Methods and compositions for the treatment of melanoma

Abstract

Disclosed herein are methods for treating melanoma, such as stage III / IV cutaneous or mucosal melanoma, in a subject in need thereof, comprising administering a therapeutically effective amount of denosumab to the subject, and optionally co-administering a therapeutically effective amount of a PD-1 inhibitor to the subject; uses of denosumab or a PD-1 inhibitor in the manufacture of a medicament adapted for use in the methods described herein; and pharmaceutical compositions comprising denosumab and / or a PD-1 inhibitor for use in the methods described herein.

Description

[Technical Field]

[0001] The present disclosure relates to materials and methods for treating melanoma in a subject in need thereof.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 409,348, filed September 23, 2022, which is incorporated herein by reference in its entirety.

[0003] Incorporation by Reference of Electronically Submitted Materials This application contains a Sequence Listing in computer-readable form (Filename: 58296_SeqListing.xml; Size: 33,764 bytes; Created September 22, 2023), which is incorporated by reference in its entirety as a separate part of this disclosure. [Background technology]

[0004] Melanoma is a type of skin cancer characterized by malignant transformation of pigment-producing cells known as melanocytes. Melanoma typically develops in the skin, but can also develop in mucous membranes (nose, vagina, rectum) or the eye. While rarer than other types of skin cancer, such as squamous cell carcinoma and basal cell carcinoma, melanoma is more likely to metastasize and disproportionately contributes to skin cancer deaths. Specifically, melanoma accounts for less than 5% of skin cancer cases but is responsible for approximately 80% of skin cancer-related deaths. While the average age of patients diagnosed with melanoma is 63, melanoma is also a common cancer in young adults.

[0005] The American Cancer Society estimated that 99,780 new cases of cutaneous melanoma (CM) and 7,650 melanoma-related deaths will occur in the United States in 2022 (Siegel RL, Miller KD, Fuchs HE, Jemal A: Cancer Statistics, 2022. CA Cancer J Clin 72:7-33, 2022). Patients diagnosed with locally advanced and / or distant metastatic melanoma have a sharp decline in overall survival (OS), so early detection and treatment significantly impact long-term prognosis. The 5-year OS rate is approximately 28%-30% for patients diagnosed with stage IV melanoma compared with patients with early-stage tumors (stages IA-IIB), which exhibit 5-year survival rates ranging from 87%-99% (Siegel RL, Miller KD, Fuchs HE, Jemal A: Cancer Statistics, 2022. CA Cancer J Clin 72:7-33, 2022). Unfortunately, some metastatic melanomas remain difficult to treat, despite the introduction of novel therapies for this disease over the past decade, and surgical resection is generally not a curative treatment option. Therefore, there is a need in the art for alternative methods of treating locally advanced (i.e., stage III) and / or distantly metastatic (i.e., stage IV) melanoma. Summary of the Invention [Means for solving the problem]

[0006] Provided herein are methods for treating melanoma, e.g., stage III / IV cutaneous or mucosal melanoma, in a subject in need thereof, comprising administering a therapeutically effective amount of denosumab to the subject, and optionally co-administering a therapeutically effective amount of a PD-1 inhibitor to the subject. Also provided herein are uses of denosumab or a PD-1 inhibitor in the manufacture of a medicament adapted for use in the methods described herein, and pharmaceutical compositions comprising denosumab and / or a PD-1 inhibitor for use in the methods described herein.

[0007] Disclosed herein is a method of treating melanoma in a subject in need thereof, comprising administering a therapeutically effective amount of denosumab to the subject, wherein the subject does not have bone metastases; and the subject does not have hypercalcemia.

[0008] Denosumab is a human IgG2 monoclonal antibody that binds to soluble RANKL and is indicated for the treatment or prevention of skeletal complications resulting from bone metastasis of any solid tumor malignancy. RANKL increases thymic tolerance by upregulating the transcription factor AIRE, an autoimmune regulator, in the thymus. In syngeneic melanoma-bearing mice, RANKL blockade increases anti-melanoma immunity by rescuing melanoma-specific T cells from thymic deletion and increases anti-tumor responses when combined with CTLA4±PD-1 blockade (Bakhru, JCI Insight 2(18):e93265, 2017).

[0009] In some embodiments, a baseline tumor tissue sample from a subject contains active tumor-infiltrating lymphocytes (TILs). TILs can be detected by various methods, including ematoxylin and eosin (HE)-stained tumor sections. In some embodiments, a baseline peripheral blood sample from a subject contains high serum-free RANKL and / or low serum osteoprotegerin (OPG) levels as assessed by flow cytometry analysis. In some embodiments, a baseline tumor tissue sample from a subject expresses two or more of Sox10, RANK, and OPG as assessed by immunohistochemistry.

[0010] In some embodiments, the subject has not previously received a PD-1 inhibitor, hi some embodiments, the subject has previously received a PD-1 inhibitor for stage III or stage IV (stage III / IV) melanoma, and the interval between the last dose of PD-1 inhibitor and the date of recurrence is at least about 1 year.

[0011] Programmed cell death ligand 1 (PD-L1) is expressed in approximately 40–50% of melanomas, with limited expression in most visceral organs except for airway epithelium and placental tissue (Johnson et al., Nivolumab in melanoma: latest evidence and clinical potential. Therapeutic Advances in Medical Oncology 7:97–106, 2015). Pembrolizumab is an IgG4κ immunoglobulin that blocks the interaction between PD-1 and PD-L1, thereby reducing T cell proliferation and cytokine production. Pembrolizumab has been approved by the United States Food and Drug Administration (FDA) for the initial treatment of unresectable stage III or distant metastatic melanoma (AJCC stage III / IV) (Robert et al., Pembrolizumab versus ipilimumab in advanced melanoma. N Engl J Med 372:2521–32, 2015). Nivolumab is an immunoglobulin IgG4 fully humanized monoclonal antibody that directly blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2. This blockade enhances the functional activity of targeted lymphocytes, promoting an antitumor immune response and leading to immune-mediated tumor regression. The FDA approved nivolumab as adjuvant treatment for patients with completely resected melanoma with lymph node involvement or metastatic disease based on findings from the phase III CheckMate-238 trial.

[0012] In some embodiments, the subject is administered one or more (e.g., 1, 2, 3, 4) loading doses of denosumab. In some embodiments, the subject is administered one loading dose of denosumab. In some embodiments, the subject is administered two loading doses of denosumab.

[0013] In some embodiments, one or more loading doses of denosumab each independently comprise one or more unit doses, each unit dose independently comprising between about 45 mg and about 120 mg of denosumab.

[0014] In some embodiments, the one or more loading doses of denosumab correspond to a first loading dose comprising about 120 mg of denosumab administered on day 1; and a second loading dose comprising about 120 mg of denosumab administered on about day 8.

[0015] In some embodiments, the one or more loading doses of denosumab include a first loading dose comprising about 120 mg of denosumab administered on day 1; and a second loading dose comprising about 120 mg of denosumab administered on about day 8.

[0016] In some embodiments, the therapeutically effective amount of denosumab comprises one or more unit doses each administered at intervals of about 4 weeks, each unit dose independently comprising from about 45 mg to about 120 mg of denosumab. In some embodiments, the therapeutically effective amount of denosumab corresponds to about 120 mg of denosumab administered once every about 4 weeks. In some embodiments, the therapeutically effective amount of denosumab is about 120 mg of denosumab administered in one or more unit doses at intervals of about 4 weeks. In some embodiments, the therapeutically effective amount of denosumab is about 120 mg of denosumab administered in one unit dose at intervals of about 4 weeks.

[0017] In some embodiments, the administration is associated with an anti-tumor immune response and / or a tumor objective response. In some embodiments, the administration is associated with an anti-tumor immune response, representative examples of which are provided below. In some embodiments, the administration is associated with a tumor objective response, representative examples of which are provided below. In some embodiments, the administration is associated with an anti-tumor immune response and a tumor objective response.

[0018] In some embodiments, the anti-tumor immune response is selected from the following: a change (e.g., an increase) in recent thymic emigrant cells (RTE) in a peripheral blood sample from the subject compared to a baseline peripheral blood sample; a change (e.g., an increase) in the density of tumor-infiltrating differentiation group (CD8+) cells (TILs) in a tumor tissue sample from the subject compared to a baseline tumor tissue sample; an increase in the number of tumor-infiltrating CD8+ cells in a tumor tissue sample from the subject compared to a baseline tumor tissue sample, as assessed by immunohistochemistry; an increase in tumor cell death in a tumor tissue sample from the subject compared to a baseline tumor tissue sample, as assessed by immunohistochemistry; CD8+ and CD4+ non-T in peripheral blood samples from subjects compared to baseline peripheral blood samples as assessed by flow cytometry analysis reg Increase in the total number of RTEs; CD4+ T in peripheral blood samples from subjects compared to baseline peripheral blood samples as assessed by flow cytometry analysis reg an increase in the total number of cells; an increase in immune cell clonal diversity in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis; RANK+T in peripheral blood samples from subjects compared to baseline peripheral blood samples as assessed by flow cytometry analysis reg an increase in the number of cells; at least partial suppression of myeloid-derived suppressor cells (MDSCs) in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis; A reduction in the number of tumor-associated macrophages in a tumor tissue sample from the subject compared to a baseline tumor tissue sample; and A decrease in the number of tumor-infiltrating MDSCs in a tumor tissue sample from the subject compared to a baseline tumor tissue sample. Any one or more of these parameters are possible.

[0019] In some embodiments, the anti-tumor immune response is an increase in recent thymic emigrant cells (RTE) in a peripheral blood sample from the subject compared to a baseline peripheral blood sample. In some embodiments, the anti-tumor immune response is a change (e.g., an increase) in the density of tumor-infiltrating cluster of differentiation (CD8+) cells (TIL) in a tumor tissue sample from the subject compared to the baseline tumor tissue sample. In some embodiments, the anti-tumor immune response is an increase in the number of tumor-infiltrating CD8+ cells in a tumor tissue sample from the subject compared to the baseline tumor tissue sample, as assessed by immunohistochemistry. In some embodiments, the anti-tumor immune response is an increase in tumor cell death in a tumor tissue sample from the subject compared to the baseline tumor tissue sample, as assessed by immunohistochemistry. In some embodiments, the anti-tumor immune response is an increase in the total number of CD8+ and CD4+ non-Treg RTE in a peripheral blood sample from the subject compared to the baseline peripheral blood sample, as assessed by flow cytometry analysis. In some embodiments, the anti-tumor immune response is an increase in the total number of CD4+ Treg cells in a peripheral blood sample from the subject compared to the baseline peripheral blood sample, as assessed by flow cytometry analysis. In some embodiments, the anti-tumor immune response is an increase in immune cell clonal diversity in a peripheral blood sample from the subject compared to the baseline peripheral blood sample, as assessed by flow cytometry analysis. In some embodiments, the anti-tumor immune response is an increase in the number of RANK+ Treg cells in a peripheral blood sample from the subject compared to the baseline peripheral blood sample, as assessed by flow cytometry analysis. In some embodiments, the anti-tumor immune response is at least partial suppression of myeloid-derived suppressor cells (MDSCs) in a peripheral blood sample from the subject compared to the baseline peripheral blood sample, as assessed by flow cytometry analysis. In some embodiments, the anti-tumor immune response is a decrease in the number of tumor-associated macrophages in a tumor tissue sample from the subject compared to the baseline tumor tissue sample. In some embodiments, the anti-tumor immune response is a decrease in the number of tumor-infiltrating MDSCs in a tumor tissue sample from the subject compared to the baseline tumor tissue sample.

[0020] In some embodiments, the tumor objective response is a complete response or a partial response as assessed using RECIST v1.1 criteria. In some embodiments, the tumor objective response is a complete response as assessed using RECIST v1.1 criteria. In some embodiments, the tumor objective response is a partial response as assessed using RECIST v1.1 criteria.

[0021] In some embodiments, the melanoma is cutaneous melanoma or mucosal melanoma. In some embodiments, the melanoma is cutaneous melanoma. In some embodiments, the melanoma is mucosal melanoma.

[0022] In some embodiments, the melanoma is stage III melanoma or stage IV melanoma. In some embodiments, the melanoma is stage III melanoma. In some embodiments, the melanoma is stage IV melanoma. In some embodiments, the melanoma is American Joint Committee on Cancer (AJCC) Stage III melanoma or AJCC Stage IV melanoma. In some embodiments, the melanoma is AJCC Stage III melanoma. In some embodiments, the melanoma is AJCC Stage IV melanoma. In some embodiments, the melanoma is unresectable. In some embodiments, the melanoma is resectable Stage III melanoma.

[0023] In some embodiments, the subject is not concomitantly administered an anti-CTLA4 agent. In some embodiments, the subject is not concomitantly administered ipilimumab.

[0024] In some embodiments, denosumab is administered to the subject by injection. In some embodiments, denosumab is administered to the subject by subcutaneous injection. In some embodiments, denosumab is administered to the subject by injection into the upper arm, upper thigh, or abdomen of the subject. In some embodiments, denosumab is administered to the subject by subcutaneous injection into the upper arm, upper thigh, or abdomen of the subject.

