WO2025167977A1 - Combined use of antibody-drug conjugate and immune checkpoint inhibitor - Google Patents

Abstract

The present invention relates to use of an antibody-drug conjugate alone or in combination in preparing a drug for preventing and/or treating cancers. Specifically, the present invention provides use of an antibody-drug conjugate or a pharmaceutically acceptable salt thereof, a metabolite thereof, or a solvate thereof alone or in combination with an immune checkpoint inhibitor in preparing a drug for preventing and/or treating cancers.

Description

Combination therapy of antibody-drug conjugates and immune checkpoint inhibitors Technical Field

This application belongs to the field of medicine and relates to the use of an antibody-drug conjugate, alone or in combination, in the preparation of a drug for the prevention and/or treatment of cancer. Specifically, the present invention provides the use of an antibody-drug conjugate, or a pharmaceutically acceptable salt, metabolite, or solvate thereof, alone or in combination with an immune checkpoint inhibitor and an optional chemotherapeutic agent, in the preparation of a drug for the prevention and/or treatment of cancer.

Background Art

Antibody-drug conjugates (ADCs) are a class of targeted biologics that link cytotoxic drugs to monoclonal antibodies via a linker. Using the monoclonal antibody as a carrier, small-molecule cytotoxic drugs are efficiently and effectively delivered to target tumor cells in a targeted manner. Tumor-specific antibodies enable ADCs to selectively deliver small-molecule cytotoxic drugs, minimizing off-target effects while retaining their anti-tumor properties, effectively improving the benefit-risk ratio of anti-tumor therapy. B7 homolog 4 is a newly discovered member of the B7 family. It plays a crucial role in multiple cellular biological processes, such as cell differentiation, proliferation, and apoptosis, and may influence tumor cell invasion and metastasis. Furthermore, the B7 family is an important co-stimulatory molecule that influences processes such as T cell proliferation and B cell activation. Studies have shown that B7 homolog 4 is highly expressed in various tumors, including cholangiocarcinoma, breast cancer, endometrial cancer, non-small cell lung cancer, ovarian cancer, gastric cancer, and pancreatic cancer, while its expression is limited in normal tissues. Therefore, B7 homolog 4 has potential as a target for ADCs.

Immune checkpoint inhibitors (ICIs) block the binding of immune checkpoints to their ligands, relieving the immune suppression caused by immune checkpoints and thereby reactivating immune cells to exert anti-tumor effects. Currently, several ICIs have achieved remarkable results in clinical anti-tumor applications, representing a breakthrough in the field of tumor treatment. Inhibitors targeting cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), programmed death-1 (PD-1), and programmed death-ligand 1 (PD-L1) have been successfully used in the clinical treatment of various malignancies. PD-1/PD-L1 antibody drugs have a broad anti-cancer spectrum and demonstrate long-lasting and potent efficacy in some tumor patients. Ten PD-1 monoclonal antibodies and three PD-L1 monoclonal antibodies have been approved for the clinical treatment of 11 cancer types, including non-small cell lung cancer, melanoma, head and neck cancer, colorectal cancer, and gastric cancer. Although ICIs have significant anti-tumor activity in many types of malignant tumors, their response rate is low in most tumors, and new regimens need to be developed to improve the response rate of ICIs treatment.

Developed in the 1960s, platinum-based drugs are non-specific cell cycle drugs that primarily form Pt-DNA adducts with DNA after entering tumor cells, thereby mediating tumor cell necrosis or apoptosis, thereby producing anti-cancer effects. Due to their unique anti-cancer mechanisms and broad anti-cancer spectrum, platinum-based drugs are currently one of the most widely used chemotherapy drugs in clinical practice. They are widely used as basic drugs in the treatment of common malignancies such as lung cancer, bladder cancer, ovarian cancer, cervical cancer, esophageal cancer, gastric cancer, colorectal cancer, and head and neck tumors. Although platinum-based drugs have become the first-line chemotherapy drugs for the clinical treatment of tumors, their severe drug resistance has greatly limited their clinical application.

In summary, there is an urgent need to develop a new combination therapy that can show better effects than any single treatment, improve the efficacy while having lower side effects of combined medication, so as to maximize the survival benefits of cancer patients.

Summary of the Invention

The present disclosure provides a use of an antibody-drug conjugate and an immune checkpoint inhibitor in combination for preparing a drug for treating cancer. The structure of the antibody-drug conjugate is shown in formula (I):

wherein n is a non-zero integer or decimal from 1 to 10, preferably a decimal or integer from 1 to 8, preferably a decimal or integer from 2 to 8, more preferably from 3 to 8, and can be an integer or a decimal, more preferably 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

Wherein Pc is an anti-B7H4 antibody or an antigen-binding fragment thereof.

The present disclosure also provides a use of an antibody-drug conjugate and a platinum drug in combination for preparing a drug for treating cancer. The structure of the antibody-drug conjugate is shown in formula (I):

wherein n is a non-zero integer or decimal from 1 to 10, preferably a decimal or integer from 1 to 8, preferably a decimal or integer from 2 to 8, more preferably from 3 to 8, and can be an integer or a decimal, more preferably 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

Wherein Pc is an anti-B7H4 antibody or an antigen-binding fragment thereof.

In some embodiments, the above-mentioned anti-B7H4 antibody or its antigen-binding fragment comprises: heavy chain HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 01, 02 and 03 amino acid sequences, respectively, and light chain LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 04, 05 and 06 amino acid sequences, respectively.

In the present invention, the amino acid sequences of the CDRs listed above are all shown according to the Kabat definition rules. However, it is well known in the art that antibody CDRs can be defined by various methods in the art. Although the scope of protection claimed in the present invention is based on the sequences shown in the Kabat definition rules, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of the present invention.

The CDR sequences are shown in Table A below:

Table A Heavy and light chain CDR sequences

Note: CDR sequences are derived from those shown in the Kabat definition.

Preferably, the anti-B7H4 antibody or antigen-binding fragment thereof is selected from a humanized antibody or a fragment thereof.

In some alternative embodiments, the anti-B7H4 antibody or antigen-binding fragment thereof described herein is an antibody fragment selected from the group consisting of Fab, Fab'-SH, Fv, scFv, and (Fab') ₂ fragments.

In some optional embodiments, the anti-B7H4 antibody or antigen-binding fragment thereof described herein comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 isotype, preferably a heavy chain constant region of IgG1 or IgG4 isotype.

In other alternative embodiments, the anti-B7H4 antibody or antigen-binding fragment thereof comprises a light chain constant region of κ or λ.

Furthermore, it is preferred that the heavy chain variable region sequence of the anti-B7H4 antibody or its antigen-binding fragment is the sequence shown in SEQ ID NO: 07 or a variant thereof, and the light chain variable region sequence is the sequence shown in SEQ ID NO: 08 or a variant thereof.

The sequences of the heavy and light chain variable regions of the aforementioned anti-B7H4 antibodies or antigen-binding fragments thereof are as follows: Heavy chain variable region sequence

Light chain variable region sequence

Note: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The italics in the sequence are FR sequences, and the underlines are CDR sequences. The CDR sequences are derived from the Kabat definition rules.

Furthermore, it is preferred that the heavy chain sequence of the anti-B7H4 antibody or its antigen-binding fragment is the sequence shown in SEQ ID NO: 09 or a variant thereof, and the light chain sequence is the sequence shown in SEQ ID NO: 10 or a variant thereof.

The sequences of the heavy and light chains of the aforementioned anti-B7H4 antibodies or antigen-binding fragments thereof are shown below:

Heavy chain (IgG1) amino acid sequence: (SEQ ID NO: 09)

Light chain (λ) amino acid sequence: (SEQ ID NO: 10)

In some embodiments, the immune checkpoint inhibitor is selected from antibodies or antigen-binding fragments thereof targeting PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, BTLA, A2aR, B7-H3, B7-H4, preferably antibodies or antigen-binding fragments thereof targeting PD-1 or PD-L1.

In some embodiments, the immune checkpoint inhibitor is selected from Adebrelimab, Camrelizumab, Dostarlimab, Toripalimab, Sintilimab, Tislelizumab, Zimberelimab, Penpulimab, Serplulimab, Pucotenlimab, Pembrolizumab , Nivolumab, Sugemalimab, Envafolimab, Atezolizumab, Durvalumab, Ipilimumab, Candonilimab, preferably Adebrelimab, Camrelizumab, Dostarlimab, Pembrolizumab, Durvalumab.

