Compositions and methods for cancer immunotherapy

Abstract

The present disclosure provides compositions and improved methods for the treatment of cancer, comprising administering to a patient a fusion protein of SEQ ID NO:1 on a less frequent dosing schedule (i.e., a single dose once every three weeks or multiple doses on non-consecutive days).

Description

[Technical field]

[0001] Cross-reference to related art This application claims the benefit of U.S. Provisional Patent Application No. 63 / 309,199, filed February 11, 2022, the entire disclosure of which is incorporated herein by reference. [Background technology]

[0002] Interleukin-2 (IL-2) is a cytokine that induces the proliferation of antigen-activated T cells and stimulates natural killer (NK) cells. The biological activity of IL-2 is mediated through the multisubunit IL-2 receptor complex (IL-2R) of three polypeptide subunits that span the cell membrane: p55 (IL-2Rα, alpha subunit, also known as CD25 in humans), p75 (IL-2R13, beta subunit, also known as CD122 in humans), and p64 ​​(IL-2Rγ, gamma subunit, also known as CD132 in humans). T cell responses to IL-2 depend on a variety of factors, including (1) the concentration of IL-2, (2) the number of IL-2R molecules on the cell surface, and (3) the number of IL-2Rs occupied by IL-2 (i.e., the affinity of the binding interaction between IL-2 and IL-2R). The IL-2:IL-2R complex is internalized upon ligand binding, and different components undergo differential sorting. IL-2Rα is recycled to the cell surface, and IL-2 associated with the IL-2:IL-2Rβγ complex is routed to lysosomes for degradation.

[0003] Outcomes of systemic IL-2 administration in cancer patients are poor. While 15-20% of patients respond objectively to high-dose IL-2, the majority do not, and many suffer severe, life-threatening side effects. Aldesleukin (recombinant human IL-2 (rhIL-2), also known as Proleukin) is approved and used for the treatment of metastatic melanoma and RCC.

[0004] Among currently used therapies, rhIL-2 is one of the few treatment regimens that induces complete and durable responses in a subset of patients, up to 12% in melanoma and 7% in RCC. High doses of rhIL-2 inhibit memory CD8 + It is required to stimulate cells that express the intermediate affinity IL-2 receptor, including T cells and natural killer (NK) cells, the primary cell type mediating anti-cancer immune responses.

[0005] A contributing factor limiting the therapeutic efficacy of rhIL-2 is the immunosuppressive CD4 receptor, which can counteract anti-cancer immune responses. + T reg It has been hypothesized that the selective activation of T reg This is due to the binding of IL-2 to the high affinity IL-2 receptor expressed on endothelial cells. Furthermore, it is hypothesized that direct interaction between rhIL-2 and the high affinity IL-2R expressed on vascular and pulmonary endothelial cells contributes to rhIL-2-mediated toxicity via capillary leak syndrome. Despite being poorly tolerated, immunotherapy with rhIL-2 remains one of the few treatment regimens for metastatic melanoma and RCC that induces complete and durable responses in a subset of patients. Thus, new IL-2 therapies are needed to more effectively combat various cancers. Summary of the Invention

[0006] The compositions, methods, and treatment regimens according to the present disclosure offer many advantages for the treatment of cancer using IL-2 immunotherapy, for example, as compared to high dose rhIL-2 therapy (eg, aldesleukin).

[0007] In one embodiment, the disclosure provides a method of treating cancer in a patient, comprising administering to the patient a fusion protein comprising an amino acid sequence set forth in SEQ ID NO:1, or a fusion protein with at least 80% identity to the amino acid sequence set forth in SEQ ID NO:1, wherein the patient is administered at least a first dose of the fusion protein and a second dose of the fusion protein on non-consecutive days.

[0008] In certain embodiments, the fusion protein is administered at a dose of about 1 μg / kg to about 60 μg / kg.

[0009] In certain embodiments, the fusion protein is administered at a concentration of about 1 μg / kg, about 2 μg / kg, about 3 μg / kg, about 4 μg / kg, about 5 μg / kg, about 6 μg / kg, about 7 μg / kg, about 8 μg / kg, about 9 μg / kg, about 10 μg / kg, about 11 μg / kg, about 12 μg / kg, about 13 μg / kg, about 14 μg / kg, about 15 μg / kg, about 16 μg / kg, about 17 μg / kg, about 18 μg / kg, about 19 μg / kg, about 20 μg / kg, about 21 μg / kg, about 22 μg / kg, about 23 μg / kg, about 24 μg / kg, about 25 μg / kg, about 26 μg / kg, about 27 μg / kg, about 28 μg / kg, about 29 μg / kg, about 30 μg / kg, Approximately 31μg / kg, approximately 32μg / kg, approximately 33μg / kg, approximately 34μg / kg, approximately 35μg / kg, approximately 36μg / kg, approximately 37μg / kg, approximately 38μg / k g, about 39μg / kg, about 40μg / kg, about 41μg / kg, about 42μg / kg, about 43μg / kg, about 44μg / kg, about 45μg / kg, about 46μg / kg The antibody is administered at a dose of about 47 μg / kg, about 48 μg / kg, about 49 μg / kg, about 50 μg / kg, about 51 μg / kg, about 52 μg / kg, about 53 μg / kg, about 54 μg / kg, about 55 μg / kg, about 56 μg / kg, about 57 μg / kg, about 58 μg / kg, about 59 μg / kg, or about 60 μg / kg.

[0010] In certain embodiments, the fusion protein is administered at a dose of about 3 μg / kg, about 6 μg / kg, about 10 μg / kg, about 15 μg / kg, about 20 μg / kg, about 25 μg / kg, about 30 μg / kg, about 35 μg / kg, or about 40 μg / kg.

[0011] In certain embodiments, the first dose and the second dose are the same. In certain embodiments, the first dose and the second dose are different.

[0012] In certain embodiments, the patient is administered a third dose of the fusion protein.

[0013] In certain embodiments, a patient is administered a first dose of the fusion protein on day 1 and a second dose of the fusion protein on any of the following days: the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, or 21st.

[0014] In a specific embodiment, the patient is administered a first dose of the fusion protein on day 1 and a second dose of the fusion protein on day 4.

[0015] In a specific embodiment, the patient is administered a first dose of the fusion protein on day 1 and a second dose of the fusion protein on day 8.

[0016] In a specific embodiment, the patient is administered a first dose of the fusion protein on day 1 and a second dose of the fusion protein on day 15.

[0017] In certain embodiments, the patient is administered the second dose of the fusion protein and the third dose of the fusion protein on consecutive days, hi certain embodiments, the patient is administered the second dose of the fusion protein and the third dose of the fusion protein on non-consecutive days.

[0018] In certain embodiments, the patient is administered a third dose of the fusion protein 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the second dose of the fusion protein.

[0019] In certain embodiments, a patient is administered a first dose of the fusion protein on day 1, a second dose of the fusion protein on any of the following days: the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, or 12th, and a third dose of the fusion protein on any of the following days: the 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, or 21st.

[0020] In certain embodiments, a patient is administered a first dose of the fusion protein on day 1, a second dose of the fusion protein on any of day 3, 4, or 5, and a third dose of the fusion protein on any of day 6, 7, 8, 9, or 10.

[0021] In certain embodiments, a patient is administered a first dose of the fusion protein on day 1, a second dose of the fusion protein on any of day 6, 7, 8, 9, or 10, and a third dose of the fusion protein on any of day 13, 14, 15, 16, or 17.

[0022] In a specific embodiment, a patient is administered a first dose of the fusion protein on day 1, a second dose of the fusion protein on day 4, and a third dose of the fusion protein on day 8.

[0023] In a specific embodiment, a patient is administered a first dose of the fusion protein on day 1, a second dose of the fusion protein on day 8, and a third dose of the fusion protein on day 15.

[0024] In certain embodiments, the patient is administered a first dose of about 10 μg / kg to about 30 μg / kg of the fusion protein on day 1, and a second dose of about 10 μg / kg to about 30 μg / kg of the fusion protein on any of the following days: day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, or day 21.

[0025] In certain embodiments, the patient is administered a first dose of about 10 μg / kg to about 15 μg / kg of the fusion protein on day 1, and a second dose of about 10 μg / kg to about 15 μg / kg of the fusion protein on any of day 3, 4, 5, or 6.

[0026] In certain embodiments, the patient is administered a first dose of about 10 μg / kg to about 25 μg / kg of the fusion protein on day 1, and a second dose of about 20 μg / kg to about 25 μg / kg of the fusion protein on any of day 6, 7, 8, 9, or 10.

[0027] In a specific embodiment, the patient is administered a first dose of about 15 μg / kg of the fusion protein on day 1, and a second dose of about 15 μg / kg of the fusion protein on day 4.

[0028] In certain embodiments, the patient is administered a first dose of about 20 μg / kg to about 25 μg / kg of the fusion protein on day 1 and a second dose of about 20 μg / kg to about 25 μg / kg of the fusion protein on day 8.

[0029] In certain embodiments, the patient is administered a first dose of about 6 μg / kg to about 25 μg / kg of the fusion protein on day 1, a second dose of about 6 μg / kg to about 25 μg / kg of the fusion protein on any of day 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and a third dose of about 6 μg / kg to about 25 μg / kg of the fusion protein on any of day 13, 14, 15, 16, 17, 18, 19, 20, or 21.

[0030] In certain embodiments, the patient is administered a first dose of about 6 μg / kg to about 15 μg / kg of the fusion protein on day 1, a second dose of about 6 μg / kg to about 15 μg / kg of the fusion protein on any of day 3, day 4, or day 5, and a third dose of about 6 μg / kg to about 15 μg / kg of the fusion protein on any of day 6, day 7, day 8, day 9, or day 10.

[0031] In certain embodiments, the patient is administered a first dose of about 10 μg / kg to about 20 μg / kg of the fusion protein on day 1, a second dose of about 10 μg / kg to about 20 μg / kg of the fusion protein on any of day 6, 7, 8, 9, or 10, and a third dose of about 10 μg / kg to about 20 μg / kg of the fusion protein on any of day 13, 14, 15, 16, or 17.

[0032] In a specific embodiment, the patient is administered a first dose of about 15 μg / kg of the fusion protein on day 1, a second dose of about 15 μg / kg of the fusion protein on day 4, and a third dose of about 15 μg / kg of the fusion protein on day 8.

[0033] In a specific embodiment, the patient is administered a first dose of about 20 μg / kg of the fusion protein on day 1, a second dose of about 20 μg / kg of the fusion protein on day 8, and a third dose of about 20 μg / kg of the fusion protein on day 15.

[0034] In certain embodiments, the first dose, the second dose, and the third dose are the same. In certain embodiments, the first dose, the second dose, and the third dose are different.

[0035] In certain embodiments, the patient is administered a fourth dose of the fusion protein.

[0036] In certain embodiments, the patient is administered a third dose of the fusion protein and a fourth dose of the fusion protein on consecutive days.

[0037] In certain embodiments, the patient is administered a third dose of the fusion protein and a fourth dose of the fusion protein on non-consecutive days.

[0038] In another aspect, the disclosure provides a method of treating cancer in a patient, comprising administering to the patient a fusion protein comprising an amino acid sequence set forth in SEQ ID NO:1, or a fusion protein with at least 80% identity to the amino acid sequence set forth in SEQ ID NO:1, wherein the patient is administered at least a first dose of the fusion protein, wherein the patient is not administered a dose of the fusion protein at least one day prior to the first dose, and wherein the patient is not administered a dose of the fusion protein one day after the first dose.

[0039] In another aspect, the disclosure provides a method of treating cancer in a patient, comprising administering to the patient a fusion protein comprising an amino acid sequence set forth in SEQ ID NO:1, or a fusion protein with at least 80% identity to the amino acid sequence set forth in SEQ ID NO:1, wherein the patient is administered a single dose of the fusion protein once a week (Q1W), once every two weeks (Q2W), once every three weeks (Q3W), or once every four weeks (Q4W).

[0040] In certain embodiments, the fusion protein is administered at a dose of about 10 μg / kg to about 60 μg / kg.

[0041] In certain embodiments, the fusion protein is administered at a dose of about 10 μg / kg, about 15 μg / kg, about 20 μg / kg, about 25 μg / kg, about 30 μg / kg, about 35 μg / kg, about 40 μg / kg, about 45 μg / kg, about 50 μg / kg, about 55 μg / kg or about 60 μg / kg.

[0042] In certain embodiments, the fusion protein is administered at a dose of about 10 μg / kg, about 15 μg / kg, about 20 μg / kg, about 25 μg / kg, about 30 μg / kg, about 35 μg / kg, or about 40 μg / kg.

[0043] In certain embodiments, administration of the fusion protein results in a dose-dependent increase in circulating NK cells and CD8+ cells in the patient in the absence of a dose-dependent increase in T regulatory (Treg) cells. In certain embodiments, the increase in circulating NK cells and CD8+ cells is at least two-fold over baseline. In certain embodiments, the increase in circulating NK cells and CD8+ cells is greater than the increase in circulating Treg cells.

[0044] In certain embodiments, the increase in circulating NK cells and CD8+ cells is greater compared to the increase in circulating Treg cells compared to the increase in circulating NK cells and CD8+ cells compared to the increase in circulating Treg cells in patients receiving high dose rhIL-2 therapy.

[0045] In certain embodiments, patients have an improved safety profile compared to patients receiving high-dose recombinant human IL-2 (rhIL-2) therapy.

[0046] In certain embodiments, the patient is at reduced risk of capillary leak syndrome or cytokine release syndrome and / or the patient is at reduced risk of cytokine release syndrome compared to patients receiving high dose rhIL-2 therapy.

[0047] In certain embodiments, the methods result in a dose-dependent expansion of circulating NK cells and CD8+ cells in the patient in the absence of a dose-dependent expansion of circulating regulatory T (Treg) cells, a greater increase in circulating NK cells and CD8+ cells compared to the increase in circulating T regulatory (Treg) cells compared to the increase in circulating Treg cells in patients receiving high dose recombinant human IL-2 (rhIL-2) therapy, and the patient is at lower risk of capillary leak syndrome.

[0048] In certain embodiments, each dose is administered by intravenous (IV) injection or infusion.

[0049] In certain embodiments, each dose is administered by intravenous (IV) injection or infusion over a period of about 30 minutes, about 60 minutes, or about 90 minutes.

[0050] In certain embodiments, the method further comprises administering to the patient a therapeutically effective amount of a therapeutic agent.

[0051] In certain embodiments, the therapeutic agent is a PARP inhibitor, an immune checkpoint inhibitor, a cytotoxic agent, or a chemotherapeutic agent.

[0052] In certain embodiments, the therapeutic agent is an immune checkpoint inhibitor.

[0053] In certain embodiments, the immune checkpoint inhibitor inhibits the interaction of PD-1 and PD-L1.

[0054] In certain embodiments, the immune checkpoint inhibitor is pembrolizumab.

[0055] In certain embodiments, pembrolizumab is administered prior to, simultaneously with, or after administration of the fusion protein of SEQ ID NO:1.

[0056] In certain embodiments, pembrolizumab is administered in a separate composition from the fusion protein of SEQ ID NO:1.

[0057] In certain embodiments, pembrolizumab is administered in an amount of 200 mg by intravenous injection or infusion.

[0058] In certain embodiments, pembrolizumab is administered once a week (Q1W), once every two weeks (Q2W), once every three weeks (Q3W), or once every four weeks (Q4W).

[0059] In certain embodiments, pembrolizumab is administered in an amount of 200 mg Q3W by intravenous injection or infusion.

[0060] In certain embodiments, pembrolizumab is administered on the same day as the first dose of the fusion protein.

[0061] In certain embodiments, the cancer to be treated is a solid tumor.

[0062] In certain embodiments, the solid tumor is a carcinoma, sarcoma, or lymphoma.

[0063] In certain embodiments, the cancer to be treated is renal cell carcinoma (RCC), melanoma, mucosal melanoma, breast cancer, pancreatic cancer, prostate cancer, non-small cell lung cancer, liver cancer, colorectal cancer, bladder cancer, cervical cancer, ovarian cancer, thyroid cancer, esophageal cancer, oral cancer, mesothelioma, non-melanoma skin cancer, and / or gastric cancer.

[0064] In certain embodiments, the cancer to be treated is a hematological cancer.

[0065] In certain embodiments, the hematological cancer is leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, and multiple myeloma.

[0066] In one embodiment, the disclosure provides a pharmaceutical composition comprising a fusion protein comprising the amino acid sequence set forth in SEQ ID NO:1, or a fusion protein having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:1, wherein the pharmaceutical composition comprises a unit dose of about 1 μg / kg to about 60 μg / kg.

[0067] In certain embodiments, the unit dose is about 1 μg / kg, about 2 μg / kg, about 3 μg / kg, about 4 μg / kg, about 5 μg / kg, about 6 μg / kg, about 7 μg / kg, about 8 μg / kg, about 9 μg / kg, about 10 μg / kg, about 11 μg / kg, about 12 μg / kg, about 13 μg / kg, about 14 μg / kg, about 15 μg / kg, about 16 μg / kg, about 17 μg / kg, about 18 μg / kg, about 19 μg / kg, about 20 μg / kg, about 21 μg / kg, about 22 μg / kg, about 23 μg / kg, about 24 μg / kg, about 25 μg / kg, about 26 μg / kg, about 27 μg / kg, about 28 μg / kg, about 29 μg / kg, about 30 μg / kg, about 31 μg / kg, about 32 μg / kg, about 33 μg / kg, about 34 μg / kg, about 35 μg / kg, about 36 μg / kg, about 37 μg / kg, about 38 μg / kg, about 39 μg / kg, about 40 μg / kg, about 41 μg / kg, about 42 μg / kg, about 43 μg / kg, about 44 μg / kg, about 45 μg / kg, about 46 μg / kg, about 47 μg / kg, about 48 μg / kg, about 49 μg / kg, about 50 μg / kg, about 51 μg / kg, about 52 μg / kg, about 53 μg / kg, about 54 μg / kg, about 55 μg / kg, about 56 μg / kg, about 57 μg / kg g, about 31 μg / kg, about 32 μg / kg, about 33 μg / kg, about 34 μg / kg, about 35 μg / kg, about 36 μg / kg, about 37 μg / kg, about 38 μg / kg, about 39 μg / kg, about 40 μg / kg, about 41 μg / kg, about 42 μg / kg, about 43 μg / kg, about 44 μg / kg, about 45 μg / kg, about 46 μg / kg, about 47 μg / kg, about 48 μg / kg, about 49 μg / kg, about 50 μg / kg, about 51 μg / kg, about 52 μg / kg, about 53 μg / kg, about 54 μg / kg, about 55 μg / kg, about 56 μg / kg, about 57 μg / kg, about 58 μg / kg, about 59 μg / kg, or about 60 μg / kg.

[0068] In certain embodiments, the unit dose is about 3 μg / kg, about 6 μg / kg, about 10 μg / kg, about 15 μg / kg, about 20 μg / kg, about 25 μg / kg, about 30 μg / kg, about 35 μg / kg, or about 40 μg / kg. [Brief description of the drawings]

[0069] The foregoing and other objects, features and advantages of the present invention will become apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views, the drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.

[0070] [Figure 1] Figure 1 shows a schematic of the study design of a less frequent dosing regimen with the fusion protein of SEQ ID NO: 1 (nembareukin). Abbreviations: D = day, IV = intravenous, n = number of patients, Q3W = every 3 weeks, MTD = maximum tolerated dose. [Figure 2A-2B] Figures 2A-B depict simulations of CD8+ T cells in blood (Figure 2A) and tumor (Figure 2B) for a 1 dose / 21 day cycle dosing regimen. Simulations were performed for doses of 15 μg / kg, 23 μg / kg, 30 μg / kg, 37 μg / kg, and 45 μg / kg, compared to 3 μg / kg and 6 μg / kg for the previous qdx5 dosing regimen. [Figure 3A-3B] Figures 3A-B depict simulations of NK cells in blood (Figure 3A) and tumor (Figure 3B) for a 1 dose / 21 day cycle dosing regimen. Simulations were performed for doses of 15 μg / kg, 23 μg / kg, 30 μg / kg, 37 μg / kg, and 45 μg / kg, compared to 3 μg / kg and 6 μg / kg for the previous qdx5 dosing regimen. [Figure 4A-4B] Figures 4A-B depict simulations of CD8+ T cells in blood (Figure 4A) and tumor (Figure 4B) for a 2 dose, day 1 and day 4 dosing / 21 day cycle dosing regimen. Simulations were performed for doses of 7.5 μg / kg, 10 μg / kg, and 15 μg / kg, compared to 3 μg / kg and 6 μg / kg for the previous qdx5 dosing regimen. [Figure 5A-5B]Figures 5A-B depict simulations of NK cells in blood (Figure 5A) and tumor (Figure 5B) for a 2 dose, day 1 and day 4 dosing / 21 day cycle dosing regimen. Simulations were performed for doses of 7.5 μg / kg, 10 μg / kg, and 15 μg / kg, compared to 3 μg / kg and 6 μg / kg for the previous qdx5 dosing regimen. [Figure 6A-6B] Figures 6A-6B depict simulations of CD8+ T cells in blood (Figure 6A) and tumor (Figure 6B) for a 2 dose, day 1 and day 8 dosing / 21 day cycle dosing regimen. Simulations were performed for doses of 7.5 μg / kg, 10 μg / kg, 15 μg / kg, and 15 μg / kg, compared to 3 μg / kg and 6 μg / kg for the previous qdx5 dosing regimen. [Figure 7A-7B] 7A-7B depict simulations of NK cells in blood (FIG. 7A) and tumor (FIG. 7B) for a 2 dose, day 1 and day 8 dosing / 21 day cycle dosing regimen. Simulations were performed for doses of 7.5 μg / kg, 10 μg / kg, 15 μg / kg, and 15 μg / kg, compared to 3 μg / kg and 6 μg / kg for the previous qdx5 dosing regimen. [Figure 8A-8B] 8A-8B depict simulations of CD8+ T cells in blood (FIG. 8A) and tumor (FIG. 8B) for a 3 dose, day 1, 4, 8 dosing / 21 day cycle dosing regimen. Simulations were performed for doses of 5 μg / kg, 7.5 μg / kg, and 10 μg / kg, compared to 3 μg / kg and 6 μg / kg for the previous qdx5 dosing regimen. [Figure 9A-9B] 9A-9B depict simulations of NK cells in blood (FIG. 9A) and tumor (FIG. 9B) for a 3 dose, day 1, 4, 8 dosing / 21 day cycle dosing regimen. Simulations were performed for doses of 5 μg / kg, 7.5 μg / kg, and 10 μg / kg, compared to 3 μg / kg and 6 μg / kg for the previous qdx5 dosing regimen. [Figure 10A-10B]10A-10B depict simulations of CD8+ T cells in blood (FIG. 10A) and tumor (FIG. 10B) for a 3 dose, day 1, 8, 15 dosing / 21 day cycle dosing regimen. Simulations were performed for doses of 5 μg / kg, 7.5 μg / kg, 10 μg / kg, and 15 μg / kg, compared to 3 μg / kg and 6 μg / kg for the previous qdx5 dosing regimen. [Figure 11A-11B] 11A-11B depict simulations of NK cells in blood (FIG. 11A) and tumor (FIG. 11B) for a 3 dose, day 1, 8, 15 dosing / 21 day cycle dosing regimen. Simulations were performed for doses of 5 μg / kg, 7.5 μg / kg, 10 μg / kg, and 15 μg / kg, compared to 3 μg / kg and 6 μg / kg for the previous qdx5 dosing regimen. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] The dosing regimens described herein are designed for less frequent administration (i.e., administration of fusion protein of SEQ ID NO:1 on non-consecutive days). These dosing regimens have the advantage of being easy to administer for patients and medical personnel without substantial reduction in safety and efficacy. These dosing regimens represent an improvement over existing dosing regimens of fusion protein of SEQ ID NO:1, which require administration on 5 consecutive days (qdx5) in each 21-day cycle of treatment (i.e., Q3W cycle).

