Anti-B7-H4 antibody-drug conjugates and their pharmaceutical uses

Abstract

The present invention provides an anti-B7-H4 antibody-drug conjugate and medical uses thereof. Specifically, the present invention provides an anti-B7-H4 antibody or an antigen-binding fragment thereof, a humanized antibody comprising the CDR region of the anti-B7-H4 antibody, and the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, as well as a pharmaceutical composition of the antibody-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, and its use as an anti-cancer drug, particularly in the preparation of a drug for treating a disease or disorder in which B7-H4 is highly expressed.

Description

[Technical field]

[0001] The present invention is in the field of biomedicine. In particular, the present invention relates to an anti-B7-H4 antibody-drug conjugate and its medical use. [Background technology]

[0002] Tumor immunotherapy is a long-lasting hotspot of research and development in the field of tumor therapy, and T cell tumor immunotherapy is in a central position. Tumor escape is a huge barrier to tumor immunotherapy. Most tumors express antigens that can be recognized by the host immune system to a greater or lesser extent. However, in many cases, inefficient activation of effector T cells causes insufficient immune responses, and thus tumor cells promote tumor growth through their inhibitory effect on the immune system. Tumor immunotherapy is to fully utilize and mobilize killer T cells and / or other immune cells in tumor-bearing patients to kill tumors.

[0003] Studies of the CD28 receptor and its ligands have led to the characterization of a group of molecules called the B7 superfamily. Members of the B7 family are a class of immunoglobulins that contain an immunoglobulin-like V domain (IgV) and an immunoglobulin-like C domain (IgC). Its members include the costimulators B7.1 (CD80) and B7.2 (CD86), inducible costimulator 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.

[0004] Human B7-H4 is a type 1 transmembrane protein consisting of 282 amino acids. Its coding gene is located in the p11.1 region of chromosome 1 (Choi IH et al., J Immunol. 2003 Nov 1; 171(9): 4650-4). B7-H4 has a negative regulatory effect on T cell immune responses. B7-H4 plays a broad inhibitory role in CD4+ and CD8+ T cell differentiation and development, cell cycle progression, and cytokine production (Sica GL et al., Immunity. 2003 Jun; 18(6): 849-61). In B7-H4 knockout mice, no immune cell damage or autoimmune phenomena were found (Zhu G et al., Blood. 2009 Feb 19; 113(8): 1759-67; Suh WK et al., Blood. Mol Cell Biol. 2006 Sep; 26(17): 6403-11). At present, the receptor for B7-H4 and its signal transduction pathway remain unknown.

[0005] Recent studies have found that B7-H4 protein is abundantly expressed in various tumor tissues, allowing tumor cells to escape from the attack of the body's immune system. Utilizing the B7-H4 molecule as a target for tumor therapy provides a new method for tumor immunotherapy.

[0006] Human B7-H4 is now known to be expressed on cancer cells, such as breast, ovarian, lung, cervical, renal, bladder, and liver cancers. B7-H4 mRNA expression has been found in spleen, lung, thymus, liver, skeletal muscle, kidney, pancreas, testis, and ovary. At the protein level, low levels of B7-H4 expression have been found in tissues such as breast (ducts and lobules), fallopian tube epithelium, and endometrial glands. 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 that macrophages constitute an important component of the tumor microenvironment and may represent as much as 50% of the tumor mass.

[0007] One strategy for cancer treatment is to use antibodies as carriers to deliver cytotoxic molecules into cancer cells, and then kill the cancer cells with dissociated small molecules. The drugs used in this strategy are called antibody-drug conjugates. Adcetris and Kadcyla are currently marketed antibody-drug conjugates. At present, many multinational pharmaceutical companies are developing monoclonal antibodies against B7-H4 and / or their drug conjugates to improve the patient's own immune system response against tumors to achieve the goal of direct tumor cell killing. Related patents include, for example, WO2013025779, US20140322129, etc. Anti-B7-H4 monoclonal antibodies from Medimmune, FivePrime, and other companies are still in preclinical development, and Genentech's anti-B7-H4 antibody-drug conjugate is also in preclinical development. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2013025779 [Patent Document 2] US Patent Application Publication No. 20140322129 [Patent Document 3] PCT International Patent Publication No. 2005081711 [Non-patent literature]

[0009] [Non-Patent Document 1] Choi IH et al., J Immunol. 2003 Nov 1; 171(9): 4650~4 [Non-Patent Document 2] Sica GL et al., Immunity. 2003 Jun; 18(6): 849~61 [Non-Patent Document 3] Zhu G et al., Blood. 2009 Feb 19; 113(8): 1759~67 [Non-Patent Document 4] Suh WK et al. Blood. Mol Cell Biol. 2006 Sep; 26(17): 6403~11 [Non-Patent Document 5] Kryczek, I. et al., J. Exp. Med. 2006, 203(4): 871~881 [Non-Patent Document 6] J. Biol. Chem, 243, p3558(1968) [Non-Patent Document 7] AbM definition criteria (http: / / bioinf.org.uk / abs / ) [Non-Patent Document 8] Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136 [Non-Patent Document 9] ImmunoGeneTics (IMGT) website: http: / / imgt.cines.fr [Non-Patent Document 10] The Immunoglobulin FactsBook, 2001ISBN012441351 [Non-Patent Document 11] Chinese Pharmacopoeia Summary of the Invention [Problem to be solved by the invention]

[0010] The objective of the present invention is to provide an anti-B7-H4 antibody-drug conjugate with high affinity, high selectivity, high endocytosis efficiency, high anti-cancer activity, high stability, high safety, and low toxicity and side effects, which is achieved by the following technical solutions: [Means for solving the problem]

[0011] General formula (A):

[0012] [ka]

[0013] (In the formula, D is a cytotoxic drug; L 1 and L 2 is a linker unit, y is a number from 1 to 20, Ab is a B7-H4 antibody or an antigen-binding fragment thereof, which comprises an antibody light chain variable region and an antibody heavy chain variable region; The antibody heavy chain variable region is SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and comprising at least one HCDR as set forth in a sequence selected from the group consisting of: The antibody light chain variable region is SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14 (including at least one LCDR as shown in the sequence selected from the group consisting of: or a pharma- ceutically acceptable salt or solvate thereof.

[0014] In one embodiment of the invention, the antibody heavy chain variable region comprises: 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 is SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45 and wherein the sequence is selected from the group consisting of: The antibody light chain variable region is 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 The LCDR comprises at least one LCDR as shown in a sequence selected from the group consisting of:

[0015] In one embodiment of the invention, the anti-B7-H4 antibody, or antigen-binding fragment thereof, comprises an antibody heavy chain variable region, the antibody heavy chain variable region comprising: HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5, Or, HCDR1 shown in SEQ ID NO:9, HCDR2 shown in SEQ ID NO:10, and HCDR3 shown in SEQ ID NO:11 The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, comprising:

[0016] In one embodiment of the invention, the anti-B7-H4 antibody, or antigen-binding fragment thereof, comprises an antibody heavy chain variable region, the antibody heavy chain variable region comprising: HCDR1 shown in SEQ ID NO: 23, HCDR2 shown in SEQ ID NO: 24, and HCDR3 shown in SEQ ID NO: 25; Or, HCDR1 shown in SEQ ID NO:29, HCDR2 shown in SEQ ID NO:30, and HCDR3 shown in SEQ ID NO:31 The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, comprising:

[0017] In one embodiment of the invention, the anti-B7-H4 antibody, or antigen-binding fragment thereof, comprises an antibody heavy chain variable region, the antibody heavy chain variable region comprising: HCDR1 shown in SEQ ID NO: 43, HCDR2 shown in SEQ ID NO: 44, and HCDR3 shown in SEQ ID NO: 25; Or, HCDR1 shown in SEQ ID NO:29, HCDR2 shown in SEQ ID NO:45, and HCDR3 shown in SEQ ID NO:31 The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, comprising:

[0018] In one embodiment of the present invention, the antibody heavy chain variable region of Ab is selected from the following (1) to (4): (1) HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5, (2) HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10, and HCDR3 shown in SEQ ID NO: 11, (3) HCDR1 shown in SEQ ID NO: 23, HCDR2 shown in SEQ ID NO: 24, and HCDR3 shown in SEQ ID NO: 25, or (4) HCDR1 shown in SEQ ID NO: 29, HCDR2 shown in SEQ ID NO: 30, and HCDR3 shown in SEQ ID NO: 31 The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, comprising any one CDR selected from the group consisting of:

[0019] The antibody heavy chain variable region of Ab is as follows: (5) to (6): (5) HCDR1 set forth in SEQ ID NO: 43, HCDR2 set forth in SEQ ID NO: 44, and HCDR3 set forth in SEQ ID NO: 25, Or, (6) HCDR1 shown in SEQ ID NO: 29, HCDR2 shown in SEQ ID NO: 45, and HCDR3 shown in SEQ ID NO: 31 It may also comprise a CDR selected from the group consisting of:

[0020] In one embodiment of the invention, the anti-B7-H4 antibody, or antigen-binding fragment thereof, comprises an antibody light chain variable region, the antibody light chain variable region comprising: 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. The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, comprising:

[0021] In some embodiments of the invention, the anti-B7-H4 antibody, or antigen-binding fragment thereof, comprises an antibody light chain variable region, the antibody light chain variable region comprising: 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. The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, comprising:

[0022] In one embodiment of the present invention, the antibody light chain variable region of Ab is selected from the following (1) to (4): (1) LCDR1 shown in SEQ ID NO: 6, LCDR2 shown in SEQ ID NO: 7, and LCDR3 shown in SEQ ID NO: 8, (2) LCDR1 shown in SEQ ID NO: 12, LCDR2 shown in SEQ ID NO: 13, and LCDR3 shown in SEQ ID NO: 14, (3) LCDR1 shown in SEQ ID NO: 26, LCDR2 shown in SEQ ID NO: 27, and LCDR3 shown in SEQ ID NO: 28, or (4) LCDR1 shown in SEQ ID NO: 32, LCDR2 shown in SEQ ID NO: 33, and LCDR3 shown in SEQ ID NO: 34 The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, comprising any one CDR selected from the group consisting of:

[0023] In one embodiment of the invention, the heavy and light chain variable regions of the anti-B7-H4 antibody, or antigen-binding fragment thereof, comprise: (1) HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5, respectively; and LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7, and LCDR3 shown in SEQ ID NO:8, or (2) HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10, and HCDR3 shown in SEQ ID NO: 11, respectively; and LCDR1 shown in SEQ ID NO:12, LCDR2 shown in SEQ ID NO:13, and LCDR3 shown in SEQ ID NO:14 The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, comprising:

[0024] In one embodiment of the invention, the heavy and light chain variable regions of the anti-B7-H4 antibody or antigen-binding fragment thereof comprise: (3) HCDR1 shown in SEQ ID NO: 23, HCDR2 shown in SEQ ID NO: 24, and HCDR3 shown in SEQ ID NO: 25, and LCDR1 shown in SEQ ID NO:26, LCDR2 shown in SEQ ID NO:27 and LCDR3 shown in SEQ ID NO:28, or (4) HCDR1 set forth in SEQ ID NO: 29, HCDR2 set forth in SEQ ID NO: 30, and HCDR3 set forth in SEQ ID NO: 31, and LCDR1 shown in SEQ ID NO: 32, LCDR2 shown in SEQ ID NO: 33, and LCDR3 shown in SEQ ID NO: 34 The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, comprising:

[0025] In one embodiment of the invention, the heavy and light chain variable regions of the anti-B7-H4 antibody or antigen-binding fragment thereof comprise: (5) HCDR1 shown in SEQ ID NO: 43, HCDR2 shown in SEQ ID NO: 44, and HCDR3 shown in SEQ ID NO: 25, respectively; and LCDR1 shown in SEQ ID NO:26, LCDR2 shown in SEQ ID NO:27 and LCDR3 shown in SEQ ID NO:28, or (6) HCDR1 as set forth in SEQ ID NO: 29, HCDR2 as set forth in SEQ ID NO: 45, and HCDR3 as set forth in SEQ ID NO: 31, respectively; and LCDR1 shown in SEQ ID NO: 32, LCDR2 shown in SEQ ID NO: 33, and LCDR3 shown in SEQ ID NO: 34 The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, comprising:

[0026] In one embodiment of the present invention, the antibody heavy chain variable region and the antibody light chain variable region of Ab are selected from the following (1) to (4): (1) HCDR1 shown in SEQ ID NO: 3, HCDR2 shown in SEQ ID NO: 4, and HCDR3 shown in SEQ ID NO: 5, and LCDR1 shown in SEQ ID NO:6, LCDR2 shown in SEQ ID NO:7, and LCDR3 shown in SEQ ID NO:8, (2) HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10, and HCDR3 shown in SEQ ID NO: 11, and LCDR1 shown in SEQ ID NO: 12, LCDR2 shown in SEQ ID NO: 13, and LCDR3 shown in SEQ ID NO: 14, (3) HCDR1 shown in SEQ ID NO: 23, HCDR2 shown in SEQ ID NO: 24, and HCDR3 shown in SEQ ID NO: 25, and LCDR1 shown in SEQ ID NO:26, LCDR2 shown in SEQ ID NO:27 and LCDR3 shown in SEQ ID NO:28, or (4) HCDR1 set forth in SEQ ID NO: 29, HCDR2 set forth in SEQ ID NO: 30, and HCDR3 set forth in SEQ ID NO: 31, and LCDR1 shown in SEQ ID NO: 32, LCDR2 shown in SEQ ID NO: 33, and LCDR3 shown in SEQ ID NO: 34 The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, comprising any one CDR selected from the group consisting of:

[0027] The antibody heavy chain variable region and the antibody light chain variable region of the Ab are the following (5) or (6): (5) HCDR1 set forth in SEQ ID NO: 43, HCDR2 set forth in SEQ ID NO: 44, and HCDR3 set forth in SEQ ID NO: 25, and LCDR1 shown in SEQ ID NO: 26, LCDR2 shown in SEQ ID NO: 27, and LCDR3 shown in SEQ ID NO: 28, (6) HCDR1 set forth in SEQ ID NO: 29, HCDR2 set forth in SEQ ID NO: 45, and HCDR3 set forth in SEQ ID NO: 31, and LCDR1 shown in SEQ ID NO: 32, LCDR2 shown in SEQ ID NO: 33, and LCDR3 shown in SEQ ID NO: 34 It may also comprise a CDR selected from the group consisting of:

[0028] In a preferred embodiment, the antibody-drug conjugate of the above general formula (A) or a pharma- ceutically acceptable salt or solvate thereof, wherein Ab is a murine antibody or a fragment thereof, a chimeric antibody or a fragment thereof, a human antibody or a fragment thereof, or a humanized antibody or a fragment thereof.