[0025] In some embodiments, the subject does not experience dose-limiting toxicity (DLT) while receiving denosumab. In some embodiments, the subject does not experience any Grade 3 or Grade 4 adverse events related to denosumab while receiving denosumab. In some embodiments, the subject is not hypocalcemia while receiving denosumab.

[0026] In some embodiments, the subject is administered denosumab for at least 12 weeks. In some embodiments, the subject is administered denosumab for at least 24 weeks. In some embodiments, the subject is administered denosumab for at least 48 weeks. In some embodiments, the subject is administered denosumab for at most 1 year.

[0027] In some embodiments, the subject is co-administered a PD-1 inhibitor. In some embodiments, the first dose of the PD-1 inhibitor is administered within about 24 hours of a third loading dose comprising about 120 mg of denosumab administered on about day 22. In some embodiments, the first dose of the PD-1 inhibitor and the combined dose of denosumab are co-administered on about day 22. In some embodiments, the combined dose of denosumab is about 120 mg of denosumab.

[0028] In some embodiments, the PD-1 inhibitor is selected from pembrolizumab, nivolumab, cemiplimab, dostallimab, atezolizumab, avelumab, and durvalumab. In some embodiments, the PD-1 inhibitor is selected from pembrolizumab and nivolumab. In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, the PD-1 inhibitor is nivolumab.

[0029] In some embodiments, when the PD-1 inhibitor and denosumab are each administered to a subject on the same day, the PD-1 inhibitor is administered to the subject before administration of denosumab.

[0030] Also disclosed herein is a method of treating melanoma in a subject in need thereof, comprising administering a therapeutically effective amount of denosumab to the subject, wherein a baseline tumor tissue sample from the subject comprises active tumor-infiltrating lymphocytes (TILs).

[0031] In some embodiments, a baseline peripheral blood sample from the subject comprises high serum-free RANKL and / or low serum OPG levels as assessed by flow cytometry analysis, hi some embodiments, a baseline tumor tissue sample from the subject expresses two or more of Sox10, RANK, and OPG as assessed by immunohistochemistry.

[0032] In some embodiments, the subject does not have bone metastases and / or the subject does not have hypercalcemia. In some embodiments, the subject does not have bone metastases. In some embodiments, the subject does not have hypercalcemia.

[0033] In some embodiments, the subject has not previously received a PD-1 inhibitor, hi some embodiments, the subject has previously received a PD-1 inhibitor for stage III or stage IV melanoma, and the interval between the last dose of PD-1 inhibitor and the date of recurrence is at least about 1 year.

[0034] In some embodiments, the subject is administered one or more (e.g., 1, 2, 3, 4) loading doses of denosumab. In some embodiments, the subject is administered one loading dose of denosumab. In some embodiments, the subject is administered two loading doses of denosumab.

[0035] In some embodiments, one or more loading doses of denosumab each independently comprise one or more unit doses, each unit dose independently comprising between about 45 mg and about 120 mg of denosumab.

[0036] In some embodiments, the one or more loading doses of denosumab are a first loading dose comprising about 120 mg of denosumab administered on day 1; and This corresponds to a second loading dose containing about 120 mg of denosumab administered on about day 8.

[0037] In some embodiments, the one or more loading doses of denosumab are a first loading dose comprising about 120 mg of denosumab administered on day 1; and and a second loading dose comprising about 120 mg of denosumab administered on about day 8.

[0038] In some embodiments, the first dose of the PD-1 inhibitor is administered within about 24 hours of a third loading dose comprising about 120 mg of denosumab administered on about day 22.

[0039] In some embodiments, the first dose of the PD-1 inhibitor and the combined dose of denosumab are co-administered on about day 22. In some embodiments, the combined dose of denosumab is about 120 mg of denosumab.

[0040] In some embodiments, the therapeutically effective amount of denosumab comprises one or more unit doses each administered at intervals of about 4 weeks, each unit dose independently comprising from about 45 mg to about 120 mg of denosumab. In some embodiments, the therapeutically effective amount of denosumab corresponds to about 120 mg of denosumab administered once every about 4 weeks. In some embodiments, the therapeutically effective amount of denosumab is about 120 mg of denosumab administered in one or more unit doses at intervals of about 4 weeks. In some embodiments, the therapeutically effective amount of denosumab is about 120 mg of denosumab administered in one unit dose at intervals of about 4 weeks.

[0041] In some embodiments, the administration is associated with an anti-tumor immune response and / or a tumor objective response. In some embodiments, the administration is associated with an anti-tumor immune response, representative examples of which are described below. In some embodiments, the administration is associated with a tumor objective response, representative examples of which are described below. In some embodiments, the administration is associated with an anti-tumor immune response and a tumor objective response.

[0042] In some embodiments, the anti-tumor immune response is selected from the following: a change (e.g., an increase) in recent thymic emigrant cells (RTE) in a peripheral blood sample from the subject compared to a baseline peripheral blood sample; a change (e.g., an increase) in the density of tumor-infiltrating differentiation group (CD8+) cells (TILs) in a tumor tissue sample from the subject compared to a baseline tumor tissue sample; an increase in the number of tumor-infiltrating CD8+ cells in a tumor tissue sample from the subject compared to a baseline tumor tissue sample, as assessed by immunohistochemistry; an increase in tumor cell death in a tumor tissue sample from the subject compared to a baseline tumor tissue sample, as assessed by immunohistochemistry; CD8+ and CD4+ non-T in peripheral blood samples from subjects compared to baseline peripheral blood samples as assessed by flow cytometry analysis reg Increase in the total number of RTEs; CD4+ T in peripheral blood samples from subjects compared to baseline peripheral blood samples as assessed by flow cytometry analysis reg an increase in the total number of cells; an increase in immune cell clonal diversity in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis; RANK+T in peripheral blood samples from subjects compared to baseline peripheral blood samples as assessed by flow cytometry analysis reg an increase in the number of cells; at least partial suppression of myeloid-derived suppressor cells (MDSCs) in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis; A reduction in the number of tumor-associated macrophages in a tumor tissue sample from the subject compared to a baseline tumor tissue sample; and A decrease in the number of tumor-infiltrating MDSCs in a tumor tissue sample from the subject compared to a baseline tumor tissue sample. Any one or more of these parameters are possible.

[0043] In some embodiments, the anti-tumor immune response is an increase in recent thymic emigrant cells (RTE) in a peripheral blood sample from the subject compared to a baseline peripheral blood sample. In some embodiments, the anti-tumor immune response is a change (e.g., an increase) in the density of tumor-infiltrating cluster of differentiation (CD8+) cells (TIL) in a tumor tissue sample from the subject compared to a baseline tumor tissue sample. In some embodiments, the anti-tumor immune response is an increase in the number of tumor-infiltrating CD8+ cells in a tumor tissue sample from the subject compared to a baseline tumor tissue sample, as assessed by immunohistochemistry. In some embodiments, the anti-tumor immune response is an increase in tumor cell death in a tumor tissue sample from the subject compared to a baseline tumor tissue sample, as assessed by immunohistochemistry. In some embodiments, the anti-tumor immune response is an increase in the number of CD8+ and CD4+ non-T cells in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis. reg In some embodiments, the anti-tumor immune response is an increase in the total number of CD4+ T cells in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis. reg In some embodiments, the anti-tumor immune response is an increase in immune cell clonal diversity in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis. In some embodiments, the anti-tumor immune response is an increase in RANK+T cells in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis. regIn some embodiments, the anti-tumor immune response is an increase in the number of myeloid-derived suppressor cells (MDSCs) in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis. In some embodiments, the anti-tumor immune response is a decrease in the number of tumor-associated macrophages in a tumor tissue sample from the subject compared to a baseline tumor tissue sample. In some embodiments, the anti-tumor immune response is a decrease in the number of tumor-infiltrating MDSCs in a tumor tissue sample from the subject compared to a baseline tumor tissue sample.

[0044] In some embodiments, the tumor objective response is a complete response (CR) or a partial response (PR) as assessed using RECIST v1.1 criteria. In some embodiments, the tumor objective response is a complete response as assessed using RECIST v1.1 criteria. In some embodiments, the tumor objective response is a partial response as assessed using RECIST v1.1 criteria.

[0045] In some embodiments, the melanoma is cutaneous melanoma (CM) or mucosal melanoma (MM). In some embodiments, the melanoma is cutaneous melanoma. In some embodiments, the melanoma is mucosal melanoma.

[0046] In some embodiments, the melanoma is stage III melanoma or stage IV melanoma. In some embodiments, the melanoma is stage III melanoma. In some embodiments, the melanoma is stage IV melanoma. In some embodiments, the melanoma is American Joint Committee on Cancer (AJCC) Stage III melanoma or AJCC Stage IV melanoma. In some embodiments, the melanoma is AJCC Stage III melanoma. In some embodiments, the melanoma is AJCC Stage IV melanoma. In some embodiments, the melanoma is unresectable. In some embodiments, the melanoma is resectable Stage III melanoma.

[0047] In some embodiments, the subject is not concomitantly administered an anti-CTLA4 agent. In some embodiments, the subject is not concomitantly administered ipilimumab.

[0048] In some embodiments, denosumab is administered to the subject by injection. In some embodiments, denosumab is administered to the subject by subcutaneous (sc) injection. In some embodiments, denosumab is administered to the subject by injection into the upper arm, upper thigh, or abdomen of the subject. In some embodiments, denosumab is administered to the subject by subcutaneous injection into the upper arm, upper thigh, or abdomen of the subject.

[0049] In some embodiments, the subject does not experience dose-limiting toxicity (DLT) while receiving denosumab. In some embodiments, the subject does not experience any Grade 3 or Grade 4 adverse events related to denosumab while receiving denosumab. In some embodiments, the subject is not hypocalcemia while receiving denosumab.

[0050] In some embodiments, the subject is administered denosumab for at least 12 weeks. In some embodiments, the subject is administered denosumab for at least 24 weeks. In some embodiments, the subject is administered denosumab for at least 48 weeks. In some embodiments, the subject is administered denosumab for at most 1 year.

[0051] In some embodiments, the subject is co-administered a PD-1 inhibitor. In some embodiments, the first dose of the PD-1 inhibitor is administered within about 24 hours of a third loading dose comprising about 120 mg of denosumab administered on about day 22. In some embodiments, the first dose of the PD-1 inhibitor and the combined dose of denosumab are co-administered on about day 22. In some embodiments, the combined dose of denosumab is about 120 mg of denosumab.

[0052] In some embodiments, the PD-1 inhibitor is selected from pembrolizumab, nivolumab, cemiplimab, dostallimab, atezolizumab, avelumab, and durvalumab. In some embodiments, the PD-1 inhibitor is selected from pembrolizumab and nivolumab. In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, the PD-1 inhibitor is nivolumab.

[0053] In some embodiments, when the PD-1 inhibitor and denosumab are each administered to a subject on the same day, the PD-1 inhibitor is administered to the subject before administration of denosumab.

[0054] Also provided is a method of treating melanoma in a subject in need thereof, comprising co-administering to the subject a therapeutically effective amount of denosumab and a therapeutically effective amount of a PD-1 inhibitor; one or more loading doses of denosumab are administered to the subject prior to the first dose of the PD-1 inhibitor; Disclosed herein are methods wherein the co-administration is associated with an anti-tumor immune response and / or tumor objective response.

[0055] In some embodiments, the PD-1 inhibitor is selected from pembrolizumab, nivolumab, cemiplimab, dostallimab, atezolizumab, avelumab, and durvalumab. In some embodiments, the PD-1 inhibitor is selected from pembrolizumab and nivolumab. In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, the PD-1 inhibitor is nivolumab.

[0056] In some embodiments, a baseline tumor tissue sample from the subject contains active tumor-infiltrating lymphocytes (TILs). In some embodiments, a baseline peripheral blood sample from the subject contains high serum-free RANKL and / or low serum OPG levels as assessed by flow cytometry analysis. In some embodiments, a baseline tumor tissue sample from the subject expresses two or more of Sox10, RANK, and OPG as assessed by immunohistochemistry.

[0057] In some embodiments, the subject has not previously received a PD-1 inhibitor, hi some embodiments, the subject has previously received a PD-1 inhibitor for stage III or stage IV melanoma, and the interval between the last dose of PD-1 inhibitor and the date of recurrence is at least about 1 year.

[0058] In some embodiments, the subject does not have bone metastases and / or the subject does not have hypercalcemia. In some embodiments, the subject does not have bone metastases. In some embodiments, the subject does not have hypercalcemia.

[0059] In some embodiments, the subject is administered one or more (e.g., 1, 2, 3, 4) loading doses of denosumab. In some embodiments, the subject is administered one loading dose of denosumab. In some embodiments, the subject is administered two loading doses of denosumab.

[0060] In some embodiments, one or more loading doses of denosumab each independently comprise one or more unit doses, each unit dose independently comprising between about 45 mg and about 120 mg of denosumab.

[0061] In some embodiments, the one or more loading doses of denosumab are a first loading dose comprising about 120 mg of denosumab administered on day 1; and This corresponds to a second loading dose containing about 120 mg of denosumab administered on about day 8.

