B7-h4 therapeutic binding molecules for treating cancer

Abstract

To provide B7-H4 therapeutic binding molecules for cancer treatment.SOLUTION: The disclosure relates to a method for treating cancer in a subject, comprising administering to a subject an antibody-drug conjugate (ADC) that specifically binds to B7-H4. In aspects, the ADC is administered to the subject in an amount from about 0.8 mg / kg to about 4.8 mg / kg.

Description

[Technical field]

[0001] Field The present disclosure provides a method for treating cancer in a subject, the method comprising administering to the subject an antibody-drug conjugate (ADC) that specifically binds to B7-H4. [Background technology]

[0002] background Surgery, radiation therapy, or chemotherapy, alone or in combination, are the most traditional and widely used cancer treatments. While these treatments are effective in removing or killing cancer cells, they often cause unwanted 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. Antibody-based cancer treatments rely 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. Antibody or ADC cancer treatments, used alone or in combination with other small molecule-based cancer treatments, can improve treatment outcomes by attacking malignant cells and tumors in one or more ways. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] U.S. Patent Application Publication No. 2022-0211863 [Patent Document 2] U.S. Patent Application Publication No. 2017-0291955 [Patent Document 3] U.S. Patent No. 7,521,541 [Patent Document 4] U.S. Patent No. 7,723,485 [Patent Document 5] International Publication No. 2009 / 052249

Non-Patent Literature

[0004]

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Summary of the Invention

Means for Solving the Problems

[0005] Summary The present disclosure provides a method of treating cancer in a subject, comprising administering to the subject an antibody-drug conjugate (ADC) that specifically binds to B7-H4 in an amount of about 0.8 mg / kg to about 4.8 mg / kg. The ADC comprises: 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; ii. a cleavable linker; iii. A cytotoxic agent.

[0006] In some embodiments, the amount of ADC administered is about 0.8 mg / kg, about 1.6 mg / kg, about 2.4 mg / kg, about 3.2 mg / kg, about 3.6 mg / kg, or about 4.8 mg / kg. In some embodiments, the amount of ADC administered is about 1.6 mg / kg. In some embodiments, the amount of ADC administered is about 2.4 mg / kg. In some embodiments, the amount of ADC administered is about 3.2 mg / kg.

[0007] In some embodiments, the cancer comprises cancer cells that express B7-H4.

[0008] In some embodiments, the ADC is administered to a subject once every three weeks.

[0009] 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 colon cancer, and lung cancer. In some embodiments, 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 embodiments, the cancer is a homologous recombination repair deficient (HRD) cancer. In some embodiments, the cancer comprises one or more cells having 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 mutation in the HRD gene is selected from BRCA1, BRCA2, and ATM.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof is 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.

[0011] In some 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.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO:45 and SEQ ID NO:34, respectively, or functional variants thereof.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof binds to the OVCAR4 cell line.

[0014] 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. 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.

[0015] 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.

[0016] 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.

[0017] In some embodiments, the cleavable linker is a mp-PEG8-val-ala linker.

[0018] In some aspects, the cytotoxic agent is a topoisomerase inhibitor.

[0019] In some embodiments, the topoisomerase inhibitor is represented by formula A * [ka] It is a compound of the formula:

[0020] In some embodiments, ii) the cleavable linker and iii) the cytotoxic agent are both selected from the following compounds: [ka] is selected from.

[0021] In some embodiments, both ii) the linker and iii) the cytotoxic agent are the compound SG3932.

[0022] In some embodiments, 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.

[0023] In some aspects, the disclosure further provides a method of treating cancer in a subject, comprising administering to a subject an antibody-drug conjugate (ADC) that specifically binds to B7-H4, the method comprising: i. an antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, comprising: 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) that comprise 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 a functional variant thereof; ii.Formula: [ka] and a cytotoxic agent, conjugated to an antibody or antigen-binding fragment thereof having the formula:

[0024] In some embodiments, the amount of ADC administered is about 0.8 mg / kg, about 1.6 mg / kg, about 2.4 mg / kg, about 3.2 mg / kg, about 3.6 mg / kg, or about 4.8 mg / kg. In some embodiments, the amount of ADC administered is about 1.6 mg / kg. In some embodiments, the amount of ADC administered is about 2.4 mg / kg. In some embodiments, the amount of ADC administered is about 3.2 mg / kg.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO:45 and SEQ ID NO:34, respectively, or functional variants thereof.

[0026] In some embodiments, the cancer comprises cancer cells that express B7-H4.

[0027] In some embodiments, the ADC is administered to a subject once every three weeks.

[0028] 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 colon cancer, and lung cancer.

[0029] In some embodiments, 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).

[0030] In some embodiments, the cancer is a homology directed repair deficient (HRD) cancer.

[0031] 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.

[0032] In some embodiments, the mutation in the HRD gene is selected from BRCA1, BRCA2, and ATM.

[0033] In some embodiments of the above methods, at least about 25% of the cancer cells in the subject are B7-H4 positive cells. In some embodiments of the above methods, the B7-H4 positive cells are analyzed using immunohistochemistry (IHC).

[0034] In some aspects, the disclosure further provides an antibody-drug conjugate (ADC) that specifically binds to B7-H4, comprising: i. an antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, comprising: 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) that comprise 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 a functional variant thereof; ii.Formula: [ka] and a cytotoxic agent, wherein the ADC is administered in an amount of about 0.8 mg / kg to about 4.8 mg / kg.

[0035] In some embodiments, the pharmaceutical composition is administered in an amount of about 0.8 mg / kg, about 1.6 mg / kg, about 2.4 mg / kg, about 3.2 mg / kg, or about 3.6 mg / kg, or about 4.8 mg / kg ADC. In some embodiments, the pharmaceutical composition is administered in an amount of about 1.6 mg / kg ADC. In some embodiments, the pharmaceutical composition is administered in an amount of about 2.4 mg / kg ADC. In some embodiments, the pharmaceutical composition is administered in an amount of about 3.2 mg / kg ADC.

[0036] In some embodiments, the antibody or antigen-binding fragment of the ADC comprises a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO:45 and SEQ ID NO:34, respectively, or functional variants thereof.

[0037] In some aspects, the disclosure further provides an antibody-drug conjugate (ADC) that specifically binds to B7-H4, 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, 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) an antibody or antigen-binding fragment thereof comprising 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; iii. A pharmaceutical composition comprising an ADC and a cytotoxic agent, the ADC being administered in an amount of about 25 mg to about 900 mg.

[0038] In some embodiments of the pharmaceutical composition, the amount of ADC administered is about 25 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 750 mg, or about 900 mg.

[0039] In some aspects of the pharmaceutical composition, the combined cleavable linker and cytotoxic agent have the formula: [ka] has.

[0040] In some embodiments of the pharmaceutical composition, the antibody or antigen-binding fragment of the ADC comprises a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO:45 and SEQ ID NO:34, respectively, or functional variants thereof.

[0041] The present disclosure further provides a kit comprising the pharmaceutical composition described above. In some embodiments, the kit further comprises instructions for administering the pharmaceutical composition.

[0042] The disclosure further provides the above pharmaceutical composition for use in treating cancer. In some embodiments, the cancer comprises cancer cells that express B7-H4.

[0043] 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 colon cancer, and lung cancer. In some embodiments, 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 embodiments, the cancer is a homologous recombination repair deficient (HRD) cancer.

[0044] In some embodiments, the cancer comprises one or more cells having 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 mutation in the HRD gene is selected from BRCA1, BRCA2, and ATM.

[0045] In some aspects, the disclosure provides a method of treating cancer in a subject, comprising administering to a subject an antibody-drug conjugate (ADC) that specifically binds to B7-H4, the 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, 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) an antibody or antigen-binding fragment thereof comprising 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; iii. administering to a subject an ADC comprising a cytotoxic agent; The method is provided, wherein at least about 25% of the cancer cells in the subject are B7-H4 positive cells.

[0046] In some aspects, B7-H4 positive cells are determined by measuring membranous B7-H4 expression prior to administration of the ADC, and optionally, B7-H4 expression is measured using immunohistochemistry (IHC), and optionally, the IHC is performed with an antibody reagent. In some aspects, the disclosure relates to a method of diagnosing a cancer sample as B7-H4 positive, comprising: analyzing the cancer sample, optionally using immunohistochemistry (IHC), and optionally, IHC performed with an antibody reagent, to determine whether at least 25% of the cancer cells express membranous B7-H4.

[0047] In some embodiments, the ADC is administered in an amount of about 0.8 mg / kg to about 4.8 mg / kg. In some embodiments, the ADC is administered to the subject once every three weeks. In some embodiments, the patient has been pre-treated with another chemotherapeutic agent. In some embodiments, the subject has been pre-treated with a platinum-based chemotherapeutic agent. 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 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). In some embodiments, the cancer is a homologous recombination repair deficient (HRD) cancer. In some embodiments, the cancer comprises one or more cells having 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 mutation in the HRD gene is selected from BRCA1, BRCA2, and ATM. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain comprising the amino acid sequence of SEQ ID NO: 45 and SEQ ID NO: 34, respectively, or a functional variant thereof. 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 is a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment thereof is a humanized monoclonal antibody. In some embodiments, the cleavable linker is a mp-PEG8-val-ala linker. In some embodiments, the cytotoxic agent is a topoisomerase inhibitor. In some embodiments, the topoisomerase inhibitor is a compound represented by formula A * [ka] It is a compound of the formula: In some embodiments, ii) the cleavable linker and iii) the cytotoxic agent are both selected from the following compounds: [ka] is selected from. In some embodiments, ii) the linker and iii) the cytotoxic agent are both the compound SG3932. In some embodiments, 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.

[0048] BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate example aspects of the present disclosure. [Brief description of the drawings]

[0049] [Figure 1] 1 is a schematic diagram showing the master protocol structure of the treatments described in the Examples. The master protocol structure consists of AZD8205 monotherapy sub-study 1 and one or more combination sub-studies using AZD8205 with another active pharmaceutical agent. [Diagram 2] FIG. 1 is a schematic diagram showing an exemplary treatment method comprising a monotherapy dose escalation phase (Part A) and a monotherapy dose expansion phase (Part B) as described in the Examples. [Diagram 3] 1 is a chart of the response of human ovarian cancer (OVCA) subjects to AZD8205 treatment at doses of 0.8 mg / kg, 1.6 mg / kg, 2.4 mg / kg, and 3.2 mg / kg, respectively. Response is confirmed by detecting the subject's cancer antigen 125 (CA-125) levels. Subjects with measurable CA-125 (>2× upper limit of normal (ULN)) are shown. Circulating tumor DNA (ctDNA) is used to detect molecular response (MR), where P-MR indicates partial molecular response (≧50% reduction in ctDNA from base), C-MR indicates complete molecular response (clearance), and N-MR indicates non-molecular response (<50% reduction, no change, or increase in ctDNA). [Figure 4]1 shows a plot of AZD8205 efficacy as measured by percent change in tumor growth versus percent of cells expressing B7-H4 in the mouse xenograft study described in Example 9. [Diagram 5] 5 is a plot of the Youden statistic versus the percentage of cells expressing B7-H4 for the mouse xenograft study described in Example 9. As can be seen in Figure 5, the Youden statistic begins to plateau at 25% B7-H4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] definition Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular.