[0029] In a preferred embodiment, the Ab further comprises light chain framework regions and heavy chain framework region sequences derived from human germline light chain and heavy chain sequences, respectively, or mutant sequences thereof, of the antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof.

[0030] In a preferred embodiment, the Ab further comprises a heavy chain constant region, which is a heavy chain constant region 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 a heavy chain constant region derived from human IgG1, IgG2 or IgG4, more preferably a heavy chain constant region of an IgG having enhanced ADCC toxicity after amino acid mutation, most preferably the heavy chain constant region shown in SEQ ID NO: 54, the antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof.

[0031] In a preferred embodiment, the Ab further comprises a light chain constant region derived from a human κ chain, λ chain or a variant thereof, preferably a light chain constant region derived from a human κ chain, more preferably a light chain constant region as shown in SEQ ID NO: 55, the antibody-drug conjugate of the above general formula (A) or a pharma- ceutically acceptable salt or solvate thereof.

[0032] In a preferred embodiment, the antibody-drug conjugate of the above general formula (A) or a pharma- ceutically acceptable salt or solvate thereof, wherein Ab comprises a light chain variable region having the following sequence: SEQ ID NO: 16 or SEQ ID NO: 18.

[0033] In a preferred embodiment, the Ab comprises a light chain variable region having the following sequences: SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:36, SEQ ID NO:38, or a light chain variable region having 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 a pharma- ceutically acceptable salt or solvate thereof.

[0034] In a preferred embodiment, the antibody-drug conjugate of the above general formula (A) or a pharma- ceutically acceptable salt or solvate thereof, wherein Ab comprises a heavy chain variable region having the following sequence: SEQ ID NO: 15 or SEQ ID NO: 17.

[0035] In a preferred embodiment, the Ab comprises a heavy chain variable region having the following sequences: SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:35, SEQ ID NO:37, or a heavy chain variable region having 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 a pharma- ceutically acceptable salt or solvate thereof.

[0036] In a preferred embodiment, the heavy and light chain variable regions of the Ab are: (1) a heavy chain variable region shown in SEQ ID NO: 15 and a light chain variable region shown in SEQ ID NO: 16, (2) a heavy chain variable region shown in SEQ ID NO: 17 and a light chain variable region shown in SEQ ID NO: 18 The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, which is any one selected from the group consisting of:

[0037] In a preferred embodiment, the heavy and light chain variable regions of the Ab are: (1) a heavy chain variable region shown in SEQ ID NO: 15 and a light chain variable region shown in SEQ ID NO: 16, (2) a heavy chain variable region shown in SEQ ID NO: 17 and a light chain variable region shown in SEQ ID NO: 18; (3) a heavy chain variable region as set forth in SEQ ID NO: 35 and a light chain variable region as set forth in SEQ ID NO: 36, or (4) A heavy chain variable region shown in SEQ ID NO: 37 and a light chain variable region shown in SEQ ID NO: 38 The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, which is any one selected from the group consisting of:

[0038] In a preferred embodiment, the antibody-drug conjugate of the above general formula (A) or a pharma- ceutically acceptable salt or solvate thereof, wherein Ab comprises a light chain having the following sequence: SEQ ID NO:20 or SEQ ID NO:22.

[0039] In a preferred embodiment, the Ab comprises a light chain having 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 having 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 a pharma- ceutically acceptable salt or solvate thereof.

[0040] In a preferred embodiment, the antibody-drug conjugate of the above general formula (A) or a pharma- ceutically acceptable salt or solvate thereof, wherein Ab comprises a heavy chain having the following sequence: SEQ ID NO: 19 or SEQ ID NO: 21.

[0041] In a preferred embodiment, the Ab comprises a heavy chain having the following sequences: SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:39, SEQ ID NO:41, or a full-length heavy chain having 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 a pharma- ceutically acceptable salt or solvate thereof.

[0042] In a preferred embodiment, Ab is (1) a light chain of SEQ ID NO: 20 and a heavy chain of SEQ ID NO: 19, or (2) a light chain of SEQ ID NO: 22 and a heavy chain of SEQ ID NO: 21 The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, comprising:

[0043] In a preferred embodiment, Ab is (1) a light chain of SEQ ID NO: 20 and a heavy chain of SEQ ID NO: 19, or (2) a light chain of SEQ ID NO: 22 and a heavy chain of SEQ ID NO: 21, or (3) a light chain of SEQ ID NO: 40 and a heavy chain of SEQ ID NO: 39, or (4) a light chain of SEQ ID NO: 42 and a heavy chain of SEQ ID NO: 41 The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, comprising:

[0044] In a preferred embodiment, the antigen-binding fragment of the anti-B7-H4 antibody is a Fab, Fab', F(ab') 2 an antibody-drug conjugate of the above general formula (A) or a pharma- ceutically acceptable salt or solvate thereof, which is selected from the group consisting of an antigen-binding fragment of a peptide comprising a CDR, a single chain antibody (scFv), a dimerized V region (diabody), a disulfide bond stabilized V region (dsFv), and a CDR-containing peptide.

[0045] In a preferred embodiment, the cytotoxic drug is selected from the group consisting of toxins, chemotherapeutic agents, antibiotics, radioisotopes, and nucleases, preferably tubulin inhibitors or DNA topoisomerase inhibitors that inhibit cell division, more preferably DM1, DM3, DM4, SN-38, MMAF or MMAE, even more preferably the tubulin inhibitors SN-38, MMAE or MMAF, the structure of MMAF and SN-38 being represented by the following formula:

[0046] [ka]

[0047] The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, which is shown in

[0048] In a preferred embodiment, the cytotoxic drug is a camptothecin derivative, preferably exatecan:

[0049] [ka]

[0050] The antibody-drug conjugate of the above general formula (A), or a pharma- ceutically acceptable salt or solvate thereof, selected from:

[0051] In a preferred embodiment, the compound of general formula (I):

[0052] [ka]

[0053] (In the formula, L 1 and L 2 is a linker unit, 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 an antigen-binding fragment thereof as defined above. or a pharma- ceutically acceptable salt or solvate thereof.

[0054] In a preferred embodiment, the compound of the general formula (II):

[0055] [ka]

[0056] (In the formula, L 1 and L 2 is a linker unit, 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 an antigen-binding fragment thereof as defined above. or a pharma- ceutically acceptable salt or solvate thereof.

[0057] In a preferred embodiment, the compound of general formula (III):

[0058] [ka]

[0059] (In the formula, L 1 and L 2 is a linker unit, 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, more preferably a number selected from 6 to 10, even more preferably a number from 7 to 9, and most preferably an integer of 7, 8, or 9; Ab is an anti-B7-H4 antibody or an antigen-binding fragment thereof as defined above. or a pharma- ceutically acceptable salt or solvate thereof.

[0060] In a preferred embodiment, L 1 is represented by the following general formula (B):

[0061] [ka]

[0062] (In the formula, M 1 -CR 1 R 2 - and R 1 and R 2 are the same or different and are independently selected from the group consisting of hydrogen, alkyl, halogen, hydroxyl, and amino; N is an integer of 0 to 5, preferably 1, 2 or 3. or a pharma- ceutically acceptable salt or solvate thereof,

[0063] In a preferred embodiment, L 2 is represented by the following general formula (C):

[0064] [ka]

[0065] (In the formula, M 2 -CR 4 R 5 - and R 3 is selected from the group consisting of a hydrogen atom, halogen, hydroxyl, amino, alkyl, alkoxyl and cycloalkyl; R 4 and R 5 are the same or different and are independently selected from the group consisting of hydrogen, alkyl, halogen, hydroxyl, and amino; m is an integer of 0 to 5, preferably 1, 2 or 3. or a pharma- ceutically acceptable salt or solvate thereof,

[0066] In a preferred embodiment, L 2 is represented by the following general formula (D): -K 1 -K 2 -K 3 -K 4 - (In the formula, K 1 teeth,

[0067] [ka]

[0068] and s is an integer of 2 to 8, more preferably an integer of 4 to 8, and even more preferably an integer of 4 to 6; K 2 -NR 1 (CH 2 CH 2 O) p CH2 CH 2 C(O)-, -NR 1 (CH 2 CH 2 O) p CH 2 C(O)-, -S(CH 2 ) p C(O)- or a single bond, and p is an integer of 1 to 20, preferably 1 to 6; R 1 is selected from the group consisting of hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl, and hydroxyalkyl; K 3 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, glutamic acid, and aspartic acid, more preferably the tetrapeptide residue GGFG; K 4 -NR 2 (CR 3 R 4 )t- and R 2 , R 3 or R 4 are each independently hydrogen, deuterium, hydroxyl, amino, alkyl, halogen, haloalkyl, deuterated alkyl, and hydroxyalkyl, and t is 1 or 2. Preferably, L 2 Regarding K 1 teeth,

[0069] [ka]

[0070] and s is 5; K 2 is a bond, K 3 is the tetrapeptide residue GGFG, K 4 -NR 2 (CR3 R 4 )t- and R 2 , R 3 or R 4 are each independently hydrogen, deuterium, hydroxyl, amino, C 1~6 Alkyl, halogen, C 1~6 Haloalkyl, C 1~6 Deuterated alkyl and C 1~6 hydroxyalkyl, and t is 1 or 2. or a pharma- ceutically acceptable salt or solvate thereof,

[0071] In a preferred embodiment, the linker unit -L 2 -K 1 The end binds to Ab and K 4 The end is L 1 or a pharma- ceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is bound to

[0072] In a preferred embodiment, L 1 But -O-(CR a R b ) m -CR 5 R 6 -C(O)-, -O-CR 5 R 6 -(CR a R b ) m -, -O-CR 5 R 6 -, -NH-(CR a R b ) m -CR 5 R 6 -C(O)- or -S-(CR a R b ) m -CR 5 R 6 -C(O)-, R a and R b are each independently selected from the group consisting of hydrogen, deuterium, halogen, and alkyl; R 5is haloalkyl or cycloalkyl; R 6 is selected from the group consisting of hydrogen, haloalkyl and cycloalkyl; Or R 5 and R 6 and the carbon atom to which they are attached form a cycloalkyl; m is 0, 1, 2, 3 or 4; The above antibody-drug conjugate or a pharma- ceutically acceptable salt or solvate thereof.

[0073] Preferably, L 1 The O-terminus of the linker unit L 2 is bound to.