[0062] In some embodiments, the one or more loading doses of denosumab are a first loading dose comprising about 120 mg of denosumab administered on day 1; and and a second loading dose comprising about 120 mg of denosumab administered on about day 8.

[0063] In some embodiments, the first dose of the PD-1 inhibitor is administered within about 24 hours of a third loading dose comprising about 120 mg of denosumab administered on about day 22. In some embodiments, the first dose of the PD-1 inhibitor and the combined dose of denosumab are administered simultaneously on about day 22. In some embodiments, the combined dose of denosumab is about 120 mg of denosumab.

[0064] In some embodiments, the therapeutically effective amount of denosumab comprises one or more unit doses each administered at intervals of about 4 weeks, each unit dose independently comprising from about 45 mg to about 120 mg of denosumab. In some embodiments, the therapeutically effective amount of denosumab corresponds to about 120 mg of denosumab administered once every about 4 weeks. In some embodiments, the therapeutically effective amount of denosumab is about 120 mg of denosumab administered in one or more unit doses at intervals of about 4 weeks. In some embodiments, the therapeutically effective amount of denosumab is about 120 mg of denosumab administered in one unit dose at intervals of about 4 weeks.

[0065] In some embodiments, the PD-1 inhibitor is pembrolizumab; and the therapeutically effective amount of the PD-1 inhibitor is equivalent to about 200 mg of pembrolizumab administered about every 3 weeks.

[0066] In some embodiments, the PD-1 inhibitor is pembrolizumab; and the therapeutically effective amount of the PD-1 inhibitor comprises about 200 mg of pembrolizumab administered about every 3 weeks.

[0067] In some embodiments, the PD-1 inhibitor is nivolumab; and the therapeutically effective amount of the PD-1 inhibitor is equivalent to about 480 mg of nivolumab administered at about four-week intervals.

[0068] In some embodiments, the PD-1 inhibitor is nivolumab; and the therapeutically effective amount of the PD-1 inhibitor comprises about 480 mg of nivolumab administered at about four-week intervals.

[0069] In some embodiments, denosumab is administered to the subject by injection. In some embodiments, denosumab is administered to the subject by subcutaneous injection. In some embodiments, denosumab is administered to the subject by injection into the upper arm, upper thigh, or abdomen of the subject. In some embodiments, denosumab is administered to the subject by subcutaneous injection into the upper arm, upper thigh, or abdomen of the subject.

[0070] In some embodiments, the PD-1 inhibitor is administered to the subject intravenously.

[0071] In some embodiments, when the PD-1 inhibitor and denosumab are each administered to a subject on the same day, the PD-1 inhibitor is administered to the subject before administration of denosumab.

[0072] In some embodiments, the co-administration is associated with an anti-tumor immune response. In some embodiments, the co-administration is associated with a tumor objective response. In some embodiments, the co-administration is associated with an anti-tumor immune response and a tumor objective response.

[0073] In some embodiments, the anti-tumor immune response is selected from the following: a change (e.g., an increase) in recent thymic emigrant cells (RTE) in a peripheral blood sample from the subject compared to a baseline peripheral blood sample; a change (e.g., an increase) in the density of tumor-infiltrating differentiation group (CD8+) cells (TILs) in a tumor tissue sample from the subject compared to a baseline tumor tissue sample; an increase in the number of tumor-infiltrating CD8+ cells in a tumor tissue sample from the subject compared to a baseline tumor tissue sample, as assessed by immunohistochemistry; an increase in tumor cell death in a tumor tissue sample from the subject compared to a baseline tumor tissue sample, as assessed by immunohistochemistry; CD8+ and CD4+ non-T in peripheral blood samples from subjects compared to baseline peripheral blood samples as assessed by flow cytometry analysis reg Increase in the total number of RTEs; CD4+ T in peripheral blood samples from subjects compared to baseline peripheral blood samples as assessed by flow cytometry analysis reg an increase in the total number of cells; an increase in immune cell clonal diversity in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis; RANK+T in peripheral blood samples from subjects compared to baseline peripheral blood samples as assessed by flow cytometry analysis reg an increase in the number of cells; at least partial suppression of myeloid-derived suppressor cells (MDSCs) in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis; A reduction in the number of tumor-associated macrophages in a tumor tissue sample from the subject compared to a baseline tumor tissue sample; and A decrease in the number of tumor-infiltrating MDSCs in a tumor tissue sample from the subject compared to a baseline tumor tissue sample. Any one or more of these parameters are possible.

[0074] In some embodiments, the anti-tumor immune response is an increase in recent thymic emigrant cells (RTE) in a peripheral blood sample from the subject compared to a baseline peripheral blood sample. In some embodiments, the anti-tumor immune response is a change (e.g., an increase) in the density of tumor-infiltrating cluster of differentiation (CD8+) cells (TIL) in a tumor tissue sample from the subject compared to a baseline tumor tissue sample. In some embodiments, the anti-tumor immune response is an increase in the number of tumor-infiltrating CD8+ cells in a tumor tissue sample from the subject compared to a baseline tumor tissue sample, as assessed by immunohistochemistry. In some embodiments, the anti-tumor immune response is an increase in tumor cell death in a tumor tissue sample from the subject compared to a baseline tumor tissue sample, as assessed by immunohistochemistry. In some embodiments, the anti-tumor immune response is an increase in the number of CD8+ and CD4+ non-T cells in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis. regIn some embodiments, the anti-tumor immune response is an increase in the total number of CD4+ T cells in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis. reg In some embodiments, the anti-tumor immune response is an increase in immune cell clonal diversity in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis. In some embodiments, the anti-tumor immune response is an increase in RANK+T cells in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis. reg In some embodiments, the anti-tumor immune response is an increase in the number of myeloid-derived suppressor cells (MDSCs) in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis. In some embodiments, the anti-tumor immune response is a decrease in the number of tumor-associated macrophages in a tumor tissue sample from the subject compared to a baseline tumor tissue sample. In some embodiments, the anti-tumor immune response is a decrease in the number of tumor-infiltrating MDSCs in a tumor tissue sample from the subject compared to a baseline tumor tissue sample.

[0075] In some embodiments, the tumor objective response is a complete response or a partial response as assessed using RECIST v1.1 criteria. In some embodiments, the tumor objective response is a complete response as assessed using RECIST v1.1 criteria. In some embodiments, the tumor objective response is a partial response as assessed using RECIST v1.1 criteria.

[0076] In some embodiments, the melanoma is cutaneous melanoma or mucosal melanoma. In some embodiments, the melanoma is cutaneous melanoma. In some embodiments, the melanoma is mucosal melanoma.

[0077] In some embodiments, the melanoma is stage III melanoma or stage IV melanoma. In some embodiments, the melanoma is stage III melanoma. In some embodiments, the melanoma is stage IV melanoma. In some embodiments, the melanoma is American Joint Committee on Cancer (AJCC) Stage III melanoma or AJCC Stage IV melanoma. In some embodiments, the melanoma is AJCC Stage III melanoma. In some embodiments, the melanoma is AJCC Stage IV melanoma. In some embodiments, the melanoma is unresectable. In some embodiments, the melanoma is resectable Stage III melanoma.

[0078] In some embodiments, the subject is not concomitantly administered an anti-CTLA4 agent. In some embodiments, the subject is not concomitantly administered ipilimumab.

[0079] In some embodiments, the subject does not experience dose-limiting toxicity (DLT) during treatment. In some embodiments, the subject does not experience any Grade 3 or Grade 4 adverse events associated with denosumab during treatment. In some embodiments, the subject is not hypocalcemia during treatment.

[0080] Also disclosed herein is the use of denosumab in the manufacture of a medicament adapted for use in a method of treating melanoma in a subject in need thereof. In some embodiments, the method has one or more of the features of the methods described above.

[0081] Also disclosed herein is the use of a PD-1 inhibitor in the manufacture of a medicament adapted for use in a method of treating melanoma in a subject in need thereof. In some embodiments, the method has one or more of the features of the methods described above.

[0082] Also disclosed herein is a pharmaceutical composition comprising denosumab for use in a method for treating melanoma in a subject in need thereof. In some embodiments, the method has one or more of the features of the methods described above.

[0083] Also disclosed herein is a pharmaceutical composition comprising a PD-1 inhibitor for use in a method for treating melanoma in a subject in need thereof. In some embodiments, the method has one or more of the features of the methods described above.

[0084] Further features and variations of the materials and methods of the present disclosure will be apparent to those skilled in the art from the entirety of this application, including the drawings and detailed description, and all such features are intended as aspects of the present disclosure. Features of the present disclosure described herein can be recombined into additional embodiments, and these are also intended as aspects of the present disclosure, regardless of whether the combination of features is specifically described as an aspect or embodiment of the present disclosure. It should be understood that the entire document is intended to relate as a unified disclosure, and that all combinations of features described herein (even if described in separate sections) are contemplated, even if the combinations of features are not found together in the same sentence, paragraph, or section of the document. Furthermore, only those limitations described herein as being essential to the present disclosure should be considered as such; variations of the present disclosure lacking limitations not described herein as being essential are intended as aspects of the present disclosure.

[0085] All references, including publications, patent applications, and patents, cited in this specification are herein incorporated by reference to the same extent as if each individual reference was individually and specifically indicated to be incorporated by reference in its entirety and set forth herein. [Brief explanation of the drawings]

[0086] [Figure 1]1 is a study schematic of the clinical trial described in Example 1. The following abbreviations are used in the schematic: SC, subcutaneous; D, day; Wk, week; RECIST, Response Evaluation Criteria in Solid Tumors; OS, overall survival; Tx, treatment; AE, adverse event; pt, patient; PBMC, peripheral blood mononuclear cell; Treg, regulatory T cell; MDSC, myeloid-derived suppressor cell; TCR, T cell receptor; OPG, osteoprotegerin; IHC, immunohistochemistry; IF, immunofluorescence. DETAILED DESCRIPTION OF THE INVENTION

[0087] Definition: As used herein, the terms "a," "an," "the," and similar referents in connection with the exemplary embodiments and claims should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0088] As used herein, the term "and / or" in reference to a selection, such as "[A] and / or [B]," includes [A] alone, [B] alone, and both [A] and [B].

[0089] As used herein, the term "anti-tumor immune response" refers to a tumor-specific innate or adaptive immune response in a subject, such as, for example, an increase in tumor-infiltrating immune cells, e.g., tumor-infiltrating dendritic cells, tumor-infiltrating antigen-presenting cells, tumor-infiltrating myeloid cells, an increase in tumor-infiltrating T cells (e.g., an increase in tumor-infiltrating CD4+ and CD8+ T cells, e.g., an increase in activated cytotoxic effector CD4+ T cells); an increase in autoreactive T cells in the blood (i.e., an increase in recent thymic emigration (RTE)). In some embodiments, the density of tumor-infiltrating T cells in tumor tissue (number of cells / mm of tumor surface area) is measured. 2 ) can be assessed by immunohistochemistry and / or immunofluorescence. In some embodiments, RTE can be measured by multiparameter flow cytometry analysis of cluster of differentiation (CD)8+ and CD4+ RTE.

[0090] As used herein, the term "tumor objective response" refers to a complete or partial response to treatment as assessed using the Response Evaluation Criteria In Solid Tumors Criteria (RECIST v1.1). RECIST response criteria for evaluation of target lesions include: Complete response (CR): disappearance of all target lesions; Partial response (PR): at least a 30% reduction in the sum of the longest diameters (LD) of the target lesions; · Stable disease (SD): based on the smallest total LD ​​since the start of treatment, there is neither sufficient shrinkage to correspond to PR nor sufficient increase to correspond to progressive disease (PD); Progressive disease (PD): At least a 20% increase in the sum of the LD of target lesions, based on the smallest total LD ​​recorded since the start of treatment or the appearance of one or more new lesions.

[0091] Furthermore, the overall response rate (ORR) was defined as (CR+PR) / total number of subjects, and a "progression event" refers to a 20% increase in the sum of the longest diameters of target lesions, or a measurable increase in non-target lesions, or the appearance of new lesions.

[0092] As used herein, the terms "comprise," "have," "include," and "containing," unless otherwise specified, are to be construed as open-ended terms including the specified components but not excluding other elements (i.e., meaning "including, but not limited to"). The present disclosure contemplates embodiments described as "comprising" a feature to encompass embodiments "consisting of" or "consisting essentially of" that feature.

[0093] As used herein, the term "about," when used in connection with a dose or amount, includes a range of values ​​encompassing the specific dose or amount, or a dose or amount that would be recognized by one of ordinary skill in the art as providing an effect equivalent to that obtained from the specific dose or amount. In some embodiments, the term "about" reflects a 10% variation of the stated value. In some embodiments, the term "about" reflects a 5% variation of the stated value. In some embodiments, the term "about" reflects a 2% variation of the stated value. In some embodiments, the term "about" reflects a 1% variation of the stated value. The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method of referring individually to each separate value and endpoint falling within the range, and each separate value and endpoint is incorporated herein as if it were individually recited herein. In any range described herein, the endpoints of the range are included within the range. However, this description also contemplates the same range excluding the lower and / or upper endpoint.

[0094] As used herein, the term "administer" and its cognates (e.g., "administering") include both self-administration and administration to a patient by another person (e.g., a medical professional or caregiver).