The sequences of the heavy and light chains of the aforementioned Adebelimumab are shown below:

Heavy chain amino acid sequence: (SEQ ID NO: 11)

Light chain amino acid sequence: (SEQ ID NO: 12)

The sequences of the heavy and light chains of the aforementioned carrelizumab are as follows:

Heavy chain amino acid sequence: (SEQ ID NO: 13)

Light chain amino acid sequence: (SEQ ID NO: 14)

Dotalizumab is a PD-1 blocking IgG4 humanized monoclonal antibody with the trade name JEMPERLI. The main structure and function of dotalizumab have been described in WO2014/179664, WO 2018/085468 and WO2018/129559.

The heavy and light chain sequences of dotalizumab are as follows:

Heavy chain amino acid sequence: (SEQ ID NO: 15)

Light chain amino acid sequence: (SEQ ID NO: 16)

In some embodiments, dotarizumab or a biosimilar thereof is administered to a patient in need thereof at a dose of 500 mg once every three weeks (Q3W).

In some embodiments, patients in need thereof are administered 500 mg of dotarizumab or a biosimilar thereof once every three weeks (Q3W) for 4-6 cycles, followed by 1000 mg of dotarizumab or a biosimilar thereof once every 6 weeks (Q6W).

Pembrolizumab is a PD-1 blocking IgG4 humanized monoclonal antibody, sold under the trade name KEYTRUDA.

The heavy and light chain sequences of pembrolizumab are as follows:

Heavy chain amino acid sequence: (SEQ ID NO: 17)

Light chain amino acid sequence: (SEQ ID NO: 18)

In some embodiments, pembrolizumab or a biosimilar thereof is administered to a patient in need thereof at a dose of 200 mg once every three weeks (Q3W); or, at a dose of 400 mg once every six weeks (Q6W).

Imfinzi is a PD-L1 blocking IgG1 monoclonal antibody marketed under the trade name IMFINZI.

The heavy and light chain sequences of durvalumab are as follows:

Heavy chain amino acid sequence: (SEQ ID NO: 19)

Light chain amino acid sequence: (SEQ ID NO: 20)

In some embodiments, durvalumab or a biosimilar thereof is administered to patients in need thereof who are ≥30 kg at a dose of 1500 mg once every three weeks (Q3W).

In some embodiments, durvalumab or a biosimilar thereof is administered to patients in need thereof who are <30 kg at a dose of 20 mg/kg once every three weeks (Q3W).

In some embodiments, durvalumab or a biosimilar thereof is administered to patients in need thereof ≥ 30 kg at a dose of 1120 mg once every three weeks (Q3W).

In some embodiments, durvalumab or a biosimilar thereof is administered to patients in need thereof weighing <30 kg at a dose of 15 mg/kg once every three weeks (Q3W).

On the other hand, the present invention discloses the use of the antibody-drug conjugate and the immune checkpoint inhibitor, further combined with a platinum drug, in the preparation of a drug for treating cancer.

In another aspect, the present invention discloses use of the antibody-drug conjugate in combination with the platinum drug in the preparation of a drug for treating cancer.

In an optional embodiment, the platinum drug is selected from: carboplatin, cisplatin, oxaliplatin, nedaplatin, lobaplatin, satraplatin, cycloplatin, miboplatin, enloplatin, iproplatin, dicycloplatin, preferably carboplatin and/or cisplatin.

In an alternative embodiment, the antibody drug conjugate and the immune checkpoint inhibitor are contained in different preparations as active ingredients and are administered simultaneously or at different times.

In an optional embodiment, the antibody-drug conjugate, the immune checkpoint inhibitor, and the platinum drug are contained in different preparations as active ingredients, and are administered simultaneously or at different times.

In an alternative embodiment, the antibody-drug conjugate and the platinum drug are contained in different preparations as active ingredients, and are administered simultaneously or at different times.

In another aspect, the antibody drug conjugate and the immune checkpoint inhibitor are contained in a single formulation as active ingredients and administered.

In another aspect, the antibody drug conjugate, the immune checkpoint inhibitor, and the platinum drug are contained in a single preparation as active ingredients and administered.

In another aspect, the antibody drug conjugate and the platinum drug are contained in a single formulation as active ingredients and administered.

In an optional embodiment, the dosage of the antibody drug conjugate is 0.1 mg/kg to 12.0 mg/kg, preferably 1.0 mg/kg to 12.0 mg/kg, more preferably 1.0 mg/kg to 10.0 mg/kg, and further preferably 1.0 mg/kg, 1.2 mg/kg, 1.4 mg/kg, 1.6 mg/kg, 1.8 mg/kg, 2.0 mg/kg, 2.2 mg/kg, 2.4 mg/kg, 2.6 mg/kg, 2.8 mg/kg, 3.0 mg/kg, 3.2 mg/kg, 3.4 mg/kg, 3.6 mg/kg, 3.8 mg/kg, 4.0 mg/kg, 4.2 mg/kg, 4.4 mg/kg, 4 .6mg/kg, 4.8mg/kg, 5.0mg/kg, 5.2mg/kg, 5.4mg/kg, 5.6mg/kg, 5.8mg/kg, 6.0mg/kg, 6.2mg/kg, 6.4mg/kg, 6.6mg/kg, 6.8mg/kg, 7.0mg/kg, 7.2mg/kg, 7 .4mg/kg, 7.6mg/kg, 7.8mg/kg, 8.0mg/kg, 8.2mg/kg, 8.4mg/kg, 8.6mg/kg, 8.8mg/kg, 9.0mg/kg, 9.2mg/kg, 9.4mg/kg, 9.6mg/kg, 9.8mg/kg or 10.0mg/kg.

In alternative embodiments, the antibody drug conjugate is administered once a week, once every two weeks, once every three weeks, or once every four weeks.

In a preferred embodiment, the antibody drug conjugate is administered at a starting dose of 2.8 mg/kg, 3.8 mg/kg, or 4.8 mg/kg, and the dosing frequency is once every three weeks.

In an optional embodiment, the dose of the immune checkpoint inhibitor is 1.0 mg/kg to 100 mg/kg, preferably 1.0 mg/kg to 40 mg/kg, more preferably 1.0 mg/kg to 30 mg/kg, and further preferably 1.0 mg/kg, 1.2 mg/kg, 1.4 mg/kg, 1.6 mg/kg, 1.8 mg/kg, 2.0 mg/kg, 2.2 mg/kg, 2.4 mg/kg, 2.6 mg/kg, 2.8 mg/kg, 3.0 mg/kg, 3.2 mg/kg, 3.4 mg/kg, 3.6 mg/kg, 3.8 mg/kg, 4.0 mg/kg, 4.2 mg/kg, 4.4 mg/kg, 4.6 mg/kg, 4.8 mg/kg kg, 5.0mg/kg, 5.2mg/kg, 5.4mg/kg, 5.6mg/kg, 5.8mg/kg, 6.0mg/kg, 6.2mg/kg, 6.4mg/kg, 6.6mg/kg, 6.8mg/kg, 7.0mg/kg, 7.2mg/kg, 7.4mg/kg, 7.6mg/kg, 7. 8mg/kg, 8.0mg/kg, 8.2mg/kg, 8.4mg/kg, 8.6mg/kg, 8.8mg/kg, 9.0mg/kg, 9.2mg/kg, 9.4mg/kg, 9.6mg/kg, 9.8mg/kg, 10.0mg/kg, 10.2mg/kg, 10.4mg/kg, 10.6m g/kg, 10.8mg/kg, 11.0mg/kg, 11.2mg/kg, 11.4mg/kg, 11.6mg/kg, 11.8mg/kg, 12.0mg/kg, 12.2mg/kg, 12.4mg/kg, 12.6mg/kg, 12.8mg/kg, 13.0mg/kg, 13.2mg /kg, 13.4mg/kg, 13.6mg/kg, 13.8mg/kg, 14.0mg/kg, 14.2mg/kg, 14.4mg/kg, 14.6mg/kg, 14.8mg/kg, 15.0mg/kg, 15.2mg/kg, 15.4mg/kg, 15.6mg/kg, 15.8mg/ kg, 16.0mg/kg, 16.2mg/kg, 16.4mg/kg, 16.6mg/kg, 16.8mg/kg, 17.0mg/kg, 17.2mg/kg, 17.4mg/kg, 17.6mg/kg, 17.8mg/kg, 18.0mg/kg, 18.2mg/kg, 18.4mg/k g, 18.6mg/kg, 18.8mg/kg, 19.0mg/kg, 19.2mg/kg, 19.4mg/kg, 19.6mg/kg, 19.8mg/kg, 20.0mg/kg, 20.2mg/kg, 20.4mg/kg, 20.6mg/kg, 20.8mg/kg, 30.0mg/kg.