[0074] In a preferred embodiment, L 1 is represented by the following general formula (E):

[0075] [ka]

[0076] (R 5 is haloalkyl or cycloalkyl; R 6 is selected from the group consisting of hydrogen, haloalkyl and cycloalkyl; Or R 5 and R 6 and the carbon atom to which they are attached form a cycloalkyl; Preferably, R 5 But, C 1~6 Haloalkyl and C 3~6 cycloalkyl; R 6 But hydrogen, C 1~6 selected from the group consisting of haloalkyl and cycloalkyl; Or R 5 and R 6 and the carbon atom to which they are bonded is C 3~6 Forming a cycloalkyl, m is an integer from 0 to 4; More preferably, the general formula (E) contains the following substituents:

[0077] [ka]

[0078] (selected from the group consisting of or a pharma- ceutically acceptable salt or solvate thereof,

[0079] In a more preferred embodiment, -L 2 -L 1 -But the following structure:

[0080] [ka]

[0081] (K 2 is a bond, K 3 is the tetrapeptide residue GGFG, R 5 haloalkyl and C 3~6 is cycloalkyl, R 6 is hydrogen, haloalkyl and C 3~6 cycloalkyl; Or R 5 and R 6 and the carbon atom to which they are bonded is C 3~6 Forming a cycloalkyl, R 2 , R 3 or R 4 are each independently 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, -L 2 -L 1 -But the following structure:

[0082] [ka]

[0083] [ka]

[0084] (selected from the group consisting of or a pharma- ceutically acceptable salt or solvate thereof,

[0085] In a preferred embodiment, the general formula (IV):

[0086] [ka]

[0087] (In the formula, W is for C 1~8 Alkyl, C 1~8 alkyl-cycloalkyl or linear heteroalkyl of 1 to 8 atoms, wherein the heteroalkyl contains 1 to 3 heteroatoms selected from the group consisting of N, O or S, where C 1~8 each alkyl, cycloalkyl, or linear heteroalkyl is independently optionally further substituted with one or more substituents selected from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxyl, and cycloalkyl; K 2 -NR 1 (CH 2 CH 2 O) p1 CH 2 CH 2 C(O)-, -NR 1 (CH 2 CH 2 O) p1 CH 2 C(O)-, -S(CH 2 )p1 C(O)- or a bond; R 1 is selected from the group consisting of a hydrogen atom, an alkyl, a haloalkyl, a deuterated alkyl, and a hydroxyalkyl; 1 is an integer from 1 to 20, K 3 is a peptide residue of 2 to 7 amino acids, which may be substituted or unsubstituted, and if substituted, the substituents may be substituted at any available point of attachment, and the substituents are one or more independently selected from the group consisting of halogen, hydroxyl, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl; R 2 are independently selected from the group consisting of hydrogen, alkyl, haloalkyl, deuterated alkyl, and hydroxyalkyl; R 3 and R 4 are each independently selected from the group consisting of a hydrogen atom, a halogen, an alkyl, a haloalkyl, a deuterated alkyl, and a hydroxyalkyl; R 5 is selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; R 6 is selected from the group consisting of a hydrogen atom, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl; Or R 5 and R 6 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 number or an integer, preferably y is a number from 2 to 10, more preferably y is a number from 4 to 10, even more preferably y is a number from 6 to 9, and most preferably 7, 8, or 9; Ab is an anti-B7-H4 antibody or an antigen-binding fragment thereof. or a pharma- ceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is an antibody-drug conjugate represented by the formula:

[0088] In a more preferred embodiment, the general formula (IA):

[0089] [ka]

[0090] (y is 1 to 10, and y is a decimal or an integer) or a pharma- ceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is an antibody-drug conjugate represented by the formula:

[0091] In a more preferred embodiment, the general formula (IB):

[0092] [ka]

[0093] (y is 1 to 10, and y is a decimal or an integer) or a pharma- ceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is an antibody-drug conjugate represented by the formula:

[0094] In a more preferred embodiment, the general formula (II-A):

[0095] [ka]

[0096] or a pharma- ceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is an antibody-drug conjugate represented by the formula:

[0097] In a more preferred embodiment, the general formula (II-B):

[0098] [ka]

[0099] or a pharma- ceutically acceptable salt or solvate thereof, The above antibody-drug conjugate or a pharma- ceutically acceptable salt or solvate thereof.

[0100] In a more preferred embodiment, the general formula (IV-A):

[0101] [ka]

[0102] (In the formula, s is an integer of 2 to 8; R 2 ~R 6 , m and y are as defined above in general formula (IV), Preferably, s is an integer of 4, 5 or 6, and y is a number from 4 to 10, preferably from 6 to 9, more preferably 7 or 8. or a pharma- ceutically acceptable salt or solvate thereof, wherein the antibody-drug conjugate is an antibody-drug conjugate represented by the formula:

[0103] In a more preferred embodiment, the following compound:

[0104] [ka]

[0105] [ka]

[0106] [ka]

[0107] or a pharma- ceutically acceptable salt or solvate thereof, selected from the group consisting of:

[0108] In the most preferred embodiment, the compound:

[0109] [ka]

[0110] [ka]

[0111] [ka]

[0112] [ka]

[0113] [ka]

[0114] [ka]

[0115] [ka]

[0116] [ka]

[0117]

change

[0118]

change

[0119]

change

[0120]

change

[0121]

change

[0122]

change

[0123]

change

[0124]

change

[0125]

change

[0126]

change

[0127]

change

[0128]

change

[0129]

change

[0130]

change

[0131]

change

[0132]

change

[0133]

change

[0134]

change

[0135]

change

[0136]

change

[0137]

change

[0138]

change

[0139] [ka]

[0140] [ka]

[0141] [ka]

[0142] (In the formula, y is a number selected from 1 to 10, preferably a number selected from 2 to 10, further a number selected from 6 to 10, more preferably a number from 7 to 9, and most preferably an integer of 7, 8, or 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, further preferably 7 or 8, and most preferably the number 8.) or a pharma- ceutically acceptable salt or solvate thereof, selected from the group consisting of:

[0143] In a preferred embodiment, the present invention comprises the steps of:

[0144] [ka]

[0145] (In the formula, Ab is an anti-B7-H4 antibody or an antigen-binding fragment thereof; W.K. 2 , K 3 , R 2 ~R 6 , m and y are as defined in general formula (IV). Including, The present invention relates to a method for preparing an antibody-drug conjugate of general formula (IV) or a pharma- ceutically acceptable salt or solvate thereof, comprising reducing Ab and then subjecting it to a coupling reaction with general formula (F) to obtain a compound of general formula (IV).

[0146] In a preferred embodiment, the general formula (F) is represented by the general formula (F-1):

[0147] [ka]

[0148] (In the formula, K 2 , K 3 , R 2 ~R 6 , s and m are the above-L 2 -L 1 - as defined in or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixed form thereof, or a pharma- ceutically acceptable salt thereof.

[0149] In a preferred embodiment, the compound of formula (F) or formula (F-1) is

[0150] [ka]

[0151] [ka]

[0152] is selected from the group consisting of:

[0153] In a preferred embodiment, the present invention relates to a pharmaceutical composition comprising an antibody-drug conjugate according to any one of the present invention or a pharma- ceutically acceptable salt or solvate thereof, and one or more pharma- ceutically acceptable excipients, diluents or carriers.

[0154] In a preferred embodiment, the present invention relates to the use of an antibody-drug conjugate of general formula (A) or a pharma- ceutically acceptable salt or solvate thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for treating a disease associated with human B7-H4, preferably for treating a cancer that highly expresses B7-H4.

[0155] In a more preferred embodiment, the cancer is selected from the group consisting of human brain astroblastoma, human pharyngeal carcinoma, adrenal tumor, AIDS-related cancer, alveolar soft part sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cancer, metastatic brain tumor, breast cancer, carotid bulb tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe cell carcinoma of the kidney, clear cell carcinoma, colon cancer, colorectal cancer, connective tissue proliferative small round cell tumor, ependymoma, Ewing's sarcoma, extraskeletal myxoid chondrosarcoma, fibrous dysplasia ossificans, fibrous dysplasia ossificans, gallbladder or cholangiocarcinoma, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, islet cell tumor, The cancer is selected from the group consisting of Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma / malignant lipoma, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroid tumor, childhood cancer, peripheral schwannoma, pinealocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, metastatic kidney 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. [Brief description of the drawings]

[0156] [Figure 1] FIG. 1 is a graph showing the in vivo efficacy of antibody-drug conjugates: hu2G6-MC-MMAF and hu2F7-MC-MMAF showed inhibitory and killing effects against MX-1 xenograft tumors at doses of 1.5 mg / kg and 3 mg / kg. [Diagram 2] FIG. 13 shows the HPLC spectra of hu2F7-MC-MMAF with drug loadings of 2 and 4, respectively. [Diagram 3] 1 is a graph showing the in vivo antitumor effects of hu2F7-MC-MMAF with drug loadings of 2 and 4, respectively. [Figure 4] 1 shows the in vivo antitumor effects of hu2F7-MC-MMAF with drug loadings of 2 and 4, respectively. The figures show the tumors in mice in each group on day 21. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0157] 1. Terminology To facilitate the understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless expressly defined otherwise elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

[0158] The three-letter and one-letter codes for amino acids used in the present invention are those described in J. Biol. Chem, 243, p. 3558 (1968).

[0159] The term "antibody" as described in the present invention refers to immunoglobulins, which are tetrapeptide chain structures consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The compositions and sequences of amino acids in the immunoglobulin heavy chain constant regions are different, and therefore their antigenicity is also different. This allows immunoglobulins to be classified into five types, also known as immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, whose corresponding heavy chains are μ, δ, γ, α and ε chains, respectively. Ig of the same type can be classified into various subclasses according to the differences in the 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 chains are classified into κ or λ chains according to the differences in the constant region. Each of the five types of Ig can have κ or λ chains.

[0160] In the present invention, the antibody light chain variable region of the present invention may further comprise a light chain constant region, including a human or mouse κ chain, λ chain, or a variant thereof.

[0161] In the present invention, the antibody heavy chain variable region of the present invention may further comprise a heavy chain constant region comprising human or mouse IgG1, 2, 3, 4, or a variant thereof.

[0162] The sequences of about 110 amino acids near the N-terminus of antibody heavy and light chains are highly variable and are the variable regions (V regions), while the remaining amino acid sequences near the C-terminus are relatively stable and are the constant regions (C regions). The variable regions contain three hypervariable regions (HVRs) and four framework regions (FRs) with relatively conserved sequences. The three hypervariable regions determine the specificity of the antibody and are also known as complementarity determining regions (CDRs). Each light chain variable region (VL) and heavy chain variable region (VH) consists of three CDR regions and four FR regions, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3. The numbers and positions of CDR amino acid residues in the VL and VH regions of an antibody or antigen-binding fragment of the invention are according to the known Kabat or Chothia numbering standards, and the Kabat or AbM definition standards (http: / / bioinf.org.uk / abs / ).

[0163] The term "antigen-presenting cell" or "APC" refers to a cell that presents foreign antigens complexed with MHC on its surface. T cells recognize such complexes using the T cell receptor (TCR). 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, allowing them to be recognized by T cells, for example, as components of MHC-I / MHC-II complexes.

[0164] 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 the functional activation of T cells. The term "B7-H4" includes any variant or isoform of B7-H4 that is naturally expressed by cells. The antibodies of the present invention may cross-react with B7-H4 from non-human species. Alternatively, the antibodies may also be specific for human B7-H4 and may not show cross-reactivity with other species. B7-H4 or any variant or isoform thereof may be isolated from cells or tissues that naturally express it or produced by recombinant techniques using techniques commonly used in the art and described herein. Preferably, the anti-B7-H4 antibody targets human B7-H4 with normal glycosylation patterns.

[0165] The term "recombinant human antibody" includes human antibodies prepared, expressed, produced, or isolated by recombinant methods, the techniques and methods involved being well known in the art. For example, it includes (1) antibodies isolated from transgenic or transchromosomic animals (e.g., mice) expressing human immunoglobulin genes, or hybridomas prepared therefrom, (2) antibodies isolated from host cells transformed to express the antibody, e.g., transfectionomas, (3) antibodies isolated from recombinant combinatorial human antibody libraries, and (4) antibodies prepared, expressed, produced, or isolated by methods of splicing human immunoglobulin gene sequences to other DNA sequences and other methods. Such recombinant human antibodies include variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, as well as subsequent rearrangements and mutations, e.g., those that occur during antibody maturation.

[0166] The term "mouse antibody" in the present invention refers to a monoclonal antibody against human B7-H4 prepared by the knowledge and skill in the art. In the preparation, a test subject is injected with B7-H4 antigen, and then a hybridoma expressing an antibody with the desired sequence or functional characteristics is isolated. In a preferred embodiment of the present invention, the mouse B7-H4 antibody or antigen-binding fragment thereof may further comprise a light chain constant region of a mouse κ chain, λ chain or variants thereof, or may further comprise a heavy chain constant region of a mouse IgG1, IgG2, IgG3 or IgG4 or variants thereof.

[0167] The term "human antibody" includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences (i.e., "humanized antibodies").

[0168] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody generated by transplanting mouse CDR sequences into the framework of human antibody variable region. This can overcome the strong immune response induced by chimeric antibodies that have a large amount of mouse protein components. To avoid the decrease in activity caused by the decrease in immunogenicity, human antibody variable region can be subjected to minimal back mutation to maintain activity.