[0072] Thus, in one embodiment, the disclosure provides a method of treating cancer in a patient comprising administering to the patient a fusion protein comprising an amino acid sequence set forth in SEQ ID NO:1, or a fusion protein having at least 80% identity to the amino acid sequence set forth in SEQ ID NO:1, wherein the patient is administered at least a first dose of the fusion protein and a second dose of the fusion protein on non-consecutive days.

[0073] In another aspect, the disclosure provides a method of treating cancer in a patient, comprising administering to the patient a fusion protein comprising an amino acid sequence set forth in SEQ ID NO:1, or a fusion protein with at least 80% identity to the amino acid sequence set forth in SEQ ID NO:1, wherein the patient is administered at least a first dose of the fusion protein, wherein the patient is not administered a dose of the fusion protein at least one day prior to the first dose, and wherein the patient is not administered a dose of the fusion protein one day after the first dose.

[0074] definition Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. The scope of the invention is not intended to be limited to the following description, but rather is as set forth in the appended claims.

[0075] In the claims, articles such as "a," "an," "the," etc. may mean one or more, unless otherwise indicated or clear from the context. A claim or description containing "or" between one or more members of a group is deemed satisfied if one, more than one, or all of the members of the group are present in, employed in, or otherwise relevant to a given product or process, unless otherwise indicated or clear from the context. The invention includes embodiments in which exactly one member is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which two or more, or all of the members of a group are present in, employed in, or otherwise relevant to a given product or process.

[0076] It should also be noted that the term "comprising" is intended to be inclusive, permitting but not requiring the inclusion of additional elements or steps. When the term "comprising" is used herein, therefore, the term "consisting of" is also included and disclosed.

[0077] Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or clear from the context and the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can assume from any particular value or subrange within the ranges set forth in different embodiments of the present disclosure to one tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0078] As used herein, the term "about" or "approximately" as applied to one or more subject values ​​refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that is within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less) of the stated reference value, unless otherwise stated or clear from the context (except where such number exceeds 100% of possible values).

[0079] The term "substantially" as used herein refers to a qualitative condition that indicates the extent or degree of the total or near total of a property or attribute of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or completeness, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness that is inherent in many biological and chemical phenomena.

[0080] As used herein, any form of administration or co-administration of a "combination," "combination therapy," and / or a "combination therapeutic regimen" refers to at least two therapeutically active agents or compositions that can be administered simultaneously, either in separate or combined formulations, or sequentially or co-administered at different times separated by minutes, hours, or days. Generally, each agent is administered at a dose and / or time schedule determined for that agent.

[0081] The term "parenteral" as used herein refers to dosage forms intended for administration by injection or infusion, and includes subcutaneous, intravenous, intraarterial, intraperitoneal, intracardiac, intrathecal, and intramuscular injections, as well as infusion injections, usually by the intravenous route.

[0082] The term "therapeutic agent" encompasses any agent that is administered in addition to, or in combination with, the fusion protein of SEQ ID NO: 1 to treat a condition or disease in an individual in need of such treatment. Such additional therapeutic agents may include any active ingredients appropriate for the particular indication being treated, preferably ingredients with complementary activities that do not adversely affect each other.

[0083] The term "chemotherapeutic agent" refers to a compound or its derivatives that can interact with cancer cells, thereby reducing the proliferation state of the cells and / or effectively targeting fast-dividing cells, for example by impairing cell division or DNA synthesis (or by damaging DNA), resulting in cell death. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate (MTX), 5-fluorouracil or its derivatives), substituted nucleotides, substituted nucleosides, DNA demethylating agents (also known as antimetabolites, e.g., azacitidine), antitumor antibiotics (e.g., mitomycin, adriamycin), plant-derived antitumor agents (e.g., vincristine, vindesine, TAXOL®, paclitaxel, abraxane), cisplatin, carboplatin, etoposide, etc. Such agents include, but are not limited to, the anticancer agents trimethotrexate (TMTX), temozolomide, raltitrexed, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benziguanidine (6-BG), nitrosoureas, nitrosoureas (rabinopyranosyl-N-methyl-N-nitrosourea (alanose), carmustine (BCNU, BiCNU), chlorozotocin, ethylnitrosourea (ENU), fotemustine, lomustine (CCNU), nimustine, N-nitroso-N-methylurea (NMU), ranimustine (MCNU), semustine, streptozocin (streptozotocin)), cytarabine, camptothecin, or any therapeutic derivatives thereof.

[0084] As used herein, a single treatment "course", such as a first course, second course, third course, etc., refers to a treatment regimen in which the fusion protein is administered for a desired period of time, such as two or three doses on non-consecutive days of treatment, followed by a rest period of a certain number of consecutive days.

[0085] The term "fusion protein" refers to a protein or peptide joined together with another protein or peptide by inserting a first protein or peptide into an internal region of the second protein or peptide by a peptide bond between their respective N-terminal and C-terminal amino acid residues or vice versa, or by two peptide bonds at the N-terminus and C-terminus of the inserted protein or peptide. A peptide bond is a covalent chemical bond formed between the carboxyl group of one amino acid and the amine group of another amino acid. A fusion protein is produced by expression of a fusion protein gene in an expression host, in which the coding sequence of the first protein or peptide is joined to the coding sequence of the second protein or peptide.

[0086] The present disclosure also contemplates the use of variants of fusion proteins of SEQ ID NO: 1 having amino acid sequences with about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity over a contiguous stretch of about 20 amino acids up to the full length of SEQ ID NO: 1. Variants of SEQ ID NO: 1 can have a defined sequence identity compared to SEQ ID NO: 1 over a defined length of contiguous amino acids (e.g., a "comparison window"). Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), or by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), or by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), or by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Madison, Wis.)), or by manual alignment and visual inspection (see, for example, Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

[0087] As an example, a variant of a fusion protein of SEQ ID NO:1 can include an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% identity of the amino acid sequence over a continuous section of SEQ ID NO:1, of at least 20, preferably from about 20 amino acids to about 40 amino acids, from about 40 amino acids to about 60 amino acids, from about 60 amino acids to about 80 amino acids, from about 80 amino acids to about 100 amino acids, from about 100 amino acids to about 120 amino acids, from about 120 amino acids to about 140 amino acids, from about 140 amino acids to about 150 amino acids, from about 150 amino acids to about 155 amino acids, from about 155 amino acids to the full length of SEQ ID NO:1.

[0088] The term "IL-2 therapy" refers to + Maintenance of regulatory T cells and CD4 expression in various subsets + and administering IL-2 and its associated biological functions as an immunotherapy, including but not limited to differentiation of CD8 T cells. + Promotes T cell and NK cytotoxic activity, regulates T cell differentiation programs in response to antigen, and mediates the differentiation of naive CD4+ into T helper-1 (Th1) and T helper-2 (Th2) cells while inhibiting T helper-17 (Th17) differentiation. + and promoting T cell differentiation. Thus, as used herein, "IL-2 therapy" includes, but is not limited to, immunotherapy with rhIL-2 or a variant of rhIL-2, such as the fusion protein of SEQ ID NO:1.

[0089] The terms "high dose IL-2" and "HD IL-2" include doses of interleukin-2 (IL-2) of about or at least about 600,000 international units (IU) per kg of body weight per dose, or about or at least about 720,000 IU / kg per dose.

[0090] The terms "low dose IL-2" and "LD IL-2" include doses of interleukin-2 (IL-2) of less than about 600,000 IU / kg body weight / dose, e.g., about 60,000 or about 72,000 IU / kg / dose, e.g., between about 60,000 and about 72,000 IU / kg / dose.

[0091] The term "subject" or "patient" as used herein refers to any organism to which a composition according to the present disclosure may be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Exemplary subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, humans, etc.) and / or plants. Preferably, "patient" refers to a human subject who may be seeking or in need of treatment, or who is undergoing or will be undergoing treatment, or a subject receiving care from a trained professional for a particular disease or condition. A "patient" may be a child (over 1 year old to 17 years old). In yet other embodiments, the patient may be an infant (1 year old or younger). In yet other embodiments, the patient may be a pediatric patient, where the term "child" is used as understood by those skilled in the art. For example, a pediatric patient includes an infant, a child, and an adolescent.

[0092] The phrase "pharmacologically acceptable" is used herein to refer to compounds, substances, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio.

[0093] The term "protein" or "peptide" as used herein refers to at least two or more amino acid residues joined together by peptide bonds. The amino acid sequence in a protein or peptide is presented in standard format, i.e., from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus).

[0094] The term "recombinant production" refers to techniques for manipulating and combining two or more DNA sequences together, including recombination, PCR (polymerase chain reaction), in vitro mutagenesis, and direct DNA synthesis. These techniques are described in numerous published books and manuals, including Current protocols in molecular biology (Ausubel eds. 2008, John Wiley & Son).

[0095] The term "substantially" as used herein refers to a qualitative condition that indicates the extent or degree of the total or near total of a property or attribute of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or completeness, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness that is inherent in many biological and chemical phenomena.

[0096] The term "therapeutically effective amount" or "effective amount" refers to the administration of an agent to a subject, either alone or as part of a pharmaceutical composition, and either in a single dose or as part of a series of doses, in an amount that can have any detectable and positive effect on any symptom, aspect, or characteristic of a disease, disorder, or condition when administered to a subject. A therapeutically effective amount can be ascertained by measuring the relevant physiological effect, and can be adjusted in conjunction with dosing regimens and diagnostic analysis of the subject's condition, and the like. As an example, measuring the amount of inflammatory cytokines produced after administration can indicate whether a therapeutically effective amount has been used. In the context of cancer or a condition associated with uncontrolled cell division, a therapeutically effective amount refers to an amount that has the effect of (1) reducing the size of a tumor (i.e., tumor regression), (2) inhibiting (i.e., slowing to some extent, preferably stopping) abnormal cell division, e.g., cancer cell division, (3) preventing or reducing metastasis of cancer cells, and / or (4) alleviating to some extent (or preferably eliminating) one or more symptoms associated with a pathology associated with or partially caused by uncontrolled or abnormal cell division (e.g., cancer, etc.). An effective amount is also an amount that results in a desirable PD and PK profile, as well as a desirable immune cell profiling, upon administration of the therapeutically active composition of the invention.

[0097] The term "treat" or "treatment" as used herein refers to inhibiting a disease, e.g., inhibiting a disease, condition or disorder (i.e., halting further development of the pathology and / or symptoms) in an individual experiencing or exhibiting the pathology or symptoms of the disease, condition or disorder, or ameliorating a disease, e.g., reducing the severity of the disease, or improving a disorder, condition or disorder (i.e., reversing the pathology and / or symptoms) in an individual experiencing or exhibiting the pathology or symptoms of the disease, condition or disorder.

[0098] The term "prevent" or "prevention" as used herein includes the prevention of at least one symptom associated with or caused by the condition, disease, or disorder being prevented.

[0099] "Progression-free survival (PFS)" as used in the context of cancer described herein refers to the length of time until a patient's objective tumor progression or death during and after cancer treatment. Treatment can be evaluated by objective or subjective parameters, including the results of physical examination, neurological examination, or psychiatric evaluation. In a preferred embodiment, PFS can be evaluated by investigator or independent central review committee.

[0100] "Overall survival (OS)" may be assessed by the OS rate at a specific time point (e.g., 1 year and 2 years) by the Kaplan-Meier method, with the corresponding 95% CI derived based on the Greenwood formula by investigational drug treatment for each tumor type. The OS rate is defined as the proportion of participants alive at that time point. A participant's OS is defined as the time from the date of first drug administration to the date of death from any cause.

[0101] As used herein, a "complete response" is either the disappearance of all signs of cancer in response to treatment or target lesions measured <5 mm or <10 mm.

[0102] As used herein, the term "partial response" refers to a reduction in the size of a tumor or a reduction in the extent of cancer in the body in response to treatment. A partial response may also be referred to herein as a "partial remission."

[0103] As used herein, the term "cancer" is intended to be given its ordinary meaning as a general term for diseases in which abnormal cells divide without control.

[0104] As used herein, the term "tumor reducing" or "tumor regression" refers to a decrease in the size or volume of a tumor mass, a decrease in the number of metastatic tumors in a subject, a decrease in the proliferative state of cancer cells (the extent to which cancer cells proliferate), and the like.

[0105] The term "enhancement" as used herein refers to enabling a subject or tumor cells to improve their ability to respond to a treatment disclosed herein. For example, an enhanced response can include an increase in responsiveness of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more. "Enhancement" as used herein can also refer to the enhancement of the number of subjects that respond to a treatment, such as chemotherapy, drug-resistant immunocompetent cells, and combination therapy including immune checkpoint inhibitors. For example, an enhanced response may refer to the total percentage of subjects that respond to a treatment, which is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more.

[0106] "Immune checkpoint proteins" regulate T cell function in the immune system. T cells play a central role in cell-mediated immunity. Immune checkpoint proteins interact with specific ligands that send signals to T cells and essentially switch or inhibit T cell function. Cancer cells exploit this system by driving high levels of expression of immune checkpoint proteins on their surface, resulting in the control of T cells that express immune checkpoint proteins on the surface of T cells that enter the tumor microenvironment, thus suppressing anti-cancer immune responses. Thus, inhibition of immune checkpoint proteins by agents referred to herein as "immune checkpoint protein inhibitors" or "immune checkpoint inhibitors" results in restoration of T cell function and immune responses against cancer cells. Examples of immune checkpoint proteins include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, OX40, B-7 family ligands, or combinations thereof. In certain embodiments, the immune checkpoint inhibitor interacts with a ligand of an immune checkpoint protein, which can be CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, OX40, A2aR, B-7 family ligands, or combinations thereof. Examples of immune checkpoint inhibitors include, but are not limited to, a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, an adenosine A2A receptor antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a KIR antagonist, a LAG3 antagonist, a TIM-3 antagonist, a VISTA antagonist, or a TIGIT antagonist.

[0107] The term "angiogenesis inhibitors" as used herein refers to drugs, compounds, antibodies, or other agents that prevent new blood vessels from forming. In cancer therapy, angiogenesis inhibitors may prevent the growth of new blood vessels that tumors need to grow. Angiogenesis inhibitors include agents that can target one or more signaling pathways associated with receptor tyrosine kinases (RTKs). RTKs include, but are not limited to, vascular endothelial growth factor receptors types 1, 2, and 3 (VEGFR1-3), platelet-derived growth factor receptors alpha and beta (PDGFRα / β), and fibroblast growth factor receptors (FGFR) types 1, 2, and 3 (FGFR1-3). In certain embodiments, angiogenesis inhibitors have broad target selectivity and are capable of simultaneous targeted inhibition of multiple RTKs, referred to herein as "multiple receptor tyrosine kinase inhibitors."

[0108] Fusion protein of SEQ ID NO: 1 The recombinant human IL-2 variant fusion protein described in WO2013 / 184942 is a circularly permuted (cp) IL-2 variant fused to the extracellular domain of the IL-2Rα portion of the IL-2 receptor, referred to herein as the "fusion protein of SEQ ID NO: 1", or "nembareukin", or "nembareukin alpha", and has the following amino acid sequence: SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG (SEQ ID NO: 1).

[0109] It is contemplated that fusion proteins closely related to SEQ ID NO: 1 may also be suitable for administration by the methods of the invention, such as fusion proteins having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity over the entire length of SEQ ID NO: 1. It is contemplated that fusion proteins closely related to SEQ ID NO: 1 may also be suitable for administration by the methods of the invention, such as fusion proteins having about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity over a contiguous sequence of at least about 20 amino acids to the entire length of SEQ ID NO: 1.

[0110] The fusion protein of SEQ ID NO:1 can be produced using a biological recombinant expression system or any protein synthesis device. Strategies for recombinant protein expression are well known in the art and typically involve transfecting cells with a DNA vector containing a gene template encoding the fusion protein of SEQ ID NO:1, and then culturing the cells so that they transcribe and translate the fusion protein. Typically, the cells are then lysed to extract the expressed protein for subsequent purification. Both prokaryotic and eukaryotic in vivo protein expression systems are widely used. The fusion protein of SEQ ID NO:1 is preferably produced in CHO cells.

[0111] The present invention provides pharmaceutical compositions of the fusion protein of SEQ ID NO:1 at a dose of at least about 1 μg / kg to about 60 μg / kg.

[0112] As used herein, a pharma- ceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, appropriate for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21 stEdition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006, incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and well-known techniques for their preparation.

[0113] Except insofar as any conventional excipient medium is incompatible with the substance or its derivatives, such as by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition, its use is contemplated within the scope of the present disclosure.

[0114] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs.In addition to active ingredients, liquid dosage forms contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, rice germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof.

[0115] In addition to inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and / or perfuming agents. In certain embodiments for parenteral administration, the compositions are mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0116] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to known techniques using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in non-toxic parenterally acceptable diluents and / or solvents, for example, as solutions in 1,3-butanediol. Acceptable vehicles and solvents that may be employed include water, Ringer's solution, USP, and isotonic sodium chloride solution. Sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any non-irritating fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid, may be used in the preparation of injectables.

[0117] Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter and / or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium before use. General considerations in the formulation and / or manufacture of pharmaceuticals can be found, for example, in Remington's The Science and Practice of Pharmacy, 21 st Edition, AR Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006, incorporated herein by reference).

[0118] The fusion protein of SEQ ID NO:1 is designed to selectively bind and activate the intermediate affinity IL-2R, but not the high affinity IL-2R. The IL-2Rα domain of the fusion protein of SEQ ID NO:1 serves to sterically inhibit the binding of the fusion protein of SEQ ID NO:1 to the high affinity IL-2R, while still allowing binding to the intermediate affinity IL-2R.

[0119] In vitro and in vivo nonclinical pharmacodynamic (PD) data support selective signaling through the intermediate affinity IL-2 receptor by the fusion protein of SEQ ID NO:1 and demonstrate immunosuppressive properties. Treg The results demonstrate that the fusion protein of SEQ ID NO:1 results in the activation and proliferation of effector cells, such as NK cells and CD8+ cells, while minimizing the activation and expansion of T cells. reg at doses that induce similar or greater proliferation of effector cells compared to rhIL-2 and show improved tolerability compared to rhIL-2.

[0120] Additional non-clinical data demonstrate that IV or SC administration of the fusion protein of SEQ ID NO:1 results in comparable tumor growth inhibition in a mouse syngeneic tumor model, as well as increased NK cell and CD8 cell proliferation following either route of administration in cynomolgus monkeys. + These results suggest that IL-16 results in similar peripheral proliferation of T cells.

[0121] Initial data in human clinical data described in Example 1 show that the fusion protein of SEQ ID NO: 1 activates the proliferation of CD8+ and NK cells in a dose-dependent manner in the absence of dose-dependent activation of Tregs. Thus, the fusion protein of SEQ ID NO: 1 can be administered in human patients at concentrations equivalent to high-dose rhIL-2 to induce comparable or greater proliferation of NK and CD8+ cells compared to high-dose rhIL-2 (but much less (at least 2-fold) relative proliferation of immunosuppressive Tregs compared to high-dose rhIL-2). This result was unexpected.

[0122] Dosing regimen The fusion protein of SEQ ID NO:1 is administered to a cancer patient according to the methods and administration regimens of the present disclosure. In certain embodiments, the route of administration is intravenous, such as intravenous injection and intravenous infusion, for example, by central venous access. Additional routes of administration include subcutaneous, intramuscular, oral, nasal, and pulmonary administration. In certain embodiments, the fusion protein of SEQ ID NO:1 may be administered as part of a pharmaceutical composition that includes at least one excipient. The administration regimens described herein are designed for less frequent administration (i.e., administration of the fusion protein of SEQ ID NO:1 on non-consecutive days). These administration regimens have the advantage of being easy to administer for patients and medical personnel without a substantial decrease in safety and efficacy. These administration regimens represent an improvement over existing administration regimens of the fusion protein of SEQ ID NO:1, which require administration for 5 consecutive days (qdx5) in each 21-day cycle of treatment (i.e., Q3W cycle).

