody includes antibodies with variable constant region(s) of human germline immunoglobulin sequences. The human antibodies of the present invention may comprise amino acid residues that are not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, the term human antibody does not include antibodies in which germline-derived CDR sequences from another mammalian species (such as mice) have been grafted onto human structural sequences (namely humanized antibodies).

[0084] The term humanized antibody, also known as CDR-grafted antibody, refers to an antibody produced by grafting murine CDR sequences into the structure of human antibody variable regions. It can overcome the strong immune response reactions induced by chimeric antibodies carrying a large number of murine protein components. In order to avoid decreased activity caused by decreased immunogenicity, the variable regions of the human antibody can undergo minimal reverse mutation to maintain activity. Petition 870260061229, dated 06 / 22 / 2026, page 78 / 414 71 / 118

[0085] The term chimeric antibody refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody. Establishing a chimeric antibody requires first establishing a hybridoma that secretes murine-specific monoclonal antibody, then cloning a variable region gene from murine hybridoma cells, and then cloning the constant region gene from the human antibody as needed, linking the murine variable region gene with the human constant region gene to form a chimeric gene, which is inserted into a human expression vector, and finally expressing the chimeric antibody molecule in an industrial eukaryotic or prokaryotic system.The constant region(s) of the human antibody may be selected from the constant heavy chain regions of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising the constant heavy chain regions of human IgG2 or IgG4, or using IgG1 with ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity enhanced after amino acid mutations.

[0086] The term antigen-binding fragment refers to antigen-binding fragments and antibody analogs of an antibody, which generally comprise at least part of the antigen-binding region or variable regions (e.g., one or more CDRs) of the parental antibody. The antibody fragment retains at least part of the binding specificity of the parental antibody. Generally, when activity is represented on a molar basis, the antibody fragment retains at least 10% of the parental binding activity. Preferably, the antibody fragment retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the parental antibody's binding affinity to the target. Examples of antibody fragments Petition 870260061229, dated 06 / 22 / 2026, page 79 / 414 72 / 118 Antigen-related antibodies include, but are not limited to: Fab, Fab', F(ab')2, Fv fragment, linear antibody, single-chain antibody, nanobody, domain antibody, and multispecific antibody. Engineered antibody variants are reviewed in Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136.

[0087] The Fab fragment consists of the variable and CH1 regions of a light chain and a heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0088] The Fc region comprises two heavy chain fragments comprising the CH1 and CH2 domains of the antibody. The two heavy chain fragments are held together by two or more disulfide bonds and through the hydrophobic interaction of the CH3 domain.

[0089] The Fab' fragment comprises a light chain and a portion of a heavy chain comprising the VH domain, the CH1 domain and the region between the CH1 and CH2 domains, such that interchain disulfide bonds can be formed between the two heavy chains of two Fab' fragments in order to form the F(ab')2 molecule.

[0090] The F(ab')2 fragment comprises two light chains and two heavy chains comprising a portion of the constant region between the CH1 and CH2 domains, thereby forming interchain disulfide bonds between the two heavy chains. Therefore, the F(ab')2 fragment consists of two Fab' fragments held together by disulfide bonds between the two heavy chains.

[0091] The Fv region comprises variable regions of both the heavy and light chains, but does not have constant regions.

[0092] The term multispecific antibody is used in its broadest sense, which encompasses antibodies with polyepitope specificity. Petition 870260061229, dated 06 / 22 / 2026, page 80 / 414 73 / 118 These multispecific antibodies include, but are not limited to: antibodies comprising a variable heavy chain (VH) region and a variable light chain (VL) region, wherein the VH-VL unit has multiepitope specificity; antibodies with two or more VL and VH regions, each VH-VL unit binding to different targets or different epitopes of the same target; antibodies with two or more unique variable regions, each unique variable region binding to different targets or different epitopes of the same target; full-length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, antibody fragments that are covalently or non-covalently linked to each other, and the like.

[0093] The term single-chain antibody refers to a recombinant single-chain protein formed by linking a variable heavy chain (VH) region and a variable light chain (VL) region of an antibody via a peptide linker. It is the smallest antibody fragment with a complete antigen-binding site.

[0094] The term domain antibody fragment is an immunoglobulin fragment with immunological functions that comprises only the variable heavy chain region or the variable light chain region. In some cases, two or more VH regions are covalently linked to a peptide linker to form a bivalent domain antibody fragment. The two VH regions of the bivalent domain antibody fragment may target different or the same antigens.

[0095] The term B7-H4 binding in the present invention refers to the ability to interact with human B7-H4. The term antigen-binding site in the present invention refers to a three-dimensional site that is spread across the antigen and is recognized by the antibody or antigen-binding fragment of the present invention.

[0096] The terms specifically link and selectively link Petition 870260061229, dated 06 / 22 / 2026, p. 81 / 414 74 / 118 terms used in the present invention refer to the binding of an antibody to an epitope of a predetermined antigen. Generally, when recombinant human B7-H4 is used as the analyte and an antibody is used as the ligand, when measured by surface plasmon resonance (SPR) technology in an instrument, the antibody binds to the predetermined antigen at an equilibrium dissociation constant (KD) of about less than 10⁻⁷ M or even less, and its binding affinity to the predetermined antigen is at least twice its binding affinity to non-specific antigens (other than the predetermined antigen or closely related antigens such as BSA and the like). The term antibody that recognizes... antigen may be used interchangeably with the term antibody that specifically binds to... herein.

[0097] The term cross-reactivity refers to the ability of the antibodies of the present invention to bind to B7-H4 from different species. For example, the antibody of the present invention that binds to human B7-H4 may also bind to B7-H4 from another species. Cross-reactivity is measured by detecting specific reactivity with purified antigens in binding assays (e.g., SPR and ELISA), or by binding or functional interaction with cells that physiologically express B7-H4. Methods for determining cross-reactivity include standard binding assays as described herein, for example, surface plasmonic resonance (SPR) analysis or flow cytometry.

[0098] The terms inhibition or blockade can be used interchangeably and encompass both partial and total inhibition / blockade. Inhibition / blockade of a ligand preferably reduces or alters the normal level or type of activity that occurs when the ligand binds without inhibition or blockade. Inhibition and blockade are also intended to include any measurable reduction in the binding affinity of the Petition 870260061229, dated 06 / 22 / 2026, page 82 / 414 75 / 118 ligand when in contact with the anti-B7-H4 antibody, compared to the binding affinity of the ligand without contact with the anti-B7-H4 antibody.

[0099] The term growth inhibition (for example, when referring to cells) is intended to include any measurable reduction in cell growth.

[00100] The terms induced immune response and enhanced immune response can be used interchangeably and refer to the immune response to stimulation by a specific antigen (i.e., passive or adaptive). The term induce used in the expression “induce CDC or ADCC” refers to a stimulus to a specific mechanism of direct cell death.

[00101] The ADCC in the present invention, that is, antibody-dependent cell-mediated cytotoxicity, means that cells expressing Fc receptors directly kill antibody-coated target cells by recognizing the Fc segment of the antibodies. The function of the ADCC effect of antibodies can be enhanced, reduced, or eliminated by modifying the Fc segment of IgG. Modification refers to mutations in a constant region of the heavy chain of an antibody.

[00102] The methods for producing antibodies and antigen-binding fragments are well known and can be found in the prior art, such as Antibodies: A Laboratory Manual, Cold Spring Harbor, chapters 5 to 8 and 15. For example, mice can be immunized with human B7-H4 or a fragment thereof, and the antibodies obtained can be renatured and purified, and amino acid sequencing can be performed by conventional methods. Antigen-binding fragments can also be prepared using conventional methods. The antibody or antigen-binding fragment of the present invention is genetically modified to introduce one or Petition 870260061229, dated 06 / 22 / 2026, p. 83 / 414 76 / 118 more human FR regions in non-human CDR regions. Human FR germline sequences can be obtained from the ImmunoGeneTics (IMGT) website http: / / imgt.cines.fr, or from The Immunoglobulin FactsBook, 2001 ISBN 012441351.

[00103] The modified antibodies or antigen-binding fragments of the present invention can be prepared and purified by conventional methods. The corresponding antibody cDNA sequences can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior technique, mammalian expression systems can lead to antibody glycosylation, especially at the highly conserved N-terminal Fc region. Stable clones are obtained by expressing antibodies that bind specifically to human antigens. Positive clones are expanded in serum-free bioreactor medium for antibody production. The culture medium into which the antibodies are secreted can be purified and collected by conventional techniques. The antibodies can be filtered and concentrated by conventional methods.Soluble mixtures and multimers can also be removed by conventional methods, for example, molecular sieves and ion exchange. The resulting product should be frozen immediately, such as at -70 °C, or freeze-dried.

[00104] The antibody of the present invention refers to a monoclonal antibody. The monoclonal antibody (mAb) described in the present invention refers to an antibody obtained from a single cloned cell strain; the cell strain is not limited to a eukaryotic, prokaryotic, or phage cloned cell strain. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombination technology, phage display technology, technology of Petition 870260061229, dated 06 / 22 / 2026, p. 84 / 414 77 / 118 synthesis (such as CDR grafting) or other existing technologies.

[00105] Administration and treatment, when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to contacting the exogenous drug, therapeutic agent, diagnostic agent, or composition with animals, humans, experimental subjects, cells, tissues, organs, or biological fluids. Administration and treatment may refer, for example, to treatment, pharmacokinetics, diagnosis, research, and experimental methods. Cell treatment includes the contact of reagents with cells, and the contact of reagents with fluids, where fluids are in contact with cells. Administration and treatment also means treating, for example, cells with reagents, diagnostics, binding compositions, or other types of cells in vitro and ex vivo.Treatment, when applied to humans, veterinary or research subjects, refers to therapeutic treatment, preventive or prophylactic measures, research, and diagnostic applications.

[00106] Treatment refers to the administration of an internal or external therapeutic agent, for example, a composition comprising any of the binding compounds of the present invention, to a patient with one or more disease symptoms where the therapeutic agent is measured to have a therapeutic effect. Generally, the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the treated individual or population, either to induce regression of such symptoms or to inhibit the development of such symptoms to any clinically measurable extent. The amount of therapeutic agent that is effective in alleviating any specific disease symptom (also referred to as a therapeutically effective amount) may vary according to a variety of factors, for example, the patient's disease status, age and Petition 870260061229, dated 06 / 22 / 2026, page 85 / 414 78 / 118 body weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the symptoms of the disease have been attenuated can be assessed by any clinical testing method commonly used by physicians or other healthcare professionals to assess the severity or progression of symptoms. Although the modalities of the present invention (e.g., treatment methods or products) may be ineffective in relieving every symptom of the disease of interest, they should reduce the symptom of the disease of interest in a statistically significant number of patients, as determined by any statistical testing methods in the art, such as Student's t-test, chi-square test, Mann-Whitney U test, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[00107] The term consisting essentially of...... or a variant thereof used throughout the specification and claims means comprising all the elements or groups of elements described, and optionally comprising other elements similar or different in nature to the elements described, which do not significantly alter the basic or novel properties of a given dosage regimen, method or composition. By way of non-limiting example, the linking compound consisting essentially of the aforementioned amino acid sequence may also comprise one or more amino acids, which do not significantly affect the properties of the linking compound.

[00108] The term naturally occurring as applied to a particular object in the present invention refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence is considered naturally occurring if it exists in organisms (including viruses) and can be isolated from natural sources, and has not been intentionally artificially modified in a laboratory. Petition 870260061229, dated 06 / 22 / 2026, page 86 / 414 79 / 118

[00109] The effective amount includes a quantity sufficient to improve or prevent a symptom or condition of a medical condition. The effective amount also refers to a quantity sufficient to allow or facilitate diagnosis. The effective amount for a given patient or veterinary individual may vary depending on the following factors: such as the condition being treated, the patient's general health status, the method, route, and dose of drug administration, and the severity of side effects. The effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxicity.