In an optional embodiment, the dose of the immune checkpoint inhibitor is 10 mg to 2000 mg, preferably 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 125 mg, 130 mg, 140 mg, 150 mg, 155 mg, 160 mg, 165 mg, 170 mg, 175 mg, 180 mg, 185 mg, 190 mg, 195 mg, 200 mg, 205 mg, 210 mg, 215 mg, 220 mg, 225 mg, 230 mg, 235 mg, 240 mg, 245 mg, 250 mg, 255 mg, 260 mg, 265 mg, 0mg, 265mg, 270mg, 275mg, 280mg, 285mg, 290mg, 295mg, 300mg, 310mg, 320mg, 325mg, 330mg, 340mg, 350mg, 360mg, 370mg, 375mg, 380mg, 390mg, 400mg, 450mg, 500 mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1000mg, 1100mg, 1120mg, 1150mg, 1200mg, 1250mg, 1300mg, 1350mg, 1400mg, 1450mg, 1500mg.

In an optional embodiment, the immune checkpoint inhibitor is administered once a week, once every two weeks, once every three weeks, once every four weeks, or once every six weeks.

In a preferred embodiment, the dose of the immune checkpoint inhibitor is 20 mg/kg, and the administration frequency is once every three weeks.

In an optional embodiment, the dosage of the platinum drug is calculated as the area under the curve (AUC) and is 1 to 20 mg/ml/min, preferably 1 to 10 mg/ml/min, more preferably 2 mg/ml/min, 3 mg/ml/min, 4 mg/ml/min, 5 mg/ml/min, 6 mg/ml/min, 7 mg/ml/min, 8 mg/ml/min, 9 mg/ml/min, and the frequency of administration is once a week, once every two weeks, once every three weeks or once every four weeks.

In an optional embodiment, the dosage of the platinum drug is 10 mg/m ² to 500 mg/m ² , preferably 10 mg/m ² to 200 mg/m ² , more preferably 25 mg/m ² , 50 mg/m ² , 75 mg/m ² , 100 mg/m ² , 125 mg/m ² , 150 mg/m ² , 175 mg/m ² or 200 mg/m ² , and the administration frequency is once a week, once every two weeks, once every three weeks or once every four weeks.

In an alternative embodiment, the platinum drug is administered for up to 6 cycles.

In a preferred embodiment, the dosage of the platinum drug is: cisplatin 75 mg/m ² or carboplatin AUC 5 mg/ml/min intravenous drip, and the administration frequency is once every three weeks.

In an optional embodiment, the cancer is selected from at least one of the following: uterine cancer, breast cancer, biliary tract cancer, lung cancer, gastric cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, ovarian cancer, bladder cancer, esophageal cancer, nasopharyngeal cancer, salivary gland cancer, head and neck cancer, skin cancer, pharyngeal cancer, laryngeal cancer, thyroid cancer, vulvar cancer, penile cancer, testicular cancer, urothelial cancer, urethral cancer, colon cancer, rectal cancer, colorectal cancer, esophageal gastric junction cancer, gastrointestinal stromal tumor, squamous cell carcinoma, peritoneal cancer, leukemia, malignant lymphoma, plasma cell neoplasm, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, mesothelioma, Paget's disease and sarcoma.

Furthermore, the uterine cancer is selected from endometrial cancer, the breast cancer is selected from triple-negative breast cancer, and the biliary tract cancer is selected from gallbladder cancer and bile duct cancer.

In alternative embodiments, the cancer is an advanced solid tumor for which adequate standard treatment has failed or is intolerant, or for which there is no effective standard treatment.

In alternative embodiments, the cancer is a recurrent, metastatic and/or drug-resistant cancer.

In an optional embodiment, the cancer is an advanced solid tumor such as advanced endometrial cancer, triple-negative breast cancer or biliary tract cancer.

In an optional embodiment, the cancer is advanced endometrial cancer for which adequate standard treatment has failed or there is no effective standard treatment; untreated advanced endometrial cancer; advanced triple-negative breast cancer for which adequate standard treatment has failed or there is no effective standard treatment; untreated advanced triple-negative breast cancer; advanced biliary tract cancer for which adequate standard treatment has failed or there is no effective standard treatment; untreated advanced biliary tract cancer.

The present disclosure also provides a pharmaceutical composition comprising the above-mentioned antibody-drug conjugate and immune checkpoint inhibitor, and one or more pharmaceutically acceptable carriers, excipients, and diluents.

In some embodiments, the pharmaceutical composition further comprises the platinum drug, and one or more pharmaceutically acceptable carriers, excipients, and diluents.

The present disclosure also provides a pharmaceutical composition comprising the above-mentioned antibody-drug conjugate and the above-mentioned platinum drug, as well as one or more pharmaceutically acceptable carriers, excipients, and diluents.

The present disclosure also provides a method for treating cancer, comprising administering the above-mentioned antibody-drug conjugate and an immune checkpoint inhibitor in combination to a subject in need thereof, wherein the combined administration can be simultaneous or at different time points.

The present disclosure also provides a method for treating cancer, comprising administering the above-mentioned antibody-drug conjugate, an immune checkpoint inhibitor, and a platinum drug in combination to a subject in need thereof, wherein the combined administration can be simultaneous or at different time points.

The present disclosure also provides a method for treating cancer, comprising administering the above-mentioned antibody-drug conjugate and a platinum drug in combination to a subject in need thereof, wherein the combined administration can be simultaneous or at different time points.

In an optional embodiment, the cancer is selected from at least one of the following: uterine cancer, breast cancer, biliary tract cancer, lung cancer, gastric cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, ovarian cancer, bladder cancer, esophageal cancer, nasopharyngeal cancer, salivary gland cancer, head and neck cancer, skin cancer, pharyngeal cancer, laryngeal cancer, thyroid cancer, vulvar cancer, penile cancer, testicular cancer, urothelial cancer, urethral cancer, colon cancer, rectal cancer, colorectal cancer, esophageal gastric junction cancer, gastrointestinal stromal tumor, squamous cell carcinoma, peritoneal cancer, leukemia, malignant lymphoma, plasma cell neoplasm, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, mesothelioma, Paget's disease and sarcoma.

In an optional embodiment, the breast cancer is selected from triple-negative breast cancer, and the biliary tract cancer is selected from gallbladder cancer and bile duct cancer.

In alternative embodiments, the cancer is an advanced solid tumor for which adequate standard treatment has failed or is intolerant, or for which there is no effective standard treatment.

In alternative embodiments, the cancer is a recurrent, metastatic and/or drug-resistant cancer.

In an optional embodiment, the cancer is an advanced solid tumor such as advanced endometrial cancer, triple-negative breast cancer or biliary tract cancer.

In an optional embodiment, the cancer is advanced endometrial cancer for which adequate standard treatment has failed or there is no effective standard treatment; untreated advanced endometrial cancer; advanced triple-negative breast cancer for which adequate standard treatment has failed or there is no effective standard treatment; untreated advanced triple-negative breast cancer; advanced biliary tract cancer for which adequate standard treatment has failed or there is no effective standard treatment; untreated advanced biliary tract cancer.

Another aspect of the present disclosure provides the aforementioned anti-B7H4 antibody-drug conjugate for use in treating cancer, wherein the anti-B7H4 antibody-drug conjugate is used in combination with the aforementioned anti-PD-1 antibody or an antigen-binding fragment thereof.

Another aspect of the present disclosure provides the aforementioned anti-B7H4 antibody-drug conjugate for use in treating cancer, wherein the anti-B7H4 antibody-drug conjugate is used in combination with the aforementioned anti-PD-L1 antibody or an antigen-binding fragment thereof.

Another aspect of the present disclosure provides the aforementioned anti-B7H4 antibody-drug conjugate for use in treating cancer, wherein the anti-B7H4 antibody-drug conjugate is used in combination with the aforementioned anti-PD-1 antibody or antigen-binding fragment thereof and the aforementioned platinum drug.

Another aspect of the present disclosure provides the aforementioned anti-B7H4 antibody-drug conjugate for use in treating cancer, wherein the anti-B7H4 antibody-drug conjugate is used in combination with the aforementioned anti-PD-L1 antibody or antigen-binding fragment thereof and the aforementioned platinum drug.

Another aspect of the present disclosure provides the aforementioned anti-B7H4 antibody-drug conjugate for use in treating cancer, wherein the anti-B7H4 antibody-drug conjugate is used in combination with the aforementioned platinum drug.

In the present disclosure, the so-called "combination" is a mode of administration, which includes various situations in which two or more drugs are administered sequentially or simultaneously.