[0051] As used herein, "a" or "an" may mean one or more. As used herein, when used in conjunction with the word "comprising," the words "a" or "an" may mean one or more. As used herein, "another" or "further" may mean at least a second or more.

[0052] Use of the term "or" in the claims is used to mean "and / or" unless expressly stated to refer to alternatives only or the alternatives are not mutually exclusive, however, the present disclosure supports a definition that refers to alternatives only and "and / or."

[0053] As used herein, the terms "comprising" (and any variant or form of "comprising", e.g., "comprise" and "comprises"), "having" (and any variant or form of "having", e.g., "have" and "has"), "including" (and any variant or form of "comprising", e.g., "includes" and "include") or "containing" (and any form of "containing", e.g., "contains" and "contain") are inclusive or open ended and do not exclude additional, unrecited elements or method steps.

[0054] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method / device used to determine the value or the variation that exists between test subjects. Typically, the term "about" is meant to encompass approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% or more variability ("greater" or "less" than the specified value), depending on the context. In some embodiments, a person skilled in the art will understand the level of variation indicated by the term "about" depending on the context in which it is used herein. It should also be understood that the use of the term "about" includes the specifically recited value.

[0055] Use of the term "for example" and its corresponding abbreviation "eg" (whether italicized or not) means that the cited defined term is representative of examples and aspects of the present disclosure that are not intended to be limited to the specific examples referenced or cited, unless expressly stated otherwise.

[0056] Ranges provided herein, of any type, include all values ​​within the particular range described, as well as values ​​near the endpoints of the particular range. As used herein, "between" refers to a range that includes both ends of the range. For example, a number between x and y explicitly includes the numbers x and y, and all numbers between x and y.

[0057] The term "antibody", as used herein, refers to a protein capable of recognizing and specifically binding to an antigen. A normal or conventional mammalian antibody comprises a tetramer, which is typically composed of two identical pairs of polypeptide chains, each pair being composed of one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). The terms "heavy chain" and "light chain", as used herein, refer to any immunoglobulin polypeptide having sufficient variable domain sequence to confer specificity for a target antigen. The amino-terminal portion of each light chain and each heavy chain typically contains a variable domain of about 100-110 or more amino acids that is typically involved in antigen recognition. The carboxyl-terminal portion of each chain typically defines a constant domain that is involved in effector function. Thus, in naturally occurring antibodies, the full-length heavy chain immunoglobulin polypeptide contains a variable domain (V H ), as well as three constant domains (C H1 , C H2 , and C H3 ), and C H1 and C. H2 and the hinge region between V H The domain is located at the amino terminus of the polypeptide and is H3 The domains are located at the carboxyl terminus, and full-length light chain immunoglobulin polypeptides contain a variable domain (V L ) and the constant domain (C L ), including V L The domain is located at the amino terminus of the polypeptide and is LThe domain is located at the carboxyl terminus. However, one skilled in the art will appreciate that the location of the domain in a naturally occurring antibody can be modified in certain antibody-like binding protein formats without losing antigen binding capacity. The classes of human light chains are referred to as kappa and lambda light chains.

[0058] In some aspects, the light chain constant region is a kappa chain. In some aspects, the light chain constant region is a lambda chain.

[0059] Within full-length light and heavy chains, the variable and constant domains are typically linked by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. The variable regions of each light / heavy chain pair typically form the antigen-binding site. The variable domains of naturally occurring antibodies typically exhibit relatively conserved framework regions (FR) of identical overall structure linked by three hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework regions, which may enable binding to a specific epitope. From the amino terminus to the carboxyl terminus, the variable domains of both light and heavy chains typically include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0060] The term "antibody fragment" refers to an intact or full-length chain or a portion of an antibody, generally the target binding or variable region. Examples of antibody fragments include F ab , F ab’ , F (ab’)2 , and F v As used herein, the term "functional fragment" is generally synonymous with "antibody fragment" and, with respect to antibodies, F v , F ab , F (ab’)2 It may refer to an antibody fragment such as.

[0061] References to the numbering of amino acid residues described herein are made according to the EU numbering system (also described in (Non-Patent Document 1)).

[0062] As used herein, the term "human antibody" includes antibodies having variable and constant regions substantially corresponding to human germline immunoglobulin sequences. In some aspects, human antibodies are produced in non-human mammals, including, but not limited to, rodents, such as mice and rats, lagomorphs, such as rabbits, and the like. In other aspects, human antibodies are produced in hybridoma cells. In yet other aspects, human antibodies are recombinantly produced. In some aspects, the bispecific binding protein is a human or humanized antibody.

[0063] The term "antigen" or "target antigen" as used herein refers to a molecule or a portion of a molecule that can be recognized and bound by a binding protein of the present disclosure. A target antigen can be used to generate antibodies in an animal that can bind to an epitope of that antigen. A target antigen can have one or more epitopes.

[0064] The term "epitope" as used herein refers to a region or structural element of an antigen that is recognized and bound by a binding protein of the present disclosure. More precisely, an epitope is a specific structure that is bound by a CDR of a binding protein. An epitope may include protein structural elements, carbohydrates, or even parts of lipid structures found in membranes. A binding protein is said to specifically bind an antigen if it preferentially recognizes an antigen target in a complex mixture of proteins and / or macromolecules. The term "specifically binds" refers to a binding protein that specifically binds to a molecule or a fragment thereof (e.g., an antigen). A binding protein that specifically binds to a molecule or a fragment thereof may bind to other molecules with lower affinity, as determined, for example, by immunoassays, BIAcore, or other assays known in the art. In particular, an antibody or fragment that specifically binds to at least one molecule or fragment thereof may compete with a molecule that binds non-specifically. The present disclosure specifically encompasses antibodies with multiple specificities (e.g., antibodies with specificity for two or more distinct antigens). For example, a bispecific antibody may bind to two adjacent epitopes on a single target antigen, or may bind to two different antigens.

[0065] The term "native Fc" as used herein refers to a molecule, whether monomeric or multimeric, that contains the sequence of a non-antigen-binding fragment resulting from the digestion of an antibody or produced by other means, and may include a hinge region. The original immunoglobulin source of the native Fc is preferably of human origin and may be any immunoglobulin. A native Fc molecule is composed of monomeric polypeptides that may be linked by covalent (i.e., disulfide bonds) and non-covalent associations into dimeric or multimeric forms. The number of intramolecular disulfide bonds between monomeric subunits of a native Fc molecule ranges from 1 to 4 depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). One example of a native Fc is a disulfide-linked dimer resulting from papain digestion of IgG. The term "native Fc" as used herein is a collective term for monomeric, dimeric, and multimeric forms.

[0066] The term "Fc variant" as used herein refers to a molecule or sequence that has been modified from a native Fc but still contains a binding site for the salvage receptor FcRn (fetal Fc receptor). Exemplary Fc variants and their interactions with the salvage receptor are known in the art. Thus, the term "Fc variant" can include a molecule or sequence that has been humanized from a non-human native Fc. Additionally, a native Fc includes regions that can be removed or mutated to produce an Fc variant that alters specific residues that provide structural features or biological activities not required for the binding proteins of the present disclosure. Thus, the term "Fc variant" includes molecules or sequences that lack or in which one or more native Fc moieties or residues have been modified that affect or are involved in: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity when expressed in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[0067] The term "Fc domain" as used herein encompasses native Fc and Fc variants and sequences as defined above. As with Fc variants and native Fc molecules, the term "Fc domain" includes monomeric or multimeric molecules, whether digested from a full-length antibody or produced by other means.

[0068] The term "treating" or "treatment" refers to administering a compound or pharmaceutical composition to a subject to effect an alteration or amelioration of a disease, disorder, or condition in the subject. As used herein, the term "treatment" or "treat" can refer to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include subjects with a disease or condition, as well as those susceptible to having a disease or condition, or those in whom a disease or condition is to be prevented.

[0069] The term "dose" refers to a specific amount of a compound or pharmaceutical agent provided in a single administration or for a specific period of time. In some embodiments, a dose can be administered by two or more boluses, tablets, or injections. For example, in some embodiments where subcutaneous administration is desired, the desired dose may require a volume that is not easily accommodated in a single injection. In such embodiments, two or more injections can be used to achieve the desired dose. In some embodiments, a dose may be administered in two or more injections to minimize injection site reactions in an individual. In other embodiments, a compound or pharmaceutical agent is administered by infusion over an extended period of time or continuously. A dose can be determined as the dose of pharmaceutical agent per hour, day, week, or month.

[0070] The terms "subject", "individual" and "patient" are used interchangeably herein to refer to a mammalian subject. In one embodiment, 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 one embodiment, the subject is a cynomolgus monkey (Macaca fascicularis). In a preferred embodiment, the subject is a human. In the methods of the invention, the subject may not have been previously diagnosed with cancer. Alternatively, the subject may have been previously diagnosed with 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 of developing it. Thus, in one embodiment, the methods of the invention may be used to determine the presence of cancer in a subject. For example, the subject may be one who has been previously diagnosed with cancer by alternative means. In one aspect, the subject has previously undergone treatment for cancer.

[0071] The term "efficacy" refers to the ability to produce a desired effect. A "therapeutically effective dose" or "therapeutic dose" is an amount sufficient to produce a desired clinical result (i.e., to achieve a therapeutic effect). A therapeutically effective dose may be administered in one or more administrations.