[0095] As used herein, the term "co-administer" and its cognates (e.g., "co-administering") refer to the administration of two or more therapeutic agents in a coordinated manner, including, but not limited to, simultaneous administration. Specifically, "co-administration" encompasses the administration of co-formulations or simultaneous administration of separate therapeutic compositions, as well as sequential or sequential administration, provided that the administration of one therapeutic agent is conditioned in some way on the administration of another therapeutic agent. Illustratively, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a predetermined period of time. Furthermore, in some embodiments, the co-administered therapeutic agents are present in a subject (PK) or otherwise induce an effect (PD) for similar, identical, or partially overlapping time periods.

[0096] As used herein, the term "combined dose" refers to a dose of one therapeutic agent administered within about 24 hours (e.g., within about ±30 minutes, within about ±1 hour, etc.) of the administration of another therapeutic agent.

[0097] As used herein, the term "antibody" refers to a protein having a conventional immunoglobulin structure, including heavy and light chains, and including a variable region and a constant region. As a non-limiting example, an antibody may be an IgG, which has a "Y-shaped" structure with two identical pairs of polypeptide chains, each pair having one "light" chain (e.g., having a molecular weight of about 25 kDa) and one "heavy" chain (e.g., having a molecular weight of about 50-70 kDa). As used herein, an "antibody" has a variable region and a constant region. In the IgG type, the variable region is generally about 100-110 or more amino acids, contains three complementarity-determining regions (CDRs), and is primarily responsible for antigen recognition and substantially differs from other antibodies that bind to different antigens. The constant region enables the antibody to recruit cells and molecules of the immune system. The variable region is made up of the N-terminal region of each light chain and heavy chain, while the constant region is made up of the C-terminal portion of each heavy chain and light chain (Janeway et al., "Structure of the Antibody Molecule and the Immunoglobulin Genes," Immunobiology: The Immune System in Health and Disease, 4 th ed.Elsevier Science Ltd. / Garland Publishing, (1999)).

[0098] The general structure and properties of antibody CDRs have been described in the art. Briefly, in the antibody framework, CDRs are embedded within the framework of the heavy and light chain variable regions, where they constitute the regions primarily responsible for antigen binding and recognition. A variable region typically contains at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, Md.; Chothia and Lesk, 1987, J. Mol. Biol. 196:901-917; see also Chothia et al., 1989, Nature 342:877-883), which are located within framework regions (referred to as framework regions 1 to 4, FR1, FR2, FR3, and FR4, by Kabat et al., 1991; see also Chothia and Lesk, 1987, supra).

[0099] Antibodies may comprise any constant region known in the art. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses (including, but not limited to, IgG1, IgG2, IgG3, and IgG4). IgM has subclasses (including, but not limited to, IgM1 and IgM2). Embodiments of the present disclosure include antibodies of all such classes or isotypes. The light chain constant region may be, for example, a kappa- or lambda-type light chain constant region, such as a human kappa- or lambda-type light chain constant region. The heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant region, such as a human alpha-, human delta-, human epsilon-, human gamma-, or human mu-type heavy chain constant region. Thus, in some embodiments, the antibody is of the isotype IgA, IgD, IgE, IgG, or IgM, including any one of IgG1, IgG2, IgG3, or IgG4.

[0100] As used herein, the term "chimeric antibody" refers to an antibody that contains domains from two or more different antibodies. A chimeric antibody may, for example, contain a constant domain from one species and a variable domain from a second species, or more commonly, may contain stretches of amino acid sequence from at least two species. A chimeric antibody may also contain domains from two or more different antibodies within the same species.

[0101] As used herein, the term "humanized," when used in reference to an antibody, refers to an antibody having at least the CDR regions from a non-human source that has been engineered to have a structure and immune function more similar to that of a true human antibody than the original source antibody. As a non-limiting example, humanization can involve grafting CDRs from a non-human antibody, such as a murine antibody, onto a human antibody. Humanization can also involve selective amino acid substitutions to make the non-human sequence more similar to human sequences.

[0102] As used herein, the term "conservative amino acid substitution" refers to the substitution of one amino acid with another amino acid having similar properties, such as size, charge, hydrophobicity, hydrophilicity, and / or aromaticity, and includes exchanges in one of the following five groups: I. Small, aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; II. Polar, negatively charged residues and their amides and esters: Asp, Asn, Glu, Gln, cysteic acid and homocysteic acid; III. Polar positively charged residues: His, Arg, Lys; ornithine (Orn) IV. Large, aliphatic, nonpolar residues: Met, Leu, Ile, Val, Cys, norleucine (Nle), homocysteine V. Large aromatic residues: Phe, Tyr, Trp, acetylphenylalanine.

[0103] As used herein, the terms "day [X]" and "week [Y]" etc. (e.g., day [X], week [Y]) refer to a specific time point measured relative to the first dose or loading dose on day 1 (e.g., relative to the first loading dose of denosumab on day 1).

[0104] The terms "at least one" and "one or more" are used interchangeably herein and include one of the indicated component and a plurality (e.g., two, three, four, etc.) of the indicated component.

[0105] As used herein, the term "baseline sample," as in "baseline tumor tissue sample" or "baseline peripheral blood sample," refers to a biological sample, such as a tumor tissue sample or a peripheral blood sample, taken from a subject prior to administration of the first dose of denosumab.

[0106] As used herein, the term "an amount of denosumab equivalent to [Dose X] of denosumab administered on [Schedule Y]" and the like refers to a dose of denosumab administered on one or more characteristics (e.g., mean serum concentration; mean plasma area under the curve (AUC 0~∞); mean plasma C at steady state max and mean plasma C at steady state min the difference between the mean plasma C at steady state max ) refers to a dosing regimen (i.e., Dose A of denosumab administered on Schedule B) that produces a pharmacokinetic profile in subjects substantially similar to the pharmacokinetic profile in subjects administered [Dose X] of denosumab on [Schedule Y].

[0107] The terms "denosumab administration" and "denosumab treatment," when used in reference to a period of time, refer to the period during which a subject is administered denosumab according to a particular schedule (i.e., from the first dose to the end of the final cycle).

[0108] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a therapeutic agent effective to treat a disease or disorder in a subject. In some embodiments, an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result (e.g., an anti-tumor response).

[0109] As used herein, the term "loading dose" refers to a dose of a therapeutic agent administered as a single dose or as part of a series of doses at the beginning of a course of treatment to initially induce a desired pharmacokinetic or pharmacodynamic effect. In some embodiments, the dose and / or administration frequency of the loading dose differs from the dosing regimen used later in the course of treatment to maintain or continue the desired pharmacokinetic or pharmacodynamic effect.

[0110] As used herein, the term "PD-1 inhibitor" refers to a therapeutic agent (such as an antibody or small molecule therapeutic agent) that blocks the binding of PD-L1 to PD-1. In some embodiments, the PD-1 inhibitor can be an anti-PD-1 agent or an anti-PD-L1 agent, so long as the agent blocks the binding of PD-L1 to PD-1. In some embodiments, the PD-1 inhibitor is selected from pembrolizumab, nivolumab, cemiplimab, dostallimab, atezolizumab, avelumab, and durvalumab. In some embodiments, the PD-1 inhibitor is selected from pembrolizumab and nivolumab. In some embodiments, the PD-1 inhibitor is pembrolizumab. In some embodiments, the PD-1 inhibitor is nivolumab. As used herein, the term "PD-1 inhibitor" is synonymous with the term "PD-L1 inhibitor," unless the context clearly dictates otherwise; in all embodiments of the present disclosure, "PD-1 inhibitor" can be replaced with "PD-L1 inhibitor," unless the context clearly dictates otherwise.

[0111] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, intraspinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the therapeutic agent may be coated in a material to protect it from the action of acids and other natural conditions that may inactivate the therapeutic agent.

[0112] As used herein, the term "progression event" refers to a 20% increase in the sum of the longest diameters of target lesions, or a measurable increase in non-target lesions, or the appearance of a new lesion.

[0113] As used herein, the terms "subject" and "patient" are used interchangeably and refer to an animal, e.g., a human. In some embodiments, a "subject" or "patient" is a human.

[0114] As used herein, the term "substantially similar" when used in connection with a particular characteristic includes a particular characteristic that is recognized by one of ordinary skill in the art as providing an equivalent effect to the particular characteristic. In some embodiments, when used in connection with a quantitative characteristic, the term "substantially similar" reflects a 10% variation in the numerical value corresponding to the quantitative characteristic. In some embodiments, when used in connection with a quantitative characteristic, the term "substantially similar" reflects a 5% variation in the numerical value corresponding to the quantitative characteristic. In some embodiments, when used in connection with a quantitative characteristic, the term "substantially similar" reflects a 2% variation in the numerical value corresponding to the quantitative characteristic. In some embodiments, when used in connection with a quantitative characteristic, the term "substantially similar" reflects a 1% variation in the numerical value corresponding to the quantitative characteristic.

[0115] As used herein, the term "treatment" and its cognates (e.g., "treat" or "treating") refer to improving at least one symptom or sign of a disease in a subject, delaying the onset of at least one symptom or sign of a disease in a subject, or reducing the severity of at least one symptom or sign of a disease in a subject. "Treatment" and its cognates do not necessarily mean 100% or complete treatment. Rather, there are various degrees of treatment that one skilled in the art will recognize as having a potential benefit or therapeutic effect. Illustratively, in the context of melanoma treatment, treatment can include, but is not limited to, an anti-tumor immune response, a partial response, a complete response, or disease stabilization.

[0116] As used herein, the term "treatment period" refers to the period during which a subject is administered denosumab and / or another therapeutic agent according to a particular schedule (i.e., from the first dose to the end of the final dose cycle).

[0117] As used herein, the term "unit dose" refers to the amount of a pharmaceutical composition, particularly a therapeutic agent therein (e.g., an anti-RANKL agent (e.g., denosumab), a PD-1 inhibitor (e.g., nivolumab, pembrolizumab)), administered to a subject in one treatment session. A treatment session can be continuous, e.g., uninterrupted parenteral administration (e.g., subcutaneous or intravenous), of a single bolus of duration (e.g., 1 hour, 2 hours). A treatment session can also be divided into two or more subsessions such that one unit dose is administered over time (e.g., 12 hours, 24 hours), with each bolus followed by a break or recovery period.

[0118] Agents targeting RANK-L Alternative aspects of the present disclosure employ at least one agent that targets RANK-L in place of denosumab in the methods, uses, or pharmaceutical compositions for use described herein. Agents that target RANK-L include RANK-L antigen binding proteins (e.g., anti-RANK-L antibodies, antigen-binding fragments thereof, and anti-RANK-L antibody protein products, some of which are disclosed in International Publication Nos. WO 2018 / 200918 and WO 03 / 002713 and U.S. Patent No. 7,364,736, each of which is incorporated herein by reference in its entirety). In some alternative embodiments, a RANK-L antigen binding protein that binds to human RANK-L and has the amino acid sequence set forth in National Center for Biotechnology Information (NCBI) Reference SEQ ID NO: NP003692 or SEQ ID NO: 1 and encoded by the polynucleotide sequence of SEQ ID NO: 2 is employed in place of denosumab in the methods, uses, or pharmaceutical compositions for use described herein.

[0119] [Table 1]

[0120] [Table 2]

[0121] In some embodiments, the RANK-L antigen binding protein is an anti-RANK-L antibody or antibody-binding fragment thereof, or an anti-RANK-L antibody protein product.

[0122] In some embodiments, the anti-RANK-L antibody is a monoclonal antibody. Accordingly, in some embodiments, the present disclosure provides a liquid composition comprising a monoclonal antibody. Alternatively, in some embodiments, the liquid composition may be a polyclonal antibody composition. In some embodiments, the antibody comprises a sequence substantially similar to a naturally occurring antibody produced by a mammal, such as a mouse, rabbit, goat, horse, chicken, hamster, human, etc. In this regard, the antibody may be considered a mammalian antibody, such as a mouse antibody, rabbit antibody, goat antibody, horse antibody, chicken antibody, hamster antibody, human antibody, etc. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a chimeric antibody or a humanized antibody.

[0123] In some embodiments, the antibody is an anti-RANK-L antibody, e.g., an anti-RANK-L monoclonal antibody. In some embodiments, the anti-RANK-L antibody is an IgG2 antibody. In some embodiments, the anti-RANK-L antibody binds to human RANK-L, which in some embodiments comprises the amino acid sequence of SEQ ID NO: 1 and is encoded by the nucleotide sequence of SEQ ID NO: 2.

[0124] In some embodiments, the antibody is denosumab or a biosimilar thereof. Denosumab is known in the art. See, e.g., International Nonproprietary Names for Pharmaceutical Substances (INN): Proposed INN:List 56, WHO Drug Information 20(3):211 (2006); CAS Registry Number 615258-40-7. Denosumab is an immunoglobulin G2 (IgG2), also known as AMG 162, and is the active pharmaceutical ingredient in Prolia® and Xgeva®.