[0169] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by mouse antibodies. The establishment of a chimeric antibody requires first establishing a hybridoma secreting a mouse-specific monoclonal antibody, then cloning the variable region gene from the mouse hybridoma cell, and then cloning the constant region gene of a human antibody as necessary, combining the mouse variable region gene with the human constant region gene to form a chimeric gene, which is inserted into a human expression vector for finally expressing the chimeric antibody molecule in a eukaryotic or prokaryotic industrial system. The human antibody constant region may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or variants thereof, and preferably, an IgG1 containing a human IgG2 or IgG4 heavy chain constant region, or with enhanced ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation may be used.

[0170] The term "antigen-binding fragment" refers to antigen-binding fragments of antibodies and antibody analogs, which typically include at least a portion of the antigen-binding or variable region (e.g., one or more CDRs) of a parent antibody. Antibody fragments retain at least a portion of the binding specificity of the parent antibody. Typically, when activity is expressed on a molar basis, antibody fragments retain at least 10% of the binding activity of the parent. Preferably, antibody fragments retain at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the binding affinity of the parent antibody for the target. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, linear antibodies, single chain antibodies, nanobodies, domain antibodies and multispecific antibodies. Engineered antibody variants are reviewed in Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136.

[0171] A "Fab fragment" consists of the CH1 and variable regions of one light chain and one heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0172] The "Fc" region comprises two antibody heavy chain fragments containing the CH1 and CH2 domains, held together by two or more disulfide bonds and through hydrophobic interactions of the CH3 domain.

[0173] A "Fab' fragment" contains a light chain and a portion of a heavy chain including the VH domain, the CH1 domain, and the region between the CH1 and CH2 domains, such that interchain disulfide bonds can form between two heavy chains of a Fab' fragment to form an F(ab')2 molecule.

[0174] An "F(ab')2 fragment" comprises two light chains and two heavy chains that contain a portion of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, an F(ab')2 fragment consists of two Fab' fragments that are linked by disulfide bonds between the two heavy chains.

[0175] The "Fv region" comprises the variable regions from both the heavy and light chains, but lacks the constant regions.

[0176] The term "multispecific antibodies" is used in its broad sense and includes antibodies with polyepitopic specificity, including, but not limited to, antibodies comprising a heavy chain variable region (VH) and a light chain variable region (VL) where the VH-VL unit has multiple epitopic specificity, antibodies with two or more VL and VH regions where each VH-VL unit binds to a different target or a different epitope on the target, antibodies with two or more single variable regions where each single variable region binds to a different target or a different epitope on the target, full length antibodies, antibody fragments, diabodies, bispecific diabodies and triabodies, covalently or non-covalently linked antibody fragments, etc.

[0177] The term "single-chain antibody" refers to a single-chain recombinant protein formed by linking the variable domains of the heavy (VH) and light (VL) chains of an antibody via a linker peptide. It is the smallest antibody fragment that contains an intact antigen-binding site.

[0178] The term "domain antibody fragment" refers to an immunologically functional immunoglobulin fragment that contains only a heavy or light chain variable 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 a bivalent domain antibody fragment can target the same or different antigens.

[0179] The term "binding to B7-H4" 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 the three-dimensional site scattered on the antigen and recognized by the antibody or antigen-binding fragment of the present invention.

[0180] The terms "specifically bind" and "selectively bind" as used herein refer to an antibody binding to an epitope on a given antigen. In general, when recombinant human B7-H4 is used as the analyte and an antibody is used as the ligand, the antibody binds to an epitope on a given antigen at a binding affinity of about 10 -7 It binds to a given antigen with an equilibrium dissociation constant (KD) of less than M or even lower, and its binding affinity to the given antigen is at least twice its binding affinity to a non-specific antigen (other than the given antigen or a closely related antigen, such as BSA). The term "antibody that recognizes an antigen" can be used interchangeably herein with the term "antibody that specifically binds to."

[0181] The term "cross-reactivity" refers to the ability of an antibody of the invention to bind to B7-H4 from various species. For example, an antibody of the invention that binds to human B7-H4 can also bind to B7-H4 from another species. Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (e.g., SPR and ELISA) or by binding to or functionally interacting with cells that physiologically express B7-H4. Methods for determining cross-reactivity include standard binding assays described herein, such as surface plasmon resonance (SPR) analysis or flow cytometry.

[0182] The terms "inhibition" or "blocking" may be used interchangeably and include both partial and complete inhibition / blocking. Inhibition / blocking of a ligand preferably reduces or alters the normal level or type of activity exhibited when ligand binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable decrease in the binding affinity of a ligand when contacted with an anti-B7-H4 antibody compared to the binding affinity of the ligand when not contacted with an anti-B7-H4 antibody.

[0183] The term "inhibition of proliferation" (eg, when referring to cells) is intended to include any measurable decrease in cell proliferation.

[0184] The terms "induction of an immune response" and "enhancement of an immune response" can be used interchangeably and can refer to an immune response (i.e., passive or adaptive) to stimulation by a specific antigen. The term "induce" as used in the phrase "induction of CDC or ADCC" refers to stimulating a specific direct cell killing mechanism.

[0185] In the present invention, "ADCC", i.e., antibody-dependent cell-mediated cytotoxicity, means that cells expressing Fc receptors directly kill target cells covered with antibodies by recognizing the Fc segment of the antibody. The function of the ADCC effect of antibodies can be enhanced, reduced or eliminated by modifying the Fc segment of IgG. The modification refers to a mutation in the heavy chain constant region of the antibody.

[0186] Methods for producing and purifying 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 5-8 and 15. For example, mice can be immunized with human B7-H4 or a fragment thereof, the resulting antibodies can be refolded and purified, and amino acid sequencing can be performed by using conventional methods. Antigen-binding fragments can also be prepared by using conventional methods. The antibodies or antigen-binding fragments of the present invention are genetically modified to introduce one or more human FR regions onto the non-human CDR regions. Human FR germline sequences can be obtained from the ImmunoGeneTics (IMGT) website http: / / imgt.cines.fr or The Immunoglobulin Facts Book, 2001 ISBN012441351.

[0187] The modified antibodies or antigen-binding fragments of the present invention can be prepared and purified by conventional methods. The cDNA sequence of the corresponding antibody can be cloned and recombined into a GS expression vector. CHO cells can be stably transfected with a recombinant immunoglobulin expression vector. As a more recommended prior art, a mammalian expression system can cause glycosylation of the antibody, especially at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing an antibody that specifically binds to a human antigen. Positive clones are grown in serum-free medium in a bioreactor to produce the antibody. The culture medium secreting the antibody can be purified and collected by conventional techniques. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and multimers can also be removed by conventional methods, such as molecular sieving and ion exchange. The resulting product must be immediately frozen or lyophilized, for example at -70°C.

[0188] 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 clone cell line, and this cell line is not limited to eukaryotic, prokaryotic, or phage clone cell line. The monoclonal antibody or antigen-binding fragment can be obtained, for example, by hybridoma technology, recombinant technology, phage display technology, synthetic technology (e.g., CDR grafting), or recombination using other existing technologies.

[0189] "Administration" and "treatment" when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contacting an exogenous pharmaceutical, therapeutic, diagnostic, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of cells includes contacting a reagent with a cell, contacting a reagent with a fluid, where the fluid is in contact with the cell. "Administration" and "treatment" can also refer to, for example, treating a cell with a reagent, diagnostic, binding composition, or another type of cell, in vitro and ex vivo. "Treatment" when provided to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic measures, research, and diagnostic applications.

[0190] "Treatment" refers to the administration of an internal or external therapeutic agent, such as a composition comprising any one of the binding compounds of the present invention, to a patient having one or more symptoms for which the therapeutic agent is known to have a therapeutic effect. In general, the therapeutic agent is given in an amount effective to reduce one or more symptoms in a subject or population to be treated, to induce regression of such symptoms or inhibit the onset of such symptoms to any clinically measurable extent. The amount of therapeutic agent effective to reduce any specific symptom (also referred to as a "therapeutically effective amount") may vary depending on various factors, such as the condition, age and weight of the patient, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the symptom is reduced can be evaluated by any clinical test method commonly used by physicians or other medical professionals to evaluate the severity or progression of symptoms. Even if an embodiment of the invention (e.g., a method of treatment or product) is ineffective in reducing each symptom of interest, there should still be a reduction in the symptom of interest in a statistically significant number of patients as determined by any statistical testing method known in the art, such as Student's t-test, chi-squared test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonkheel-Tapstra test, and Wilcoxon test.

[0191] As used throughout this specification and claims, the term "consisting essentially of" or variants thereof means including all recited elements or elements, and optionally including other elements similar or different in nature to the recited elements, that do not significantly alter the basic or novel properties of a given dosing regimen, method, or composition. As a non-limiting example, a binding compound consisting essentially of a recited amino acid sequence may also include one or more amino acids that do not significantly affect the properties of the binding compound.

[0192] 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 sequence or a polynucleotide sequence is considered to be naturally occurring and not intentionally modified artificially in a laboratory if it is present in an organism (including viruses) and can be isolated from a natural source.

[0193] An "effective amount" includes an amount sufficient to ameliorate or prevent signs or symptoms of a physical disease. Effective amount also refers to an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on the following factors: the condition being treated, the general health of the patient, the method, route and dose of drug administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0194] "Exogenous" refers to a substance that is produced outside an organism, cell, or body according to a background. "Endogenous" refers to a substance that is produced inside a cell, organism, or body according to a background.

[0195] "Homology" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. If the positions of two aligned sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, the molecules are considered to be homologous at this position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of aligned positions multiplied by 100%. For example, in optimal sequence alignment, if 6 out of 10 positions in the two sequences are matched or homologous, the two sequences are 60% homologous. Generally, alignment is performed when two sequences are aligned to obtain the maximum homology percentage.

[0196] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably, and all such designations include their progeny. Thus, the words "transformant" and "transformed cell" include the primary test cell and cultures derived therefrom, regardless of the number of passages. It should also be understood that all progeny may not be exactly identical in DNA content due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as screened for in the original transformed cell are included. This will be clear from the context when referring to the various designations.

[0197] "Optional" or "optionally" means that the event or circumstance following the "optional" or "optionally" expression may, but need not, occur, and the description includes cases where the event or circumstance occurs or does not occur. For example, "optionally comprising one to three antibody heavy chain variable regions" means that an antibody heavy chain variable region of a particular sequence may, but need not be present.

[0198] "Pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein, or physiologically / pharmaceutical acceptable salts or prodrugs thereof, and other chemical and other components, such as physiologically / pharmaceutical acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate drug administration to an organism and enhance the absorption of the active ingredient, thereby resulting in biological activity. The preparation of conventional pharmaceutical compositions is described in the Chinese Pharmacopoeia.

[0199] "Pharmaceutically acceptable salt" refers to a salt of the antibody-drug conjugate of the present invention, which is safe and effective for use in mammals and has the desired biological activity. The antibody-drug conjugate of the present invention contains at least one amino group, and thus can react with an acid to produce a salt. Non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, hydrogen sulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, and p-toluenesulfonate.

[0200] "Solvate" refers to a pharma-ceutically acceptable solvate formed by an 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.

[0201] A "cytotoxic drug", as used herein, refers to a substance that inhibits the function of cells and / or causes cell death or destruction.

[0202] "Tubulin inhibitors" refers to a class of compounds that interfere with the process of cell mitosis by inhibiting tubulin polymerization or promoting tubulin aggregation, thereby resulting in an anti-tumor effect, non-limiting examples of which include maytansinoids, calicheamicins, taxanes, vincristine, colchicine, dolastatins / auristatins / monomethylauristatin E (MMAE) / monomethylauristatin F (MMAF).

[0203] "Linker" refers to a chemical moiety that covalently attaches an antibody to a covalent bond or chain of atoms of a drug. Non-limiting examples of linkers include arylene, heteroarylene, PEG, polymethyleneoxy, succinic acid, succinamide, diglycolic acid, malonic acid, and caproic acid amide.

[0204] "Drug loading" (DAR) is represented in formula (A) by y, which is the average number of cytotoxic drugs per antibody. The range of drug loading in the present invention can be 1-20 cytotoxic drugs (D) per antibody. The antibody-drug conjugate of general formula (A) is a collection of antibodies conjugated to a specific range (1-20) of cytotoxic drugs. The drug loading (DAR) of the antibody-drug conjugates by the coupling reaction can be characterized by conventional means, such as mass spectrometry, HPLC and ELISA. These means allow the determination of the quantitative distribution of the antibody-drug conjugates based on the y value.

[0205] 2. Abbreviations MC = 6-maleimidohexanoyl VC=Valine-Citrulline PAB = p-aminobenzyloxycarbonyl MMAE = monomethylauristatin E (MW718) MMAF = a variant of monomethylauristatin E (MW 731.5) that has a phenylalanine at the C-terminus of the molecule.

[0206] For further explanation of the present invention, examples are incorporated below, but these examples do not limit the scope of the present invention. The experimental methods with unspecified conditions 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 follow the conditions recommended by the manufacturers of raw materials or products. The reagents with unspecified sources are conventional reagents purchased on the market.