[00110] Exogenous refers to substances produced outside of organisms, cells, or human bodies according to the background. Endogenous refers to substances produced within cells, organisms, or human bodies according to the background.

[00111] Homology refers to the similarity of sequences between two polynucleotide sequences or between two polypeptides. When the positions in the two aligned sequences are occupied by the same base or amino acid monomer subunit, for example, if each position in two DNA molecules is occupied by adenine, then the molecules are considered homologous at that position. The percentage of homology between two sequences is a function of the number of paired or homologous positions shared by the two sequences divided by the number of aligned positions χ 100%. For example, in ideal sequence alignment, if 6 out of 10 positions in the two sequences are paired or homologous, then the two sequences are 60% homologous. Generally, alignment is performed when two sequences are aligned to obtain the maximum percentage of homology.

[00112] The terms cell, cell line and cell culture Petition 870260061229, dated 06 / 22 / 2026, p. 87 / 414 The terms 80 / 118 used here can be used interchangeably, and all these names include progeny from them. Therefore, the words transformant and transformed cell include primary test cells and cultures derived from them, regardless of the number of passages. It should also be understood that due to intentional or unintentional mutations, not all descendants can be exactly the same in terms of DNA content. Mutant progeny with the same biological function or activity as those analyzed in the original transformed cells is included. This can be clearly understood from the context when different names are used.

[00113] Optionally or optionally means that: an event or circumstance that follows the expression optionally or optionally may occur, but does not have to occur, and the description includes occasions when the event or circumstance does or does not occur. For example, optionally comprising 1 to 3 variable phosphorus heavy chain regions means that a variable heavy chain region of particular sequences may, but does not have to, be present.

[00114] Pharmaceutical composition means a mixture containing one or more of the compounds described herein, or a physiologically / pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiologically / pharmaceutically acceptable vehicles and excipients. The purpose of the pharmaceutical composition is to promote the administration of drugs to organisms, to facilitate the absorption of the active ingredient and thus exert biological activity. The preparation of conventional pharmaceutical compositions is described in the Chinese Pharmacopoeia.

[00115] Pharmaceutically acceptable salt refers to a salt of the antibody-drug conjugate of the present invention, which is safe and Petition 870260061229, dated 06 / 22 / 2026, page 88 / 414 81 / 118 effective for use in mammals and possesses the desired biological activity. The antibody-drug conjugate of the present invention comprises at least one amino group, therefore it can form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, iodide, 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.

[00116] The solvate refers to a pharmaceutically acceptable solvate formed by the antibody-drug conjugate compound of the present invention and one or more solvent molecules. Non-limiting examples of solvent molecules include: water, ethanol, acetonitrile, isopropanol, and ethyl acetate.

[00117] The cytotoxic drug, when used in the present invention, refers to a substance that inhibits cellular function and / or causes cell death or destruction.

[00118] Tubulin inhibitor refers to a class of compounds that interfere with the process of cell mitosis by inhibiting tubulin polymerization or promoting tubulin aggregation, thus exerting an antitumor effect. Non-limiting examples include: maytansinoids, calicheamicin, taxanes, vincristine, colchicine, dolastatin / auristatin / monomethyl auristatin E (MMAE) / monomethyl auristatin F (MMAF).

[00119] The linker refers to a chemical moiety by which an antibody is covalently linked to a covalent bond or atomic chain of a drug. Non-limiting examples of linkers include: arylene, heteroarylene, PEG, polymethylene oxy, succinate, succinamide, diglycolate, malonate, and caproamide.

[00120] Drug loading (DAR) is represented by y, which is the Petition 870260061229, dated 06 / 22 / 2026, page 89 / 414 82 / 118 average number of cytotoxic drugs per antibody in formula (A). The drug loading range in the present invention can be from 1 to 20 cytotoxic drugs (D) per antibody. The antibody-drug conjugate of general formula (A) is a collection of antibodies conjugated to a given range (1 to 20) of cytotoxic drugs. The drug loading (DR) in an antibody-drug conjugate from the coupling reaction can be characterized by conventional means, for example, mass spectrometry, HPLC and ELISA. In this way, the quantitative distribution of the antibody-drug conjugate in the value y can be determined. 2. Abbreviations MC = 6-maleimido-hexanoyl VC = valine-citrulline PAB = p-aminobenzyloxycarbonyl MMAE = monomethyl auristatin E (MW 718) MMAF = a variant of monomethyl auristatin E, which has phenylalanine at the C-terminal of the molecule (MW 731.5)

[00121] The examples are incorporated below for a more detailed description of the present invention, but these examples do not limit the scope of the present invention. Experimental methods with conditions not specified in the examples of the present invention generally follow conventional conditions, such as Antibodies: A Laboratory Manual and Molecular Cloning: A Laboratory Manual, Cold Spring Harbor; or according to the conditions recommended by the manufacturer of the raw material or the commodity. Reagents of unspecified origin are conventional reagents acquired on the market. Example 1: Antigen preparation and stable cell line construction

[00122] The sequence encoding human B7-H4 labeled with his (huB7-H4-His) and the sequence encoding human B7-H4 labeled with Petition 870260061229, dated 06 / 22 / 2026, page 90 / 414 83 / 118 Fc (huB7-H4-Fc) proteins were synthesized by Integrated DNA Technology (IDT) from CRO (the template sequences of the recombinant B7-H4 proteins above were all designed by the present invention) and respectively cloned into pTT5 vectors (Biovector). After the recombinant B7-H4 proteins were expressed in 293T cells, they were purified by the following experimental methods, and the purified proteins could be used in the following example experiments. huB7-H4-His sequence: MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGK GNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKADYKDDDDKGSHHHHHHHH SEQ ID NO: 1 huB7-H4-Fc sequence: FGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFESEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKAGSGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 2 Purification stages of huB7-H4-His: Petition 870260061229, de 22 / 06 / 2026, pág. 91 / 414 84 / 118

[00123] The HEK293 cell expression supernatant sample was centrifuged at high speed to remove impurities. The buffer was replaced with PBS and added with imidazole to a final concentration of 5 mM. The nickel column was equilibrated with 5 mM imidazole-containing PBS solution and washed with 2 to 5 times the column volume. The supernatant sample obtained after replacement was loaded onto the column. The column was rinsed with 5 mM imidazole-containing PBS solution until the A280 reading fell to the reference line. Then, the chromatography column was rinsed with 10 mM PBS + imidazole to remove impurities from non-specifically bound proteins, and the effluent was collected. The target protein was eluted with 300 mM imidazole-containing PBS solution, and the elution peak was collected. The collected eluate was further purified by ion exchange (SP column). Preparing a solution: PB at 0.01 M, pH 8.0. Preparation of solution B: A solution + NaCl at 1 M.First, the imidazole PBS solution with eluted target protein was replaced with solution A, the SP column was equilibrated using solution A, and the sample was loaded. The concentration gradient of solution B was 0 to 100%. The sample was eluted with 10 times the column volume, and each elution peak was collected. The obtained protein was identified as corrected by electrophoresis, peptide mapping, and liquid chromatography-mass spectrometry (LC-MS) and then aliquoted for use. Purification steps for huB7-H4-Fc:

[00124] The HEK293 cell expression supernatant sample was centrifuged at high speed to remove impurities. The buffer was replaced with PBS. The Protein A affinity column was equilibrated with 10 mM PBS buffer and washed 2 to 5 times the column volume. The supernatant sample obtained after the replacement was loaded onto the column. The column was washed with 25 buffer Petition 870260061229, dated 06 / 22 / 2026, page 92 / 414 The column volume was reduced by 85 / 118 times until the A280 reading fell to the reference line. The target protein was eluted with 0.8% acetate buffer pH 3.5, and elution peaks were collected. After aliquoting, 1 M Tris-Cl buffer pH 8.0 was immediately added for neutralization. The solution was then replaced with PBS pH 6.9 using the Millipore Amico-15 filter column. The obtained protein was identified by electrophoresis, peptide mapping, and liquid chromatography-mass spectrometry (LC-MS) and then aliquoted for use. Construction of a CHO-S cell pool:

[00125] Full-length sequences encoding human or cynomolgus monkey B7-H4 protein (huB7-H4 or cyB7-H4) were synthesized by Integrated DNA Technology (IDT) (the template sequences of the recombinant B7-H4 proteins above were all designed by the present invention), and respectively cloned into the modified pcDNA3.1 vector, pcDNA3.1 / pure (Invitrogen #V79020). CHO-S (ATCC) cells were cultured in CD-CHO medium (Life Technologies, #10743029) at 0.5 x 10⁶ cells / ml. 10 μg of vector encoding the huB7-H4 or cyB7-H4 gene were mixed with 50 μl of LF-LTX (Life Technologies, # A12621) in 1 ml of Opti-MEM medium (Life Technologies, # 31985088). The mixture was incubated at room temperature for 20 minutes and added to CHO cell culture medium. The cells were placed in a carbon dioxide incubator for culture. After 24 hours, the culture medium was replaced with fresh medium and 10 μg / ml of puromycin was added.After that, the culture medium was replaced with a new medium every 2 to 3 days, and a stable pool of CHO-S cells was obtained after 10 to 12 days of selection. Example 2: Obtaining mouse hybridoma and antibody sequences

[00126] A total of 5 female Balb / ce 5 A / J mice with 10 Petition 870260061229, dated 06 / 22 / 2026, page 93 / 414 Mouse aged 86-118 weeks were immunized with the human huB7-H4-Fc antigen. Freund's Complete Sigma Adjuvant (CFA) and Freund's Incomplete Sigma Adjuvant (IFA) were used. The immunogen and the immunological adjuvant were thoroughly mixed and emulsified in a 1:1 ratio to form a stable water-in-oil liquid. The injection dose was 25 μg / 200 μL / mouse.

[00127] Day 1: First immunization, complete Freund's adjuvant.

[00128] Day 21: Second immunization, incomplete Freund's adjuvant.

[00129] Day 35: Third immunization, Freund's adjuvant incomplete.

[00130] Day 42: Blood collection and serum titer test (blood after 3 immunizations).

[00131] Day 49: Fourth immunization, Freund's adjuvant incomplete.

[00132] Day 56: Blood collection and serum titer test (blood after 4 immunizations).

[00133] The indirect ELISA method was used to detect antibody or serum affinity: the huB7-H4-His protein was diluted to a concentration of ^g / ml with PBS pH 7.4, and added at 100 μl / well in a 96-well high-affinity ELISA plate and refrigerated at 4 °C for overnight incubation (16 to 20 hours). The plate was washed 4 times with PBST (pH 7.4 PBS containing 0.05% Tween-20). A 3% bovine serum albumin (BSA) blocking solution diluted with PBST was added at 150 μl / well and incubated at room temperature for 1 hour for blocking. After blocking was complete, the blocking solution was discarded and the plate was washed 4 times with PBST buffer. The antibody or serum to be tested was diluted with PBST containing 3% BSA to obtain a 10-dose gradient (10-fold dilution) from 1 μM and added to the microtiter plate at 100 μl / well and incubated at room temperature. Petition 870260061229, dated 06 / 22 / 2026, page 94 / 414 87 / 118 for 1 hour. After incubation was complete, the plate was washed 4 times with PBST, HRP-labeled goat anti-human secondary antibody (Abcam, cat # ab97225) diluted with PBST containing 3% BSA was added at 100 μl / well, and incubated for 1 hour at room temperature. The plate was washed 4 times with PBST, then TMB chromogenic substrate (Cell Signaling Technology, cat #7004S) was added at 100 μl / well and incubated at room temperature in the dark for 1 minute. Stop solution (Cell Signaling Technology, cat # 7002S) was added at 100 μl / well to terminate the reaction. The absorbance value was read at 450 nm with a microplate reader (BioTek, model Synergy H1). The data were analyzed to determine the binding affinity of the antibody or serum to the human B7-H4 antigen.