[0125] In some embodiments, the antibody comprises a light chain comprising the CDR1, CDR2, and CDR3 set forth in Table 2. In some embodiments, the antibody comprises a heavy chain comprising the CDR1, CDR2, and CDR3 set forth in Table 2. In some embodiments, the antibody comprises a VH and VL sequence listed in Table 2 or a sequence comprising the VH-IgG2 and VL-IgGkappa sequences listed in Table 2. In some embodiments, the RANK-L antibody binding protein is an antibody comprising the amino acid sequence of SEQ ID NOs: 3-8. In some embodiments, the anti-RANK-L antibody comprises six CDR amino acid sequences of SEQ ID NOs: 3-8. In some embodiments, the anti-RANK-L antibody comprises the heavy chain (HC) complementarity-determining region (CDR) 1 amino acid sequence of SEQ ID NO: 3, the HC CDR2 amino acid sequence of SEQ ID NO: 4, the HC CDR3 amino acid sequence of SEQ ID NO: 5, the light chain (LC) CDR1 amino acid sequence of SEQ ID NO: 6, the LC CDR2 amino acid sequence of SEQ ID NO: 7, and the LC CDR3 amino acid sequence of SEQ ID NO: 8. In some embodiments, the anti-RANK-L antibody comprises a RANK-L binding domain comprising: (a) a heavy chain variable region (VH) comprising: (i) a VH complementarity-determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 3; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5; and (b) a light chain variable region (VL) comprising: (i) a VL complementarity-determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 6; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) a VL comprising the amino acid sequence of SEQ ID NO: 8. In some embodiments, the RANK-L binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 9 and a VL comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-RANK-L antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 9 and a VL comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-RANK-L antibody comprises an HC comprising the amino acid sequence of SEQ ID NO:11 or 13 and an LC comprising the amino acid sequence of SEQ ID NO:12.

[0126] [Table 3]

[0127] [Table 4]

[0128] In some embodiments, the antibody comprises: i. a heavy chain (HC) CDR1 comprising the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 3, or a variant amino acid sequence of SEQ ID NO: 3 having one or two amino acid substitutions; ii. HC CDR2 comprising the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 4, or a variant amino acid sequence of SEQ ID NO: 4 having one or two amino acid substitutions; iii. HC CDR3 comprising the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 5, or a variant amino acid sequence of SEQ ID NO: 5 having one or two amino acid substitutions; iv. a light chain (LC) CDR1 comprising the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 6, or a variant amino acid sequence of SEQ ID NO: 6 having one or two amino acid substitutions; v. an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 7, or a variant amino acid sequence of SEQ ID NO: 7 having one or two amino acid substitutions; vi. An LC CDR3 comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 8, or a variant amino acid sequence of SEQ ID NO: 8 having one or two amino acid substitutions.

[0129] In some embodiments, the antibody comprises an HC variable region comprising the amino acid sequence of SEQ ID NO: 9, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 9, or a variant amino acid sequence of SEQ ID NO: 9 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0130] In some embodiments, the antibody comprises an LC variable region comprising the amino acid sequence of SEQ ID NO: 10, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 10, or a variant amino acid sequence of SEQ ID NO: 10 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0131] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 11, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 11, or a variant amino acid sequence of SEQ ID NO: 11 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0132] In some embodiments, the terminal lysine may be absent. In some embodiments, the terminal lysine may be present. In some embodiments, the terminal glycine-lysine may be absent. In some embodiments, the terminal glycine-lysine may be present. C-terminal lysine clipping is a common phenomenon that occurs during the bioproduction of monoclonal antibodies. Often, lysine residues are removed via carboxypeptidase D (CpD), resulting in a mixture of antibody isoforms with zero or one C-terminal lysine residue on each heavy chain. Furthermore, after C-terminal lysine cleavage, peptidylglycine α-amidating monooxygenase (PAM) catalyzes the hydroxylation of glycine and removal of glyoxylate from the glycine residue, leaving an amidated C-terminal proline. Thus, during recombinant production of monoclonal antibodies, the product is often a mixture of C-terminal processing variants in which the heavy chain C-terminus is (amidated) proline, glycine, or lysine.

[0133] In some embodiments, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 12, or a variant amino acid sequence of SEQ ID NO: 12 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0134] In some embodiments, the amino acid substitutions are conservative amino acid substitutions.

[0135] In an alternative embodiment of the present disclosure, a biosimilar of denosumab may be used in place of denosumab in the methods, uses, or pharmaceutical compositions described herein.

[0136] PD-1 inhibitors In some embodiments of the present disclosure, the PD-1 inhibitor is co-administered with at least one agent that targets RANK-L (e.g., denosumab). In some embodiments, the PD-1 inhibitor is an anti-PD-1 or anti-PD-L1 antibody, or an antibody-binding fragment thereof, or an anti-PD-1 or anti-PD-L1 antibody protein product.

[0137] In some embodiments, the PD-1 inhibitor is a monoclonal antibody. Accordingly, in some embodiments, the present disclosure provides a liquid composition comprising a monoclonal antibody. Alternatively, in some embodiments, the liquid composition can be a polyclonal antibody composition. In some embodiments, the antibody comprises a sequence substantially similar to a naturally occurring antibody produced by a mammal, such as a mouse, rabbit, goat, horse, chicken, hamster, human, etc. In this regard, the antibody can be considered a mammalian antibody, such as a mouse antibody, rabbit antibody, goat antibody, horse antibody, chicken antibody, hamster antibody, human antibody, etc. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a chimeric antibody or a humanized antibody.

[0138] In some embodiments, the antibody is an anti-PD-1 or anti-PD-L1 antibody, such as, for example, an anti-PD-1 or anti-PD-L1 monoclonal antibody.

[0139] In some embodiments, the antibody is selected from pembrolizumab, nivolumab, cemiplimab, dostallimab, atezolizumab, avelumab, durvalumab, and biosimilars of any of the foregoing. In some embodiments, the PD-1 inhibitor is selected from pembrolizumab, nivolumab, and biosimilars of any of the foregoing. In some embodiments, the PD-1 inhibitor is pembrolizumab or a biosimilar thereof. In some embodiments, the PD-1 inhibitor is nivolumab or a biosimilar thereof.

[0140] In some embodiments, the PD-1 inhibitor is an antibody comprising a light chain comprising a CDR1, CDR2, and CDR3 as set forth in Table 3. In some embodiments, the PD-1 inhibitor is an antibody comprising a heavy chain comprising a CDR1, CDR2, and CDR3 as set forth in Table 3. In some embodiments, the PD-1 inhibitor is an antibody comprising a VH and VL sequence listed in Table 3. In some embodiments, the PD-1 inhibitor is an antibody comprising an amino acid sequence of SEQ ID NOs: 13-18. In some embodiments, the PD-1 inhibitor comprises six CDR amino acid sequences of SEQ ID NOs: 13-18. In some embodiments, the PD-1 inhibitor comprises a heavy chain (HC) complementarity determining region (CDR) 1 amino acid sequence of SEQ ID NO: 13, a HC CDR2 amino acid sequence of SEQ ID NO: 14, a HC CDR3 amino acid sequence of SEQ ID NO: 15, a light chain (LC) CDR1 amino acid sequence of SEQ ID NO: 16, a LC CDR2 amino acid sequence of SEQ ID NO: 17, and a LC CDR3 amino acid sequence of SEQ ID NO: 18. In some embodiments, the PD-1 inhibitor comprises a PD-1 binding domain comprising: (a) a heavy chain variable region (VH) comprising: (i) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 13; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; and (b) a light chain variable region (VL) comprising: (i) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 16; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17; and (iii) a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the PD-1 binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the PD-1 inhibitor comprises a VH comprising the amino acid sequence of SEQ ID NO: 19 and a VL comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the PD-1 inhibitor comprises a HC comprising the amino acid sequence of SEQ ID NO:21 and a LC comprising the amino acid sequence of SEQ ID NO:22.

[0141] [Table 5]

[0142] [Table 6]

[0143] In some embodiments, the PD-1 inhibitor comprises: i. a heavy chain (HC) CDR1 comprising the amino acid sequence of SEQ ID NO: 13, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 13, or a variant amino acid sequence of SEQ ID NO: 13 having one or two amino acid substitutions; ii. HC CDR2 comprising the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 14, or a variant amino acid sequence of SEQ ID NO: 14 having one or two amino acid substitutions; iii. A HC CDR3 comprising the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 15, or a variant amino acid sequence of SEQ ID NO: 15 having one or two amino acid substitutions. iv. a light chain (LC) CDR1 comprising the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 16, or a variant amino acid sequence of SEQ ID NO: 16 having one or two amino acid substitutions; v. an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 17, or a variant amino acid sequence of SEQ ID NO: 17 having one or two amino acid substitutions; vi. An LC CDR3 comprising the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 18, or a variant amino acid sequence of SEQ ID NO: 18 having one or two amino acid substitutions.

[0144] In some embodiments, the PD-1 inhibitor comprises an HC variable region comprising the amino acid sequence of SEQ ID NO: 19, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 19, or a variant amino acid sequence of SEQ ID NO: 19 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2) amino acid substitutions.

[0145] In some embodiments, the PD-1 inhibitor comprises an HC variable region comprising the amino acid sequence of SEQ ID NO:20, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:20, or a variant amino acid sequence of SEQ ID NO:20 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2) amino acid substitutions.

[0146] In some embodiments, the PD-1 inhibitor comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:21, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:21, or a variant amino acid sequence of SEQ ID NO:21 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0147] In some embodiments, the PD-1 inhibitor comprises a light chain comprising the amino acid sequence of SEQ ID NO:22, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:22, or a variant amino acid sequence of SEQ ID NO:22 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0148] In some embodiments, the amino acid substitutions are conservative amino acid substitutions.

[0149] In some embodiments, the PD-1 inhibitor is an antibody comprising a light chain comprising a CDR1, CDR2, and CDR3 as set forth in Table 4. In some embodiments, the PD-1 inhibitor is an antibody comprising a heavy chain comprising a CDR1, CDR2, and CDR3 as set forth in Table 4. In some embodiments, the PD-1 inhibitor is an antibody comprising a VH and VL sequence listed in Table 4. In some embodiments, the PD-1 inhibitor is an antibody comprising the amino acid sequence of SEQ ID NOs: 23-28. In some embodiments, the PD-1 inhibitor comprises six CDR amino acid sequences of SEQ ID NOs: 23-28. In some embodiments, the PD-1 inhibitor comprises a heavy chain (HC) complementarity determining region (CDR) 1 amino acid sequence of SEQ ID NO: 23, a HC CDR2 amino acid sequence of SEQ ID NO: 24, a HC CDR3 amino acid sequence of SEQ ID NO: 25, a light chain (LC) CDR1 amino acid sequence of SEQ ID NO: 26, a LC CDR2 amino acid sequence of SEQ ID NO: 27, and a LC CDR3 amino acid sequence of SEQ ID NO: 28. In some embodiments, the PD-1 inhibitor comprises a PD-1 binding domain comprising: (a) a heavy chain variable region (VH) comprising: (i) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 23; (ii) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 24; and (iii) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 25; and (b) a light chain variable region (VL) comprising: (i) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 26; (ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 27; and (iii) a VL comprising the amino acid sequence of SEQ ID NO: 28. In some embodiments, the PD-1 binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 29 and a VL comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the PD-1 inhibitor comprises a VH comprising the amino acid sequence of SEQ ID NO: 29 and a VL comprising the amino acid sequence of SEQ ID NO: 30. In some embodiments, the PD-1 inhibitor comprises an HC comprising the amino acid sequence of SEQ ID NO:31 and an LC comprising the amino acid sequence of SEQ ID NO:32.

[0150] [Table 7]

[0151] [Table 8]

[0152] In some embodiments, the PD-1 inhibitor comprises: i. a heavy chain (HC) CDR1 comprising the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 23, or a variant amino acid sequence of SEQ ID NO: 23 having one or two amino acid substitutions; ii. HC CDR2 comprising the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 24, or a variant amino acid sequence of SEQ ID NO: 24 having one or two amino acid substitutions; iii. A HC CDR3 comprising the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 25, or a variant amino acid sequence of SEQ ID NO: 25 having one or two amino acid substitutions. iv. a light chain (LC) CDR1 comprising the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 26, or a variant amino acid sequence of SEQ ID NO: 26 having one or two amino acid substitutions; v. an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 27 or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 27, or a variant amino acid sequence of SEQ ID NO: 27 having one or two amino acid substitutions; vi. An LC CDR3 comprising the amino acid sequence of SEQ ID NO: 28 or an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 28, or a variant amino acid sequence of SEQ ID NO: 28 having one or two amino acid substitutions.

[0153] In some embodiments, the PD-1 inhibitor comprises an HC variable region comprising the amino acid sequence of SEQ ID NO:29, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:29, or a variant amino acid sequence of SEQ ID NO:29 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2) amino acid substitutions.

[0154] In some embodiments, the PD-1 inhibitor comprises an HC variable region comprising the amino acid sequence of SEQ ID NO: 30, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 30, or a variant amino acid sequence of SEQ ID NO: 30 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2) amino acid substitutions.

[0155] In some embodiments, the PD-1 inhibitor comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 31, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 31, or a variant amino acid sequence of SEQ ID NO: 31 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0156] In some embodiments, the PD-1 inhibitor comprises a light chain comprising the amino acid sequence of SEQ ID NO: 32, an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 32, or a variant amino acid sequence of SEQ ID NO: 32 having 1 to 10 (e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1, or 2) amino acid substitutions.