[0207] Example 1: Preparation of antigen and construction of stable cell lines The sequence encoding his-tagged human B7-H4 (huB7-H4-His) and the sequence encoding Fc-tagged human B7-H4 (huB7-H4-Fc) were synthesized by CRO's Integrative DNA Technology (IDT) (the template sequences of the above B7-H4 recombinant proteins were all designed according to the present invention), and cloned into pTT5 vector (Biovector), respectively. After the recombinant B7-H4 proteins were expressed in 293T cells, they were purified according to the following experimental method, and the purified proteins could be used in the following experiments in the examples.

[0208] The sequence of huB7-H4-His: MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGK GNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKADYKDDDDKGSHHHHHHHH SEQ ID NO:1 Sequence of huB7-H4-Fc: FGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTVVWASQVDQGANFESEVSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLNSKAGSGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 2

[0209] huB7-H4-His purification process: HEK293 cell expression supernatant samples were centrifuged at high speed to remove impurities. The buffer was exchanged against PBS and imidazole was added to a final concentration of 5 mM. The nickel column was equilibrated with PBS solution containing 5 mM imidazole and washed with 2-5 column volumes. The supernatant sample obtained after exchange was loaded onto the column. The column was rinsed with PBS solution containing 5 mM imidazole until the A280 reading dropped to baseline. The chromatography column was then rinsed with PBS + 10 mM imidazole to remove non-specifically bound protein impurities and the flow-through was collected. The target protein was eluted with PBS solution containing 300 mM imidazole and the elution peak was collected. The collected eluate was further purified by ion exchange (SP column). Preparation of solution A: 0.01 M PB, pH 8.0. Preparation of solution B: Solution A + 1 M NaCl. First, the PBS solution of imidazole with the eluted target protein was exchanged against solution A, and the SP column was equilibrated by using solution A and loaded with the sample. The concentration gradient of solution B was 0-100%. The sample was eluted with 10 column volumes, and each elution peak was collected. The obtained protein was identified as the correct one by electrophoresis, peptide map, and liquid chromatography-mass spectrometry (LC-MS), and then divided into aliquots for use.

[0210] Purification process of huB7-H4-Fc: HEK293 cell expression supernatant samples were centrifuged at high speed to remove impurities. The buffer was exchanged against PBS. A Protein A affinity column was equilibrated with 10 mM PBS buffer and washed with 2-5 column volumes. The supernatant sample obtained after exchange was loaded onto the column. The column was washed with 25 column volumes of buffer until the A280 reading dropped to baseline. The target protein was eluted with 0.8% acetate buffer pH 3.5 and the elution peak was collected. After aliquoting, 1 M Tris-Cl buffer pH 8.0 was immediately added to neutralize. The solution was then exchanged against PBS pH 6.9 using a Millipore Amico-15 filter column. The obtained proteins were identified by electrophoresis, peptide map and liquid chromatography mass spectrometry (LC-MS) and then aliquoted for use.

[0211] Construction of a stable CHO-S cell pool: The full-length sequences encoding human or cynomolgus B7-H4 proteins (huB7-H4 or cyB7-H4) were synthesized by Integrated DNA Technology (IDT) (the template sequences of the above B7-H4 recombinant proteins were all designed according to the present invention) and cloned into a modified pcDNA3.1 vector, pcDNA3.1 / puro (Invitrogen, #V79020), respectively. CHO-S (ATCC) cells were cultured in CD-CHO medium (Life Technologies, #10743029) to obtain 0.5 × 10 6 The concentration was adjusted to cells / ml. 10 μg of vectors encoding huB7-H4 or cyB7-H4 genes were mixed in 50 μl of LF-LTX (Life Technologies, #A12621) and 1 ml of Opti-MEM medium (Life Technologies, #31985088). The mixture was incubated at room temperature for 20 minutes and added to the culture medium of CHO cells. The cells were seeded and cultured in a carbon dioxide incubator. After 24 hours, the culture medium was replaced with fresh medium and 10 μg / ml of puromycin was added. Thereafter, the culture medium was replaced with fresh medium every 2-3 days, and a stable CHO-S cell pool was obtained 10-12 days after selection.

[0212] Example 2: Obtaining mouse hybridoma and antibody sequences A total of five Balb / c and five 10-week-old female A / J mice were immunized with the human antigen huB7-H4-Fc. Complete Freund's Adjuvant (CFA) from Sigma and Incomplete Freund's Adjuvant (IFA) from Sigma were used. The immunogen and immunoadjuvant were thoroughly mixed and emulsified in a 1:1 ratio to create a stable "water-in-oil" solution. The injection dose was 25 μg / 200 μL / mouse. Day 01: First immunization, complete Freund's adjuvant Day 21: Second immunization, incomplete Freund's adjuvant Day 35: Third immunization, incomplete Freund's adjuvant Day 42: Blood collection and serum titer test (blood after 3 immunizations) Day 49: 4th immunization, incomplete Freund's adjuvant Day 56: Blood collection and serum titer test (blood after 4 immunizations)

[0213] An indirect ELISA method was used to detect the affinity of antibodies or sera. huB7-H4-His protein was diluted to a concentration of 1 μg / ml in PBS pH 7.4, added at 100 μl / well into a 96-well high affinity ELISA plate, and incubated overnight (16-20 hours) refrigerated at 4°C. The plate was washed four times with PBST (PBS pH 7.4 containing 0.05% Tween-20). Blocking was performed by adding 150 μl / well of 3% bovine serum albumin (BSA) blocking solution diluted in PBST and incubating at room temperature for 1 hour. After completion of blocking, the blocking solution was discarded and the plate was washed four times with PBST buffer. Antibodies or sera to be tested were diluted in PBST containing 3% BSA to obtain a gradient of 10 doses (10-fold dilutions) starting from 1 μM, added at 100 μl / well into the microtiter plate, and incubated at room temperature for 1 hour. After the incubation was completed, the plate was washed 4 times with PBST, and HRP-conjugated goat anti-human secondary antibody (Abcam, cat#ab97225) diluted in PBST containing 3% BSA was added at 100 μl / well and incubated at room temperature for 1 hour. The plate was washed 4 times with PBST, and 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. The reaction was terminated by adding stop solution (Cell Signaling Technology, cat#7002S) at 100 μl / well. The absorbance value was read at 450 nm by a microplate reader (BioTek, Synergy H1 model). The data was analyzed to obtain the binding affinity of the antibody or serum to the human B7-H4 antigen.

[0214] Serum titer and the ability of immunized mouse serum to bind to cell surface antigens were evaluated by using indirect ELISA. Cell fusion was performed according to the titer detection results (dilution of more than 100,000 times). Immunized mice with high serum titer, affinity and FACS binding were selected for one final immunization and then sacrificed. Spleen cells and SP2 / 0 myeloma cells were fused and seeded to obtain hybridomas. Target hybridomas were screened by indirect ELISA, and monoclonal cell lines were established by limiting dilution. Among the obtained positive antibody lines, hybridoma lines expressing non-specific binding antibodies were further excluded by comparing CHO-S cells stably expressing huB7-H4 and cyB7-H4 with blank CHO-S cells. Target hybridomas were screened by using a method similar to that of the in vitro cell binding experiment in Example 5, and monoclonal cell lines were established by limiting dilution. Hybridoma cells in logarithmic growth phase were harvested. 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 using a mouse Ig primer set (Novagen, TB326 Rev.B 0503) and sequenced. Finally, the sequences of four strains of mouse antibodies were obtained for humanization and construction of antibody-drug conjugates.

[0215] The heavy and light chain variable region sequences of the mouse monoclonal antibody 2G6 are as follows: 2G6 HCVR EVQLVESGGGLVKPGGSLKLSCAASGFTFSRYGMSWVRQTPEKRLEWVAGINGGGSYTYYLDTVKGRFTISRDNSRNTLYLQMSSLRSEDTAMYYCVSQGSNYYFDYWGQGTTLTVSS SEQ ID NO:46 2G6 LCVR DIRMTQSPSSMSVSLGDTVSITCHASQGISSNIGWLQQKPGKSFKALIYHGTNLEDGVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPYTFGGGTKLEIK SEQ ID NO:47

[0216] The heavy and light chain variable regions of the murine monoclonal antibody 2G6 contain the following CDR sequences:

[0217] [Table 1]

[0218] The sequences of the heavy and light chain variable regions of the mouse monoclonal antibody 2F7 are as follows: 2F7 HCVR EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYYMSWVRQTPEKRLEWVAYVSSGGGSTYYSDSVKGRFTISRDNAKNTLYLQMSSLKPEDTAMYYCTRESYSQGNYFDYWGQGTTLTVSS SEQ ID NO:48 2F7 LCVR DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKFASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFSLTFGAGTKLELK SEQ ID NO:49

[0219] The heavy and light chain variable regions of the murine monoclonal antibody 2F7 contain the following CDR sequences:

[0220] [Table 2]

[0221] The sequences of the heavy and light chain variable regions of mouse monoclonal antibody 2F8 are as follows: 2F8 HCVR QVQLQQPGSVLVRPGASVKLSCKASGYTFTNSWMNWAKLRPGQGLEWIGGIYPNSGNIEYNEKFKGKATLTVDTSSSTAYMDLTSLTSEDSAVYYCARDSRFSYWGQGTLVTVSA SEQ ID NO:50 2F8 LCVR DIVMTQSHKFMSTSVGDRVSITCKASQDVRTAVAWYQQKPGQSPKLLISSTSYRYTGVPDRFTGSGSGTDFTFIISSVQAEDLAVYYCQQHYSTPLTFGAGTKLELK SEQ ID NO:51

[0222] The heavy and light chain variable regions of the murine monoclonal antibody 2F8 contain the following CDR sequences:

[0223] [Table 3]

[0224] The sequences of the heavy and light chain variable regions of the mouse monoclonal antibody 1C9 are as follows: 1C9 HCVR QVQLQQPGSVLVRPGASVKLSCKASGDTFTTYWMNWVKQRPGQGLEWIGGIYLNSGSSEYNEKFKGKATLSVDTSSSTAYMDLSSLTSEDSAVYYCARDSRFSYWGQGTLVTVSA SEQ ID NO:52 1C9 LCVR DIVMTQSHKFLSTSVGDRVSITCKASQDVSTAVAWYQQKPGQSPELLISSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYNTPLTFGAGTQLELK SEQ ID NO:53

[0225] The heavy and light chain variable regions of the murine monoclonal antibody 1C9 contain the following CDR sequences:

[0226] [Table 4]

[0227] [Example 3: Humanization experiment of mouse antibody] Humanization of mouse anti-human B7-H4 monoclonal antibody was performed by using methods that have been widely documented in the art. Briefly, the parent (mouse antibody) constant domain was replaced with a human constant domain. In the present invention, human germline antibody sequences were selected according to the homology between mouse and human antibodies, and mouse antibodies 2G6, 2F7, 2F8 and 1C9 were humanized. The CDR regions of mouse antibodies 2G6 and 2F7 were grafted onto the selected corresponding humanized templates to replace the humanized variable regions, and then recombined with IgG constant regions (preferably IgG1 for heavy chain and κ for light chain). Then, based on the three-dimensional structure of the mouse antibody, buried residues, residues that directly interact with the CDR region, and residues that have significant effects on the three-dimensional structures of VL and VH were subjected to back mutation to optimize the chemically unstable amino acid residues in the CDR region, in which HCDR1 of mouse monoclonal antibody 2F8 was optimized against GYTFTSSWMN (sequence number 43), HCDR2 was optimized against GIYPNRGNIEYNEKFKG (sequence number 44), and HCDR2 of mouse monoclonal antibody 1C9 was optimized against YLNRGS (sequence number 45), resulting in the design of a humanized antibody comprising the following combination of light chain and heavy chain variable region sequences:

[0228] The heavy and light chain variable regions of humanized antibody hu2G6 are as follows: hu2G6 HCVR EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQAPGKGLEWVSGINGGGSYTYYLDTVKGRFTISRDNARNTLYLQMSSLRAEDTAVYYCVSQGSNYYFDYWGQGTLVTVSS SEQ ID NO:15 hu2G6 LCVR DIRMTQSPSSLSASVGDRVTITCHASQGISSNIGWLQQKPGKAPKALIYHGTNLEDGVPSRFSGSGSGADYTLTISSLQPEDFATYYCVQYAQFPYTFGGGTKVEIK SEQ ID NO:16

[0229] The heavy and light chain variable regions of humanized antibody hu2F7 are as follows: hu2F7 HCVR EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVAYVSSGGGSTYYSDSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCTRESYSQGNYFDYWGQGTTVTVSS SEQ ID NO:17 hu2F7 LCVR EIVMTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQSPRLLIKFASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFSLTFGQGTKLEIK SEQ ID NO:18

[0230] The heavy and light chain variable regions of humanized antibody hu2F8 are as follows: hu2F8 HCVR EVQLVQSGAEVKKPGASVKVSCKASGYTFTSSWMNWVRQAPGQRLEWMGGIYPNRGNIEYNEKFKGRVTLTVDTSASTAYMELSSLRSEDTAVYYCARDSRFSYWGQGTLVTVSS SEQ ID NO:35 hu2F8 LCVR DIQMTQSPSSLSASVGDRVTITCKASQDVRTAVAWYQQKPGKAPKLLISSTSYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPLTFGGGTKVEIK SEQ ID NO:36

