Combination Therapies for the Treatment of Cancer Comprising B7-H4 Antibody Drug Conjugates - Patent application

Abstract

A method of treating cancer in a subject is provided, the method comprising administering to the subject i) an antibody-drug conjugate (ADC), ii) a cytotoxic agent, and iii) an additional agent, wherein the additional agent is a PARP1 inhibitor or an ATR inhibitor or a pharma- ceutically acceptable salt thereof.The present disclosure further provides a kit comprising i) an antibody-drug conjugate (ADC), ii) a cytotoxic agent, and iii) an additional agent, wherein the additional agent is a PARP1 inhibitor or an ATR inhibitor or a pharma- ceutically acceptable salt thereof.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 310,967, filed February 16, 2022, and U.S. Provisional Patent Application No. 63 / 378,295, filed October 4, 2022, which are incorporated by reference in their entireties for all purposes.

[0002] Incorporation by Reference of Electronically Submitted Materials The computer readable nucleotide / amino acid sequence listing submitted concurrently herewith is incorporated by reference in its entirety and is identified as follows: One 54,907 byte XML file entitled "B7H4-101-WO-PCT_SeqList.xml" created on February 8, 2023.

[0003] The present disclosure provides a method of treating cancer in a human subject, the method comprising administering to the subject i) an antibody-drug conjugate (ADC), ii) a cytotoxic agent, and iii) an additional agent, where the additional agent is a PARP1 inhibitor or an ATR inhibitor or a pharma- ceutically acceptable salt thereof.The present disclosure further provides a kit comprising i) an antibody-drug conjugate (ADC), ii) a cytotoxic agent, and iii) an additional agent, where the additional agent is a PARP1 inhibitor or an ATR inhibitor or a pharma- ceutically acceptable salt thereof. [Background technology]

[0004] Surgery, radiotherapy or chemotherapy, alone or in combination, are the most conventional and widely used treatments for cancer.While these treatments are effective in removing or killing cancer cells, they often cause undesirable side effects in treated patients, such as hair loss, anemia, severe nausea and the death of healthy cells.Due to these limitations, there is an urgent need for innovative and less harmful cancer treatments.

[0005] Antibody-based cancer therapy relies on the recognition and binding of antibody-drug conjugates to specific proteins on cancer cells. Antibody-drug conjugates (ADCs) can exploit the specificity of the antibody portion of the conjugate to deliver highly toxic drugs directly to cells to kill them. The use of antibody or ADC cancer therapy in combination with other small molecule-based cancer therapies can improve treatment outcomes by attacking malignant cells and tumors in more than one way. Summary of the Invention [Means for solving the problem]

[0006] In some aspects, the present disclosure is directed to a method of treating cancer in a human subject in need thereof, the method comprising: A) An antibody-drug conjugate (ADC), i. An antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively, or functional variants thereof; b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, or functional variants thereof; c) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively, or functional variants thereof; d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively, or functional variants thereof; or e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively, or functional variants thereof. an antibody or antigen-binding fragment thereof comprising ii. a cleavable linker, and iii. Cytotoxic agents and an antibody-drug conjugate (ADC) comprising B) a further agent which is a PARP1 inhibitor or an ATR inhibitor or a pharma- ceutically acceptable salt thereof; to a human subject. In some embodiments, the additional agent is AZD5305. In some embodiments, the additional agent is AZD6738.

[0007] In some aspects of the method, the cancer comprises cancer cells that express B7-H4. In some aspects, the cancer further comprises cancer cells that do not express B7-H4. In some aspects, the cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, hematological cancer, endometrial cancer, cholangiocarcinoma, NSCLC (squamous and / or adenocarcinoma), gastrointestinal cancer such as gastric cancer and colorectal cancer, and lung cancer. In some aspects, the cancer is breast cancer selected from hormone receptor positive (HR+) breast cancer, human epidermal growth factor receptor 2 positive (HER2+) breast cancer, and triple negative breast cancer (TNBC). In some aspects, the cancer is a homologous recombination repair deficient (HRD) cancer. In some embodiments, the cancer comprises one or more cells with a mutation in an HRD gene selected from BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L. In some embodiments, the mutated HRD gene is selected from BRCA1, BRCA2, and ATM.

[0008] In some aspects of the method, the antibody or antigen-binding fragment thereof comprises: i. a variable heavy (VH) chain and a variable light (VL) chain comprising the amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 34, respectively, or a functional variant thereof; ii. a variable heavy (VH) chain and a variable light (VL) chain comprising the amino acid sequences of SEQ ID NO: 33 and SEQ ID NO: 34, respectively, or a functional variant thereof; iii. A variable heavy (VH) chain and a variable light (VL) chain comprising the amino acid sequences of SEQ ID NO: 43 and SEQ ID NO: 34, respectively, or a functional variant thereof; iv. A variable heavy (VH) chain and a variable light (VL) chain comprising the amino acid sequences of SEQ ID NO: 46 and SEQ ID NO: 34, respectively, or a functional variant thereof; v. A variable heavy (VH) chain and a variable light (VL) chain comprising the amino acid sequences of SEQ ID NO: 47 and SEQ ID NO: 34, respectively, or a functional variant thereof; vi. A VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 31 and SEQ ID NO: 32, respectively, or a functional variant thereof; vii. A VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 36, respectively, or a functional variant thereof; viii. A VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 37 and SEQ ID NO: 38, respectively, or a functional variant thereof; or ix. A VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 39 and SEQ ID NO: 40, respectively, or functional variants thereof. Includes.

[0009] In some aspects, the antibody or antigen-binding fragment thereof comprises: i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively, or functional variants thereof. Includes.

[0010] In some aspects, the antibody or antigen-binding fragment thereof comprises: i. A VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 34, respectively, or a functional variant thereof. Includes.

[0011] In some embodiments of the method, the antibody or antigen-binding fragment thereof binds to the OVCAR4 cell line.

[0012] In some embodiments of the methods, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 51 and a light chain comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 48 and a light chain comprising the amino acid sequence of SEQ ID NO: 44.

[0013] In some aspects, the antibody or antigen-binding fragment thereof is a monoclonal antibody. In some aspects, the antibody or antigen-binding fragment thereof is a humanized monoclonal antibody.

[0014] In some embodiments, the cleavable linker is a mp-PEG8-val-ala linker.

[0015] In some embodiments, the cytotoxic agent is a topoisomerase inhibitor. In some embodiments, the topoisomerase inhibitor is represented by the formula A* [ka] It is a compound of the formula:

[0016] In some embodiments of the method, ii) the linker and iii) the cytotoxic agent are the following compounds: [ka] are selected together.

[0017] In some aspects of the method, ii) the linker and iii) the cytotoxic agent together are the compound SG3932.

[0018] In some embodiments of the methods, the ADC has a drug-to-antibody ratio (DAR) of about 1 to about 8. In some embodiments, the ADC has a DAR of about 8.

[0019] In some aspects, the present disclosure provides a method for producing a method for treating a cancer cell comprising: A) An antibody-drug conjugate (ADC), i. An antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively, or functional variants thereof; b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, or functional variants thereof; c) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively, or functional variants thereof; d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively, or functional variants thereof; or e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively, or functional variants thereof. an antibody or antigen-binding fragment thereof comprising ii. a cleavable linker, and iii. Cytotoxic agents and an antibody-drug conjugate (ADC) comprising B) and a further agent which is a PARP1 inhibitor or an ATR inhibitor or a pharma- ceutically acceptable salt thereof. In some embodiments, the additional agent is AZD5305. In some embodiments, the additional agent is AZD6738.

[0020] In some aspects of the kit, the antibody or antigen-binding fragment thereof comprises: i. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively, or functional variants thereof. Includes.

[0021] In some aspects of the kit, the antibody or antigen-binding fragment thereof comprises: i. A VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 34, respectively, or a functional variant thereof. Includes.

[0022] In some embodiments of the kit, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 41. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 52. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO: 42. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 51 and a light chain comprising the amino acid sequence of SEQ ID NO: 44. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 48 and a light chain comprising the amino acid sequence of SEQ ID NO: 44.

[0023] In some embodiments of the kit, the cleavable linker is an mp-PEG8-val-ala linker.

[0024] In some embodiments of the kit, the cytotoxic agent is a topoisomerase inhibitor. In some embodiments, the topoisomerase inhibitor is represented by the formula A* [ka] It is a compound of the formula:

[0025] In some embodiments of the kit, ii) the linker and iii) the cytotoxic agent are the following compounds: [ka] are selected together.

[0026] In some embodiments of the kit, ii) the linker and iii) the cytotoxic agent together are the compound SG3932.

[0027] In some embodiments of the kit, the ADC has a drug-to-antibody ratio (DAR) of about 1 to about 8. In some embodiments, the ADC has a DAR of about 8.

[0028] In some aspects, the present disclosure is directed to a method of treating cancer in a human subject in need thereof, the method comprising: A) An antibody-drug conjugate (ADC), i. An antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively, or functional variants thereof; b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, or functional variants thereof; c) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively, or functional variants thereof; d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively, or functional variants thereof; or e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively, or functional variants thereof. and ii.Formula: [ka] A cleavable linker and a cytotoxic agent conjugated to an antibody or antigen-binding fragment thereof, comprising: and an antibody-drug conjugate (ADC) comprising B) AZD5305 or a pharma- ceutically acceptable salt thereof; to a human subject.

[0029] In some aspects, the present disclosure is directed to a method of treating cancer in a human subject in need thereof, the method comprising: A) An antibody-drug conjugate (ADC), i. An antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, f) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively, or functional variants thereof; g) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, or functional variants thereof; h) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively, or functional variants thereof; i) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively, or functional variants thereof; or j) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30, respectively, or functional variants thereof. and ii.Formula: [ka] and a cytotoxic agent conjugated to an antibody or antigen-binding fragment thereof, comprising: and an antibody-drug conjugate (ADC) comprising B) AZD6738 or a pharma- ceutically acceptable salt thereof; to a human subject.

[0030] In some aspects of the method, the cancer comprises cancer cells that express B7-H4. In some aspects, the cancer further comprises cancer cells that do not express B7-H4. In some aspects, the cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, hematological cancer, endometrial cancer, cholangiocarcinoma, NSCLC (squamous and / or adenocarcinoma), gastrointestinal cancer such as gastric cancer and colorectal cancer, and lung cancer. In some aspects, the cancer is breast cancer selected from hormone receptor positive (HR+) breast cancer, human epidermal growth factor receptor 2 positive (HER2+) breast cancer, and triple negative breast cancer (TNBC). In some aspects, the cancer is a homologous recombination repair deficient (HRD) cancer. In some embodiments, the cancer comprises one or more cells with a mutation in an HRD gene selected from BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L. In some embodiments, the mutated HRD gene is selected from BRCA1, BRCA2, and ATM.

[0031] The following drawings form part of the present specification and are included to further demonstrate illustrative embodiments of certain aspects of the present disclosure. [Brief description of the drawings]

[0032] [Figure 1] 1 shows the results of a cytotoxicity assay using E02-GL-SG3932 and AZD5305 treatment in DLD-1-BRCA wild-type cells engineered to express B7-H4. [Diagram 2]FIG. 1 shows the Bliss synergy score matrix of DLD-1-BRCA wild type cells treated with E02-GL-SG3932 and AZD5305 combination therapy. [Diagram 3] 1 shows the results of a cytotoxicity assay using E02-GL-SG3932 and AZD5305 treatment on DLD-1-XMAN-BRCA2- / - cells engineered to express B7-H4. [Figure 4] FIG. 1 shows the Bliss synergy score matrix of DLD-1-XMAN-BRCA2− / − cells treated with E02-GL-SG3932 and AZD5305 combination therapy. [Diagram 5] 1 shows the results of a cytotoxicity assay using E02-GL-SG3932 and AZD5305 treatment in MX-1 cells. [Figure 6] FIG. 1 shows the Bliss synergy score matrix of MX-1 cells treated with the combination therapy of E02-GL-SG3932 and AZD5305. [Figure 7] FIG. 1 shows the in vivo antitumor efficacy of individual E02-GL-SG3932 and AZD5305 treatments in a mouse model. [Figure 8] 1 shows the results of in vivo antitumor efficacy experiments of E02-GL-SG3932 and AZD5305 combination therapy in a mouse model. [Figure 9] 1 shows the mean tumor volumes of in vivo antitumor efficacy experiments of E02-GL-SG3932 and AZD5305 individual and combination therapy in a mouse model. [Figure 10] 1 shows the mean tumor volumes of in vivo antitumor efficacy experiments of E02-GL-SG3932 and AZD6738 individual and combination therapy in a mouse model. [Figure 11]Results of B7-H4 expression in human tumors as described in Example 6 are shown. Figure 11A: IHC was used to assess the expression of B7-H4 across multiple tumor types. The distribution of B7-H4 expression in the display is shown by the percentage of cells expressing B7-H4 positive at any intensity. Figure 11B: Representative images of B7-H4 IHC staining in endometrial cancer, cholangiocarcinoma, ER+ breast cancer or TNBC and ovarian cancer. Figure 11C: Quantitative image analysis showing the distribution of mean optical density (OD) of B7-H4 expression in each tumor cell membrane in primary tumor surgically resected, formalin-fixed, paraffin-embedded (FFPE) samples of TNBC (n=196). Each truncated violin plot corresponds to an individual donor sample. The line corresponds to the median value. [Figure 12A] Figures 12A-12B: Antitumor efficacy results from E02-GL-SG3932 in combination with PARP1 selective inhibitor AZD5305 in TNBC PDX models as described in Example 6. Tumors were established as described in Example 6 and treatments were administered at the doses indicated in each panel. ADC was delivered as a single bolus IV injection and AZD5305 was delivered by oral gavage once daily for 28 days. Figures 12A-12B: Antitumor efficacy results from (A) 1.25 mg / kg or (B) 3.5 mg / kg ADC treatment alone or in combination with AZD5305 in the B7-H4 high expressing BRCA WT HBCx-39 model. Figures 12C-12D: Activity resulting from treatment in (C) B7-H4 high expressing BRCA1-mutant HBCx-24 and (D) BRCA1-downexpressing HBCx-11 models. 12E-12F: Efficacy resulting from treatment of (E) HBCx-8, a BRCA1-mutant model, and (F) HBCx-2, a BRCA WT model with low B7-H4 expression. Data represent mean tumor volume ± standard error of the mean (n=4 or 5 animals / group). [Figure 12B] This is a continuation of Figure 12A. [Figure 12C] This is a continuation of Figure 12B. [Figure 12D] This is a continuation of Figure 12C. [Figure 12E] This is a continuation of Figure 12D. [Figure 12F] Continuation of Figure 12E. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] Disclosed herein are methods, combinations, and kits that include administering an antibody-drug conjugate (ADC) together with a second agent, such as a small molecule drug. The method combinations and kits can be used to treat cancer in a subject, as described herein.

[0034] In some aspects, the disclosure provides a method of treating cancer in a human subject in need thereof, comprising administering to the human subject A) an antibody-drug conjugate (ADC) comprising i. an antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, ii. a cleavable linker, and iii. a cytotoxic agent; and B) a PARP1 (poly(ADP-ribose) polymerase 1) inhibitor. In some aspects, the disclosure provides a method of treating cancer in a human subject in need thereof, comprising administering to the human subject A) an antibody-drug conjugate (ADC) comprising i. an antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, ii. a cleavable linker, and iii. a cytotoxic agent; and B) an ATR (FRAP-associated protein 1, FRP1, MEC1, SCKL, SECKL1R) inhibitor.

[0035] In some aspects, the disclosure provides a method of treating cancer in a human subject in need thereof, the method comprising administering to the human subject A) an ADC comprising i. an antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, ii. a cleavable linker, and iii. a cytotoxic agent, and B) AZD5305 or a pharmaceutically acceptable salt thereof. In some aspects, the disclosure provides a method of treating cancer in a human subject in need thereof, the method comprising administering to the human subject A) an ADC comprising i. an antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, ii. a cleavable linker, and iii. a cytotoxic agent, and B) AZD6738 or a pharmaceutically acceptable salt thereof.

[0036] In some aspects, the disclosure provides a method of treating cancer in a human subject in need thereof, the method comprising: A) an ADC, an antibody or antigen-binding fragment thereof that binds to an i.B7-H4 polypeptide, the antibody or antigen-binding fragment thereof comprising: a) a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), a heavy chain CDR3 (HCDR3), a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variants thereof; b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, or functional variants thereof; c) an ADC, an antibody or antigen-binding fragment thereof that binds to an i.B7-H4 polypeptide, the antibody or antigen-binding fragment thereof comprising: 6. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:17 and SEQ ID NO:18, or functional variants thereof, respectively; d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, or functional variants thereof, respectively; or e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, or functional variants thereof, respectively; ii. a cleavable linker; and iii. a cytotoxic agent; and B) AZD5305 or a pharma- ceutically acceptable salt thereof to a human subject.

[0037] In some aspects, the disclosure provides a method of treating cancer in a human subject in need thereof, the method comprising: A) an ADC, an antibody or antigen-binding fragment thereof that binds to an i.B7-H4 polypeptide, the antibody or antigen-binding fragment thereof comprising: a) a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), a heavy chain CDR3 (HCDR3), a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variants thereof; b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, or functional variants thereof; c) an ADC, an antibody or antigen-binding fragment thereof that binds to an i.B7-H4 polypeptide, the antibody or antigen-binding fragment thereof comprising: 6. HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:17 and SEQ ID NO:18, or functional variants thereof, respectively; d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, or functional variants thereof, respectively; or e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, or functional variants thereof, respectively; ii. a cleavable linker; and iii. a cytotoxic agent; and B) AZD6738 or a pharma- ceutically acceptable salt thereof to a human subject.

[0038] Cancer Treatment In some embodiments, disclosed herein are methods of treating cancer associated with expression of B7-H4. In some embodiments, the cancer comprises cancer cells that express B7-H4. In some embodiments, the cancer is a tumor or other mass of malignant cells that comprises cancer cells that express B7-H4. In some embodiments, the cancer further comprises cancer cells that do not express B7-H4.

[0039] B7-H4 (also known as V-set domain-containing T-cell activation inhibitor 1, encoded by the VTCN1 gene) is a transmembrane polypeptide of the B7 family of costimulatory proteins. It is understood that B7-H4 is expressed on the surface of antigen-presenting cells for interaction with ligands of immune cells (e.g., T-lymphocytes, of which CD28 is a potential ligand). B7-H4 has been observed to be highly expressed on cells of various cancer types and is considered to be a tumor-associated antigen. Furthermore, B7-H4 expression is not limited to a particular cancer type, such that it represents a target antigen for treating a wide range of cancer types.

[0040] In a preferred embodiment, the cancer referred to herein is a cancer characterized by expression (preferably overexpression) of the B7-H4 molecule. In other words, the cancer referred to herein may comprise cancerous cells expressing B7-H4. The cancerous cells may be contained within a tumor. In another embodiment, the B7-H4 molecule is expressed in cancer cells at a level similar to that of expression in non-cancer cells. In another embodiment, the B7-H4 molecule is expressed in cancer cells at a level lower than that of expression in non-cancer cells.