[00134] Serum titer and the ability of immunized mouse serum to bind to cell surface antigens were evaluated by the indirect ELISA method. Cell fusion was performed according to the titer detection results (greater than 100,000-fold dilution). Mice immunized with high serum titer, affinity, and binding to FACS were selected for final immunization and then sacrificed. Spleen cells and SP2 / 0 myeloma cells were fused and seeded to obtain hybridomas. Target hybridomas were selected by indirect ELISA, and monoclonal cell strains were established by the dilution-limiting method. Among the antibody-positive strains obtained, hybridoma strains expressing non-specific binding antibodies were also compared, as were CHO-S cells that stably express huB7-H4 and cyB7-H4, with blank CHO-S cells.The target hybridomas were analyzed using methods similar to those of the in vitro cell ligation experiment in Example 5, and established as monoclonal cell strains by the limiting method of. Petition 870260061229, dated 06 / 22 / 2026, page 95 / 414 88 / 118 dilution. Hybridoma cells in the logarithmic growth phase were collected. RNA was extracted with Trizol (Invitrogen, 15596-018) and subjected to reverse transcription (PrimeScript™ Reverse Transcriptase, Takara # 2680A). The cDNA obtained by reverse transcription was amplified by PCR with a pool of mouse Ig primers (Novagen, TB326 Rev.B 0503) and sequenced. Finally, sequences of 4 murine antibody strains for humanization and construction of antibody-drug conjugates were obtained.

[00135] The variable region sequences of the murine monoclonal antibody 2G6 heavy chain and light chain are as follows: 2G6 HCVR EVQLVESGGGLVKPGGSLKLSCAASGFTFSRYGMSWVRQTPEKRLEWVAGINGGGSYTYYLDTVKGRFTISRDNSRNTLYLQMSSLRSEDTAMYYCVSQGSNYYFDYWGQGTTLTVSS SEQ ID NO: 2G6 LCVR DIRMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQKPGKSFKALIYHGTNLEDGVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPYTFGGGTKLEIK SEQ ID NO: 47

[00136] Variable regions of the heavy chain and light chain of murine monoclonal antibody 2G6 comprise the following CDR sequences: Name Sequence No. HCDR1 GFTFSRYGMS SEQ ID NO: 3 HCDR2 GINGGGSYTYYLDTVKG SEQ ID NO: 4 HCDR3 QGSNYYFDY SEQ ID NO: 5 LCDR1 HASQGISSNIG SEQ ID NO: 6 LCDR2 HGTNLED SEQ ID NO: 7 LCDR3 VQYAQFPYT SEQ ID NO: 8

[00137] The sequences of the variable heavy chain regions and Petition 870260061229, dated 06 / 22 / 2026, page 96 / 414 89 / 118 murine monoclonal antibody light chain 2F7 are as follows: 2F7 HCVR EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYYMSWVRQTPEKRLEWVAYVSSGGGSTYYSDSVKGRFTISRDNAKNTLYLQMSSLKPEDTAMYYCTRESYSQGNYFDYWGQGTTLTVSS SEQ ID NO: 48 2F7 LCVR DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKFASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFSLTFGAGTKLELK SEQ ID NO: 49

[00138] Variable regions of the heavy chain and light chain of murine monoclonal antibody 2F7 comprise the following CDR sequences: Name Sequence No. HCDR1 GFTFSNYYMS SEQ ID NO: 9 HCDR2 YVSSGGGSTYYSDSVKG SEQ ID NO: 10 HCDR3 ESYSQGNYFDY SEQ ID NO: 11 LCDR1 RASQSISDYLH SEQ ID NO: 12 LCDR2 FASQSIS SEQ ID NO: 13 LCDR3 QNGHSFSLT SEQ ID NO: 14

[00139] The sequences of the variable regions of the murine monoclonal antibody 2F8 heavy chain and light chain are as follows: 2F8 HCVR QVQLQQPGSVLVRPGASVKLSCCASGYTFTNSWMNWAKLRPGQGLEWIGGIYPNSGNIEYNEKFKGKATLTVDTSSSTAYMDLTSLTSEDSAVYYCARDSRFSYWGQGTLVTVSA SEQ ID NO: 50 2F8 LCVR DIVMTQSHKFMSTSVGDRVSITCKASQDVRTAVAWYQQKPGQSPKLLISSTSYRYTGVPDRFTGSGSGTDFTFIISSVQAEPetition 870260061229, of 06 / 22 / 2026, p. 97 / 414 90 / 118 DLAVYYCQQHYSTPLTFGAGTKLELK SEQ ID NO: 51

[00140] Variable regions of the heavy chain and light chain of murine monoclonal antibody 2F8 comprise the following CDR sequences: Name Sequence No. HCDR1 GYTFTNSWMN SEQ ID NO: 23 HCDR2 GIYPNSGNIEYNEKFKG SEQ ID NO: 24 HCDR3 DSRFSY SEQ ID NO: 25 LCDR1 KASQDVRTAVA SEQ ID NO: 26 LCDR2 STSYRYT SEQ ID NO: 27 LCDR3 QQHYSTPLT SEQ ID NO: 28

[00141] The sequences of the variable regions of the murine monoclonal antibody 1C9 heavy chain and light chain are as follows: 1C9 HCVR QVQLQQPGSVLVRPGASVKLSCKASGDTFTTYWMNWVKQRPGQGLEWIGGIYLNSGSSEYNEKFKGKATLSVDTSSSTAYMDLSSLTSEDSAVYYCARDSRFSYWGQGTLVTVSA SEQ ID NO: 52 1C9 LCVR DIVMTQSHKFLSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPELLISSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYNTPLTFGAGTQLELK SEQ ID NO: 53

[00142] Variable regions of the heavy chain and light chain of murine monoclonal antibody 1C9 comprise the following CDR sequences: Name Sequence No. HCDR1 GDTFTTY SEQ ID NO: 29 HCDR2 YLNSGS SEQ ID NO: 30 Petition 870260061229, dated 06 / 22 / 2026, page 98 / 414 91 / 118 Name Sequence No. HCDR3 DSRFSY SEQ ID NO: 31 LCDR1 KASQDVSTAVA SEQ ID NO: 32 LCDR2 SASYRYT SEQ ID NO: 33 LCDR3 QQHYNTPLT SEQ ID NO: 34 Example 3: Murine Antibody Humanization Experiment

[00143] The humanization of murine anti-B7-H4 monoclonal antibodies was performed using methods described in various documents in the art. Briefly, the parental constant domains (murine antibody) were replaced with human constant domains. In the present invention, human germline antibody sequences were selected based on homology between murine and human antibodies, and murine antibodies 2G6, 2F7, 2F8, and 1C9 were humanized. The CDR regions of murine antibodies 2G6 and 2F7 were grafted onto the corresponding humanized templates selected to replace the humanized variable regions, and then recombined with IgG constant regions (preferably IgG1 for the heavy chain and κ for the light chain).Next, based on the three-dimensional structure of murine antibodies, embedded residues, residues directly interacting with CDR regions, and residues with significant influence on VL and VH conformation were subjected to reverse mutation, and chemically unstable amino acid residues of the CDR regions were optimized, whereby HCDR1 of the murine monoclonal antibody 2F8 was optimized to GYTFTSSWMN (SEQ ID NO: 43), HCDR2 was optimized to GIYPNRGNIEY NEKFKG (SEQ ID NO: 44), HCDR2 of the murine monoclonal antibody 1C9 was optimized to YLNRGS (SEQ ID NO: 45), and humanized antibodies designed comprising the following combination of variable light and heavy chain region sequences were obtained. Petition 870260061229, dated 06 / 22 / 2026, page 99 / 414 92 / 118

[00144] Variable regions of the heavy and light chains of the humanized antibody hu2G6 are as follows: hu2G6 HCVR EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQAPGKGLEWVSGINGGGSYTYYLDTVKGRFTISRDNARNTLYLQMSSLRAEDTAVYYCVSQGSNYYFDYWGQGTLVTVSS SEQ ID NO: 15 hu2G6 LCVR DIRMTQSPSSLSASVGDRVTITCHASQGISSNIGWLQQKPGKAPKALIYHGTNLEDGVPSRFSGSGSGADYTLTISSLQPEDFATYYCVQYAQFPYTFGGGTKVEIK SEQ ID NO: 16

[00145] Variable regions of the heavy and light chains of the humanized antibody hu2F7 are as follows: hu2F7 HCVR EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVAYVSSGGGSTYYSDSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCTRESYSQGNYFDYWGQGTTVTVSS SEQ ID NO: 17 hu2F7 LCVR EIVMTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQSPRLLIKFASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFSLTFGQGTKLEIK SEQ ID NO: 18

[00146] Variable regions of the heavy and light chains of the humanized antibody hu2F8 are as follows: hu2F8 HCVR Petition 870260061229, dated 06 / 22 / 2026, page 100 / 414 93 / 118 DTSASTAYMELSSLRSEDTAVYYCARDSRFSYWGQGTLVTVSS SEQ ID NO: 35 hu2F8 LCVR DIQMTQSPSSLSASVGDRVTITCKASQDVRTAVAWYQQKPGKAPKLLISSTSYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPLTFGGGTKVEIK SEQ ID NO:36

[00147] Variable regions of the heavy and light chains of the humanized antibody hu1C9 are as follows: hu1C9 HCVR EVQLVQSGAEVKKPGASVKVSCKASGDTFTTYWMNWVRQAPGQRLEWMGGIYLNRGSSEYNEKFKGRVTLTVDTSASTAYMELSSLRSEDTAVYYCARDSRFSYWGQGTLVTVSS SEQ ID NO:37 hu1C9 LCVR DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLISSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYNTPLTFGGGTKVEIK SEQ ID NO:38;

[00148] The variable regions were recombined with the constant regions of IgG (preferably, IgG1 for the heavy chain, and κ for the light chain). The exemplary constant region sequences of light and heavy chains are shown below, IgG1 C: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNPetition 870260061229, 22 / 06 / 2026, page 101 / 414 94 / 118 GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 54 Ig kappa C: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 55;

[00149] Humanized antibodies consisting of the following exemplary light and heavy chain sequences were obtained after ligation: hu2G6 HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQAPGKGLEWVSGINGGGSYTYYLDTVKGRFTISRDNARNTLYLQMSSLRAEDTAVYYCVSQGSNYYFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 19 hu2G6 LC DIRMTQSPSSLSASVGDRVTITCHASQGISSNIGWLQQKPGKAPKALIYHGTNLEDGVPSRFSGSGSGADYTLTISSLQPEDFATYYCVQYAQFPYTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRPetition 870260061229, of 06 / 22 / 2026, p. 102 / 414 95 / 118 GEC SEQ ID NO: 20 hu2F7 HC EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVAYVSSGGGSTYYSDSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCTRESYSQGNYFDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 21 hu2F7 LC EIVMTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQSPRLLIKFASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFSLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 22 hu2F8 HC EVQLVQSGAEVKKPGASVKVSCKASGYTFTSSWMNWVRQAPGQRLEWMGGIYPNRGNIEYNEKFKGRVTLTVDTASTAYMELSSLRSEDTAVYCARDSRFSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEPetição 870260061229, 22 / 06 / 2026, p. 103 / 414 96 / 118 QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:39 hu2F8 LC DIQMTQSPSSLSASVGDRVTITCKASQDVRTAVAWYQQKPGKAPKLLISSTSSYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:40 hu1C9HC EVQLVQSGAEVKKPGASVKVSCKASGDTFTTYWMNWVRQAPGQRLEWMGGIYLNRGSSEYNEKFKGRVTLTVDTSASTAYMELSSLRSEDTAvyYCARDSRFSYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEKPKSCDKTH TCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK SEQ ID NO:41 hu1C9 LC DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLISSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYNTPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKPetição 870260061229, de 22 / 06 / 2026, pág. 104 / 414 97 / 118 DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:42. Example 4: Expression of anti-B7-H4 humanized antibodies

[00150] The recombinant vector was designed according to the sequences of the humanized antibodies. The heavy chain vector was designed as follows: signal peptide + humanized heavy chain. The light chain vector was designed as follows: signal peptide + humanized light chain.