[0123] In one embodiment, the disclosure provides a method of treating cancer in a patient, comprising administering to the patient a fusion protein comprising an amino acid sequence set forth in SEQ ID NO:1, or a fusion protein with at least 80% identity to the amino acid sequence set forth in SEQ ID NO:1, wherein the patient is administered at least a first dose of the fusion protein and a second dose of the fusion protein on non-consecutive days.

[0124] In another aspect, the disclosure provides a method of treating cancer in a patient, comprising administering to the patient a fusion protein comprising an amino acid sequence set forth in SEQ ID NO:1, or a fusion protein with at least 80% identity to the amino acid sequence set forth in SEQ ID NO:1, wherein the patient is administered at least a first dose of the fusion protein, wherein the patient is not administered a dose of the fusion protein at least one day prior to the first dose, and wherein the patient is not administered a dose of the fusion protein one day after the first dose.

[0125] In certain embodiments, the fusion protein is administered at a dose of about 1 μg / kg to about 60 μg / kg.

[0126] In certain embodiments, the fusion protein is administered at a concentration of about 1 μg / kg, about 2 μg / kg, about 3 μg / kg, about 4 μg / kg, about 5 μg / kg, about 6 μg / kg, about 7 μg / kg, about 8 μg / kg, about 9 μg / kg, about 10 μg / kg, about 11 μg / kg, about 12 μg / kg, about 13 μg / kg, about 14 μg / kg, about 15 μg / kg, about 16 μg / kg, about 17 μg / kg, about 18 μg / kg, about 19 μg / kg, about 20 μg / kg, about 21 μg / kg, about 22 μg / kg, about 23 μg / kg, about 24 μg / kg, about 25 μg / kg, about 26 μg / kg, about 27 μg / kg, about 28 μg / kg, about 29 μg / kg, about 30 μg / kg, Approximately 31μg / kg, approximately 32μg / kg, approximately 33μg / kg, approximately 34μg / kg, approximately 35μg / kg, approximately 36μg / kg, approximately 37μg / kg, approximately 38μg / k g, about 39μg / kg, about 40μg / kg, about 41μg / kg, about 42μg / kg, about 43μg / kg, about 44μg / kg, about 45μg / kg, about 46μg / kg The antibody is administered at a dose of about 47 μg / kg, about 48 μg / kg, about 49 μg / kg, about 50 μg / kg, about 51 μg / kg, about 52 μg / kg, about 53 μg / kg, about 54 μg / kg, about 55 μg / kg, about 56 μg / kg, about 57 μg / kg, about 58 μg / kg, about 59 μg / kg, or about 60 μg / kg.

[0127] In certain embodiments, the fusion protein is administered at a dose of about 3 μg / kg, about 6 μg / kg, about 10 μg / kg, about 15 μg / kg, about 20 μg / kg, about 25 μg / kg, about 30 μg / kg, about 35 μg / kg, or about 40 μg / kg.

[0128] In certain embodiments, the first dose and the second dose are the same (i.e., the first dose is about 10 μg / kg and the second dose is about 10 μg / kg). In other embodiments, the first dose and the second dose are different (i.e., the first dose is about 15 μg / kg and the second dose is about 10 μg / kg).

[0129] In certain embodiments, the patient is administered a third dose of the fusion protein.

[0130] In certain embodiments, a patient is administered a first dose of the fusion protein on day 1 and a second dose of the fusion protein on any of the following days: the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, or 21st.

[0131] In certain embodiments, a patient receives a first dose of the fusion protein on day 1 and a second dose of the fusion protein on day 4 (ie, a 1-4 dosing regimen).

[0132] In certain embodiments, a patient receives a first dose of the fusion protein on day 1 and a second dose of the fusion protein on day 8 (ie, a 1-8 dosing regimen).

[0133] In certain embodiments, a patient receives a first dose of the fusion protein on day 1 and a second dose of the fusion protein on day 15 (ie, a 1-15 dosing regimen).

[0134] In certain embodiments, the patient is administered the second dose of the fusion protein and the third dose of the fusion protein on consecutive days (i.e., the second dose on day 4 and the third dose on day 5). In other embodiments, the patient is administered the second dose of the fusion protein and the third dose of the fusion protein on non-consecutive days (i.e., the second dose on day 4 and the third dose on day 8). In certain embodiments, the patient is administered the third dose of the fusion protein 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days after the second dose of the fusion protein.

[0135] In certain embodiments, a patient is administered a first dose of the fusion protein on day 1, a second dose of the fusion protein on any of the following days: the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, or 12th, and a third dose of the fusion protein on any of the following days: the 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, or 21st.

[0136] In certain embodiments, a patient is administered a first dose of the fusion protein on day 1, a second dose of the fusion protein on any of day 3, 4, or 5, and a third dose of the fusion protein on any of day 6, 7, 8, 9, or 10.

[0137] In certain embodiments, a patient is administered a first dose of the fusion protein on day 1, a second dose of the fusion protein on any of day 6, 7, 8, 9, or 10, and a third dose of the fusion protein on any of day 13, 14, 15, 16, or 17.

[0138] In certain embodiments, a patient is administered a first dose of the fusion protein on day 1, a second dose of the fusion protein on day 4, and a third dose of the fusion protein on day 8 (i.e., a 1-4-8 dosing regimen).

[0139] In certain embodiments, a patient is administered a first dose of the fusion protein on day 1, a second dose of the fusion protein on day 8, and a third dose of the fusion protein on day 15 (i.e., a 1-8-15 dosing regimen).

[0140] In certain embodiments, the patient is administered a first dose of about 10 μg / kg to about 30 μg / kg of the fusion protein on day 1, and a second dose of about 10 μg / kg to about 30 μg / kg of the fusion protein on any of the following days: day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, or day 21.

[0141] In certain embodiments, the patient is administered a first dose of about 10 μg / kg to about 15 μg / kg of the fusion protein on day 1, and a second dose of about 10 μg / kg to about 15 μg / kg of the fusion protein on any of day 3, 4, 5, or 6.

[0142] In certain embodiments, the patient is administered a first dose of about 10 μg / kg to about 25 μg / kg of the fusion protein on day 1, and a second dose of about 20 μg / kg to about 25 μg / kg of the fusion protein on any of day 6, 7, 8, 9, or 10.

[0143] In a specific embodiment, the patient is administered a first dose of about 15 μg / kg of the fusion protein on day 1, and a second dose of about 15 μg / kg of the fusion protein on day 4.

[0144] In certain embodiments, the patient is administered a first dose of about 20 μg / kg to about 25 μg / kg of the fusion protein on day 1 and a second dose of about 20 μg / kg to about 25 μg / kg of the fusion protein on day 8.

[0145] In certain embodiments, the patient is administered a first dose of about 6 μg / kg to about 25 μg / kg of the fusion protein on day 1, a second dose of about 6 μg / kg to about 25 μg / kg of the fusion protein on any of day 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and a third dose of about 6 μg / kg to about 25 μg / kg of the fusion protein on any of day 13, 14, 15, 16, 17, 18, 19, 20, or 21.

[0146] In certain embodiments, the patient is administered a first dose of about 6 μg / kg to about 15 μg / kg of the fusion protein on day 1, a second dose of about 6 μg / kg to about 15 μg / kg of the fusion protein on any of day 3, day 4, or day 5, and a third dose of about 6 μg / kg to about 15 μg / kg of the fusion protein on any of day 6, day 7, day 8, day 9, or day 10.

[0147] In certain embodiments, the patient is administered a first dose of about 10 μg / kg to about 20 μg / kg of the fusion protein on day 1, a second dose of about 10 μg / kg to about 20 μg / kg of the fusion protein on any of day 6, 7, 8, 9, or 10, and a third dose of about 10 μg / kg to about 20 μg / kg of the fusion protein on any of day 13, 14, 15, 16, or 17.

[0148] In a specific embodiment, the patient is administered a first dose of about 15 μg / kg of the fusion protein on day 1, a second dose of about 15 μg / kg of the fusion protein on day 4, and a third dose of about 15 μg / kg of the fusion protein on day 8.

[0149] In a specific embodiment, the patient is administered a first dose of about 20 μg / kg of the fusion protein on day 1, a second dose of about 20 μg / kg of the fusion protein on day 8, and a third dose of about 20 μg / kg of the fusion protein on day 15.

[0150] In certain embodiments, the first dose, second dose, and third dose are the same (i.e., a first dose of about 10 μg / kg, a second dose of about 10 μg / kg, and a third dose of about 10 μg / kg). In other embodiments, the first dose, second dose, and third dose are different (i.e., a first dose of about 20 μg / kg, a second dose of about 15 μg / kg, and a third dose of about 10 μg / kg).

[0151] In certain embodiments, the patient is administered a fourth dose of the fusion protein. In certain embodiments, the patient is administered a third dose of the fusion protein and a fourth dose of the fusion protein on consecutive days. In certain embodiments, the patient is administered a third dose of the fusion protein and a fourth dose of the fusion protein on non-consecutive days.

[0152] In yet another aspect, the disclosure provides a method of treating cancer in a patient, comprising administering to the patient a fusion protein comprising an amino acid sequence set forth in SEQ ID NO:1, or a fusion protein with at least 80% identity to the amino acid sequence set forth in SEQ ID NO:1, wherein the patient is administered a single dose of the fusion protein once per week (Q1W), once every two weeks (Q2W), once every three weeks (Q3W), or once every four weeks (Q4W).

[0153] In certain embodiments, the fusion protein is administered at a dose of about 10 μg / kg to about 60 μg / kg. In certain embodiments, the fusion protein is administered at a dose of about 10 μg / kg, about 15 μg / kg, about 20 μg / kg, about 25 μg / kg, about 30 μg / kg, about 35 μg / kg, about 40 μg / kg, about 45 μg / kg, about 50 μg / kg, about 55 μg / kg, or about 60 μg / kg. In certain embodiments, the fusion protein is administered at a dose of about 10 μg / kg, about 15 μg / kg, about 20 μg / kg, about 25 μg / kg, about 30 μg / kg, about 35 μg / kg, or about 40 μg / kg.

[0154] In certain embodiments, the fusion protein of SEQ ID NO: 1 is administered daily as a single intravenous infusion. The single intravenous infusion may take from 5 minutes to 2 hours. In certain embodiments, each dose is administered by intravenous (IV) injection or infusion over a period of about 30 minutes, about 60 minutes, or about 90 minutes.

[0155] In certain embodiments, the dosing regimen for administration of the fusion protein of SEQ ID NO: 1 provides one or more therapeutic courses (i.e., therapeutic cycles). A single therapeutic course may be administered for a number of days ranging from 1 to 90 days. Preferably, a single therapeutic course extends for 14 or 21 days.

[0156] In certain embodiments, the first course of treatment involves administering the fusion protein of SEQ ID NO:1 by intravenous infusion on non-consecutive days, followed by a rest period of a selected number of days over a first course of treatment lasting about 21 days (i.e., 21 days counting from day 1 on which the first dose of the fusion protein of SEQ ID NO:1 is administered). Generally, the second course of treatment follows the first course of treatment. The second course of treatment may be initiated at any time after the first course of treatment, but is preferably initiated within about 24 hours or more after the first course of treatment has ended.

[0157] In certain embodiments, the fusion protein of SEQ ID NO: 1 is administered together with another therapeutic agent and / or anti-cancer agent as described below. Preferably, the therapeutic agent is pembrolizumab, an immune checkpoint inhibitor. Preferably, pembrolizumab is administered in a separate composition from the fusion protein of SEQ ID NO: 1, and is preferably administered by intravenous infusion before, after, or simultaneously with the infusion of the fusion protein of SEQ ID NO: 1. In certain embodiments, pembrolizumab is administered at a dose of about 200 mg once every two weeks (Q2W), once every three weeks (Q3W), once every four weeks (Q4W), or according to standard prescription recommendations. In certain embodiments, pembrolizumab is administered on the first day of each course of treatment with the fusion protein of SEQ ID NO: 1. Preferably, when the fusion protein is co-administered with pembrolizumab, the first course of treatment with the fusion protein and all subsequent courses of treatment (e.g., courses 2, 3, 4, and 5) are generally about 21-day courses.

[0158] All of the above-described dosing regimens of the invention preferably result in a dose-dependent increase in circulating NK cells and CD8+ cells in the patient in the absence of a dose-dependent increase in T regulatory (Treg) cells, and preferably result in an increase in circulating NK cells and CD8+ cells compared to an increase in circulating Treg cells in the patient. Compared to high-dose or low-dose rhIL-2 therapy, all dosing regimens of the invention preferably require less frequent dosing, e.g., a once-daily administration of the fusion protein of SEQ ID NO:1, compared to three times-daily administration of high-dose or low-dose rhIL-2.

[0159] The increase in circulating CD8+ T cells resulting from administration of the fusion protein of SEQ ID NO:1 is preferably at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, about 9-fold, about 10-fold, or more compared to baseline. The rate of increase in circulating CD8+ T cells resulting from administration of the fusion protein of SEQ ID NO:1 is preferably greater than the rate of increase in circulating regulatory T cells.

[0160] Preferably, the fusion protein of SEQ ID NO: 1 and its pharmaceutical compositions in combination with one or more immune checkpoint inhibitors for treating and / or preventing various diseases, disorders, and conditions (e.g., cancer) are affected by utilizing specific administration parameters that serve to minimize any side effects associated with the administration of the individual therapy by itself. By way of example, the addition of the administration of the fusion protein of SEQ ID NO: 1 in a treatment regimen that includes an immune checkpoint inhibitor (e.g., pembrolizumab) may allow for a reduction in the amount of immune checkpoint inhibitor required to achieve the therapeutic goal, thus reducing (or even eliminating) the severe and fatal immune-mediated adverse reactions that prompted the FDA to request a "black box" warning for certain immune checkpoint inhibitors (e.g., pembrolizumab).

[0161] In general, the administration parameters of monotherapy using the fusion protein of SEQ ID NO:1 or any of the combination therapies described herein dictate that the dosage is less than the amount that would be irreversibly toxic to the subject (i.e., the maximum tolerated dose, "MTD") and less than the amount required to produce a measurable effect in the subject. Such amounts are determined by pharmacokinetic and pharmacodynamic parameters relevant to ADME, taking into account, for example, the route of administration and other factors.

[0162] Effective dose (ED) is the dose or amount of a drug that produces a therapeutic response or desired effect in some percentage of subjects who take it. The "median effective dose" or ED50 of a drug is the dose or amount of a drug that produces a therapeutic response or desired effect in 50% of the population to which it is administered. Although ED50 is commonly used as a measure of the reasonably expected effect of a drug, it is not necessarily the dose that a clinician may consider appropriate, taking into account all relevant factors. Thus, in some situations, the effective amount may be greater than the calculated ED50, in other situations, the effective amount may be less than the calculated ED50, and in still other situations, the effective amount may be the same as the calculated ED50.

[0163] Furthermore, an effective dose of the fusion protein of SEQ ID NO: 1 can be an amount that, when administered to a subject in one or more doses, produces a desired result relative to a healthy subject. For example, for a subject experiencing a particular disorder, an effective dose can be a dose that improves a diagnostic parameter, measurement scale, marker, etc. of that disorder by at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, with 100% being defined as the diagnostic parameter, measurement scale, marker, etc. exhibited by a normal subject.

[0164] Preferably, if the cancer recurs after initial treatment, the patient is again administered the fusion protein of SEQ ID NO: 1. For example, if the patient is initially treated for a solid tumor and the tumor recurs or more tumors develop, the patient is administered SEQ ID NO: 1, for example as another course or series of courses of SEQ ID NO: 1.

[0165] Preferably, the fusion protein of SEQ ID NO: 1 is administered to cancer patients according to the method and administration regimen of the present invention.Preferred administration route is intravenous, such as intravenous injection and intravenous infusion, for example, by central venous access.Additional preferred administration routes include subcutaneous, intramuscular, oral, nasal, and pulmonary administration.

[0166] The therapeutic regimen of the present invention is administered to the patient until the patient is cured or until the patient no longer benefits from the therapeutic regimen.

[0167] Improved safety profile The toxicity of rhIL-2 in humans and animals is well documented. At the high doses of rhIL-2 used in most cancer trials, only minor toxicity has been documented with only occasional tumor responses. One of the major dose-limiting toxicities of human recombinant interleukin-2 (rhIL-2) is capillary leak syndrome (CLS), also referred to herein as vascular leak syndrome (VLS). CLS is characterized by increased vascular permeability with extravasation of fluid and protein resulting in interstitial edema and organ failure. Symptoms of CLS include fluid retention, weight gain, peripheral edema, pleural and pericardial effusions, ascites, generalized edema, and in severe forms, signs of pulmonary and cardiovascular failure. Symptoms vary widely between patients and the causes are poorly understood.

[0168] The pathogenesis of endothelial cell (EC) injury is complex and may involve activation or injury of ECs and leukocytes, release of cytokines and inflammatory mediators, alterations in cell-cell and cell-matrix adhesion and cytoskeletal function.CLS limits the dose of IL-2 that can be administered to humans and in some cases requires cessation of therapy.

[0169] The methods of the invention reduce the risk of side effects often associated with high-dose therapy while maintaining the desired therapeutic activity of IL-2 therapy, including but not limited to CLS and cytokine release syndrome (CRS), another syndrome associated with cytokine-based immunotherapy that often accompanies and / or overlaps with CLS.

[0170] Dose escalation studies in human clinical trials described in the Examples surprisingly revealed that administering the fusion polypeptide of SEQ ID NO: 1 to patients at concentrations equivalent to those of high-dose rh-IL-2 did not result in the frequency and severity of certain side effects (e.g., capillary leak syndrome) that are often associated with high-dose rhIL-2 therapy. As described in the Examples outlining the human clinical trials, dose-limiting toxicities (DLTs) have not yet been reached at doses of 6 μg / kg / day or higher, despite exceeding the EC50 values ​​for natural killer cell and CD8+ T cell activation. Thus, the methods of the present invention may provide an improved safety profile for patients with cancer and in need of IL-2 therapy, for example, compared to standard rhIL-2 therapy, and particularly compared to high-dose rhIL-2 therapy.

[0171] As used herein, "improved safety profile" or "reduced risk of side effects" or "reduced frequency or severity of side effects" for example with standard rhIL-2 therapy, and especially compared to high dose rhIL-2 therapy, can be assessed in several ways. Side effects or symptoms of IL-2 therapy can be quantified. Side effects or symptoms of IL-2 therapy may be quantified on a semi-quantitative scale, for example 0-5, with 0 representing absence, 1-4 representing a discernible increase in severity, and 5 representing maximum severity. Clinical trials often use a scale of 1-5, with 1 representing a mild adverse event (side effect), 2 representing a moderate adverse event (side effect), 3 representing a severe adverse event (side effect), 4 representing a life-threatening or disabling adverse event (side effect), and 5 representing death associated with the adverse event (side effect). Alternatively, side effects or symptoms of IL-2 therapy can be quantified as a binary event, i.e., presence or absence (0 or 1). Other semi-quantitative measures will be readily apparent to those skilled in the art. In another embodiment, the side effects or symptoms of IL-2 therapy may be quantified on a quantitative scale, for example mass per volume, such as mass of cytokine per volume of tissue fluid, temperature, duration, rate, enzyme activity, oxygen saturation, etc. Those skilled in the art will readily understand how to assess and quantify any side effects or symptoms of IL-2 therapy, and will be able to do so without difficulty or undue burden. For example, those skilled in the art will be able to measure plasma or serum cytokine concentrations, temperature (fever), heart rate (tachycardia), blood pressure (hypotension), cardiac dysfunction, renal impairment, serum or plasma enzyme concentrations (liver function), etc. Any quantification of side effects or symptoms of IL-2 therapy may be compared to a control, for example a healthy control subject not receiving IL-2 therapy, or may be compared to other control subjects receiving, for example, standard high-dose rhIL-2 therapy.

[0172] A "reduced risk" of a side effect of IL-2 therapy can be, for example, about a 1% reduction, about a 2% reduction, about a 3% reduction, about a 4% reduction, about a 5% reduction, about a 6% reduction, about a 7% reduction, about a 8% reduction, about a 9% reduction, about a 10% reduction, about a 20% reduction, about a 30% reduction, about a 40% reduction, about a 50% reduction, about a 60% reduction, about a 70% reduction, about a 80% reduction, about a 90% reduction, about a 100% reduction in the occurrence of a side effect or symptom of IL-2 therapy, as compared to high dose rhIL-2 therapy. Alternatively, treating a side effect or symptom of IL-2 therapy can be about a 2-fold, about a 3-fold, about a 4-fold, about a 5-fold, about a 6-fold, about a 7-fold, about a 8-fold, about a 9-fold, about a 10-fold or greater reduction in the side effect or symptom of IL-2 therapy. Thus, "less severe side effects" refers to such a reduction in the side effects or symptoms of IL-2 therapy.

[0173] Preferably, an administration regimen of a fusion protein according to the present invention reduces the frequency and severity of capillary leak syndrome (CLS), also referred to herein as vascular leak syndrome (VLS).