Administration by simultaneous administration, independent formulation and co-administration, or independent formulation and sequential administration all fall within the scope of combined administration described herein. "Simultaneously" herein refers to administering at least one dose of an anti-PD-1 or PD-L1 antibody or antigen-binding fragment thereof, and an anti-B7H4 antibody-drug conjugate within a certain time period, optionally within 3 days, 2 days, or 1 day, wherein both or more drugs exhibit pharmacological effects. "Sequential" administration includes administering an anti-PD-1 or PD-L1 antibody or antigen-binding fragment thereof, and an anti-B7H4 antibody-drug conjugate separately within different dosing cycles. The time period may be within one dosing cycle, optionally within 4 weeks, 3 weeks, 2 weeks, or 1 week. This period includes treatments in which the anti-PD-1 or PD-L1 antibody or antigen-binding fragment thereof, and the anti-B7H4 antibody-drug conjugate are administered by the same route of administration or by different routes of administration.

the term

In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

The present disclosure incorporates all the contents of application WO2020244657 into the present application.

The term "antibody drug conjugate" refers to an antibody linked to a biologically active drug via a stable linker. In the present disclosure, "antibody drug conjugate" refers to a monoclonal antibody or antibody fragment linked to a biologically active toxic drug via a stable linker.

The term "antibody" refers to immunoglobulins, which are tetrapeptide chains composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and order of the constant region of immunoglobulins' heavy chains vary, resulting in different antigenicity. Consequently, immunoglobulins can be divided into five classes, or isotypes, namely IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being μ, δ, γ, α, and ε, respectively. Within the same class, Igs are further divided into subclasses based on the amino acid composition of their hinge regions and the number and location of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as either kappa or lambda chains based on differences in their constant regions. Each of the five Ig classes can have either kappa or lambda chains.

The approximately 110 amino acids near the N-terminus of an antibody's heavy and light chains vary greatly in sequence, forming the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable, forming the constant region. The variable region comprises three hypervariable regions (HVRs) and four framework regions (FRs), whose sequences are relatively conserved. These three hypervariable regions determine the antibody's specificity and are also known as complementarity-determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, arranged in the following order from amino to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDR regions of the light chain are LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are HCDR1, HCDR2, and HCDR3.

In the present disclosure, the amino acid sequences of the above CDRs are shown in accordance with the Kabat definition rules. However, it is well known to those skilled in the art that the CDRs of antibodies can be defined in the art by a variety of methods, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loop (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on the variability of antibody sequences (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the International ImMunoGeneTics database (IMGT) (world wide web imgt.cines.fr/), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to cover the complementarity determining region defined by any of the above-mentioned known schemes described by the present invention. Although the scope of protection claimed in the present invention is based on the sequences shown in the Kabat definition rules, amino acid sequences corresponding to the definition rules of other CDRs should also fall within the scope of protection of the present invention.

The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Examples of binding fragments included in "antigen-binding fragments" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab') ₂ fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) a single domain or dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) or (vii) a combination of two or more isolated CDRs, optionally connected by a synthetic linker.

The term "drug loading" refers to the average number of cytotoxic drugs loaded per ligand in a molecule of Formula (I), and can also be expressed as the ratio of the amount of drug to the amount of antibody. The drug loading can range from 0 to 12, preferably 1 to 10, cytotoxic drugs (D) attached per antibody (Pc). In the embodiments of the present disclosure, the drug loading is expressed as n, also known as the DAR value, and exemplary values are 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The average number of drug products per ADC molecule after the conjugation reaction can be determined by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization.

The term "immune checkpoint inhibitor" refers to agents that inhibit the immunosuppressive system to activate tumor immunity.

The term "anti-PD-L1 antibody or antigen-binding fragment thereof" refers to an antibody that specifically binds to PD-L1 (programmed cell death ligand 1; CD274; B7-H1) and has the activity of reducing, inhibiting, and/or interfering with signal transduction caused by the interaction between PD-L1 and PD-1 or B7.1 (CD80) as a binding partner. The anti-PD-L1 antibody used in the present disclosure is not particularly limited as long as its clinical efficacy and safety have been demonstrated.

The term "pharmaceutical composition" is a product comprising one or more active ingredients (e.g., antibodies, ADCs) in optionally specified amounts, as well as any product produced directly or indirectly by combining one or more active ingredients in optionally specified amounts. The different active ingredients in the pharmaceutical composition can be administered independently in separate formulations, including administration simultaneously or at different time points for combined synergistic effect. In the present disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

The term "treating" means administering an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present disclosure, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measurable degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other health care professional to assess the severity or progression of the symptoms. Although the embodiments of the present disclosure (e.g., treatment methods or products) may not be effective in alleviating every symptom of the target disease, they should alleviate the symptoms of the target disease in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: In vitro proliferation inhibitory activity of drug A and drug B combined on OVCAR-3 cells

Figure 2: In vitro proliferation inhibitory activity of drug A and drug C combined on OVCAR-3 cells

Figure 3: In vitro proliferation inhibitory activity of drug A and drug B combined on RL92-5 cells

Figure 4: Effects of Drug A, Drug D, and Drug E alone or in combination on tumor volume in the mouse MC38-hB7H4 homograft tumor model

Figure 5: Effects of Drug A, Drug D, and Drug E alone or in combination on body weight changes in the mouse MC38-hB7H4 homograft tumor model

Figure 6: Effects of Drug A, Drug D, and Drug E alone or in combination on tumor volume in the mouse CT26-hB7H4 homograft tumor model

Figure 7: Effects of Drug A, Drug D, and Drug E alone or in combination on body weight changes in the CT26-hB7H4 homograft tumor model in mice

Figure 8: Effects of drug A, drug E, and drug B alone or in combination on tumor volume in the mouse MC38-hB7H4 homograft tumor model

Figure 9: Effects of Drug A, Drug E, and Drug B alone or in combination on body weight changes in the mouse MC38-hB7H4 homograft tumor model

DETAILED DESCRIPTION

The present application will be explained in more detail below with reference to the embodiments. The embodiments of the present application are only used to illustrate the technical solutions of the present application and are not intended to limit the essence and scope of the present application.

Example 1. Preparation of anti-B7H4 antibody drug conjugates

According to the production method described in WO2020244657, hu2F7 (an anti-B7H4 antibody) and an isotecan analog were used to prepare the anti-B7H4 antibody-drug conjugate shown in the following structure. The average value calculated by the HIC method was: y = 6.1. The hu2F7 heavy chain sequence is shown in SEQ ID NO: 09, and the light chain sequence is shown in SEQ ID NO: 10.

Example 2. Inhibitory Effects of Anti-B7H4 Antibody-Drug Conjugates Combined with Platinum Drugs on Proliferation of Human Ovarian Cancer Cells, Human Breast Cancer Cells, and Human Endometrial Cancer Cells

1. Experimental Materials

1.1 Test drug

1) Drug A: an anti-B7H4 antibody-drug conjugate prepared by the method of Example 1, using physiological saline for drug preparation.

2) Drug B: Cisplatin API, purchased from MCE, product number HY-17394, prepared with double-distilled water.

3) Drug C: Carboplatin API, purchased from MCE, product number HY-17393, prepared with double-distilled water.

1.2 Experimental instruments

Biosafety cabinet (BSC-1300IIA ₂ , Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory); CO ₂ incubator (Thermo311); centrifuge (Eppendorf 5810R); microplate reader (BioTek Synergy H1 or PerkinElmer Envision); pipette (Eppendorf or Rainin)

1.3 Experimental Reagents

OVCAR-3 cells were purchased from ATCC; RL95-2 cells were purchased from Nanjing Kebai; Cell Titer-Glo was purchased from Promega Company with the catalog number G7573; RPMI 1640 was purchased from Gibco with the catalog number 22400-089; DMEM was purchased from Gibco with the catalog number 11995-065; FBS was purchased from Gibco with the catalog number 10091148; PBS was purchased from Gibco with the catalog number 10010023; trypsin was purchased from Gibco with the catalog number 25200056; Insulin-Transferrin-Se was purchased from Gibco with the catalog number 51500-056; and cell culture plates were purchased from thermo company with the catalog number 165306.

2 Experimental methods

When OVCAR-3 and RL95-2 cells were cultured to the appropriate cell density using RPMI1640 medium containing 20% FBS and MEM medium containing 10% FBSD, respectively, the cells were collected and adjusted to the appropriate cell concentration using complete medium. The cell suspension was plated in a 96-well plate with 180 μL per well and placed in a 37°C, 5% _CO2 incubator to adhere overnight.

A solvent control (double-distilled water) or a fixed concentration of drug B or drug C solution was added to a 96-well plate. Then, different concentrations of drug A solution were prepared using culture medium. Drug A was added to the 96-well plate at 10 μL per well of each compound. The plate was cultured in a 37°C, 5% _CO2 incubator for 6 days. CellTiter-Glo solution was then added, the plates were shaken to mix evenly, and incubated in the dark for 10 minutes. The plates were read using a Synergy H1 microplate reader.