[0072] The term "side effects" refers to physiological disorders and / or symptoms resulting from treatment other than the desired effects. In some embodiments, side effects include injection site reactions, liver function test abnormalities, renal function abnormalities, hepatotoxicity, nephrotoxicity, central nervous system abnormalities, myopathy, and fatigue. For example, an increase in aminotransferase levels in serum may indicate hepatotoxicity or hepatic function abnormalities. For example, an increase in bilirubin may indicate hepatotoxicity or hepatic function abnormalities. "Disease" or "pathological condition" refers to any condition that would benefit from treatment using the methods of the present disclosure. "Disease" and "pathological condition" are used interchangeably herein and include chronic and acute disorders or diseases, such as pathological conditions that predispose a patient to the disorder in question. In some embodiments, the disease is a tumor. In some embodiments, the disease is a solid tumor. In some embodiments, the disease is a cancer. In some aspects, the cancer is one or more of ovarian cancer, breast cancer, colon cancer, prostate cancer, cervical cancer, uterine cancer, testicular cancer, bladder cancer, head and neck cancer, melanoma, pancreatic cancer, renal cell carcinoma, and lung cancer.

[0073] The term "administration" or "administering" as used herein refers to providing, contacting, and / or delivering a compound by any route appropriate to achieve a desired effect. Administration may include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intralesional, or intracranial injection), transdermal, topical, buccal, rectal, vaginal, intranasal, ophthalmic, via inhalation, and implant.

[0074] As used herein, the term "pharmaceutical composition" or "therapeutic composition" refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a subject. In some aspects, the present disclosure provides a pharmaceutical composition comprising a pharma- ceutical acceptable carrier and a therapeutically effective amount of a binding protein of the present disclosure.

[0075] As used herein, the term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" refers to one or more formulation materials suitable for achieving or enhancing delivery of one or more binding proteins of the present disclosure.

[0076] Methods of Treating Cancer The present disclosure relates to a method of treating cancer in a subject, comprising administering to the subject an antibody-conjugate (ADC) that specifically binds to B7-H4 in an amount of about 0.8 mg / kg to about 4.8 mg / kg. In some embodiments, the ADC is administered in an amount of about 25 mg to about 900 mg per subject. The present disclosure also provides compositions, including pharmaceutical compositions, and kits comprising such ADCs.

[0077] In some embodiments, the ADC comprises: 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) an antibody or antigen-binding fragment thereof comprising 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; iii. A cytotoxic agent.

[0078] 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 on 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 restricted to a particular cancer type, such that it represents a target antigen for treating a wide range of cancer types.

[0079] 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.

[0080] In some embodiments, the antibody or antigen-binding fragment thereof may bind to B7-H4 molecules across species, e.g., the antibody or fragment may bind to mouse B7-H4, rat B7-H4, rabbit, human B7-H4, and / or cynomolgus B7-H4. In some embodiments, the antibody or fragment may bind to human B7-H4 and cynomolgus B7-H4. In some embodiments, the antibody or antigen-binding fragment may also bind to mouse B7-H4.

[0081] 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.

[0082] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC comprises 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: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. An antibody or antigen-binding fragment thereof comprising the above sequences may be referred to herein as "ZY0EQD-E02" or "EQD-E02."

[0083] 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: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. The antibody or antigen-binding fragment thereof comprising the above sequences may be referred to herein as "ZY0EPQ-E02" or "EPQ-E02".

[0084] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC 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. An antibody or antigen-binding fragment thereof comprising the above sequences may be referred to herein as "ZY0EOB-F05" or "EOB-F05."

[0085] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC 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. An antibody or antigen-binding fragment thereof comprising the above sequences may be referred to herein as "ZY0EO5-E07" or "EO5-E07."

[0086] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC 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. An antibody or antigen-binding fragment thereof comprising the above sequences may be referred to herein as "ZY0EP0-C07" or "EP0-C07."

[0087] In some embodiments, the antibodies or antigen-binding fragments thereof having CDRs with the amino acid sequences disclosed above have beneficial advantages such as high targeting specificity or avidity for the B7-H4 peptide on the surface of cancer cells.

[0088] Additionally or alternatively, the antibodies or antigen-binding fragments thereof described herein may be described by their variable heavy chain (VH) and variable light chain (VL).

[0089] In some embodiments, 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: 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: 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: 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: 38, respectively, or a functional variant thereof; VH chains and VL chains comprising the amino acid sequences of SEQ ID NO: 34 or functional variants thereof; VH chains and VL chains comprising the amino acid sequences of SEQ ID NO: 31 and SEQ ID NO: 32, respectively, or functional variants thereof; VH chains and VL chains comprising the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 36, respectively, or functional variants thereof; VH chains and VL chains comprising the amino acid sequences of SEQ ID NO: 37 and SEQ ID NO: 38, respectively, or functional variants thereof; or VH chains and VL chains comprising the amino acid sequences of SEQ ID NO: 39 and SEQ ID NO: 40, respectively, or functional variants thereof.

[0090] For example, in one embodiment, the antibody or antigen-binding fragment thereof of the ADC 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; and (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.

[0091] In some 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.

[0092] In some embodiments, the antibody or antigen-binding fragment thereof comprises 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. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 45. In some embodiments, the antibody or antigen-binding fragment thereof comprises a VL chain having an amino acid sequence that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO:34.

[0093] 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 κλ light chain constant region, e.g., a human κ constant region or a human λ constant region.

[0094] 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. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO:41.

[0095] 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 heavy chain constant region that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO:52.

[0096] 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 light chain constant region that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO:42.

[0097] 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 that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 51. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 44.

[0098] 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. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO: 48. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO:44.

[0099] 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.

[0100] Advantageously, the present disclosure reveals that the above-mentioned antibodies or antigen-binding fragments can target a broader spectrum of B7-H4 expressing cells when compared to existing (commercially available) antibodies reported to target B7-H4. Thus, the present disclosure not only provides the antibodies (or antigen-binding fragments) with affinity and specificity for clinically relevant targets, but also reveals unique advantages (e.g., unexpected technical effects) associated therewith.

[0101] 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 colon cancer, and lung cancer.

[0102] In some embodiments, 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).

[0103] In some embodiments, the cancer is a homology directed repair deficient (HRD) cancer.

[0104] In some embodiments, the cancer comprises one or more cells having 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 mutation in the HRD gene is selected from BRCA1, BRCA2, and ATM.

[0105] In some embodiments, the antibody or antigen-binding fragment thereof of the ADC binds to an OVCAR4 cell line. In some embodiments, the antibody or antigen-binding fragment thereof described herein may bind to an OVCAR4 cell line and / or a CHO cell line (e.g., which may lack exogenous nucleic acid encoding B7-H4). For example, the antibody or antigen-binding fragment thereof binds to B7-H4 (e.g., the B7-H4 epitope) on an OVCAR4 cell line and / or a CHO cell line (e.g., which may lack exogenous nucleic acid encoding B7-H4).

[0106] In some embodiments, the antibody or antigen-binding fragment thereof binds to an OVCAR4 cell line and / or a CHO cell line (which may lack exogenous nucleic acid encoding B7-H4, for example) with greater affinity 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. Affinity (eg, binding affinity) can be measured by any suitable method for measuring binding affinity as described herein.

[0107] The OVCAR4 cell line is a human ovarian cancer cell line, and the CHO cell line is an epithelial cell line derived from the Chinese hamster ovary and is widely available.

[0108] In some embodiments, at least about 25% of the cancer cells are B7-H4 positive cells. In some embodiments, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% of the cancer cells are B7-H4 positive cells. In some embodiments, the B7-H4 positive cells are cells that express B7-H4 on their cell surface. In some embodiments, the percentage of B7-H4 positive cells is determined by analyzing a sample, e.g., a biopsy of cancer tissue from a subject. In some embodiments, the percentage of B7-H4 positive cells is determined using an antibody for detecting B7-H4.

[0109] In some embodiments, the percentage of cancer cells that are B7-H4 positive is determined by immunohistochemistry (IHC). IHC is a test that uses antibodies to detect antigens (biomarkers) in tissue samples. In embodiments, the antibodies are conjugated to an enzyme or fluorescent dye. Once the antibody binds to the antigen in the tissue sample, the enzyme or dye is activated and the antigen can be detected using a microscope or other visualization device.

[0110] In some embodiments, the antibody or antigen-binding fragment thereof is linked to the cytotoxic agent by a linker. In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to the cytotoxic agent by a linker. As used herein, "conjugated" means linked via a covalent or ionic bond. In some embodiments, the linker is cleavably linked (e.g., conjugated) to an amino residue, e.g., an amino acid of the antibody or antigen-binding fragment of the ADC. In some embodiments, the cleavable linker of the ADC is a mp-PEG8-val-ala linker.

[0111] A cytotoxic agent may be referred to herein as a "drug" or an "active agent." In some embodiments, a cytotoxic agent is a drug. A cytotoxic agent or cytotoxin may 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, combretatstatin, calicheamicin, maytansine, DM-1, vinblastine, methotrexate, and netropsin, and derivatives and analogs thereof.

[0112] In some embodiments, the cytotoxic agent is a drug. In some embodiments, the cytotoxic agent of the ADC is a topoisomerase inhibitor, a tubulysin derivative, a pyrrolobenzodiazepine, or a combination thereof. In some embodiments, the cytotoxic agent is one of the cytotoxic agents disclosed in U.S. Patent No. 5,993,331 and U.S. Patent No. 5,993,332, both of which are incorporated by reference in their entirety.

[0113] In a preferred embodiment, the cytotoxic agent is a topoisomerase inhibitor.Topoisomerase inhibitors are chemical compounds that block the action of topoisomerase (topoisomerase I and II), which is a type of enzyme that controls the change in DNA structure by catalyzing the breakage and recombination of the phosphodiester backbone of DNA strands during normal cell cycle.Topoisomerase I inhibitors can be, for example, SG3932, SG4010, SG4057, or SG4052, as provided below.

[0114] The present disclosure provides a cytotoxic agent (drug unit) of an ADC that is connected to a Ligand unit of the ADC via a linker (linking unit) as described above. The Ligand unit is preferably an antibody or an antigen-binding fragment thereof. In some embodiments, the present disclosure provides the following topoisomerase inhibitor derivatives (A * , drug unit): [ka] The present invention provides a conjugate comprising:

[0115] In some embodiments, the cytotoxic agent is a topoisomerase inhibitor, where the cleavable linker and the cytotoxic agent of the ADC together have Formula I: [ka] R L is the cleavable linker described above.

[0116] In some embodiments, the linker and the cytotoxic agent are both selected from the following compounds: [ka] Contains one of the following:

[0117] In some embodiments, both the linker and the cytotoxic agent are the compound SG3932.

[0118] Cytotoxic agents are typically linked to or "loaded onto" the antibody or antigen-binding fragment. Drug loading (p) is the average number of drugs per antibody or antigen-binding fragment (e.g., ligand unit).