[0174] Other side effects often associated with rhIL-2 immunotherapy that may be reduced by the therapeutic regimen of the present invention include, but are not limited to, cytokine release syndrome (CRS). CRS is a serious side effect of immunotherapy with symptoms that may clinically overlap with those of CLS, but may cause symptoms quite different from CRS. CRS is believed to result from proliferating T cells releasing large amounts of cytokines, including IL-6, IFN-γ, TNF, IL-2, IL-2 receptor, IL-8, IL-10, and GMCSF. Patients with CRS may experience cardiovascular symptoms including fever, tachycardia; pulmonary symptoms including hypotension, arrhythmias, reduced ventricular ejection fraction, edema, hypoxia, dyspnea, and pneumonia; acute kidney injury, usually caused by reduced renal perfusion; hepatic and gastrointestinal symptoms including elevated serum transaminases and bilirubin; diarrhea, colitis, nausea, and abdominal pain; cytopenias such as grade 3–4 anemia; hematologic symptoms including thrombocytopenia, leukopenia, neutropenia, and lymphopenia; prolonged prothrombin time and activated partial thrombocytopenia; Patients may experience one or more of the following symptoms: breakdown of clots including prolonged prothrombin time (PTT), elevated D-dimers, hypofibrinogen, disseminated intravascular coagulation, macrophage activation syndrome (MAS), hemorrhage, B-cell dysplasia, and infections including hypogammaglobulinemia, bacteremia, viral infections including Salmonella, urinary tract infections, influenza, respiratory syncytial virus, and herpes simplex virus; musculoskeletal symptoms including elevated creatine kinase; and neurological symptoms including pain or weakness, delirium, confusion, and seizures.

[0175] Administration of SEQ ID NO:1 has been shown to induce lower levels of pro-inflammatory cytokines compared to, for example, rhIL-2 in mice.

[0176] Administration of SEQ ID NO:1 has also been shown to induce higher levels of desirable cytokines such as IFN, while inducing lower levels of pro-inflammatory cytokines such as IL-6 in humans.

[0177] MAS overlaps clinically with CRS, in that subjects may experience hepatosplenomegaly, lymphadenopathy, pancytopenia, liver dysfunction, disseminated intravascular coagulation, hypofibrinogenemia, hyperferritinemia, and hypertriglyceridemia. Similar to CRS, subjects with MAS exhibit elevated levels of cytokines, including IFN-γ and GMCSF.

[0178] Another side effect of immunotherapy, including IL-2 therapy, is tumor lysis syndrome (TLS), which occurs when cellular contents are released as a result of therapy causing cell death and is most often associated with lymphomas and leukemias. TLS is characterized by an imbalance of blood ions and metabolites, and symptoms include nausea, vomiting, acute urate nephropathy, acute renal failure, seizures, cardiac arrhythmias, and death.

[0179] Neurotoxicity can result from immunotherapy, including IL-2 therapy, and symptoms can include cerebral edema, delirium, hallucinations, aphasia, akinetic mutism, headache, confusion, altered alertness, ataxia, insomnia, facial paralysis, tremor, dysmetria, and seizures.

[0180] Patients receiving IL-2 immunotherapy may experience the following side effects: anemia, aphasia, arrhythmias, joint pain, back pain, blood and bone marrow disorders, blood and lymphatic system disorders, cardiac disorders, chills, coagulation disorders, colitis, confusion, constitutional symptoms, cough, loss of appetite, diarrhea, disorientation, dizziness, dyspnea, encephalopathy, fatigue, fever, gastrointestinal disorders, general cardiovascular disorders, bleeding, liver disorders, hyperglycemia, hypokalemia, hypothyroidism, increased ALT, increased AST, increased C-reactive protein, infections, febrile neutropenia, leukopenia, malaise, abnormal metabolic laboratory results, Patients may experience one or more side effects or symptoms not necessarily caused by CLS, CRS, MAS, or TLS, including, but not limited to, metabolic and nutritional disorders, mucosal inflammation, musculoskeletal disorders, myalgia, nausea, nervous system disorders, neuropathy, neutropenic edema, pain, palmar-plantar erythrodysesthesia syndrome, dysesthesias, pneumonia, pruritus, pulmonary disorders, fever, rash, renal and genitourinary disorders, respiratory disorders, skin and subcutaneous tissue disorders, drowsiness, speech disorders, sweating, thoracic and mediastinal disorders, thrombocytopenia, tremor, tumor relapse, tumor lysis syndrome, vascular disorders, and vomiting.

[0181] Cancer indications The therapeutic regimen of the present invention using the fusion protein of SEQ ID NO: 1 is useful for the treatment of many types of cancer. The term "cancer" as used herein shall be given its ordinary meaning as a general term for diseases in which abnormal cells divide without control. In particular, and in the context of the present embodiment, cancer refers to cancers associated with angiogenesis. Cancer cells can invade nearby tissues and spread to other parts of the body through the bloodstream and lymphatic system. There are several main types of cancer, for example, carcinoma is cancer that begins in the skin, or tissues that fill or cover the internal organs. Sarcoma is cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissues. Leukemia is cancer that begins in blood-forming tissues, such as bone marrow, and causes a large number of abnormal blood cells to be produced and enter the bloodstream. Lymphoma is cancer that begins in cells of the immune system.

[0182] Tumors form when normal cells lose their ability to behave as a specified, controlled, coordinated unit. A solid tumor is generally an abnormal mass of tissue that does not usually contain cysts or liquid areas (some brain tumors have fluid-filled cysts and central necrotic areas). A single tumor may even have different cell populations within it, with different processes going awry. Solid tumors can be benign (not cancerous) or malignant (cancerous). Different types of solid tumors are named for the type of cells that form them. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) do not generally form solid tumors.

[0183] Representative cancers include, but are not limited to, adult acute lymphoblastic leukemia, childhood acute lymphoblastic leukemia, adult acute myeloid leukemia, adrenocortical cell carcinoma, childhood adrenocortical cell carcinoma, AIDS-related lymphoma, AIDS-related malignancies, anal cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, extrahepatic bile duct cancer, bladder cancer, childhood bladder cancer, bone cancer osteosarcoma / malignant fibrous histiocytoma, childhood glioblastoma, adult glioblastoma, childhood brain stem glioma, adult brain tumor, childhood brain stem glioma brain tumor, childhood cerebellar astrocytoma brain tumor, childhood cerebral astrocytoma / malignant glioma brain tumor, childhood ependymoma brain tumor, childhood medulloblastoma brain tumor, childhood supratentorial primitive neuroectodermal tumor brain tumor, childhood visual pathway and hypothalamic glioma brain tumor, childhood brain tumor (other), breast cancer, breast cancer and pregnancy pregnancy, pediatric breast cancer, male breast cancer, pediatric bronchial adenoma / carcinoid, pediatric carcinoid tumor, gastrointestinal carcinoid tumor, adrenal cortical cell carcinoma, carcinoma, islet cell carcinoma, cancer of unknown primary, primary central nervous system lymphoma, pediatric cerebellar astrocytoma, pediatric cerebral astrocytoma / malignant glioma, cervical cancer, pediatric cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, clear cell sarcoma of tendon sheath, colon cancer, pediatric tuberculosis Colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, childhood ependymoma, epithelial ovarian cancer, esophageal cancer, childhood esophageal cancer, Ewing's tumor, childhood extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, intraocular melanoma which is eye cancer, retinoblastoma which is eye cancer, gallbladder cancer, stomach cancer, childhood stomach cancer, gastrointestinal carcinoid tumor, childhood extracranial germ cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor, glioma. childhood brain stem, glioma. Childhood Visual Pathway and Hypothalamic Glioma, Hairy Cell Leukemia, Head and Neck Cancer, Adult Hepatocellular (Liver) Cancer (Primary), Childhood Hepatocellular (Liver) Cancer (Primary), Adult Hodgkin's Lymphoma, Childhood Hodgkin's Lymphoma, Hodgkin's Lymphoma During Pregnancy, Hypopharyngeal Cancer, Childhood Hypothalamic and Visual Pathway Glioma, Intraocular Melanoma, Islet Cell Carcinoma (Endocrine Pancreas), Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Childhood Laryngeal Cancer, Adult Acute Lymphoblastic Leukemia, Childhood Acute Lymphoblastic Leukemia, Adult Acute Myeloid Leukemia, Childhood Acute Myeloid Leukemia, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Hairy Cell Leukemia, Lip and Oral Cancer, Adult Liver Cancer (Primary), Childhood Liver Cancer (Primary), Non-Small Cell Lung Cancer, Small Cell Lung Cancer, Adult Acute Lymphoblastic Leukemia, Childhood Acute Lymphoblastic Leukemia, Chronic Lymphocytic Leukemia,AIDS-related lymphoma, Central nervous system lymphoma (primary), Cutaneous T-cell lymphoma, Adult Hodgkin lymphoma, Childhood Hodgkin lymphoma, Hodgkin lymphoma during pregnancy, Adult non-Hodgkin lymphoma, Childhood non-Hodgkin lymphoma, Non-Hodgkin lymphoma during pregnancy, Primary central nervous system lymphoma, Waldenstrom's macroglobulinemia, Male breast cancer, Adult malignant mesothelioma, Childhood malignant mesothelioma, Malignant thymoma, Childhood medulloblastoma, Melanoma, Intraocular melanoma, Merkel cell carcinoma, Malignant mesothelioma, Metastatic squamous cell carcinoma of the neck of unknown primary, Childhood multiple endocrine neoplasia syndrome, Multiple myeloma / plasma cell neoplasm, Mycosis fungoides Myelodysplastic syndromes, chronic myeloid leukemia, childhood acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, childhood nasopharyngeal cancer, neuroblastoma, neurofibroma, adult non-Hodgkin's lymphoma, childhood non-Hodgkin's lymphoma, non-Hodgkin's lymphoma during pregnancy, non-small cell lung cancer, childhood oral cavity cancer, oral cavity and lip cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, childhood ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumors, ovarian low malignant potential tumors, pancreatic cancer, childhood pancreatic cancer, pancreatic islet cell carcinoma, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pheochromocytoma, childhood pineal and supratentorial tumours Primitive neuroectodermal tumors, Pituitary tumors, Plasmacytoma / multiple myeloma, Pleuropulmonary blastoma, Pregnancy and breast cancer, Pregnancy and Hodgkin's lymphoma, Pregnancy and non-Hodgkin's lymphoma, Primary central nervous system lymphoma, Adult primary liver cancer, Pediatric primary liver cancer, Prostate cancer, Rectal cancer, Renal cell (kidney) cancer, Pediatric renal cell carcinoma, Transitional cell carcinoma of the renal pelvis and ureter, Retinoblastoma, Pediatric rhabdomyosarcoma, Salivary gland cancer, Pediatric salivary gland cancer, Ewing's sarcoma family of tumors, Kaposi's sarcoma, Sarcoma of bone (osteosarcoma) / malignant fibrous histiocytoma, Pediatric rhabdomyosarcoma, Adult soft tissue sarcoma, Pediatric soft tissue sarcoma, Sezary syndrome, Skin cancer, Pediatric skin cancer, Skin cancer (melanoma), Merkel cell skin cancer, small cell lung cancer, small intestine cancer, adult soft tissue sarcoma, pediatric soft tissue sarcoma, metastatic cervical squamous cell carcinoma of unknown primary site, gastric cancer, pediatric gastric cancer, pediatric supratentorial primitive neuroectodermal tumor, cutaneous T-cell lymphoma, testicular cancer, pediatric thymoma, malignant thymoma, thyroid cancer, pediatric thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, gestational trophoblastic tumor, pediatric cancer of unknown primary site, unusual pediatric cancer, transitional cell carcinoma of the urinary tract and renal pelvis, urethral cancer, uterine sarcoma, vaginal cancer, pediatric visual pathway and hypothalamic glioma, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor.

[0184] Tumors can be classified as malignant or benign. In both cases, there is an abnormal aggregation and proliferation of cells. In the case of malignant tumors, these cells behave more aggressively and acquire increased invasive properties. Eventually, tumor cells may even gain the ability to detach from the microscopic environment in which they originated and spread to another area of ​​the body (which has a very different environment and is not normally conducive to proliferation) and continue their rapid proliferation and division in this new location. This is called metastasis. Once malignant cells have metastasized, a cure is more difficult to achieve. Benign tumors are less prone to invasion and less likely to metastasize.

[0185] As used herein, the term "reducing a tumor" refers to a decrease in the size or volume of a tumor mass, a decrease in the number of metastasized tumors in a subject, a decrease in the proliferative state of cancer cells (the extent to which cancer cells proliferate), and the like.

[0186] The therapeutic regimen of the present invention is particularly suitable for treating solid tumors, including but not limited to lymphoma, melanoma, renal cell carcinoma (RCC), advanced solid tumors, tumors that have been previously treated with curative therapy but remain refractory to the previous therapy.Preferably, the therapeutic regimen of the present invention is particularly suitable for treating solid tumors, including but not limited to lymphoma, melanoma, renal cell carcinoma (RCC), hepatocellular carcinoma (HCC), non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), squamous cell carcinoma of the head and neck (SCCHN), advanced solid tumors, and tumors that have been previously treated with anticancer therapy but remain refractory to the previous therapy.

[0187] Complementary immunotherapy and other combination therapies While the fusion protein of SEQ ID NO: 1 may be used as a monotherapy in the therapeutic regimen according to the present invention, the combination of the fusion protein of SEQ ID NO: 1 with other anti-cancer treatments in the context of the present invention is also contemplated.Other therapeutic treatment regimens include adoptive cell transfer regimens, antigen-specific vaccination, inhibition of DNA repair proteins (e.g., nucleic acid enzyme poly(adenosine 5'-diphospho-ribose) polymerase ["poly(ADP-ribose) polymerase" PARP inhibitors"), and inhibition of immune checkpoint inhibitory molecules, such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1) antibodies, and other therapeutic immunotherapies.

[0188] Immune checkpoint proteins regulate T cell function in the immune system. T cells play a central role in cell-mediated immunity. Immune checkpoint proteins interact with specific ligands that send signals to T cells and essentially switch or inhibit T cell function. Cancer cells exploit this system by driving high levels of expression of immune checkpoint proteins on their surface, which results in the control of T cells that express immune checkpoint proteins on the surface of T cells that enter the tumor microenvironment, thus suppressing anti-cancer immune responses. Thus, inhibition of immune checkpoint proteins by agents referred to herein as "immune checkpoint protein (ICP) inhibitors" results in the restoration of T cell function and immune responses against cancer cells. Examples of immune checkpoint proteins include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, OX40, B-7 family ligands, or combinations thereof. Preferably, the immune checkpoint inhibitor interacts with a ligand of an immune checkpoint protein, which may be CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, OX40, A2aR, B-7 family ligands, or combinations thereof. Preferably, the immune checkpoint inhibitor is a biological therapeutic or a small molecule. Preferably, the immune checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof. Preferably, the PD1 immune checkpoint inhibitor comprises one or more anti-PD-1 antibodies, including nivolumab and pembrolizumab.

[0189] The combination therapy methods described herein include administering at least one immune checkpoint inhibitor in combination with a fusion protein of SEQ ID NO: 1. The present invention is not limited to any particular immune checkpoint inhibitor, so long as the immune checkpoint inhibitor inhibits one or more activities of the target immune checkpoint protein when administered in an effective amount, either as a monotherapy or in combination with a fusion protein of SEQ ID NO: 1. In some cases, for example due to synergistic effects, minimal inhibition of immune checkpoint protein by immune checkpoint inhibitor may be sufficient in the presence of SEQ ID NO: 1. Many immune checkpoint inhibitors are known in the art.

[0190] Exemplary PD-1 / PD-L1 immune checkpoint inhibitors include antibody-based therapeutics. Exemplary therapeutic methods using PD-1 / PD-L1 immune checkpoint inhibition are described in U.S. Patent Nos. 8,728,474 and 9,073,994, and EP 1537878B1, and include, for example, the use of anti-PD-1 antibodies. Exemplary anti-PD-1 antibodies are described, e.g., in U.S. Patent Nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Exemplary anti-PD-1 antibodies include, for example, nivolumab (OPDIVO®, Bristol-Myers Squibb Co.), pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), pidilizumab (CT-011, Cure Tech). Exemplary anti-PD-L1 antibodies are described, for example, in U.S. Patent Nos. 9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and 8,217,149. Exemplary anti-PD-L1 antibodies include, for example, atezolizumab (TECENTRIQ®, Genentech), durvalumab (AstraZeneca), MEDI4736, avelumab, BMS 936559 (Bristol Myers Squibb Co.).

[0191] In certain embodiments, the methods or compositions described herein are administered in combination with CTLA-4 inhibitors.In the CTLA-4 pathway, the interaction of CTLA-4 on T cells with its ligand (e.g., CD80, also known as B7-1 and CD86) on the surface of antigen-presenting cells (not cancer cells) leads to T cell inhibition.Exemplary CTLA-4-based immune checkpoint inhibition methods are described in U.S. Patent Nos. 5,811,097, 5,855,887, and 6,051,227. Exemplary anti-CTLA-4 antibodies are described in U.S. Patent Nos. 6,984,720, 6,682,736, 7,311,910, 7,307,064, 7,109,003, 7,132,281, 6,207,156, 7,807,797, 7,824,679, 8,143,379, 8,263,073, 8,318,916, 8,017,114, 8,784,815, and 8,883,984; International (PCT) Publication Nos. WO 98 / 42752, WO 00 / 37504, and WO 01 / 14424; and European Patent No. EP 1212422 B1. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.

[0192] Preferably, a method or composition of the invention is administered in combination with (i) a PD-1 or PD-L1 inhibitor (e.g., a PD-1 or PD-L1 inhibitor disclosed herein), and (ii) a CTLA-4 inhibitor (e.g., a CTLA-4 inhibitor disclosed herein).

[0193] Examples of FDA-approved immune checkpoint protein inhibitors include: Ipilimumab (YERVOY®) Pembrolizumab (KEYTRUDA®) Atezolizumab (TECENTRIQ®) Durvalumab (IMFINZ®) Avelumab (BAVENCIO®) Nivolumab (OPDIVO®).

[0194] In the preferred treatment regimen of the present invention, the fusion protein of SEQ ID NO: 1 administered according to the present invention is combined with the immune checkpoint inhibitor pembrolizumab.Pembrolizumab is preferably administered on the first day of each treatment cycle of the treatment regimen according to the present invention.Preferably, 200 mg of pembrolizumab is administered generally once every 3 weeks or once every 21 days according to the manufacturer's recommendations.

[0195] The therapeutic regimen using the fusion protein of SEQ ID NO:1 according to the present invention may also be combined with other therapeutic and / or anti-cancer agents in addition to or instead of immune checkpoint inhibitors. Preferably, the therapeutic and / or anti-cancer agent is an antibody. Preferably, the therapeutic agent is a therapeutic protein. Preferably, the therapeutic agent is a small molecule. Preferably, the anti-cancer agent is an antigen. Preferably, the therapeutic agent is a cell population. Preferably, the therapeutic agent is a therapeutic antibody. Preferably, the therapeutic agent is another cytotoxic agent and / or a chemotherapeutic agent. As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and / or causes cell death or destruction. "Chemotherapeutic agent" includes compounds useful in the treatment of cancer.

[0196] antibody Preferably, administration of SEQ ID NO:1 may be combined with therapeutic antibodies. Methods for generating antibodies and their antigen-binding fragments are well known in the art and are described, for example, in U.S. Patent No. 7,247,301, US2008 / 0138336, and U.S. Patent No. 7,923,221, all of which are incorporated herein by reference in their entirety. Therapeutic antibodies that can be used in the methods of the present invention include, but are not limited to, any of the art-recognized therapeutic antibodies that have been approved for use or are undergoing clinical trials or are being developed for clinical use. In some embodiments, two or more therapeutic antibodies can be included in the combination therapy of the present invention.

[0197] Non-limiting examples of therapeutic antibodies include, but are not limited to: trastuzumab (HERCEPTIN™ by Genentech, South San Francisco, Calif.), used to treat HER-2 / neu positive or metastatic breast cancer; Bevacizumab (AVASTIN™ by Genentech), used to treat colorectal cancer, metastatic colorectal cancer, breast cancer, metastatic breast cancer, non-small cell lung cancer, or renal cell cancer; Rituximab (RITUXAN(TM) by Genentech), used to treat non-Hodgkin's lymphoma or chronic lymphocytic leukemia; Pertuzumab (OMNITARG™ by Genentech), used to treat breast cancer, prostate cancer, non-small cell lung cancer, or ovarian cancer cetuximab (ERBITUX™ by ImClone Systems Incorporated, New York, NY), which can be used to treat colorectal cancer, metastatic colorectal cancer, lung cancer, head and neck cancer, colon cancer, breast cancer, prostate cancer, gastric cancer, ovarian cancer, brain cancer, pancreatic cancer, esophageal cancer, renal cell cancer, prostate cancer, cervical cancer, or bladder cancer; IMC-1C11 (ImClone Systems Incorporated), used to treat colorectal cancer, head and neck cancer, as well as other potential cancer targets; Tositumomab and tositumomab and iodine I, used to treat non-Hodgkin's lymphoma 131 (BEXXAR™ by Corixa Corporation, Seattle, Washington) (this can be CD20-positive follicular non-Hodgkin's lymphoma with or without transformation, the disease is refractory to rituximab and has relapsed after chemotherapy); In 111 Ibritumomab Tiuxetan, Y 90 Ibritumomab tiuxetan, I 111Ibritumomab tiuxetan, and Y 90 ibritumomab tiuxetan (ZEVALIN™ by Biogen Idec, Cambridge, Massachusetts), which is used to treat lymphoma or non-Hodgkin's lymphoma, which can include relapsed follicular lymphoma, relapsed or refractory low-grade or follicular non-Hodgkin's lymphoma, or transformed B-cell non-Hodgkin's lymphoma; EMD 7200 (EMD Pharmaceuticals, Durham, North Carolina), used to treat non-small cell lung cancer or cervical cancer; SGN-30, a genetically engineered monoclonal antibody targeting the CD30 antigen from Seattle Genetics (Bothell, Washington), which is used to treat Hodgkin's or non-Hodgkin's lymphoma; SGN-15 (a genetically engineered monoclonal antibody targeting Lewis gamma-associated antigen conjugated to doxorubicin by Seattle Genetics), which is used to treat non-small cell lung cancer; SGN-33 (a humanized antibody targeting the CD33 antigen by Seattle Genetics), which is used to treat acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS); SGN-40 (a humanized monoclonal antibody by Seattle Genetics that targets the CD40 antigen, which is used to treat multiple myeloma or non-Hodgkin's lymphoma); SGN-35 (a genetically engineered monoclonal antibody targeting the CD30 antigen conjugated to auristatin E by Seattle Genetics), which is used to treat non-Hodgkin's lymphoma; SGN-70 (a humanized antibody targeting the CD70 antigen from Seattle Genetics) for the treatment of renal and nasopharyngeal cancer; SGN-75 (a conjugate of the SGN70 antibody and an auristatin derivative from Seattle Genetics), and · SGN-17 / 19 (a fusion protein containing an antibody and an enzyme conjugated to a melphalan prodrug, by Seattle Genetics), which is used to treat melanoma or metastatic melanoma.