[0041] "Treating" refers to a therapeutic measure that cures, slows, relieves symptoms, and / or stops the progression of a diagnosed pathological condition or disorder. Thus, those in need of treatment include those who already have the disorder. In some embodiments, a subject is successfully "treated" according to the methods provided herein for a disease or disorder (preferably cancer) when the patient shows, for example, a complete, partial, or transient reduction or disappearance of symptoms associated with the disease or disorder (preferably cancer).

[0042] In some embodiments, the methods of the present disclosure may be used to prevent the onset of cancers involving cancer cells expressing B7-H4. "Preventing" refers to prophylactic or defensive measures that prevent and / or delay the onset of the targeted pathological condition or disorder. Thus, those in need of prevention include those who are prone or susceptible to developing the disorder. In some embodiments, a disease or disorder (preferably cancer) is successfully prevented according to the methods provided herein if the patient develops, for example, fewer or less severe symptoms associated with the disease or disorder than a patient who has not been subjected to the methods of the present disclosure, either transiently or permanently, or develops symptoms associated with the disease or disorder with a delayed onset.

[0043] The terms "subject", "individual" and "patient" are used interchangeably herein to refer to a mammalian subject. In some embodiments, the "subject" is a human, a farm animal, a livestock animal, a sport animal and a zoo animal, such as a human, a non-human primate, a dog, a cat, a guinea pig, a rabbit, a rat, a mouse, a horse, a cow, etc. In some embodiments, the subject is a cynomolgus monkey (Macaca fascicularis). In a preferred embodiment, the subject is a human. In the methods of the present disclosure, the subject may not have been previously diagnosed as having cancer. Alternatively, the subject may have been previously diagnosed as having cancer. The subject may also be one who exhibits disease risk factors or is asymptomatic for cancer. The subject may also be one who is suffering from cancer or at risk for developing cancer. Thus, in some embodiments, the methods of the present disclosure may be used to determine the presence of cancer in a subject. For example, the subject may be one who has previously been diagnosed with cancer by alternative means. In some embodiments, the subject has previously undergone cancer treatment.

[0044] In some embodiments, disclosed herein is a method for treating a cancer selected from ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, hematological cancer, endometrial cancer, cholangiocarcinoma, NSCLC (squamous and / or adenocarcinoma), gastrointestinal cancer such as gastric cancer and colorectal cancer, and lung cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments of the method for treating breast cancer, the breast cancer is hormone receptor positive (HR+) breast cancer, human epidermal growth factor receptor 2 positive (HER2+) breast cancer, or triple negative breast cancer (TNBC). In some embodiments, the breast cancer is TNBC.

[0045] In some aspects of the method, the cancer is a homologous recombination repair deficient (HRD) cancer. In some aspects, the cancer comprises one or more cells with a mutation in an HRD gene selected from BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L. In some aspects, the mutated HRD gene is selected from BRCA1, BRCA2, and ATM. In some aspects, the mutated HRD gene is BRCA1. In some aspects, the mutated HRD gene is BRCA2. In some aspects, the mutated HRD gene is ATM.

[0046] Antibody or antigen-binding fragment The antibody drug conjugates provided herein comprise an antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide. The RNA, DNA and amino acid sequences of B7-H4 are known to those skilled in the art and can be found in many databases, such as the National Center for Biotechnology Information (NCBI) and UniProt databases. Examples of these sequences found in UniProt are Q7Z7D3 (VTCN1_HUMAN) for human B7-H4 and Q7TSP5 (VTCN1_MOUSE) for mouse B7-H4. The nucleotide sequence encoding human B7-H4 can be SEQ ID NO:53, more preferably SEQ ID NO:54. The polypeptide sequence of human B7-H4 is preferably SEQ ID NO:55.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively, or functional variants thereof. The antibody or antigen-binding fragment thereof comprising said sequences may be referred to herein as "ZY0EPQ-E02" or "EPQ-E02".

[0048] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively, or functional variants thereof. The antibody or antigen-binding fragment thereof comprising said sequences may be referred to herein as "ZY0EOB-F05" or "EOB-F05".

[0049] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively, or functional variants thereof. The antibody or antigen-binding fragment thereof comprising said sequences may be referred to herein as "ZY0EO5-E07" or "EO5-E07".

[0050] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively, or functional variants thereof. The antibody or antigen-binding fragment thereof comprising said sequences may be referred to herein as "ZY0EP0-C07" or "EP0-C07".

[0051] In certain aspects, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12, respectively, or functional variants thereof.

[0052] In other words, the antibody or antigen-binding fragment thereof preferably comprises: i. HCDR1 comprising the amino acid sequence of SEQ ID NO: 7 or a functional variant thereof; ii. HCDR2 comprising the amino acid sequence of SEQ ID NO: 8 or a functional variant thereof; iii. HCDR3 comprising the amino acid sequence of SEQ ID NO: 9 or a functional variant thereof; iv. LCDR1 comprising the amino acid sequence of SEQ ID NO: 10 or a functional variant thereof; v. LCDR2 comprising the amino acid sequence of SEQ ID NO: 11 or a functional variant thereof; and vi. LCDR3 comprising the amino acid sequence of SEQ ID NO: 12 or a functional variant thereof may include.

[0053] In some aspects, the antibody or antigen-binding fragment thereof comprises: i. HCDR1 comprising the amino acid sequence of SEQ ID NO: 1 or a functional variant thereof; ii. HCDR2 comprising the amino acid sequence of SEQ ID NO:2 or a functional variant thereof; iii. HCDR3 comprising the amino acid sequence of SEQ ID NO: 3 or a functional variant thereof; iv. LCDR1 comprising the amino acid sequence of SEQ ID NO: 4 or a functional variant thereof; v. LCDR2 comprising the amino acid sequence of SEQ ID NO:5 or a functional variant thereof; and vi. LCDR3 comprising the amino acid sequence of SEQ ID NO:6 or a functional variant thereof Includes.

[0054] In some aspects, the antibody or antigen-binding fragment thereof comprises: i. HCDR1 comprising the amino acid sequence of SEQ ID NO: 13 or a functional variant thereof; ii. HCDR2 comprising the amino acid sequence of SEQ ID NO: 14 or a functional variant thereof; iii. HCDR3 comprising the amino acid sequence of SEQ ID NO: 15 or a functional variant thereof; iv. LCDR1 comprising the amino acid sequence of SEQ ID NO: 16 or a functional variant thereof; v. LCDR2 comprising the amino acid sequence of SEQ ID NO: 17 or a functional variant thereof; and vi. LCDR3 comprising the amino acid sequence of SEQ ID NO: 18 or a functional variant thereof Includes.

[0055] In some aspects, the antibody or antigen-binding fragment thereof comprises: i. HCDR1 comprising the amino acid sequence of SEQ ID NO: 19 or a functional variant thereof; ii. HCDR2 comprising the amino acid sequence of SEQ ID NO: 20 or a functional variant thereof; iii. HCDR3 comprising the amino acid sequence of SEQ ID NO: 21 or a functional variant thereof; iv. LCDR1 comprising the amino acid sequence of SEQ ID NO: 22 or a functional variant thereof; v. LCDR2 comprising the amino acid sequence of SEQ ID NO: 23 or a functional variant thereof; and vi. LCDR3 comprising the amino acid sequence of SEQ ID NO: 24 or a functional variant thereof Includes.

[0056] In some aspects, the antibody or antigen-binding fragment thereof comprises: i. HCDR1 comprising the amino acid sequence of SEQ ID NO: 25 or a functional variant thereof; ii. HCDR2 comprising the amino acid sequence of SEQ ID NO: 26 or a functional variant thereof; iii. HCDR3 comprising the amino acid sequence of SEQ ID NO: 27 or a functional variant thereof; iv. LCDR1 comprising the amino acid sequence of SEQ ID NO: 28 or a functional variant thereof; v. LCDR2 comprising the amino acid sequence of SEQ ID NO: 29 or a functional variant thereof; and vi. LCDR3 comprising the amino acid sequence of SEQ ID NO: 30 or a functional variant thereof Includes.

[0057] Additionally or alternatively, the antibodies or antigen-binding fragments thereof described herein may be described by their variable heavy (VH) chain and variable light (VL) chain.

[0058] Suitable variable heavy (VH) chain sequences (which the antibody or antigen-binding fragment thereof may comprise) are outlined individually below: - SEQ ID NO: 31 or a functional variant thereof, - SEQ ID NO: 33 or a functional variant thereof, - SEQ ID NO: 43 or a functional variant thereof, - SEQ ID NO: 45 or a functional variant thereof, - SEQ ID NO: 46 or a functional variant, - SEQ ID NO: 47 or a functional variant thereof, - SEQ ID NO: 35 or a functional variant thereof, - SEQ ID NO: 37 or a functional variant thereof, - SEQ ID NO: 39 or a functional variant thereof.

[0059] Particularly suitable variable heavy (VH) chain sequences (which the antibody or antigen-binding fragment thereof may comprise) are outlined individually below: - SEQ ID NO: 45 or a functional variant thereof, - SEQ ID NO: 33 or a functional variant thereof, - SEQ ID NO: 43 or a functional variant thereof, - SEQ ID NO: 46 or a functional variant thereof, - SEQ ID NO: 47 or a functional variant thereof.

[0060] Suitable variable light (VL) chain sequences (which the antibody or antigen-binding fragment thereof may comprise) are outlined individually below: - SEQ ID NO: 32 or a functional variant thereof, - SEQ ID NO: 34 or a functional variant thereof, - SEQ ID NO: 36 or a functional variant thereof, - SEQ ID NO: 38 or a functional variant thereof, - SEQ ID NO: 40 or a functional variant thereof.

[0061] A preferred variable light (VL) chain sequence (which the antibody or antigen-binding fragment thereof may comprise) may comprise the amino acid sequence of SEQ ID NO: 34 (or a functional variant thereof).

[0062] For example, in some embodiments, the antibody or antigen-binding fragment thereof comprises: i. a variable heavy chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 31, 33, 35, 37 or 39, or a functional variant thereof; ii. a variable light chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 32, 34, 36, 38 or 40, or a functional variant thereof; Includes.

[0063] For example, in some embodiments, the antibody or antigen-binding fragment thereof comprises: i. a variable heavy chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 31, 33, 35, 37, 39, 43, 45, 46 or 47, or a functional variant thereof; ii. a variable light chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 32, 34, 36, 38 or 40, or a functional variant thereof; Includes.

[0064] Suitably the antibody or antigen-binding fragment thereof comprises: i. a variable heavy chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 33, or a functional variant thereof; ii. a variable light chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 34, or a functional variant thereof; may include.

[0065] More suitably, the antibody or antigen-binding fragment thereof comprises: i. a variable heavy chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 45, or a functional variant thereof; ii. a variable light chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 34, or a functional variant thereof; may include.

[0066] In some aspects, the antibody or antigen-binding fragment thereof comprises: - a variable heavy (VH) chain and a variable light (VL) chain comprising the amino acid sequences of SEQ ID NO: 31 and SEQ ID NO: 32, respectively, or a functional variant thereof, - a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 33 and SEQ ID NO: 34, respectively, or a functional variant thereof, - a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 43 and SEQ ID NO: 34, respectively, or a functional variant thereof, - a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 34, respectively, or a functional variant thereof, - a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 46 and SEQ ID NO: 34, respectively, or a functional variant thereof, - a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 47 and SEQ ID NO: 34, respectively, or a functional variant thereof, - a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 36, respectively, or a functional variant thereof, - a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 37 and SEQ ID NO: 38, respectively, or a functional variant thereof, or - a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 39 and SEQ ID NO: 40, respectively, or a functional variant thereof Includes.

[0067] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a variable heavy (VH) chain comprising the amino acid sequence of SEQ ID NO: 45, 33, 43, 46 or 47 (or a functional variant thereof) and a variable light (VL) chain comprising the amino acid sequence of SEQ ID NO: 34 (or a functional variant thereof). For example, the VHs of SEQ ID NO: 33, 45, 46 and / or 47 may correspond to "germlined" versions of the VH of SEQ ID NO: 33 (e.g., all with the same CDR sequences but with framework diversity). Advantageously, each variant retains equivalent binding characteristics.

[0068] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 31, or a functional variant thereof, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 32, or a functional variant thereof. The antibody or antigen-binding fragment thereof comprising said sequences may be referred to as "ZY0EPD-E02" or "EPD-E02".

[0069] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 35, or a functional variant thereof, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 36, or a functional variant thereof. The antibody or antigen-binding fragment thereof comprising said sequences may be referred to as "ZY0EOB-F05" or "EOB-F05".

[0070] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 37, or a functional variant thereof, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 38, or a functional variant thereof. The antibody or antigen-binding fragment thereof comprising said sequences may be referred to as "ZY0EO5-E07" or "EO5-E07".

[0071] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 39, or a functional variant thereof, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 40, or a functional variant thereof. The antibody or antigen-binding fragment thereof comprising said sequences may be referred to as "ZY0EP0-C07" or "EP0-C07".

[0072] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 33, or a functional variant thereof, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 34, or a functional variant thereof.

[0073] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:43, or a functional variant thereof, and a variable light chain comprising the amino acid sequence of SEQ ID NO:34, or a functional variant thereof.

[0074] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:46, or a functional variant thereof, and a variable light chain comprising the amino acid sequence of SEQ ID NO:34, or a functional variant thereof.

[0075] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:47, or a functional variant thereof, and a variable light chain comprising the amino acid sequence of SEQ ID NO:34, or a functional variant thereof.

[0076] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 45, or a functional variant thereof, and a variable light chain comprising the amino acid sequence of SEQ ID NO: 34, or a functional variant thereof. The antibody or antigen-binding fragment thereof comprising said sequences may be referred to as "EQD-E02_GL".

[0077] In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95% or 100% sequence identity to the reference amino acid sequence of SEQ ID NO: 43. In some embodiments, the antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 43. For example, the antibody or antigen-binding fragment thereof may comprise a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 43 and a variable light chain comprising the amino acid sequence of SEQ ID NO:34.

[0078] Additionally or alternatively, the antibodies or antigen-binding fragments thereof described herein may be described by their heavy and / or light chains.

[0079] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain (e.g., including a VL and a constant light chain) comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 44. In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a light chain (e.g., including a VL and a constant light chain) comprising the amino acid sequence of SEQ ID NO:44.

[0080] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain (e.g., including a VH and a constant heavy chain) comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95% or 100% sequence identity to the reference amino acid sequence of SEQ ID NO: 48. For example, the antibody or antigen-binding fragment thereof may comprise a heavy chain (e.g., including a VH and a constant heavy chain) comprising the amino acid sequence of SEQ ID NO: 48. Such a heavy chain may be referred to as "E02-GL-Maia-heavy chain."

[0081] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain (e.g., including a VH and a constant heavy chain) comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95% or 100% sequence identity to the reference amino acid sequence of SEQ ID NO: 49. For example, the antibody or antigen-binding fragment thereof may comprise a heavy chain (e.g., including a VH and a constant heavy chain) comprising the amino acid sequence of SEQ ID NO: 49. Such a heavy chain may be referred to as an "E02-GLY-Maia-heavy chain."

[0082] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain (e.g., including a VH and a constant heavy chain) comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95% or 100% sequence identity to the reference amino acid sequence of SEQ ID NO: 50. For example, the antibody or antigen-binding fragment thereof may comprise a heavy chain (e.g., including a VH and a constant heavy chain) comprising the amino acid sequence of SEQ ID NO: 50. Such a heavy chain may be referred to as an "E02-GLQ-Maia-heavy chain."

[0083] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain (e.g., including a VH and a constant heavy chain) comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95% or 100% sequence identity to the reference amino acid sequence of SEQ ID NO: 51. In a more preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain (e.g., including a VH and a constant heavy chain) comprising the amino acid sequence of SEQ ID NO: 51. Such a heavy chain may be referred to as "E02-GL-WT-heavy chain."

[0084] In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain constant region comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, 95% or 100% sequence identity to the reference amino acid sequence of SEQ ID NO: 42. In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO:42.

[0085] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 41. More preferably, the antibody, or antigen-binding fragment thereof, comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 52.

[0086] In a preferred embodiment, the antibody or antigen-binding fragment thereof comprises a light chain (e.g., comprising a VL and a constant light chain) comprising the amino acid sequence of SEQ ID NO: 44, and a heavy chain (e.g., comprising a VH and a constant heavy chain) comprising the amino acid sequence of SEQ ID NO: 51.

[0087] Advantageously, the claimed antibodies or antigen-binding fragments have been found to be capable of targeting a broader spectrum of B7-H4 expressing cells when compared to existing (commercially available) antibodies reported to target B7-H4. Thus, not only have the antibodies (or antigen-binding fragments thereof) disclosed herein been found to have affinity and specificity for clinically relevant targets, but they have also been found to have unique advantages (e.g., unexpected technical effects) associated therewith.

[0088] Preferably, the antibodies or antigen-binding fragments thereof described herein are capable of binding to B7-H4 as an integral component of cancer cells (eg, B7-H4 as an integral component of the cell membrane of cancer cells).

[0089] The antibodies or antigen-binding fragments thereof described herein may bind to OVCAR4 cell lines and / or CHO cell lines (e.g., which may lack exogenous nucleic acid encoding B7-H4). For example, the antibodies or antigen-binding fragments thereof bind to B7-H4 (e.g., the B7-H4 epitope) in OVCAR4 cell lines and / or CHO cell lines (e.g., which may lack exogenous nucleic acid encoding B7-H4). Suitably, the antibodies or antigen-binding fragments thereof described herein may bind to OVCAR4 cell lines and CHO cell lines (e.g., which may lack exogenous nucleic acid encoding B7-H4).

[0090] The term "epitope" refers to a region of a target protein (e.g., a polypeptide) capable of binding (e.g., bound by) an antibody or antigen-binding fragment of the disclosure.

[0091] In some embodiments, the antibody or antigen-binding fragment thereof binds with higher affinity to an OVCAR4 cell line and / or a CHO cell line (which may, for example, lack exogenous nucleic acid encoding B7-H4) when compared to one or more antibodies selected from E Biosciences 14-5949 anti-human B7H4 mouse IgG, US biological B0000-35B anti-human B7H4 mouse IgG, R and D systems AF2514 anti-mouse B7H4 goat IgG1, Sigma SAB2500141 anti-B7H4 goat IgG1, isotype 1 CAT004 SP06-003, isotype 2 R and D normal goat IgG control (AB-108C), AdD serotec MCA2632, Epitomics 2516-1, eBiosciences, 145972-82, eBioscience 145970-85, or a combination thereof. For example, the antibody or antigen-binding fragment thereof may bind with higher affinity to an OVCAR4 cell line and / or a CHO cell line (e.g., which may lack exogenous nucleic acid encoding B7-H4) when compared to one or more antibodies selected from E Biosciences 14-5949 anti-human B7H4 mouse IgG, US biological B0000-35B anti-human B7H4 mouse IgG, R and D systems AF2514 anti-mouse B7H4 goat IgG1, and Sigma SAB2500141 anti-B7H4 goat IgG1, or a combination thereof.

[0092] In a preferred embodiment, the antibody or antigen-binding fragment thereof binds to the OVCAR4 cell line with higher affinity when compared to E Biosciences 14-5949 anti-human B7H4 mouse IgG.

[0093] Reference to "E Biosciences 14-5949 anti-human B7H4 mouse IgG" may be used interchangeably herein with the term "B7-H4 monoclonal antibody (H74), eBioscience." Said antibody is available from ThermoFisher Scientific (catalog #14-5949-82).