[0119] The average number of drugs per antibody when preparing ADCs from conjugation reactions can be characterized by conventional methods 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. The average value of p in a particular preparation of ADCs can be determined by ELISA (Non-Patent Document 2). However, the distribution of p(drug) values ​​cannot be distinguished due to the detection limits of antibody-antigen binding and ELISA. In addition, ELISA assays for detecting antibody-drug conjugates do not reveal where the drug moiety is attached on the antibody, e.g., to the heavy or light chain fragment, or to a particular amino acid residue. 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.

[0120] Typically, fewer than the theoretical maximum number of drug moieties are conjugated to the antibody during the conjugation reaction. An antibody may contain, for example, many lysine residues that do not react with a drug linker. Only the most reactive lysine groups can react with an amine-reactive linker reagent. Also, only the most reactive cysteine ​​thiol groups can react with a thiol-reactive linker reagent. In general, an antibody does not contain many, if any, free reactive cysteine ​​thiol groups that can be linked to a drug moiety. Most cysteine ​​thiol residues in the compound's antibody exist as disulfide bridges and must be reduced with a reducing agent (such as dithiothreitol (DTT) or TCEP) under partial or total reducing conditions. The loading capacity (drug / antibody ratio) of an ADC can be controlled in several different ways, including: (i) limiting the molar excess of drug linker relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limited reducing conditions of cysteine ​​thiol modification.

[0121] Certain antibodies have interchain disulfides, i.e. cysteine ​​bridges, that can be reduced. Antibodies can be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Thus, each cysteine ​​bridge theoretically becomes two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies by converting amines to thiols by reaction of lysine with 2-iminothiolane (Traut's reagent). Reactive thiol groups can be introduced into antibodies (or fragments thereof) by engineering one, two, three, four or more cysteine ​​residues (e.g., preparing mutant antibodies containing one or more non-natural cysteine ​​amino acid residues). (Patent Document 3) teaches antibody engineering by introduction of reactive cysteine ​​amino acids.

[0122] Cysteine ​​amino acids that are in the reactive sites of antibodies and do not form interchain or intermolecular disulfide bridges can be engineered (Non-Patent Document 3; Patent Document 3; Patent Document 4; Patent Document 5). The engineered cysteine ​​thiols can react with the drug linkers of the invention bearing thiol-reactive electrophilic groups (such as maleimides or α-haloamides) to form ADCs with cysteine ​​engineered antibodies. Thus, the placement of the drug units can be designed, controlled, and known. Drug loading can be controlled because the engineered cysteine ​​thiol groups react with drug linker reagents, typically in high yield. An IgG antibody is engineered to introduce a cysteine ​​amino acid by substitution at one site on the heavy or light chain, providing two new cysteines on a symmetric antibody.

[0123] In some embodiments, the average number of drugs per antibody (or antigen-binding fragment thereof) ranges from 1 to 20. In some embodiments, the ranges are 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 referred to as the drug-to-antibody ratio (DAR). The 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 ranges of the DAR are selected from 1 to 10, 2 to 10, 2 to 8, 2 to 6, and 4 to 10. In some embodiments, the DAR is between about 1 and 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.

[0124] In some embodiments, the ADC is AZD8205. AZD8205 is an anti-B7H4 Ab conjugated to a topoisomerase inhibitor (TOPO) warhead. In some embodiments, AZD8205 targets ovarian cancer and cholangiocarcinoma (CCA). In some embodiments, AZD8205 targets triple-negative breast cancer (TNBC).

[0125] In some embodiments, the amount of ADC administered is about 0.5 mg / kg to about 8 mg / kg. In some embodiments, the amount of ADC administered is about 0.8 mg / kg to about 4.8 mg / kg. In some embodiments, the amount of ADC administered is about 1.2 mg / kg to about 3.6 mg / kg. In some embodiments, the amount of ADC administered is about 1.4 mg / kg to about 3.2 mg / kg. In some embodiments, the amount of ADC administered is about 1.6 mg / kg to about 2.4 mg / kg. In some embodiments, the amount of ADC administered is about 1.8 mg / kg to about 2.2 mg / kg.

[0126] In some embodiments, the amount of ADC administered is about 0.5 mg / kg, about 0.8 mg / kg, about 1.0 mg / kg, about 1.2 mg / kg, about 1.4 mg / kg, about 1.6 mg / kg, about 1.8 mg / kg, about 2.0 mg / kg, about 2.2 mg / kg, about 2.4 mg / kg, about 2.6 mg / kg, about 2.8 mg / kg, about 3.0 mg / kg, about 3.2 mg / kg, about 3.6 mg / kg, about 4.0 mg / kg, about 4.4 mg / kg, about 4.8 mg / kg, about 5.5 mg / kg, about 6.0 mg / kg, about 7.0 mg / kg, or about 8 mg / kg.

[0127] In some embodiments, the amount of ADC administered is about 1.2 mg / kg, about 1.6 mg / kg, about 1.8 mg / kg, about 2.0 mg / kg, about 2.2 mg / kg, about 2.4 mg / kg, or about 3.2 mg / kg.

[0128] In some embodiments, the amount of ADC administered is about 1.6 mg / kg. In some embodiments, the amount of ADC administered is about 2.0 mg / kg. In some embodiments, the amount of ADC administered is about 2.4 mg / kg. In some embodiments, the amount of ADC administered is about 3.2 mg / kg.

[0129] In some embodiments, the amount of ADC administered is about 25 mg to about 900 mg. In some embodiments, the amount of ADC administered is about 40 mg to about 600 mg. In some embodiments, the amount of ADC administered is about 60 mg to about 450 mg. In some embodiments, the amount of ADC administered is about 80 mg to about 300 mg. In some embodiments, the amount of ADC administered is about 100 mg to about 240 mg. In some embodiments, the amount of ADC administered is about 120 mg to about 200 mg. In some embodiments, the amount of ADC administered is about 140 mg to about 180 mg.

[0130] In some embodiments, the amount of ADC administered is about 25 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 750 mg, or about 900 mg.

[0131] In some embodiments, the amount of ADC administered is about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 240 mg, about 280 mg, or about 320 mg.

[0132] In some embodiments, the amount of ADC administered is about 120 mg. In some embodiments, the amount of ADC administered is about 160 mg. In some embodiments, the amount of ADC administered is about 200 mg.

[0133] In some embodiments, the ADC is administered to the subject once every three weeks (Q3W). In some embodiments, the ADC is administered to the subject once every week (Q1W), once every two weeks (Q2W), once every four weeks (Q4W), once every five weeks (Q5W), or once every six weeks (Q6W).

[0134] In some embodiments, the disclosure further provides a method of treating cancer in a subject, comprising administering to the subject an antibody-drug conjugate (ADC) that specifically binds B7-H4 in an amount of about 0.8 mg / kg to about 4.8 mg / kg. In some embodiments, the ADC comprises: i. an antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, the antibody or antigen-binding fragment thereof comprising: 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 a functional variant thereof; and ii. [ka] and a cleavable linker conjugated to an antibody or antigen-binding fragment thereof having the formula:

[0135] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO:45 and SEQ ID NO:34, respectively, or functional variants thereof.

[0136] 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 colon cancer, and lung cancer.

[0137] In some embodiments, 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). In some embodiments, the cancer is a homologous recombination repair deficient (HRD) cancer.

[0138] In some embodiments, the cancer comprises one or more cells having 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 mutation in the HRD gene is selected from BRCA1, BRCA2, and ATM.

[0139] In some embodiments, the amount of ADC administered is about 0.5 mg / kg to about 8 mg / kg. In some embodiments, the amount of ADC administered is about 0.8 mg / kg to about 4.8 mg / kg. In some embodiments, the amount of ADC administered is about 1.2 mg / kg to about 3.6 mg / kg. In some embodiments, the amount of ADC administered is about 1.4 mg / kg to about 3.2 mg / kg. In some embodiments, the amount of ADC administered is about 1.6 mg / kg to about 2.4 mg / kg. In some embodiments, the amount of ADC administered is about 1.8 mg / kg to about 2.2 mg / kg.

[0140] In some embodiments, the amount of ADC administered is about 0.5 mg / kg, about 0.8 mg / kg, about 1.0 mg / kg, about 1.2 mg / kg, about 1.4 mg / kg, about 1.6 mg / kg, about 1.8 mg / kg, about 2.0 mg / kg, about 2.2 mg / kg, about 2.4 mg / kg, about 2.6 mg / kg, about 2.8 mg / kg, about 3.0 mg / kg, about 3.2 mg / kg, about 3.6 mg / kg, about 4.0 mg / kg, about 4.4 mg / kg, about 4.8 mg / kg, about 5.5 mg / kg, about 6.0 mg / kg, about 7.0 mg / kg, or about 8 mg / kg.

[0141] In some embodiments, the amount of ADC administered is about 1.2 mg / kg, about 1.6 mg / kg, about 1.8 mg / kg, about 2.0 mg / kg, about 2.2 mg / kg, about 2.4 mg / kg, or about 3.2 mg / kg.

[0142] In some embodiments, the amount of ADC administered is about 1.6 mg / kg. In some embodiments, the amount of ADC administered is about 2.0 mg / kg. In some embodiments, the amount of ADC administered is about 2.4 mg / kg. In some embodiments, the amount of ADC administered is about 3.2 mg / kg.

[0143] In some embodiments, the amount of ADC administered is about 25 mg to about 900 mg. In some embodiments, the amount of ADC administered is about 40 mg to about 600 mg. In some embodiments, the amount of ADC administered is about 60 mg to about 450 mg. In some embodiments, the amount of ADC administered is about 80 mg to about 300 mg. In some embodiments, the amount of ADC administered is about 100 mg to about 240 mg. In some embodiments, the amount of ADC administered is about 120 mg to about 200 mg. In some embodiments, the amount of ADC administered is about 140 mg to about 180 mg.

[0144] In some embodiments, the amount of ADC administered is about 25 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 750 mg, or about 900 mg.

[0145] In some embodiments, the amount of ADC administered is about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 240 mg, about 280 mg, or about 320 mg.

[0146] In some embodiments, the amount of ADC administered is about 120 mg. In some embodiments, the amount of ADC administered is about 160 mg. In some embodiments, the amount of ADC administered is about 200 mg.

[0147] In some embodiments, the ADC is administered to the subject once every three weeks (Q3W). In some embodiments, the ADC is administered to the subject once every week (Q1W), once every two weeks (Q2W), once every four weeks (Q4W), once every five weeks (Q5W), or once every six weeks (Q6W).