[0198] The therapeutic antibody used in the method of the present invention is not limited to those described herein.For example, the following approved therapeutic antibodies can also be used in the method of the present invention: brentuximab vedotin (ADCETRIS ™) for anaplastic large cell lymphoma and Hodgkin's lymphoma, ipilimumab (MDX-101, YERVOY ™) for melanoma, ofatumumab (ARZERRA ™) for chronic lymphocytic leukemia, panitumumab (VECTIBIX ™) for colorectal cancer, alemtuzumab (CAMPATH ™) for chronic lymphocytic leukemia, ofatumumab (ARZERRA ™) for chronic lymphocytic leukemia, gemtuzumab ozogamicin (MYLOTARG ™) for acute myeloid leukemia.

[0199] Antibodies for use according to the invention may also target molecules expressed by immune cells, including, but not limited to, tremelimumab (CP-675206) and ipilimumab (MDX-010), which target CTLA4 and have tumor rejection, protection from rechallenge, and enhanced tumor-specific T cell responses; OX86, which targets OX40 and increases antigen-specific CD8+ T cells at the tumor site and enhances tumor rejection; CT-011, which targets PD 1 and has the effect of maintaining and expanding tumor-specific memory T cells and activates NK cells; BMS-663513, which targets CD137 and causes regression of established tumors and the expansion and maintenance of CD8+ T cells; and daclizumab (ZENAPAX™), which targets CD25 and causes a transient depletion of CD4+CD25+FOXP3+Tregs, enhancing tumor regression and increasing the number of effector T cells. A more detailed discussion of these antibodies can be found, for example, in Weiner et al., Nature Rev. Immunol 2010; 10:317-27.

[0200] Preferably, the antibody is a pro-inflammatory and / or pro-tumorigenic cytokine targeting antibody, including but not limited to anti-TNF antibody, anti-IL-1Ra receptor targeting antibody, anti-IL-1 antibody, anti-IL-6 receptor antibody, and anti-IL-6 antibody. Preferably, the antibody comprises an antibody that targets pro-inflammatory T helper type 17 cells (TH17).

[0201] The therapeutic antibody can be an antigen-binding fragment of the antibody, a complex comprising the antibody, or a conjugate comprising the antibody. The antibody can optionally be chimeric or humanized, or fully human.

[0202] Therapeutic Proteins and Polypeptides Preferably, the method of the present invention comprises the administration of the fusion protein of SEQ ID NO: 1 in combination with a therapeutic protein or peptide according to the therapeutic regimen of the present invention. Therapeutic proteins effective in the treatment of cancer are well known in the art. Preferably, the therapeutic polypeptide or protein is a "suicide protein" that induces cell death by itself or in the presence of other compounds.

[0203] A representative example of such a suicide protein is the thymidine kinase of herpes simplex virus. Additional examples include the thymidine kinase of vesicular zoster virus, the bacterial gene cytosine deaminase (which converts 5-fluorocytosine into the highly toxic compound 5-fluorouracil), p450 oxidoreductase, carboxypeptidase G2, beta-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, beta-lactamase, nitroreductase, carboxypeptidase A, renamerase (also called β-glucosidase), the E. coli gpt gene, and the E. coli Deo gene, although others are known in the art. In some embodiments, the suicide protein converts a prodrug into a toxic compound.

[0204] As used herein, "prodrug" refers to any compound useful in the method of the present invention that can be converted into a toxic product, i.e., toxic to tumor cells. The prodrug is converted into a toxic product by a suicide protein. Representative examples of such prodrugs include ganciclovir, acyclovir, and FIAU (1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-iodo-uracil) for thymidine kinase, ifosfamide for oxidoreductase, 6-methoxypurine arabinoside for VZV-TK, 5-fluorocytosine for cytosine deaminase, doxorubicin for beta-glucuronidase, CB 1954 and nitrofurazone for nitroreductase, and N-(cyanoacetyl)-L-phenylalanine or N-(3-chloropropionyl)-L-phenylalanine for carboxypeptidase A. Prodrugs can be easily administered by those skilled in the art. One of ordinary skill in the art would be able to readily determine the most appropriate dosage and route for administration of the prodrug.

[0205] Preferably, the therapeutic protein or polypeptide is a tumor suppressor, such as p53 or Rb, or a nude acid encoding such a protein or polypeptide. Those skilled in the art are aware of a wide variety of such tumor suppressors and methods for obtaining them and / or the nucleic acids encoding them.

[0206] Other examples of anti-cancer / therapeutic proteins or polypeptides include pro-apoptotic therapeutic proteins and polypeptides, such as p15, p16, or p21 WAF-1 Includes:

[0207] Cytokines, and the nucleic acids encoding them, may also be used as therapeutic proteins and polypeptides. Examples include interferons, including but not limited to GM-CSF (granulocyte macrophage colony stimulating factor), TNF-alpha (tumor necrosis factor alpha), IFN-alpha and IFN-gamma, interleukin-1 (IL-1), interleukin-beta (IL-beta), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8. Interleukins include, but are not limited to, interleukin-8, interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-14 (IL-14), interleukin-15 (IL-15), interleukin-16 (IL-16), interleukin-18 (IL-18), interleukin-23 (IL-23), and interleukin-24 (IL-24), although other embodiments are known in the art.

[0208] Further examples of cytocidal genes include, but are not limited to, mutant cyclin G1 genes. By way of example, the cytocidal gene may be a dominant-negative mutation of the cyclin G1 protein (e.g., WO / 01 / 64870).

[0209] vaccine Preferably, the therapeutic regimen of the present invention comprises the administration of the fusion protein of SEQ ID NO: 1 in combination with the administration of a cancer vaccine to stimulate a cancer-specific immune response, such as innate and adaptive immune response, to generate host immunity against cancer (see, for example, Overwijk, et al. Journal of Experimental Medicine 2008;198:569-80). Exemplary vaccines include, but are not limited to, antigen vaccines, whole cell vaccines, dendritic cell vaccines, and DNA vaccines. Depending on the specific type of vaccine, the vaccine composition may comprise one or more suitable adjuvants known to enhance the subject's immune response to the vaccine.

[0210] Vaccines may be, for example, cell-based, i.e., made using cells from the patient's own cancer cells to recognize and acquire antigens. Exemplary vaccines include tumor cell-based and dendritic cell-based vaccines, where activated immune cells from a subject are delivered to the same subject along with other proteins to further facilitate immune activation of immune cells primed with these tumor antigens. Tumor cell-based vaccines include whole tumor cells and genetically modified tumor cells. Whole tumor cell vaccines may optionally be treated to enhance antigen presentation, for example, by irradiation of either the tumor cells or tumor lysates). Vaccine administration may also be accompanied by adjuvants such as bacillus Calmette-Guerin (SIP) or keyhole limpet hemocyanin (KLH), depending on the type of vaccine used. Plasmid DNA vaccines may also be used and can be administered by direct injection or with biolistic bombardment. Also contemplated are peptide vaccines, viral gene transfer vector vaccines, and the use of antigen-modified dendritic cells (DCs).

[0211] The vaccine is preferably a therapeutic cancer peptide-based vaccine. Peptide vaccines can be made using known sequences or from isolated antigens from the subject's own tumor, including neo- and modified antigens. Exemplary antigen-based vaccines include vaccines in which the antigen is a tumor-specific antigen. For example, tumor-specific antigens can be selected from cancer-testis antigens, differentiation antigens, and widely occurring overexpressed tumor-associated antigens, among others. Recombinant peptide vaccines based on peptides derived from tumor-associated antigens, when used in the present method, may be administered or formulated with adjuvants or immunomodulatory agents. Exemplary antigens for use in peptide-based vaccines include, but are not limited to, the following, since this list is intended to be merely exemplary. For example, peptide vaccines may include cancer-testis antigens such as MAGE, BAGE, NY-ESO-1, and SSX-2, which are encoded by genes that are normally silenced in adult tissues but transcriptionally reactivated in tumor cells. Alternatively, peptide vaccines may include tissue differentiation-associated antigens, i.e., antigens that are of normal tissue origin and shared by both normal and tumor tissues. For example, the vaccine may include melanoma-associated antigens such as gp100, Melan-A / Mart-1, MAGE-3, or tyrosinase, or may include prostate cancer antigens such as PSA or PAP. The vaccine may include breast cancer-associated antigens such as mammaglobin-A. Other tumor antigens that may be included in the vaccine used in the present method include, for example, CEA, MUC-1, HER1 / Nue, hTERT, ras, and B-raf. Other suitable antigens that may be used in the vaccine include SOX-2 and OCT-4, which are associated with cancer stem cells or EMT processes.

[0212] Antigen vaccines include multi-antigen vaccines and single antigen vaccines. Exemplary cancer antigens may include peptides having about 5 to about 30 amino acids, or about 6 to 25 amino acids, or about 8 to 20 amino acids.

[0213] As mentioned above, immune stimulating adjuvants (different from RSLAIL-2) can be used in vaccines, especially tumor-associated antigen-based vaccines, to help generate an effective immune response. For example, the vaccine can incorporate pathogen-associated molecular patterns (PAMPs) to help improve immunity. Additional suitable adjuvants include monophosphoryl lipid A or other lipopolysaccharides, Toll-like receptor (TLR) agonists such as imiquimod, resiquimod (R-848), TLR3, IMO-8400, lintatorimod, etc. Additional adjuvants suitable for use include heat shock proteins.

[0214] Genetic vaccines typically use viral or plasmid DNA vectors carrying expression cassettes. Upon administration, the vector transfects somatic or dendritic cells as part of the inflammatory response, thereby resulting in cross-priming or direct antigen presentation. Preferably, genetic vaccines provide for the delivery of multiple antigens in a single immunization. Genetic vaccines include DNA vaccines, RNA vaccines, and virus-based vaccines.

[0215] The DNA vaccine used in this method is a bacterial plasmid that is constructed to deliver and express tumor antigen.The DNA vaccine can be administered by any suitable administration method, for example, subcutaneous or intradermal injection, but can also be directly injected into lymph nodes.Additional delivery methods include, for example, gene gun, electroporation, ultrasound, laser, liposome, microparticle, nanoparticle.

[0216] Preferably, the vaccine comprises a neoantigen, or multiple neoantigens. Preferably, the vaccine is a neoantigen-based vaccine. Preferably, the neoantigen-based vaccine (NBV) composition may encode multiple cancer neoantigens in tandem, where each neoantigen is a polypeptide fragment derived from a protein mutated in the cancer cell. For example, the neoantigenic vaccine may comprise a first vector comprising a nucleic acid construct encoding multiple immunogenic polypeptide fragments (respective immunogenic polypeptide fragments of proteins mutated in the cancer cell), each immunogenic polypeptide fragment comprising one or more mutated amino acids adjacent to a varying number of wild-type amino acids from the original protein, each polypeptide fragment being joined head-to-tail in a face-to-face manner to form an immunogenic polypeptide. The length of each of the immunogenic polypeptide fragments forming the immunogenic polypeptide may vary.

[0217] Viral gene transfer vector vaccines may also be used, in which recombinantly engineered viruses, yeast, bacteria, etc. are used to introduce cancer-specific proteins into the patient's immune cells. In vector-based approaches, which can be oncolytic or non-oncolytic, the vector can increase the efficiency of the vaccine, for example, due to its inherent immune stimulatory properties. Exemplary viral vectors include vectors derived from the poxvirus family, such as vaccinia, modified vaccinia strain Ankara, and avipoxvirus. Also suitable for use is PROSTVAC, a cancer vaccine containing a replication-competent vaccinia priming vector and a replication-incompetent Fowlbox boosting vector. Each vector contains a transgene for PSA and three costimulatory molecules, CD80, CD54, CD58 (collectively referred to as TRICOM). Other suitable vector-based cancer vaccines include Trovax and TG4010 (encoding MUC1 antigen and IL-2). Additional vaccines of use include bacterial and yeast-based vaccines, such as recombinant Listeria monocytogenes and Saccharomyces cerevisiae.

[0218] The aforementioned vaccines may be combined and / or formulated with adjuvants and other immune boosters to increase efficacy. Depending on the particular vaccine, administration may be either intratumoral or non-intratumoral (i.e., systemic).

[0219] Other cancer antigens that can be used for vaccination include, but are not limited to, (i) tumor-specific antigens, (ii) tumor-associated antigens, (iii) cells expressing tumor-specific antigens, (iv) cells expressing tumor-associated antigens, (v) embryonic antigens on tumors, (vi) autologous tumor cells, (vii) tumor-specific membrane antigens, (viii) tumor-associated membrane antigens, (ix) growth factor receptors, (x) growth factor ligands, and (xi) any other type of antigen or antigen-presenting cell or substance associated with cancer.

[0220] The cancer antigen may be an epithelial cancer antigen (e.g., breast, gastrointestinal, lung), including prostate-specific cancer antigen (PSA) or prostate-specific membrane antigen (PSMA), bladder cancer antigen, lung (e.g., small cell lung) cancer antigen, colon cancer antigen, ovarian cancer antigen, brain cancer antigen, gastric cancer antigen, renal cell cancer antigen, pancreatic cancer antigen, liver cancer antigen, esophageal cancer antigen, head and neck cancer antigen, or colorectal cancer antigen.

[0221] In another embodiment, the cancer antigen is a lymphoma antigen (e.g., non-Hodgkin's lymphoma or Hodgkin's lymphoma), a B-cell lymphoma cancer antigen, a leukemia antigen, a myeloma (i.e., multiple myeloma or plasma cell myeloma) antigen, an acute lymphoblastic leukemia antigen, a chronic myelogenous leukemia antigen, or an acute myelogenous leukemia antigen. The cancer antigens listed are merely exemplary, and any cancer antigen can be targeted in the present invention.

[0222] Preferably, the cancer antigen is mucin-1 protein or peptide (MUC-1), which is found in all human adenocarcinomas (pancreas, colon, breast, ovary, lung, prostate, head and neck), including multiple myeloma and some B-cell lymphomas. Patients with inflammatory bowel disease, either Crohn's disease or ulcerative colitis, are at high risk of developing colorectal cancer. MUC-1 is a type I transmembrane glycoprotein. The major extracellular portion of MUC-1 has multiple tandem repeats of 20 amino acids that contain immunogenic epitopes. In some cancers, it is exposed in a non-glycosylated form that is recognized by the immune system (Gendler et al., J Biol Chem 1990;265:15286-15293).

[0223] In another embodiment, the cancer antigen is a mutated B-Raf antigen, which is associated with melanoma and colon cancer. The majority of these mutations represent a single nucleotide change of TA at nucleotide 1796, resulting in a change from valine to glutamic acid at residue 599 within the activation segment of B-Raf. Raf proteins are also indirectly associated with cancer as effectors of activated Ras proteins, the oncogenic forms of which are present in roughly one-third of all human cancers. Normal, non-mutated B-Raf participates in cell signaling, relaying signals from the cell membrane to the nucleus. The protein is usually active only when necessary to relay signals. In contrast, mutant B-Raf is reported to be constantly active and disrupts signaling relays (Mercer and Pritchard, Biochim Biophys Acta(2003)1653(1):25-40; Sharkey et al., Cancer Res.(2004)64(5):1595-1599).

[0224] Preferably, the cancer antigen is the human epidermal growth factor receptor-2 (HER-2 / neu) antigen. Cancers with cells that overexpress HER-2 / neu are known to be HER-2 / neu-specific. + Cancer is called. Exemplary HER-2 / neu +Cancers include prostate cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, skin cancer, liver cancer (eg, hepatocellular adenocarcinoma), intestinal cancer, and bladder cancer.

[0225] HER-2 / neu has an extracellular binding domain (ECD) of approximately 645 aa with 40% homology to the epidermal growth factor receptor (EGFR), a highly hydrophobic transmembrane anchor domain (TMD), and a carboxy-terminal intracellular domain (ICD) of approximately 580 aa with 80% homology to EGFR. The nucleotide sequence of HER-2 / neu is available in GENBANK™ under accession numbers AH002823 (human HER-2 gene, promoter region and exon 1), M16792 (human HER-2 gene, exon 4), M16791 (human HER-2 gene, exon 3), M16790 (human HER-2 gene, exon 2), and M16789 (human HER-2 gene, promoter region, and exon 1). The amino acid sequence of the HER-2 / neu protein is available in GENBANK™ under accession number AAA58637. Based on these sequences, one of skill in the art can develop HER-2 / neu antigens using known assays to find appropriate epitopes that will generate an effective immune response.

[0226] Exemplary HER-2 / neu antigens include p369-377 (a HER-2 / neu derived HLA-A2 peptide), dHER2 (Corixa Corporation), li-Key MHC class II epitope hybrid (Generex Biotechnology Corporation), peptide P4 (amino acids 378-398), peptide P7 (amino acids 610-623), a mixture of peptide P6 (amino acids 544-560) and P7, a mixture of peptides P4, P6, and P7, and HER2[9 754 ] etc.

[0227] Preferably, the cancer antigen is epidermal growth factor receptor (EGFR) antigen. The EGFR antigen can be EGFR variant 1 antigen, EGFR variant 2 antigen, EGFR variant 3 antigen, and / or EGFR variant 4 antigen. The cancer with cells that overexpress EGFR is called EGFR cancer. Exemplary EGFR cancers include lung cancer, head and neck cancer, colon cancer, colorectal cancer, breast cancer, prostate cancer, gastric cancer, ovarian cancer, brain cancer, and bladder cancer.

[0228] Preferably, the cancer antigen is a vascular endothelial growth factor receptor (VEGFR) antigen. VEGFR is believed to be a regulator of cancer-induced angiogenesis. Cancers with cells that overexpress VEGFR are known to be associated with VEGFR-associated angiogenesis. + Cancer. Exemplary VEGFR + Cancers include breast cancer, lung cancer, small cell lung cancer, colon cancer, colorectal cancer, renal cancer, leukemia, and lymphocytic leukemia.

[0229] Preferably, the cancer antigen is prostate-specific antigen (PSA) and / or prostate-specific membrane antigen (PSMA), which are commonly expressed in androgen-independent prostate cancer.

[0230] Preferably, the cancer antigen is Gp-100 glycoprotein 100 (gp 100), a tumor-specific antigen associated with melanoma.

[0231] Preferably, the cancer antigen is carcinoembryonic antigen (CEA). Cancers with cells that overexpress CEA are characterized by the presence of CEA antigen. + Cancer is called. Exemplary CEA + Cancers include colorectal cancer, gastric cancer, and pancreatic cancer. Exemplary CEA antigens include CAP-1 (i.e., CEA aa 571-579), CAP1-6D, CAP-2 (i.e., CEA aa 555-579), CAP-3 (i.e., CEA aa 87-89), CAP-4 (CEA aa 1-11), CAP-5 (i.e., CEA aa 345-354), CAP-6 (i.e., CEA aa 19-28), and CAP-7.

[0232] Preferably the cancer antigen is carbohydrate antigen 10.9 (CA19.9), which is an oligosaccharide related to Lewis A blood group material and is associated with colorectal cancer.

[0233] Preferably, the cancer antigen is a melanoma cancer antigen. The melanoma cancer antigen is useful for treating melanoma. Exemplary melanoma cancer antigens include MART-1 (e.g., MART-1 26-35 peptide, MART-1 27-35 peptide), MART-1 / MelanA, pMel17, pMel17 / gp100, gp100 (e.g., gp 100 peptide 280-288, gp 100 peptide 154-162, gp 100 peptide 457-467), TRP-1, TRP-2, NY-ESO-1, p16, beta-catenin, mum-1, etc.

[0234] Preferably, the cancer antigen is a mutant or wild-type ras peptide. The mutant ras peptide can be a mutant K-ras peptide, a mutant N-ras peptide, and / or a mutant H-ras peptide. The mutations in ras protein typically occur at position 12 (e.g., arginine or valine substituted for glycine), position 13 (e.g., asparagine to glycine), position 61 (e.g., glutamine to leucine), and / or position 59. The mutant ras peptide can be useful as a lung cancer antigen, gastrointestinal cancer antigen, hepatoma antigen, bone marrow cancer antigen (e.g., acute leukemia, myelodysplasia), skin cancer antigen (e.g., melanoma, basal cell, squamous cell), bladder cancer antigen, colon cancer antigen, colorectal cancer antigen, renal cell carcinoma antigen.

[0235] In another embodiment of the present invention, the cancer antigen is a mutant and / or wild-type p53 peptide. The p53 peptide can be used as a colon cancer antigen, a lung cancer antigen, a breast cancer antigen, a hepatocellular carcinoma antigen, a lymphoma cancer antigen, a prostate cancer antigen, a thyroid cancer antigen, a bladder cancer antigen, a pancreatic cancer antigen, an ovarian cancer antigen.

[0236] The cancer antigen can be a cell, a protein, a peptide, a fusion protein, a DNA encoding a peptide or a protein, an RNA encoding a peptide or a protein, a glycoprotein, a lipoprotein, a phosphoprotein, a carbohydrate, a lipopolysaccharide, a lipid, a chemically bonded combination of two or more thereof, a fusion of two or more thereof, or a mixture of two or more thereof, or a virus encoding two or more thereof, or an oncolytic virus encoding two or more thereof. In another embodiment, the cancer antigen is a peptide comprising about 6 to about 24 amino acids, about 8 to about 20 amino acids, about 8 to about 12 amino acids, about 8 to about 10 amino acids, or about 12 to about 20 amino acids. In one embodiment, the cancer antigen is a peptide having an MHC class I binding motif or an MHC class II binding motif. In another embodiment, the cancer antigen comprises a peptide corresponding to one or more cytotoxic T lymphocyte (CTL) epitopes.