[00151] The preceding sequences were inserted into the pCEP4 vector (ThermoFisher # V04450). The expression vector was synthesized according to the above design. The plasmid vector was obtained and extracted on a large scale and sent for sequencing for verification. The qualified plasmid was transfected into human 293F cells (ThermoFisher #R79007) with PEI (ThermoFisher# BMS1003-A). The 293F cells were continuously cultured with serum-free medium (Shanghai OPM Biosciences, OPM-293 CD03) to the logarithmic growth phase and used for cell transfection. 21.4 μg of humanized antibody light chain plasmid and 23.6 μg of humanized antibody heavy chain plasmid were dissolved in 10 ml of Opti-MEM® I Reduced Serum Medium (GIBCO, 31985-070) and thoroughly mixed. Then, 200 μg of PEI were added and thoroughly mixed, incubated at room temperature for 15 min. 50 ml of cells were then added. Cell culture conditions: 5% CO2, 37 °C, 125 rpm.During culture, supplements were added on day 1 and day 3 until cell viability was less than 70%. The cell supernatant was collected, centrifuged, and filtered. The filtered and centrifuged cell culture medium was loaded onto an antibody affinity purification column. The column was washed with phosphate buffer. The sample was eluted with glycine hydrochloride buffer (pH 2.7, 0.1 M Gly-HCl), neutralized with 1 M Tris hydrochloride pH 9.0, and dialyzed. Petition 870260061229, dated 06 / 22 / 2026, page 105 / 414 98 / 118 with phosphate buffer to finally obtain the purified humanized antibody, which could be used in experiments for each Example. Example 5: Detection of in vitro binding capacity of humanized antibodies.

[00152] The engineered humanized antibodies were tested in in vitro experiments as follows: 1. In vitro cell binding experiment:

[00153] Cultured MX-1 cells expressing human B7-H4 (CLS Cell Lines Service GmbH # 300296) were collected, cell density was adjusted with PBS at pH 7.4, and cells were seeded in a 96-well V-bottom plate at 1 χ¹⁰⁵ cells per well. The plate was centrifuged at 2,000 rpm for 5 minutes and the supernatant was removed. 100 μl of gradient-diluted humanized antibody solution (diluted with PBS containing 0.5% BSA, starting from 1 μm, a 3-fold gradient of 10 doses) was added to each well, well mixed, and incubated at 4 °C on a shaker for 1 hour. The plate was centrifuged at 2,000 rpm for 5 minutes and the supernatant was removed. Cells were washed twice with PBS. 100 μl of FITC-labeled goat anti-human secondary antibody (Abcam, cat # ab97224) diluted with 0.5% BSA in PBS were added to each well, mixed well, and incubated for 30 minutes at 4 °C on a shaker.The plate was centrifuged at 2,000 rpm for 5 minutes and the supernatant was removed. The cells were washed twice with PBS and then resuspended in PBS. The signal was detected using a flow cytometer (BECKMAN COULTER, model DxFLEX), and each concentration curve was plotted for analysis of the results. The results are presented in Table 1. The humanized antibodies hu2G6 and hu2F7 bind positively to MX-1 cells with high expression of B7-H4.

[00154] Table 1. Affinity (EC50) of each humanized antibody to Petition 870260061229, dated 06 / 22 / 2026, page 106 / 414 99 / 118 cells MX-1 Name of the FACS-binding EC50 antibody (nM) with MX-1 cells: hu2G6 14.3, hu2F8 7.26, hu1C9 7.25, hu2F7 7.32 2. Affinity kinetics experiment:

[00155] The Biacore method is a generally known method for objectively detecting the affinity and kinetics between proteins. The affinity and binding kinetics of the antibodies to be tested in the present invention were analyzed by Biacore T200 (GE). The anti-B7-H4 antibodies to be tested in the present invention were covalently linked to CM5 chips (GE) using the kit provided by Biacore and the standard NHS amino coupling method. Then, human huB7-H4-His protein at 50 nM diluted in the same buffer was injected at a flow rate of 10 µL / min. After injection, all samples were regenerated with the kit's regeneration reagent. Antigen-antibody binding kinetics were monitored for 3 minutes and dissociation kinetics were monitored for 10 minutes. The data obtained were analyzed with a 1:1 binding model (Langmuir) using GE's BIAevaluation software.The affinity kinetics data for the humanized antibodies calculated by this method are shown in Table 2. The humanized antibodies hu2G6, hu2F7, hu2F8, and hu1C9 exhibit strong affinity for the human B7-H4 antigen protein. Table 2. Affinity and kinetic characterization of each humanized antibody. Petition 870260061229, dated 06 / 22 / 2026, page 107 / 414 100 / 118 Antibody Binding rate ka (1 / M*s) Dissociation rate kd (1 / s) Affinity Kd hu2G6 7.41e+05 1.00e-05 1.35e-11 hu2F8 4.47e+05 1.12e-04 2.50e-10 hu1C9 3.35e+05 1.00e-05 2.98e-11 hu2F7 3.29e+05 2.49e-04 7.57e-10 Example 6: Endocytosis of anti-B7-H4 antibodies

[00156] In order to test whether the antibodies of the present invention can be endocytosed together with human B7-H4 after binding to B7-H4, MX-1 cells were used for evaluation. MX-1 cells were trypsinized (first washed with PBS once at 37 °C for approximately 2 min), collected, and resuspended in pre-chilled FACS buffer. The cell concentration was adjusted to 1 x 10⁶ cells / mL. 1 mL of the cell suspension was added to an EP tube, centrifuged at 1500 rpm for 5 minutes, and the supernatant was removed. 1 mL of the prepared antibody to be tested was added to resuspend the cells, and the final antibody concentration was 20 μg / mL. The cells were incubated on a shaker at 4 °C for 1 hour, centrifuged, and the supernatant was discarded (4 °C, 1500 rpm x 5 min). The cells were washed twice with FACS buffer and the supernatant was removed.100 μL of fluorescent secondary antibody working solution were added to each tube to resuspend the cells. The cells were incubated on a shaker at 4 °C for 30 min, centrifuged, and the supernatant was discarded (4 °C, 1500 rpm x 5 min). The cells were washed twice with FACS buffer and the supernatant was removed. 1.0 mL of complete pre-warmed MX-1 cell medium was added to each tube to resuspend the cells and mixed. The cell suspension was aliquoted into 4 tubes at 200 μL per tube, which were respectively the 0 min group, the blank group, the 30 min group, and the 2 h group. The blank and 0 min groups were placed on ice, en. Petition 870260061229, dated 06 / 22 / 2026, page 108 / 414 Groups 101 / 118 and the other groups were placed in an incubator at 37 °C for endocytosis for 30 min and 2 h, respectively. At the corresponding time point, the EP tube was removed and placed on ice to pre-cool for 5 min. All treatment groups were centrifuged and the supernatant discarded (4 °C, 1500 rpm χ 5 min). The cells were washed once with FACS buffer and the supernatant was removed. 250 μL of strip buffer were added to the EP tubes of all treatment groups except the 0 min group and incubated for 8 min at room temperature. The cells were centrifuged and the supernatant discarded (4 °C, 1500 rpm χ 5 min). The cells were washed twice with FACS buffer and the supernatant was removed. All treatment groups were treated with 100 μL of immunolabeling fixative, placed at 4 °C for more than 30 min, and detected by DxFlex flow cytometry.The percentage of B7-H4 antibody endocytosis (%) = (mean fluorescence intensity value at each time point - mean fluorescence intensity value of the blank group) / (mean fluorescence intensity value at point zero - mean fluorescence intensity value of the blank group) χ² 100%. The data are shown in Table 3. Table 3. Efficiency of humanized antibody-mediated endocytosis of B7-H4 protein. Antibody 0 hours (%) 0.5 hours (%) 2 hours (%) IgG Control 0 0.1 0.9 hu2G6 0 18.6 28.7 hu2F7 0 13.9 25.9 Example 7: Antibody conjugation to MC-MMAF

[00157] The antibodies of the present invention have cell affinity activity and endocytosis activity, making them suitable for coupling with drugs to form antibody-drug conjugates for the treatment of B7-H4 mediated diseases. The coupling process is shown in the following equation, where Ab represents Petition 870260061229, dated 06 / 22 / 2026, page 109 / 414 102 / 118 hu2G6 or hu2F7: the MC-MMAF

[00158] In the first step, S-(3-hydroxypropyl)thioacetate (0.7 mg, 5.3 mol) was dissolved in 0.9 mL of acetonitrile to form a solution for later use. The acetonitrile solution pre-prepared above with S-(3-hydroxypropyl)thioacetate was added to the antibody in pH = 4.3 sodium acetate buffer (10.35 mg / mL, 9.0 mL, 0.97 mol). Then, 1.0 mL of aqueous sodium cyanoborohydride solution (14.1 mg, 224 mol) was added dropwise to the reaction mixture and reacted under stirring at 25 °C for 2 hours. After the reaction was complete, the reaction mixture was desalinated and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution pH 6.5) to obtain a solution of product 1f. The solution was concentrated to 10 mg / mL and used directly in the subsequent reaction.

[00159] In the second step, solution 1f (11.0 mL) was added with 0.35 mL of 2.0 M carboxiamine hydrochloride solution and reacted under stirring at 25 °C for 30 minutes. Then, the reaction solution was desalinated and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution pH 6.5) to obtain a solution of product 2f (concentration 6.17 mg / mL, 14.7 mL).

[00160] In the third step, the MC-MMAF compound (1.1 mg, 1.2 mol, prepared by the method described in PCT patent WO2005081711) was dissolved in 0.3 mL of acetonitrile, added to solution 2f (con Petition 870260061229, dated 06 / 22 / 2026, page 110 / 414 103 / 118 concentration 6.17 mg / mL, 3.0 mL) and reacted under stirring at 25 °C for 4 hours. The reaction solution was then desalinated and purified by a Sephadex G25 gel column (elution phase: 0.05 M PBS solution at pH 6.5), and filtered under sterile conditions with a filter to obtain the antibody-drug conjugate product Ab-MC-MMAF in PBS buffer (3.7 mg / mL, 4.7 mL), which was refrigerated at 4 °C. The mean y-value of the hu2G6-MC-MMAF product determined by HIC-HPLC was 3.8, and hu2G6-MC-MMAF samples (y = 4) were obtained by HIC-HPLC purification. The average y value of the hu2F7-MC-MMAF product determined by HIC-HPLC was 3.2, and the hu2F7-MC-MMAF (y = 2) and hu2F7-MC-MMAF (y = 4) samples were obtained by purification using HIC-HPLC. Example 8: Antibody conjugation for SN-38

[00161] The drugs conjugated with p antibody were prepared through the following coupling process, where Ab represents hu2F7: Ab-SN-38

[00162] In the first step, S-(3-hydroxypropyl)thioacetate (0.7 mg, 5.3 mol) was dissolved in 0.9 mL of acetonitrile to form a solution for later use. The acetonitrile solution pre-prepared above with S-(3-hydroxypropyl)thioacetate was added to the antibody at pH = 4.3 Petition 870260061229, dated 06 / 22 / 2026, page 111 / 414 104 / 118 acetate / sodium acetate buffer (10.35 mg / mL, 9.0 mL, 0.97 mol). Then, 1.0 mL of aqueous sodium cyanoborohydride solution (14.1 mg, 224 mol) was added dropwise to the reaction mixture and reacted under stirring at 25 °C for 2 hours. After the reaction was complete, the reaction mixture was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution pH 6.5) to obtain a 1h product solution. The solution was concentrated to 10 mg / mL and used directly in the subsequent reaction.