[0157] In some embodiments, the amino acid substitutions are conservative amino acid substitutions.

[0158] In alternative embodiments of the present disclosure, biosimilars of pembrolizumab, nivolumab, cemiplimab, dostallimab, atezolizumab, avelumab, and durvalumab may be used in place of pembrolizumab, nivolumab, cemiplimab, dostallimab, atezolizumab, avelumab, and durvalumab, respectively, in the methods, uses, or pharmaceutical compositions described herein.

[0159] subject In some embodiments of the methods of the present disclosure, the subject is a human subject. In some embodiments, the human subject is about 18 years of age or older. In some embodiments, the human subject is about 30 years of age or older. In some embodiments, the human subject is about 60 years of age or older.

[0160] In some embodiments, a baseline tumor tissue sample from the subject contains active tumor-infiltrating lymphocytes (TILs). In some embodiments, a baseline peripheral blood sample from the subject contains high serum-free RANKL and / or low serum OPG levels as assessed by flow cytometry analysis. In some embodiments, a baseline tumor tissue sample from the subject expresses two or more of Sox10, RANK, and OPG as assessed by immunohistochemistry.

[0161] In some embodiments, the subject has not previously received a PD-1 inhibitor, hi some embodiments, the subject has previously received a PD-1 inhibitor for stage III or stage IV melanoma, and the interval between the last dose of PD-1 inhibitor and the date of recurrence is at least about 1 year.

[0162] In some embodiments, the subject does not have bone metastases. In some embodiments, the subject does not have hypercalcemia.

[0163] In some embodiments, the subject meets at least one of the inclusion criteria for the clinical trial described in Example 1 of this application.

[0164] In some embodiments, the subject does not meet any of the trial exclusion criteria described in Example 1 of this application.

[0165] melanoma Some embodiments of the present disclosure provide methods of treating melanoma in a subject in need thereof. In some embodiments, the melanoma is cutaneous melanoma or mucosal melanoma. In some embodiments, the melanoma is cutaneous melanoma. In some embodiments, the melanoma is mucosal melanoma.

[0166] In some embodiments, the melanoma is stage III melanoma or stage IV melanoma. In some embodiments, the melanoma is stage III cutaneous melanoma or stage IV cutaneous melanoma. In some embodiments, the melanoma is stage III mucosal melanoma or stage IV mucosal melanoma.

[0167] In some embodiments, the melanoma is stage III melanoma. In some embodiments, the melanoma is stage III cutaneous melanoma. In some embodiments, the melanoma is stage III mucosal melanoma.

[0168] In some embodiments, the melanoma is stage IV melanoma. In some embodiments, the melanoma is stage IV cutaneous melanoma. In some embodiments, the melanoma is stage IV mucosal melanoma.

[0169] In some embodiments, the melanoma is American Joint Committee on Cancer (AJCC) Stage III melanoma or AJCC Stage IV melanoma. In some embodiments, the melanoma is AJCC Stage III cutaneous melanoma or AJCC Stage IV cutaneous melanoma. In some embodiments, the melanoma is AJCC Stage III mucosal melanoma or AJCC Stage IV mucosal melanoma.

[0170] In some embodiments, the melanoma is AJCC Stage III melanoma. In some embodiments, the melanoma is AJCC Stage III cutaneous melanoma. In some embodiments, the melanoma is AJCC Stage III mucosal melanoma.

[0171] In some embodiments, the melanoma is AJCC Stage IV melanoma. In some embodiments, the melanoma is AJCC Stage IV cutaneous melanoma. In some embodiments, the melanoma is AJCC Stage IV mucosal melanoma.

[0172] In some embodiments, the melanoma is unresectable.

[0173] In some embodiments, the melanoma is resectable stage III melanoma.

[0174] Routes of Administration and Pharmaceutical Compositions An agent targeting RANK-L, such as an anti-RANK-L antibody, e.g., denosumab, can be administered to a subject via any suitable route of administration. For example, an anti-RANK-L antibody (e.g., denosumab) can be administered to a subject via parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration. In some embodiments, an anti-RANK-L antibody (e.g., denosumab) is administered to a subject by injection. In some embodiments, an anti-RANK-L antibody (e.g., denosumab) is administered to a subject by subcutaneous injection. In some embodiments, an anti-RANK-L antibody (e.g., denosumab) is administered to a subject by injection into the subject's upper arm, upper thigh, or abdomen. In some embodiments, an anti-RANK-L antibody (e.g., denosumab) is administered to a subject by subcutaneous injection into the subject's upper arm, upper thigh, or abdomen.

[0175] Similarly, the PD-1 inhibitor, e.g., nivolumab or pembrolizumab, can be administered to a subject via any suitable route of administration. For example, the PD-1 inhibitor (e.g., nivolumab or pembrolizumab) can be administered to a subject via parenteral, nasal, oral, pulmonary, topical, vaginal, or rectal administration. In some embodiments, the PD-1 inhibitor (e.g., nivolumab or pembrolizumab) is administered to a subject intravenously.

[0176] The following discussion of routes of administration is provided merely to illustrate non-limiting exemplary embodiments and should not be construed as limiting the scope of the present disclosure in any way.

[0177] Formulations suitable for parenteral administration include, but are not limited to, aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The term "parenteral" means not through the digestive tract, but by some other route such as subcutaneous, intramuscular, intrathecal, or intravenous. Anti-RANK-L antibodies (e.g., denosumab) and / or PD-1 inhibitors (e.g., nivolumab, pembrolizumab) may be administered with a physiologically acceptable diluent in a pharmaceutical carrier such as water, saline, aqueous dextrose and related sugar solutions, alcohols such as ethanol or hexadecyl alcohol, glycols such as propylene glycol or polyethylene glycol, dimethyl sulfoxide, glycerol, ketals such as 2,2-dimethyl-153-dioxolane-4-methanol, ethers, poly(ethylene glycol) 400, oils, fatty acids, fatty acid esters, or glycerides, or acetylated fatty acid glycerides, with or without the addition of a pharmaceutically acceptable surfactant such as a soap or detergent, pectin, carbomer, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose, or suspending agents such as emulsifiers, and other pharmaceutical adjuvants.

[0178] Oils that can be used in parenteral formulations include, but are not limited to, petroleum, animal, vegetable, or synthetic oils. Specific, non-limiting examples of oils include peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petrolatum oil, and mineral oil. Fatty acids suitable for use in parenteral formulations include, but are not limited to, oleic acid, stearic acid, and isostearic acid. Ethyl oleate and isopropyl myristate are non-limiting examples of suitable fatty acid esters.

[0179] In some embodiments, parenteral formulations contain about 0.5% to about 25% by weight of an anti-RANK-L antibody (e.g., denosumab) in solution. Preservatives and buffers may be used. To minimize or eliminate irritation at the injection site, such compositions may contain one or more non-ionic surfactants with a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. In some embodiments, the amount of surfactant in such formulations ranges from about 5% to about 15% by weight. Suitable surfactants include, but are not limited to, polyethylene glycol sorbitan fatty acid esters, e.g., sorbitan monooleate and a high molecular weight adduct of ethylene oxide with a hydrophobic base formed by the condensation of propylene oxide with propylene glycol. In some embodiments, parenteral formulations are presented in unit-dose or multi-dose hermetically sealed containers, such as ampoules and vials, and can be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid excipient for injection, e.g., water, immediately prior to use. In some embodiments, extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets.

[0180] Injectable formulations are subject to the present disclosure. The requirements of effective pharmaceutical carriers for injectable compositions are well known to those skilled in the art (see, for example, Pharmaceutics and Pharmacy Practice, J.B. Lippincott Company, Philadelphia, PA, Banker and Chalmers, eds., pages 238-250 (1982) and ASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

[0181] safety In some embodiments, the method comprises administering an anti-RANK-L antibody (e.g., denosumab) in an amount that does not result in dose-limiting toxicities (DLTs) during treatment with the anti-RANK-L antibody (e.g., denosumab). In some embodiments, the method comprises administering a PD-1 inhibitor (e.g., nivolumab, pembrolizumab) in an amount that does not result in dose-limiting toxicities (DLTs) during treatment with the PD-1 inhibitor (e.g., nivolumab, pembrolizumab). In some embodiments, the subject does not experience any DLTs during administration of the anti-RANK-L antibody (e.g., denosumab) and / or PD-1 inhibitor (e.g., nivolumab, pembrolizumab). In some embodiments, the subject does not experience any grade 3 or grade 4 adverse events associated with treatment with the anti-RANK-L antibody (e.g., denosumab) and / or PD-1 inhibitor (e.g., nivolumab or pembrolizumab) during the treatment period. In some embodiments, the subject does not experience any grade 3 or grade 4 adverse events associated with treatment with the anti-RANK-L antibody (e.g., denosumab) and / or PD-1 inhibitor (e.g., nivolumab or pembrolizumab) during the treatment period. In some embodiments, the treatment period is at least 1 month (e.g., 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year). In some embodiments, the treatment period is at most 1 year.

[0182] In some embodiments, the subject is not hypocalcemic. In some embodiments, the subject is not hypocalcemic during administration of an anti-RANK-L antibody (e.g., denosumab) and / or a PD-1 inhibitor (e.g., nivolumab, pembrolizumab). In some embodiments, the subject is not hypocalcemic prior to administration of an anti-RANK-L antibody (e.g., denosumab) and / or a PD-1 inhibitor (e.g., nivolumab, pembrolizumab).

[0183] Dose response In some embodiments, the methods disclosed herein are associated with an anti-tumor immune response and / or a tumor objective response. In some embodiments, the methods disclosed herein are associated with an anti-tumor immune response. In some embodiments, the methods disclosed herein are associated with a tumor objective response. In some embodiments, the methods disclosed herein are associated with an anti-tumor immune response and a tumor objective response.

[0184] In some embodiments, the methods disclosed herein are associated with an anti-tumor immune response within about 3 weeks of administering a first loading dose of denosumab. In some embodiments, the methods disclosed herein are associated with an anti-tumor immune response within about 16 weeks of administering a first loading dose of denosumab.

[0185] In some embodiments, the methods disclosed herein are associated with a tumor objective response within about 16 weeks of administering a first loading dose of denosumab. In some embodiments, the methods disclosed herein are associated with a tumor objective response within about 6 months of administering a first loading dose of denosumab.

[0186] In some embodiments, the anti-tumor immune response is selected from the following: a change (e.g., an increase) in recent thymic emigrant cells (RTE) in a peripheral blood sample from the subject compared to a baseline peripheral blood sample; a change (e.g., an increase) in the density of tumor-infiltrating differentiation group (CD8+) cells (TILs) in a tumor tissue sample from the subject compared to a baseline tumor tissue sample; an increase in the number of tumor-infiltrating CD8+ cells in a tumor tissue sample from the subject compared to a baseline tumor tissue sample, as assessed by immunohistochemistry; an increase in tumor cell death in a tumor tissue sample from the subject compared to a baseline tumor tissue sample, as assessed by immunohistochemistry; an increase in the total number of CD8+ and CD4+ non-Treg RTE in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis; an increase in the total number of CD4+ Treg cells in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis; an increase in immune cell clonal diversity in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis; an increase in the number of RANK+ Treg cells in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis; at least partial suppression of myeloid-derived suppressor cells (MDSCs) in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis; A reduction in the number of tumor-associated macrophages in a tumor tissue sample from the subject compared to a baseline tumor tissue sample; and A decrease in the number of tumor-infiltrating MDSCs in a tumor tissue sample from the subject compared to a baseline tumor tissue sample. Any one or more of these parameters are possible.

[0187] In some embodiments, the anti-tumor immune response is an increase in recent thymic emigrant cells (RTE) in a peripheral blood sample from the subject compared to a baseline peripheral blood sample.

[0188] In some embodiments, the anti-tumor immune response is a change (e.g., an increase) in the density of tumor-infiltrating differentiation group (CD8+) cells (TILs) in a tumor tissue sample from the subject compared to a baseline tumor tissue sample.

[0189] In some embodiments, the anti-tumor immune response is an increase in the number of tumor-infiltrating CD8+ cells in a tumor tissue sample from the subject compared to a baseline tumor tissue sample, as assessed by immunohistochemistry.

[0190] In some embodiments, the anti-tumor immune response is an increase in tumor cell death in a tumor tissue sample from the subject compared to a baseline tumor tissue sample, as assessed by immunohistochemistry.

[0191] In some embodiments, the anti-tumor immune response is an increase in the total number of CD8+ and CD4+ non-Treg RTE in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis.

[0192] In some embodiments, the anti-tumor immune response is an increase in the total number of CD4+ Treg cells in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis.

[0193] In some embodiments, the anti-tumor immune response is an increase in immune cell clonal diversity in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis.

[0194] In some embodiments, the anti-tumor immune response is an increase in the number of RANK+ Treg cells in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis.

[0195] In some embodiments, the anti-tumor immune response is at least partial suppression of myeloid-derived suppressor cells (MDSCs) in a peripheral blood sample from the subject compared to a baseline peripheral blood sample, as assessed by flow cytometry analysis.