[0237] cell therapy The disclosed method includes administration of a fusion protein of SEQ ID NO: 1 in combination with administration of a therapeutic cell therapy. Cell therapy useful for treating cancer is well known and is disclosed, for example, in U.S. Patent No. 7,402,431. In a preferred embodiment, the cell therapy is T cell transplantation. In a preferred method, T cells are expanded ex vivo with IL-2 before transplantation into a subject. Methods of cell therapy are disclosed, for example, in U.S. Patent No. 7,402,431, U.S. Patent No. 2006 / 0057121, U.S. Patent No. 5,126,132, U.S. Patent No. 6,255,073, U.S. Patent No. 5,846,827, U.S. Patent No. 6,251,385, U.S. Patent No. 6,194,207, U.S. Patent No. 5,443,983, U.S. Patent No. 6,040,177, U.S. Patent No. 5,766,920, and U.S. Patent No. 2008 / 0279836.

[0238] Radiation therapy The disclosed method includes administration of a fusion protein of SEQ ID NO:1 further combined with radiation therapy. The term "radiotherapy", which may be used interchangeably with the term radiotherapy, is a type of cancer treatment that uses beams of intense energy to kill cancer cells. Radiation therapy often uses X-rays, but can also use gamma rays, electron beams, or protons. The term "radiotherapy" often refers to external beam radiation therapy. During this type of radiation, the high-energy beam comes from a machine outside the patient's body, which directs the beam to a precise point on the body. Each session is quick and painless, lasting about 15 minutes. As used herein, the term "session" or "session of treatment" refers to each radiation therapy treatment. A radiation therapy "regimen" or "schedule" usually consists of a specific number of treatments given over a period of time, depending on the type and stage of the cancer.

[0239] low molecule The disclosed method includes administration of a fusion protein of SEQ ID NO: 1 in combination with administration of an anti-cancer small molecule. Small molecules effective in treating cancer are well known in the art and include antagonists of factors involved in tumor growth, such as EGFR, ErbB2 (also known as Her2), ErbB3, ErbB4 or TNF. Non-limiting examples include small molecule receptor tyrosine kinase inhibitors (RTKIs) that target one or more tyrosine kinase receptors, such as VEGF receptors, FGF receptors, EGF receptors, PDGF receptors, etc.

[0240] Many therapeutic small molecule RTKIs are known in the art, including lenvatinib (LENVIMA), lucitanib, axitinib (INLYTA), vatalanib (PTK787), erlotinib (TARCEVA™), OSI-7904, ZD6474 (ZACTIMA™), ZD6126 (ANG453), ZD1839, sunitinib (SUTENT™), ), Semaxanib (SU5416), AMG706, AG013736, Imatinib (GLEEVEC™), MLN-518, CEP-701, PKC-412, Lapatinib (GSK572016), VELCADE™, AZD2171, Sorafenib (NEXAVAR™), XL880, CHIR-265. Small molecule protein tyrosine phosphatase inhibitors (such as those disclosed in Jiang et al., Cancer Metastasis Rev. 2008;27:263-72) are also useful in carrying out the methods of the present invention. Such inhibitors can target, for example, HSP2, PRL, PTP1B, or Cdc25 phosphatases.

[0241] Small molecules that target Bcl-2 / Bcl-XL (such as those disclosed in US 2008 / 0058322) are also useful for carrying out the methods of the present invention. Further exemplary small molecules for use in the present invention are disclosed in Zhang et al. Nature Reviews: Cancer 2009;9:28-39. In particular, chemotherapeutic agents that cause immunogenic cell death, such as anthracyclines (Kepp et al., Cancer and Metastasis Reviews 2011;30:61-9), may be well suited for synergistic effects with extended PK IL-2.

[0242] Other cytotoxic and chemotherapeutic agents The methods of the present disclosure include administration of a fusion protein of SEQ ID NO:1 in combination with chemotherapeutic agents, including, but not limited to, alkylating agents, antitumor antibiotics, antimetabolites, other antitumor antibiotics, and plant-derived agents.

[0243] Alkylating agents are agents that impair cell function by forming covalent bonds with amino, carboxyl, sulfhydryl, and phosphate groups in biologically important molecules. The most important sites of alkylation are DNA, RNA, and proteins. Alkylating agents depend on cell proliferation for activity, but are not cell cycle phase specific. Alkylating agents suitable for use in the present invention include, but are not limited to, bischloroethylamines (nitrogen mustards, e.g., chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracil mustard), aziridines (e.g., thiotepa), alkylalkonesulfonates (e.g., busulfan), nitrosoureas (e.g., BCNU, carmustine, lomustine, streptozocin), non-classical alkylating agents (e.g., altretamine dacarbazine, procarbazine), and platinum compounds (e.g., carboplatin, oxaliplatin, cisplatin).

[0244] Antitumor antibiotics such as adriamycin intercalate DNA at guanine-cytosine and guanine-thymine sequences, resulting in spontaneous oxidation and formation of free oxygen radicals that cause strand breaks.Other antibiotics suitable for use in the present invention include, but are not limited to, anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, anthracenedione), mitomycin C, bleomycin, dactinomycin, and plicatomycin.

[0245] Anti-metabolites suitable for use in the present invention include, but are not limited to, floxuridine, fluorouracil, methotrexate, leucovorin, hydroxyurea, thioguanine, mercaptopurine, cytarabine, pentostatin, fludarabine phosphate, cladribine, asparaginase, gemcitabine.

[0246] Plant-derived agents include taxanes, which are semi-synthetic derivatives of precursors extracted from the needles of the yew plant. These drugs have a novel 14-membered ring, which is the taxane. Unlike vinca alkaloids, which cause microtubule polymerization, taxanes (e.g., taxol) promote microtubule polymerization and stability, thus blocking the cell cycle in mitosis. Other plant-derived agents include, but are not limited to, vincristine, vinblastine, vindesine, vinzolidine, vinorelbine, etoposide, teniposide, and docetaxel.

[0247] Combination Therapeutic Compositions In certain embodiments, the fusion protein of SEQ ID NO:1 is administered together (concurrently or sequentially) with one or more additional therapeutic agents or other therapeutic agents, such as a therapeutic antibody. The fusion protein of SEQ ID NO:1 is preferably administered prior to administration of one or more therapeutic agents, such as a therapeutic antibody. The fusion protein of SEQ ID NO:1 is preferably administered simultaneously with administration of one or more therapeutic agents, such as a therapeutic antibody. The fusion protein of SEQ ID NO:1 is preferably administered after administration of one or more therapeutic agents, such as a therapeutic antibody. SEQ ID NO:1 and one or more therapeutic agents, such as a therapeutic antibody, are preferably administered simultaneously. In other embodiments, the one or more therapeutic agents, such as a therapeutic antibody, are administered sequentially. The fusion protein of SEQ ID NO:1 and one or more therapeutic agents, such as a therapeutic antibody, are preferably administered within 1, 2, or 3 days of each other.

[0248] The one or more therapeutic agents may serve as adjunctive therapy for cancer, such as cytokines, chemotherapeutic agents, small molecules, antigens, or therapeutic antibodies, which are well known in the art and discussed above. Additional non-limiting examples of additional agents include GM-CSF (expands monocyte and neutrophil populations), IL-7 (important for memory T cell generation and survival), interferon alpha, tumor necrosis factor alpha, IL-12, and therapeutic antibodies, such as anti-PD-1, anti-PD-L, anti-CTLA4, anti-CD40, anti-OX40, anti-CD137, PARP inhibitors, PARP antibodies, etc. In some embodiments, the subject receives the fusion protein of SEQ ID NO: 1 and one or more therapeutic agents during the same period of prevention, disorder development, and / or treatment.

[0249] Preferably, the invention provides a separate pharmaceutical composition comprising a fusion protein of SEQ ID NO:1 and a pharma- ceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant, and another pharmaceutical composition comprising one or more therapeutic agents, such as a therapeutic antibody, and a pharma- ceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0250] Preferably, the present invention provides a pharmaceutical composition comprising a fusion protein of SEQ ID NO:1 and one or more therapeutic or anti-cancer agents in the same composition, together with a pharma- ceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0251] kit Also provided is a kit comprising the fusion protein of SEQ ID NO:1 formulated for SC administration, and optionally any other chemotherapeutic or anticancer agent. The kit is generally in the form of a physical structure housing various components, as described below, and can be utilized, for example, in carrying out the above-mentioned methods. The kit can include the fusion protein of SEQ ID NO:1 (e.g., provided in a sterile container), which can be in the form of a pharmaceutical composition suitable for administration to a subject. The pharmaceutical composition can be provided in a ready-to-use form or in a form that requires, for example, reconstitution or dilution before administration. If the composition is in a form that requires reconstitution by the user, the kit can also include buffers, pharma- ceutically acceptable excipients, etc., packaged together with or separately from the fusion protein of SEQ ID NO:1. If a combination therapy (e.g., a fusion protein of SEQ ID NO:1 and an immune checkpoint inhibitor) is contemplated, the kit can contain the several agents separately, or the agents can be already combined in the kit. Similarly, if an additional complementary therapy (e.g., a fusion protein of SEQ ID NO:1, an immune checkpoint inhibitor, and an additional complementary therapy or agent) is required, the kit can contain the several agents separately, or two or more of the agents can be already combined in the kit.

[0252] The kits of the invention can be designed for conditions (e.g., refrigeration or freezing) necessary to properly maintain the components contained therein. The kits can include labels or packaging inserts containing the identity of the components therein and instructions for their use (e.g., clinical pharmacology of the active ingredients, including administration parameters, mechanism of action, pharmacokinetics and pharmacodynamics, side effects, contraindications, etc.).

[0253] Each component of the kit can be enclosed within an individual container, and all of the various containers can be placed within a single package. The label or insert can include manufacturer information such as lot number and expiration date. The label or packaging insert can, for example, be incorporated into the physical structure that houses the component, can be separately contained within the physical structure, or can be affixed to the kit component (e.g., an ampoule, syringe, or vial).

[0254] The label or insert may additionally comprise or be incorporated into a computer readable medium such as a disk (e.g., hard disk, card, memory disk), optical disk such as CD-ROM / RAM or DVD-ROM / RAM, DVD, MP3, magnetic tape, or electronic storage medium such as RAM and ROM, or hybrids thereof such as magnetic / optical storage media, flash media, or memory type cards, etc. In some embodiments, the actual instructions are not present in the kit, but a means is provided for obtaining the instructions from a remote source, for example via an internet site.

[0255] Equivalence and Scope Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or clear from the context and the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can assume from any particular value or subrange within the ranges described in different embodiments of the invention to one tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0256] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Such embodiments may be excluded because they are deemed to be known to those of skill in the art, even if the exclusion is not expressly set forth herein.

[0257] Any particular embodiment of the compositions of the present invention, any method of manufacture, or any method of use may be excluded from any one or more claims for any reason, whether or not related to the existence of prior art.

[0258] EXAMPLES

[0259] Example 1 - A less frequent dosing regimen of the fusion protein of SEQ ID NO:1 administered intravenously as monotherapy and in combination with pembrolizumab in subjects with advanced solid tumors. The fusion protein of SEQ ID NO:1 is a cytotoxic CD8 + It is a fusion of circularly permuted IL-2 and IL-2 receptor alpha (IL-2Rα) designed to selectively activate the intermediate affinity IL-2R, consisting of IL-2Rβ and γ, for the activation of T cells and NK cells. The intermediate affinity IL-2R is expressed primarily on effector lymphocytes, which play an important role in driving antitumor immune responses. Wild-type IL-2 binds IL-2Rα, β, and γ c and activates the high-affinity IL-2R, which consists of IL-2 receptors, at concentrations below those at which effector cells bearing intermediate-affinity IL-2R are activated, and inhibits the immunosuppressive CD4 + Adjustability T(T reg Selective activation of the intermediate affinity IL-2R can enhance tumor killing and has been shown to have enhanced antitumor activity compared to IL-2 in mouse models.

[0260] Previous clinical trials have relied on administering the fusion protein of SEQ ID NO:1 for 5 consecutive days at the start of therapy. This administration schedule is cumbersome for patients and presents logistical difficulties. A less frequent administration schedule (e.g., 1 administration per treatment cycle, 2 administrations per treatment cycle, or 3 administrations per treatment cycle) would be preferable. To that end, an optimized administration regimen was developed using quantitative systems pharmacology (QSP) modeling to predict the dose that would result in similar proliferation of CD8+ T cells and NK cells as previously achieved with 3 μg / kg / day and 6 μg / kg / day qdx5 (administration for 5 consecutive days).

[0261] method A QSP model of the fusion protein of SEQ ID NO:1 was developed in MATLAB / Simbiology by leveraging published literature data and calibrated with in vitro pSTAT5 data and in vivo clinical data. The model was validated by comparing simulated cell counts in peripheral blood with observed clinical data and applied to predict changes in CD8+ T cells and NK cells in peripheral blood and tumors in response to IV administration of the fusion protein of SEQ ID NO:1 given once (day 1), twice (days 1 and 8, or days 1 and 4), or three times (days 1, 8 and 15, or days 1, 4 and 8) in a 21-day cycle.

[0262] result Model-predicted time courses of circulating CD8+ T cells and NK cells explained the observed clinical data from the ARTISTRY-1 trial reasonably well. Application of the QSP model predicted that CD8+ T cell and NK cell expansion could be equivalent to that achieved with 6 μg / kg / day qdx5 for each of a) a single dose of 30-40 μg / kg, b) two doses of 20-25 μg / kg / dose on days 1 and 8, c) two doses of 15 μg / kg / dose on days 1 and 4, d) three doses of 20 μg / kg / dose on days 1, 8, and 15, and e) three doses of 15 μg / kg / dose on days 1, 4, and 8. Lower dose levels would be required to achieve CD8+ T cell and NK cell expansion equivalent to that achieved with 3 μg / kg / day qdx5.

[0263] Simulations with the model were performed to match the area under the curve (AUC) or maximum cell count (Rmax) of CD8+ T cells and NK cells in blood and tumor with 3 μg / kg or 6 μg / kg IV qdx5 (5 days on, 16 days off) in a 21 day cycle. The simulations assumed that 3 μg / kg and 6 μg / kg represented a "stable disease" state. Simulations were performed over the following 21 day cycles: -3 doses / 21 day cycle: 1) doses on days 1, 4, and 8, or 2) doses on days 1, 8, and 15. -2 doses / 21-day cycle: 1) doses on days 1 and 4, or 2) doses on days 1 and 8. -Single dose / 21-day cycle: Administer on day 1.

[0264] Simulation graphs depicting CD8+ T cell and NK cell counts in blood and tumor are shown in Figures 2-11. The graphs begin at "Day 50," which corresponds to the day 1 dosing described above for each condition. The results show that each of the new less frequent dosing regimens demonstrated similar or better activity than the previous 3 μg / kg or 6 μg / kg qdx5 dosing regimens. This was observed in both blood and tumor for maximum cell counts (Rmax) and AUC values ​​for both CD8+ T cells and NK cells, respectively.

[0265] Example 2 - Cohort 2 of the ARTISRY-3 clinical trial: A less frequent dosing regimen of the fusion protein of SEQ ID NO: 1 administered intravenously as monotherapy and in combination with pembrolizumab in subjects with advanced solid tumors. Given the modeling data predicting a less frequent dosing regimen, we plan to add a new cohort (cohort 2) for the ARTISRY-3 clinical trial (NCT04592653).

[0266] The current dosing schedule requires patients to receive the fusion protein of SEQ ID NO:1 by infusion for 5 consecutive days in a 21-day cycle (Q21D), which limits dosing flexibility and can be a burden for patients and staff at treatment facilities. A longer dosing interval period can minimize patient burden and reduce risks associated with more frequent dosing (e.g., infusion reactions), as well as exposure to infectious diseases (e.g., SARS-CoV-2) associated with visits to hospitals or infusion centers. It is hypothesized that higher doses of the fusion protein of SEQ ID NO:1 administered in less frequent dosing will be safe and effective.

[0267] [Table 1-1] [Table 1-2] [Table 1-3]

[0268] Cohort 2 (less frequent IV administration) The dose selection for Cohort 2 (less frequent IV administration) is supported by preclinical and clinical evidence.

[0269] 1. Preclinical Data: Various preclinical studies provide sufficient support for testing higher doses of the fusion protein of SEQ ID NO:1 administered once, twice, or three times during a 21-day cycle in clinical settings. 1. Safety assessment for systemic toxicity and nonclinical exposure margins support dose selection for Cohort 2 (less frequent IV administration). Maximum observed concentration (C) during 24-hour exposure on Day 1 at the no observed adverse effect level (NOAEL, 0.1 mg / kg / day) from a repeated-dose (28-day) Standard Clinical Practice IV toxicity study in mice (using daily dosing) and a 5-week Standard Clinical Practice IV study in cynomolgus monkeys (5 days on, 9 days off cycling dosing regimen).max ) and area under the curve (AUC 0-24h ) was administered as a single dose of 40 μg / kg (C of 920 ng / mL) max and AUC of 3808 ng-hours / mL. 0-24hr ) were comparable to or somewhat higher than the clinical exposures expected in humans after 28-day and 5-week IV toxicity studies. In addition, the calculated AUCs at the NOAEL in mice and monkeys from 28-day and 5-week IV toxicity studies were 0-21day The exposure of 100 mg / kg / day was measured over a 21-day period (AUC 0-21day ) The exposure in animals at the NOAEL was at least 9.5-fold higher than the estimated human exposure (5554 ng h / mL). 2. According to the International Council for Harmonisation (ICH) guidance, the highest dose or exposure tested in nonclinical studies does not limit the dose escalation or highest dose investigated in clinical trials in cancer patients. However, these observations in monkeys and mice suggest that there may be an appropriate exposure multiple between the planned clinical dose under evaluation and the dose in animals that was systemically well tolerated. 3. A safety evaluation of local toxicity was performed. The NOAEL in the definitive 5-week IV monkey study was 0.1 mg / kg / day (cycling dosing regimen) based on systemic toxicity findings. Because there were no adverse events at the IV injection site in this study, a higher dose of 0.3 mg / kg / day was used for the safety evaluation of local tolerability. The 0.3 mg / kg / day dose was converted to approximately 0.75 mg / injection site based on an average monkey weight of approximately 2.5 kg. Doses of 25 and 40 μg / kg in humans correspond to approximately 1.5 mg and 2.4 mg per injection site, which are 2.0- and 3.2-fold higher than the IV NOAEL in cynomolgus monkeys, respectively. In a dose-ranging IV monkey study, no adverse injection site findings were observed at doses up to 0.6 mg / kg (the highest dose for which histopathology data were available). This dose in monkeys (approximately 1.5 mg / injection site) is equivalent to the human dose of 25 μg / kg on a mg / injection site basis, and slightly lower than the human dose of 40 μg / kg. These non-clinical findings, together with the acceptable local tolerability profile of the fusion protein of SEQ ID NO:1 following IV administration in humans, support the local safety of the planned clinical doses of the fusion protein of SEQ ID NO:1. 4. Furthermore, it is possible to perform less frequent IV administration of the fusion protein of SEQ ID NO:1 with higher dose levels without causing weight loss while achieving comparable antitumor efficacy based on results from the B16F10 mouse model of lung metastasis (Lopes et al. Journal for Immunotherapy of Cancer. 2020:E000673).

[0270] 2. Clinical Data: In the ongoing ARTISTRY-1 study, the fusion protein of SEQ ID NO:1 was shown to be generally well tolerated at doses up to 8 μg / kg / day for monotherapy and up to 6 μg / kg / day when administered in combination with pembrolizumab. Treatment-related adverse events (AEs) were manageable and those associated with the expected targeted effects (e.g., fever, chills, reduction in white blood cell subsets) were transient. Systemic exposure to the fusion protein of SEQ ID NO:1 increased with increasing dose levels evaluated in the ongoing ARTISTRY-1 study. In patients with a variety of solid tumors, serum concentrations of the fusion protein of SEQ ID NO:1 achieved target exposures in excess of levels required for activation of NK cells and CD8+ T cells as determined by preclinical pharmacology studies. Furthermore, clinical administration of the fusion protein of SEQ ID NO:1 at dose levels up to 8 μg / kg / day demonstrated a significant improvement in T reg Resulted in selective dose-dependent increases in circulating NK cells and CD8+ T cells combined with minimal and non-dose-dependent changes in IL-6 levels. Transient dose-dependent increases in serum IL-6 levels occurred within hours of dosing and were associated with transient fever and chills. Both the 6 μg / kg / day monotherapy dose level in combination with pembrolizumab (monotherapy RP2D) and the 3 μg / kg / day dose induced the desired mechanistic effects, and AEs were expected based on the mechanism of action and were manageable in both inpatient and outpatient settings. In ARTISTRY-1, clinical efficacy was observed in patients treated with fusion protein of SEQ ID NO:1 in monotherapy at RP2D of 6 μg / kg (Part B) and in patients treated with fusion protein of SEQ ID NO:1 at 3 μg / kg and 6 μg / kg in combination with pembrolizumab (Part C). Based on the hypothesized mechanism of action of fusion protein of SEQ ID NO:1, activation and proliferation of tumor-killing effector cells such as CD8+ T cells and NK cells are used as biomarkers to predict clinical efficacy. Therefore, PD modeling was performed to predict dose levels with less frequent IV dosing regimens (e.g., 1 dose, 2 doses, or 3 doses in a 21-day treatment cycle) to achieve a degree of CD8+ T cell and NK cell proliferation comparable to that achieved with fusion protein of SEQ ID NO:1 at 3 μg / kg and 6 μg / kg administered daily for 5 days in a 21-day treatment cycle.