[0148] In some aspects, the disclosure further provides a method of treating cancer in a subject, comprising administering to a subject an antibody-drug conjugate (ADC) that specifically binds to B7-H4, the ADC comprising: 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) an antibody or antigen-binding fragment thereof comprising 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; iii. administering to a subject an ADC comprising a cytotoxic agent; The method is provided, wherein at least about 25% of the cancer cells in the subject are B7-H4 positive cells.

[0149] Cleavable linkers and cytotoxic agents of the ADCs for use in the methods are described herein. Cancers treatable by the methods are described herein.

[0150] In some embodiments, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75% of the cancer cells are B7-H4 positive cells. In some embodiments, the B7-H4 positive cells are cells that express B7-H4 on their cell surface. In some embodiments, the percentage of B7-H4 positive cells is determined by analyzing a sample, e.g., a biopsy of cancer tissue from a subject. In some embodiments, the percentage of B7-H4 positive cells is determined using an antibody for detecting B7-H4.

[0151] In some embodiments of the method, the ADC is administered in an amount of about 0.8 mg / kg to about 4.8 mg / kg. In embodiments, the amount of ADC administered is about 0.8 mg / kg, about 1.6 mg / kg, about 2.4 mg / kg, about 3.2 mg / kg, about 3.6 mg / kg, or about 4.8 mg / kg. In embodiments, the amount of ADC administered is about 1.6 mg / kg. In embodiments, the amount of ADC administered is about 2.4 mg / kg. In embodiments, the amount of ADC administered is about 3.2 mg / kg.

[0152] In other aspects of the methods, the ADC is administered in an amount disclosed herein.

[0153] In some embodiments, the ADC is administered to the subject once every three weeks (Q3W). In some embodiments, the ADC is administered to the subject once every week (Q1W), once every two weeks (Q2W), once every four weeks (Q4W), once every five weeks (Q5W), or once every six weeks (Q6W).

[0154] In any of the embodiments of the methods or uses described herein, the subject has been pre-treated with another chemotherapeutic agent. In some embodiments, the subject has been pre-treated with a platinum-based chemotherapeutic agent. In some embodiments, the platinum-based chemotherapeutic agent is cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, or heptaplatin.

[0155] Pharmaceutical Compositions In some embodiments, the disclosure further provides pharmaceutical compositions comprising an antibody-drug conjugate (ADC) that specifically binds to B7-H4, the ADC being administered in an amount of about 0.8 mg / kg to about 4.8 mg / kg. In some embodiments, the ADC comprises: i. an antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, the antibody or antigen-binding fragment thereof comprising: 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 a functional variant thereof; and ii. [ka] and a cleavable linker conjugated to an antibody or antigen-binding fragment thereof having the formula:

[0156] In some embodiments, the amount of ADC administered is about 0.5 mg / kg to about 8 mg / kg. In some embodiments, the amount of ADC administered is about 0.8 mg / kg to about 4.8 mg / kg. In some embodiments, the amount of ADC administered is about 1.2 mg / kg to about 3.6 mg / kg. In some embodiments, the amount of ADC administered is about 1.4 mg / kg to about 3.2 mg / kg. In some embodiments, the amount of ADC administered is about 1.6 mg / kg to about 2.4 mg / kg. In some embodiments, the amount of ADC administered is about 1.8 mg / kg to about 2.2 mg / kg.

[0157] In some embodiments, the amount of ADC administered is about 0.5 mg / kg, about 0.8 mg / kg, about 1.0 mg / kg, about 1.2 mg / kg, about 1.4 mg / kg, about 1.6 mg / kg, about 1.8 mg / kg, about 2.0 mg / kg, about 2.2 mg / kg, about 2.4 mg / kg, about 2.6 mg / kg, about 2.8 mg / kg, about 3.0 mg / kg, about 3.2 mg / kg, about 3.6 mg / kg, about 4.0 mg / kg, about 4.4 mg / kg, about 4.8 mg / kg, about 5.5 mg / kg, about 6.0 mg / kg, about 7.0 mg / kg, or about 8 mg / kg.

[0158] In some embodiments, the amount of ADC administered is about 1.2 mg / kg, about 1.6 mg / kg, about 1.8 mg / kg, about 2.0 mg / kg, about 2.2 mg / kg, about 2.4 mg / kg, or about 3.2 mg / kg.

[0159] In some embodiments, the amount of ADC administered is about 1.6 mg / kg. In some embodiments, the amount of ADC administered is about 2.0 mg / kg. In some embodiments, the amount of ADC administered is about 2.4 mg / kg. In some embodiments, the amount of ADC administered is about 3.2 mg / kg.

[0160] In some embodiments, the amount of ADC administered is about 25 mg to about 900 mg. In some embodiments, the amount of ADC administered is about 40 mg to about 600 mg. In some embodiments, the amount of ADC administered is about 60 mg to about 450 mg. In some embodiments, the amount of ADC administered is about 80 mg to about 300 mg. In some embodiments, the amount of ADC administered is about 100 mg to about 240 mg. In some embodiments, the amount of ADC administered is about 120 mg to about 200 mg. In some embodiments, the amount of ADC administered is about 140 mg to about 180 mg.

[0161] In some embodiments, the amount of ADC administered is about 25 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 600 mg, about 750 mg, or about 900 mg.

[0162] In some embodiments, the amount of ADC administered is about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 240 mg, about 280 mg, or about 320 mg.

[0163] In some embodiments, the amount of ADC administered is about 120 mg. In some embodiments, the amount of ADC administered is about 160 mg. In some embodiments, the amount of ADC administered is about 200 mg.

[0164] In some embodiments of the use of the pharmaceutical compositions disclosed herein for the treatment of cancer, the ADC is administered to the subject once every three weeks (Q3W). In some embodiments of the use of the pharmaceutical compositions disclosed herein for the treatment of cancer, the ADC is administered to the subject once every week (Q1W), once every two weeks (Q2W), once every four weeks (Q4W), once every five weeks (Q5W), or once every six weeks (Q6W).

[0165] In some aspects of the use of the pharmaceutical compositions disclosed herein for the treatment of cancer, the patient with cancer has been pre-treated with another chemotherapeutic agent. In some aspects, the subject has been pre-treated with a platinum-based chemotherapeutic agent. In some aspects, the platinum-based chemotherapeutic agent is cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, or heptaplatin.

[0166] In some embodiments, the antibody or antigen-binding fragment of the ADC comprises a VH chain and a VL chain comprising the amino acid sequences of SEQ ID NO:45 and SEQ ID NO:34, respectively, or functional variants thereof. In some embodiments, the antibody or antigen-binding fragment of the ADC comprises a VH chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:45, and a VL chain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:34.

[0167] In some embodiments, the pharmaceutical compositions disclosed herein may be formulated with a pharma- ceutically acceptable carrier, excipient, or stabilizer. In certain embodiments, such pharmaceutical compositions are suitable for administration to humans or non-human animals by any one or more routes of administration using methods known in the art. The term "pharmaceutically acceptable carrier" refers to one or more non-toxic substances that do not interfere with the effectiveness of the biological activity of the active ingredient. Such preparations may always include salts, buffers, preservatives, compatible carriers, and optional other therapeutic agents. Such pharma- ceutically acceptable preparations may also include compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans. Other contemplated carriers, excipients, and / or additives that may be utilized in the formulations described herein include, for example, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients, such as serum albumin, gelatin, casein, salt-forming counterions, such as sodium, and the like. These and further known pharmaceutical carriers, excipients, and / or additives suitable for use in the formulations described herein are known in the art and are described, for example, in (Non-Patent Document 4) and (Non-Patent Document 5). Pharmaceutically acceptable carriers suitable for the desired or required mode of administration, solubility, and / or stability may be selected.

[0168] The present disclosure further provides the above pharmaceutical composition for use in treating cancer. In some embodiments, the cancer comprises cancer cells expressing B7-H4. 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 colon cancer, and lung cancer. In some embodiments, 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 embodiments, the cancer is homologous recombination repair deficient (HRD) cancer. In some embodiments, the cancer comprises one or more cells having 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 mutation in the HRD gene is selected from BRCA1, BRCA2, and ATM.

[0169] kit In some aspects, the disclosure further provides a kit comprising any of the above pharmaceutical compositions. In some aspects, the kit comprises instructions for administering the pharmaceutical composition. In some aspects, the kit comprises an additional anti-cancer agent as described herein.

[0170] Antibody or antigen-binding fragment sequence of the ADC Embodiments of the present disclosure include antibodies or antigen-binding fragments in the DuetMab format that bind to B7-H4 polypeptides generated using the sequences in Table 1 below. The CDRs in Table 1 are determined according to the Kabat system.

[0171] [Table 1]

[0172] [Table 2]

[0173] [Table 3]

[0174] [Table 4]

[0175] [Table 5]

[0176] All references cited herein, e.g., patents, patent applications, articles, textbooks, etc., and the references cited therein, are incorporated by reference in their entirety, unless they have already been cited.

[0177] Without limiting the disclosure, several aspects of the disclosure are described herein for purposes of illustration. EXAMPLES

[0178] Working Example The following examples illustrate specific embodiments of the present disclosure and various uses thereof, they are set forth for illustrative purposes only and should not be construed as limiting the scope of the disclosure in any way.

[0179] AZD8205 is provided for treatment in the examples. AZD8205 is an ADC that targets B7-H4 (VTCN1) and consists of a human anti-B7-H4 antibody ("INT016") conjugated to a TOP1i warhead ("AZ14170132") via a cleavable maleimide-PEG8-valine-alanine linker. AZD8205 has a DAR of about 8. The antibody component of AZD8205 binds to B7-H4 on the cell surface, promoting internalization of the ADC as well as trafficking to lysosomes. When the linker is enzymatically cleaved, the TOP1i warhead is released, and TOP1 is trapped on DNA, forming a double-stranded DNA break. If not repaired, the double-stranded break eventually leads to programmed cell death.

[0180] Preclinical pharmacological studies suggest that the primary mechanism of action of AZD8205 is the intracellular delivery of TOP1i warheads to B7-H4-positive tumor cells. In vitro assays demonstrated that AZD8205 specifically binds and exhibits cytotoxicity to B7-H4-expressing tumor cells, but not to B7-H4-negative cells. In vivo pharmacokinetic results using human tumor xenograft mouse models showed dose-dependent accumulation of AZD8205 and increased positive staining for γH2AX foci (an indicator of DNA double-strand breaks), elevated cleaved caspase-3, and an overall decrease in epithelial cell density over time, suggesting that AZD8205 binds to B7-H4 in tumor cells, causing DNA damage and apoptotic cell death, consistent with the mechanism of action of TOP1i warheads.