[0094] In another preferred embodiment, the antibody or antigen-binding fragment thereof binds to the OVCAR4 cell line with higher affinity when compared to US biological B0000-35B anti-human B7H4 mouse IgG.

[0095] The affinity (eg, binding affinity) can be measured by any suitable method for measuring binding affinity as described herein.

[0096] The OVCAR4 cell line is a human ovarian cancer cell line. The OVCAR4 cell line is available from the National Cancer Institute due to transfer of cell lines from the Division of Cancer Treatment and Diagnosis Tumor Repository. The Chinese Hamster Ovary (CHO) cell line is an epithelial cell line derived from the ovary of the Chinese hamster and is widely available.

[0097] In some aspects, the antibody or antigen-binding fragment thereof is a monoclonal antibody.

[0098] A "monoclonal antibody" (mAb) refers to a homogeneous antibody population involved in highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to a polyclonal antibody, which typically contains different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv) mutants, fusion proteins containing antibody portions, and any other modified immunoglobulin molecule containing an antigen recognition site. Furthermore, "monoclonal antibody" refers to antibodies as produced in a number of ways, including, but not limited to, hybridoma, phage selection, recombinant expression, and transgenic animals.

[0099] In a preferred aspect, the antibody or antigen-binding fragment thereof (e.g., mAb) of the present disclosure is a humanized antibody or antigen-binding fragment thereof. Suitably, the humanized antibody or antigen-binding fragment thereof is an IgG.

[0100] The term "humanized antibody" refers to an antibody derived from a non-human (e.g., mouse) immunoglobulin that has been modified to contain minimal non-human (e.g., mouse) sequences. Typically, a humanized antibody is a human immunoglobulin in which residues from a complementarity determining region (CDR) are replaced by residues from a CDR of a non-human species (e.g., mouse, rat, rabbit, or hamster) having the desired specificity, affinity, and capacity (Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some instances, Fv framework region (FW) residues of a human immunoglobulin are replaced by the corresponding residues in an antibody derived from a non-human species having the desired specificity, affinity, and capacity.

[0101] Humanized antibodies can be further modified to refine and optimize antibody specificity, affinity and / or capacity by substitution of additional residues in the Fv framework regions and / or within the substituted non-human residues. Generally, a humanized antibody contains substantially all of at least one, typically two or three, variable domains that contain all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are of human immunoglobulin consensus sequences. Humanized antibodies can also contain at least a portion of an immunoglobulin constant region or domain (Fc), typically of a human immunoglobulin. Examples of methods used to make humanized antibodies are described in U.S. Pat. Nos. 5,225,539 and 5,639,641.

[0102] The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chains each consist of four framework regions (FWs) connected by three complementarity determining regions (CDRs), also known as hypervariable regions. The CDRs in each chain are held together adjacent to each other by the FW regions and contribute, together with the CDRs from the other chain, to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) an approach based on sequence variability between species (i.e., Kabat et al., Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948)). In addition, the art sometimes uses a combination of these two approaches to determine CDRs.

[0103] The "Kabat numbering system" is commonly used to refer to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0104] The numbering of amino acid positions as in Kabat refers to the numbering system used for the heavy or light chain variable domains of the antibody compilation in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). When using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FW or CDR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and may contain inserted residues after heavy chain FW residue 82 (e.g., residues 82a, 82b and 82c according to Kabat, etc.).

[0105] The Kabat numbering of residues can be determined for a given antibody by aligning the antibody's sequence with the "standard" Kabat numbered sequence at the regions of homology. Chothia instead refers to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The ends of the Chothia CDR-H1 loops when numbered using the Kabat numbering convention vary from H32 to H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The table below lists the positions of the amino acids that comprise the variable regions of the antibodies in each system.

[0106] [Table 1]

[0107] ImMunoGeneTics (IMGT) also provides a numbering system for immunoglobulin variable regions, including CDRs. See, e.g., Lefranc, MP et al., Dev. Comp. Immunol. 27:55-77 (2003). The IMGT numbering system is based on the alignment of over 5,000 sequences, structural data, and characterization of hypervariable loops, allowing easy comparison of variable and CDR regions for all species. According to the IMGT numbering scheme, VH-CDR1 is at positions 26-35, VH-CDR2 is at positions 51-57, VH-CDR3 is at positions 93-102, VL-CDR1 is at positions 27-32, VL-CDR2 is at positions 50-52, and VL-CDR3 is at positions 89-97.

[0108] As used throughout the specification, the VH CDR sequences described correspond to classical Kabat numbering positions, i.e. Kabat VH-CDR1 is at positions 31-35, VH-CDR2 is at positions 50-65 and VH-CDR3 is at positions 95-102. VL-CDR1, VL-CDR2 and VL-CDR3 also correspond to classical Kabat numbering positions, i.e. positions 24-34, 50-56 and 89-97, respectively.

[0109] In some aspects, the antibodies of the disclosure are human antibodies.

[0110] The term "human antibody" refers to an antibody produced in a human or an antibody having an amino acid sequence corresponding to an antibody produced in a human, made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies that comprise at least one human heavy and / or light chain polypeptide, such as an antibody that comprises a murine light chain and a human heavy chain polypeptide.

[0111] In some aspects, the antibodies of the present disclosure are chimeric antibodies.

[0112] The term "chimeric antibody" refers to an antibody in which the amino acid sequences of the immunoglobulin molecule are derived from two or more species. Typically, the variable regions of both the light and heavy chains correspond to the variable regions of antibodies with the desired specificity, affinity and capacity derived from one species of mammal (e.g., mouse, rat, rabbit, etc.), while the constant regions are homologous to sequences in antibodies derived from that species (usually human) to avoid eliciting an immune response in another species.

[0113] The term "YTE" or "YTE mutant" refers to a mutation in IgG1 Fc that results in increased binding to human FcRn and improves the serum half-life of antibodies carrying the mutation. The YTE mutant contains a combination of three mutations, M252Y / S254T / T256E (EU numbering Kabat et al. (1991) Sequences of Proteins of Immunological Interest, US Public Health Service, National Institutes of Health, Washington, DC), introduced into the heavy chain of IgG1. See U.S. Patent No. 7,658,921, incorporated herein by reference. YTE mutants have been shown to increase the serum half-life of antibodies by approximately 4-fold when compared to the wild-type version of the same antibody (Dall'Acqua et al., J. Biol. Chem. 281:23514-24 (2006); Robbie et al., (2013) Antimicrob. Agents Chemother. 57, 6147-6153). See also U.S. Patent No. 7,083,784, which is incorporated herein by reference in its entirety.

[0114] Suitably, the antibodies or antigen-binding fragments of the present disclosure bind to the B7-H4 molecule with sufficient affinity such that the antibodies are useful as therapeutic agents or diagnostic reagents in targeting B7-H4.

[0115] In some embodiments, the antibody or antigen-binding fragment thereof binds to B7-H4 (preferably human B7-H4) with a dissociation constant (KD) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦10 pM, ≦1 pM, or ≦0.1 pM. In some embodiments, the antibody or antigen-binding fragment thereof binds to B7-H4 (preferably human B7-H4) with a KD of about 0.1 nM to about 40 nM, about 0.5 nM to about 30 nM, about 1 nM to about 20 nM, or about 1.5 nM to about 20 nM.

[0116] In a preferred embodiment, the antibody or antigen-binding fragment thereof binds to B7-H4 (preferably human B7-H4) with a KD of about 23 nM to about 27 nM. In a more preferred embodiment, the antibody or antigen-binding fragment thereof binds to B7-H4 (preferably human B7-H4) with a KD of about 1 nM to about 1.5 nM.

[0117] KD measurements (binding affinity) can be performed by any suitable assay known in the art. Suitable assays include affinity assays that can be performed via the KinExA system (e.g., KinExA 3100, KinExA 3200 or KinExA 4000) (Sapidyne Instruments, Idaho) or the ForteBio Octet system.

[0118] In some embodiments, the extent of binding of an antibody or antigen-binding fragment thereof of the present disclosure to an unrelated non-B7-H4 protein is less than about 10%, 5%, 2%, or 1% (preferably less than about 10%) of the binding of the antibody (or antigen-binding fragment thereof) to B7-H4 (preferably human B7-H4). The binding can be measured, for example, by radioimmunoassay (RIA), BIACORE® (using recombinant B7-H4 as the analyte and an antibody as the ligand or vice versa), KINEXA®, ForteBio Octet system, or other binding assays known in the art.

[0119] In some embodiments, the antibody or antigen-binding fragment thereof does not bind to one or more selected from human B7-H1 molecule, human B7-H2 molecule, human B7-H3 molecule, human BTN1A1 molecule, human HHLA2 molecule, human BTN3A2 molecule, or a combination thereof. In a preferred embodiment, the antibody or antigen-binding fragment thereof does not bind to one or more selected from human B7-H1 molecule, human B7-H2 molecule, human B7-H3 molecule, or a combination thereof.

[0120] The term "does not bind" means that an antibody or antigen-binding fragment thereof described herein does not substantially bind to one or more of the molecules (e.g., human B7-H1 molecule, human B7-H2 molecule, human B7-H3 molecule, human BTN1A1 molecule, human HHLA2 molecule, human BTN3A2 molecule, or a combination thereof). The term "substantially free" as used herein in relation to binding can mean that less than 5%, 2%, 1%, 0.5% or 0.1% of cells expressing one or more of the molecules in a cell culture become bound by (upon contact with) an antibody or antigen-binding fragment thereof described herein. Suitably, the term "substantially free" as used herein in relation to binding can mean that no such cells become bound.

[0121] In some embodiments, the antibody or antigen-binding fragment thereof does not bind to a human B7-H1 molecule, a human B7-H2 molecule, a human B7-H3 molecule, a human BTN1A1 molecule, a human HHLA2 molecule, or a human BTN3A2 molecule. In preferred embodiments, the antibody or antigen-binding fragment thereof does not bind to a human B7-H1 molecule, a human B7-H2 molecule, or a human B7-H3 molecule.

[0122] In some embodiments, the B7-H4 polypeptide is contained within a B7-H4 polypeptide sequence or a fragment thereof.

[0123] A "B7-H4 polypeptide" may include the full-length polypeptide sequence of B7-H4 (e.g., SEQ ID NO:55) or may include a fragment of B7-H4 of any length (e.g., including 5%, 15%, 25%, 35%, 45%, 55%, 65%, 75%, 85% or 95% of the polypeptide sequence of the full-length polypeptide sequence of B7-H4) that includes an epitope that can bind (e.g., can be bound by) an antibody or antigen-binding fragment of the disclosure. A B7-H4 polypeptide may include a sequence having 75%, 80%, 85%, 90% or 90% sequence identity to the sequence of SEQ ID NO:55. Preferably, a B7-H4 polypeptide includes the sequence of SEQ ID NO:55.

[0124] Antibody and antigen-binding fragment characteristics The present antibodies or antigen-binding fragments have high affinity for B7-H4 both in vitro and in vivo and therefore may be advantageously used in methods for detecting B7-H4 epitopes and related diagnostic methods.

[0125] The term "antibody" encompasses monoclonal antibodies and fragments thereof (e.g., that exhibit a desired biological activity). In preferred aspects, the antibodies of the present disclosure are monoclonal antibodies. In more preferred aspects, the antibodies are fully human monoclonal antibodies. In some aspects, the methods of the present disclosure may use polyclonal antibodies.

[0126] In particular, an antibody is a protein that comprises at least one or two heavy (H) chain variable regions (abbreviated herein as VHC) and at least one or two light (L) chain variable regions (abbreviated herein as VLC). The VHC and VLC regions can be further subdivided into regions of hypervariability called "complementarity determining regions" ("CDRs"), interspersed with regions that are more conserved, called "framework regions" (FRs). The extent of the framework regions and CDRs have been precisely defined (see Kabat, EA, et al. Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242, 1991, and Chothia, C. et al, J. MoI. Biol. 196:901-917, 1987). Preferably, each VHC and VLC is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, DR2, FR3, CDR3, FR4. The VHC or VLC chain of the antibody may further comprise all or a portion of a heavy or light chain constant region. In some embodiments, the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, the heavy and light immunoglobulin chains being linked between each other, for example, by disulfide bonds. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. The light chain constant region is composed of one domain, CL. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The term "antibody" includes intact immunoglobulins of the types IgA, IgG, IgE, IgD, IgM (and their subtypes), and the light chains of the immunoglobulins may be of the kappa or lambda type. The term antibody, as used herein, also refers to a portion of an antibody that binds to one of the above-mentioned markers, for example a molecule in which one or more immunoglobulin chains are not full length but which binds to the marker.Examples of binding moieties encompassed within the term antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VLC, VHC, CL, and CH1 domains; (ii) an F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fc fragment consisting of the VHC and CH1 domains; (iv) an Fv fragment consisting of the VLC and VHC domains of a single arm of an antibody; (v) a dAb fragment consisting of the VHC domain (Ward et al, Nature 341:544-546, 1989); and (vi) an isolated complementarity determining region (CDR) having sufficient framework for binding, for example, to an antigen-binding portion of the variable region. The antigen-binding portion of the light chain variable region and the antigen-binding portion of the heavy chain variable region, e.g., the two domains of the Fv fragment, VLC and VHC, can be linked by a synthetic linker that allows them to be produced using recombinant techniques as a single protein chain in which the VLC and VHC regions pair to form a monovalent molecule (known as single-chain Fv (scFv), see, e.g., Bird et al. (1988) Science lAl-ATi-Alβ and Huston et al., (1988) Proc Natl. Acad. ScL USA 85:5879-5883). Such single-chain antibodies are also encompassed within the term antibody. They can be obtained using conventional techniques known to those skilled in the art, and the portions are screened for utility in the same manner as intact antibodies.

[0127] In some embodiments, the antibody or antigen-binding fragment is one or more selected from a murine antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a multispecific antibody, or a combination thereof.

[0128] In some embodiments, the antigen-binding fragment is one or more selected from an Fv fragment, a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a dsFv fragment, a scFv fragment, a sc(Fv)2 fragment, or a combination thereof.

[0129] In some aspects, an antibody or antigen-binding fragment thereof (e.g., mAb) of the disclosure is an scFV.

[0130] In some embodiments, the antibodies or antigen-binding fragments thereof may bind to B7-H4 molecules across species, e.g., the antibodies or fragments may bind to mouse B7-H4, rat B7-H4, rabbit, human B7-H4, and / or cynomolgus B7-H4. In some embodiments, the antibodies or fragments may bind to human B7-H4 and cynomolgus B7-H4. In some embodiments, the antibodies or antigen-binding fragments may also bind to mouse B7-H4.

[0131] In some embodiments, the antibody or antigen-binding fragment thereof may specifically bind to B7-H4, e.g., human B7-H4 and cynomolgus monkey B7-H4, but does not specifically bind to human B7-H1, B7-H2 and / or B7-H3.

[0132] In some embodiments, the antibody or antigen-binding fragment thereof may comprise a heavy chain constant region or fragment thereof in addition to VH and VL. In some embodiments, the heavy chain constant region is a human heavy chain constant region, e.g., a human IgG constant region, e.g., a human IgG1 constant region. In some embodiments (preferably when the antibody or antigen-binding fragment thereof is conjugated to an agent, such as a cytotoxic agent), a cysteine ​​residue is inserted between amino acids S239 and V240 in the CH2 region of IgG1. This cysteine ​​is referred to as a "239 insertion" or "239i".

[0133] In some embodiments, the antibody or antigen-binding fragment thereof may comprise a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 41. Preferably, the antibody or antigen-binding fragment thereof may comprise a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 52.

[0134] The "E02-GL" antibody has CDR sequences of (e.g., corresponding to) ZY0EQD-E02 ("GL" means that the antibody is germlined). For example, E02-GL may comprise a VH chain sequence of SEQ ID NO: 45, e.g., a germlined version of SEQ ID NO: 43, and a VL chain sequence of SEQ ID NO: 34. E02-GL may also comprise a heavy chain sequence of SEQ ID NO: 51 and a light chain sequence of SEQ ID NO: 44. Thus, "E02-GL" conjugated to topoisomerase I payload SG3932 as described herein is referred to as "E02-GL-SG3932". In one embodiment, clone E02-GL is conjugated to topoisomerase I payload SG3932 at an average drug-antibody ratio (DAR) of 8.

[0135] In some embodiments, the heavy chain constant region or fragment thereof, e.g., a human IgG constant region or fragment thereof, can comprise one or more amino acid substitutions compared to a wild-type IgG constant domain, such that the modified IgG has an increased half-life compared to the half-life of an IgG having a wild-type IgG constant domain. For example, the IgG constant domain can contain one or more amino acid substitutions at amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, where the numbering of the amino acid positions is according to the EU index as defined in Kabat. In some embodiments, the IgG constant domain is substituted with the amino acid at Kabat position 252 by tyrosine (Y), phenylalanine (F), tryptophan (W), or threonine (T); the amino acid at Kabat position 254 by threonine (T); the amino acid at Kabat position 256 by serine (S), arginine (R), glutamine (Q), glutamic acid (E), aspartic acid (D), or threonine (T); the amino acid at Kabat position 257 by leucine (L); the amino acid at Kabat position 309 by proline (P); In a preferred embodiment, the IgG constant domain may contain one or more of the following amino acid substitutions when compared to a wild-type human IgG constant domain: substitution of the amino acid at Kabat position 311 with serine (S), substitution of the amino acid at Kabat position 428 with threonine (T), leucine (L), phenylalanine (F) or serine (S), substitution of the amino acid at Kabat position 433 with arginine (R), serine (S), isoleucine (I), proline (P) or glutamine (Q), or substitution of the amino acid at Kabat position 434 with tryptophan (W), methionine (M), serine (S), histidine (H), phenylalanine (F) or tyrosine. In a preferred embodiment, the IgG constant domain may contain amino acid substitutions when compared to a wild-type human IgG constant domain, including substitution of the amino acid at Kabat position 252 with tyrosine (Y), substitution of the amino acid at Kabat position 254 with threonine (T) and substitution of the amino acid at Kabat position 256 with glutamic acid (E). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain, wherein the heavy chain is a human IgG1 YTE mutant.

[0136] In some embodiments, the antibody or antigen-binding fragment thereof can include, in addition to the VH and VL, optionally a heavy chain constant region or fragment thereof, a light chain constant region or fragment thereof, in some embodiments, the light chain constant region is a kappa-lambda light chain constant region, such as a human kappa constant region or a human lambda constant region.

[0137] In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO:42.

[0138] In some embodiments, the VH and / or VL amino acid sequences may have 85%, 90%, 95%, 96%, 97%, 98% or 99% similarity to the sequences shown herein. In some embodiments, the VH and / or VL amino acid sequences may include one, two, three, four, five or more substitutions, e.g., conservative substitutions, compared to the sequences shown herein. B7-H4 antibodies having VH and VL regions with a particular percent similarity to the VH or VL regions or having one or more substitutions, e.g., conservative substitutions, may be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis) of nucleic acid molecules encoding the VH and / or VL regions described herein and then testing the encoded modified antibodies for binding to B7-H4 and, optionally, for retention of function using the functional assays described herein.