[0196] In some embodiments, the anti-tumor immune response is a decrease in the number of tumor-associated macrophages in a tumor tissue sample from the subject compared to a baseline tumor tissue sample.

[0197] In some embodiments, the anti-tumor immune response is a decrease in the number of tumor-infiltrating MDSCs in a tumor tissue sample from the subject compared to a baseline tumor tissue sample.

[0198] In some embodiments, the tumor objective response is a complete response or a partial response as assessed using RECIST v1.1 criteria. In some embodiments, the tumor objective response is a complete response as assessed using RECIST v1.1 criteria. In some embodiments, the tumor objective response is a partial response as assessed using RECIST v1.1 criteria.

[0199] In some embodiments, a subject treated according to the methods described herein does not experience a progression event or a death event within about 1 month of administration of the first loading dose of denosumab. In some embodiments, a subject treated according to the methods described herein does not experience a progression event or a death event within about 2 months of administration of the first loading dose of denosumab. In some embodiments, a subject treated according to the methods described herein does not experience a progression event or a death event within about 3 months of administration of the first loading dose of denosumab. In some embodiments, a subject treated according to the methods described herein does not experience a progression event or a death event within about 4 months of administration of the first loading dose of denosumab. In some embodiments, a subject treated according to the methods described herein does not experience a progression event or a death event within about 5 months of administration of the first loading dose of denosumab. In some embodiments, a subject treated according to the methods described herein does not experience a progression event or a death event within about 6 months of administration of the first loading dose of denosumab. In some embodiments, a subject treated according to the methods described herein does not experience a progression event or a death event within about 7 months of administering the first loading dose of denosumab. In some embodiments, a subject treated according to the methods described herein does not experience a progression event or a death event within about 8 months of administering the first loading dose of denosumab. In some embodiments, a subject treated according to the methods described herein does not experience a progression event or a death event within about 9 months of administering the first loading dose of denosumab. In some embodiments, a subject treated according to the methods described herein does not experience a progression event or a death event within about 10 months of administering the first loading dose of denosumab. In some embodiments, a subject treated according to the methods described herein does not experience a progression event or a death event within about 11 months of administering the first loading dose of denosumab. In some embodiments, a subject treated according to the methods described herein does not experience a progression event or a death event within about 1 year of administering the first loading dose of denosumab.

[0200] The disclosed subject matter is not intended to be limited in scope by the specific embodiments described herein, but instead as non-limiting exemplifications of particular aspects of the present disclosure. Functionally equivalent methods and components are within the scope of the present disclosure. Indeed, various modifications of the disclosed subject matter in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing and accompanying drawings. Such modifications are intended to be within the scope of the disclosed subject matter.

[0201] The descriptions of various embodiments and / or examples of the disclosed subject matter are presented for purposes of illustration and are not intended to be exhaustive or limiting in any way. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein has been selected to best explain the principles of the embodiments, practical applications, or technical improvements to technology found in the marketplace, and / or to enable those skilled in the art to understand the disclosed subject matter. [Example]

[0202] In order that the present disclosure may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the disclosure in any manner.

[0203] Example 1: Phase 2 Study of Denosumab in Combination with a PD-1 Inhibitor in Subjects with Stage III / IV Melanoma This ongoing, multicenter, open-label, single-arm Phase II trial (ClinicalTrials.gov Identifier: NCT03620019) was designed to investigate the pharmacodynamic (PD) and antitumor effects of denosumab alone and in combination with anti-PD-1 agents (pembrolizumab or nivolumab) in patients with unresectable, PD-1 / PD-L1 inhibitor-naïve, locally and distantly metastatic melanoma (AJCC stage III / IV). Patients must have available baseline tumor tissue and undergo study biopsies at day 22 (mandatory) and week 15 (optional) to participate. Translational research will be conducted on peripheral blood and tumor tissue collected before and during treatment with denosumab alone and in combination with anti-PD-1 therapy to investigate the pharmacodynamic and antitumor effects.

[0204] Up to 25 subjects received denosumab at the FDA-approved dose (120 mg subcutaneously (sc) in the upper arm, upper thigh, or abdomen every 4 weeks) with additional loading doses of 120 mg denosumab administered sc on days 8 and 22 (Figure 1). Nivolumab, 480 mg, was administered intravenously (IV) every 4 weeks, starting 21 days after the first dose of denosumab. In subjects enrolled before Amendment 1, pembrolizumab, 200 mg, was administered intravenously (IV) every 3 weeks, starting 21 days after the first dose of denosumab. On days when denosumab is administered on the same day as the anti-PD-1 agent (e.g., 21 days after the first dose of denosumab), the sc injection should be administered after the anti-PD-1 agent infusion is completed. Combination therapy with both drugs will continue for up to one year, as long as the subject is benefiting from the therapy. Study treatment may be discontinued for unacceptable toxicity, disease progression, or other reasons, at the discretion of the investigator. If the subject is not withdrawn early, the subject's final dose of study drug will be administered approximately 49 weeks after starting denosumab.

[0205] [Table 9]

[0206] The Phase 2 study has a combined primary objective: to evaluate the pharmacodynamic effects (immunomodulatory and / or antitumor effects) of denosumab alone, and to evaluate the pharmacodynamic effects of denosumab in combination with an anti-PD-1 agent. To investigate any potential direct antitumor and indirect immunomodulatory effects of denosumab alone, concomitant anti-PD-1 treatment will not begin until Day 22 of study treatment initiation. Any tumor tissue will be collected from any procedures performed prior to study enrollment. Mandatory tumor biopsies and peripheral blood will be collected on Day 22, immediately prior to the first anti-PD-1 agent infusion. Blood samples will be collected at Weeks 16, 28, and 40. Any tumor tissue will also be collected at the end of Week 16 and at any procedures performed after study completion or withdrawal due to tumor progression.

[0207] The resected tumors were formalin-fixed and paraffin-embedded (FFPE). Hematoxylin and eosin (H&E)-stained tissue sections were generated to assess the quality (necrosis) and quantity of tumor cells present. In addition, the amount of TILs was scored as previously described (Cancer Genome Atlas N: Genomic Classification of Cutaneous Melanoma. Cell 161:1681-96, 2015). Furthermore, 5 μm-thick tissue sections were prepared for tumor imaging analysis (e.g., CD8, RIP3, Apoptag®, Sox10, RANK, RANKL, OPG, PD-1, CD68, CD163, CD33, CD11b, HLA-DR).

[0208] Serum levels of RANKL (free and OPG-bound) and OPG in peripheral blood samples are measured by ELISA. Additionally, the following peripheral blood mononuclear cell populations are enumerated using multiparameter flow cytometry: · Bone marrow-derived double-negative early thymocytes (baseline, week 4, week 16, week 28, and week 40) CD34+CD2+CD4-CD5+CD7+CD8-CD45RA+; RTE by flow cytometry analysis (baseline, week 4, week 16, week 28, and week 40): CD4+CD45RO-CD45RA+CD62L+CD31+, CD4+Foxp3+CD45RO-CD45RA+CD62L+CD31+, and CD8+CD45RO-CD45RA+CD62L+CD31+; · RTE by sjTREC analysis of FACS-sorted CD4+ and CD8+ PBMCs (baseline, weeks 4, 16, 28, and 40); Immune cell receptor repertoire analysis on FACS-sorted CD4+ and CD8+ PBMCs (baseline, weeks 4, 16, 28, and 40); Effector T cell subtypes (baseline, week 4, week 16, week 28, and week 40), e.g., cytotoxic (CD3+CD4-CD8+Granzyme B+) and exhausted (CD3+CD4-CD8+PD-1+TIM3+LAG3+); · Treg cells (baseline, week 4, week 16, week 28, and week 40) CD4+CD25high+FoxP3+(RANKL); MDSCs (baseline, week 4, week 16, week 28, and week 40; Su's lab), monocytes (HLA-DRlow CD14+), other monocytes (lin1 - HLA-DR - CD33+CD11b+), lymphocytes (lin1 - HLA-DR - CD33+CD11b+).

[0209] The study's coprimary secondary endpoints include evaluation of the pharmacodynamic effects of denosumab and anti-PD-1 combination therapy in peripheral blood samples collected during the study (i.e., at weeks 16, 28, and 40) compared to denosumab alone. An optional tumor biopsy may be obtained at week 16. Secondary endpoints include measurement of the clinical efficacy of denosumab in combination with an anti-PD-1 agent based on response rate by Response Evaluation Criteria in Solid Tumors version 1.1 (RECIST v1.1) at week 16, progression-free survival (PFS) at 6 months, and overall survival (OS) at 1 year of study treatment, in addition to evaluations to determine the safety of the combination in subjects with metastatic melanoma. Safety of the combination will be assessed according to the NCI Common Terminology Criteria for Adverse Events (NCI-CTCAE). Predefined Pocock discontinuation criteria indicate early study discontinuation if excessive toxicity or other clinically significant adverse effects (AEs) are observed with this combination.

[0210] Inclusion criteria for the Phase 2 trial include: 1. Signed written informed consent and HIPAA authorization for the release of personal health information. 2.Age 18 or older at the time of obtaining consent. Eastern Cooperative Oncology Group (ECOG) performance status of 3.0–2. 4. Histologically confirmed melanoma of cutaneous or mucosal primary (e.g., sinus, vagina, anus, gastrointestinal tract); metastatic melanoma of unknown primary is acceptable because melanoma of unknown primary is biologically similar to cutaneous melanoma. 5. AJCC Stage III / IV unresectable (or resectable) disease, both measurable by RECIST v1.1 criteria. Patients with bulky resectable Stage IIIB, IIIC, or IIID melanoma (tumor-involved lymph node(s) ≥ 2 cm in shortest diameter and tumor-involved non-lymph node(s) ≥ 2 cm in longest diameter) may also be enrolled in the study at the investigator's discretion. 6. Patients must be available and consent to have a preserved tumor mass harvested from a previous surgery to confirm or treat metastatic disease (e.g., radical lymph node dissection); if not available or of insufficient quantity (e.g., tumor <2 mm2 in size) or quality (>50% necrosis, <30% tumor cells), they may be enrolled in the study if they agree to undergo a tumor biopsy before initiating treatment. 7. Must agree to undergo one on-treatment biopsy at week 4 of the study; the week 16 biopsy is optional. 8. You must consent to 100 mL of blood being collected for testing at week 1, day 20±2 (week 4), week 16, week 28, week 40, and at the end of treatment. 9. Demonstrate adequate organ function as defined in the table; all screening tests obtained within 21 days prior to enrollment. 10. Women of childbearing potential must have a negative serum pregnancy test within 72 hours prior to study treatment. Note: Women are considered of childbearing potential unless they are surgically sterile (have had a hysterectomy, bilateral tubal ligation, or bilateral oophorectomy) or have been naturally postmenopausal for at least 12 consecutive months. 11. Females of childbearing potential must be willing to use appropriate contraceptive methods as outlined in section 4.5.2 - oral contraception is required from 14 days prior to initiation of study drug until 120 days after discontinuation of treatment. Note: Abstinence is acceptable if this is the subject's usual lifestyle and preferred method of contraception. 12. Male patients with female partners must have had a previous vasectomy or agree to use appropriate contraception as outlined in Section 4.5.2 - Contraception starting with the first dose of study treatment and continuing until 120 days after the last dose of study treatment. Note: Abstinence is acceptable if this is the subject's usual lifestyle and preferred method of contraception. 13. The subject is willing and able to understand and comply with the study procedures as determined by the registered physician or protocol designee. 14. Prior radiation therapy is permitted if completed ≥ 14 days before starting denosumab and the patient has sufficiently recovered from any associated toxicity (Grade ≤ 1 or Grade ≤ 2 that has been stable for ≥ 3 months). 15. If patients receive adjuvant treatment, especially ipilimumab and high-dose interferon, any toxicities must resolve to Grade 1 or less. Grade 2 toxicities due to ipilimumab resulting from autoimmune endocrinopathy requiring permanent hormone replacement therapy are acceptable as long as they are adequately treated. This means patients should discontinue systemic steroids for the treatment of any of these or other autoimmune toxicities (e.g., colitis, rash).

[0211] Additionally, subjects who have previously received a PD-1 inhibitor in Stage III (adjuvant) or Stage IV disease may enroll in a Phase 2 trial as long as: 1. The interval between the last dose of adjuvant PD-1 inhibitor and the date of relapse (clinical or radiographic) is at least 1 year; 2. Subjects treated for stage IV disease had an anti-tumor response (partial or complete response) according to RECIST version 1.1, but at the subject's / investigator's option, there was a 1-year discontinuation between the last dose of PD-1 inhibitor and the date of recurrence (clinical or radiographic). The inclusion of these subjects who previously received PD-1 inhibitors in the adjuvant setting (i.e., without knowledge of clinical efficacy) or after a clear anti-tumor response in the metastatic setting is based on a recent series of subjects who responded to re-administration of PD-1 inhibitors if they previously responded to PD-1 inhibitors. This suggests that a decline in anti-tumor immunity in the absence of costimulation with PD-1 inhibitors (i.e., >1 year) may be the reason for cancer recurrence, rather than primary resistance to PD-1 inhibitors. 3. Any side effects that may have occurred during previous exposure to a PD-1 inhibitor are not severe (i.e., grade 1 or 2 according to CTCAE version 5.0 criteria).