[0231] The heavy and light chain variable regions of humanized antibody hu1C9 are as follows: hu1C9 HCVR EVQLVQSGAEVKKPGASVKVSCKASGDTFTTYWMNWVRQAPGQRLEWMGGIYLNRGSSEYNEKFKGRVTLTVDTSASTAYMELSSLRSEDTAVYYCARDSRFSYWGQGTLVTVSS SEQ ID NO:37 hu1C9 LCVR DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLISSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYNTPLTFGGGTKVEIK SEQ ID NO:38;

[0232] The variable regions were recombined with IgG constant regions (preferably IgG1 for the heavy chain and κ for the light chain). Exemplary heavy and light chain constant region sequences are shown below. IgG1 C: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:54 Ig Kappa C: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:55;

[0233] A humanized antibody consisting of the following exemplary light and heavy chain sequences was obtained after combination: hu2G6 HC EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQAPGKGLEWVSGINGGGSYTYYLDTVKGRFTISRDNARNTLYLQMSSLRAEDTAVYYCVSQGSNYYFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:19 hu2G6 LC DIRMTQSPSSLSASVGDRVTITCHASQGISSNIGWLQQKPGKAPKALIYHGTNLEDGVPSRFSGSGSGADYTLTISSLQPEDFATYYCVQYAQFPYTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:20 hu2F7HC EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVAYVSSGGGSTYYSDSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCTRESYSQGNYFDYWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:21 hu2F7 LC EIVMTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQSPRLLIKFASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFSLTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:22 hu2F8HC EVQLVQSGAEVKKPGASVKVSCKASGYTFTSSWMNWVRQAPGQRLEWMGGIYPNRGNIEYNEKFKGRVTLTVDTASTAYMELSSLRSEDTAVYYCARDSRFSYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:39 hu2F8 LC DIQMTQSPSSLSASVGDRVTITCKASQDVRTAVAWYQQKPGKAPKLLISSTSYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:40 hu1C9HC EVQLVQSGAEVKKPGASVKVSCKASGDTFTTYWMNWVRQAPGQRLEWMGGIYLNRGSSEYNEKFKGRVTLTVDTSASTAYMELSSLRSEDTAVYYCARDSRFSYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:41 hu1C9 LC DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLISSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYNTPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:42

[0234] [Example 4: Expression of B7-H4 humanized antibody] The recombinant vectors were designed according to the sequences of the humanized antibodies. The heavy chain vector was designed as: signal peptide + humanized heavy chain. The light chain vector was designed as: signal peptide + humanized light chain.

[0235] The above sequence was inserted into pCEP4 vector (ThermoFisher #V04450). The expression vector was synthesized according to the design above. The vector plasmid was obtained, extracted on a large scale, and sent for sequencing to be verified. The certified plasmid was transfected into human 293F cells (ThermoFisher #R79007) with PEI (ThermoFisher #BMS1003-A). 293F cells were continuously cultured in serum-free medium (Shanghai OPM Biosciences, OPM-293 CD03) to 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 mixed thoroughly, then 200 μg of PEI was added, mixed thoroughly, and incubated at RT for 15 minutes. Then, 50 mL of cells were added. Cell culture conditions: 5% CO 2 , 37°C, 125 rpm / min. During the culture, supplements were added on the first and third days until cell viability was less than 70%. The cell supernatant was collected, centrifuged and filtered. The centrifuged and filtered cell culture medium was loaded onto an antibody purification affinity column. The column was washed with phosphate buffer. The sample was eluted with glycine-HCl buffer (pH 2.7 0.1M Gly-HCl), neutralized with 1M Tris-HCl pH 9.0, and dialyzed against phosphate buffer to finally obtain purified humanized antibody, which could be used in the experiments of each example.

[0236] [Example 5: Detection of binding ability of humanized antibodies in vitro] The designed humanized antibodies were tested in in vitro experiments as follows.

[0237] 1. In vitro cell binding experiments: Cultured MX-1 cells (CLS Cell Lines Service GmbH #300296) expressing human B7-H4 were harvested, the cell concentration was adjusted with PBS, pH 7.4, and the cells were diluted to 1 × 10 per well.5 Cells were seeded on a V-bottom 96-well plate. The plate was centrifuged at 2000 rpm for 5 min and the supernatant was removed. 100 μl of gradient diluted humanized antibody solution (diluted in PBS with 0.5% BSA starting at 1 μM with a 3-fold gradient of 10 doses) was added to each well, mixed thoroughly and incubated on a shaker at 4° C. for 1 h. The plate was centrifuged at 2000 rpm for 5 min and the supernatant was removed. The cells were washed twice with PBS. 100 μl of FITC-labeled goat anti-human secondary antibody (Abcam, cat#ab97224) diluted in 0.5% BSA in PBS was added to each well, mixed thoroughly and incubated on a shaker at 4° C. for 30 min. The plate was centrifuged at 2000 rpm for 5 min and the supernatant was removed. The cells were washed twice with PBS and then resuspended in PBS. The signals were detected using a flow cytometer (BECKMAN COULTER, DxFLEX model), and the concentration curves were plotted to obtain the analysis results. The results are shown in Table 1. Both humanized antibodies hu2G6 and hu2F7 actively bind to MX-1 cells that highly express B7-H4.

[0238] [Table 5]

[0239] 2. Affinity kinetics experiments: The Biacore method is a commonly accepted method for objectively detecting affinity and kinetics between proteins. The affinity and binding kinetics of the tested antibodies of the present invention were analyzed by Biacore T200 (GE). The tested anti-B7-H4 antibodies of the present invention were covalently coupled to a CM5 (GE) chip by using the kit provided by Biacore and the NHS standard amino coupling method. Then, 50 nM of human huB7-H4-His protein diluted in the same buffer was injected at a flow rate of 10 μL / min. After injection, all samples were regenerated with the regeneration reagent in the kit. The antigen-antibody binding kinetics was followed for 3 minutes, and the dissociation kinetics was followed for 10 minutes. The obtained data was analyzed by using GE's BIAevaluation software with a 1:1 (Langmuir) binding model. The affinity kinetic data of the humanized antibodies calculated by this method are shown in Table 2. The humanized antibodies hu2G6, hu2F7, hu2F8 and hu1C9 all exhibit strong affinity for the human B7-H4 antigen protein.

[0240] [Table 6]

[0241] [Example 6: Endocytosis of anti-B7-H4 antibody] To test whether the antibodies of the present invention can be endocytosed with human B7-H4 after binding to B7-H4, they were evaluated using MX-1 cells. MX-1 cells were trypsinized (first washed once with PBS at 37° C. for approximately 2 min), harvested, and resuspended in pre-chilled FACS buffer. The cell concentration was 1×10 6The cells were adjusted to 100 cells / mL. 1 mL of cell suspension was added to an EP tube, centrifuged at 1500 rpm for 5 min, and the supernatant was removed. 1 mL of prepared antibody to be tested was added to resuspend the cells, resulting in a final antibody concentration of 20 μg / mL. The cells were incubated for 1 hour on a 4°C shaker, 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 was added to each tube, and the cells were resuspended. The cells were incubated for 30 minutes on a 4°C shaker, 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 pre-warmed MX-1 cell complete medium was added to each tube, and the cells were resuspended and mixed. The cell suspension was divided into 4 tubes with 200 μL aliquots per tube, which were 0 min, blank, 30 min and 2 h groups, respectively. The 0 min and blank groups were placed on ice, while the other groups were placed in a 37° C. incubator to allow endocytosis for 30 min and 2 h, respectively. At the corresponding time points, the EP tubes were removed and placed on ice to pre-cool for 5 min. All treatment groups were centrifuged and the supernatant was discarded (4° C., 1500 rpm×5 min). The cells were washed once with FACS buffer and the supernatant was removed. 250 μL of strip buffer was added to the EP tubes of all groups except the 0 min group and incubated at room temperature for 8 min. The cells were centrifuged and the supernatant was discarded (4° C., 1500 rpm×5 min). The cells were washed twice with FACS buffer and the supernatant was removed. All treatment groups were added with 100 μL of immunostaining fixative, placed at 4° C. for more than 30 minutes, and detected by flow cytometer DxFlex. The percentage of endocytosis of B7-H4 antibody (%)=(mean fluorescence intensity value at each time point-mean fluorescence intensity value of blank group) / (mean fluorescence intensity value at zero point-mean fluorescence intensity value of blank group)×100%. The data are shown in Table 3.

[0242] [Table 7]

[0243] [Example 7: Conjugation of antibodies to MC-MMAF] The antibodies of the present invention have cytotropic and endocytic activities, which makes them suitable for coupling with drugs to form antibody-drug conjugates for treating B7-H4 mediated diseases. The coupling process is shown in the following equation, where Ab represents hu2G6 or hu2F7.

[0244] [ka]

[0245] In the first step, S-(3-hydroxypropyl)thioacetic acid (0.7 mg, 5.3 mol) was dissolved in 0.9 mL of acetonitrile to make a solution for later use. The above pre-prepared acetonitrile solution of S-(3-hydroxypropyl)thioacetic acid was added to the antibody in acetic acid / sodium acetate buffer at pH=4.3 (10.35 mg / mL, 9.0 mL, 0.97 mol). Then, 1.0 mL of an aqueous solution of sodium cyanoborohydride (14.1 mg, 224 mol) was added dropwise into the reaction mixture and reacted under shaking at 25° C. for 2 hours. After completion of the reaction, the reaction mixture was desalted and purified by using a Sephadex G25 gel column (elution phase: pH 6.5 0.05 M PBS solution) to obtain a solution of product 1f. The solution was concentrated to 10 mg / mL and used directly in the next reaction.

[0246] In the second step, 0.35 mL of 2.0 M carboxyamine hydrochloride solution was added to the 1f solution (11.0 mL) and reacted for 30 min under shaking at 25° C. The reaction solution was then desalted and purified by using a Sephadex G25 gel column (elution phase: pH 6.5 0.05 M PBS solution) to obtain a solution of product 2f (concentration 6.17 mg / mL, 14.7 mL).

[0247] In the third step, compound MC-MMAF (1.1 mg, 1.2 mol, prepared by the method disclosed in PCT Patent Publication WO2005081711) was dissolved in 0.3 mL of acetonitrile and added into 2f solution (concentration 6.17 mg / mL, 3.0 mL), and reacted under shaking at 25°C for 4 hours. The reaction solution was then desalted and purified by Sephadex G25 gel column (elution phase: pH 6.5 0.05 M PBS solution), and filtered under sterile conditions to obtain product Ab-MC-MMAF antibody-drug conjugate in PBS buffer (3.7 mg / mL, 4.7 mL), which was refrigerated at 4°C. The average value y of product hu2G6-MC-MMAF determined by HIC-HPLC was 3.8, and a sample of hu2G6-MC-MMAF (y=4) was obtained by HIC-HPLC purification. The average y value of the product hu2F7-MC-MMAF determined by HIC-HPLC was 3.2, and samples of hu2F7-MC-MMAF (y=2) and hu2F7-MC-MMAF (y=4) were obtained by HIC-HPLC purification.

[0248] [Example 8: Conjugation of antibody to SN-38] The antibody conjugate drug was prepared by the following coupling process: where Ab represents hu2F7.

[0249] [ka]

[0250] In the first step, S-(3-hydroxypropyl)thioacetic acid (0.7 mg, 5.3 mol) was dissolved in 0.9 mL of acetonitrile to make a solution for later use. The above pre-prepared acetonitrile solution of S-(3-hydroxypropyl)thioacetic acid was added to the antibody in acetic acid / sodium acetate buffer at pH=4.3 (10.35 mg / mL, 9.0 mL, 0.97 mol). Then, 1.0 mL of an aqueous solution of sodium cyanoborohydride (14.1 mg, 224 mol) was added dropwise into the reaction mixture and reacted under shaking at 25° C. for 2 hours. After completion of the reaction, the reaction mixture was desalted and purified by using a Sephadex G25 gel column (elution phase: pH 6.5 0.05 M PBS solution) to obtain a solution of product 1h. The solution was concentrated to 10 mg / mL and used directly in the next reaction.

[0251] In the second step, 0.35 mL of 2.0 M carboxyamine hydrochloride solution was added to the 1h solution (11.0 mL) and reacted under shaking at 25° C. for 30 minutes. The reaction solution was then desalted and purified by using a Sephadex G25 gel column (elution phase: pH 6.5 0.05 M PBS solution) to obtain a solution of product 2h (concentration 6.2 mg / mL, 15.0 mL). The 2h solution was concentrated to about 10 mg / mL and used directly in the next reaction.