[0139] The affinity or avidity of an antibody or antigen-binding fragment thereof for an antigen can be determined experimentally using any suitable method known in the art, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA) or kinetics (e.g., KINEXA® or BIACORE® analysis). Direct binding assays as well as competitive binding assay formats can be readily used. (See, e.g., Berzofsky et al., Antibody-Antigen Interactions, In Fundamental Immunology, Paul, WE, Ed., Raven Press: New York, NY (1984); Kuby, Immunology, WH Freeman and Company: New York, NY (1992) and the methods described herein). The measured affinity of a particular antibody-antigen interaction can vary when measured under different conditions (e.g., salt concentration, pH, temperature). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD or Kd, Kon, Koff) are performed with standardized solutions of antibody and antigen and standardized buffers, as known in the art.

[0140] In some embodiments, the antibodies or antigen-binding fragments thereof may bind to B7-H4 expressing cells with an IC50 of less than about 500 nM, less than about 350 nM, less than about 250 nM, less than about 150 nM, less than about 100 nM, less than about 75 nM, less than about 60 nM, less than about 50 nM, less than about 40 nM, less than about 30 nM, less than about 20 nM, less than about 15 nM, less than about 10 nM, less than about 5 nM, less than about 1 nM, less than about 500 pM, less than about 350 pM, less than about 250 pM, less than about 150 pM, less than about 100 pM, less than about 75 pM, less than about 60 pM, less than about 50 pM, less than about 40 pM, less than about 30 pM, less than about 20 pM, less than about 15 pM, less than about 10 pM, or less than about 5 pM. Preferably, the IC50 is measured by flow cytometry.

[0141] Cleavable Linkers and Cytotoxic Agents In some aspects of the antibody-drug conjugates disclosed herein, the antibody or antigen-binding fragment thereof is linked to a cytotoxic agent by a linker. In some aspects, the antibody or antigen-binding fragment thereof is conjugated to a cytotoxic agent by a linker. As used herein, "conjugated" means linked via a covalent or ionic bond. The cytotoxic agent may be referred to herein as a "drug" or an "active agent." In some aspects, the cytotoxic agent is a drug.

[0142] In some embodiments, the cytotoxic agent (sometimes referred to as a "warhead") on the ADC is one of the cytotoxic agents disclosed in published international application WO 2020 / 200880, which is incorporated by reference in its entirety.

[0143] A cytotoxic agent or cytotoxin can be any molecule known in the art that inhibits or prevents the function of a cell and / or causes destruction of a cell (cell death) and / or exerts an anti-neoplastic / anti-proliferative effect. Several classes of cytotoxic agents are known to have potential utility in ADC molecules. These include, but are not limited to, topoisomerase I inhibitors, amanitins, auristatins, daunomycins, doxorubicins, duocarmycins, dolastatins, enediynes, lexitropsins, taxanes, puromycins, maytansinoids, vinca alkaloids, tubulysins, and pyrrolobenzodiazepines (PBDs). Examples of such cytotoxic agents are AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatostatin, calicheamicin, maytansine, DM-1, vinblastine, methotrexate and netropsin and derivatives and analogs thereof. Further disclosure regarding suitable cytotoxins for use in ADCs can be found, for example, in WO 2015 / 155345 and WO 2015 / 157592, which are incorporated by reference in their entireties.

[0144] In some embodiments, the antibody or antigen-binding fragment is conjugated to one or more heterologous agents selected from the group consisting of a topoisomerase I inhibitor, a tubulysin derivative, a pyrrolobenzodiazepine, an antibacterial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a xenogenous antibody, a fragment of a xenogenous antibody, a detectable label, polyethylene glycol (PEG), a radioisotope, or a combination thereof.

[0145] In some embodiments, the antibody antigen-binding fragment is conjugated to one or more cytotoxins selected from a topoisomerase I inhibitor, a tubulysin derivative, a pyrrolobenzodiazepine, or a combination thereof. For example, the antibody or antigen-binding fragment thereof is conjugated to one or more cytotoxins selected from the group consisting of the topoisomerase I inhibitors SG3932, SG4010, SG4057, or SG4052 (the structures of which are provided below); tubulysin AZ1508, the pyrrolobenzodiapezine SG3315, the pyrrolobenzodiapezine SG3249, or a combination thereof.

[0146] In a preferred embodiment, the antibody or antigen-binding fragment thereof is conjugated to a topoisomerase I inhibitor. Topoisomerase inhibitors are chemical compounds that block the action of topoisomerases (topoisomerase I and II), a class of enzymes that control changes in DNA structure by catalyzing the cleavage and rejoining of the phosphodiester backbone of DNA strands during the normal cell cycle.

[0147] The general formula for a suitable topoisomerase I inhibitor is compound A* below: [ka] which may be referred to herein as a "drug unit."

[0148] The compound (e.g., A*) is preferably provided with a linker for linking (preferably conjugating) to an antibody or antigen-binding fragment described herein (which may be referred to as a "ligand unit"). Suitably, the linker is cleavably attached (e.g., conjugated) to an amino residue, such as an amino acid of an antibody or antigen-binding fragment described herein.

[0149] More specifically, examples of suitable topoisomerase I inhibitors are the following compounds having formula "I": [ka] and salts and solvates thereof, wherein R L is a linker for linking to an antibody or antigen-binding fragment thereof (e.g., a ligand unit) described herein, said linker preferably being (ia): [ka] (In the formula, Q is, [ka] Wherein, Q X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue, X is [ka] In the formula, a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, and d=0 to 5, where at least b1 or b2=0 (i.e., only one of b1 and b2 may not be 0), and at least c1 or c2=0 (i.e., only one of c1 and c2 may not be 0), G L is a linker for linking to an antibody or antigen-binding fragment thereof (e.g., a ligand unit) described herein; or (ib): [ka] (In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1. is selected from.

[0150] It will be understood by one of skill in the art that more than one of the above agents (eg, topoisomerase I inhibitors) can be conjugated to the antibody or antigen-binding fragment thereof.

[0151] For example, a conjugate (e.g., an antibody-drug conjugate) of the disclosure may be represented by the general formula IV: L-(D L ) p (IV) or a pharma- ceutically acceptable salt or solvate thereof, in which L is an antibody or an antigen-binding fragment thereof (e.g., a ligand unit) as described herein; and D L is represented by formula III: [ka] R is a topoisomerase I inhibitor (e.g., a drug linker unit) having a linker of LL is a linker that is linked to an antibody or antigen-binding fragment thereof (e.g., a ligand unit) described herein, the linker preferably being (ia'): [ka] (wherein Q and X are as defined above; G LL is a linker that is linked to an antibody or antigen-binding fragment thereof (e.g., a ligand unit) described herein, and (ib'): [ka] (In the formula, R L1 and R L2 is as defined above) and p is an integer from 1 to 20.

[0152] Drug loading is represented by p, which is the number of topoisomerase I inhibitors (e.g., drug units) per antibody or antigen-binding fragment thereof (e.g., ligand unit). Drug loading can range from 1 to 20 drug units (D) per ligand unit. In the case of a composition, p represents the average drug loading of the complexes in the composition, and p ranges from 1 to 20.

[0153] Accordingly, embodiments of the ADCs disclosed herein include conjugates comprising an antibody or antigen-binding fragment thereof (e.g., a ligand unit) described herein covalently attached to at least one topoisomerase I inhibitor (e.g., a drug unit such as A* as exemplified above). The inhibitor is preferably linked to a linker (e.g., a linker unit), such as R L and / or R LL The antibody or antigen-binding fragment thereof is linked to the antibody or antigen-binding fragment thereof by a linker such as those described above. In other words, the embodiments of the ADCs disclosed herein preferably include an antibody or antigen-binding fragment thereof (e.g., a ligand unit) described herein to which one or more topoisomerase I inhibitors are linked via a linker (e.g., a drug-linker unit). The antibody or antigen-binding fragment thereof (representing a ligand unit) described in more detail above is a targeting agent that binds to a targeting moiety. More specifically, the ligand unit can, for example, specifically bind to B7-H4 on a target cell, and thus the drug unit is delivered thereto. Thus, the methods described herein can be used for the treatment of, for example, various cancers and other disorders (e.g., cancers / disorders linked to the presence of cells, preferably cancer cells, expressing B7-H4) with ADCs, as discussed elsewhere herein.

[0154] Certain properties of the above topoisomerase I inhibitors are particularly preferred and can be defined in more detail as set out below. By way of example, property Q X Preferred embodiments of (eg, within the linker of 1a above) are outlined below.

[0155] The following preferences may apply to all aspects of the methods, combinations and kits as described herein or may relate to a single aspect. The preferences may be combined together in any combination.

[0156] Q X In some embodiments, Q is an amino acid residue. The amino acid may be a natural or unnatural amino acid. For example, Q may be selected from Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp, where Cit is citrulline.

[0157] In some embodiments, Q comprises a dipeptide residue. The amino acids in the dipeptide can be any combination of natural and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. If the linker is a cathepsin-labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. In that case, the dipeptide is the recognition site for cathepsin.

[0158] In some embodiments, Q is NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O , NH -Val-Cit- C=O , NH -Phe-Cit- C=O , NH -Leu-Cit- C=O , NH -Ile-Cit- C=O , NH -Phe-Arg- C=O , NH -Trp-Cit- C=O , and NH -Gly-Val- C=O wherein Cit is citrulline.

[0159] Preferably, Q is NH -Phe-Lys- C=O , NH -Val-Ala- C=O , NH -Val-Lys- C=O , NH -Ala-Lys- C=O , and NH -Val-Cit- C=O is selected from.

[0160] More preferably, Q is NH -Phe-Lys- C=O , NH -Val-Cit- C=O or NH -Val-Ala- C=O is selected from.

[0161] Other suitable dipeptide combinations include: NH -Gly-Gly- C=O , NH -Gly-Val- C=O , NH -Pro-Pro- C=O , and NH -Val-Glu- C=O Examples include:

[0162] Other dipeptide combinations may be used, including those described in Dubowchik et al., Bioconjugate Chemistry, 2002, 13, 855-869, incorporated herein by reference.

[0163] In some embodiments, Q is a tripeptide residue. The amino acids in the tripeptide can be any combination of natural and non-natural amino acids. In some embodiments, the tripeptide comprises natural amino acids. If the linker is a cathepsin-labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. In that case, the tripeptide is the recognition site for cathepsin. Particularly interesting tripeptide linkers are: NH -Glu-Val-Ala- C=O , NH -Glu-Val-Cit- C=O , NH -αGlu-Val-Ala- C=O , NH -αGlu-Val-Cit- C=O It is.

[0164] In some embodiments, Q is a tetrapeptide residue. The amino acids in the tetrapeptide can be any combination of natural and non-natural amino acids. In some embodiments, the tetrapeptide comprises natural amino acids. If the linker is a cathepsin-labile linker, the tetrapeptide is the site of action for cathepsin-mediated cleavage. The tetrapeptide is then the recognition site for cathepsin. Tetrapeptide linkers of particular interest are: NH -Gly-Gly-Phe-Gly C=O , and NH -Gly-Phe-Gly-Gly C=O It is.

[0165] In some embodiments, the tetrapeptide is NH -Gly-Gly-Phe-Gly C=O It is.

[0166] In the above representation of peptide residues: NH - represents the N-terminus of the residue, - C=Orepresents the C-terminus of the residue. The C-terminus is bound to the NH of A*.

[0167] Glu is a glutamic acid residue, i.e. [ka] and αGlu represents the residue of glutamic acid when bound via the α chain, i.e. [ka] Represents.

[0168] In some embodiments, where appropriate, amino acid side chains are chemically protected. Side chain protecting groups can be groups as discussed above. Protected amino acid sequences can be cleaved by enzymes. For example, dipeptide sequences containing Boc side chain protected Lys residues can be cleaved by cathepsin.

[0169] Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem catalogue, as discussed above.

[0170] G L G L may be selected from the following:

[0171] [Table 2]

[0172] [Table 3]

[0173] In the formula, Ar is C 5~6 represents an arylene group, e.g., phenylene; X is C 1~4 Represents alkyl.

[0174] In some embodiments, G L is G L1-1 and GL1-2 In some of these embodiments, G L is G L1-1 It is.

[0175] G LL G LL may be selected from the following:

[0176] [Table 4]

[0177] [Table 5]

[0178] In the formula, Ar is C 5~6 represents an arylene group, e.g., phenylene; X is C 1~4 Represents alkyl.

[0179] In some embodiments, G LL is G LL1-1 and G LL1-2 In some of these embodiments, G LL is G LL1-1 It is.

[0180] X X is preferably [ka] (In the formula, a=0 to 5, b1=0 to 16, b2=0 to 16, c=0 or 1, and d=0 to 5, where at least b1 or b2=0, and at least c1 or c2=0.) a can be 0, 1, 2, 3, 4, or 5. In some embodiments, a is 0 to 3. In some of these embodiments, a is 0 or 1. In further embodiments, a is 0. b1 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b1 is 0 to 12. In some of these embodiments, b1 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8. b2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b2 is 0 to 12. In some of these embodiments, b2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8. Preferably, only one of b1 and b2 may not be 0. c1 can be 0 or 1. c2 can be 0 or 1. Preferably, only one of c1 and c2 is not 0. d can be 0, 1, 2, 3, 4, or 5. In some embodiments, d is 0 to 3. In some of these embodiments, d is 1 or 2. In a further embodiment, d is 2. In a further embodiment, d is 5.

[0181] In some embodiments of X, a is 0, b1 is 0, c1 is 1, c2 is 0, d is 2, and b2 can be 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8. In some embodiments of X, a is 1, b2 is 0, c1 is 0, c2 is 0, d is 0, and b1 can be 0 to 8. In some of these embodiments, b1 is 0, 2, 3, 4, 5, or 8. In some embodiments of X, a is 0, b1 is 0, c1 is 0, c2 is 0, d is 1, and b2 can be 0 to 8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8. In some embodiments of X, b1 is 0, b2 is 0, c1 is 0, c2 is 0, and one of a and d is 0. The other of a and d is 1-5. In some of these embodiments, the other of a and d is 1. In others of these embodiments, the other of a and d is 5. In some embodiments of X, a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 can be 0-8. In some of these embodiments, b2 is 0, 2, 3, 4, 5, or 8.

[0182] In some embodiments, R L is of formula Ib. In some embodiments, R LL is of formula Ib'.

[0183] R L1 and R L2 may be independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group.

[0184] In some embodiments, R L1 and R L2 and R are H. L1 is H and R L2 is methyl. In some embodiments, R L1 and R L2 Both are methyl.

[0185] In some embodiments, R L1 and R L2 together with the carbon atom to which they are attached form a cyclopropylene group. In some embodiments, R L1 and R L2 together with the carbon atom to which they are attached form a cyclobutylene group.

[0186] In group Ib, in some embodiments, e is 0. In other embodiments, e is 1 and the nitro group can be at any available position on the ring. In some of these embodiments, it is at the ortho position. In others of these embodiments, it is at the para position.

[0187] In some embodiments where the compounds described herein are provided in a single enantiomer or in an enantiomerically enriched form, the enantiomerically enriched form has an enantiomeric ratio of greater than 60:40, 70:30, 80:20, or 90:10. In further embodiments, the enantiomeric ratio is greater than 95:5, greater than 97:3, or greater than 99:1.

[0188] In some embodiments, R L is selected from the following: [ka] [ka]

[0189] In some embodiments, R LL is the above R L It is a group derived from the group.

[0190] Having outlined these preferences above, certain preferred Topoisomerase I-linker (eg, Drug-Linker unit) formulas are now described.

[0191] In some embodiments, the compound of formula I is P: [ka] and salts and solvates thereof, wherein R LP is a linker for linking to an antibody or antigen-binding fragment thereof described herein, said linker comprising: (ia): [ka] (In the formula, Q P teeth, [ka] Wherein, Q XP Q P is an amino acid residue, a dipeptide residue or a tripeptide residue, X P teeth, [ka] In the formula, aP=0 to 5, bP=0 to 16, cP=0 or 1, and dP=0 to 5; G L is a linker for linking to an antibody or antigen-binding fragment thereof (e.g., a ligand unit) described herein; (ib): [ka] (In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1. aP can be 0, 1, 2, 3, 4 or 5. In some embodiments, aP is 0-3. In some of these embodiments, aP is 0 or 1. In further embodiments, aP is 0. bP can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In some embodiments, b is 0-12. In some of these embodiments, bP is 0-8, and can be 0, 2, 4 or 8. cP can be 0 or 1. dP can be 0, 1, 2, 3, 4 or 5. In some embodiments, dP is 0-3. In some of these embodiments, dP is 1 or 2. In further embodiments, dP is 2.

[0192] X P In some embodiments, aP is 0, cP is 1, dP is 2, and bP can be 0 to 8. In some of these embodiments, bP is 0, 4, or 8.

[0193] Q above for compounds of formula I X Preferred for (e.g., where appropriate) Q XP can be applied to.

[0194] For compounds of formula I, L , R L1 , R L2 Preferred for e are those of formula I P The present invention can be applied to compounds of the above formula (I).

[0195] In some embodiments, the compound of formula IV is P : L-(D LP ) p (IV P ) or a pharma- ceutically acceptable salt or solvate thereof, wherein L is an antibody or an antigen-binding fragment thereof (e.g., a ligand unit) as described herein; and D LP is represented by formula III P : [ka] and wherein: R LLP is a linker that is linked to the antibody or antigen-binding fragment thereof (e.g., a ligand unit), said linker comprising: (ia'): [ka] (In the formula, Q P and X P is as defined above, and G LL is a linker that is linked to an antibody or antigen-binding fragment thereof (e.g., a ligand unit) described herein, and (ib'): [ka] (In the formula, R L1 and R L2 is as defined above) and p is an integer from 1 to 20.

[0196] In some embodiments, the compound of formula I is P2 : [ka] and salts and solvates thereof, wherein R LP2 is a linker for linking to an antibody or antigen-binding fragment thereof described herein, said linker comprising: (ia): [ka] (In the formula, Q is, [ka] Wherein, Q X is such that Q is an amino acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue, X P2 teeth, [ka] In the formula, aP2=0 to 5, b1P2=0 to 16, b2P2=0 to 16, cP2=0 or 1, and dP2=0 to 5, where at least b1P2 or b2P2=0 (i.e., only one of b1 and b2 may not be 0), G L is a linker for linking to an antibody or antigen-binding fragment thereof (e.g., a ligand unit) described herein; (ib): [ka] (In the formula, R L1 and R L2 are independently selected from H and methyl, or together with the carbon atom to which they are attached form a cyclopropylene or cyclobutylene group; e is 0 or 1. is selected from.

[0197] aP2 can be 0, 1, 2, 3, 4, or 5. In some embodiments, aP2 is 0 to 3. In some of these embodiments, aP2 is 0 or 1. In further embodiments, aP2 is 0.

[0198] b1P2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b1P2 is 0 to 12. In some of these embodiments, b1P2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

[0199] b2P2 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, b2P2 is 0 to 12. In some of these embodiments, b2P2 is 0 to 8, and can be 0, 2, 3, 4, 5, or 8.

[0200] Preferably, only one of b1P2 and b2P2 may be non-zero.

[0201] cP2 can be 0 or 1.

[0202] dP2 can be 0, 1, 2, 3, 4 or 5. In some embodiments, dP2 is 0 to 3. In some of these embodiments, dP2 is 1 or 2. In further embodiments, dP2 is 2. In further embodiments, dP2 is 5.