[0212] Exclusion criteria for Phase 2 trials include: 1. History of previous malignancies, except for the following: Non-melanoma skin cancer, non-invasive bladder cancer, and cervical intraepithelial neoplasia, History of prostate cancer (provided the patient is not on active systemic treatment other than hormone therapy and has documented undetectable prostate-specific antigen (PSA < 0.2 ng / mL)), Chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL) (provided that the patient has isolated lymphocytosis (Rai stage O) and does not require systemic treatment) [in the case of "B" symptoms, Richter transformation, lymphocyte doubling time (<6 months), lymphadenopathy, or hepatosplenomegaly], Lymphoma or any type or hairy cell leukemia (provided that the patient is not receiving active systemic treatment and is in complete remission as documented by positron emission tomography (PET) / CT scan and bone marrow biopsy for at least three months), History of other malignancies (provided the patient has completed treatment or is not requiring treatment and has been disease-free for ≥ 2 years). If a patient has had another malignancy within the past 2 years that could have been completely cured with surgery alone or has not required any treatment other than observation at the discretion of a specialist, the patient may be considered for enrollment provided that the risk of developing recurrent or distant metastatic disease is less than 30% according to the American Joint Committee in Cancer (AJCC) staging system within 3 years of the initial diagnosis of the other malignancy. 2. Have known active central nervous system (CNS) metastases that are symptomatic and require antiepileptic drugs or corticosteroids. Subjects with previously treated brain metastases may participate if they have been stable (no evidence of progression by imaging) for at least 2 weeks prior to the first dose of study treatment, have returned to baseline neurological symptoms, have no evidence of new or growing brain metastases, and have been steroid-free for at least 7 days prior to study treatment. This exception does not include carcinomatous meningitis, which is excluded regardless of clinical stability. Patients with leptomeningeal disease detected by either brain MRI or cytology (e.g., lumbar puncture) or who have leptomeningeal disease are further excluded. 3. Treatment with any investigational agent, immunotherapy, or chemotherapy within 28 days prior to study treatment (i.e., initiation of denosumab). Treatment with any targeted therapy (e.g., mitogen-activated protein kinase (MAPK) inhibitors) is permitted as long as at least 15 days have elapsed since the last dose of drug. 4. Patients who have discontinued previous treatment with a tyrosine kinase inhibitor for melanoma should discontinue these medications for at least 15 days before starting study treatment. 5. Prior PD-1 / PD-L1 therapy in the adjuvant setting; targeted therapy or prior ipilimumab in the adjuvant setting is acceptable. 6. Any condition, including laboratory abnormalities, that, in the opinion of the Investigator, places the subject at unacceptable risk if the Investigator is participating in the study, including, but not limited to, any serious medical condition or psychiatric illness that may prevent participation in this clinical trial. 7. Has been diagnosed with an immunodeficiency or is receiving systemic steroid therapy equivalent to a daily dose of 10 mg or more of prednisone (or an equivalent dose of other corticosteroids), or any other form of immunosuppressive therapy, within 7 days prior to the first dose of study treatment. 8. Have a history of active tuberculosis (Mycobacterium Bacillus Tuberculosis). 9. Pregnant or nursing, or intending to conceive or father a child within the planned study period starting from the prescreening or screening visit through 120 days after the last dose of study treatment. 10. Hypersensitivity to nivolumab, pembrolizumab, or denosumab or any of their excipients. 11. Have an active autoimmune disease that has required systemic treatment (i.e., with the use of disease-modifying agents, corticosteroids, or immunosuppressants) within the past two years. Replacement therapy (e.g., thyroxine, insulin, or physiological corticosteroid replacement therapy for adrenal or pituitary insufficiency) is not considered a form of systemic treatment. 12. History of non-infectious pneumonia requiring systemic corticosteroids or evidence of interstitial lung disease or current active non-infectious pneumonia. Recurrent short-term (<7 days) exposure to systemic corticosteroids (e.g., poison ivy poisoning or steroid tapering for chronic obstructive pulmonary disease (COPD) exacerbations) is permitted. 13. Has a history of acute coronary event (e.g., myocardial infarction) within 3 months of study enrollment, uncontrolled symptomatic coronary artery disease, or New York Heart Association class III / IV congestive heart failure. 14. Have an active infection requiring systemic treatment within 7 days prior to starting treatment. 15. Have a known history of human immunodeficiency virus (HIV1 / 2 antibodies). 16. Known serologic status reflecting active hepatitis B or hepatitis C infection. Patients who are hepatitis B core antibody positive but antigen negative will require a negative polymerase chain reaction (PCR) prior to enrollment. [Note: Hepatitis B antigen or PCR positive patients will be excluded]. 17. Received a live vaccine within 30 days of the planned start of study treatment. Note: Injectable seasonal influenza vaccines are generally inactivated influenza vaccines and are acceptable; however, intranasal influenza vaccines (e.g., Flu-Mist®) are live attenuated vaccines and are not acceptable. 18. Known active metabolic bone disease, e.g., Paget's disease, Cushing's disease, hyperprolactinemia over the past 1 year 12 months, known history of symptomatic osteoporosis (e.g., history of fractures, bone pain), or any type of hypercalcemia / hypocalcemia (serum free calcium >1.1 times the upper limit of normal (ULN) and <0.9 times the lower limit of normal LLN) over the past 2 weeks from study initiation requiring treatment beyond calcium and vitamin D supplementation. 19. Prior treatment with denosumab. Use of bisphosphonates for the treatment of metastatic bone disease is permitted, but use for hypercalcemia of malignancy is not permitted. 20. History of osteonecrosis or osteomyelitis of the jaw, active dental or jaw disorder requiring known invasive dental procedure during the study, or current evidence of unhealed dental or oral surgery. Note: Patients should be referred to a dentist prior to the start of study treatment for inadequate teeth or other dental problems that, in the treating physician's opinion, may increase the risk of osteonecrosis of the jaw.

[0213] Twenty-two patients treated with denosumab and nivolumab were evaluable (median age, 60 years; 12 males; 19 cutaneous melanoma; 5 stage IIIB-C; 3 with bone metastases). Immune-mediated fatigue and arthralgia attributed to denosumab alone (first 3 weeks) were observed in 10 and 4 patients, respectively. Six patients developed tumor pain during denosumab ± nivolumab treatment. Additionally, one subject developed colitis and another developed pneumonia; these adverse events (AEs) were attributed to nivolumab, which was permanently discontinued. Two patients each developed grade 2 pneumonia and colitis upon rechallenge with denosumab alone. The overall response rate (RR) (partial response (PR) + complete response (CR)) was 50%. Two patients had antitumor responses before starting nivolumab; both patients had active (3+) TILs in their baseline tumors.

[0214] With a median follow-up of 19.9 months (range, 5-44.1 months), median PFS was 13.3 months (range, 1.1-41.4+ months). Eleven patients did not progress. Five patients died, four from metastatic melanoma (one from COVID-19 complications).

[0215] With a median follow-up of 21.8 months, five patients died of melanoma and one patient died of complications from COVID-19 infection; the latter had a complete radiographic response. Seven patients are currently alive and without evidence of melanoma.

[0216] The median progression-free survival for the denosumab-nivolumab combination was 14.9 months (range 1.1 months, 57.4+ months). A recently published study of nivolumab plus leratolimab (Tawbi et al., N Engl J Med 386:24-24, 2022) showed a median progression-free survival of 10.1 months, suggesting that the nivolumab-denosumab combination is effective in metastatic melanoma. The median overall survival for the denosumab-nivolumab combination was 21.8 months (range 4.4 months, 57.4+ months).

[0217] Based on patients evaluated to date, denosumab alone has rare antitumor activity and induces mild immune-mediated AEs. Denosumab in combination with a PD-1 inhibitor, such as nivolumab, appears to be well tolerated and has a higher RR than previous studies with single-agent PD-1 inhibitors.

Claims

1. 1. A method of treating melanoma in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of denosumab; the subject does not have bone metastases; The method, wherein the subject does not have hypercalcemia.

2. 1. A method of treating melanoma in a subject in need thereof, comprising administering a therapeutically effective amount of denosumab to the subject, wherein a baseline tumor tissue sample from the subject comprises active tumor-infiltrating lymphocytes (TILs).

3. The method of claim 2, wherein the subject does not have bone metastases.

4. The method of claim 2 or 3, wherein the subject does not have hypercalcemia.

5. the subject has not previously received a PD-1 inhibitor; or the subject has previously received a PD-1 inhibitor for stage III or stage IV melanoma, and the interval between the last dose of the PD-1 inhibitor and the date of recurrence is at least about 1 year; The method according to any one of claims 1 to 4.

6. 6. The method of any one of claims 1 to 5, wherein the subject is administered one or more loading doses of denosumab.

7. The method of any one of claims 1 to 6, wherein said administration is associated with an anti-tumor immune response and / or tumor objective response.

8. 1. A method of treating melanoma in a subject in need thereof, comprising co-administering to said subject a therapeutically effective amount of denosumab and a therapeutically effective amount of a PD-1 inhibitor; one or more loading doses of denosumab are administered to the subject prior to the first dose of the PD-1 inhibitor; The method, wherein said co-administration is associated with an anti-tumor immune response and / or tumor objective response.

9. 9. The method of claim 8, wherein the PD-1 inhibitor is nivolumab or pembrolizumab.

10. 10. The method of claim 8 or 9, wherein the subject has not previously received a PD-1 inhibitor.

11. 10. The method of claim 8 or 9, wherein the subject has previously received a PD-1 inhibitor for stage III or stage IV melanoma, and the interval between the last dose of the PD-1 inhibitor and the date of recurrence is at least 1 year.

12. 12. The method of any one of claims 1-11, wherein the therapeutically effective amount of denosumab comprises one or more unit doses, each administered at intervals of about four weeks, each unit dose independently comprising from about 45 mg to about 120 mg of denosumab.

13. 13. The method of any one of claims 1-12, wherein the therapeutically effective amount of denosumab is about 120 mg of denosumab administered in one or more unit doses at about four week intervals.

14. 14. The method of any one of claims 1-13, wherein the therapeutically effective amount of denosumab is about 120 mg of denosumab administered in one unit dose at about four week intervals.

15. 15. The method of any one of claims 1-14, wherein the one or more loading doses of denosumab each independently comprise one or more unit doses, each unit dose independently comprising from about 45 mg to about 120 mg of denosumab.

16. the one or more loading doses of denosumab a first loading dose comprising about 120 mg of denosumab administered on day 1; and a second loading dose comprising about 120 mg of denosumab administered on about day 8; The method according to any one of claims 6 to 15.

17. 17. The method of any one of claims 8-16, wherein the first dose of the PD-1 inhibitor is administered within about 24 hours of a third loading dose comprising about 120 mg of denosumab administered on about day 22.

18. the PD-1 inhibitor is pembrolizumab; the therapeutically effective amount of the PD-1 inhibitor comprises about 200 mg of pembrolizumab administered at about three-week intervals. The method according to any one of claims 8 to 17.

19. the PD-1 inhibitor is nivolumab; the therapeutically effective amount of the PD-1 inhibitor comprises about 480 mg of nivolumab administered at about four week intervals. The method according to any one of claims 8 to 17.

20. 20. The method of any one of claims 8 to 19, wherein the baseline tumor tissue sample from the subject comprises active tumor infiltrating lymphocytes (TILs).

21. the subject does not have bone metastases; and / or the subject does not have hypercalcemia; The method according to any one of claims 8 to 20.

22. 22. The method of any one of claims 1 to 21, wherein the subject is not concurrently administered an anti-CTLA4 agent.

23. 23. The method of any one of claims 1-22, wherein the subject is not concurrently administered ipilimumab.

24. 24. The method of any one of claims 1 to 23, wherein the melanoma is stage III melanoma or stage IV melanoma.

25. 25. The method of treatment of any one of claims 1 to 24, wherein the melanoma is cutaneous or mucosal melanoma.

26. The method of any one of claims 1 to 25, wherein the melanoma is unresectable.

27. a baseline peripheral blood sample from the subject containing high serum-free RANKL and / or low serum OPG levels; and / or a baseline tumor tissue sample from the subject expresses two or more of Sox10, RANK, and OPG; 27. The method according to any one of claims 1 to 26.

28. wherein the administering or co-administration is associated with an anti-tumor immune response; the anti-tumor immune response is a change in recent thymic egress cells in a peripheral blood sample from the subject compared to a baseline peripheral blood sample; and / or the anti-tumor immune response is a change in density of tumor-infiltrating differentiation group (CD8+) cells (TILs) in a tumor tissue sample from the subject compared to a baseline tumor tissue sample; 28. The method according to any one of claims 1 to 27.

29. 29. The method of any one of claims 1 to 28, wherein said administering or co-administering is associated with a complete or partial response as assessed using RECIST v1.1 criteria.

30. 30. The method of any one of claims 1 to 29, wherein said administering or co-administering is associated with stable disease as assessed using RECIST v1.1 criteria.

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