[0252] In the third step, compound MC-VC-PAB-SN-38 (1.3 mg, 1.2 mol) was dissolved in 0.3 mL of acetonitrile and added to the solution for 2 h (concentration 6.2 mg / mL, 3.0 mL), and reacted under shaking at 25 ° C for 4 hours. The reaction solution was then desalted and purified by Sephadex G25 gel column (elution phase: pH 6.5 0.05 M PBS solution), and filtered under sterile conditions to obtain the product hu2F7-SN-38 antibody-drug conjugate in PBS buffer (3.7 mg / mL, 4.7 mL), which was refrigerated at 4 ° C. The average value y was determined by ultraviolet method. A cuvette filled with sodium succinate buffer was placed in the reference absorbance cell and the sample judgment absorbance cell, respectively, and the cuvette filled with the test solution was placed in the sample judgment absorbance cell after subtracting the solvent blank. The absorbance was measured at 280 nm and 370 nm.

[0253] Data Processing: Antibody amount C mab was determined by establishing a standard curve and measuring the absorbance at a wavelength of 280 nm. Drug was determined by measuring the absorbance at a wavelength of 370 nm.

[0254] Average drug load y=C Drug / C mab The average value of product hu2F7-SN-38, y=3.7, was determined by the method described above. A sample of hu2F7-SN-38 (y=4) was obtained by UV-HPLC purification.

[0255] Example 9: Conjugation of antibodies to exatecan

[0256] [ka]

[0257] In the first step, 2a (2 g, 17.2 mmol) was dissolved in 75 mL of acetonitrile, and potassium carbonate (9.27 g, 67.2 mmol), benzyl bromide (20 mL, 167.2 mmol), and tetrabutylammonium iodide (620 mg, 1.68 mmol) were added successively. The reaction solution was stirred at room temperature for 48 h and filtered through diatomaceous earth. The filter cake was rinsed with ethyl acetate (20 ml). The filtrate was pooled and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography developing with solvent system C to give product 5a (3.2 g, yield: 90.1%).

[0258] In the second step, 5a (181.3 mg, 0.879 mmol) and 4b (270 mg, 0.733 mmol) were added to a reaction flask, 6 mL of tetrahydrofuran was added, and the atmosphere was replaced with argon three times. The reaction mixture was cooled to 0-5°C in an ice-water bath, potassium tert-butoxide (164 mg, 1.46 mmol) was added, and then the mixture was warmed to room temperature by removing the ice-water bath and stirred for 40 min. The reaction mixture was added with 15 mL of ice water and extracted with ethyl acetate (40 mL x 2) and chloroform (20 mL x 5). The organic phase was pooled and concentrated. The resulting residue was dissolved in 6 mL of dioxane, and 3 mL of water, sodium bicarbonate (73.8 mg, 0.879 mmol), and 9-fluorene methyl chloroformate (190 mg, 0.734 mmol) were added, and the mixture was stirred at room temperature for 2 h. The reaction solution was added with 30 mL of water and extracted with ethyl acetate (20 mL x 3). The organic phase was washed with saturated sodium chloride solution (30 mL), dried by using anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography using solvent system C to obtain product 5b benzyl 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-ate (73 mg, yield: 19.4%). MS m / z(ESI): 515.0[M+1]

[0259] 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), palladium was added on carbon (18 mg, content 10%, anhydrous basis), and hydrogen was replaced three times, and the reaction was allowed to proceed under stirring at room temperature for 1 h. The reaction solution was filtered through diatomaceous earth. The filter cake was rinsed with ethyl acetate. The filtrate was concentrated to obtain crude product 5c, 10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundec-11-oic acid (20 mg), which was used directly in the next reaction without purification. MSm / z(ESI):424.9[M+1]

[0260] In the fourth step, 1b (15 mg, 28.2 μmol) was added into the reaction flask, 1.5 mL of N,N-dimethylformamide was added, and the atmosphere was replaced with argon three times. The reaction mixture was cooled to 0-5°C in an ice-water bath, a drop of triethylamine was added, the crude product 5c (20 mg, 47.1 μmol) was added, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylchloromorpholine (25.4 mg, 86.2 μmol) was added, and the mixture was reacted for 40 minutes under stirring in an ice-water bath. 15 mL of water was added to the reaction mixture, and it was extracted with ethyl acetate (20 mL x 3). The organic phase was pooled. The organic phase was washed with saturated sodium chloride solution (20 mL x 2), dried by using anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography developing solvent system B to give the title product 5d (9H-fluoren-9-yl)methyl(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)carbamic acid (23.7 mg, yield: 78.9%). MSm / z(ESI):842.1[M+1]

[0261] In the fifth step, 5d (30 mg, 35.7 μmol) was dissolved in 3 mL of dichloromethane, and 1.5 mL of diethylamine was added and stirred 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, which was repeated twice. 4.5 mL of n-hexane was added to the residue and triturated. After standing, the supernatant was poured off to keep the solid. The solid residue was concentrated under reduced pressure and dried at the pump to give the crude product 5e, 2-((2-aminoacetamido)methoxy)-2-cyclopropyl-N-((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)acetamide (23 mg), which was used directly in the next reaction without purification. MSm / z(ESI):638.0[M+18]

[0262] In the sixth step, the crude product 5e (20 mg, 32.3 μmol) was dissolved in 1 mL of N,N-dimethylformamide and purged with argon three times. The reaction mixture was cooled to 0-5°C in an ice-water bath, 0.5 mL of a solution of 4g (31.8 mg, 76.3 μmol) in N,N-dimethylformamide was added, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylchloromorpholine (27.8 mg, 94.3 μmol) was added and reacted for 10 minutes under stirring in an ice-water bath. The reaction mixture was warmed to room temperature by removing the ice-water bath and reacted for 1 hour under stirring to produce compound 5. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5μm 19*250mm; mobile phase: A-water (10mmol NH4OAc): B-acetonitrile, gradient elution, flow rate: 18mL / min). The corresponding components were collected and concentrated under reduced pressure to give products 5-A and 5-B (3.6mg, 2.6mg). MSm / z(ESI):1074.4[M+1]

[0263] Single structure compound 5-A (short retention time) UPLC analysis: retention time 1.14 min, purity: 85% (column: ACQUITY UPLC BEHC18 1.7 μm 2.1 * 50 mm, mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile)

[0264] 1 H NMR (400 MHz, DMSO - d 6 ): 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)

[0265] Single - structure compound 5 - B (long retention time): UPLC analysis: retention time 1.16 min, purity: 89% (column: ACQUITY UPLC BEHC18 1.7 μm 2.1 * 50 mm, mobile phase: A - water (5 mmol NH4OAc), B - acetonitrile)

[0266] 1 H NMR (400 MHz, DMSO - d 6): δ8.68~8.60(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, 2) H), 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)

[0267] The preparation method of other intermediates was based on the method of Intermediate 5.

[0268] The prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.347 mL, 3.47 μmol) was added to an aqueous solution of antibody hu2F7 in PBS buffer (pH=6.5 0.05M aqueous PBS buffer; 7.3 ml, 13.8 mg / ml, 0.681 μmol) at 37° C. The reaction mixture was placed in a water bath shaker and reacted for 3 hours under shaking at 37° C. The reaction was terminated, the reaction solution was cooled to 25° C. in a water bath, diluted to 14.0 ml, and 3.3 ml of the solution was removed for the next reaction.

[0269] Compound 5-A (3.0 mg, 3.72 μmol) was dissolved in 0.15 mL of DMSO and added to 3.3 mL of the above solution. The reaction mixture was placed in a water bath shaker and reacted for 3 hours under shaking at 25°C. The reaction was terminated. The reaction solution was desalted and purified by using a Sephadex G25 gel column (elution phase: pH 6.5 0.05 M PBS buffer aqueous solution, containing 0.001 M EDTA) to obtain the example 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 value y was determined by ultraviolet light method. A cuvette filled with sodium succinate buffer was placed in the reference absorbance cell and the sample judgment absorbance cell, respectively, and after subtracting the solvent blank, a cuvette filled with the test solution was placed in the sample judgment absorbance cell. The absorbance was measured at 280 nm and 370 nm.

[0270] Data Processing: Antibody amount C mab was determined by establishing a standard curve and measuring the absorbance at a wavelength of 280 nm. Drug was determined by measuring the absorbance at a wavelength of 370 nm.

[0271] Average drug load y=C Drug / C mab For the exemplary product hu2F7-exatecan (compound 34), y was determined to be 7.6 by the methods described above. A sample of hu2F7-exatecan (y=8) was obtained by UV-HPLC purification.

[0272] The preparation method of other antibody conjugates was referred to the method for compound 34.

[0273] [Example 10: Killing activity of antibody-drug conjugates against tumor cells] To test the killing effect of the antibody-drug conjugate of the present invention on tumor cells, it was evaluated using MX-1 breast cancer cells. MX-1 cells were harvested, centrifuged, and counted. The cell concentration was 0.44×10 in complete medium. 6The cells were adjusted to 100,000 cells / mL and plated in 60 wells of a white 96-well plate at 40,000 cells per well in 90 μL. 100 μL of PBS was added to the remaining surrounding wells. The cell plate was incubated at 37 °C and 5% CO 2 The cells were placed in a 37°C incubator and incubated overnight. On the second day of the experiment, antibody-drug conjugate solutions were prepared in PBS in 96-well V-bottom plates starting at a concentration of 1000 nM (3-fold dilutions and 9 concentrations). After preparation was complete, the solutions were added in duplicate to white 96-well plates at 10 μL per well. The cell plates were incubated at 37°C, 5% CO 2 The plates were placed in an incubator at 37 °C and the culture was allowed to continue for 72 hours. On the fifth day of the experiment, the plates were detected and read. The cell culture plate was removed. Two control wells were set up. 100 μL of medium containing 40,000 cells was added to each well, and after equilibration to room temperature, 50 μL of CTG solution (Promega G7573) was added to each well. The mixture was shaken and mixed, left in the dark for 10 minutes, and then detected by using the luminescence program of the microplate reader. Maximum killing rate = (fluorescence value of 1-1000 nM well / fluorescence value of control well)%. The experimental results are shown in Table 4.

[0274] [Table 8]

[0275] [Example 11: Inhibitory effect of antibody-drug conjugates on tumor cell proliferation] To test the killing effect of the antibody-drug conjugate of the present invention on tumor cells, it was evaluated using SK-BR-3 (ATCC#HTB30) breast cancer cells. SK-BR-3 cells were harvested, centrifuged, and counted. The cell concentration was 0.44×10 in complete medium. 6 The cells were adjusted to 100,000 cells / mL and plated in 60 wells of a white 96-well plate at 40,000 cells per well in 90 μL. 100 μL of PBS was added to the remaining surrounding wells. The cell plate was incubated at 37 °C and 5% CO 2The cells were placed in a 37°C incubator and cultured overnight. On the second day of the experiment, antibody-drug conjugate solutions were prepared in PBS in 96-well V-bottom plates starting at a concentration of 1000 nM (3-fold dilutions and 9 concentrations). After preparation was completed, the solutions were added to a white 96-well plate at 10 μL per well in duplicate. Two other control wells were set up and 10 μL of PBS was added to each well. The cell plates were incubated at 37°C, 5% CO 2 The plates were placed in an incubator at 37 °C and the culture was allowed to continue for 72 hours. On the fifth day of the experiment, the plates were detected and read. The cell culture plate was taken out. After equilibration to room temperature, 50 μL of CTG solution (Promega G7573) was added to each well. The mixture was shaken and mixed, left in the dark for 10 minutes, and then detected by using the luminescence program of the microplate reader. Maximum inhibition rate = (fluorescence value of 1-1000 nM well / fluorescence value of control well)%. The experimental results are shown in Table 5.

[0276] [Table 9]

[0277] Example 12: Evaluation of in vivo efficacy of antibody-drug conjugates conjugated to MMAF. After formation of transplanted tumors in mice using MX-1 cells, the antitumor effect of the antibody-drug conjugate of the present invention was evaluated. 6The cells were subcutaneously injected into immunodeficient nude mice (BALB / cNude). After 2 weeks, intravenous injection of antibody-drug conjugates hu2G6-MC-MMAF and hu2F7-MC-MMAF was performed at a frequency of once a week and at a dose of 1.5 mg / kg or 3 mg / kg. Human IgG protein was used as a control at a dose of 3 mg / kg. There were 5 mice in each control or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume. Tumor inhibition rate = 100% - (tumor volume of treatment group on day 21 - tumor volume of treatment group on day 0) / (tumor volume of control group on day 21 - tumor volume of 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 dose-dependent antitumor effects. Both hu2G6-MC-MMAF and hu2F7-MC-MMAF showed 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 have a killing effect on already formed tumors.