[0203] X P2 In some embodiments, aP2 is 0, b1P2 is 0, cP2 is 1, dP2 is 2, and b2P2 can be 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5, or 8. P2 In some embodiments, aP2 is 1, b2P2 is 0, cP2 is 0, dP2 is 0, and b1P2 can be 0 to 8. In some of these embodiments, b1P2 is 0, 2, 3, 4, 5, or 8. P2 In some embodiments, aP2 is 0, b1P2 is 0, cP2 is 0, and dP2 is 1, and b2P2 can be 0 to 8. In some of these embodiments, b2P2 is 0, 2, 3, 4, 5, or 8. P2 In some embodiments, b1P2 is 0, b2P2 is 0, cP2 is 0, and one of aP2 and dP2 is 0. The other of aP2 and d is 1 to 5. In some of these embodiments, the other of aP2 and d is 1. In others of these embodiments, the other of aP2 and dP2 is 5.

[0204] Q above for compounds of formula I X Preferred for (e.g., where appropriate) are compounds of formula Ia P2 Q X can be applied to.

[0205] For compounds of formula I, L , R L1 , R L2 Preferred for e are those of formula I P2The present invention can be applied to compounds of the above formula (I).

[0206] In some embodiments, the compound of formula IV is P2 : L-(D LP2 ) p (IV P2 ) or a pharma- ceutically acceptable salt or solvate thereof, wherein L is an antibody or an antigen-binding fragment thereof (e.g., a ligand unit) as described herein; and D LP2 is represented by formula III P2 : [ka] R is a topoisomerase I inhibitor (e.g., a drug linker unit) LLP2 is a linker that is linked to the antibody or antigen-binding fragment thereof (e.g., a ligand unit), said linker comprising: (ia'): [ka] (Wherein, Q and X P2 is as defined above, and G LL is a linker attached to the antibody or antigen-binding fragment thereof, and (ib'): [ka] (In the formula, R L1 and R L2 is as defined above) and p is an integer from 1 to 20.

[0207] In some embodiments, the linker is a mp-PEG8-val-ala linker. As indicated in the name, the mp-PEG8-val-ala linker has eight consecutive polyethylene glycol units followed by a valine-alanine (val-ala) dipeptide that is linked to a cytotoxic agent.

[0208] In some embodiments, the linker and the cytotoxic agent are the following compounds: [ka] Includes one of the following together.

[0209] In some embodiments, the linker and cytotoxic agent are SG3932: [ka] together.

[0210] Drug-antibody ratio Drugs are generally linked to or "loaded onto" an antibody or antigen-binding fragment. Drug loading (p) is the average number of drugs per antibody or antigen-binding fragment (e.g., ligand unit).

[0211] The average number of drugs per antibody (or antigen-binding fragment) when preparing ADCs from conjugation reactions can be characterized by conventional means such as UV, reversed-phase HPLC, HIC, mass spectrometry, ELISA assays and electrophoresis. The quantitative distribution of ADCs with respect to p can also be determined. ELISA can determine the average value of p in a particular preparation of ADCs (Hamblett et al. (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843-852). In some cases, separation, purification and characterization of homogeneous ADCs with a particular value of p from ADCs with different drug loadings can be achieved by means such as reversed-phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugates.

[0212] Cysteine ​​amino acids can be engineered at reactive sites of antibodies (or antigen-binding fragments thereof) such that intrachain or intermolecular disulfide linkages are preferably not formed (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al. (2009) Blood 114(13):2721-2729; U.S. Pat. No. 7,521,541; U.S. Pat. No. 7,723,485; WO 2009 / 052249). The engineered cysteine ​​thiols can react with linkers in drugs (e.g., Formula I below) that may bear thiol-reactive electrophilic groups, such as maleimides or α-haloamides, to form ADCs with cysteine-engineered antibodies. Thus, the placement of the drug unit can be designed, controlled, and known. Drug loading can be controlled because engineered cysteine ​​thiol groups typically react with drug linker reagents in high yield. Engineering an IgG antibody to introduce a cysteine ​​amino acid by substitution at one site on the heavy or light chain provides two new cysteines on the symmetric antibody. Drug loadings approaching 2 can be achieved with near uniformity of the conjugated product ADC.

[0213] When multiple nucleophilic or electrophilic groups on an antibody or antigen-binding fragment thereof react with a drug, the resulting product can be a mixture of ADC compounds with a distribution of drug units attached to the antibody, for example, 1, 2, 3, etc. Liquid chromatography methods such as polymeric reversed phase (PLRP) and hydrophobic interaction (HIC) can separate compounds in a mixture by drug loading value. Preparations of ADCs with a single drug loading value (p) can be isolated.

[0214] Thus, the antibody drug conjugate compositions of the disclosure may include a mixture of antibody drug conjugates in which the antibodies or antigen-binding fragments thereof bear one or more drug moieties, and the drug moieties may be linked to the antibody or antigen-binding fragment thereof at different amino acid residues.

[0215] In some embodiments, the average number of drugs per antibody (or antigen-binding fragment thereof) ranges from 1 to 20. In some embodiments, the range is selected from 1 to 10, 2 to 10, 2 to 8, 2 to 6, and 4 to 10. In some embodiments, there is one drug per antibody (or antigen-binding fragment thereof). In some embodiments, the number of drugs per antibody (or antigen-binding fragment thereof) can be expressed as a ratio of drugs (i.e., drugs) to antibodies. This ratio is called the drug-to-antibody ratio (DAR). "DAR is the average number of drugs (i.e., drugs) linked to each antibody. In some embodiments of the present disclosure, the DAR ranges from 1 to 20. In some embodiments, the DAR ranges from 1 to 10, 2 to 10, 2 to 8, 2 to 6, and 4 to 10. In some embodiments, the DAR is about 1 to about 8. In certain embodiments of the present disclosure, the DAR is about 8. In certain embodiments of the present disclosure, the DAR is 8.

[0216] Dosing and Pharmaceutically Acceptable Compounds for ADC In some embodiments, the ADC is delivered directly to the site of the harmful cell population (e.g., thereby increasing exposure of the diseased tissue to the therapeutic agent), hi some embodiments, administration is directly to the airways, e.g., by inhalation or intranasal administration.

[0217] The ADCs described herein can be included in pharmaceutical compositions. The pharmaceutical compositions can include one or more pharma- ceutically acceptable excipients. In some embodiments, the pharmaceutical compositions of the present disclosure can include pharma- ceutically acceptable non-toxic sterile carriers, such as saline, non-toxic buffers, preservatives, and the like. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012).

[0218] In some embodiments, the ADC pharmaceutical compositions of the disclosure may be contained within one or more formulations selected from a capsule, a tablet, an aqueous suspension, a solution, a nasal aerosol, or a combination thereof.

[0219] In some embodiments, an ADC pharmaceutical composition comprises more than one type of ADC, for example, a pharmaceutical composition can include two or more ADCs with different antibodies, antigen-binding fragments, linkers, or cytotoxic agents, or different combinations thereof.

[0220] The term "pharmacologically effective amount" of an antibody or antigen-binding fragment means an amount sufficient to achieve effective binding to a target and to achieve a benefit, e.g., ameliorating the symptoms of a disease or condition or detecting a substance or cell.

[0221] In some aspects, the pharmaceutical composition may include a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), optionally a stabilizer (e.g., human albumin), and the like.

[0222] antibody preparation The antibodies of the present disclosure can be obtained using conventional techniques known to those skilled in the art, and their usefulness is confirmed by conventional binding tests, an exemplary method of which is described in Example 2. As an example, a convenient binding assay is to incubate cells expressing the antigen with the antibody. If the antibody is tagged with a fluorophore, the binding of the antibody to the antigen can be detected by FACS analysis.

[0223] The antibodies of the present disclosure can be produced in a variety of animals, including mice, rats, rabbits, goats, sheep, monkeys, or horses. Antibodies can be produced after immunization with individual capsular polysaccharides or multiple capsular polysaccharides. Blood isolated from these animals contains polyclonal antibodies (multiple antibodies that bind to the same antigen). Antigens can also be injected into chickens for the production of polyclonal antibodies in egg yolk. To obtain monoclonal antibodies specific to a single epitope of an antigen, antibody-secreting lymphocytes are isolated from the animal and immortalized by fusing them with a cancer cell line. The fused cells, called hybridomas, grow continuously in culture and secrete antibodies. Single hybridoma cells are isolated by dilution cloning to generate cell clones that all produce the same antibody, and these antibodies are called monoclonal antibodies. Methods for producing monoclonal antibodies are conventional techniques known to those skilled in the art (see, for example, Making and Using Antibodies: A Practical Handbook. GC Howard. CRC Books. 2006. ISBN 0849335280). Polyclonal and monoclonal antibodies are often purified using Protein A / G or antigen affinity chromatography.

[0224] The antibodies or antigen-binding fragments thereof of the present disclosure may be prepared as monoclonal anti-B7-H4 antibodies, which may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature 256:495 (1975). Using the hybridoma method, a mouse, hamster, or other suitable host animal is immunized as described above to induce lymphocyte production of antibodies that specifically bind to the immunizing antigen. Lymphocytes may also be immunized in vitro. After immunization, lymphocytes may be isolated and fused with a suitable myeloma cell line, for example using polyethylene glycol, to form hybridoma cells, which may then be selected away from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies specifically directed against the selected antigen as determined by immunoprecipitation, immunoblotting, or in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)) can then be grown in in vitro culture using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites tumors in animals. The monoclonal antibodies can then be purified from the culture medium or ascites fluid using known methods.

[0225] Alternatively, the antibodies or antigen-binding fragments thereof (e.g., as monoclonal antibodies) can be produced using recombinant DNA methods as described in U.S. Pat. No. 4,816,567. Polynucleotides encoding the monoclonal antibodies are isolated from mature B cells or hybridoma cells, such as by RT-PCR using oligonucleotide primers that specifically amplify genes encoding the heavy and light chains of the antibody, and sequenced using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into an appropriate expression vector, which when transferred into host cells that do not normally produce immunoglobulin proteins, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, the monoclonal antibodies are produced by the host cells. Recombinant monoclonal antibodies or antigen-binding fragments thereof of the desired species can also be isolated from phage display libraries expressing the CDRs of the desired species, such as those described in McCafferty et al., Nature 348:552-554 (1990); Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0226] Polynucleotides encoding the antibodies or antigen-binding fragments thereof of the present disclosure may be further modified in many different ways using recombinant DNA technology to generate alternative antibodies. In some aspects, for example, the constant domains of the light and heavy chains of a mouse monoclonal antibody may be (1) substituted, for example, with those regions of a human antibody to generate a chimeric antibody, or (2) substituted with a non-immunoglobulin polypeptide to generate a fusion antibody. In some aspects, the constant regions are truncated or removed to generate the desired antibody fragment of the monoclonal antibody. Site-directed or high-density mutagenesis of the variable regions may be used to optimize the specificity, affinity, etc. of the monoclonal antibody.

[0227] In some embodiments, the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof. Human antibodies can be prepared directly using various techniques known in the art. Immortalized human B lymphocytes can be produced by in vitro immunization or isolated from immunized individuals that produce antibodies against target antigens. See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., J. Immunol. 147(1): 86-95 (1991); U.S. Patent No. 5,750,373.

[0228] In some aspects, the antibodies or antigen-binding fragments thereof can be selected from phage libraries that express human antibodies, e.g., as described in Vaughan et al., Nat. Biotech. 14:309-314 (1996); Sheets et al., Proc. Natl. Acad. Sci. USA, 95:6157-6162 (1998); Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991), and Marks et al., J. Mol. Biol. 222:581 (1991). Techniques for the production and use of antibody phage libraries are also described in U.S. Pat. Nos. 5,969,108, 6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484 and 7,264,963, each of which is incorporated by reference in its entirety, and in Rothe et al., J. Molec. Biol. 376:1182-1200 (2008).

[0229] Affinity maturation and chain shuffling strategies are known in the art and can be utilized to generate high affinity human antibodies or antigen-binding fragments thereof, see Marks et al., BioTechnology 10:779-783 (1992), incorporated by reference in its entirety.

[0230] In some embodiments, the antibody or antigen-binding fragment thereof (e.g., monoclonal antibody) may be a humanized antibody. Methods of modifying, humanizing, or resurfacing non-human or human antibodies may also be used and are well known in the art. A humanized, resurfacing, or similarly modified antibody may have one or more amino acid residues derived from a non-human source, such as, but not limited to, mouse, rat, rabbit, non-human primate, or other mammalian source. These non-human amino acid residues are often replaced by residues referred to as "import" residues, which are typically taken from "import" variable, constant, or other domains of known human sequences. Such imported sequences may be used to reduce immunogenicity, or to reduce, enhance, or alter binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable property as known in the art. Suitably, the CDR residues may be directly and most substantially involved in influencing B7-H4 binding. Thus, while it is preferable to maintain some or all of the non-human or human CDR sequences, the non-human sequences of the variable and constant regions may be replaced with human or other amino acids.

[0231] The antibodies may optionally be humanized, resurfacing, engineered or human antibodies that have been modified while retaining high affinity for the antigen B7-H4 and other favorable biological properties. To achieve this goal, humanized (or human) or engineered anti-B7-H4 antibodies and resurfacing antibodies may optionally be prepared by a process of analysis of the parental sequences and various conceptual humanized and engineered products using three-dimensional models of the parental, engineered and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display predicted three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind to its antigen, such as B7-H4. In this way, FW residues can be selected and combined from consensus and import sequences to achieve desired antibody properties, such as increased affinity for the target antigen.

[0232] The anti-B7-H4 antibodies or antigen-binding fragments thereof of the present disclosure can be humanized, resurfaced, or modified by any known method, such as those described in Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988); Sims et al., J. Immunol. 151:2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al. al., J. Immunol. 151:2623 (1993); US Patent No. 5,639,641, US Patent No. 5,723,323; US Patent No. 5,976,862; , 824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,76 Specification No. 6,886; Specification No. 5,714,352; Specification No. 6,204,023; Specification No. 6,180,370; Specification No. 5,693,762; Specification No. 5,530,1 Specification No. 01; Specification No. 5,585,089; Specification No. 5,225,539; Specification No. 4,816,567; Specification No. 7,557,189; Specification No. 7,538,195 and 7,342,110; International Application Nos. PCT / US98 / 16280; PCT / US96 / 18978; PCT / US91 / 09630; PCT / US91 / 05939; PCT / US94 / 01234; PCT / GB89 / 01334; PCT / GB91 / 01134; PCT / GB92 / 01755; WO 90 / 14443; WO 90 / 14424; WO 90 / 14430, and EP 229246, each of which is incorporated by reference in its entirety, including the references cited therein.

[0233] Anti-B7-H4 humanized antibodies and antigen-binding fragments thereof can also be generated in transgenic mice containing human immunoglobulin loci that are capable of producing a full repertoire of human antibodies upon immunization without producing endogenous immunoglobulins. This approach is described in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425 and 5,661,016.

[0234] In some embodiments, fragments (e.g., antibody fragments) of antibodies (e.g., anti-B7-H4 antibodies) are provided. Various techniques are known for the production of antibody fragments. Traditionally, these fragments are obtained via proteolytic digestion of intact antibodies, for example, as described by Morimoto et al., J. Biochem. Biophys. Meth. 24:107-117 (1993) and Brennan et al., Science 229:81 (1985). In some embodiments, anti-B7-H4 antibody fragments are produced recombinantly. Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E. coli or other host cells, allowing for the production of large amounts of these fragments. Such anti-B7-H4 antibody fragments can also be isolated from the antibody phage libraries discussed above. Anti-B7-H4 antibody fragments can also be linear antibodies, as described in U.S. Pat. No. 5,641,870. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.

[0235] According to the present disclosure, techniques may be adapted for the production of single chain antibodies specific for B7-H4. See, e.g., U.S. Pat. No. 4,946,778). Additionally, methods may be adapted for the construction of Fab expression libraries to allow for the rapid and efficient identification of monoclonal Fab fragments with the desired specificity for B7-H4 or derivatives, fragments, analogs or homologs thereof. See, e.g., Huse et al., Science 246:1275-1281 (1989). Antibody fragments may be produced by techniques known in the art, including, but not limited to, pepsin digestion of antibody molecules to produce F(ab')2 fragments, reduction of disulfide bridges of F(ab')2 fragments to produce Fab fragments, treatment of antibody molecules with papain and a reducing agent to produce Fab fragments, or Fv fragments.

[0236] In some aspects, the antibody or antigen-binding fragment thereof of the present disclosure may be modified to extend its serum half-life. This may be accomplished, for example, by incorporating a salvage receptor binding epitope into the antibody or antibody fragment by mutating the appropriate region in the antibody or antibody fragment, or by incorporating the epitope into a peptide tag which is then fused to the antibody or antibody fragment at either end or in the middle (e.g., by DNA or peptide synthesis), or by YTE mutation. Other methods for extending the serum half-life of an antibody or antigen-binding fragment thereof, such as conjugation with a heterologous molecule such as PEG, are known in the art.

[0237] The modified antibodies or antigen-binding fragments thereof as provided herein may comprise any type of variable region that provides for the association of the antibody or polypeptide with B7-H4. In this regard, the variable region may comprise or be derived from any type of mammal that can be induced to mount a humoral response to a desired antigen and produce immunoglobulins. As such, the variable region of an anti-B7-H4 antibody or antigen-binding fragment thereof may be of, for example, human, murine, non-human primate (e.g., cynomolgus monkey, macaque, etc.) or wolf origin. In some embodiments, both the variable and constant regions of the modified antibody or antigen-binding fragment thereof are human. In some embodiments, the variable region of a matched antibody (usually from a non-human source) may be modified or specifically tailored to improve the binding properties or reduce the immunogenicity of the molecule. In this regard, the variable regions useful in the present disclosure may be humanized or otherwise modified by including imported amino acid sequences.

[0238] In some embodiments, both the heavy and light chain variable domains of the antibody or antigen-binding fragment thereof are modified by at least partial replacement of one or more CDRs and / or partial framework region replacement and sequence alteration. The CDRs may be derived from antibodies of the same class or even subclass as the antibody from which the framework regions are derived, but it is envisaged that the CDRs are derived from antibodies of different classes, and in some particular embodiments from different species. To transfer the antigen-binding capacity of one variable domain to another, it is not necessary to replace all CDRs with the complete CDRs of the donor variable region. Rather, it is sufficient to transfer only those residues necessary to maintain the activity of the antigen-binding site. Given the explanations in US Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it is well within the capabilities of the skilled artisan to carry out routine experimentation to obtain functional antibodies with reduced immunogenicity.

[0239] Notwithstanding the modifications to the variable regions, one of skill in the art will understand that the modified antibodies or antigen-binding fragments thereof of the present disclosure include antibodies (e.g., full-length antibodies or antigen-binding fragments thereof) in which at least a portion of one or more of the constant region domains has been deleted or otherwise modified to provide a desired biochemical property, such as improved tumor localization or reduced serum half-life, when compared to an antibody of substantially the same immunogenicity that includes a native or unmodified constant region. In some embodiments, the constant region of the modified antibody includes a human constant region. Modifications to the constant region that are compatible with the present disclosure include the addition, deletion, or substitution of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein may include modifications or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and / or the light chain constant domain (CL). In some embodiments, modified constant regions in which one or more domains are partially or completely deleted are contemplated. In some embodiments, the modified antibodies include domain-deleted constructs or variants in which the entire CH2 domain has been removed (ΔCH2 constructs). In some embodiments, the deleted constant region domain may be replaced with a short amino acid spacer (e.g., 10 residues) that typically provides some of the molecular flexibility conferred by the missing constant region.