In OVCAR-3 cells, drug B was administered at three fixed concentrations: 500 nM, 167 nM, and 56 nM; drug C was administered at three fixed concentrations: 2000 nM, 667 nM, and 222 nM; in RL95-2 cells, drug B was administered at three fixed concentrations: 2000 nM, 667 nM, and 222 nM. In both OVCAR-3 and RL95-2 cells, drug A was administered at a starting concentration of 1000 nM, diluted 1:3, and administered across a total of nine concentration gradients. The specific drug application schedule is shown in Table 1. Six days after combined drug application, the in vitro proliferation inhibitory effects of drug A combined with drug B, or drug A combined with drug C, on human ovarian and endometrial cancer cells were assessed using the CTG assay.

Table 1 Dosage regimens of drug A combined with drug B and drug C

Experimental data processing method:

According to the signal value measured by the microplate reader, the cell inhibition rate was calculated according to the following formula:

Inhibition rate (%) = (1 - (sample well signal value - average blank control well signal value) / (average control well signal value - average blank control well signal value)) × 100%. Sample wells are cell culture wells that receive the test drug combination. Control wells are cell culture wells that receive only vehicle control or fixed concentrations of Drug B and Drug C. Blank control wells are wells that receive only culture medium.

3 Experimental Conclusions

The in vitro proliferation inhibitory activity of drug A alone or in combination with drugs B or C on tumor cells is shown in Table 2-4 and Figure 1-3. The results show that the _IC50 of drug A on tumor cell proliferation decreased when drug A was used in combination with drug B or drug C, indicating that the combination therapy can enhance the growth inhibitory effect of drug A on tumor cells, and the combination of drug A and drug B or drug C has a synergistic effect.

Table 2 Evaluation of the inhibitory effect of drug A and drug B on the growth of OVCAR-3 cells

Table 3 Evaluation of the growth inhibitory effect of drug A and drug C on OVCAR-3 cells

Table 4 Evaluation of the growth inhibitory effect of drug A and drug B on RL95-2 cells

Example 3. Efficacy of anti-B7H4 antibody drug conjugates combined with immune checkpoint inhibitors in the mouse MC38-hB7H4 allograft model

1. Experimental Materials

1.1 Test drug

1) Drug A: anti-B7H4 antibody-drug conjugate, prepared using the method described in Example 1, with normal saline used for drug preparation.

2) Drug D: Anti-Mouse PD-1 Antibody (mPD-1) monoclonal antibody, purchased from MCE, product number HY-P99144, the drug was prepared with normal saline.

3) Drug E: Anti-Mouse PD-L1 Antibody (mPD-L1) monoclonal antibody, purchased from MCE, product number HY-P99145, the drug was prepared with normal saline.

1.2 Mouse MC38-hB7H4 CDX Model Information

MC38-hB7H4 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 5 μg/mL Puromycin at 37°C in a 5% _CO₂ incubator. MC38-hB7H4 cells in the logarithmic growth phase were harvested and counted. Resuspend the cells in PBS to 5 × ^10⁶ cells/mL before plating.

1.3 Experimental animals

C57BL/6J female mice, weighing 17–24 g, were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.

1.4 Experimental Instruments

Table 5. Instruments used in the experiment

1.5 Experimental reagents and consumables

Table 6. Reagents and consumables used in the experiment

2. Experimental Methods

0.1 mL of MC38-hB7H4 cell suspension (containing 5×10 ⁵ cells) was subcutaneously inoculated into the right dorsal region of C57BL/6J mice. Tumor growth was observed and animals were randomly grouped based on tumor volume and body weight. The average tumor volume at the time of grouping was 98 mm ³ . The day of grouping was defined as day 0, or PG-D0. All animals were dosed starting from PG-D0 based on their body weight at grouping. The remaining animals were euthanized at the end of the experiment. Specific dosages and dosing schedules are shown in Table 7. Tumor volume was measured, mice were weighed, and the data were recorded.

Table 7. Dosage regimen and grouping

a. Dosing volume: 10 μl/g of mouse body weight. Stop dosing when the body weight drops by more than 15% and return to normal.
Resume dosing when the dose is less than 10%.

Experimental indicators are used to examine the effect of drugs on tumor growth, specifically relative tumor proliferation rate (T/C) (%) or tumor inhibition rate (TGI) (%). Tumor volume measurement: Tumor volume was measured twice a week using a vernier caliper. The tumor volume was calculated using the formula V = 0.5a × b ² , where a and b represent the length and width of the tumor, respectively.

Calculation of TGI (%): If the tumor did not regress, TGI (%) = [1 - (average tumor volume at the end of dosing for a given treatment group - average tumor volume at the time of grouping for that treatment group) / (average tumor volume at the end of treatment for the vehicle control group - average tumor volume at the time of grouping for the vehicle control group)] × 100%. If the tumor regressed, TGI (%) = [1 - (average tumor volume at the end of dosing for a given treatment group - average tumor volume at the time of grouping for that treatment group) / average tumor volume at the time of grouping for that treatment group] × 100%.

Calculation of T/C (%): T/C (%) = average tumor volume of a treatment group at the end of drug administration / average tumor volume of the vehicle control group at the end of treatment × 100%.

At the end of the experiment (PG-D14), all animals were euthanized by _CO2 asphyxiation in the order of their groups. After euthanasia, the tumor masses were removed, weighed, and photographed.

All data are expressed as mean ± SEM. Based on the tumor volume data of each group at different time points, Dunnett's multiple comparisons test in two-way ANOVA was used to statistically analyze and assess the differences between the groups. Dunnett's multiple comparisons test in one-way ANOVA was used to analyze the differences in tumor volume between the groups. The t-test was used to analyze the differences in tumor volume between the two groups. All data were analyzed using GraphPad Prism 10, and p < 0.05 was considered to be significant.

3. Experimental Results

The results of drug A, drug D and drug E alone or in combination for the MC38-hB7H4 homograft tumor model in mice are shown in Table 8, Figures 4 and 5.

Table 8. Evaluation of the antitumor efficacy of drug A alone or in combination therapy in the MC38-hB7H4 homograft tumor model

a. Mean ± SEM;
b. Comparison with the Vehicle group - Statistical analysis was performed using Dunnett's multiple comparisons test in a two-way ANOVA.

In the MC38-hB7H4 cell line homograft tumor model, the average tumor volume of tumor-bearing mice in the Vehicle group reached 2661 mm ³ on day 14 after administration. Throughout the experiment, no animals discontinued the drug due to weight loss, and no animals became ill or died.

The tumor inhibition effects of each treatment group were compared based on the tumor volume on the 14th day after administration. Statistics showed that tumor-bearing mice were tolerant to drug A monotherapy or combined treatment with drug D and drug E. The anti-tumor efficacy of the drug A_5mg/kg and drug D_10mg/kg combination treatment group was significantly better than that of each single-drug treatment group. The anti-tumor efficacy of the drug A_5mg/kg and drug E_30mg/kg combination treatment group was significantly better than that of the drug A_5mg/kg monotherapy group and better than that of the drug E_30mg/kg monotherapy group.

Example 4. Efficacy of anti-B7H4 antibody drug conjugates combined with immune checkpoint inhibitors in the mouse CT26-hB7H4 homograft model

1. Experimental Materials

1.1 Test drug

1) Drug A: anti-B7H4 antibody-drug conjugate, prepared using the method described in Example 1, with normal saline used for drug preparation.

2) Drug D: Anti-Mouse PD-1 Antibody (mPD-1) monoclonal antibody, purchased from MCE, product number HY-P99144, the drug was prepared with normal saline.

3) Drug E: Anti-Mouse PD-L1 Antibody (mPD-L1) monoclonal antibody, purchased from MCE, product number HY-P99145, the drug was prepared with normal saline.

1.2 Mouse CT26-hB7H4 CDX Model Information

CT26-hB7H4 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 5 μg/mL Puromycin at 37°C in a 5% _CO₂ incubator. CT26-hB7H4 cells in the logarithmic growth phase were harvested and counted. Resuspend the cells in PBS to 7.5 × ^10⁶ cells/mL before plating.

1.3 Experimental animals

Balb/c female mice, weighing 18-24 g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.

1.4 Experimental Instruments

Table 9. Instruments used in the experiment

1.5 Experimental reagents and consumables

Table 10. Reagents and consumables used in the experiment

2. Experimental Methods

Balb/c mice were subcutaneously inoculated with 0.1 mL of CT26-hB7H4 cell suspension (containing 7.5 × 10 ⁵ cells) on the right back. Tumor growth was observed and animals were randomly grouped based on tumor volume and body weight. The average tumor volume at the time of grouping was 115 mm ³ . The day of grouping was defined as day 0, or PG-D0. All animals were dosed starting from PG-D0 according to their body weight at grouping. The remaining animals were euthanized at the end of the experiment. Specific dosage and dosing schedule are shown in Table 11. Tumor volume was measured, mice were weighed, and the data were recorded.