[00163] In the second step, a 1h solution (11.0 mL) was added with 0.35 mL of 2.0 M carboxiamine hydrochloride solution and reacted under stirring at 25 °C for 30 minutes. The reaction solution was then desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS solution pH 6.5) to obtain a 2h product solution (concentration 6.2 mg / mL, 15.0 mL). The 2h solution was concentrated to approximately 10 mg / mL and used in the subsequent reaction.

[00164] In the third step, the compound MC-VC-PAB-SN-38 (1.3 mg, 1.2 mol) was dissolved in 0.3 mL of acetonitrile, added to a 2h solution (concentration 6.2 mg / mL, 3.0 mL) and reacted under stirring at 25 °C for 4 hours. The reaction solution was then desalted and purified by a Sephadex G25 gel column (elution phase: 0.05 M PBS solution pH 6.5), and filtered under sterile conditions with a filter to obtain the antibody-drug conjugate product hu2F7-SN-38 in PBS buffer (3.7 mg / mL, 4.7 mL), which was refrigerated at 4 °C. The average y value was determined by the ultraviolet method. Cuvettes filled with sodium succinate buffer were placed in the reference absorption cell and the sample determination absorption cell, respectively, and after deduction of the solvent blank, cuvettes filled with the test solution were placed in the sample determination absorption cell. Petition 870260061229, dated 06 / 22 / 2026, page 112 / 414 105 / 118 absorbance was measured at 280 nm and 370 nm. Data processing:

[00165] The Cmab antibody content was determined by establishing a standard curve and measuring absorption at a wavelength of 280 nm. The CFdrug small molecule content was determined by measuring absorption at a wavelength of 370 nm. Average drug loading y=CFrug / Cmab

[00166] The average value y=3.7 of hu2F7-SN-38 was determined by the above method. Samples of hu2F7-SN-38 (y=4) were obtained by UV-HPLC purification. Example 9: Antibody conjugation for Exatecan

[00167] In the first step, 2a (2 g, 17.2 mmol) was dissolved in 75 mL of acetonitrile and successively added with potassium carbonate (9.27 g, 67.2 mmol), benzyl bromide (20 mL, 167.2 mmol), and tetrabutylammonium iodide (620 mg, 1.68 mmol). The reaction solution was stirred at room temperature for 48 hours and filtered through diatomaceous earth. The filtered mass was rinsed with ethyl acetate (20 mL). The filtrate was mixed and concentrated under reduced pressure. The residues obtained were purified by carbon chromatography. Petition 870260061229, dated 06 / 22 / 2026, page 113 / 414 106 / 118 silica gel with solvent system under development C to obtain product 5a (3.2 g, yield: 90.1%).

[00168] In the second step, 5a (181.3 mg, 0.879 mmol) and 4b (270 mg, 0.733 mmol) were added to a reaction flask, supplemented with 6 mL of tetrahydrofuran and replaced with argon three times. The reaction mixture was cooled to 0–5°C in an ice bath and supplemented with potassium tert-butoxide (164 mg, 1.46 mmol), then heated to room temperature by removing the ice bath and stirred for 0 minutes. The reaction mixture was supplemented with 15 mL of ice-cold water and extracted with ethyl acetate (40 mL χ²) and chloroform (20 mL χ²). The organic phases were mixed and concentrated. The residues obtained were dissolved in 6 mL of dioxane, added with 3 mL of water, sodium bicarbonate (73.8 mg, 0.879 mmol) and 9-fluorene methyl chloroformate (190 mg, 0.734 mmol) and stirred at room temperature for 2 hours. The reaction solution was added with 30 mL of water and extracted with ethyl acetate (20 mL x 3).The organic phases were washed with a saturated sodium chloride solution (30 mL), dried with anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The residues obtained were purified by silica gel column chromatography with the developing solvent system C to obtain the product 5b 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-benzyl ate (73 mg, yield: 19.4%).

[00169] EM m / z (ESI): 515.0 [M +1].

[00170] In the third step, 5b (30 mg, 0.058 mmol) was dissolved in 6.75 mL of a mixed solvent of tetrahydrofuran and ethyl acetate (V:V = 2:1), added with palladium on carbon (18 mg, 10% content, dry basis), replaced by hydrogen three times, and reacted under stirring at room temperature for 1 hour. The reaction solution was filPetition 870260061229, dated 06 / 22 / 2026, page 114 / 414 107 / 118 treated with diatomaceous earth. The filter mass was rinsed with ethyl acetate. The filtrate was concentrated to obtain the crude product 5c 10-cyclopropyl-1-(9H-fluoren-9-yl)3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-acid (20 mg), which was used directly in the following reaction without purification.

[00171] EM m / z (ESI): 424.9 [M+1].

[00172] In the fourth step, 1b (15 mg, 28.2 μmol) was added to a reaction flask, supplemented with 1.5 mL of N,N-dimethylformamide and replaced with argon three times. The reaction mixture was cooled to 0–5 °C in an ice water bath, supplemented with a drop of triethylamine, supplemented with crude product 5c (20 mg, 47.1 μmol), supplemented with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylchloromorpholine (25.4 mg, 86.2 μmol), and reacted under stirring in an ice bath for 40 minutes. The reaction mixture was supplemented with 15 mL of water and extracted with ethyl acetate (20 mL χ³). The organic phases were pooled. The organic phases were washed with a saturated sodium chloride solution (20 mL χ 2), dried with anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure.The residues obtained were purified by thin-layer chromatography with the developing solvent system B to obtain the title product 5d (2-(((1-cyclopropyl-2-(((1S,)9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolozino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate of (9H-fluoren-9-yl)methyl (23.7 mg, yield: 78.9%).

[00173] EM m / z (ESI): 842.1 [M + 1].

[00174] In the fifth step, 5d (30 mg, 35.7 μmol) was dissolved in 3 mL of dichloromethane, added with 1.5 mL of diethylamine and stirred. Petition 870260061229, dated 06 / 22 / 2026, page 115 / 414 108 / 118 at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, 1.5 mL of toluene was added, and concentrated under reduced pressure again, repeating twice. The residues were added with 4.5 mL of n-hexane and depulped. The supernatant was discarded after standing, and the solid was retained. The solid residues were concentrated under reduced pressure and pumped dry to obtain the crude product 5e 2-((2-aminoacetamido)methoxy)-2-cyclopropyl-N-((1 S , S )-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3', 4': 6,7] indolozine [1,2-b]quinolin-1-yl)acetamide (23 mg), which was used directly in the following reaction without purification.

[00175] EM m / z (ESI): 638.0 [M + 18].

[00176] In the sixth step, the crude product 5e (20 mg, 32.3 μmol) was dissolved in 1 mL of N,N-dimethylformamide and replaced with argon three times. The reaction mixture was cooled to 0–5 °C in an ice water bath, 0.5 mL of 4 g (31.8 mg, 67.3 μmol) N,N-dimethylformamide solution was added, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylchloromorpholine (27.8 mg, 94.3 μmol) was added, and reacted under stirring in an ice bath for 10 minutes. The reaction mixture was heated to room temperature by removing the ice bath and reacted under stirring for 1 hour to produce compound 5. The reaction solution was purified by high-performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A - water (10 mmol NH4OAc); B - acetonitrile; elution gradient; flow rate: 18 mL / min).The corresponding components were collected and concentrated under reduced pressure to obtain products 5-A and 5-B (3.6 mg, 2.6 mg). Petition 870260061229, dated 06 / 22 / 2026, page 116 / 414 109 / 118

[00177] EM m / z (ESI): 1074.4 [M + 1]. Single Configuration Compound 5-A (shortest retention time)

[00178] UPLC analysis: retention time 1.14 minutes, purity: 85% (column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A-water (5 mmols NH4OAc), B-acetonitrile).

[00179] 1H NMR (400 MHz, DMSO-d6): δ 8.60 (t, 1H), 8.51-8.49 (d, 1H), 8.32-8.24 (m, 1H), 8.13-8.02 (m, 2H), 8.02-7.96 (m, 1H), 7.82-7.75 (m, 1H), 7.31 (s, 1H), 7.26-7.15 (m, 4H), 6.99 (s, 1H), 6.55-6.48 (m, 1H), 5.65-5.54 (m, 1H), 5.41 (s, 2H), 5.35-5.15 (m, 3H), 4.74-4.62 (m, 2H), 4.54-4.40 (m, 2H), 3.76-3.64 (m, 4H), 3.62-3.48 (m, 2H), 3.20-3.07 (m, 2H), 3.04-2.94 (m, 2H), 2.80-2.62 (m, 2H), 2.45-2.30 (m, 3H), 2.25-2.15 (m, 2H), 2.15-2.04 (m, 2H), 1.93-1.78 (m, 2H), 1.52-1.39 (m, 3H), 1.34-1.12 (m, 5H), 0.87 (t, 3H), 0.64-0.38 (m, 4H). Simple 5-B configuration compound (longer retention time):

[00180] UPLC analysis: retention time 1.16 minutes, purity: 89% (column: ACQUITY UPLC BEHC18 1.7 μm 2.1*50 mm, mobile phase: A-water (5 mmol NH4OAc), B-acetonitrile).

[00181] 1H NMR (400 MHz, DMSO-d6): δ 8.68-8.60 (m, 1H), 8.58-8.50 (m, 1H), 8.32-8.24 (m, 1H), 8.13-8.02 (m, 2H), 8.02-7.94 (m, 1H), 7.82-7.75 (m, 1H), 7.31 (s, 1H), 7.26-7.13 (m, 4H), 6.99 (s, 1H), 6.55-6.48 (m, 1H), 5.60-5.50 (m, 1H), 5.41 (s, 2H), 5.35-5.15 (m, 3H), 4.78-4.68 (m, 1H), 4.60-4.40 (m, 2H), 3.76-3.58 (m, 4H), 3.58-3.48 (m, 1H), 3.20-3.10 (m, 2H), 3.08-2.97 (m, 2H), 2.80-2.72 (m, 2H), 2.45-2.30 (m, 3H), 2.25-2.13 (m, 2H), 2.13-2.04 (m, 2H), 2.03-1.94 (m, 2H), 1.91-1.78 (m, 2H), 1.52-1.39 (m, 3H), 1.34-1.12 (m, 5H), 0.91-0.79 (m, 3H), 0.53-0.34 (m, 4H).

[00182] The methods of preparation of other intermediates were with reference to those of intermediate 5. Petition 870260061229, dated 06 / 22 / 2026, page 117 / 414 110 / 118

[00183] A PBS-buffered aqueous solution of the antibody hu2F7 (pH = 6.5, 0.05 M PBS-buffered aqueous solution; 7.3 mL, 13.8 mg / mL, 0.681 μmol) was added to a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.347 mL, 3.47 pmol) at 37 °C. The reaction mixture was placed in a stirrer in a water bath and reacted under stirring at 37 °C for 3 hours. The reaction was terminated, and the reaction solution was cooled to 25 °C in a water bath, diluted to 14.0 mL, and 3.3 mL of the solution were retained for the subsequent reaction.

[00184] Compound 5-A (3.0 mg, 3.72 μmol) was dissolved in 0.15 mL of DMSO and added to the 3.3 mL solution above. The reaction mixture was placed in a stirrer in a water bath and reacted under stirring at 25 °C for 3 hours. The reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: pH 6.5 aqueous solution buffered by 0.05 M PBS, containing 0.001 M EDTA) to obtain an exemplary product of Ab-Exatecan, hu2F7-Exatecan (Compound 34) in PBS Buffer (1.35 mg / mL, 13 mL), which was stored at 4 °C. The average y value was determined by the ultraviolet method. Cuvettes filled with sodium succinate buffer were placed in the reference absorption cell and the sample determination absorption cell, respectively. After removing the blank solvent, cuvettes filled with the test solution were placed in the sample determination absorption cell. Absorbance at 280 nm and 370 nm was measured. Processing Data:

[00185] The Cmab antibody content was determined by establishing a standard curve and measuring absorption at a wavelength of 280 nm. The small molecule CFdrug content was determined by measuring absorption at a wavelength of 370 nm. Average drug loading y = Cdrug / Cmab Petition 870260061229, dated 06 / 22 / 2026, page 118 / 414 111 / 118

[00186] As for the exemplary product hu2F7-Exatecan (compound 34), y was determined to be 7.6 by the above method. Samples of hu2F7-Exatecan (y = 8) were obtained by UV-HPLC purification.