[0240] In addition to their structure, it is known in the art that the constant region mediates several effector functions. For example, antibodies bind to cells via the Fc region, and Fc receptor sites on the antibody Fc region bind to Fc receptors (FcR) on the cells. There are a number of Fc receptors specific for different antibody classes, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors), and IgM (mu receptors). Binding of antibodies to Fc receptors on the cell surface elicits a number of diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, placental passage, and regulation of immunoglobulin production.

[0241] In some embodiments, the antibody or antigen-binding fragment thereof provides altered effector functions, which then affect the biological profile of the antibody or antigen-binding fragment thereof when administered. For example, constant region domains may be deleted or inactivated (by point mutation or other means) to reduce Fc receptor binding of the modified antibody in circulation. In other cases, constant region modifications may modulate complement binding, thus reducing serum half-life and non-specific association of conjugated cytotoxins, consistent with the present disclosure. Still other modifications of the constant region may be used to remove disulfide bonds or oligosaccharide moieties that allow for enhanced localization by improving antigen specificity or antibody mobility. Similarly, modifications to the constant region according to the present disclosure may be readily made using well-known biochemical or molecular engineering techniques well within the purview of one of skill in the art.

[0242] In some embodiments, the antibody or antigen-binding fragment thereof does not have one or more effector functions. For example, in some embodiments, the antibody or antigen-binding fragment thereof does not have antibody-dependent cellular cytotoxicity (ADCC) activity and / or complement-dependent cytotoxicity (CDC) activity. In some embodiments, the antibody or antigen-binding fragment thereof does not bind to Fc receptors and / or complement factors. In some embodiments, the antibody or antigen-binding fragment thereof does not have effector functions.

[0243] In some embodiments, the antibodies or antigen-binding fragments thereof may be modified to fuse the CH3 domain directly to the hinge region of the individual modified antibodies or fragments thereof. In other constructs, a peptide spacer may be inserted between the hinge region and the modified CH2 and / or CH3 domain. For example, a compatible construct may be expressed in which the CH2 domain is deleted and the remaining CH3 domain (modified or unmodified) is linked to the hinge region with a spacer of 5-20 amino acids. Adding such a spacer may ensure, for example, that the regulatory elements of the constant domain remain free and available or that the hinge region remains mobile. In some cases, amino acid spacers may prove to be immunogenic and induce undesirable immune responses against the construct. In some embodiments, any spacers added to the construct may be relatively non-immunogenic or even omitted entirely to maintain the desired biochemical qualities of the modified antibodies.

[0244] In addition to the deletion of the entire constant region domain, the antibodies or antigen-binding fragments thereof provided herein may be modified by partial deletion or substitution of several or even single amino acids in the constant region. For example, the mutation of a single amino acid in a selected region of the CH2 domain may be sufficient to substantially reduce Fc binding and thereby increase tumor localization. Similarly, one or more constant region domains that control effector functions (e.g., complement C1Q binding) may be completely or partially deleted. Such partial deletion of the constant region may improve selected properties of the antibody or antigen-binding fragment thereof (e.g., serum half-life) while leaving other desirable functions associated with the subject constant region domain intact. Furthermore, the constant regions of the antibodies and antigen-binding fragments thereof may be modified by mutation or substitution of one or more amino acids that enhance the profile of the resulting construct. In this regard, it is possible to disrupt the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the configuration and immunogenicity profile of the modified antibody or antigen-binding fragment thereof. In some embodiments, there may be the addition of one or more amino acids to the constant region to enhance a desired property, such as reduced or increased effector function, or to provide for greater cytotoxin or carbohydrate binding. In some embodiments, it may be desirable to insert or duplicate a particular sequence from a selected constant region domain.

[0245] The methods, combinations, and kits described herein encompass substantially homologous variants and equivalents of the antibodies or antigen-binding fragments of the present disclosure (e.g., murine, chimeric, humanized, or human antibodies or antigen-binding fragments thereof). These may contain, for example, conservative substitution mutations, i.e., the replacement of one or more amino acids with a similar amino acid. For example, a conservative substitution refers to the replacement of an amino acid with another amino acid within the same general class, such as the replacement of one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. What is intended by conservative amino acid substitution is well known in the art.

[0246] In some aspects, the antibody or antigen-binding fragment thereof may be further modified to contain additional chemical moieties that are not normally part of the protein. These derivatized moieties may improve the solubility, biological half-life, or absorption of the protein. These moieties may also reduce or eliminate any undesirable side effects of the protein. A review of such moieties can be found in Remington's Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012).

[0247] definition The following definitions may relate specifically to the description of Topoisomerase I inhibitors above, and more specifically to the section entitled "Additional Preferences."

[0248] C 5~6 Arylene: "C 5~6 The term "arylene," as used herein, pertains to a divalent moiety obtained by removing two hydrogen atoms from an aromatic ring atom of an aromatic compound.

[0249] In this context, prefixes (e.g., C 5~6 ) indicates the number of ring atoms or range of number of ring atoms, whether carbon atoms or heteroatoms.

[0250] The ring atoms may be all carbon atoms, as in a "carbarylene group," in which case the group is phenylene (C6).

[0251] Alternatively, the ring atoms may contain one or more heteroatoms, as in a "heteroarylene group." Examples of heteroarylene groups include, but are not limited to, those derived from: N1: Pyrrole (azole) (C5), pyridine (azine) (C6), O1: Furan (oxol) (C5), S1: thiophene (thiol) (C5), N1O1: Oxazole (C5), Isoxazole (C5), Isoxazine (C6), N2O1: Oxadiazole (Furazan) (C5), N3O1: Oxatriazole (C5), N1S1: Thiazole (C5), isothiazole (C5), N2: imidazole (1,3-diazole) (C5), pyrazole (1,2-diazole) (C5), pyridazine (1,2-diazine) (C6), pyrimidine (1,3-diazine) (C6) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C6), and N3: Triazole (C5), triazine (C6).

[0252] C 1~4 Alkyl: As used herein, “C 1~4 The term "alkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 4 carbon atoms, which may be aliphatic or alicyclic, and may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated). 1~n The term "alkyl" refers to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having 1 to n carbon atoms, which may be aliphatic or alicyclic, and may be saturated or unsaturated (e.g., partially unsaturated, fully unsaturated). Thus, the term "alkyl" includes the subclasses alkenyl, alkynyl, cycloalkyl, etc., discussed below.

[0253] Examples of saturated alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), propyl (C3), and butyl (C4).

[0254] Examples of saturated straight chain alkyl groups include, but are not limited to, methyl (C1), ethyl (C2), n-propyl (C3), and n-butyl (C4).

[0255] Examples of saturated branched alkyl groups include iso-propyl (C3), iso-butyl (C4), sec-butyl (C4) and tert-butyl (C4).

[0256] C 2~4 Alkenyl: As used herein, the term "C 2~4 The term "alkenyl" refers to an alkyl group having one or more carbon-carbon double bonds.

[0257] Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, -CH=CH2), 1-propenyl (-CH=CH-CH3), 2-propenyl (allyl, -CH-CH=CH2), isopropenyl (1-methylvinyl, -C(CH3)=CH2), and butenyl (C4).

[0258] C 2~4 Alkynyl: As used herein, the term "C 2~4 The term "alkynyl" refers to an alkyl group having one or more carbon-carbon triple bonds.

[0259] Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (-C≡CH) and 2-propynyl (propargyl, -CH2-C≡CH).

[0260] C 3~4 Cycloalkyl: As used herein, "C 3~4 The term "cycloalkyl" refers to an alkyl group that is also a cyclyl group, i.e., a monovalent moiety obtained by removing one hydrogen atom from one alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.

[0261] Examples of cycloalkyl groups include: Saturated monocyclic hydrocarbon compounds: Cyclopropane (C3) and cyclobutane (C4), and Unsaturated monocyclic hydrocarbon compounds: Cyclopropene (C3) and Cyclobutene (C4) Examples of such compounds include, but are not limited to, those derived from

[0262] Connecting indicator: Expression [ka] So, superscript signs C(=O) and NH indicates the group to which the atom is attached. For example, an NH group is shown attached to a carbonyl (not part of the depicted moiety) and a carbonyl is shown attached to an NH group (not part of the depicted moiety).

[0263] salt It may be convenient or desirable to prepare, purify, and / or handle the corresponding salt of the active compound / agent, e.g., a pharma- ceutically acceptable salt. Examples of pharma-ceutically acceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).

[0264] For example, the compound may be anionic or have a functional group that may be anionic (e.g., -COOH may be -COO - If so, salts can be formed with suitable cations. Examples of suitable inorganic cations include Na + and K + Alkali metal ions such as Ca 2+ and Mg 2+ Alkaline earth cations such as Al +3 Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH + ) and substituted ammonium ions (e.g., NHR + , NH2R2 + , NHR3 + , NR4 +) are included, but are not limited to. Some suitable examples of substituted ammonium ions include ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as those derived from amino acids such as lysine and arginine. One common example of a quaternary ammonium ion is N(CH3)4 + It is.

[0265] The compound is cationic or has a functional group that can be cationic (e.g., -NH2 can be -NH3 + If so, salts can be formed with suitable anions. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

[0266] Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, phenylsulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, trifluoroacetic acid, and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

[0267] solvate It may be convenient or desirable to prepare, purify and / or handle the corresponding solvate of active compound.The term "solvate" is used herein in the conventional sense to refer to a complex of solute (e.g., active compound, salt of active compound) and solvent.When the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a monohydrate, a dihydrate, a trihydrate, etc.

[0268] Isomers Certain compounds / agents of the present disclosure may exist in one or more particular geometric isomers, optical isomers, enantiomers, diastereomers, epimers, atropes, stereoisomers, tautomers, conformers or anomeric forms, including, but not limited to, cis and trans forms; E and Z forms; c, t and r forms; endo and exo forms; R, S and meso forms; D and L forms; d and l forms; (+) and (-) forms; keto, enol and enolate forms; syn and anti forms; synclinal and anticlinal forms; α and β forms; axial and equatorial forms; boat, chair, twist, envelope and half-chair forms and combinations thereof, hereinafter collectively referred to as "isomers" (or isomeric forms).

[0269] The term "chiral" refers to a molecule that has the property of not being superimposable on its mirror image partner, while the term "achiral" refers to a molecule that is superimposable on its mirror image partner.

[0270] The term "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

[0271] "Diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers can be separated by high resolution analytical procedures such as electrophoresis and chromatography.

[0272] "Enantiomers" refers to two stereoisomers of a compound which are non-superimposable mirror images of one another.

[0273] The stereochemical definitions and conventions used herein generally follow those of S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds of the present disclosure may contain asymmetric or chiral centers and therefore may exist in different stereoisomeric forms. All stereoisomeric forms of the compounds of the present disclosure, including but not limited to diastereomers, enantiomers and atropisomers and mixtures thereof, such as racemic mixtures, are intended to be part of the present disclosure. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the chiral centers of the molecule. The prefixes d and l or (+) and (-) are used to designate the sign of rotation of plane polarized light by the compound, with (-) or l meaning that the compound is levorotatory. Compounds preceded by (+) or d are dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often referred to as an enantiomeric mixture. A 50:50 mixture of enantiomers is called a racemic mixture or racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, which is devoid of optical activity.

[0274] "Enantiomerically enriched form" refers to a sample of a chiral substance in which the enantiomeric ratio is greater than 50:50 but less than 100:0.

[0275] It should be noted that, except as described below with respect to tautomeric forms, when the term "isomer" is used herein, structural (or constitutional) isomers (i.e., isomers that differ not only in the arrangement of the atoms in space but also in the connections between the atoms) are specifically excluded. For example, a reference to a methoxy group, -OCH3, is not to be considered a reference to its structural isomer, a hydroxymethyl group, -CH2OH. Similarly, a reference to ortho-chlorophenyl is not to be considered a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may fully include structural isomeric forms that fall within that class (e.g., C 1~7 Alkyl includes n-propyl and iso-propyl, butyl includes n-, iso-, sec- and tert-butyl, methoxyphenyl includes ortho-, meta- and para-methoxyphenyl).

[0276] The above exclusion does not pertain to tautomeric forms, such as keto, enol and enolate forms, such as the following tautomeric pairs: keto / enol (as illustrated below), imine / enamine, amide / iminoalcohol, amidine / enediamine, nitroso / oxime, thioketone / enethiol, N-nitroso / hydroxyazo and nitro / aci-nitro. [ka]

[0277] The term "tautomer" or "tautomeric form" refers to structural isomers of different energies that are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by reorganization of some of the bonding electrons.

[0278] It should be noted that the term "isomer" specifically includes compounds with one or more isotopic substitutions. For example, H is 1 H, 2 H(D) and 3 H(T) and C can be any isotopic form, including 12 C. 13 C and 14 It can be any isotopic form including C, and O is 16 O and 18 It may be in any isotopic form, including O, etc.

[0279] Examples of isotopes that may be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, e.g., 2 H (deuterium, D), 3 H (tritium), 11 C. 13 C. 14 C. 15 N, 18 F, 31 P, 32 P, 35 S, 36 Cl and 125I and the like. The compounds of the present disclosure are labeled with various isotopes, such as those incorporating radioisotopes such as 3H, 13C and 14C. Such isotopically labeled compounds may be useful in metabolic studies, kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), including drug or substrate tissue distribution assays, or in radiation therapy of patients. The deuterium labeled or substituted therapeutic compounds of the present disclosure may have improved DMPK (drug metabolism and pharmacokinetics) properties in relation to distribution, metabolism and excretion (ADME). Substitution with heavier isotopes such as deuterium may provide certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements. 18F labeled compounds may be useful in PET or SPECT studies. Isotopically labeled compounds of the present disclosure and their prodrugs can generally be prepared by replacing nonisotopically labeled reagents with readily available isotope-labeled reagents, following the schemes described below or by carrying out the procedures disclosed in the Examples and Preparations. Furthermore, substitution with heavier isotopes, particularly deuterium (i.e., 2H or D), may provide certain therapeutic advantages due to greater metabolic stability, such as extended in vivo half-life or reduced dosage requirements or improved therapeutic index. In this context, deuterium is understood to be considered as a substituent. The concentration of such heavy isotopes, particularly deuterium, may be defined by the isotopic enrichment factor. In the compounds of the present disclosure, any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom.

[0280] Unless otherwise specified, reference to a particular compound encompasses all such isomeric forms, including (fully or partially) racemic and other mixtures thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallization and chromatographic means) of such isomeric forms are either known in the art or readily obtained by application of the methods taught herein or known methods in a known manner.

[0281] PARP1 inhibitors In some embodiments, the ADCs described herein are administered in combination with an inhibitor of PARP1 (poly(ADP-ribose) polymerase 1). In some embodiments, the inhibitor of PARP1 is AZD5305. The terms "inhibit," "inhibition," or "inhibiting" include a decrease in the baseline activity of a biological activity or process.

[0282] The term "AZD5305" refers to the compound having the chemical name 5-[4-[(7-ethyl-6-oxo-5H-1,5-naphthyridin-3-yl)methyl]piperazin-1-yl]-N-methyl-pyridine-2-carboxamide and the following: [ka] It refers to a compound having the structure shown below.

[0283] Preparations of AZD5305 are disclosed in US Patent Publication No. 2021 / 0040084A1, the disclosure of which is incorporated by reference in its entirety. In some embodiments, a free base of AZD5305 is administered to the subject. In some embodiments, a pharma- ceutically acceptable salt of AZD5305 is administered to the subject. In some embodiments, crystalline AZD5305 is administered to the subject. In some embodiments, crystalline form A of AZD5305 is administered to the subject.

[0284] A "pharmaceutical composition" comprising AZD5305 includes a composition comprising an active ingredient and a pharma- ceutically acceptable excipient, carrier or diluent, wherein the active ingredient is AZD5305 or a pharma- ceutically acceptable salt thereof.

[0285] ATR inhibitors In some embodiments, the ADCs described herein are administered in combination with an inhibitor of ATR (also known as FRAP-associated protein 1, FRP1, MEC1, SCKL, SECKL1R). In some embodiments, the inhibitor of ATR is AZD6738. The terms "inhibit," "inhibition," or "inhibiting" include a decrease in the baseline activity of a biological activity or process.

[0286] The term "AZD6738" refers to the ATR (also known as FRAP-related protein 1, FRP1, MEC1, SCKL, SECKL1R) inhibitor, 4-{4-[(3R)-3-methylmorpholin-4-yl]-6-[1-((R)-S-methylsulfonimidoyl)cyclopropyl]pyrimidin-2-yl}-1H-pyrrolo[2,3-b]pyridine, having the following formula: [ka] or a pharma- ceutically acceptable salt thereof. Further examples of ATR inhibitors can be found in U.S. Patent No. 8,252,802, which is incorporated herein by reference in its entirety for all purposes.

[0287] A "pharmaceutical composition" comprising AZD6738 includes a composition comprising an active ingredient and a pharma- ceutically acceptable excipient, carrier or diluent, wherein the active ingredient is AZD6738 or a pharma- ceutically acceptable salt thereof.

[0288] combination The disclosure further provides a combination of an ADC and a PARP1 inhibitor for the treatment of cancer. Or an ADC and an ATR inhibitor for the treatment of cancer.

[0289] In some aspects, the disclosure provides a combination of A) an antibody-drug conjugate (ADC) comprising: i. an antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, ii. a cleavable linker, and iii. a cytotoxic agent; and B) a PARP1 (poly(ADP-ribose) polymerase 1) inhibitor, for treating cancer in a human subject in need thereof. In some aspects, the disclosure provides a combination of A) an antibody-drug conjugate (ADC) comprising: i. an antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, ii. a cleavable linker, and iii. a cytotoxic agent; and B) an ATR inhibitor, for treating cancer in a human subject in need thereof.

[0290] In some aspects, the disclosure provides a combination of A) an ADC comprising: i. an antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, ii. a cleavable linker, and iii. a cytotoxic agent; and B) AZD5305 or a pharma- ceutically acceptable salt thereof, for the treatment of cancer in a human subject in need thereof.

[0291] In some aspects, the disclosure provides A) an ADC, comprising an antibody or antigen-binding fragment thereof that binds to an i.B7-H4 polypeptide, comprising: a) a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), a heavy chain CDR3 (HCDR3), a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variants thereof; b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, or functional variants thereof; c) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively, or functional variants thereof; 1. HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or functional variants thereof; d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or functional variants thereof comprising the amino acid sequences of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, respectively; or e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or functional variants thereof comprising the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively; ii. a cleavable linker; and iii. a cytotoxic agent; and B) AZD5305 or a pharma- ceutically acceptable salt thereof is provided in combination for the treatment of cancer in a human subject in need thereof.

[0292] In some aspects, the disclosure provides A) an ADC, comprising an antibody or antigen-binding fragment thereof that binds to an i.B7-H4 polypeptide, the antibody or antigen-binding fragment thereof comprising: a) a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), a heavy chain CDR3 (HCDR3), a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variants thereof; b) a HCDR1, a HCDR2, a HCDR3, a LCDR1, a LCDR2, and a LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, or functional variants thereof; c) a HCD comprising the amino acid sequences of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18, respectively. R1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or functional variants thereof, d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, respectively, or functional variants thereof, or e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively, or functional variants thereof, ii. a cleavable linker, and iii. a cytotoxic agent; and B) AZD6738 or a pharma- ceutically acceptable salt thereof, are provided in combination for treating cancer in a human subject in need thereof.