Table 11. Dosage and grouping

b. Dosing volume: 10 μl/g of mouse body weight. Stop dosing when the body weight drops by more than 15% and return to normal.
Resume dosing when the dose is less than 10%.

Experimental indicators are used to examine the effect of drugs on tumor growth, specifically relative tumor proliferation rate (T/C) (%) or tumor inhibition rate (TGI) (%). Tumor volume measurement: Tumor volume was measured twice a week using a vernier caliper. The tumor volume was calculated using the formula V = 0.5a × b ² , where a and b represent the length and width of the tumor, respectively.

Calculation of TGI (%): If the tumor did not regress, TGI (%) = [1 - (average tumor volume at the end of dosing for a given treatment group - average tumor volume at the time of grouping for that treatment group) / (average tumor volume at the end of treatment for the vehicle control group - average tumor volume at the time of grouping for the vehicle control group)] × 100%. If the tumor regressed, TGI (%) = [1 - (average tumor volume at the end of dosing for a given treatment group - average tumor volume at the time of grouping for that treatment group) / average tumor volume at the time of grouping for that treatment group] × 100%.

Calculation of T/C (%): T/C (%) = average tumor volume of a treatment group at the end of drug administration / average tumor volume of the vehicle control group at the end of treatment × 100%.

At the end of the experiment (PG-D14), all animals were euthanized by _CO2 asphyxiation in the order of their groups. After euthanasia, the tumor masses were removed, weighed, and photographed.

All data are expressed as mean ± SEM. Based on the tumor volume data of each group at different time points, Dunnett's multiple comparisons test in two-way ANOVA was used to statistically analyze and assess the differences between the groups. Dunnett's multiple comparisons test in one-way ANOVA was used to analyze the differences in tumor volume between the groups. The t-test was used to analyze the differences in tumor volume between the two groups. All data were analyzed using GraphPad Prism 10, and p < 0.05 was considered to be significant.

3. Experimental Results

The results of drug A, drug B and drug C alone or in combination for the CT26-hB7H4 homograft tumor model in mice are shown in Table 12 and Figures 6-7.

Table 12. Evaluation of the antitumor efficacy of drug A alone or in combination therapy in the CT26-hB7h4 homograft tumor model

a. Mean ± SEM;
b. Comparison with the Vehicle group - Statistical analysis was performed using Dunnett's multiple comparisons test in a two-way ANOVA.

In the CT26-hB7H4 cell line homograft tumor model, the average tumor volume of tumor-bearing mice in the vehicle group reached 2021 mm ³ on day 14 after administration. Compared with the vehicle group, the average tumor volume of each treatment group decreased to varying degrees. Throughout the experiment, no animals discontinued the drug due to weight loss, and no animals became ill or died.

In summary, tumor-bearing mice tolerated drug A alone or in combination with drugs D and E. Compared with the vehicle group, the mean tumor volume decreased to varying degrees in each treatment group, with a statistically significant difference observed in the drug A 5 mg/kg combined with drug D 10 mg/kg group. The antitumor efficacy of the drug A 5 mg/kg plus drug D 10 mg/kg combination group was superior to that of the monotherapy groups, while the antitumor efficacy of the drug A 5 mg/kg plus drug E 30 mg/kg combination group was superior to that of the monotherapy groups.

Example 5. Efficacy of anti-B7H4 antibody-drug conjugates combined with immune checkpoint inhibitors and platinum drugs in the mouse MC38-hB7H4 homograft model

1. Experimental Materials

1.1 Test drug

1) Drug A: anti-B7H4 antibody-drug conjugate, prepared using the method described in Example 1, with normal saline used for drug preparation.

2) Drug E: Anti-Mouse PD-L1 Antibody (mPD-L1) monoclonal antibody, purchased from MCE, product number HY-P99145, the drug was prepared with normal saline.

3) Drug B: Cisplatin API, purchased from MCE, product number HY-17394, ultrapure water was used for drug preparation.

1.2 Experimental instruments

Table 13. Instruments used in the experiment

1.3 Experimental Reagents

Table 14. Reagents and consumables used in the experiment

1.4 Experimental Animals

C57BL/6J female mice, weighing 20-21 g, were purchased from SPIEF (Suzhou) Biotechnology Co., Ltd.

1.5 Mouse MC38-hB7H4 CDX Model Information

MC38-hB7H4 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin (P-streptomycin), and 5 μg/mL puromycin at 37°C in a 5% _CO₂ incubator. MC38-hB7H4 cells in the logarithmic growth phase were harvested and counted. Resuspend the cells in PBS to 5 × ^10⁶ cells/mL before plating.

2 Experimental methods

0.1 mL of MC38-hB7H4 cell suspension (containing 5×10 ⁵ cells) was subcutaneously inoculated into the right dorsal region of C57BL/6J mice. Tumor growth was observed and the mice were randomly grouped based on tumor volume and body weight. The average tumor volume at the time of grouping was 106 mm ³ . The day of grouping was defined as day 0 (PG-D0).

All animals were dosed starting on PG-D0 based on their grouping weight. The remaining animals were euthanized at the end of the experiment. Specific dosages and dosing schedules are shown in Table 15. Tumor volumes were measured, mice were weighed, and data were recorded.

Table 15. Dosage regimen and grouping

a. Dosing volume: 10 μl/g of mouse body weight. Stop dosing when body weight drops by more than 15% and return to 10%.
Resume dosing within 24 hours.

Experimental indicators are used to examine the effect of drugs on tumor growth, specifically relative tumor proliferation rate (T/C) (%) or tumor inhibition rate (TGI) (%). Tumor volume measurement: Tumor volume was measured twice a week using a vernier caliper. The tumor volume was calculated using the formula V = 0.5a × b ² , where a and b represent the length and width of the tumor, respectively.

Calculation of TGI (%): If the tumor did not regress, TGI (%) = [1 - (average tumor volume at the end of dosing for a given treatment group - average tumor volume at the time of grouping for that treatment group) / (average tumor volume at the end of treatment for the vehicle control group - average tumor volume at the time of grouping for the vehicle control group)] × 100%. If the tumor regressed, TGI (%) = [1 - (average tumor volume at the end of dosing for a given treatment group - average tumor volume at the time of grouping for that treatment group) / average tumor volume at the time of grouping for that treatment group] × 100%.

Calculation of T/C (%): T/C (%) = average tumor volume of a treatment group at the end of drug administration / average tumor volume of the vehicle control group at the end of treatment × 100%.

At the end of the experiment (PG-D14), all animals were euthanized by _CO2 asphyxiation in the order of their groups. After euthanasia, the tumor masses were removed, weighed, and photographed.

All data are expressed as mean ± SEM. Based on the tumor volume data of each group at different time points, Dunnett's multiple comparisons test in two-way ANOVA was used to statistically analyze and assess the differences between the groups. Dunnett's multiple comparisons test in one-way ANOVA was used to analyze the differences in tumor volume between the groups. The t-test was used to analyze the differences in tumor volume between the two groups. All data were analyzed using GraphPad Prism 10, and p < 0.05 was considered to be significant.

3 Experimental Conclusions

The results of drug A, drug B and drug C alone or in combination for the MC38-hB7H4 homograft tumor model in mice are shown in Table 16 and Figures 8-9.

Table 16. Evaluation of the antitumor efficacy of drug A alone or in combination therapy in the MC38-hB7H4 homograft tumor model

a. Mean ± SEM;
b. Based on the tumor volume on day 14 after administration, compared with the Vehicle group, Dunnett's
Statistical analysis was performed using multiple comparisons test. In addition, statistical analysis was performed using t-test based on the tumor volume on day 14 after administration.

In the MC38-hB7H4 cell line homograft tumor model, the average tumor volume of tumor-bearing mice in the Vehicle group reached 3297 mm ³ on day 14 after administration. Throughout the experiment, no animals discontinued the drug due to weight loss, and no animals became ill or died.

Comparison of the tumor inhibition effects between the treatment groups based on tumor volume on day 14 after administration showed that the combined treatment group of drug A 5 mg/kg and drug E 30 mg/kg had significantly better tumor inhibition than the drug A 5 mg/kg treatment group and the drug E 30 mg/kg treatment group. The combined treatment group of drug A 5 mg/kg and drug B 5 mg/kg had significantly better tumor inhibition than the drug A 5 mg/kg treatment group and the drug B 5 mg/kg treatment group. The combined treatment group of drug A 5 mg/kg, drug E 30 mg/kg, and drug B 5 mg/kg had significantly better tumor inhibition than each monotherapy group, and significantly better than the combined treatment group of drug A 5 mg/kg and drug E 30 mg/kg, and the combined treatment group of drug A 5 mg/kg and drug B 5 mg/kg.