[0271] Doses of 15 μg / kg and 30-40 μg / kg administered once (day 1) in a 21-day cycle are predicted to achieve CD8+ T cell and NK cell proliferation levels comparable to those achieved with 3 μg / kg and 6 μg / kg fusion protein of SEQ ID NO:1, respectively, administered once daily for 5 days in a 21-day cycle. Two scenarios for two doses in a 21-day cycle were evaluated: dosing on days 1 and 8 and dosing on days 1 and 4. Doses of 10 μg / kg and 20-25 μg / kg administered twice (days 1 and 8) in a 21-day cycle are predicted to achieve CD8+ T cell and NK cell proliferation levels comparable to those achieved with 3 μg / kg and 6 μg / kg fusion protein of SEQ ID NO:1, respectively, administered once daily for 5 days in a 21-day cycle. Doses of 10 μg / kg and 15 μg / kg administered twice (days 1 and 4) in a 21 day cycle are predicted to achieve CD8+ T cell and NK cell proliferation levels comparable to those achieved with 3 μg / kg and 6 μg / kg, respectively, of the fusion protein of SEQ ID NO:1 administered once daily for 5 days in a 21 day cycle.

[0272] In Part A of ARTISTRY-1, 43 patients received the fusion protein of SEQ ID NO:1 as monotherapy at up to 10 μg / kg. To date, at least seven patients have received the fusion protein of SEQ ID NO:1 at 10 μg / kg on Day 1 of the ARTISTRY-1 study. All seven patients tolerated the Day 1 dose, and no dose-limiting toxicities (DLTs) were reported within 24 hours of the Day 1 dose (prior to the Day 2 dose). One DLT was reported in a 70-year-old male patient with a history of RCC and left nephrectomy after Day 4 of continuous IV dosing of 10 μg / kg of the fusion protein of SEQ ID NO:1. After receiving four doses of the fusion protein of SEQ ID NO:1 in cycle 1, the patient developed grade 4 acute kidney injury requiring hemodialysis after hypotension (responsive to fluids) and the use of nonsteroidal anti-inflammatory drugs. This event led to the patient's discontinuation of treatment.

[0273] Enrollment in the ARTISTRY-1 trial is now closed, and the Safety Review Committee (SRC) concluded that the MTD had not been reached. The 8 μg / kg dose level administered on days 1-5 of a 21-day cycle (cumulative dose of 40 μg / kg / 21 days) was also well tolerated, with no DLTs reported.

[0274] PD modeling and clinical data from the ARTISTRY-1 trial support begins at 10 μg / kg administered on day 1 of a 21-day cycle.

[0275] Cohort 2 Study Objectives Main purpose: 1) To investigate the safety and tolerability of a less frequent IV administration schedule of the fusion protein of SEQ ID NO:1 to identify and determine the RP2D of the fusion protein of SEQ ID NO:1 as monotherapy. 2) To determine the MTD of the fusion protein of SEQ ID NO:1 as monotherapy and in combination with pembrolizumab. Each primary objective will be assessed by measuring the incidence of DLT from the first dose to the end of the DLT observation period. Secondary Objectives: 1) To evaluate clinical antitumor activity (ORR, DOR). 2) To evaluate the safety and tolerability of protein fusions of SEQ ID NO:1 as monotherapy and in combination with pembrolizumab. 3) To evaluate the clinical PK of protein fusions of SEQ ID NO:1. 4) To evaluate the immunogenicity of protein fusions of SEQ ID NO:1. 5) To evaluate the effect of protein fusion of SEQ ID NO:1 as monotherapy and in combination with pembrolizumab on circulating leukocyte and peripheral blood soluble protein profiles in patients with various advanced malignant solid tumors. Secondary objectives are evaluated as follows: 1) Clinical antitumor activity: ORR and DOR based on RECIST v1.1 and iRECIST. 2) Occurrence, nature, and severity of AEs, including absolute values ​​and changes from baseline in vital signs, laboratory tests, and ECG. 3) Concentrations of the fusion protein of SEQ ID NO:1 in the serum of patients throughout the treatment period, and descriptive PK parameters. 4) Presence of the anti-fusion protein of SEQ ID NO:1 antibody in the patient's serum throughout the treatment period. 5) Changes in absolute cell counts (including total T cells, CD8+ T cells, CD56+ cells, and Treg cells) and ratios (including T / Treg, CD8+ / Treg, CD56+ / Treg) between serial peripheral blood samples before and during treatment obtained from patients treated with the fusion protein of SEQ ID NO:1 as monotherapy and with the combination of the fusion protein of SEQ ID NO:1 and pembrolizumab.

[0276] Patient selection criteria Each patient must meet all of the following inclusion criteria to be eligible to participate in the study. 1. The patient or the patient's legal guardian is willing and able to provide written informed consent prior to the start of the study. 2. The patient is aged 18 years or older. 3. Patients have a histologically or cytologically confirmed diagnosis of a malignant solid tumor of the type included in the study (see inclusion criterion 4 below) and have at least one accessible lesion for biopsy. 4. All patients must have advanced solid malignancies as defined below. Histologically or cytologically confirmed epithelial tumors of the fallopian tube, peritoneum, or ovary, cervical carcinoma, endometrial carcinoma, non-small cell lung adenocarcinoma, small cell lung carcinoma, gastric and gastroesophageal junction adenocarcinoma, esophageal carcinoma (squamous and adenocarcinoma types), pancreatic carcinoma, biliary tract tumors (including intrahepatic and extrahepatic cholangiocarcinoma, gallbladder, dilated carcinoma), cutaneous melanoma, mucosal melanoma, squamous cell carcinoma of the head and neck, or metastatic or advanced breast cancer after failure or poor tolerance of 1 to 3 prior established indication-specific therapies. NOTE: Patients must have received 1-3 prior FDA-approved targeted therapies. Failure of adjuvant and neoadjuvant therapy is considered a treatment option. 5. All patients must agree to have a baseline biopsy used to evaluate the MSI status of their malignancy. 6. Baseline biopsies for all patients must be obtained within 3 months prior to screening and at least 4 weeks after completion of the last anti-tumor therapy. 7. Patients must have at least one lesion that qualifies as a target lesion based on Response Evaluation Criteria in Solid Tumors (RECIST) version (v) 1.1 as determined by the treating physician and is not confounded by prior therapy such as radiation. A lesion will not be considered eligible as a target lesion if it has been previously irradiated. Patients must be able to provide an adequate tissue sample for planned biopsy, which will not affect the measurement of tumor size by RECIST v1.1 criteria. 8. Patients must be ambulatory with an Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 or 1 and an estimated life expectancy of at least 3 months. 9. Patients must have adequate hemocompetence as evidenced by the following: Absolute neutrophil count ≥ 1000 / μL Absolute lymphocyte count ≥ 500 / μL within 2 weeks of starting study drug - Platelet count ≥ 75,000 / μL without platelet transfusion support within 2 weeks of starting study drug Hemoglobin ≥ 9g / dL (may be transfused to this level if necessary) 10. Patients must have adequate hepatic and renal function as evidenced by the following: Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels ≤ 3 x upper limit of normal (ULN; ≤ 5 x ULN if liver is known to be involved with metastatic disease) Serum total bilirubin level ≤ 1.5 x ULN (≤ 2 x ULN for patients with known Gilbert syndrome) Reference laboratory serum creatinine ≤ 1.5 x ULN or calculated creatinine clearance ≥ 60 mL / min by Cockcroft-Gault equation 11. For Cohort 2, Part A, prior treatment with immunotherapy is permitted as long as the patient has not previously experienced grade ≥3 autoimmune toxicity or drug-related toxicity requiring discontinuation. Patients in Cohort 2, Part B (less frequent IV dosing) who have previously received anti-PD-(L)1 for at least 3 months may be enrolled if they have a stable disease response or better. 12. Patients must have recovered from side effects / toxicity from any prior chemotherapy, immunotherapy, other prior systemic anti-cancer treatment, radiation therapy, or surgery (i.e., Grade 1 or less toxicity [Grade 2 alopecia and treatment-related peripheral neuropathy are acceptable] unless otherwise specified). 13. For Part A of Cohort 2, patients receiving prior anti-PD-1 directed therapy must wait at least 4 weeks from the last dose of such therapy before a screening biopsy is taken. Patients receiving other standard or investigational agents must wait at least 5 half-lives or 4 weeks (whichever is shorter) before a screening biopsy is taken, or 4 weeks if the half-life of the investigational agent is not known. For cytotoxic chemotherapy, patients must wait at least 2 weeks to allow bone marrow function to recover to baseline levels. A shorter waiting period may be acceptable after discussion with the medical monitor. 14. Women of childbearing potential (WOCBP) must have a negative pregnancy test (serum or urine) within 7 days of initiating treatment and on Day 1 prior to the first dose of study drug.

[0277] Patient Exclusion Criteria To be eligible to participate in this study, each patient must not have the following conditions: 1. Patient is currently pregnant or breastfeeding, or intends to become pregnant during the study period. 2. Patients with active infection or fever of 38.5°C or higher within 3 days after the first scheduled day of Cycle 1 administration. 3.Patients with primary central nervous system malignant tumors. 4. Patients with active or symptomatic central nervous system metastases have been tapered to a dose of 10 mg or less of the corticosteroid prednisone (or equivalent) for at least 2 weeks prior to the first dose, unless the metastases have been treated by surgery and / or radiation therapy and / or gamma knife, and the patient is neurologically stable. Patients with leptomeningeal disease are excluded. 5. Patients with known hypersensitivity to the fusion protein of SEQ ID NO:1 or any component of pembrolizumab. 6.Patients with uncontrollable bleeding disorders 7. Patients with a prolonged partial thromboplastin time >1.5 × ULN and / or an international normalized ratio >1.5. Patients receiving anticoagulants must have normalized clotting times or undergo appropriate interruption and / or reversal of anticoagulation therapy prior to undergoing biopsy. 8. Patients who require pharmacologic administration of systemic corticosteroids (>10 mg prednisone daily or equivalent). However, topical, ophthalmic, or inhaled steroids are acceptable. 9. Patients with a mean QT interval corrected by the Fridericia formula value >470 ms (females) or >450 ms (males) at screening, patients known to have congenital long QT syndrome, or patients taking medications known to cause QT interval prolongation on electrocardiogram (ECG). Patients whose ECG is isometric or borderline for QT prolongation must undergo appropriate cardiological evaluation and consultation and be considered at low risk for arrhythmias and / or other cardiac events before receiving study drug. 10. Patients with active autoimmune disease requiring systemic treatment within the past 2 years or a documented history of clinically severe autoimmune disease that has required chronic or frequent systemic steroids. Replacement therapy (e.g., thyroxine, insulin, or physiological corticosteroid replacement therapy for adrenal or pituitary insufficiency) is permitted. Vitiligo is not an exclusion. 11. Patient is known to be HIV positive and / or has a history of hepatitis B or C infection or is known to be positive for hepatitis B antigen / hepatitis B virus DNA or hepatitis C antibody or RNA. Active hepatitis C is defined by a known positive hepatitis C antibody result and a known quantitative hepatitis C virus RNA result greater than the lower limit of detection of the assay. 12. Subjects who are investigational site staff directly involved in the conduct of the study and their immediate family members, investigational site staff separately supervised by the investigator, or subjects who are employees of Alkermes or Syneos Health directly involved in the conduct of the study (immediate family members are defined as spouse, parent, child, or sibling, whether biological or adopted) 13. Patients with a known additional malignancy within 2 years of screening initiation. Exceptions include stable basal cell carcinoma of the skin, cutaneous squamous cell carcinoma that has undergone presumed curative therapy, and adequately treated cervical intraepithelial neoplasia. Other exceptions may be allowed with the sponsor's medical monitor and investigator approval. Patients with a completely treated prior malignancy and no evidence of disease for ≥2 years are eligible. 14. Patients with active autoimmune disease who have required systemic treatment within the past 2 years (i.e., treatment with the use of disease-modifying agents, corticosteroids, and / or other immunosuppressants). Replacement therapy (e.g., thyroxine) is not considered an excluded form of systemic treatment. 15. Patients who have received a live vaccine within 30 days prior to the first dose of study treatment 16. Patients with underlying chronic pulmonary disease, primary or metastatic pulmonary disease, pleural effusion, and / or interstitial lung disease will be excluded unless biopsy can be safely performed, preferably on a nonpulmonary lesion, and room oxygen saturation is ≥92%. 17. Patient has other uncontrolled medical conditions, including psychiatric or substance abuse disorders, that may interfere with the patient's ability to cooperate and participate in the study. Other examples of such conditions include cardiopulmonary disease defined as unstable, uncontrolled, or severe hypertension, clinically significant pericardial effusion, New York Heart Association class III or IV congestive heart failure, unstable angina, myocardial infarction, or cerebrovascular accident within 6 months of first dose, chronic obstructive pulmonary disease or diabetes that required 2 or more hospitalizations in the last year, severe peripheral vascular disease, or recent severe trauma. 18. Patients who have difficulty breathing at rest or require oxygen therapy 19. Previous solid organ transplant and / or non-autologous hematopoietic stem cell or bone marrow transplant 20. Patients who have received radiotherapy within the past 4 weeks prior to the start of study treatment, except for limited regional palliative radiotherapy at the discretion of the medical monitor. 21. Patients who have received a systemic immunomodulator within 4 weeks or 5 half-lives, whichever is shorter, prior to Day 1 of Cycle 1, except as described in #22 below. Exceptions may be made on a case-by-case basis by the medical monitor. 22.Patients who have previously received IL-2 or IL-15 soluble protein therapy will be excluded.

[0278] Overall study design and planning The trial will be conducted in two parts: Part A (monotherapy with the fusion protein of SEQ ID NO:1), and Part B (combination therapy with the fusion protein of SEQ ID NO:1 and pembrolizumab).

[0279] Cohort 2 (less frequent IV dosing) is a multicenter, open-label, sequential-arm study consisting of two parts that will evaluate IV dosing schedules to identify optimal less frequent IV dosing schedules of both SEQ ID NO:1 fusion protein as monotherapy and SEQ ID NO:1 fusion protein in combination with pembrolizumab by investigating the safety and tolerability of SEQ ID NO:1 fusion protein at various doses and schedules.

[0280] In this cohort, patients with advanced histologically or cytologically confirmed epithelial tumors of the fallopian tube, peritoneum, or ovary, cervical cancer, endometrial cancer, non-small cell lung adenocarcinoma, small cell lung cancer, gastric and gastroesophageal junction adenocarcinoma, esophageal cancer (squamous and adenocarcinoma types), pancreatic cancer, biliary tract tumors (including intrahepatic and extrahepatic cholangiocarcinoma, gallbladder, dilated tumors), any melanoma (excluding uveal melanoma), cervical cancer, squamous cell carcinoma of the head and neck, or metastatic or advanced breast cancer after failure or intolerance of up to three prior established indication-specific therapies will be enrolled.

[0281] Cohort 2 (less frequent IV dosing) will follow the BOIN design for dose escalation with modifications and accommodate open enrollment. Open enrollment is defined as the allocation of timed slots for enrollment at all available sites, maximizing utilization of patient resources. The SRC will be composed of the investigator, sponsor's medical monitor, sponsor's global safety officer, and additional representatives from the study team. The SRC will review safety data from Cohort 2 (less frequent IV dosing) and other relevant information to support dose escalation / deescalation decisions, as well as start or stop schedules.

[0282] In cohort 2 (less frequent IV dosing), the less frequent IV dosing RP2D will be selected and, after consultation with the sponsor, intrapatient dose escalation will be permitted.

[0283] Part A (monotherapy) In Part A, 1 to 3 different dosing schedules may be evaluated. A 1 dose per cycle dosing schedule will be evaluated first. Two doses per cycle (days 1 and 8, or days 1 and 4) may be evaluated depending on tolerability assessment of the 1 dose per cycle schedule. The MTD will be determined for 1 to 2 dosing schedules.

[0284] The three dosing schedules for Part A are as follows: · Schedule 1: Dosing on day 1 of a 21 day cycle (one dose per cycle schedule), planned dose levels of the fusion protein of SEQ ID NO:1 include 10, 20, 30, 35, and 40 μg / kg. · Schedule 2: Administered on days 1 and 8 of a 21 day cycle (administered on days 1 and 8 per cycle schedule), planned dose levels of the fusion protein of SEQ ID NO:1 include 15, 20, and 25 μg / kg. · Schedule 3:Administered on days 1 and 4 of a 21 day cycle (administered on days 1 and 4 per cycle schedule), planned dose levels of the fusion protein of SEQ ID NO:1 include 10, 15, and 20 μg / kg.

[0285] The maximum sample size was 30 patients per schedule.

[0286] Schedule 1 (one dose per cycle) of fusion protein of SEQ ID NO:1 will be tested first, starting with a dose of 10 μg / kg administered on day 1 of a 21-day cycle, escalating to doses of 20, 30, 35, and 40 μg / kg. If DLT criteria are met at doses above any of these respective doses and tapering is recommended based on dose escalation / tapering rules or by the SRC, dose levels of 15 μg / kg and / or 25 μg / kg may be allowed to facilitate tapering. Up to six patients may be enrolled at the two lower doses of this schedule (10 μg / kg and 20 μg / kg fusion protein of SEQ ID NO:1). If at least one patient completes the DLT evaluation period without experiencing a DLT, subsequent patients will be enrolled at the next dose level, starting initially with 10-20 μg / kg fusion protein of SEQ ID NO:1, then 20-30 μg / kg fusion protein of SEQ ID NO:1. In this schedule, up to nine patients may be enrolled at each of the higher doses (i.e., 30, 35, and 40 μg / kg of the fusion protein of SEQ ID NO:1). At least three patients must have complete DLT information (based on the definitions described at dose levels ≧30 μg / kg, completing DLT assessment and confirmation to allow for further dose de-escalation decisions).

[0287] More frequent dosing schedules (Schedule 2 and / or Schedule 3) may be allowed after the MTD has been determined, or after all expected dose levels are deemed tolerable on the Day 1 schedule, especially if the MTD of the Day 1 schedule is below the highest planned dose level and the highest planned dose level cannot be tested, or earlier based on SRC recommendation. Additionally, if evaluation of Schedule 1 reaches the maximum tolerated dose without reaching the MTD, a decision is made in conjunction with the SRC to allow Schedule 2 or 3 for alternative dosing twice per cycle. A second dose escalation is performed on Schedule 2 or 3, with dose levels starting at approximately half the MTD from Schedule 1 and escalating to the highest dose planned for the selected schedule.

[0288] For all schedules, additional higher dose levels may be administered, if necessary, at least one or two dose levels in increments of 5 μg / kg from the maximum dose.

[0289] Part B (Combination Therapy) Part B will begin when the highest dose of one of the three Part A dosing schedules has been tested and a decision has been made to stop testing the additional schedules. The combination dose may be initiated at a dose below the highest dose level being tested in Part A and titrated to the highest dose if the highest dose is deemed tolerable as monotherapy. A total of 12-15 patients will be enrolled in Part B in Cohort 2 (less frequent IV dosing) according to the BOIN design guidelines. If the tested combination dose is not tolerated, dose de-escalation to a dose level below the current dose will be considered. For Part B, the lowest dose of fusion protein of SEQ ID NO:1 that may be tested in combination with pembrolizumab is 15 μg / kg (if schedule 1 is selected) or 10 μg / kg (if schedule 2 or schedule 3 is selected). All patients in Part B of Cohort 2 will receive pembrolizumab at the approved dose of 200 mg Q3W.

[0290] A schematic diagram of the Cohort 2 study design is provided in Figure 1.

[0291] Dose-limiting toxicity DLT is defined by any of the following events observed during the interval from cycle 1 day 1 to cycle 1 day 21 and considered possibly, possibly, or definitely related to the fusion protein of SEQ ID NO: 1. DLT period is defined as the duration from the first dose to the end of the first cycle (21 days), although longer follow-up may be necessary to confirm DLT. To ensure patient safety, study enrollment will be paused (investigational sites will be notified by email or during regularly scheduled team meetings) if three or more participants have incomplete DLT information.

[0292] Definition of DLT: Hematological: Clinically significant grade 4 drug-related hematological AEs, except: - Grade 4 lymphopenia lasting 7 days or more and not associated with clinical symptoms; Non-haematological: Any Grade ≥ 3 non-haematological AE including blood chemistry / electrolyte imbalance or abnormality that does not resolve to Grade ≤ 2 within 7 days and is considered related to the study drug. - Drug-induced liver injury of any grade, - Any grade ≥2 myocarditis, - Any grade ≥3 hypotension / cytokine release syndrome occurring within 24-48 hours after study drug administration, persisting for >72 hours, and manageable with supportive care; - Capillary leak syndrome of any grade ≥ 3, - Treatment-related events leading to discontinuation or requiring a delay in initiating Cycle 2 of more than 7 days, - Exceptions: medically manageable fatigue, nausea, vomiting, diarrhea, and alopecia. Tumor relapse reported as a related AE is not considered a DLT. Patients may continue treatment until criteria for discontinuation are met.

[0293] Dose escalation and / or tapering Decisions to dose escalate and / or decrement, or to declare a dose unsafe, will be made in collaboration with the study SRC based on the following BOIN guidelines.

[0294] Data from the current dose will be used to determine the dosing schedule assignment for new patients entering the study, and this decision will be based on the estimated probability of DLT at the current dose.

number

number

number

[0295] The lower and upper bounds are derived using non-informative prior distributions following BOIN design calculations to minimize the chance of inaccurate dose allocation.

[0296] The dose levels are:

number

number

number

[0297] If the escalation / tapering rules recommend tapering from the lowest dose level and fewer than three patients are assigned to that dose, patients may continue to be assigned to the lowest dose level. The purpose of this rule is to ensure that sufficient data is collected at the lowest dose level. DLT decision criteria are summarized in Table 2 below.