[0293] In some embodiments of the combination provided herein, the cancer to be treated is a cancer as described herein. In some embodiments, the cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, hematological cancer, endometrial cancer, cholangiocarcinoma, NSCLC (squamous and / or adenocarcinoma), gastrointestinal cancer such as gastric cancer and colorectal cancer, and lung cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments of the method for treating breast cancer, the breast cancer is hormone receptor positive (HR+) breast cancer, human epidermal growth factor receptor 2 positive (HER2+) breast cancer, or triple negative breast cancer (TNBC). In some embodiments, the breast cancer is TNBC.

[0294] In some embodiments, the cancer is a homologous recombination repair deficient (HRD) cancer. In some embodiments, the cancer comprises one or more cells with a mutation in an HRD gene selected from BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L. In some embodiments, the mutated HRD gene is selected from BRCA1, BRCA2, and ATM. In some embodiments, the mutated HRD gene is BRCA1. In some embodiments, the mutated HRD gene is BRCA2. In some embodiments, the mutated HRD gene is ATM.

[0295] In some embodiments of the combinations provided herein, the ADC may comprise any antibody or antigen-binding fragment thereof as described herein. In some embodiments of the combinations provided herein, the ADC may comprise any linker as described herein. In some embodiments of the combinations provided herein, the ADC may comprise any cytotoxic agent as described herein.

[0296] In some embodiments of the combinations provided herein, the ADC comprises an antibody or antigen-binding fragment thereof as described herein conjugated to SG3932, SG4010, SG4057, SG4052, or a combination thereof. In some embodiments of the combinations provided herein, the ADC comprises an antibody or antigen-binding fragment thereof as described herein conjugated to SG3932. The linker-cytotoxic agent molecules SG3932, SG4010, SG4057, and SG4052 are described elsewhere herein.

[0297] kit In one aspect, a kit is provided comprising an ADC as described herein and a PARP1 inhibitor as described herein. In one aspect, a kit is provided comprising an ADC as described herein and an ATR inhibitor as described herein. Further encompassed is the use of said kit in the methods of the disclosure.

[0298] In some aspects, the disclosure provides A) an antibody-drug conjugate (ADC), comprising an antibody or antigen-binding fragment thereof that binds to an i.B7-H4 polypeptide, comprising: a) a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), a heavy chain CDR3 (HCDR3), a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variants thereof; b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, or functional variants thereof; c) an antibody or antigen-binding fragment thereof that binds to an i.B7-H4 polypeptide, comprising: or functional variants thereof comprising the amino acid sequences of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively; or e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, comprising the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; an antibody-drug conjugate (ADC) comprising: ii. a cleavable linker; and iii. a cytotoxic agent; and B) AZD5305 or a pharma- ceutically acceptable salt thereof.

[0299] In some aspects, the disclosure provides A) an antibody-drug conjugate (ADC), comprising an antibody or antigen-binding fragment thereof that binds to an i.B7-H4 polypeptide, comprising: a) a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), a heavy chain CDR3 (HCDR3), a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively, or functional variants thereof; b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively, or functional variants thereof; c) an antibody or antigen-binding fragment thereof that binds to an i.B7-H4 polypeptide, comprising: or functional variants thereof comprising the amino acid sequences of SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively; or e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, comprising the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively; an antibody-drug conjugate (ADC) comprising: ii. a cleavable linker; and iii. a cytotoxic agent; and B) AZD6738 or a pharma- ceutically acceptable salt thereof.

[0300] In some embodiments of the kits provided herein, the ADC may comprise any antibody or antigen-binding fragment thereof as described herein. In some embodiments of the combinations provided herein, the ADC may comprise any linker as described herein. In some embodiments of the combinations provided herein, the ADC may comprise any cytotoxic agent as described herein.

[0301] In some embodiments of the kits provided herein, the ADC comprises an antibody or antigen-binding fragment thereof as described herein conjugated to SG3932, SG4010, SG4057, SG4052, or a combination thereof. In some embodiments of the combinations provided herein, the ADC comprises an antibody or antigen-binding fragment thereof as described herein conjugated to SG3932. The linker-cytotoxic agent molecules SG3932, SG4010, SG4057, and SG4052 are described elsewhere herein.

[0302] In some embodiments, the kit comprises an isolated (e.g., purified) antigen or antibody binding fragment as described herein. In some embodiments, the kit comprises an isolated (e.g., purified) ADC as described herein. In some embodiments, the kit comprises one or more containers. The kit may provide the antigen or antibody binding fragment and the linker and / or cytotoxic agent individually (e.g., the agent is not conjugated to the antigen or antibody binding fragment but is in a suitable form for conjugation thereto), and optionally, the kit is further provided with instructions and / or reagents for conjugating the agent to the antigen or antibody binding fragment. In some embodiments, the kit comprises all of the components necessary and / or sufficient to administer a combination of an ADC and a PARP1 inhibitor to a subject. In some embodiments, the kit comprises all of the instructions necessary and / or sufficient to administer a combination of an ADC and a PARP1 inhibitor to a subject. EXAMPLES

[0303] The combination therapies disclosed herein will now be further described with reference to the following non-limiting examples.

[0304] Example 1 – Cytotoxicity assay using DLD-1-BRCA WT cells engineered to express B7-H4 A cytotoxicity assay was performed to determine the efficacy of combined B7-H4-TOP1i ADC (E02-GL-SG3932) and PARP1 selective inhibitor (AZD5305) therapy in killing DLD-1-BRCA wild-type (WT) cells expressing B7-H4. DLD-1-BRCA WT cells (Horizon Discovery) modified to express B7-H4 were seeded in 96-well plates at a seeding density of 1,000 cells / well in RPMI medium containing 10% heat-inactivated FBS. The next day, cells were treated with increasing concentrations of E02-GL-SG3932, isotype-matched control ADC (0.003052-200 nM) or AZD5305 (0.4-10 μM), either individually or in combination. Cells were incubated for 7 days after treatment and cell viability was measured using the CellTiter-Glo assay (Promega), which quantifies cell viability based on the amount of adenosine triphosphate (ATP) present. Results show that combined E02-GL-SG3932 and AZD5305 treatment at increasing concentrations each killed more WT cells expressing B7-H4 at a faster rate than the other treatments: E02-GL-SG3932 individually, AZD5305 individually, isotype-matched control ADC, and isotype-matched control ADC and AZD5305 (Figure 1).

[0305] The Bliss synergy model was used to estimate the effect of the two-drug combination and the pharmacological synergistic (Bliss score > 0) or antagonistic (Bliss score < 0) response of AZD5305 in combination with E02-GL-SG3932 ADC, as determined using Combenefit software (Cancer Research UK Cambridge Institute). Veroli et al., Bioinformatics. 2016 Sep 15; 32 (18): 2866-2868. As shown in Figure 2, increasing doses of the combination treatment resulted in a Bliss energy score greater than 10 (blue), indicating treatment synergy between E02-GL-SG3932 and AZD5305 at increasing doses.

[0306] Example 2 - Cytotoxicity assay using DLD-1-XMAN-BRCA2- / - cells engineered to express B7-H4 DLD-1-XMAN-BRCA2- / - cells modified to express B7-H4 were used to evaluate the efficacy of combined E02-GL-SG3932 and AZD5305 treatment in killing target cells. DLD-1-XMAN-BRCA2- / - cells lack the BRCA2 gene, which provides instructions for producing proteins that act as tumor suppressors. DLD-1-XMAN-BRCA2- / - cells modified to express B7-H4 (Horizon Discovery) were seeded in 96-well plates at a seeding density of 1,000 cells / well in RPMI medium containing 10% heat-inactivated FBS. The next day, cells were treated with increasing concentrations of E02-GL-SG3932, isotype-matched control ADC (0.0001-6.7 nM) or AZD5305 (0.04-1 nM), either individually or in combination. Cells were incubated for 7 days after treatment and cell viability was measured using the CellTiter-Glo assay (Promega). As can be seen in Figure 3, the 1 nM combination treatment killed more DLD-1-XMAN-BRCA2- / - cells expressing B7-H4 at a faster rate than the other treatments: E02-GL-SG3932 individually, AZD5305 individually, isotype-matched control ADC, and isotype-matched control ADC and AZD5305.

[0307] The Bliss synergy model was used to estimate the combined effect of the two drugs and the pharmacological synergistic (Bliss score >0) or antagonistic (Bliss score <0) response of AZD5305 in combination with E02-GL-SG3932 ADC, determined using Combenefit software (Cancer Research UK Cambridge Institute). As shown in Figure 4, doses of E02-GL-SG3932 (0.02 nM) in combination with AZD5305 (0.04 nM, 0.2 nM and 1 nM) gave Bliss energy scores greater than 10 (blue), indicating processing synergy between E02-GL-SG3932 and AZD5305.

[0308] Example 3 – Cytotoxicity assay using MX-1 cells engineered to express B7-H4 MX-1 cells, established as in vitro cultures from the MX-1 tumor xenograft model of breast carcinoma tissue, were used to evaluate the efficacy of combined E02-GL-SG3932 and AZD5305 treatment in killing target cells. MX-1 cells were seeded in 96-well plates at a seeding density of 1,500 cells / well in DMEM medium containing 10% heat-inactivated FBS. The next day, cells were treated with increasing concentrations of E02-GL-SG3932, isotype-matched control ADC (0.001-400 nM) or AZD5305 (0.2 or 1 nM), either individually or in combination. Cells were incubated for 7 days after treatment and cell viability was measured using the CellTiter-Glo assay (Promega). FIG. 5 shows the ability of combined E02-GL-SG3932 and AZD5305 treatment at 0.2 nM and 1 nM concentrations to kill MX-1 cells to a greater extent than other treatments: E02-GL-SG3932 individually, AZD5305 individually, isotype-matched control ADC, and isotype-matched control ADC and AZD5305.

[0309] The Bliss synergy model was used to estimate the combined effect of the two drugs and the pharmacological synergistic (Bliss score >0) or antagonistic (Bliss score <0) response of AZD5305 in combination with E02-GL-SG3932 ADC, as determined using Combenefit software (Cancer Research UK Cambridge Institute). Figure 6 shows that doses of E02-GL-SG3932 (0.128-3.2 nM and 400 nM) in combination with AZD5305 (1 nM) yielded Bliss energy scores of ≦16 and above 10 (blue), indicating processing synergy between E02-GL-SG3932 and AZD5305 in these combinations.

[0310] Example 4 – MDA-MB-468 breast cancer xenograft model In vivo experiments were performed to determine the antitumor efficacy of E02-GL-SG3932 and AZD5305 treatment in mice bearing xenografts of human estrogen-dependent MDA-MB-468 breast cancer. MDA-MB-468 cells were implanted at 5 × 10 6 The tumors were injected at 150-200 mm 3 Dosing with isotype-ADC, E02-GL-SG3932, AZD5305 or combination was initiated when tumor size reached 100%. Isotype control ADC and E02-GL-SG3932 were administered as a single IV tail vein injection on day 1 only, and for combination studies, this injection was followed by oral AZD5305 once daily for 28 days. Tumor and body weight measurements were recorded twice weekly for the duration of the study. Tumors measured over time in mice treated with vehicle (control), individual AZD5305 (0.1 mg / kg and 1 mg / kg), isotype-matched control ADC (0.5 mg / kg), isotype-matched control ADC and AZD5305 (1 mg / kg) and individual E02-GL-SG3932 (0.1 mg / kg and 1 mg / kg) either increased in size or remained the same size, indicating that these treatment regimens have little to no anti-tumor activity in vivo (Figure 7).

[0311] Treatment with E02-GL-SG3932 (0.5 mg / kg) and AZD5305 (0.1 mg / kg) and E02-GL-SG3932 (0.5 mg / kg) and AZD5305 (1 mg / kg) showed high antitumor activity. As shown in FIG. 8, combined treatment with E02-GL-SG3932 and AZD5305 was effective in inhibiting the growth of approximately 67% of tumors in mice. Further comparative analysis of the efficacy of various treatment combinations in mice is shown in FIG. 9. The average tumor volume in mice treated with E02-GL-SG3932 (0.5 mg / kg) and AZD5305 (0.1 mg / kg) and E02-GL-SG3932 (0.5 mg / kg) and AZD5305 (1 mg / kg) was significantly smaller than the average tumor volume of the other treatment methods.

[0312] Example 5 – MDA-MB-468 breast cancer xenograft model In vivo experiments were performed to measure the antitumor efficacy of E02-GL-SG3932 and AZD6738 treatment in mice bearing xenografts of human estrogen-independent MDA-MB-468 breast cancer. MDA-MB-468 cells were cultured at 5 × 10 6 10 / mouse into the mammary fat pad of female CB-17 SCID mice. Isotype control ADC and E02-GL-SG3932 were administered as a single IV tail vein injection on day 1 only, and for combination studies, this injection was followed by oral administration of AZD6738 twice daily (BID) for 14 days. Tumor and body weight measurements were recorded twice weekly for the duration of the study. Tumors measured over time in mice treated with untreated (control), AZD6738 individually (25 mg / kg), isotype-matched control ADC (0.5 mg / kg), E02-GL-SG3932 individually (0.5 mg / kg), and E02-GL-SG3932 (0.5 mg / kg) and AZD6738 (25 mg / kg) are shown in Figure 10.

[0313] As shown in Figure 10, combined treatment with E02-GL-SG3932 and AZD6738 was effective in further inhibiting tumor growth in mice. The mean tumor volumes in mice treated with E02-GL-SG3932 (0.5 mg / kg) and AZD6738 (25 mg / kg) were significantly smaller than those of the other treatment methods.

[0314] Example 6 –Preclinical evaluation of antibody-drug conjugates directed at B7-H4 in combination with PARP1 selective inhibitors This study evaluated the activity of E02-GL-SG3932, an antibody-drug conjugate (ADC) against B7-H4 carrying a novel topoisomerase I inhibitor (TOP1i) payload, in combination with the poly-ADP ribose polymerase 1 (PARP1) selective inhibitor AZD5305 in preclinical models. A comprehensive analysis of B7-H4 distribution rates and assessment of intratumor heterogeneity in human tumors was performed. The addition of the next-generation PARP1 selective inhibitor AZD5305 to E02-GL-SG3932 treatment sensitized B7-H4 low-expressing PDX tumors, including models with homology capabilities that represent acquired or natural mechanisms of resistance to PARP inhibition. These findings support the feasibility of combining anti-B7H4 ADCs and PARP inhibitors in subjects who have already been treated with PARP inhibitors and in subjects who are resistant to treatment with PARP inhibitors.

[0315] Materials and Methods antibody Antibodies targeting human B7-H4 were generated in VelocImmune V2 mice by engineered immunization with SKBR3 cells and mouse B7-H4 extracellular domain chemically conjugated to keyhole limpet hemocyanin. Lead antibodies were selected based on specificity and cross-reactivity to human and cynomolgus B7-H4, and the antibody frameworks were matured to the closest human germline relative.

[0316] cell line HT29 and MDA-MB-468 cells were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen, GmbH (Braunschweig, Germany) and American Type Culture Collection (Rockville, MD, USA), respectively, and cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA). MX-1 cells were obtained from the National Cancer Institute (Bethesda, MD, USA) and cultured in Dulbecco's modified essential / F12 medium containing 10% FBS and 2 mM L-glutamine (Thermo Fisher Scientific). HT29 clonal cell lines stably expressing human B7-H4 (HT29-huB7-H4) were generated by using lentiviral expression vector prepared with pPACK H1 HIV Lentivector Packaging Kit (System Biosciences, Palo Alto, CA, USA), followed by transduction, geneticin selection, and isolation with fluorescence-activated cell selection through direct staining with anti-B7-H4-PE antibody (US Biological, Salem, MA, USA). All cell lines were cultured at a temperature of 37°C in a humidified atmosphere of 5% CO2, confirmed by short tandem repeat DNA profiling (IDEXX BioResearch Laboratories, Columbia, MO, USA), and found negative for mycoplasma by polymerase chain reaction and MycoSEQ Mycoplasma Detection Assay Kit (Thermo Fisher Scientific).

[0317] immunohistochemistry Immunohistochemistry (IHC) detection of B7-H4 was performed on 4 μm formalin-fixed paraffin-embedded tumor or tissue sections using an automated Leica Bond RX IHC staining platform (Leica Biosystems, Nussloch, Germany). Human normal and tumor tissue sections were stained with anti-human B7-H4 mouse immunoglobulin G1 (IgG1) (clone ZY0EYT-D11; AstraZeneca, Cambridge, UK), and tumor tissue sections derived from patient-derived xenografts (PDXs) were stained with anti-human B7-H4 rabbit IgG1. To ensure specificity and sensitivity, both antibodies were thoroughly validated for use in IHC with a series of known B7-H4-expressing cell lines, a B7-H4 transfected cell line (HT29-huB7H4), and human breast tumor tissues (MX-1 and MDA-MB-468) (Table 1). B7-H4-negative HT29 cells were used as a negative control. For orthogonal analysis of B7-H4 expression by IHC, formalin-fixed, paraffin-embedded cell pellets were prepared from the same cells. After cover slipping and mounting, slides were scanned using a Leica Aperio Scanscope AT2 slide scanner and then examined and scored by a pathologist. Antibody binding, a measure of B7-H4 receptor density, was measured by quantitative flow cytometry with the Quantum Simply Cellular kit from Bangs Laboratories (Fisher, IN, USA).

[0318] [Table 6]

[0319] Preparation of ADCs Various TOP1i warheads were covalently linked to native cysteines on the antibodies through val-ala (VA) or Gly-Gly-Phe-Gly (GGFG) peptide linkers with or without a PEG8 spacer, resulting in four individual ADCs with drug-to-antibody ratios (DARs) of approximately 8: SG3932, SG4057, SG4010, and SG4052. The preparation of these ADCs is also described in WO2022053650, which is incorporated herein by reference in its entirety. Non-targeting isotype control ADCs (Iso-ADCs) were similarly synthesized by using an isotype-matched antibody with comparable DAR. The E02-GL-SG3932 antibody drug conjugate is the E02-GL antibody conjugated to the SG3932 warhead.

[0320] A 100 mM solution of TCEP [tris(2-carboxyethyl)phosphine] in PBS (12.5 molar equivalents / antibody, 0.3 mmol) was added to E02-GL antibody (3.6 g, 24.0 micromoles) in reduction buffer containing PBS and 1 mM ethylenediaminetetraacetic acid and a final antibody concentration of 10 mg / mL. The reduction mixture was heated at 37°C in an orbital shaker with gentle (60 rpm) shaking for 2 h (or until complete reduction was observed by ultra-performance liquid chromatography [UPLC]). The reduced antibody was removed from incubation and allowed to cool to room temperature. To this mixture was added SG3932, SG4057, SG4010 or SG4052 linker-payload as a dimethyl sulfoxide (DMSO) solution (11-14 molar equivalents / antibody, 26.4-33.6 micromoles) for a final DMSO concentration of 10% (v / v). The solution was shaken for 1 h at 25 °C, and then the conjugation reaction was quenched with N-acetylcysteine ​​(5x excess compared to payload as a 100 mM stock solution). mPES, surface area 375-cm 2Excess free drug was removed via a tangential flow filtration unit using a MidiKros 30-kDa fiber filter. The neat conjugate was used to monitor the extent of free drug removal by UPLC-RP. After complete removal of free drug (11–16 DV), antibody-drug conjugates (ADCs) were formulated in 20 mM histidine, 240 mM sucrose, pH 6.0 (5 DV) and the resulting ADCs were filtered using a 0.22 μm filter under a sterile atmosphere. The reduced conjugates were used to calculate the drug-to-antibody ratio (DAR) by UPLC-RP, and the neat conjugates were used to measure the purity of the monomer by UPLC-size exclusion chromatography (SEC).