[00187] The methods of preparation of other antibody conjugates were with reference to those of compound 34. Example 10: The killing activity of human antibody conjugates on tumor cells

[00188] In order to test the killing effect of the antibody-drug conjugates of the present invention on tumor cells, MX-1 breast cancer cells were used for evaluation. MX-1 cells were collected, centrifuged, and counted. The cell density was adjusted to 0.44 χ106 cells / mL with complete medium, and the cells were seeded into 60 wells of a 96-well white plate at 90 μL per well with a cell count of 40,000. The remaining peripheral wells were filled with 100 μL of PBS. The cell plate was placed in an incubator at 37 °C, 5% Cu2, and cultured overnight. On the second day of the experiment, the antibody-drug conjugate was prepared with PBS in a 96-well V-bottom plate, starting from a concentration of 1000 nM (3-fold dilution and 9 concentrations). After preparation was complete, the solution was added to a 96-well blank plate at 10 μL per well in duplicate.The cell plate was placed in an incubator at 37 °C with 5% CO2, and the culture lasted 72 hours. On the fifth day of the experiment, the plate was detected and read. The cell culture plate was removed. Two control wells were set up. 100 μL of medium containing 40,000 cells were added to each well, and after equilibration to room temperature, 50 μL of CTG solution (Promega G7573) were added to each well. The mixture was stirred and mixed, placed in the dark and left to stand for 10 minutes, and then detected using the luminescence program. Petition 870260061229, dated 06 / 22 / 2026, page 119 / 414 112 / 118 microplate reader. Maximum death rate = (1000 nM cavity fluorescence value / control cavity fluorescence value)%. Experimental results are shown in Table 4: Table 4: Evaluation of the killing activity of antibody-drug conjugates on tumor cells. Antibody-drug conjugate IC50 (nM) Kill effect at maximum dose IgG-MC-MMAF (control) ND ND hu2G6-MC-MMAF (compound 1, y=4) 1.1 48.2% hu2F7-MC-MMAF (compound 2, y=4) 3.4 53.9% hu2F7-SN-38 (compound 6, y=4) 78.85 56.4% hu2F7-Exatecan (compound 34, y=8) 80.28 60.39% ND: No detectable activity. Example 11: The inhibitory effect of antibody-drug conjugates on the growth of tumor cells.

[00189] In order to test the killing effect of the antibody-drug conjugates of the present invention on tumor cells, SK-BR-3 breast cancer cells (ATCC # HTB30) were used for evaluation. SK-BR-3 cells were collected, centrifuged, and counted. The cell density was adjusted to 0.44*10⁶ cells / mL with complete medium, and the cells were seeded into 60 wells of a 96-well white plate at 90 μL per well with a cell count of 40,000. The remaining peripheral wells were filled with 100 μL of PBS. The cell plate was placed in an incubator at 37°C, 5% CO₂, and cultured overnight. On the second day of the experiment, the antibody-drug conjugate was prepared with PBS in a 96-well V-bottom plate, starting from a concentration of 1000 nM (3-fold dilution and 9 concentrations). After preparation was complete, the solution was added to a 96-well blank plate. Petition 870260061229, dated 06 / 22 / 2026, page 120 / 414 113 / 118 cells were added to 10 μL per well in duplicate. Two other control wells were prepared and 10 μL of PBS was added to each well. The cell plate was placed in an incubator at 37 °C with 5% CO2 and the culture lasted 72 hours. On the fifth day of the experiment, the plate was detected and read. The cell culture plate was removed. After equilibration to room temperature, 50 μL of CTG solution (Promega G7573) was added to each well. The mixture was stirred and mixed, placed in the dark and left to stand for 10 minutes, and then detected using the microplate reader's luminescence program. Maximum inhibition rate = (1-fluorescence value of 1000 nM well / fluorescence value of control well)%. The experimental results are shown in Table 5: Table 5: Evaluation of the inhibitory activity of antibody-drug conjugates on tumor cells Antibody-drug conjugate IC50 (nM) Maximum inhibition rate IgG-MC-MMAF (control) ND ND hu2G6-MC-MMAF (compound 1, y=4) 1.0 90.3% hu2F7-MC-MMAF (compound 2, y=4) 9.5 91.8% ND: No detectable activity. Example 12: Evaluation of the in vivo efficacy of antibody-drug conjugates conjugated to MMAF.

[00190] After the formation of transplanted tumors with MX-1 cells in mice, the antitumor effect of the antibody-drug conjugate of the present invention was evaluated. 5x10⁶ MX-1 cells were injected subcutaneously into immunodeficient naked hairless mice (BALB / c naked mice). After 2 weeks, intravenous injection of the antibody-drug conjugate hu2G6-MCMMAF and hu2F7-MC-MMAF was performed once a week at a dose of 1.5 mg / kg or 3 mg / kg. Human IgG1 protein was used. Petition 870260061229, dated 06 / 22 / 2026, page 121 / 414 114 / 118 as a control at a dose of 3 mg / kg. There were 5 mice in each group of the control group or the administration group. The tumor inhibition rate was calculated by measuring the tumor volume. Tumor inhibition rate = 100% - (tumor volume of the administration group on day 21 - tumor volume of the administration group on day 22) 0) / (tumor volume of the control group on day 21 - tumor volume of the control group on day 0). The experimental results are shown in Figure 1 and Table 6. Both hu2G6-MC-MMAF and hu2F7-MC-MMAF show a dose-dependent antitumor effect. Both hu2G6-MC-MMAF and hu2F7-MC-MMAF show a tumor inhibition rate of more than 100% at a dose of 3 mg / kg, which means that the antibody-drug conjugates of the present invention can not only inhibit tumor growth but also exert a scavenging effect on already formed tumors. Table 6. Efficacy of administered compounds on MX-1 tumor transplanted in naked mice carrying tumor. Administration Group | Tumor Inhibition Rate | hu2G6-MC-MMAF (compound 1, y=4) | 1.5 mg / kg | 38.0% | hu2G6-MC-MMAF (compound 1, y=4) | 3 mg / kg | 115.6% | hu2F7-MC-MMAF (compound 2, y=4) | 1.5 mg / kg | 90.3% | hu2F7-MC-MMAF (compound 2, y=4) | 3 mg / kg | 163.8% Example 13: Evaluation of the in vivo efficacy of antibody-drug conjugates with different drug loadings.

[00191] In order to also study antibody-drug conjugates with different drug loadings, antibody-drug conjugates were prepared by the method of Example 7 and purified by HPLC to obtain hu2F7-MC-MMAF (y = 2) and hu2F7-MC-MMAF (y = 4) (Figure 2). After the formation of MX-1 transplanted tumors in mice, the antitumor effect of the antibody-drug conjugates of the present invention was evaluated. 5x106 MX-1 cells were injected Petition 870260061229, dated 06 / 22 / 2026, p. 122 / 414 115 / 118 subcutaneously in immunodeficient nude mice. After 2 weeks, intravenous injection of the antibody-drug conjugates hu2F7-MC-MMAF (y = 2) and hu2F7-MC-MMAF (y = 4) was performed once a week at a dose of 3 mg / kg. Human IgG1 protein was used as a control at a dose of 3 mg / kg. There were 5 mice in each group, either the control group or the administration group. The tumor inhibition rate was calculated by measuring the tumor volume. Tumor inhibition rate = 100% - (tumor volume of the administration group on day 21 - tumor volume of the administration group on day 0) / (tumor volume of the control group on day 21 - tumor volume of the control group on day 0). The experimental results are shown in Figure 3 and Table 7. Both hu2F7-MC-MMAF (y = 2) and hu2F7-MC-MMAF (y = 4) show an antitumor effect.hu2F7-MC-MMAF (y = 4) has a stronger antitumor effect than hu2F7-MC-MMAF (y = 2) at a dose of 3 mg / kg, showing a tumor inhibition rate of more than 100%, which means that hu2F7-MC-MMAF (y = 4) can not only inhibit tumor growth but also exert a scavenging effect on already formed tumors (Figure 4). Table 7. Efficacy of antibody-drug conjugates with different drug loadings on MX-1 transplanted tumor in tumor-bearing nude mice. Administration group | Tumor inhibition rate | hu2F7-MC-MMAF (y=2) 3 mg / kg | 87.99% | hu2F7-MC-MMAF (y=4) 3 mg / kg | 197.87% Example 14: Evaluation of the in vivo efficacy of antibody-drug conjugates conjugated to Exatecan.

[00192] To study antibody-drug conjugates with Exatecan, antibody-drug conjugates were prepared by the method of Example 9 and purified by HPLC to obtain hu2F7-Exatecan (compound 34, y = 8). After the formation of MX-1 transplanted tumors in mice, the anti- Petition 870260061229, dated 06 / 22 / 2026, page 123 / 414 116 / 118 tumor inhibition of the antibody-drug conjugates of the present invention. MX-1 5 x 10⁶ cells were injected subcutaneously into immunodeficient nude mice. After 2 weeks, intravenous injection of the antibody-drug conjugate hu2F7-Exatecan (y = 8) was performed, at a frequency of once / week and at a dose of 5 mg / kg and 10 mg / kg. Human IgG1 protein was used as a control at a dose of 5 mg / kg. There were 5 mice in each group, either the control group or the administration group. The tumor inhibition rate was calculated by measuring the tumor volume. Tumor inhibition rate = 100% - (tumor volume of the administration group on day 18 - tumor volume of the administration group on day 0) / (tumor volume of the control group on day 18 - tumor volume of the control group on day 0). The experimental results are shown in Table 8.hu2F7-Exatecan shows a tumor inhibition rate of over 100% at doses of 5 mg / kg and 10 mg / kg, which means that hu2F7-Exatecan (y = 8) can not only inhibit tumor growth but also exert a scavenging effect on already formed tumors. Table 8. Efficacy of antibody-drug conjugates to Exatecan on MX-1 transplanted tumor in tumor-bearing nude mice. Administration group | Tumor inhibition rate | hu2F7-Exatecan (compound 34, y=8) 5 mg / kg | 173.55% | hu2F7-Exatecan (compound 34, y=8) 10 mg / kg | 183.48% Example 15: Spectator death activity experiment of antibody-drug conjugates conjugated to Exatecan