[0181] Subsequent in vivo efficacy studies of AZD8205 in human tumor cell line-derived xenografts and patient-derived xenograft models of triple-negative breast cancer further demonstrated that a single IV dose of AZD8205 produced antitumor effects in both a target- and dose-dependent manner. In studies evaluating patient-derived xenograft models of triple-negative breast cancer (N=26), a single IV dose of 1.25 mg / kg or 3.5 mg / kg produced effective tumor regression (≥30% reduction in tumor volume from baseline) in 12 of 26 models (46%) and 18 of 26 models (69%), respectively. To elucidate the relationship between B7-H4 expression and AZD8205 response, immunohistochemical staining and image analysis techniques were used to quantify B7-H4 expression in each patient-derived xenograft model. A statistically significant association between B7-H4 expression and antitumor efficacy was observed following treatment with 1.25 mg / kg AZD8205, suggesting that elevated B7-H4 levels are associated with AZD8205 response.

[0182] Example 1: Starting Dose Justification As described in the Examples below, AZD8205 monotherapy will be investigated intravenously Q3W at dose levels ranging from 0.8 mg / kg to 3.2 mg / kg. The recommended starting dose of 0.8 mg / kg for single agent AZD8205 is based on the results of a Good Laboratory Practice (GLP) toxicity study in cynomolgus monkeys, scaling of cynomolgus monkey to human pharmacokinetics (PK), and FDA regulatory guidelines. Dose increments and a clinically effective dose range (0.8-2.4 mg / kg) were determined and modeled exposures estimated. The highest non-severely toxic dose (HNSTD) of AZD8205 was identified in a GLP study in cynomolgus monkeys as 15 mg / kg for a total of two doses every 3 weeks (Q3W), and the human equivalent dose (HED) of this HNSTD was calculated to be 4.8 mg / kg based on the U.S. FDA Guidance for Industry: Estimation of the Maximum Safe Starting Dose for Initial Clinical Trials of Therapeutic Agents in Adult Healthy Volunteers. The selection of the clinical starting dose of 0.8 mg / kg Q3W in the first-in-human (FTIH) study was based on one-sixth of the HED of 4.8 mg / kg. When administered as a 1-hour intravenous infusion at a dose of 0.8 mg / kg Q3W, the steady-state plasma exposure of AZD8205 was 16 μg / mL. max , AUC of 77 μg.d / mL 0-21d It is predicted that.

[0183] This approach to identifying the starting dose of an ADC has generally been shown to provide an acceptable balance of safety and efficient dose escalation in FTIH studies. A human starting dose of 0.8 mg / kg provides a safety margin based on the HNSTD: 6.05 based on the HED, C max It is predicted to have a β-blocking coefficient of 20.4 based on β-blocking coefficient, and 11.2 based on AUC.

[0184] Example 2: Study Design and Definitions This first-in-human (FTIH) study will be conducted within the context of a master protocol to evaluate the safety, tolerability, and preliminary efficacy of AZD8205 initially as monotherapy and, after defining a recommended Phase 2 dose (RP2D), in combination with other anti-cancer agents in subjects with B7-H4 expressing advanced or metastatic solid malignancies. The protocol will also explore the potential biological activity of AZD8205 by characterizing pharmacokinetics (PK), pharmacodynamics (PDx), exploratory biomarkers, and anti-tumor activity.

[0185] The design of the master protocol is shown in Figure 1. As shown in Figure 1, study-wide information such as study objectives, rationale, general inclusion and exclusion criteria, safety assessments, and adverse event (AE) reporting common to all substudies is described in the master protocol. Substudy-specific information such as planned dose levels and their rationale, toxicity management, and dose modifications can be found in the relevant substudies.

[0186] The master protocol consists of several sub-studies evaluating AZD8205. The combination treatment sub-studies will be added after defining the recommended phase 2 dose (RP2D) of monotherapy based on new supporting preclinical and / or clinical data and scientific rationale. Each sub-study includes a dose escalation (e.g., Part A) and a dose expansion (e.g., Part B). The objective of the dose escalation (Part A) of each sub-study is to determine the safety, tolerability, RP2D and / or maximum tolerated dose (MTD) of AZD8205 in subjects with B7-H4-expressing tumors. As described in Example 9, all subjects selected for this study had tumors in which at least 25% of the cells in their tumor biopsies were B7-H4 positive. The RP2D will be selected by pooling and evaluating all available data, including appropriate PK, PD, efficacy, safety, and tolerability. The objective of the dose expansion (Part B) of each sub-study is to evaluate the antitumor activity of AZD8205. Initiation of Part B of each substudy will depend on evaluation of the safety results from Part A and determination of the RP2D for AZD8205 monotherapy and each combination.

[0187] Figure 2 shows the study flow for sub-study 1, which is outlined in the Examples below. The pre-screening selection process for sub-study 1 and key aspects of the study, such as starting dose of AZD8205, dose escalation for Part A of all sub-studies, stopping criteria, and cohort size, are based on accepted methodology for Phase I / II oncology studies. Additionally, blood samples will be collected to allow characterization of AZD8205 PK and investigation of additional metabolites, if necessary.

[0188] As part of the clinical drug development program for AZD8205, PDx and exploratory biomarker (DNA, RNA, protein or metabolite) profiles will be investigated and their relationship to drug efficacy will be studied. Potential benefits of this exploratory study include: (1) identifying subjects most likely to benefit from treatment; (2) predicting which subjects may not respond to treatment; (3) identifying side effects associated with drug exposure; and (4) characterizing new toxicities.

[0189] In summary, the Examples provide the information and understanding necessary to deliver safe and effective new drugs to subjects with tumors that express B7-H4.

[0190] Definitions - The following terms are used in the studies described in the Examples below. Dose-limiting toxicity (DLT). DLTs will be assessed during Part A (dose escalation). The DLT assessment period is 21 days from the first dose of AZD8205 on Day 1 of Cycle 1 or up to (and including) the planned end of Cycle 1 if alternative dosing schedules are being considered. A DLT is defined as any Grade 3 or higher treatment-emergent AE, including modifications or exceptions, occurring during the DLT assessment period and not attributable to the primary disease or disease-related process or intercurrent illness.

[0191] Maximum tolerated dose (MTD). The MTD is defined as the highest dose at which the target DLT probability is 30%. The MTD is determined by isotonic regression analysis applied to the DLT rates observed during the dose escalation phase using the modified toxicity probability interval-2 (mTPI-2) method (6).

[0192] Efficacy assessment. Tumor response will be assessed by RECIST v1.1 (7) according to a schedule.

[0193] Tumor assessment. Tumor assessment will use computed tomography (CT, preferred) or magnetic resonance imaging (MRI) imaging of the chest, abdomen, and pelvis with IV contrast to investigate areas of possible involvement based on individual subject's signs and symptoms, collected during screening / baseline, and at regular (follow-up) intervals during study intervention. A brain scan (MRI preferred) is mandatory at screening / baseline for all subjects in this study. Post-baseline brain MRI should only be performed in subjects with brain metastases at baseline, whereas subjects without brain metastases do not require additional brain scans for subsequent tumor assessments unless clinically indicated. The imaging modality used for baseline tumor assessment will be kept consistent and the same for each subsequent follow-up assessment throughout the study, if possible.

[0194] Screening / baseline imaging will be performed ≥28 days prior to initiation of study intervention, ideally as close as possible to and prior to initiation of study intervention. As part of standard clinical practice, scans will be obtained prior to informed consent (although scans obtained within 28 days will be acceptable). Tumor assessments will be performed according to each substudy schedule until objective disease progression as defined by RECIST v1.1 and assessed by the investigator, or withdrawal of consent. Responses (complete response (CR) or partial response (PR)) will be confirmed by repeat serial scans at least 4 weeks after documentation of the first response.

[0195] Per RECIST v1.1, tumor markers will not be used to assess tumor response. Tumor markers will be collected for separate analysis. However, the results will not contribute to tumor response based on RECIST v1.1 assessment.

[0196] Eastern Cooperative Oncology Group (ECOG) performance status (PS). Subject performance status will be assessed using the ECOG PS scale as detailed in each substudy schedule.

[0197] Adverse Event. An adverse event (AE) is any undesirable medical occurrence in a subject administered a medicinal product or in a clinical trial subject that does not necessarily have a causal relationship to this treatment. Thus, an AE can be any undesirable and unintended sign (such as an abnormal laboratory finding), symptom (e.g., nausea, chest pain), or illness temporarily associated with the use of a medicinal product, regardless of whether it is considered related to the medicinal product.

[0198] The term AE is used to include both serious and nonserious AEs and may include a worsening of a pre-existing medical event. AEs may occur at any time, including during the lead-in or washout periods, even when the study intervention is not being administered.

[0199] Serious Adverse Events. A serious adverse event (SAE) is an AE occurring during any study phase (i.e., induction, treatment, washout, follow-up) that meets one or more of the following criteria: (1) results in death; (2) is immediately life-threatening; (3) requires hospitalization of the subject or an extension of an existing hospitalization; (4) results in persistent or significant impairment or incapacity; (5) is a congenital anomaly or birth defect; (6) is a significant medical event that may endanger the subject or require medical treatment to prevent any of the above outcomes.

[0200] Disease progression. Disease progression may be considered as a worsening of the subject's condition due to the disease for which the investigational drug is being tested. It may be an increase in disease severity and / or an increase in symptoms of the disease during the study. The occurrence of new metastases or progression of existing metastases to the primary cancer during the study should be considered as disease progression and not as an AE. Events that are clearly due to disease progression should not be reported as AEs during the study.

[0201] New cancer. The occurrence of a new cancer should be considered an SAE. A new primary cancer is one that is not the primary reason for administering the study intervention and is identified after the subject enters the study. Metastases of the original cancer are not included.

[0202] Example 3: Formulation and administration of AZD8205 This example illustrates the formulation and administration of AZD8205. AZD8205 is supplied in Part A and Part B as either a lyophilized or liquid product.

[0203] Lyophilized AZD8205 is supplied as a sterile lyophilized solid containing 100 mg AZD8205 (nominal) per vial in 20R amber vials. AZD8205 Investigational Medicinal Product (IP) should be stored at 2°C to 8°C (refrigerated) and protected from exposure to light prior to use. AZD8205 vials should not be frozen.