[0298] [Table 2]

[0299] If a dose level is unsafe, all dose levels above it are also considered unsafe. New patients cannot be enrolled at the unsafe dose, but new patients may be enrolled at the current dose level or lower dose levels, upon recommendation of the SRC, if maximum patient enrollment at these dose levels has not yet been reached.

[0300] As additional information becomes available, the decision to declare a dose unsafe may change. For example, if three patients are enrolled at a dose and two patients with complete data have a DLT (2 / 2), the dose is considered unsafe. However, when the third patient completes the DLT observation period, if that patient does not experience a DLT (2 / 3), the dose is no longer considered unsafe.

[0301] The MTD will be determined when enrollment in the study is stopped and all patients have complete DLT information. The MTD will be determined using a Bayesian logistic regression method.

[0302] Fusion Protein of SEQ ID NO: 1 1. Dosing and Administration Patients are considered enrolled after providing written informed consent, completing all screening assessments, and providing a pretreatment biopsy. Study drug should be initiated as close as possible to the date and time of enrollment, but no later than 72 hours after enrollment.

[0303] In cohort 2 (less frequent IV dosing), the fusion protein of SEQ ID NO:1 will be administered via 30 minute (-5 min / +10 min) IV infusion on the assigned scheduled days. The length of each cycle is 21 days. In part B, the fusion protein of SEQ ID NO:1 will be administered as a 30 minute IV infusion of the assigned dose, followed by pembrolizumab 200 mg as a 30 minute IV infusion (-5 min / +10 min). If a 30 minute infusion of the fusion protein of SEQ ID NO:1 at a dose >10 μg / kg is not well tolerated, a longer infusion time (i.e., 60 or 90 min) may be considered if recommended by the SRC. The investigational site will be notified of this change.

[0304] Antipyretics should be administered prior to the fusion protein of SEQ ID NO: 1. The fusion protein of SEQ ID NO: 1 may be infused by peripheral or central venous access. Patients receiving treatment with the fusion protein of SEQ ID NO: 1 should have a large bore peripheral IV catheter in place to facilitate fluid management. Patients without adequate peripheral venous access should be provided with a central venous access device.

[0305] In cohort 2 (less frequent IV dosing), patients should be monitored for 6-8 hours post-infusion after dosing in cycles 1 and 2. Patients experiencing unmanageable / non-resolving AEs at the end of the observation period must be admitted to hospital for overnight observation and given supportive care. From cycle 3 onwards, the investigator may, at his discretion, shorten the post-infusion observation period to 2 hours, except for patients who required inpatient observation in the first 2 cycles. For patients who required inpatient observation in the first 2 cycles, the mandatory observation period in future cycles may be shortened to 2 hours after consultation with the sponsor's medical monitor.

[0306] Patients undergoing investigational treatment with the fusion protein of SEQ ID NO:1 in combination with pembrolizumab will continue to receive treatment for up to 2 years of total exposure (starting on day 1 of cycle 1 of this study) if, at the discretion of the treating investigator, the patient is experiencing clinical benefit (i.e., stable disease or better) and is deemed tolerable to the therapy (whichever is shorter). Treatment with the fusion protein of SEQ ID NO:1 in combination with pembrolizumab may be allowed for up to 35 treatments (35 cycles, approximately 2 years) if well tolerated and continues to show benefit. Patients receiving combination therapy with the fusion protein of SEQ ID NO:1 in combination with pembrolizumab may continue the fusion protein of SEQ ID NO:1 as monotherapy without pembrolizumab beyond 35 cycles, in discussion with the sponsor and if no other criteria for treatment discontinuation are met.

[0307] Pembrolizumab Dosing and Administration Pembrolizumab is administered at a dose of 200 mg q21d by IV infusion over 30 minutes (-5 minutes / +10 minutes) for up to 35 cycles or 2 years, as long as the patient is experiencing clinical benefit (i.e., stable disease or better) and tolerates the therapy, per the pembrolizumab prescribing information. Patients are monitored for at least 1 hour prior to administration of the fusion protein of SEQ ID NO:1, as described above, for potential acute reactions to pembrolizumab.

[0308] Pembrolizumab is available as single-dose vials of either lyophilized powder or solution and should be administered according to the pembrolizumab prescribing information.

[0309] The patents and scientific literature referred to herein establish knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated by reference. All other published references, documents, manuscripts, and scientific literature cited herein are incorporated by reference.

[0310] While the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims. It should also be understood that the embodiments described herein are not mutually exclusive, and that features of various embodiments may be combined in whole or in part in accordance with the present invention.

Claims

1. 1. A method of treating cancer in a patient, comprising administering to the patient a fusion protein comprising an amino acid sequence set forth in SEQ ID NO:1, or a fusion protein with at least 80% identity to the amino acid sequence set forth in SEQ ID NO:1, wherein the patient is administered at least a first dose of the fusion protein and a second dose of the fusion protein on non-consecutive days.

2. 10. The method of claim 1, wherein the fusion protein is administered at a dose of about 1 μg / kg to about 60 μg / kg.

3. The fusion protein is at least about 1 μg / kg, about 2 μg / kg, about 3 μg / kg, about 4 μg / kg, about 5 μg / kg, about 6 μg / kg, about 7 μg / kg, about 8 μg / kg, about 9 μg / kg, about 10 μg / kg, about 11 μg / kg, about 12 μg / kg, about 13 μg / kg, about 14 μg / kg, about 15 μg / kg, about 16 μg / kg, kg, about 17 μg / kg, about 18 μg / kg, about 19 μg / kg, about 20 μg / kg, about 21 μg / kg, about 22 μg / kg, about 23 μg / kg, about 24 μg / kg, about 25 μg / kg, about 26 μg / kg, about 27 μg / kg, about 28 μg / kg, about 29 μg / kg, about 30 μg / kg, about 31 μg / kg, about 32 μg 3. The method of claim 1 or claim 2, wherein the antibody is administered at a dose of about 33 μg / kg, about 34 μg / kg, about 35 μg / kg, about 36 μg / kg, about 37 μg / kg, about 38 μg / kg, about 39 μg / kg, about 40 μg / kg, about 41 μg / kg, about 42 μg / kg, about 43 μg / kg, about 44 μg / kg, about 45 μg / kg, about 46 μg / kg, about 47 μg / kg, about 48 μg / kg, about 49 μg / kg, about 50 μg / kg, about 51 μg / kg, about 52 μg / kg, about 53 μg / kg, about 54 μg / kg, about 55 μg / kg, about 56 μg / kg, about 57 μg / kg, about 58 μg / kg, about 59 μg / kg, or about 60 μg / kg.

4. 4. The method of any one of claims 1-3, wherein the fusion protein is administered at a dose of about 3 μg / kg, about 6 μg / kg, about 10 μg / kg, about 15 μg / kg, about 20 μg / kg, about 25 μg / kg, about 30 μg / kg, about 35 μg / kg, or about 40 μg / kg.

5. The method of any one of claims 1 to 4, wherein the first dose and the second dose are the same.

6. The method of any one of claims 1 to 4, wherein the first dose and the second dose are different.

7. The method of any one of claims 1 to 6, wherein the patient is administered a third dose of the fusion protein.

8. 8. The method of any one of claims 1-7, wherein the patient is administered the first dose of the fusion protein on day 1 and the second dose of the fusion protein on any of the following days: day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, or day 21.

9. 8. The method of any one of claims 1 to 7, wherein the patient is administered the first dose of the fusion protein on day 1 and the second dose of the fusion protein on day 4.

10. 8. The method of any one of claims 1 to 7, wherein the patient is administered the first dose of the fusion protein on day 1 and the second dose of the fusion protein on day 8.

11. 8. The method of any one of claims 1 to 7, wherein the patient is administered the first dose of the fusion protein on day 1 and the second dose of the fusion protein on day 15.

12. The method of any one of claims 7 to 11, wherein the patient is administered the second dose of the fusion protein and the third dose of the fusion protein on consecutive days.

13. The method of any one of claims 7 to 11, wherein the patient is administered the second dose of the fusion protein and the third dose of the fusion protein on non-consecutive days.

14. 14. The method of any one of claims 7 to 13, wherein the patient is administered the third dose of the fusion protein 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the second dose of the fusion protein.

15. 15. The method of any one of claims 7-14, wherein the patient is administered the first dose of the fusion protein on day 1, the second dose of the fusion protein on any of days 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and the third dose of the fusion protein on any of days 13, 14, 15, 16, 17, 18, 19, 20, or 21.

16. 15. The method of any one of claims 7-14, wherein the patient is administered the first dose of the fusion protein on day 1, the second dose of the fusion protein on any of day 3, day 4, or day 5, and the third dose of the fusion protein on any of day 6, day 7, day 8, day 9, or day 10.

17. 15. The method of any one of claims 7-14, wherein the patient is administered the first dose of the fusion protein on day 1, the second dose of the fusion protein on any of days 6, 7, 8, 9, or 10, and the third dose of the fusion protein on any of days 13, 14, 15, 16, or 17.

18. 15. The method of any one of claims 7-14, wherein the patient is administered the first dose of the fusion protein on day 1, the second dose of the fusion protein on day 4, and the third dose of the fusion protein on day 8.

19. 15. The method of any one of claims 7-14, wherein the patient is administered the first dose of the fusion protein on day 1, the second dose of the fusion protein on day 8, and the third dose of the fusion protein on day 15.

20. 15. The method of any one of claims 1-14, wherein the patient is administered a first dose of about 10 μg / kg to about 30 μg / kg of the fusion protein on day 1, and a second dose of about 10 μg / kg to about 30 μg / kg of the fusion protein on any of days 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21.

21. 15. The method of any one of claims 1-14, wherein the patient is administered a first dose of about 10 μg / kg to about 15 μg / kg of the fusion protein on day 1 and a second dose of about 10 μg / kg to about 15 μg / kg of the fusion protein on any of days 3, 4, 5, or 6.

22. 15. The method of any one of claims 1-14, wherein the patient is administered a first dose of about 10 μg / kg to about 25 μg / kg of the fusion protein on day 1, and a second dose of about 20 μg / kg to about 25 μg / kg of the fusion protein on any of days 6, 7, 8, 9, or 10.

23. 15. The method of any one of claims 1-14, wherein the patient is administered a first dose of about 15 μg / kg of the fusion protein on day 1 and a second dose of about 15 μg / kg of the fusion protein on day 4.

24. 15. The method of any one of claims 1-14, wherein the patient is administered a first dose of about 20 μg / kg to about 25 μg / kg of the fusion protein on day 1, and a second dose of about 20 μg / kg to about 25 μg / kg of the fusion protein on day 8.

25. 25. The method of any one of claims 7-24, wherein the patient is administered a first dose of about 6 μg / kg to about 25 μg / kg of the fusion protein on day 1, a second dose of about 6 μg / kg to about 25 μg / kg of the fusion protein on any of days 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and a third dose of about 6 μg / kg to about 25 μg / kg of the fusion protein on any of days 13, 14, 15, 16, 17, 18, 19, 20, or 21.

26. 25. The method of any one of claims 7-24, wherein the patient is administered a first dose of about 6 μg / kg to about 15 μg / kg of the fusion protein on day 1, a second dose of about 6 μg / kg to about 15 μg / kg of the fusion protein on any of days 3, 4, or 5, and a third dose of about 6 μg / kg to about 15 μg / kg of the fusion protein on any of days 6, 7, 8, 9, or 10.

27. 25. The method of any one of claims 7-24, wherein the patient is administered a first dose of about 10 μg / kg to about 20 μg / kg of the fusion protein on day 1, a second dose of about 10 μg / kg to about 20 μg / kg of the fusion protein on any of days 6, 7, 8, 9, or 10, and a third dose of about 10 μg / kg to about 20 μg / kg of the fusion protein on any of days 13, 14, 15, 16, or 17.

28. 25. The method of any one of claims 7-24, wherein the patient is administered a first dose of about 15 μg / kg of the fusion protein on day 1, a second dose of about 15 μg / kg of the fusion protein on day 4, and a third dose of about 15 μg / kg of the fusion protein on day 8.

29. 25. The method of any one of claims 7-24, wherein the patient is administered a first dose of about 20 μg / kg of the fusion protein on day 1, a second dose of about 20 μg / kg of the fusion protein on day 8, and a third dose of about 20 μg / kg of the fusion protein on day 15.

30. 20. The method of any one of claims 7 to 19, wherein the first dose, the second dose, and the third dose are the same.

31. The method of any one of claims 7 to 19, wherein the first dose, the second dose, and the third dose are different.

32. The method of any one of claims 7 to 31, wherein the patient is administered a fourth dose of the fusion protein.

33. 33. The method of claim 32, wherein the patient is administered the third dose of the fusion protein and the fourth dose of the fusion protein on consecutive days.

34. 33. The method of claim 32, wherein the patient is administered the third dose of the fusion protein and the fourth dose of the fusion protein on non-consecutive days.

35. 1. A method of treating cancer in a patient, comprising administering to the patient a fusion protein comprising an amino acid sequence set forth in SEQ ID NO:1, or a fusion protein that has at least 80% identity to the amino acid sequence set forth in SEQ ID NO:1, wherein the patient is administered at least a first dose of the fusion protein, the patient is not administered a dose of the fusion protein at least one day prior to the first dose, and the patient is not administered a dose of the fusion protein one day after the first dose.

36. 1. A method of treating cancer in a patient, comprising administering to the patient a fusion protein comprising an amino acid sequence set forth in SEQ ID NO:1, or a fusion protein with at least 80% identity to the amino acid sequence set forth in SEQ ID NO:1, wherein the patient is administered a single dose of the fusion protein once a week (Q1W), once every two weeks (Q2W), once every three weeks (Q3W), or once every four weeks (Q4W).

37. 37. The method of claim 35 or claim 36, wherein the fusion protein is administered at a dose of about 10 μg / kg to about 60 μg / kg.

38. 38. The method of any one of claims 35-37, wherein the fusion protein is administered at a dose of about 10 μg / kg, about 15 μg / kg, about 20 μg / kg, about 25 μg / kg, about 30 μg / kg, about 35 μg / kg, about 40 μg / kg, about 45 μg / kg, about 50 μg / kg, about 55 μg / kg, or about 60 μg / kg.

39. 39. The method of any one of claims 35-38, wherein the fusion protein is administered at a dose of about 10 μg / kg, about 15 μg / kg, about 20 μg / kg, about 25 μg / kg, about 30 μg / kg, about 35 μg / kg, or about 40 μg / kg.

40. 40. The method of any one of claims 1-39, wherein administration of the fusion protein results in a dose-dependent expansion of circulating NK and CD8+ cells in the patient, in the absence of a dose-dependent expansion of T regulatory (Treg) cells.

41. 41. The method of claim 40, wherein the increase in circulating NK cells and CD8+ cells is at least 2-fold over baseline.

42. 41. The method of claim 40, wherein said increase in circulating NK cells and CD8+ cells is greater compared to said increase in circulating Treg cells.

43. The method of claim 40, wherein the increase in circulating NK cells and CD8+ cells is greater relative to the increase in circulating Treg cells compared to the increase in circulating NK cells and CD8+ cells compared to the increase in circulating Treg cells in patients receiving high dose rhIL-2 therapy.

44. 44. The method of any one of claims 1 to 43, wherein the patient has an improved safety profile compared to patients receiving high-dose recombinant human IL-2 (rhIL-2) therapy.

45. 45. The method of claim 44, wherein the patient is at reduced risk for capillary leak syndrome or cytokine release syndrome and / or the patient is at reduced risk for cytokine release syndrome as compared to patients receiving high dose rhIL-2 therapy.

46. 46. ​​The method of any one of claims 1-45, which results in a dose-dependent expansion of circulating NK and CD8+ cells in the patient in the absence of a dose-dependent expansion of circulating regulatory T (Treg) cells, wherein the increase in circulating NK and CD8+ cells compared to the increase in circulating T regulatory (Treg) cells is greater compared to the increase in circulating NK and CD8+ cells compared to the increase in circulating Treg cells in patients receiving high dose recombinant human IL-2 (rhIL-2) therapy, and wherein the patient is at lower risk of capillary leak syndrome.

47. The method of any one of claims 1 to 46, wherein each dose is administered by intravenous (I.V.) injection or infusion.

48. 47. The method of any one of claims 1-46, wherein each dose is administered by intravenous (I.V.) injection or infusion over a period of about 30 minutes, about 60 minutes, or about 90 minutes.

49. 49. The method of any one of claims 1 to 48, further comprising administering to the patient a therapeutically effective amount of a therapeutic agent.

50. 50. The method of claim 49, wherein the therapeutic agent is a PARP inhibitor, an immune checkpoint inhibitor, a cytotoxic agent, or a chemotherapeutic agent.

51. 50. The method of claim 49, wherein the therapeutic agent is an immune checkpoint inhibitor.

52. The method of claim 50 or claim 51, wherein the immune checkpoint inhibitor inhibits the interaction of PD-1 and PD-L1.

53. 53. The method of any one of claims 50-52, wherein the immune checkpoint inhibitor is pembrolizumab.

54. 54. The method of claim 53, wherein the pembrolizumab is administered before, simultaneously with, or after administration of the fusion protein of SEQ ID NO:

1.

55. 55. The method of claim 53 or claim 54, wherein the pembrolizumab is administered in a separate composition from the fusion protein of SEQ ID NO:

1.

56. 56. The method of any one of claims 53-55, wherein the pembrolizumab is administered in an amount of 200 mg by intravenous injection or infusion.

57. 57. The method of any one of claims 53-56, wherein the pembrolizumab is administered once a week (Q1W), once every two weeks (Q2W), once every three weeks (Q3W), or once every four weeks (Q4W).

58. 58. The method of any one of claims 53-57, wherein the pembrolizumab is administered in an amount of 200 mg by intravenous injection or infusion once every three weeks (Q3W).

59. 59. The method of any one of claims 53-58, wherein the pembrolizumab is administered on the same day as the first dose of the fusion protein.

60. 60. The method of any one of claims 1 to 59, wherein the cancer to be treated is a solid tumor.

61. 61. The method of claim 60, wherein the solid tumor is a carcinoma, sarcoma, or lymphoma.

62. 61. The method of claim 60, wherein the cancer being treated is renal cell carcinoma (RCC), melanoma, mucosal melanoma, breast cancer, pancreatic cancer, prostate cancer, non-small cell lung cancer, liver cancer, colorectal cancer, bladder cancer, cervical cancer, ovarian cancer, thyroid cancer, esophageal cancer, oral cancer, mesothelioma, non-melanoma skin cancer, and / or gastric cancer.

63. 60. The method of any one of claims 1 to 59, wherein the cancer to be treated is a hematological cancer.

64. 64. The method of claim 63, wherein the hematological cancer is leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, and multiple myeloma.

65. 1. A pharmaceutical composition comprising a fusion protein comprising the amino acid sequence set forth in SEQ ID NO:1, or a fusion protein with at least 80% identity to the amino acid sequence set forth in SEQ ID NO:1, wherein said pharmaceutical composition comprises a unit dose of about 1 μg / kg to about 60 μg / kg.

66. The unit dose is about 1 μg / kg, about 2 μg / kg, about 3 μg / kg, about 4 μg / kg, about 5 μg / kg, about 6 μg / kg, about 7 μg / kg, about 8 μg / kg, about 9 μg / kg, about 10 μg / kg, about 11 μg / kg, about 12 μg / kg, about 13 μg / kg, about 14 μg / kg, about 15 μg / kg, about 16 μg / kg, about 17 μg / kg, about 18 μg / kg, about 19 μg / kg, about 20 μg / kg, about 21 μg / kg, about 22 μg / kg, about 23 μg / kg, about 24 μg / kg, about 25 μg / kg, about 26 μg / kg, about 27 μg / kg, about 28 μg / kg, about 29 μg / kg, about 30 μg / kg, about 31 μg / kg, about 32 μg / kg, about 33 μg / kg, about 34 μg / kg, about 35 μg / kg, about 36 μg / kg, about 37 μg / kg, about 38 μg / kg, about 39 μg / kg, about 40 μg / kg, about 41 μg / kg, about 42 μg / kg, about 43 μg / kg, about 44 μg / kg, about 45 μg / kg, about 46 μg / kg, about 47 μg / kg, about 48 μg / kg, about 49 μg / kg, about 50 μg / kg, about 51 μg / kg, about 52 μg / kg, about 53 μg / kg, about 54 μg / kg, about 55 μg / kg, about 56 μg / kg, about 57 μg / kg, about 5 kg, about 17 μg / kg, about 18 μg / kg, about 19 μg / kg, about 20 μg / kg, about 21 μg / kg, about 22 μg / kg, about 23 μg / kg, about 24 μg g / kg, about 25 μg / kg, about 26 μg / kg, about 27 μg / kg, about 28 μg / kg, about 29 μg / kg, about 30 μg / kg, about 31 μg / kg, about 3 66. The pharmaceutical composition of claim 65, wherein the dose is 2 μg / kg, about 33 μg / kg, about 34 μg / kg, about 35 μg / kg, about 36 μg / kg, about 37 μg / kg, about 38 μg / kg, about 39 μg / kg, about 40 μg / kg, about 41 μg / kg, about 42 μg / kg, about 43 μg / kg, about 44 μg / kg, about 45 μg / kg, about 46 μg / kg, about 47 μg / kg, about 48 μg / kg, about 49 μg / kg, about 50 μg / kg, about 51 μg / kg, about 52 μg / kg, about 53 μg / kg, about 54 μg / kg, about 55 μg / kg, about 56 μg / kg, about 57 μg / kg, about 58 μg / kg, about 59 μg / kg, or about 60 μg / kg.

67. 67. The pharmaceutical composition of claim 65 or claim 66, wherein the unit dose is about 3 μg / kg, about 6 μg / kg, about 10 μg / kg, about 15 μg / kg, about 20 μg / kg, about 25 μg / kg, about 30 μg / kg, about 35 μg / kg, or about 40 μg / kg.

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