[00193] In order to study the effect of antibody-drug conjugates conjugated to Exatecan of the present invention on the death of B7-H4 negative cells through the bystander effect, the killing activity of antibody-drug conjugates in a mi cell line Petition 870260061229, dated 06 / 22 / 2026, p. 124 / 414 117 / 118 eloma with negative B7-H4 expression was detected using the ONE-Glo™ Luciferase Assay detection kit. MX-1 cells were collected, centrifuged, counted, adjusted to a cell suspension of 7.5χ10⁶ cells / mL with complete medium, and seeded in a 96-well white plate. 33 μL of MX-1 cell suspension were added to each well in the mixed cell group, and 33 μL of PBS were added to each well in the single cell group. NCI-H929-LUC tumor cells with negative B7-H4 expression (Cobioer catalog number CBP30061L) were collected. Cell density was adjusted to 1.5χ10⁶ cells / mL. 33 μL of NCI-H929-LUC cell suspension were added to each well in both the mixed cell group and the single cell group. The cell plate was placed in an incubator at 37 °C, 5% CO2 for one overnight.On the second day of the experiment, the highest concentration of the hu2F7-Exatecan antibody-drug conjugate (compound 34, y = 8) to be tested was uniformly adjusted to 3000 nM and diluted to obtain a 5-fold gradient of 9 doses. 33.3 μL of the working solution of the diluted drug-antibody conjugate was added to each well of the experimental plate and mixed gently. The cell plate was placed in an incubator at 37°C, 5%, and incubated for 48 hours. The cell culture plate was seized and equilibrated to room temperature. Then, 100 μL of ONE-Glo™ Luciferase reagent (Promega E6120) was added to each well. The cells were agitated and lysed at room temperature in the dark for 10 minutes. The cell lysate was centrifuged at 1000 rpm / min for 1 minute, and then transferred to a 96-well transparent-bottom plate at 180 μL per well. The plate was read at a wavelength of 490 nm using a microplate reader.The data were analyzed using Graphpad Prism software, and the experimental results are shown in Table 9. Petition 870260061229, dated 06 / 22 / 2026, page 125 / 414 118 / 118 ONE-Glo™ Luciferase reagent can specifically detect the viability of NCI-H929-LUC cells. hu2F7-Exatecan effectively kills NCI-H929-LUC cells in the mixed cell group, indicating that in the presence of B7-H4 positive cells, hu2F7-Exatecan has a bystander killing effect, and can not only kill B7-H4 positive cells, but can also kill insensitive B7-H4 negative cells (in the single cell group). Table 9. Evaluation of death activity by bystander. Experimental group IC50 (nM) Maximum inhibition rate Mixed cell group (MX-1:NCI-LUC=5:1) 70.34 76.8% Single cell group (NCI-H929-LUC) ND ND ND: No detectable activity Petition 870260061229, dated 06 / 22 / 2026, page 126 / 414

Claims

1. Antibody-drug conjugate, characterized in that it is represented by the general formula (A) or a pharmaceutically acceptable salt or solvate thereof, Ab-(L2-L1-D)y (A) wherein: D is a cytotoxic drug; Li and L2 are linker units; y is a number from 1 to 20; Ab is a B7-H4 antibody or antigen-binding fragment thereof, comprising a variable antibody light chain region and a variable antibody heavy chain region, the variable antibody heavy chain region comprising at least one HCDR, as shown in the selected sequence from the group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, the variable antibody light chain region comprising at least one LCDR, as shown in the selected sequence from the group consisting of: SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:

34.

2. Antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof, according to claim 1, characterized in that the variable heavy chain region of the antibody comprises the CDRs of any one selected from the group consisting of the following (1) to (4): (1) HCDR1, as shown in SEQ ID NO:3, HCDR2, as shown in SEQ ID NO:4 and HCDR3, as shown in SEQ ID NO:5; (2) HCDR1, as shown in SEQ ID NO:9, HCDR2, as shown in SEQ ID NO:10 and HCDR3, as shown in SEQ ID NO:11; (3) HCDR1, as shown in SEQ ID NO:23, HCDR2, as shown in SEQ ID NO:24 and HCDR3, as shown in SEQ ID NO:25; or, (4) HCDR1, as shown in SEQ ID NO:29, HCDR2, as shown in SEQ ID NO:30 and HCDR3, as shown in SEQ ID NO:31; or wherein the antibody light chain variable region of Ab comprises the CDRs of any one selected from the group consisting of the following (1) to (4): (1) LCDR1, as shown in SEQ ID NO:6, LCDR2, as shown in SEQ ID NO:7 and LCDR3, as shown in SEQ ID NO:8; (2) LCDR1, as shown in SEQ ID NO:12, LCDR2, as shown in SEQ ID NO:13 and LCDR3, as shown in SEQ ID NO:14; (3) LCDR1, as shown in SEQ ID NO:26, LCDR2, as shown in SEQ ID NO:27 and LCDR3, as shown in SEQ ID NO:28; or, (4) LCDR1, as shown in SEQ ID NO:32, LCDR2, as shown in SEQ ID NO:33 and LCDR3,as shown in SEQ ID NO:34; or wherein the antibody heavy chain variable region and antibody light chain variable region of Ab comprise the CDRs of any one selected from the group consisting of the following (1) to (4): (1) HCDR1, as shown in SEQ ID NO:3, HCDR2, as shown in SEQ ID NO:4 and HCDR3, as shown in SEQ ID NO:5; and LCDR1, as shown in SEQ ID NO:6, LCDR2, as shown in SEQ ID NO:7 and LCDR3, as shown in SEQ ID NO:8; (2) HCDR1, as shown in SEQ ID NO:9, HCDR2, as shown in SEQ ID NO:10 and HCDR3, as shown in SEQ ID NO:11; and LCDR1, as shown in SEQ ID NO:12, LCDR2, as shown in SEQ ID NO:13 and LCDR3, as shown in SEQ ID NO:14; (3) HCDR1, as shown in SEQ ID NO:23, HCDR2, as shown in SEQ ID NO:24 and HCDR3, as shown in SEQ ID NO:25; and LCDR1, as shown in SEQ ID NO:26, LCDR2, as shown in SEQ ID NO:27 and LCDR3, as shown in SEQ ID NO:28; or,(4) HCDR1, as shown in SEQ ID NO:29, HCDR2, as shown in SEQ ID NO:30 and HCDR3, as shown in SEQ ID NO:31; and LCDR1, as shown in SEQ ID NO:32, LCDR2, as shown in SEQ ID NO:33 and LCDR3, as shown in SEQ ID NO:34; or wherein the Ab is a murine antibody or fragment thereof, a chimeric antibody or fragment thereof, a human antibody or fragment thereof, and a humanized antibody or fragment thereof; or wherein the Ab also comprises any Petition 870260061229, dated 06 / 22 / 2026, p. 129 / 414 4 / 57 one of (a), (b) and (c) or a combination thereof: (a) light chain structural regions and heavy chain structural regions derived from human germline light chain and heavy chain sequences or mutant sequence(s) thereof; (b) heavy chain constant regions derived from human IgG1 or a variant thereof, IgG2 or a variant thereof, IgG3 or a variant thereof, or IgG4 or a variant thereof,(a) heavy chain constant regions derived from human IgG1, IgG2 or IgG4, more preferably heavy chain constant regions of IgG1 with enhanced ADCC toxicity after amino acid mutation, most preferably the heavy chain constant region as shown in SEQ ID NO: 54; (b) light chain constant region(s) derived from human κ chain, λ chain or variant thereof, preferably light chain constant region(s) derived from human κ chain, more preferably the light chain constant region as shown in SEQ ID NO: 55; or wherein Ab comprises variable light chain region(s) of the following sequences: SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 36, SEQ ID NO: 38, or variable light chain region(s) with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology to SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 36, SEQ ID NO: 38; or wherein Ab comprises variable heavy chain region(s) of the following sequences: SEQ ID NO: 15,SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 37, or region(s) heavy chain variables with at least 70%, 75%, 80%, 85%, 90%, 95% or 99% homology to SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 35, SEQ ID NO: 37; or wherein the Ab light chain is selected from the group that Petition 870260061229, dated 06 / 22 / 2026, p. 130 / 414 5 / 57 consists of the following sequences: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 40, SEQ ID NO: 42, or a full-length light chain with at least 80%, 85%, 90%, 95% or 99% homology to SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 40, SEQ ID NO: 42; or where the Ab heavy chain is selected from the group consisting of the following sequences: SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 39, SEQ ID NO: 41, or the full-length heavy chain with at least 80%, 85%, 90%, 95% or 99% homology to SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 39,SEQ ID NO: 41; or wherein the variable heavy chain region and variable light chain region of Ab is any one selected from the group consisting of the following: (1) the variable heavy chain region as shown in SEQ ID NO: 15 and the variable light chain region as shown in SEQ ID NO: 16; (2) the variable heavy chain region as shown in SEQ ID NO: 17 and the variable light chain region as shown in SEQ ID NO: 18; (3) the variable heavy chain region as shown in SEQ ID NO: 35 and the variable light chain region as shown in SEQ ID NO: 36; or, (4) the variable heavy chain region as shown in SEQ ID NO: 37 and the variable light chain region as shown in SEQ ID NO: 38; or wherein Ab is any one selected from the group consisting of the following: (1) the light chain, as shown in SEQ ID NO: 20 and the heavy chain, as shown in SEQ ID NO: 19; (2) the light chain, as shown in SEQ ID NO: 22 and Petition 870260061229, dated 22 / 06 / 2026,pg. 131 / 414 6 / 57 heavy chain, as shown in SEQ ID NO: 21; (3) the light chain, as shown in SEQ ID NO: 40 and the heavy chain, as shown in SEQ ID NO: 39; or, (4) the light chain, as shown in SEQ ID NO: 42 and the heavy chain, as shown in SEQ ID NO: 41; or wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, single-chain antibody, dimerized V region, disulfide-linked stabilized V region and antigen-binding fragments of a peptide comprising CDRs; or wherein the cytotoxic drug is selected from the group consisting of toxin, chemotherapeutic, antibiotic, radioisotope and nucleolytic enzyme; or in which the cytotoxic drug is selected from the group consisting of tubulin inhibitors or DNA topoisomerase inhibitors that inhibit cell division; preferably DM1, DM3, DM4, SN-38, MMAF or MMAE; more preferably the tubulin inhibitor SN-38,MMAE or MMAF.

3. Antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof, according to any one of claims 1 and 2, characterized in that the cytotoxic drug is selected from camptothecin derivatives, preferably Exatecan:

4. Antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof, according to any one of claims 1 to 2, characterized in that the cytotoxic drug is selected from the group consisting of: MMAF 5. Antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof, according to claim 1, characterized in that it is as shown in general formula (I), (I) wherein: Li and L2 are linking units; y is a number selected from 1 to 8, preferably a number selected from 2 to 4; Ab is the B7-H4 antibody or antigen-binding fragment thereof,according to any one of claims 1 to 2.

6. Antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof, according to claim 1, characterized in that it is as shown in general formula (II): Petition 870260061229, dated 22 / 06 / 2026, page 133 / 414 8 / 57 where: Li and L2 are linking units; y is a number selected from 1 to 8, preferably a number selected from 2 to 4; Ab is the B7-H4 antibody or antigen-binding fragment thereof, according to any one of claims 1 to 2.

7. Antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof, according to claim 1, characterized in that it is as shown in general formula (III): L1 and L2 are linking units; y is a number selected from 1 to 10, preferably a number selected from 2 to 8, more preferably a number from 4 to 8, also preferably a number from 6 to 8,and more preferably 8; Ab is the antibody B7-H4 or antigen-binding fragment thereof, according to any one of claims 1 to 12.

8. Antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof, according to any one of claims 5 to 7, characterized in that L1 is as shown in the general formula (B): oo (B) wherein: Petition 870260061229, dated 22 / 06 / 2026, p. 134 / 414 9 / 57 Mi is -CR1R2-; Ri and R2 are the same or different, and are independently selected from the group consisting of hydrogen, alkyl, halogen, hydroxyl and amino; n is an integer from 0 to 5, preferably 1, 2 or 3.

9. Antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof, according to any one of claims 5 to 7, characterized in that L2 is as shown in the general formula (C): wherein: M2 is -CR4R5-; R3 is selected from the group consisting of hydrogen, halogen, hydroxyl, amino, alkyl,alkoxyl and cycloalkyl: R4 and R5 are the same or different, and are independently selected from the group consisting of hydrogen, alkyl, halogen, hydroxyl and amino; m is an integer from 0 to 5, preferably 1, 2 or 3.

10. Antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof, according to any one of claims 5 to 7, characterized in that L2 is as shown in the general formula (D): -K1-K2-K3-K4- (D) wherein: .o K1 is the , s is an integer from 2 to 8; K2 is selected from the group consisting of Petition 870260061229, dated 22 / 06 / 2026, page. 135 / 414 10 / 57 -NR1(CH2CH2O)pCH2CH2C(O)-, -NR1(CH2CH2O)pCH2C(O)-, -S(CH2)pC(O)- and single linkage, p is an integer from 1 to 20, preferably 1 to 6; R1 is selected from the group consisting of hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl and hydroxyalkyl; K3 is a tetrapeptide residue, preferably,The tetrapeptide residue is a peptide residue formed from amino acids selected from the group consisting of two or more of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamate, and aspartate; more preferably the tetrapeptide residue GGFG; K4 is -NR2(CR3R4)t-, R2, R3, or R4 are each independently hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl, and hydroxyalkyl, et is 1 or 2.

11. Antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof, according to claim 10, characterized in that the terminal K1 of the ligand unit -L2- is linked to Ab, and the terminal K4 is linked to L1.

12. Antibody-drug conjugate or pharmaceutically acceptable salt or solvate thereof, according to any one of claims 5 to 7, characterized in that: L1 is selected from the group consisting of -O-(CRaRb)m-CR5R6-C(O)-, -O-CR5R6-(CRaRb)m-, -O-CR5R6-,-NH-(CRaRb)m-CR5R6-C(O)- and -S-(CRaRb)m-CR5R6-C(O)-; Ra and Rb are each independently selected from the group consisting of hydrogen, deuterium, halogen and alkyl; R5 is haloalkyl or cycloalkyl; R6 is selected from the group consisting of hydrogen, haloalkyl and cycloalkyl; or, R5 and R6 and the carbon atom to which they are attached, Petition 870260061229, 2026-06-22, p. 136 / 414 11 / 57 form a cycloalkyl; m is 0, 1, 2, 3 or 4.

13. Antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof, according to claim 12, characterized in that the terminal of L1 is linked to the L2 ligand unit.

14. Antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof, according to claim 12, characterized in that L1 is as shown in the general formula (E): r5R6 (E), R5 is selected from the group consisting of haloalkyl and cycloalkyl, R6 is selected from the group consisting of hydrogen,haloalkyl and cycloalkyl, or, R5 and R6 and the carbon atom to which they are attached form a cycloalkyl; preferably, R5 is selected from the group consisting of C1-6 haloalkyl and C3-6 cycloalkyl, R6 is selected from the group consisting of hydrogen, C1-6 haloalkyl and C3-6 cycloalkyl, or, R5 and R6 and the carbon atom to which they are attached form a C3-6 cycloalkyl; m is an integer from 0 to 4; more preferably, general formula (E) is selected from the group consisting of the following substituents: Petition 870260061229, dated 22 / 06 / 2026, page. 137 / 414 12 / 57 oe ο 15. Antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof, according to any one of claims 1 to 14, characterized in that the -L2-L1- is as shown in the following structure: K2 is a linkage; K3 is the tetrapeptide residue GGFG; R5 is selected from the group consisting of haloalkyl and C3-6 cycloalkyl; R6 is selected from the group consisting of hydrogen,haloalkyl and C3-6 cycloalkyl; or, R5 and R6 and the carbon atom to which they are attached form a C3-6 cycloalkyl; R2, R3 or R4 are each independently hydrogen or alkyl; s is an integer from 2 to 8; m is an integer from 0 to 4; preferably, -L2-L1- is selected from the group consisting of the following structures: Petition 870260061229, 22 / 06 / 2026, p. 138 / 414 13 / 57 Petition 870260061229, 22 / 06 / 2026, p. 139 / 414 14 / 57 16. Antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof, according to any one of claims 1 to 2, characterized in that the antibody-drug conjugate is as shown in the general formula (IV): wherein: W is selected from the group consisting of C1-8 alkyl, C1-8 alkyl-cycloalkyl and linear heteroalkyl of 1 to 8 atoms, the heteroalkyl comprises 1 to 3 heteroatom(s) selected from the group consisting of N, O and S, where C1-8 alkyl,Linear cycloalkyl and heteroalkyl are optionally also substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxyl and cycloalkyl; K2 is selected from the group consisting of -NR1(CH2CH2O)p1CH2CH2C(O)-, -NR1(CH2CH2O)p1CH2C(O)-, -S(CH2)piC(O)- or linkage, R1 is selected from the group consisting of hydrogen atom, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl, and pi is an integer from 1 to 20; K3 is a peptide residue consisting of 2 to 7 amino acids, the amino acids may be substituted or unsubstituted; If substituted, the substituents may be substituted at any available bonding point, and the substituents are one or more independently selected from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxyl. Petition 870260061229, dated 22 / 06 / 2026.pg. 140 / 414 15 / 57 and cycloalkyl; R2 is independently selected from the group consisting of hydrogen atom, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl; R3 and R4 are each independently selected from the group consisting of hydrogen atom, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl; R5 is selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; R6 is selected from the group consisting of hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; or, R5 and R6 and the carbon atom to which they are attached form a cycloalkyl or heterocyclyl; m is an integer from 0 to 4; y is 1 to 10,y is a decimal or an integer; Ab is an anti-B7-H4 antibody or antigen-binding fragment thereof.

17. Antibody-drug conjugate of general formula (I) or the pharmaceutically acceptable salt or solvate thereof, according to claim 5, characterized in that it is as shown in general formula (IA): (IA).

18. Antibody-drug conjugate of general formula (I) or the pharmaceutically acceptable salt or solvate thereof, according to claim 5, characterized in that it is as shown in general formula (IB): Petition 870260061229, dated 22 / 06 / 2026, p. 141 / 414 16 / 57 19. Antibody-drug conjugate of general formula (II) or the pharmaceutically acceptable salt or solvate thereof, according to claim 6, characterized in that it is as shown in general formula (II-A):

20. Antibody-drug conjugate of general formula (II) or the pharmaceutically acceptable salt or solvate thereof, according to claim 6,Antibody-drug conjugate characterized in that it is as shown in the general formula (II-B):

21. Antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof, according to claim 16, antibody-drug conjugate characterized in that it is as shown in the general formula (IV-A): Petition 870260061229, dated 22 / 06 / 2026, p. 142 / 414 17 / 57 preferably, the antibody-drug conjugate is shown as follows: Petition 870260061229, dated 22 / 06 / 2026, p. 143 / 414 18 / 57 Petition 870260061229, dated 22 / 06 / 2026, p. 144 / 414 19 / 57 y or 22. Antibody-drug conjugate of general formula (A) or the pharmaceutically acceptable salt or solvate thereof, according to any one of claims 1 to 21, characterized in that it is selected from the group consisting of the following compounds: Petition 870260061229, dated 22 / 06 / 2026, p. 145 / 414 20 / 57 (Compound 2) (Compound 3) y (Compound 4) (Compound 5) Petition 870260061229, dated 22 / 06 / 2026,p. 146 / 414 21 / 57 (Compound 8) Petition 870260061229, dated 06 / 22 / 2026, p. 147 / 414 22 / 57 (Compound 12) Petition 870260061229, dated 06 / 22 / 2026, p. 148 / 414 23 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 149 / 414 24 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 150 / 414 25 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 151 / 414 26 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 152 / 414 27 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 153 / 414 28 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 154 / 414 29 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 155 / 414 30 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 156 / 414 31 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 157 / 414 32 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 158 / 414 33 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 159 / 414 34 / 57 (Compound 58) (Compound 60) Petition 870260061229, dated 06 / 22 / 2026, p. 160 / 414 35 / 57 Η2νΛο (Compound 61) -1 y (Compound 62) η2νΛ0 (Compound 63) (Compound 64) — y Petition 870260061229, of 06 / 22 / 2026,Page 161 / 414 36 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 162 / 414 37 / 57 (Compound 72) Petition 870260061229, dated 06 / 22 / 2026, p. 163 / 414 38 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 164 / 414 39 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 165 / 414 40 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 166 / 414 41 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 167 / 414 42 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 168 / 414 43 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 169 / 414 44 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 170 / 414 45 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 171 / 414 46 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 172 / 414 47 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 173 / 414 48 / 57 Petition 870260061229, dated 06 / 22 / 2026, p. 174 / 414 49 / 57 (Compound 122) and (Compound 123) (Compound 124) Petition 870260061229, dated 06 / 22 / 2026, p. 175 / 414 50 / 57 hu1C9 hu2G6 (Compound 132) (Compound 133) Petition 870260061229, dated 06 / 22 / 2026,pg. 176 / 414 51 / 57 hu2F7 hu2F8 (Compound 143) and Petition 870260061229, dated 06 / 22 / 2026, pg. 177 / 414 52 / 57 hu2G6 hu2F7 hu2F8 hu1C9 hu2G6 hu2F7 hu2F8 hu1C9 (Compound 145) (Compound 146) (Compound 147) (Compound 148) (Compound 149) (Compound 150) (Compound 151) (Compound 152) Legend: Compound = compound where y is selected from 2 to 10, preferably 4 to 8, more preferably 6 to 8, also preferably 7 to 8, and most preferably 8.

23. Method for preparing an antibody-drug conjugate of general formula (IV) or the pharmaceutically acceptable salt or solvate thereof, characterized in that it comprises the following step: Petition 870260061229, dated 22 / 06 / 2026, pg. 178 / 414 53 / 57 Ab Since Ab is reduced, it is subjected to the coupling reaction with general formula (F) to obtain the compound of general formula (IV); wherein: Ab is an anti-B7-H4 antibody or antigen-binding fragment thereof; W, K2, K3, R2 to R6, mey are as defined in claim 29.

24. Method,According to claim 23, characterized in that the general formula (F) is a compound of general formula (F-1): either a tautomer, mesomer, racemate, enantiomer, diastereomer or a mixture thereof, or the pharmaceutically acceptable salt thereof, wherein K2, K3, R2 to R6 are as defined in claim 28. Petition 870260061229, dated 22 / 06 / 2026, p. 179 / 414 54 / 57 25. Compound of general formula (F) or general formula (F-1), characterized in that it is selected from the group consisting of: Petition 870260061229, dated 22 / 06 / 2026, p. 180 / 414 55 / 57 Petition 870260061229, dated 06 / 22 / 2026, page 181 / 414 56 / 57 26. Pharmaceutical composition, characterized in that it comprises the antibody-drug conjugate, as defined in any of claims 1 to 22, or the pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable carriers.

27. Use of antibody-drug conjugate or the pharmaceutically acceptable salt or solvate thereof,as defined in any one of claims 1 to 22 and pharmaceutical composition, as defined in claim 26, characterized in that it is for the manufacture of a medicament for the treatment of a disease related to human B7-H4.

28. Use, according to claim 27, characterized in that it is for the manufacture of a medicament for the treatment of a cancer with elevated B7-H4 expression, wherein the cancer is selected from the group consisting of human brain astroblastoma, human pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar sarcoma of soft tissues, astrocytoma, bladder cancer, bone cancer, brain and spinal cord cancer, metastatic brain tumor, breast cancer, carotid body tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe cell carcinoma of the kidney, clear cell carcinoma, colon cancer, colorectal cancer, proliferative round cell tumor of connective tissue, ependymoma,Ewing's sarcoma, extraosseous mucoid chondrosarcoma, fibrogenesis imperfecta of bone, fibrous dysplasia of bone, gallbladder or cholangiocarcinoma, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, Kaposi's sarcoma, renal cancer, leukemia, liposarcoma / malignant lipomatous tumor, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, mi Petition 870260061229, dated 06 / 22 / 2026, page. 182 / 414 57 / 57 multiple cancer, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid adenoma, pediatric cancer, peripheral schwannoma, pheocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, metastatic renal cancer, rhabdoid tumor, rhabdomyosarcoma, sarcoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma,Synovial sarcoma, testicular cancer, thymic cancer, metastatic thyroid cancer, and uterine cancer.

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