[0204] Liquid AZD8205 is supplied as an intravenous bag protectant (IVBP) solution to ensure compatibility of AZD8205 with IV infusion components and diluents. IVBPs were stored at 2°C to 8°C (refrigerated). Lyophilized AZD8205 was not reconstituted in IVBP solution. Sterile water for injection is used for reconstitution.

[0205] All IP should be stored in a secure, dry place. Vials should be stored at 2°C to 8°C (refrigerated) and protected from light; they should not be frozen.

[0206] Weight-based doses are calculated using the following formula:

number

[0207] Example 4: Early risk / benefit assessment Studies will be conducted to assess the risks of AZD8205 administration and to provide appropriate AZD8205 doses for Part A and Part B.

[0208] Preclinical toxicology studies evaluating the safety of AZD8205 demonstrated that exposure to AZD8205 in cynomolgus monkeys at levels above the expected clinical efficacy range induced changes consistent with other marketed TOP1 iADCs. The primary target organs for toxicity were those with rapid cell turnover (e.g., bone marrow and testes).

[0209] In a non-GLP dose-ranging study, male cynomolgus monkeys were exposed to AZD8205 (IV Q3W×2) at dose levels of 15, 20, 22.5, and 25 mg / kg. Results were as expected and consistent with a cytotoxic TOP1 inhibitor. Dose levels ≥20 mg / kg were not tolerated. At dose levels ≥20 mg / kg, in addition to signs of moribundity, there were moderate to marked effects on the hematopoietic system (most notably reductions in lymphocytes, neutrophils, and reticulocytes) with histologic correlation to moderate to marked bone marrow hypocellularity and decreased cellularity in other lymphoid organs; gastrointestinal toxicity was noted in the large and small intestinal mucosa (minimal to marked degeneration and regeneration) and was believed to be the primary cause of moribundity in the animals.

[0210] Other organs showing AZD8205 treatment-related pharmacological effects included reversible mild to moderate histopathological findings in the kidney, liver, pancreatic islets, and testes. Some of these treatment-related findings were also indicative of stress / deconditioning and clinical deterioration.

[0211] Clinical chemistry changes were noted in the liver and kidney, including mild, transient elevations in liver enzymes (2x control ALT / AST levels on days 4-6 only, but with no histopathological correlation) and marked increases in renal enzymes that correlated with clinical observations of dehydration. All changes were reversible prior to scheduled necropsy on day 43. Hematopoietic effects were also noted in surviving animals (2 / 3 at 22.5 mg / kg and 3 / 3 at 15 mg / kg), but these were minimal to mild. The only treatment-related microscopic findings were noted in animals receiving 22.5 mg / kg IV Q3W×2, again consistent with the mechanism of action (MOA) of the TOP1 inhibitor. At 15 mg / kg, there were no histopathological findings in any organs examined.

[0212] A pivotal GLP 6-week repeat-dose (Q3W×2) IV study in cynomolgus monkeys with a 6-week recovery period showed no adverse findings at dose levels up to 15 mg / kg (maximum dose tested and HNSTD). All major organs and functions (respiratory, cardiovascular, renal, hepatic, and nervous systems) were assessed and no AZD8205-related clinical signs or effects were noted with respect to viability, local (skin) tolerance, body weight, electrocardiogram, blood pressure, respiratory rate, ophthalmoscopy, neurological endpoints, clinical chemistry, urinalysis, systemic cytokine release or coagulation parameters. The only treatment-related changes were observed at dose levels ≥10 mg / kg and were associated with the expected pharmacological activity of AZD8205 on the hematopoietic system, including minimal, transient effects on some erythroid parameters that were reversible during the 6-week recovery phase.

[0213] The pivotal GLP AZD8205 toxicity study in male and female cynomolgus monkeys showed significant adverse events at dose levels up to 15 mg / kg (gender-mixed mean AUC of 963 μg.d / mL and C of 323 μg / mL). max No evidence of liver enzyme elevations, hypersensitivity / anaphylaxis, or infusion-related reactions was found at the starting dose of 100 μg.d / mL. These values ​​are consistent with the minimum clinical efficacy AUC (77 μg.d / mL) and C maxThese are expected to provide a safety margin of approximately 13-fold and 20-fold, respectively, above the 16 μg / mL (16 μg / mL) level. Where changes in liver enzymes were observed in dose-ranging studies, they were minimal (approximately 2-fold), transient (seen over 1-2 days in some animals), not associated with hepatocellular necrosis, and fully reversible by the end of dosing of animals with 15 mg / kg AZD8205.

[0214] Example 5: Test Subjects This example provides criteria for selecting subjects for the Substudy 1 Part A and Part B studies.

[0215] Inclusion Criteria for Part A and Part B: 1. You must be 18 years of age or older to register for the exam. 2. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 at the time of enrollment. 3. Subjects with recurrent / metastatic solid tumors must have received sufficient prior SoC therapy if a clinical trial is the best option for next treatment based on tumor type and stage, or response and / or tolerability to previous treatment. 4. Subjects must have measurable disease according to RECIST v1.1, defined by at least one lesion that is accurately measurable at baseline as ≥10 mm in longest diameter on CT or MRI (excluding lymph nodes, which must have a short axis of ≥15 mm) and amenable to accurate repeat measurement. Previously irradiated lesions or lesions in radiation fields should not be used as target lesions unless the lesion(s) have demonstrated unequivocal disease progression by RECIST v1.1. Target lesions should not be used for baseline tumor biopsy unless there are no other lesions suitable for biopsy and the prescribed requirements are met. 5. Predicted life expectancy ≥ 12 weeks. 6. Adequate organ function as defined in Table 2.

[0216] [Table 6]

[0217] Example 6: Part A Dose Escalation This example describes a Part A dose escalation study to determine the dose of AZD8205 for Part B.

[0218] This substudy is being conducted to investigate the safety and tolerability of AZD8205 as monotherapy and to evaluate the antitumor activity of AZD8205 in breast, ovarian, endometrial, and biliary tract cancers. AZD8205 will be administered intravenously Q3W. The study will be conducted in the following phases, which will apply to both Parts A and B: 1. Pre-screening phase: All subjects provide a baseline tissue sample for B7-H4 analysis. Subjects with tumors expressing B7-H4 enter the next phase. 2. Screening Phase: Subjects are assessed for eligibility for the study intervention. Only subjects who meet all inclusion criteria and none of the exclusion criteria will be assigned to the study. 3. Treatment Phase: Subjects continue to receive study intervention until one of the discontinuation criteria is met, including any AE or AEs contraindicating further dosing, or radiological progression. 4. Follow-up Phase: All subjects are followed up for survival.

[0219] Part A (dose escalation) will evaluate AZD8205 in approximately 60 subjects with positive levels of B7-H4 tumor expression to determine the MTD and / or RP2D. The RP2D will be selected by pooling and evaluating all available data, including adequate PK, PD, efficacy, safety, and tolerability. Based on safety, PK, and PDx data, the SRC may recommend intermediate dose levels and / or alternative dosing schedules and longer treatment intervals. Based on new data, any dose level not exceeding the MTD may be expanded to up to 18 additional subjects (referred to as the PDx backfill cohort, for a total of 36 subjects). Subjects enrolled in the PDx backfill cohort will not impact the dosing decision made by the SRC based on the mTPI-2 algorithm. The PDx backfill cohort may enroll certain tumor types for which pre- and on-treatment tumor biopsies are mandatory. These PDx backfill cohorts will provide additional PDx and safety data that will inform the selection of optimal dose levels for expansion based on available data.

[0220] In Part A, four dose levels were administered via IV Q3W: 0.8 mg / kg, 1.6 mg / kg, 2.4 mg / kg, and 3.2 mg / kg.

[0221] Following an initial screening period of up to 28 days, the intervention period in Part A was planned to include at least 20 cycles, each of which was 21 or 28 days, with AZD8205 administered every 3 weeks (Q3W), i.e., once every 21-day cycle or once every 28-day cycle.

[0222] Eligible subjects will receive AZD8205 Q3W by intravenous (IV) infusion at the selected dose for ≥20 cycles starting on day 1 of cycle 1. Subjects will be treated with study intervention until disease progression, unacceptable toxicity, investigator decision, completion of maximum treatment cycles, or withdrawal of consent. All subjects will be followed for survival until study end.

[0223] After the period of the present invention, subjects are further evaluated for disease progression, end of treatment status, and follow-up status.

[0224] Example 7: Part B Dose Expansion The Part B study phase of this Example was conducted using a 2.4 mg / kg dose of AZD8205 to study dose escalation.

[0225] The objectives and endpoints are described in Example 6 above.

[0226] Part B (dose expansion) will be initiated based on preliminary efficacy and safety data in approximately 220 subjects from specific populations. Pending review of new data from Part A, four expansion cohorts are planned: Approximately 40 subjects from the BTC cohort (Cohort B1) Approximately 60 subjects from the ovarian cancer cohort (Cohort B2) - Approximately 30 subjects with PRR (Cohort B2A) - Approximately 30 subjects with PSR (cohort B2B) Approximately 90 subjects across the HER2-negative (IHC: 0, 1+, 2+ / ISH-, as defined by the latest ASCO / CAP guidelines) breast cancer cohort (Cohort B3): - Approximately 30 subjects with HR+, HER2-negative breast cancer (Cohort B3A) - Approximately 30 subjects with TNBC (Cohort B3B) - Approximately 30 subjects with HR±, HER2-negative breast cancer who have been pre-treated with a TOP1i ADC (Cohort B3C) Approximately 30 subjects from the endometrial cancer cohort (cohort B4).

[0227] Each expansion cohort may be initiated at multiple dose levels or schedules depending on new data. Expansion cohorts may be initiated simultaneously or separately in a parallel or staggered approach. If a new dosing schedule is considered, dose modifications will be agreed upon and modified based on previously established safe exposure ranges. If it is decided to investigate two dose levels / schedules within the expansion cohort, the overall sample size of Part B may increase up to a maximum of 440 subjects. In such cases, subjects may be randomized to the selected dose level / schedule; allocation will not be blinded. Randomization will occur on Day 1.

[0228] Safety and tolerability data for all subjects will be evaluated regularly and subjects' clinical response status will be classified according to RECIST v1.1.

[0229] Example 8: Preliminary Part A and Part B Results Thirty-five subjects have been evaluated in the Part A and Part B portions of the study described in the Examples above, with evaluation of 16 subjects ongoing.

[0230] Table 3 shows the demographics of subjects evaluated in Parts A and B.