In summary, tumor-bearing mice tolerated drug A alone or in combination with drug E or drug B. The combination of drug A 5 mg/kg and drug E 30 mg/kg had significantly better tumor inhibition than either the single-drug groups, as did the combination of drug A 5 mg/kg and drug B 5 mg/kg. The triple-drug combination of drug A, drug E, and drug B exhibited significantly better antitumor efficacy than either the single-drug treatment groups, as did the combination of drug A, drug E, or drug A and drug B.

Example 6. Clinical Trial of Anti-B7H4 Antibody Drug Conjugate Combined with Immune Checkpoint Inhibitors ± Platinum Drugs for the Treatment of Advanced Solid Tumors

1. Research Objectives

Main research objectives:

To evaluate the safety and tolerability of anti-B7H4 antibody-drug conjugate combination therapy in subjects with advanced solid tumors.

Secondary study objectives:

1. Evaluate the PK characteristics of anti-B7H4 antibody-drug conjugate combination therapy in subjects with advanced solid tumors;

2. Evaluate the efficacy of anti-B7H4 antibody-drug conjugate combination therapy in subjects with advanced solid tumors;

3. Evaluate the immunogenicity of anti-B7H4 antibody-drug conjugate combination therapy in subjects with advanced solid tumors.

Exploratory research objectives:

1. Explore the relationship between exposure and effect of anti-B7H4 antibody-drug conjugates;

2. Explore biomarkers that predict or influence the efficacy of anti-B7H4 antibody-drug conjugate combination therapy.

2. Name of investigational drug:

(1) Anti-B7H4 Antibody-Drug Conjugates

Dosage form: Sterile powder for injection, Specification: 50 mg/bottle, Manufacturer: Shanghai Hansoh Biopharmaceutical Technology Co., Ltd.

(2) Immune checkpoint inhibitors

Adebelimumab, properties: colorless to light yellow clear liquid, specifications: 600mg (12ml)/bottle, manufacturer: Suzhou Shengdia Biopharmaceutical Co., Ltd.

(3) Platinum drugs

Carboplatin, Appearance: White or off-white freeze-dried loose blocks or powder, Manufacturer: Qilu Pharmaceutical Co., Ltd.

Cisplatin, Appearance: Light yellow-green to light yellow to slightly viscous clear liquid, Manufacturer: Jiangsu Hausen Pharmaceutical Co., Ltd.

3. Target group:

Patients with pathologically confirmed advanced solid tumors, specifically as follows:

(1) Patients with cytologically or histologically confirmed advanced endometrial cancer who have failed adequate standard treatment or have no effective standard treatment. (2) Patients with histologically or cytologically confirmed advanced triple-negative breast cancer based on the most recent biopsy who have failed adequate standard treatment or have no effective standard treatment. (3) Patients with cytologically or histologically confirmed advanced biliary tract cancer who have failed adequate standard treatment or have no effective standard treatment. (4) Patients with cytologically or histologically confirmed recurrent/metastatic/locally advanced inoperable endometrial cancer. (5) Patients with histologically or cytologically confirmed recurrent/metastatic triple-negative breast cancer. (6) Patients with histologically or cytologically confirmed recurrent/metastatic/locally advanced inoperable biliary tract cancer.

4. Dosage regimen:

In this study, every 3 weeks (21 days) was a treatment cycle (C).

The study is designed to have two combination therapy cohorts, each consisting of a dose-finding phase and a dose-expansion phase:

Cohort 1 (1A/1B): Anti-B7H4 antibody-drug conjugate combined with adebelimab

Cohort 2 (2A/2B): Anti-B7H4 antibody-drug conjugate combined with adebelimumab and platinum

The starting dose of the anti-B7H4 antibody-drug conjugate for the dual therapy cohort (i.e., cohort 1) was 4.8 mg/kg, with two pre-defined dose groups (3.8 mg/kg and 4.8 mg/kg). If the starting dose was not tolerated, the dose was reduced to 3.8 mg/kg. The starting dose of the anti-B7H4 antibody-drug conjugate for the triple therapy cohort (i.e., cohort 2) was 3.8 mg/kg, with three pre-defined dose groups (2.8 mg/kg, 3.8 mg/kg, and 4.8 mg/kg). If the starting dose was not tolerated, the dose was reduced to 2.8 mg/kg.

Cohort 1A: Anti-B7H4 antibody-drug conjugate administered every 3 weeks at the entry dose level until disease progression or other discontinuation criteria are met. Adebelimumab 20 mg/kg administered every 3 weeks until disease progression or other discontinuation criteria are met.

Cohort 2A: Anti-B7H4 antibody-drug conjugate every 3 weeks, continuing at the entry dose level until disease progression or other discontinuation criteria are met. Adebelimumab 20 mg/kg every 3 weeks, continuing until disease progression or other discontinuation criteria are met. Cisplatin 75 mg/m² or carboplatin AUC 5 mg/ml/min every 3 weeks is recommended for up to 6 cycles.

Cohort 1B: Anti-B7H4 antibody-drug conjugate every 3 weeks, expanded dose/recommended dose, continued until disease progression or other discontinuation criteria are met. Adebelimumab 20 mg/kg every 3 weeks, continued until disease progression or other discontinuation criteria are met.

Cohort 2B: Anti-B7H4 antibody-drug conjugate every 3 weeks, expanded dose/recommended dose, continued until disease progression or other discontinuation criteria are met. Adebelimumab 20 mg/kg every 3 weeks, continued until disease progression or other discontinuation criteria are met. Cisplatin or carboplatin, select the corresponding recommended dose and number of cycles according to the indication (see Table 17)

Table 17. Recommended platinum doses and cycles for each indication during the dose expansion phase

5. Study endpoints:

Primary study endpoint:

The maximum tolerated dose (MTD) or maximum applicable dose (MAD) of anti-B7H4 antibody-drug conjugate combination therapy.

Secondary study endpoints:

1. Safety of anti-B7H4 antibody-drug conjugate combination therapy: incidence of adverse events (AEs); incidence of serious adverse events (SAEs); proportion of subjects with dose adjustments and treatment discontinuations due to AEs; changes in physical examination, ophthalmological examination, vital signs, body weight, laboratory tests (blood count, urine count, blood biochemistry, coagulation function), ECG, echocardiogram, and ECOGPs;

2. PK characteristics of anti-B7H4 antibody-drug conjugate combination therapy;

3. Effectiveness of anti-B7H4 antibody-drug conjugate combination therapy: Investigator-assessed objective response rate (ORR), disease control rate (DCR), duration of response (DoR), and progression-free survival (PFS) according to RECIST v1.1 criteria; overall survival (OS);

4. Immunogenicity of anti-B7H4 antibody-drug conjugates: anti-drug antibody (ADA) detection rate and ADA titer.

Exploratory study endpoints:

1. The relationship between exposure and effect of anti-B7H4 antibody-drug conjugates;

2. Explore biomarkers that predict or influence the efficacy of anti-B7H4 antibody-drug conjugate combination therapy.

6. Research results:

As of November 20, 2024, a total of 23 patients received anti-B7H4 antibody-drug conjugate (4.8 mg/kg, every 3 weeks) combined with adebelimumab (20 mg/kg, every 3 weeks). Among them, 11 patients (47.8%) had breast cancer, 6 patients (26.1%) had endometrial cancer, 5 patients (21.7%) had biliary tract cancer, and 1 patient (4.3%) had other solid tumors. The number of previous lines of anti-cancer drug therapy was: first-line therapy for 8 patients, second-line therapy for 7 patients, third-line therapy for 4 patients, and fourth-line therapy or higher for 3 patients, with a median number of lines of treatment (range) of 2.0 (1.0, 3.0).

In 12 evaluable subjects (6 with advanced endometrial cancer, 3 with advanced cholangiocarcinoma, and 3 with advanced triple-negative breast cancer), the drug demonstrated a clear anti-tumor effect. Efficacy data are shown in Table 18. A total of 3 PRs, 6 SDs, 2 PDs, and 1 NE were achieved, resulting in an ORR of 25% and a DCR of 75%. Efficacy data for advanced endometrial cancer and advanced triple-negative breast cancer are shown in Tables 19 and 20, respectively.

Table 18. Efficacy data of 12 subjects who could be evaluated for efficacy

Table 19. Efficacy data for advanced endometrial cancer

Table 20. Efficacy data for advanced triple-negative breast cancer

As of November 20, 2024, among 23 patients treated with an anti-B7H4 antibody-drug conjugate (4.8 mg/kg, Q3W) combined with adebelimumab (20 mg/kg, Q3W), there were 7 (30.4%) adverse events with CTCAE ≥ Grade 3 related to the study drug and 5 (21.7%) adverse events with CTCAE ≥ Grade 3 related to adebelimumab. There were 4 (17.4%) serious adverse events related to the study drug and 3 (13.0%) serious adverse events related to adebelimumab. There were no adverse events leading to interruption of dosing, permanent discontinuation of treatment, or death.