[0278] [Table 10]

[0279] Example 13: Evaluation of in vivo efficacy of antibody-drug conjugates with different drug loadings. To further test the antibody-drug conjugates with different drug loadings, the antibody-drug conjugates were prepared according to 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 transplanted tumors in mice using MX-1, the antitumor effects of the antibody-drug conjugates of the present invention were evaluated. MX-1 cells 5×10 6The cells were subcutaneously injected into immunodeficient nude mice. After 2 weeks, intravenous injection of antibody-drug conjugates hu2F7-MC-MMAF (y=2) and hu2F7-MC-MMAF (y=4) was performed at a frequency of once a week and a dose of 3 mg / kg. Human IgG protein was used as a control at a dose of 3 mg / kg. There were 5 mice in each control or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume. Tumor inhibition rate=100%-(tumor volume of treatment group on day 21-tumor volume of treatment group on day 0) / (tumor volume of control group on day 21-tumor volume of control group on day 0). The experimental results are shown in FIG. 3 and Table 7. Both hu2F7-MC-MMAF (y=2) and hu2F7-MC-MMAF (y=4) show antitumor effects. At a dose of 3 mg / kg, hu2F7-MC-MMAF (y=4) had a stronger antitumor effect than hu2F7-MC-MMAF (y=2), showing a tumor inhibition rate of over 100%, which means that hu2F7-MC-MMAF (y=4) could not only inhibit tumor growth but also have a killing effect on already formed tumors (Figure 4).

[0280] [Table 11]

[0281] Example 14: Evaluation of in vivo efficacy of antibody-drug conjugates conjugated to exatecan. To test the antibody-drug conjugate conjugated to exatecan, the antibody-drug conjugate was prepared according to the method of Example 9 and purified by HPLC to obtain hu2F7-exatecan (compound 34, y=8). After the formation of transplanted tumors in mice using MX-1, the antitumor effect of the antibody-drug conjugate of the present invention was evaluated. MX-1 cells 5×10 6The cells were injected subcutaneously into immunodeficient nude mice. After 2 weeks, intravenous injection of antibody-drug conjugate hu2F7-exatecan (y=8) was performed at a frequency of once a week and at doses of 5 mg / kg and 10 mg / kg. Human IgG protein was used as a control at a dose of 5 mg / kg. There were 5 mice in each control or treatment group. The tumor inhibition rate was calculated by measuring the tumor volume. Tumor inhibition rate=100%-(tumor volume of treatment group on day 18-tumor volume of treatment group on day 0) / (tumor volume of control group on day 18-tumor volume of control group on day 0). The experimental results are shown in Table 8. hu2F7-exatecan showed a tumor inhibition rate of over 100% at both doses of 5 mg / kg and 10 mg / kg, which means that hu2F7-exatecan (y=8) can not only inhibit tumor growth but also have a killing effect on already formed tumors.

[0282] [Table 12]

[0283] [Example 15: Experiment on bystander killing activity of antibody-drug conjugates conjugated to exatecan] To test the effect of the antibody-drug conjugate of the present invention conjugated to exatecan on the killing of B7-H4 negative cells through bystander effect, the killing activity of the antibody-drug conjugate against a myeloma cell line negative for B7-H4 expression was detected by using the ONE-Glo™ Luciferase Assay Detection Kit. MX-1 cells were harvested, centrifuged, counted, and cultured at 7.5×10 in complete medium. 6 The cell suspension was adjusted to 1.5 × 10 cells / mL and seeded on a white 96-well plate. 33 μL of the MX-1 cell suspension was added to each well of the "mixed cell group" and 33 μL of PBS was added to each well of the "single cell group". B7-H4 expression negative NCI-H929-LUC tumor cells (Cobioer catalog number CBP30061L) were harvested. The cell concentration was 1.5 × 10 633 μL of the NCI-H929-LUC cell suspension was added to each well of the "mixed cell group" and "single cell group." The cell plate was incubated at 37°C, 5% CO 2 The cells were placed in a 37°C incubator and incubated overnight. On the second day of the experiment, the highest concentration of antibody-drug conjugate to be tested, hu2F7-exatecan (compound 34, y=8), was uniformly adjusted to 3000 nM and diluted to obtain a 5-fold gradient of 9 doses. 33 μL of diluted antibody-drug conjugate working solution was added to each well of the experimental plate and mixed gently. The cell plates were incubated at 37°C, 5% CO 2 The plates were placed in a 300-well incubator and incubated for 48 hours. The cell culture plates were removed and equilibrated to room temperature. Then 100 μL of ONE-Glo™ Luciferase Reagent (Promega E6120) was added to each well. The cells were permeabilized and lysed for 10 minutes in the dark at room temperature. The cell lysates were centrifuged at 1000 rpm / min for 1 minute and then transferred to a clear-bottom 96-well plate at 180 μL per well. The plates were read at 490 nm wavelength on a microplate reader. The data was analyzed using Graphpad Prism software. The experimental results are shown in Table 9. The ONE-Glo™ Luciferase Reagent can specifically detect the viability of NCI-H929-LUC cells. Hu2F7-exatecan efficiently killed NCI-H929-LUC cells in the "mixed cell population," 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 also non-sensitive B7-H4-negative cells (in the "single cell population").

[0284] [Table 13]

Claims

1. General formula (A): 【Chemistry 1】 (In the formula, D is for exatecan: 【Chemistry 2】 and L 1 and L 2 is the following: 【Chemistry 3】 and y is a number between 1 and 20, Ab is a B7-H4 antibody or an antigen-binding fragment thereof comprising an antibody light chain variable region and an antibody heavy chain variable region; the antibody heavy chain variable region comprises HCDR1 shown in SEQ ID NO: 9, HCDR2 shown in SEQ ID NO: 10, and HCDR3 shown in SEQ ID NO: 11; The antibody light chain variable region comprises LCDR1 shown in SEQ ID NO: 12, LCDR2 shown in SEQ ID NO: 13, and LCDR3 shown in SEQ ID NO:

14. or a pharma- ceutically acceptable salt or solvate thereof.

2. The antibody-drug conjugate of claim 1, or a pharma- ceutically acceptable salt or solvate thereof, wherein Ab is a murine antibody or fragment thereof, a chimeric antibody or fragment thereof, or a humanized antibody or fragment thereof.

3. Ab is (a), (b) and (c): (a) light chain and heavy chain framework regions derived from human germline light chain and heavy chain sequences or variants thereof; (b) a heavy chain constant region derived from human IgG1 or a variant thereof, IgG2 or a variant thereof, IgG3 or a variant thereof, or IgG4 or a variant thereof; (c) a light chain constant region derived from a human κ chain, λ chain, or a variant thereof 3. The antibody-drug conjugate of claim 1 or 2, or a pharma- ceutically acceptable salt or solvate thereof, further comprising any one of the following:

4. The antibody-drug conjugate or a pharma- ceutically acceptable salt or solvate thereof described in claim 3, wherein the heavy chain constant region is a heavy chain constant region derived from human IgG1, IgG2 or IgG4, or a heavy chain constant region of an IgG having enhanced ADCC toxicity after amino acid mutation, or a heavy chain constant region shown in SEQ ID NO:

54.

5. The antibody-drug conjugate or a pharma- ceutically acceptable salt or solvate thereof described in claim 3, wherein the light chain constant region is a light chain constant region derived from human κ chain, or a light chain constant region shown in SEQ ID NO:

55.

6. 6. The antibody-drug conjugate of any one of claims 1 to 5, wherein the Ab comprises a light chain variable region of SEQ ID NO: 18, or a light chain variable region that is at least 90%, 95% or 99% identical to SEQ ID NO: 18, or a pharma- ceutically acceptable salt or solvate thereof.

7. 7. The antibody-drug conjugate of any one of claims 1 to 6, wherein the Ab comprises a heavy chain variable region of SEQ ID NO: 17, or a heavy chain variable region that is at least 90%, 95% or 99% identical to SEQ ID NO: 17, or a pharma- ceutically acceptable salt or solvate thereof.

8. The antibody-drug conjugate of any one of claims 1 to 7, or a pharma- ceutically acceptable salt or solvate thereof, wherein the light chain of the Ab is selected from the group consisting of SEQ ID NO:22 and a full-length light chain that is at least 90%, 95% or 99% identical to SEQ ID NO:

22.

9. 9. The antibody-drug conjugate of any one of claims 1 to 8, or a pharma- ceutically acceptable salt or solvate thereof, wherein the heavy chain of the Ab is selected from the group consisting of SEQ ID NO:21 and a full-length heavy chain that is at least 90%, 95% or 99% identical to SEQ ID NO:

21.

10. The antibody-drug conjugate of any one of claims 1 to 9, or a pharma- ceutically acceptable salt or solvate thereof, wherein the heavy chain variable region and light chain variable region of the Ab are the heavy chain variable region shown in SEQ ID NO: 17 and the light chain variable region shown in SEQ ID NO:

18.

11. 11. The antibody-drug conjugate of claim 10, or a pharma- ceutically acceptable salt or solvate thereof, wherein the Ab has a light chain as set forth in SEQ ID NO:22 and a heavy chain as set forth in SEQ ID NO:

21.

12. The antibody or antigen-binding fragment thereof is Fab, Fab', F(ab') 2 12. The antibody-drug conjugate of claim 1 , or a pharma- ceutically acceptable salt or solvate thereof, selected from the group consisting of an antigen-binding fragment of a peptide comprising a single chain antibody, a dimerized V region, a disulfide bond stabilized V region, and a CDR.

13. -L 2 -L 1 -But the following structure: 【Chemistry 4】 13. The antibody-drug conjugate of any one of claims 1 to 12, or a pharma- ceutically acceptable salt or solvate thereof, selected from the group consisting of:

14. -L 2 -L 1 -But the following structure: 【Chemistry 5】 13. The antibody-drug conjugate according to any one of claims 1 to 12, or a pharma- ceutically acceptable salt or solvate thereof,

15. 5. The antibody-drug conjugate comprising: 【Chemistry 6】 15. The antibody-drug conjugate of claim 1, or a pharma- ceutically acceptable salt or solvate thereof, wherein:

16. The antibody-drug conjugate may comprise the compound: 【Chemistry 7】 wherein y is selected from 2 to 10; hu2F7 is an antibody having a light chain shown in SEQ ID NO: 22 and a heavy chain shown in SEQ ID NO:

21.

16. The antibody-drug conjugate of any one of claims 1 to 15, or a pharma- ceutically acceptable salt or solvate thereof, selected from the group consisting of:

17. The antibody-drug conjugate of claim 16, or a pharma- ceutically acceptable salt or solvate thereof, wherein y is selected from 4 to 8.

18. The antibody-drug conjugate of claim 17, or a pharma- ceutically acceptable salt or solvate thereof, wherein y is selected from 6 to 8.

19. The antibody-drug conjugate of claim 18, or a pharma- ceutically acceptable salt or solvate thereof, wherein y is 7 or 8.

20. The antibody-drug conjugate of claim 19, or a pharma- ceutically acceptable salt or solvate thereof, wherein y is 8.

21. The process is as follows: 【Chemistry 8】 21. A method for producing the antibody-drug conjugate of any one of claims 1 to 20, or a pharma- ceutically acceptable salt or solvate thereof, comprising:

22. 21. A pharmaceutical composition comprising the antibody-drug conjugate of any one of claims 1 to 20, or a pharma- ceutically acceptable salt or solvate thereof, and one or more pharma- ceutically acceptable carriers.

23. A pharmaceutical for treating a disease associated with human B7-H4, comprising an antibody-drug conjugate described in any one of claims 1 to 20 or a pharma- ceutically acceptable salt or solvate thereof, or a pharmaceutical composition described in claim 22.

24. The present invention is characterized in that it is a medicine for treating cancers that highly express B7-H4, and the cancers are selected from the group consisting of human brain astroblastoma, human pharyngeal cancer, adrenal tumor, AIDS-related cancer, alveolar soft part sarcoma, astrocytoma, bladder cancer, bone cancer, brain and spinal cancer, metastatic brain tumor, breast cancer, carotid ball tumor, cervical cancer, chondrosarcoma, chordoma, chromophobe cell carcinoma of the kidney, clear cell carcinoma, colon cancer, colorectal cancer, connective tissue proliferative small round cell tumor, ependymoma, Ewing's sarcoma, extraskeletal myxoid chondrosarcoma, fibrous dysplasia ossificans, fibrous dysplasia ossificans, gallbladder or cholangiocarcinoma, gastric cancer, gestational trophoblastic disease, germ cell tumor, head and neck cancer, hepatocellular carcinoma, and insular cancer.

24. The pharmaceutical composition according to claim 23, wherein the therapeutic agent is selected from the group consisting of cell tumors, Kaposi's sarcoma, kidney cancer, leukemia, liposarcoma / malignant lipoma, liver cancer, lymphoma, lung cancer, medulloblastoma, melanoma, meningioma, multiple endocrine neoplasia, multiple myeloma, myelodysplastic syndrome, neuroblastoma, neuroendocrine tumor, ovarian cancer, pancreatic cancer, papillary thyroid cancer, parathyroidoma, childhood cancer, peripheral schwannoma, pinealocytoma, pituitary tumor, prostate cancer, posterior uveal melanoma, metastatic kidney 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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