[0321] In vivo efficacy testing All animal studies were performed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care under the guidelines of AstraZeneca's Institutional Animal Care and Use Committee and appropriate animal research approvals. Cell line xenograft models were established through subcutaneous implantation of cells in 50% Matrigel (Corning, Corning, NY, USA) in either the right mammary fat pad (MX-1, MDA-MB-468) or left flank (HT29, HT29-huB7-H4) of female CB-17 mice (Envigo, Frederick, MD, USA). Tumor volumes were 150–200 mm 3 Upon reaching 1 h, mice were randomized and received a single intravenous (IV) injection of vehicle control (20 mM histidine, pH 6; 240 mM sucrose; 0.02% PS-80) or ADC. Body weights and tumor measurements were taken once or twice weekly, and tumor volumes were calculated using the following formula:

number

[0322] PDX models of human TNBC were established through subcutaneous implantation of tumor fragments into the flank of athymic nude mice (Envigo, Riom, France) and tested at Xentech (Evry, France). Animals were matched by tumor volume and then administered vehicle control or ADC as a single IV injection. When used, the PARP1 selective inhibitor AZD5305 (AstraZeneca) was formulated in water / HCl pH 3.5-4 and administered by oral gavage once daily for 28 days at a final dose volume of 10 mL / kg. Antitumor activity was determined on the final day, at which time all mice in the untreated control group remained on study. If the final tumor volume (ETV) in the treatment group was less than the initial tumor volume (ITV), the formula

number

[0323] Otherwise, the formula was significantly lower than that of the untreated control arm.

number

[0324] For pharmacodynamic and pharmacokinetic studies, HT29 or HT29-huB7-H4 tumors (250–300 mm 3 Mice bearing either the IgG1 or IgG2-specific mAb were given a single IV injection of vehicle control or ADC. Plasma and tumor samples were collected at the indicated time points, a portion of the tumor was fixed in formalin and embedded in a paraffin block for IHC analysis, and the remaining portion was snap frozen for analysis of total monoclonal antibody, ADC, and free warhead by liquid chromatography-mass spectrometry.

[0325] statistical analysis Data are presented as mean ± standard deviation or mean ± standard error of the mean. To assess the association between response vs. non-response in PDX models, Wilcoxon tests for group comparisons were performed in the R statistical programming environment (version 4.1). P<0.05 was considered statistically significant.

[0326] B7-H4 expression and distribution in tumor tissues Several tumor types express B7-H4, most commonly in endometrial carcinoma (94%), cholangiocarcinoma (89%), and breast cancer (HER2 + 78%, TNBC 74%, ER + In contrast, B7-H4 was detected in a limited number of normal human tissues, including fallopian tube, lung, breast, and prostate, generally found in <10% of the total cells in the samples, restricted to ductal or renal tubular epithelium, and primarily localized to the apical luminal membrane (Table 2).

[0327] To understand B7-H4 intratumoral heterogeneity within individual tumors, an important factor influencing ADC activity, a separate collection of histologically annotated samples of TNBC (n=196) were stained and digitally analyzed using a deep learning-based image analysis algorithm to detect and segment individual tumor epithelial cells and quantitate the level of B7-H4 expression at the membrane of each cell. This approach allowed examination of the distribution of B7-H4 expression on a cell-by-cell basis, indicating the extent of heterogeneous B7-H4 expression within each patient sample across the cohort (Figure 11C). B7-H4 expression and expression heterogeneity were weakly correlated (r=-0.84, P<0.01).

[0328] [Table 7]

[0329] [Table 8]

[0330] Combination of E02-GL-SG3932 with AZD5305, a next-generation PARP1 selective inhibitor PARP1 limits the cytotoxicity of TOP1i by promoting the resection and repair of the TOP1 cleavage complex. Therefore, we tested E02-GL-SG3932 in combination with AZD5305, a next-generation PARP1-selective inhibitor, in a panel of HRP PDX models, including BRCA WT tumors and models representing post-PARP resistance mechanisms. In the B7-H4-high expressing BRCA WT HBCx-39 model, AZD5305 sensitized tumors to a single suboptimal dose of 1.25 mg / kg E02-GL-SG3932, resulting in tumor regression in 5 of 5 mice compared to 24.5% TGI observed in the E02-GL-SG3932 monotherapy arm (Figure 12A). Higher activity of the combination treatment was also observed at a higher dose (3.5 mg / kg) of E02-GL-SG3932, with longer duration of response over E02-GL-SG3932 monotherapy (Figure 12B). Similar results were obtained in other high B7-H4 expressing models, including the BRCA1-mutant HBCx-24 model (Figure 12C), where PARPi resistance is likely driven through BRCA1 promoter methylation, and the HBCx-11 model (Figure 12D), which has reduced BRCA1 expression and is resistant to PARPi. The combination benefit observed in HBCx-39 and HBCx-11 is interesting, since these models not only represent BRCA WT (HBCx-39) or PARPi-resistant settings (HBCx-11), but are also SLFN11 negative. The robust combination activity observed in these models is unexpected, since SLFN11 deletion is a proposed resistance biomarker for both TOP1i and PARPi. The combination benefit was also surprisingly extended to HBCx-8, a BRCA1-mutated HRP model with very low B7-H4 expression and resistance to PARPi (Figure 12E). In this model, a single dose of 1.25 mg / kg E02-GL-SG3932 resulted in modest antitumor activity (32.9% TGI), while combination with AZD5305 resulted in tumor regression in 5 of 5 mice.A similar combination benefit was observed in a low B7-H4 expressing BRCA WT model, HBCx-2 (Figure 12F), as improved activity was observed with escalating doses of the ADC.

[0331] Pharmacological inhibition of DNA damage response pathways is an attractive approach to maximize response to TOP1i-ADC and result in greater antitumor activity. This study evaluated the combination of E02-GL-SG3932 and AZD5305, a potent PARP1 selective inhibitor that shows reduced hematologic toxicity in preclinical models compared to nonselective PARPi. The combination of E02-GL-SG3932 and AZD5305 resulted in greater antitumor activity than monotherapy, resulting in greater tumor regression or improved duration of response, even in models with low B7-H4 expression. This combination benefit was observed at low, suboptimal doses of E02-GL-SG3932 as well as in HRP BRCA1 WT or BRCA1-mutated models, representing a mechanism of PARPi resistance. These findings suggest that this combination may be effective in the setting of PARPi resistance, and does not need to achieve maximum ADC activity to exploit this mechanism.

[0332] array

[0333] [Table 9]

[0334] SEQ ID NO:31 (ZY0EPQ-E02, variable heavy chain) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWNWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTILVDTSKNQFSLKLSSVTAADTAVYYCARNLYNWNLDSWGQGTLVTVSS SEQ ID NO:32 (ZY0EPQ-E02, variable light chain) DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGRAPKRLIYVASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPRTFGQGTKVEIK SEQ ID NO:33 (ZY0EQD-E02, variable heavy chain, e.g. pre-germlined) QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWNWIRQPPGKGLEWIGEINHSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVLYNWNVDSWGQGTLVTVSS SEQ ID NO:34 (ZY0EQD-E02, variable light chain) DIQMTQSPSSLSASVGDRVTITCRASQDIRNDVGWYQQKPGKAPKRLIYAASRLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYPRTFGQGTKVEIK SEQ ID NO:35 (ZY0EOB-F05, variable heavy chain) [ka] SEQ ID NO:36 (ZY0EOB-F05, variable light chain) DIQLTQSPSFLSASVGDRVTITCWASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHLNSYPLTFGGGTKVEIK SEQ ID NO:37 (ZY0EO5-E07, variable heavy chain) [ka] SEQ ID NO:38 (ZY0EO5-E07, variable light chain) DIQLTQSPSFLSASVGGRVTITCWASQGIAGYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQHLNSYPLTFGGGTKVEIK SEQ ID NO:39: (ZY0EP0-C07, variable heavy chain) QVQLVESGGVLVKPGGSLRLSCAASGFTLSDYYMSWIRQAPGMGLEWVSYISSSGSTIYYTDSVKGRFTISRDSAKNSLYLQMNSLRAEDTAVYYCARDGVGFDYWGQGTLVTVSS SEQ ID NO: 40 (ZY0EP0-C07, variable light chain) EIVLTQSPGTLSLFPGERATLSCRASQSVSSSYLAWYQQKPGQSPRLLIYAASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLYTFGQGTKLEIK SEQ ID NO:41 (Maia heavy chain constant region, cysteine ​​insertion underlined): [ka] SEQ ID NO: 42 (light chain constant region) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:43 (ZY0EQD-E02, variable heavy chain, e.g. pre-germlined, e.g. variant of SEQ ID NO:33 / SEQ ID NO:45) QVQLQQWGAGLLKPSETLSLTCTVYGGSFSGYYWNWIRQPPGRGLEWIGEINHSGSTSYNPSLKSRITISIDTSKNQFSLKLSSVTAADTAVYYCARVLYNWNVDSWGQGTLVTVSS SEQ ID NO:44 (ZY0EQD-E02, light chain): [ka] SEQ ID NO: 45 (EQD-E02_GL, variable heavy chain, GL=germlined) QVQLQQWGAGLLKPSETLSLACTVYGGSFSGYYWNWIRQPPGKGLEWIGEINHSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVLYNWNVDSWGQGTLVTVSS SEQ ID NO: 46 (EQD-E02-GLY, variable heavy chain, germlined with GLY=Y substitution) QVQLQQWGAGLLKPSETLSLACTVYGGSFSGYYWNWIRQPPGKGLEWIGEIYHSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVLYNWNVDSWGQGTLVTVSS SEQ ID NO: 47 (EQD-E02-GLQ, variable heavy chain, germlined with GLQ=Q substitution) QVQLQQWGAGLLKPSETLSLACTVYGGSFSGYYWNWIRQPPGKGLEWIGEIQHSGSTSYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARVLYNWNVDSWGQGTLVTVSS SEQ ID NO: 48 (E02-GL-Maia-heavy chain, cysteine ​​insertion underlined) [ka] SEQ ID NO: 49 (E02-GLY-Maia-heavy chain, germlined with GLY=Y substitution) [ka] SEQ ID NO:50 (E02-GLQ-Maia-heavy chain, germlined with GLQ=Q substitution) [ka] SEQ ID NO:51 (E02-GL-WT-heavy chain) [ka] SEQ ID NO: 52 (heavy chain constant region) [ka] SEQ ID NO:53 (Human B7H4 nucleic acid sequence, including 5' and 3' UTRs) [ka] SEQ ID NO:54 (Human B7H4 nucleic acid sequence, coding sequence) [ka] SEQ ID NO:55 (Human B7H4 polypeptide sequence; UniProt accession number Q7Z7D3) [ka]

Claims

1. 1. A pharmaceutical composition for treating cancer in a human subject in need thereof, comprising: An antibody-drug conjugate (ADC), comprising: i. An antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; c) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively an antibody or antigen-binding fragment thereof comprising ii. a cleavable linker, and iii. Cytotoxic Agents an antibody-drug conjugate (ADC) comprising Including, The pharmaceutical composition, wherein the antibody-drug conjugate (ADC) is administered to the human subject in combination with an additional agent that is a PARP1 inhibitor or an ATR inhibitor.

2. 2. The pharmaceutical composition of claim 1, wherein the additional agent is AZD5305, AZD6738 or a pharmaceutically acceptable salt thereof.

3. The pharmaceutical composition according to claim 1 or 2, wherein the cancer comprises cancer cells that express B7-H4.

4. i) the cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, hematological cancer, endometrial cancer, cholangiocarcinoma, NSCLC (squamous and / or adenocarcinoma), gastrointestinal cancer such as gastric cancer and colorectal cancer, and lung cancer; or ii) the cancer is a breast cancer selected from hormone receptor positive (HR+) breast cancer, human epidermal growth factor receptor 2 positive (HER2+) breast cancer and triple negative breast cancer (TNBC); iii) the cancer is a homologous recombination repair deficient (HRD) cancer; or iv) The pharmaceutical composition of claim 1 or 2, wherein the cancer comprises one or more cells with a mutation in an HRD gene selected from BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D and RAD54L.

5. The antibody or antigen-binding fragment thereof i. a variable heavy (VH) chain and a variable light (VL) chain comprising the amino acid sequences of SEQ ID NO:45 and SEQ ID NO:34, respectively; ii. a variable heavy (VH) chain and a variable light (VL) chain comprising the amino acid sequences of SEQ ID NO: 33 and SEQ ID NO: 34, respectively; iii. A variable heavy (VH) chain and a variable light (VL) chain comprising the amino acid sequences of SEQ ID NO: 43 and SEQ ID NO: 34, respectively; iv. a variable heavy (VH) chain and a variable light (VL) chain comprising the amino acid sequences of SEQ ID NO: 46 and SEQ ID NO: 34, respectively; v. a variable heavy (VH) chain and a variable light (VL) chain comprising the amino acid sequences of SEQ ID NO:47 and SEQ ID NO:34, respectively; vi. a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 31 and SEQ ID NO: 32, respectively; vii. a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 36, respectively; viii. a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO: 37 and SEQ ID NO: 38, respectively; or ix. VH and VL chains comprising the amino acid sequences of SEQ ID NO: 39 and SEQ ID NO: 40, respectively.

3. The pharmaceutical composition of claim 1 or 2, comprising:

6. 3. The pharmaceutical composition of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 41 or 52, or the antibody or antigen-binding fragment thereof comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO:

42.

7. The pharmaceutical composition of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 48 or 51 and a light chain comprising the amino acid sequence of SEQ ID NO:

44.

8. The pharmaceutical composition of claim 1 or 2, wherein the antibody or antigen-binding fragment thereof is a humanized monoclonal antibody.

9. The pharmaceutical composition according to claim 1 or 2, wherein the cleavable linker is an mp-PEG8-val-ala linker.

10. The pharmaceutical composition of claim 1 or 2, wherein the cytotoxic agent is a topoisomerase inhibitor.

11. The topoisomerase inhibitor has the formula A* 【Chemistry 1】 11. The pharmaceutical composition of claim 10, wherein the compound is

12. The ii) cleavable linker and iii) cytotoxic agent may be the following compound: 【Chemistry 2】 The pharmaceutical composition according to claim 1 or 2, wherein the

13. 13. The pharmaceutical composition of claim 12, wherein said ii) linker and iii) cytotoxic agent together is said compound SG3932.

14. 3. The pharmaceutical composition of claim 1, wherein the ADC has a drug-to-antibody ratio (DAR) of about 1 to about 8.

15. 3. The pharmaceutical composition of claim 1, wherein the ADC has a DAR of about 8.

16. A) Antibody-drug conjugates (ADCs), i. An antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; c) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively an antibody or antigen-binding fragment thereof comprising ii. a cleavable linker; iii. Cytotoxic Agents an antibody-drug conjugate (ADC) comprising: B) an additional agent that is a PARP1 inhibitor or an ATR inhibitor; Kit including:

17. 17. The kit of claim 16, wherein the additional agent is AZD5305, AZD6738 or a pharmaceutically acceptable salt thereof.

18. The antibody or antigen-binding fragment thereof i. VH and VL chains comprising the amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 34, respectively 18. The kit of claim 16 or 17, comprising:

19. The kit of claim 16 or 17, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 41 or 52, or the antibody or antigen-binding fragment thereof comprises a light chain constant region comprising the amino acid sequence of SEQ ID NO:

42.

20. The kit of claim 16 or 17, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 48 or 51 and a light chain comprising the amino acid sequence of SEQ ID NO:

44.

21. The kit of claim 16 or 17, wherein the cleavable linker is an mp-PEG8-val-ala linker.

22. 18. The kit of claim 16 or 17, wherein the cytotoxic agent is a topoisomerase inhibitor.

23. The topoisomerase inhibitor has the formula A* 【Transformation 3】 22. The kit of claim 21, wherein the compound is

24. The ii) cleavable linker and iii) cytotoxic agent may be the following compound: 【Chemistry 4】 18. The kit of claim 16 or 17, wherein the components are selected from the following:

25. 25. The kit of claim 24, wherein said ii) linker and iii) cytotoxic agent together are said compound SG3932.

26. 18. The kit of claim 16 or 17, wherein the ADC has a drug-to-antibody ratio (DAR) of about 1 to about 8.

27. 18. The kit of claim 16 or 17, wherein the ADC has a DAR of about 8.

28. 1. A pharmaceutical composition for treating cancer in a human subject in need thereof, comprising: An antibody-drug conjugate (ADC), comprising: i. An antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; c) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively an antibody or antigen-binding fragment thereof comprising ii. formula: 【Transformation 5】 a cleavable linker and a cytotoxic agent conjugated to the antibody or antigen-binding fragment thereof, and an antibody-drug conjugate (ADC) comprising: The pharmaceutical composition wherein the antibody-drug conjugate (ADC) is administered to the human subject in combination with a further agent, which is AZD5305, AZD6738 or a pharmaceutically acceptable salt thereof.

29. The pharmaceutical composition of claim 28, wherein the cancer comprises cancer cells that express B7-H4.

30. i) the cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, hematological cancer, endometrial cancer, cholangiocarcinoma, NSCLC (squamous and / or adenocarcinoma), gastrointestinal cancer such as gastric cancer and colorectal cancer, and lung cancer; or ii) the cancer is a breast cancer selected from hormone receptor positive (HR+) breast cancer, human epidermal growth factor receptor 2 positive (HER2+) breast cancer and triple negative breast cancer (TNBC); iii) the cancer is a homologous recombination repair deficient (HRD) cancer; or iv) The pharmaceutical composition of claim 28 or 29, wherein the cancer comprises one or more cells with a mutation in an HRD gene selected from BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D and RAD54L.

31. 1. A pharmaceutical composition for treating cancer in a human subject in need thereof, comprising: including agents that are PARP1 inhibitors or ATR inhibitors, the agent is administered to the human subject in combination with an antibody-drug conjugate (ADC); The antibody-drug conjugate (ADC) i. An antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; c) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively an antibody or antigen-binding fragment thereof comprising ii. a cleavable linker, and iii. Cytotoxic Agents A pharmaceutical composition comprising:

32. 1. A pharmaceutical composition for treating cancer in a human subject in need thereof, comprising: comprising an agent which is AZD5305, AZD6738 or a pharmaceutically acceptable salt thereof; the agent is administered to the human subject in combination with an antibody-drug conjugate (ADC); The antibody-drug conjugate (ADC) i. An antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, a) heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2), heavy chain CDR3 (HCDR3), light chain CDR1 (LCDR1), light chain CDR2 (LCDR2), and light chain CDR3 (LCDR3) comprising the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12, respectively; b) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6, respectively; c) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; d) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively; or e) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30, respectively an antibody or antigen-binding fragment thereof comprising ii. formula: 【Transformation 6】 a cleavable linker and a cytotoxic agent conjugated to the antibody or antigen-binding fragment thereof, A pharmaceutical composition comprising:

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