In summary, the anti-B7H4 antibody-drug conjugate (4.8 mg/kg, Q3W) combined with adebelimumab (20 mg/kg, Q3W) has definite and superior anti-tumor efficacy in advanced endometrial cancer, advanced triple-negative breast cancer, and advanced cholangiocarcinoma, with predictable and manageable safety events.

Claims (27)

The use of an antibody drug conjugate and an immune checkpoint inhibitor in combination in the preparation of a drug for treating cancer, wherein the structure of the antibody drug conjugate is shown in formula (I):
in: n is 1 to 10, preferably 2 to 8, more preferably 3 to 8, and n is a decimal or an integer; Pc is an anti-B7H4 antibody or an antigen-binding fragment thereof. The use according to claim 1, wherein the anti-B7H4 antibody or its antigen-binding fragment comprises: heavy chain HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 01, 02 and 03 amino acid sequences, respectively, and light chain LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 04, 05 and 06 amino acid sequences, respectively. The use according to claim 1 or 2, wherein the anti-B7H4 antibody or antigen-binding fragment thereof is selected from a humanized antibody or a fragment thereof. According to the use according to claim 3, the anti-B7H4 antibody or its antigen-binding fragment comprises a heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 isotype, and a light chain constant region comprising κ or λ; preferably, the anti-B7H4 antibody or its antigen-binding fragment comprises a heavy chain constant region of IgG1 or IgG4 isotype. The use according to claim 3, wherein the heavy chain variable region sequence of the anti-B7H4 antibody or its antigen-binding fragment is such as the sequence shown in SEQ ID NO: 07 or its variant, and the light chain variable region sequence is such as the sequence shown in SEQ ID NO: 08 or its variant. The use according to any one of claims 1 to 5, wherein the heavy chain sequence of the anti-B7H4 antibody or its antigen-binding fragment is such as the sequence shown in SEQ ID NO: 09 or a variant thereof, and the light chain sequence is such as the sequence shown in SEQ ID NO: 10 or a variant thereof. The use according to any one of claims 1 to 6, wherein the immune checkpoint inhibitor is selected from antibodies or antigen-binding fragments thereof targeting PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, BTLA, A2aR, B7-H3, B7-H4, preferably antibodies or antigen-binding fragments thereof targeting PD-1 or PD-L1. The method according to any one of claims 1 to 7, wherein the immune checkpoint inhibitor is selected from Adebrelimab, Camrelizumab, Dostarlimab, Toripalimab, Sintilimab, Tislelizumab, Zimberelimab, Penpulimab, Serplulimab, Pucotenlimab, Pembrolizumab, and Glutathione. zumab), Nivolumab, Sugemalimab, Envafolimab, Atezolizumab, Durvalumab, Ipilimumab, Candonilimab, preferably Adebrelimab, Camrelizumab, Dostarlimab, Pembrolizumab, Durvalumab. The use according to any one of claims 1 to 8, further combined with a platinum drug. Use of the antibody-drug conjugate according to any one of claims 1 to 8 in combination with a platinum drug in the preparation of a medicament for treating cancer. The use according to claim 9 or 10, wherein the platinum drug is selected from: carboplatin, cisplatin, oxaliplatin, nedaplatin, lobaplatin, satraplatin, cycloplatin, miboplatin, enloplatin, iproplatin, dicycloplatin, preferably carboplatin and/or cisplatin. The use according to any one of claims 1 to 9, wherein the antibody drug conjugate and the immune checkpoint inhibitor are contained in different preparations as active ingredients and are administered simultaneously or at different times. The use according to claim 9, wherein the antibody-drug conjugate, the immune checkpoint inhibitor, and the platinum drug are respectively contained in different preparations as active ingredients and are administered simultaneously or at different times. The use according to claim 10, wherein the antibody-drug conjugate and the platinum drug are contained in different preparations as active ingredients and are administered simultaneously or at different times. The use according to any one of claims 9 to 11, 13 to 14, wherein the dosage of the platinum drug is calculated as the area under the curve (AUC) and is 1 to 10 mg/ml/min, preferably 3 mg/ml/min, 4 mg/ml/min, 5 mg/ml/min, 6 mg/ml/min, 7 mg/ml/min, 8 mg/ml/min, and the administration frequency is once a week, once every two weeks, once every three weeks or once every four weeks. The use according to any one of claims 9 to 11, 13 to 14, wherein the dosage of the platinum drug is 10 mg/m ² to 500 mg/m ² , preferably 10 mg/m ² to 200 mg/m ² , more preferably 25 mg/m ² , 50 mg/m ² , 75 mg/m ² , 100 mg/m ² , 125 mg/m ² , 150 mg/m ² , 175 mg/m ² or 200 mg/m ² , and the administration frequency is once a week, once every two weeks, once every three weeks or once every four weeks. The use according to any one of claims 1 to 9, 12 to 13, wherein the dose of the immune checkpoint inhibitor is 1.0 mg/kg to 100 mg/kg, preferably 1.0 mg/kg to 40 mg/kg, more preferably 1.0 mg/kg to 30 mg/kg, and the administration frequency is once a week, once every two weeks, once every three weeks or once every four weeks. The use according to any one of claims 1 to 17, wherein the dose of the antibody drug conjugate is 0.1 mg/kg to 12.0 mg/kg, preferably 1.0 mg/kg to 12.0 mg/kg, and the administration frequency is once a week, once every two weeks, once every three weeks or once every four weeks. The method according to any one of claims 1 to 18, wherein the cancer is selected from at least one of the following: uterine cancer, breast cancer, biliary tract cancer, lung cancer, gastric cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, ovarian cancer, bladder cancer, esophageal cancer, nasopharyngeal cancer, salivary gland cancer, head and neck cancer, skin cancer, pharyngeal cancer, laryngeal cancer, thyroid cancer, vulvar cancer, penile cancer, testicular cancer, urothelial cancer, urethral cancer, colon cancer, rectal cancer, colorectal cancer, esophageal gastric junction cancer, gastrointestinal stromal tumor, squamous cell carcinoma, peritoneal cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, mesothelioma, Paget's disease and sarcoma. The use according to claim 19, wherein the uterine cancer is selected from endometrial cancer, the breast cancer is selected from triple-negative breast cancer, and the biliary tract cancer is selected from gallbladder cancer and bile duct cancer. The use according to any one of claims 1 to 18, wherein the cancer is an advanced solid tumor for which adequate standard treatment has failed or is intolerant, or for which there is no effective standard treatment. A pharmaceutical composition comprising an antibody-drug conjugate as defined in any one of claims 1 to 9 and an immune checkpoint inhibitor, and one or more pharmaceutically acceptable carriers, excipients, and diluents. A method for treating cancer, comprising administering to a subject in need thereof a combination of an antibody drug conjugate as defined in any one of claims 1 to 21, an immune checkpoint inhibitor as defined in any one of claims 1 to 9 and 17, and optionally a platinum drug as defined in any one of claims 9 to 11 and 15 to 16, wherein the combined administration may be simultaneous or at different time points. A method for treating cancer, comprising administering to a subject in need thereof an antibody-drug conjugate as defined in any one of claims 1 to 21 and a platinum drug as defined in any one of claims 9 to 11, 15 to 16, wherein the combined administration may be simultaneous or at different time points. The method of claim 23 or 24, wherein the cancer is selected from at least one of the following: uterine cancer, breast cancer, biliary tract cancer, lung cancer, stomach cancer, liver cancer, kidney cancer, pancreatic cancer, prostate cancer, ovarian cancer, bladder cancer, esophageal cancer, nasopharyngeal cancer, salivary gland cancer, head and neck cancer, skin cancer, pharyngeal cancer, laryngeal cancer, thyroid cancer, vulvar cancer, penile cancer, testicular cancer, urothelial cancer, urethral cancer, colon cancer, rectal cancer, colorectal cancer, esophageal gastric junction cancer, gastrointestinal stromal tumor, squamous cell carcinoma, peritoneal cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, neuroepithelial tissue tumor, nerve sheath tumor, mesothelioma, Paget's disease and sarcoma. The method according to claim 25, wherein the uterine cancer is selected from endometrial cancer, the breast cancer is selected from triple-negative breast cancer, and the biliary tract cancer is selected from gallbladder cancer and bile duct cancer. The method according to claim 23 or 24, wherein the cancer is an advanced solid tumor for which adequate standard treatment has failed or is intolerant, or for which there is no effective standard treatment.