[0231] [Table 7]

[0232] Preliminary PK data are provided. Data are available from n=3 pts (0.8mg / kg), n=9 pts (1.6mg / kg), n=9 (2.4mg / Kg), and n=5 pts (3.2mg / Kg). PK data show that total AZD8205 ADC and total antibody concentrations were comparable at all dose levels tested, indicating that warhead binding is stable in subjects. Notably, AZD8205 exposure is consistent with preclinical predictions, increasing proportionally with dose, with a half-life = 4.9-7.2 days. Additionally, 1.6 and 2.4mg / kg (AUC(0-21) 143.5±39.2 and 190.9±10.1 days, respectively) were significantly longer than baseline. * AZD8205 exposure at 1 μg / mL was within the predicted effective range (80-230 days) * The exposure of unconjugated warhead in plasma is within 1.2-fold of the predicted AUC.

[0233] Computed tomography data suggest that AZD8205 has activity in breast cancer (BC), ovarian cancer (OC), and endometrial cancer (EC). Notably, 4 subjects demonstrated both tumor marker reduction + target lesion (1uPR and 3cPR) shrinkage (>30%). 18 subjects demonstrated SD as BoR by RECIST (including 4 subjects with ongoing tumor shrinkage of ≥20%). Tumor markers decreased >50% in 10 / 24 evaluable pts across all dose levels in OC, BC, and EC. Molecular ctDNA measurements demonstrated responses in N=14 / 21 (~67%) evaluable subjects - N=5 / 8 subjects (63%) in dose level 2 (DL2), N=6 / 9 subjects (67%) in dose level 3 (DL3), and 3 / 4 subjects (75%) in dose level 4 (DL4), a pattern generally consistent with clinical responses and tumor markers.

[0234] OvCa subjects with measurable CA125 (>2×ULN) are plotted in FIG. 3, where responses to AZD8205 at 1.6, 2.4, and 3.2 mg / kg are confirmed. ctDNA molecular responses were observed in approximately 67% of evaluable subjects (n=14 / 21) at 1.6, 2.4, and 3.2 mg / kg. ctDNA changes primarily reflect clinical responses, with greater ctDNA reductions observed in subjects experiencing benefit. New preclinical / clinical efficacy results are shown in Table 6.

[0235] [Table 8]

[0236] Example 9: Determination of B7-H4 cutoff in selected subjects In the early stages of the first-in-human trials described in the Examples above, it was observed that higher overall response rates (ORR) were seen in subjects whose tumors had higher levels of B7-H4 expression and responded better to treatment with AZD8205. The ORR for all subjects versus B7-H4 expressing subjects is summarized in Table 7 below.

[0237] [Table 9]

[0238] To determine the B7-H4 expression cutoff for ongoing human trials, mouse xenograft studies were performed for CCA, ovarian cancer, and TNBC. Athymic nude mice (Fox1 nu Tumors were xenografted into either 16- or 24-cell-expressing (SCID) or NOD-SCID mice. AZD8205 was administered as a single intravenous dose on day 0. Changes in tumor growth were monitored 28 days after treatment. A plot of the change in percent tumor growth versus the percentage of cells expressing B7-H4 is shown in FIG. 4.

[0239] Receiver operating characteristic (ROC) curves were determined for each dose and the curves were used to calculate the Youden statistic at each dose. The Youden statistic is plotted against the percentage of B7-H4 cells as shown in Figure 5. A Youden statistic of 1 represents an intact biomarker, while a statistic of 0 means the biomarker is undifferentiated. As seen in Figure 5, the Youden statistic plateaued at B7-H4 percentages above 25%. Therefore, a 25% B7-H4 expression cutoff is used to assess the dose used.

[0240] Example 10: Overall response rate in heavily pretreated subjects. A total of 44 heavily pretreated subjects from the study described in the above examples were evaluated for objective response rate (ORR). As shown in Table 8 as part of the second data cut considering Table 6, the 44 subjects include 18 subjects with ovarian cancer, 9 subjects with cholangiocarcinoma (CCA), 12 subjects with breast cancer (BC), and 5 subjects with endometrial cancer (EC). Of these 44 subjects, 40 were evaluated for ORR. Some of the subjects had been previously treated with platinum chemotherapy, as shown in Table 8. As shown in Table 8, for subjects with a significant reduction in tumor markers and / or a reduction in ctDNA, the average ORR is about 25%.

[0241] [Table 10]

Claims

1. A pharmaceutical composition for treating a target cancer, comprising an antibody-drug conjugate (ADC) that specifically binds to B7-H4, The aforementioned ADC is administered to the subject in an amount of approximately 0.8 mg / kg to approximately 4.8 mg / kg. The aforementioned ADC is 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), or functional variants thereof, each containing 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, or functional variants thereof, each containing 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, or functional variants thereof, each containing 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, or functional variants thereof, each containing 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) an antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, or functional variants thereof, each containing 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. Cutting linker and; iii. Cytotoxic agents and A pharmaceutical composition containing the following:

2. The pharmaceutical composition according to claim 1, wherein the amount of ADC administered is approximately 0.8 mg / kg, approximately 1.6 mg / kg, approximately 2.4 mg / kg, approximately 3.2 mg / kg, approximately 3.6 mg / kg, or approximately 4.8 mg / kg.

3. The pharmaceutical composition according to claim 1, wherein the ADC is administered to the subject once every three weeks.

4. The aforementioned antibody or its antigen-binding fragment: i. Variable heavy (VH) chains and variable light (VL) chains, or functional variants thereof, containing the amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 34, respectively; ii. VH chains and VL chains, or functional variants thereof, containing the amino acid sequences of SEQ ID NO: 33 and SEQ ID NO: 34, respectively; iii. VH chains and VL chains, or functional variants thereof, containing the amino acid sequences of SEQ ID NO: 43 and SEQ ID NO: 34, respectively; iv. VH chains and VL chains, or functional variants thereof, containing the amino acid sequences of SEQ ID NO: 46 and SEQ ID NO: 34, respectively; v. VH chains and VL chains, or functional variants thereof, containing the amino acid sequences of SEQ ID NO: 47 and SEQ ID NO: 34, respectively; vi. VH chains and VL chains, or functional variants thereof, containing the amino acid sequences of SEQ ID NO: 31 and SEQ ID NO: 32, respectively; vii. VH chains and VL chains, or functional variants thereof, containing the amino acid sequences of SEQ ID NO: 35 and SEQ ID NO: 36, respectively; viiii. VH chains and VL chains containing the amino acid sequences of SEQ ID NO: 37 and SEQ ID NO: 38, respectively, or functional variants thereof; or ix. The pharmaceutical composition according to claim 1, comprising a VH chain and a VL chain, or functional variants thereof, each containing the amino acid sequences of SEQ ID NO: 39 and SEQ ID NO: 40, respectively.

5. The pharmaceutical composition according to claim 1, wherein the antibody or its antigen-binding fragment is bound to the OVCAR4 cell line.

6. The pharmaceutical composition according to claim 1, wherein the antibody or its antigen-binding fragment comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 48 or 51 and a light chain containing the amino acid sequence of SEQ ID NO:

44.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the cleavage linker is an mp-PEG8-val-ala linker.

8. The pharmaceutical composition according to any one of claims 1 to 6, wherein the cytotoxic agent is a topoisomerase inhibitor.

9. The topoisomerase inhibitor is, Formula A* 【Chemistry 1】 The pharmaceutical composition according to claim 8, wherein the compound is [the compound].

10. The above ii) cleavage linker and iii) cytotoxic agent are both the following compounds: 【Chemistry 2】 A pharmaceutical composition according to any one of claims 1 to 6, selected from the above.

11. The pharmaceutical composition according to claim 10, wherein both the ii) linker and the iii) cytotoxic agent are compound SG3932.

12. The pharmaceutical composition according to any one of claims 1 to 6, wherein the ADC has a drug-to-antibody ratio (DAR) of about 1 to about 8.

13. The pharmaceutical composition according to any one of claims 1 to 6, wherein the ADC has a DAR of about 8.

14. A pharmaceutical composition for treating a target cancer, comprising an antibody-drug conjugate (ADC) that specifically binds to B7-H4, The aforementioned ADC is administered to the subject in an amount of approximately 0.8 mg / kg to approximately 4.8 mg / kg. The aforementioned ADC is i. An antibody or antigen-binding fragment thereof that binds to a B7-H4 polypeptide, comprising: 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), or functional variants thereof, each containing 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; ii. formula: 【Transformation 3】 A pharmaceutical composition comprising the antibody having the above-mentioned characteristic or an antigen-binding fragment thereof, a cleavage linker complexed with the antibody, and a cytotoxic agent.

15. The pharmaceutical composition according to claim 14, wherein the amount of ADC administered is approximately 0.8 mg / kg, approximately 1.6 mg / kg, approximately 2.4 mg / kg, approximately 3.2 mg / kg, approximately 3.6 mg / kg, or approximately 4.8 mg / kg.

16. The pharmaceutical composition according to claim 14, wherein the antibody or its antigen-binding fragment comprises a VH chain and a VL chain, each containing the amino acid sequences of SEQ ID NO: 45 and SEQ ID NO: 34, or functional variants thereof.

17. The pharmaceutical composition according to any one of claims 1 to 6 and 14 to 16, wherein the cancer comprises cancer cells expressing B7-H4.

18. The pharmaceutical composition according to any one of claims 14 to 16, wherein the ADC is administered to the subject once every three weeks.

19. The pharmaceutical composition according to any one of claims 1 to 6 and 14 to 16, wherein the cancer is selected from ovarian cancer, breast cancer, pancreatic cancer, prostate cancer, hematological cancer, endometrial cancer, bile duct cancer, NSCLC (squamous and / or adenocarcinoma), gastrointestinal cancers such as gastric cancer and colorectal cancer, and lung cancer.

20. The pharmaceutical composition according to any one of claims 1 to 6 and 14 to 16, wherein 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).

21. The pharmaceutical composition according to any one of claims 1 to 6 and 14 to 16, wherein the cancer is homologous recombination repair deficiency (HRD) cancer.

22. The pharmaceutical composition according to claim 21, wherein the cancer comprises one or more cells having a mutation in the HRD gene selected from BRCA1, BRCA2, ATM, BRIP1, BARD1, CDK12, CHEK1, CHEK2, FANCL, PALB2, PPP2R2A, RAD51B, RAD51C, RAD51D, and RAD54L.

23. The pharmaceutical composition according to any one of claims 1 to 6 and 14 to 16, wherein at least about 25% of the cancer cells in the subject are B7-H4 positive cells, and optionally the cancer cells are analyzed using immunohistochemistry (IHC).

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