Compositions and methods for cancer immunotherapy

Abstract

To provide novel IL-2 therapies to more effectively combat various cancers.SOLUTION: The invention provides compositions and improved methods for the treatment of cancer using IL-2 immunotherapy. The methods include a step of administering to a patient a fusion protein of SEQ ID NO: 1 at a dose of about 6 μg / kg / day to about 70 μg / kg / day and preferably at a dose of at least about 6 μg / kg / day to about 15 μg / kg / day or at a corresponding fixed daily dose based, for example, on an average adult human of about 60 to about 70 kg or based, for example, on a child of about 12 kg to about 50 kg or more. The administration 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 circulating immunosuppressive T regulatory (Treg) cells. Preferably, the increase in circulating NK cells and CD8+ cells is greater than the increase in circulating T Treg cells.SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 860,182, filed Jun. 11, 2019; U.S. Provisional Application No. 62 / 932,160, filed Nov. 7, 2019; and U.S. Provisional Application No. 62 / 924,356, filed Oct. 22, 2019. The entire teachings of the above applications are incorporated herein by reference.

Background Art

[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 by a multi-subunit IL-2 receptor complex (IL-2R) consisting of three polypeptide subunits that traverse the cell membrane, namely, p55 (IL-2Rα, the alpha subunit, also known as CD25 in humans), p75 (IL-2Rβ, the beta subunit, also known as CD122 in humans), and p64 (IL-2Rγ, the gamma subunit, also known as CD132 in humans). The T cell response to IL-2 depends on various 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 internalizes upon ligand binding, and different components undergo different sorting. IL-2Rα is recycled to the cell surface, whereas IL-2 that associates with the IL-2:IL-2Rβγ complex is transported to lysosomes and degraded.

[0003] The outcome of systemic IL-2 administration in cancer patients is poor. 15-20 percent of patients respond objectively to high-dose IL-2, but the majority do not respond 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] Of the therapies currently in use, rhIL-2 is one of the few treatment regimens that result in complete and durable responses in a subset of patients, namely up to 12% in melanoma and up to 7% in RCC. High-dose rhIL-2 is required to stimulate cells expressing intermediate-affinity IL-2 receptors, including memory CD8 + T cells and natural killer (NK) cells, which are the major cell types mediating the anti-cancer immune response.

[0005] Contributing factors that limit the therapeutic efficacy of rhIL-2 are hypothesized to preferentially activate immunosuppressive CD4 + T reg cells and induce their expansion. This preferential activation is due to the binding of IL-2 to the high-affinity IL-2 receptor expressed on T reg cells. Furthermore, direct interaction between rhIL-2 and the high-affinity IL-2R expressed on vascular and lung endothelial cells is hypothesized to contribute to rhIL-2-mediated toxicity via capillary leak syndrome. Despite the inadequate tolerability of immunotherapy with rhIL-2, rhIL-2 remains one of the few treatment regimens for metastatic melanoma and RCC that result in complete and durable responses in a subset of patients. Therefore, new IL-2 therapies are needed to more effectively combat various cancers.

Prior Art Documents

Patent Documents

[0006]

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[0007] [Non-Patent Document 1] Wang et al., Science. 2005;310(5751):1159 - 1163. doi:10.1126 / science.1117893 [Non-Patent Document 2] Smith and Waterman, Adv. Appl. Math. 2:482(1981) [Non-Patent Document 3] Needleman and Wunsch, J. Mol. Biol. 48:443(1970) [Non-Patent Document 4] Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444(1988) [Non-Patent Document 5] Current Protocols in Molecular Biology (edited by Ausubel et al., 1995 supplement) [Non-Patent Document 6] "Current protocols in molecular biology" (edited by Ausubel, 2008, John Wiley & Son) [Non-Patent Document 7] Allen, Jr., L. V. (editor), Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press, London, UK (2012) [Non-Patent Document 8] Remington's The Science and Practice of Pharmacy, 21st edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006 [Non-Patent Document 9] Weiner et al., Nature Rev. Immunol 2010;10:317~27 [Non-Patent Document 10] Overwijk et al., Journal of Experimental Medicine 2008;198:569~80 [Non-Patent Document 11] Gendler et al., J Biol Chem 1990;265:15286~15293 [Non-Patent Document 12] Mercer and Pritchard, Biochim Biophys Acta (2003) 1653(1):25 - 40

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Summary of the Invention

Means for Solving the Problems

[0008] The compositions, methods, and treatment regimens of the present invention provide a number of advantages for the treatment of cancer using IL-2 immunotherapy, for example, as compared to high-dose rhIL-2 therapy (e.g., Aldesleukin). Administration of the fusion protein of SEQ ID NO:1 is at a dose of about 6 μg / kg / day to about 70 μg / kg / day, preferably about 6 μg / kg / day to about 15 μg / kg / day, or a corresponding fixed daily dose based on, for example, an adult human of average 60 - 70 kg (e.g., 0.4 mg / day to about 1 - 4 mg), or a corresponding fixed daily dose based on a pediatric patient, for example, about 12 kg to 50 kg or more, and it has been discovered that it achieves a dose-dependent increase in circulating NK cells and CD8+ cells in a patient in the absence of a dose-dependent increase in regulatory T (Treg) cells. Preferably, the increase in circulating NK cells and CD8+ cells compared to the increase in regulatory T (Treg) in patients administered the fusion protein of SEQ ID NO:1 according to the method of the present invention is greater, for example, as compared to the increase in circulating NK cells and CD8+ cells compared to the increase in regulatory T (Treg) in patients receiving high-dose recombinant human IL-2 (rhIL-2) treatment. It has also been discovered that administration of the fusion protein of SEQ ID NO:1 at a dose equal to or higher than the dose of high-dose IL-2 is not accompanied by the toxicity and side effects often associated with administration of high-dose rhIL-2, such as capillary leak syndrome (CLS).

[0009] Preferably, the present invention provides a method for treating cancer in a patient, the method comprising administering to the patient a dose of at least about 6 μg / kg / day to about 15 μg / kg / day, or its corresponding fixed dose based on, for example, an adult of 60-70 kg (e.g., about 0.4 mg / day to about 1.0 mg / day), or its corresponding fixed dose based on a pediatric patient, e.g., a pediatric patient of about 12 kg to about 50 kg. Preferably, the dose of the fusion protein of SEQ ID NO:1 in units of μg / kg / day is a dose of about 6 μg / kg / day, 8 μg / kg / day, 10 μg / kg / day, 12 μg / kg / day, 14 μg / kg / day, or 15 μg / kg / day, or its corresponding fixed dose based on, for example, an adult of 60-70 kg or a pediatric patient, e.g., a pediatric patient of about 12 kg to 50 kg or more. Preferably, the administration of the fusion protein of SEQ ID NO:1 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 regulatory T (Treg) cells. Preferably, the increase in circulating NK cells and CD8+ cells is at least 2-fold relative to the baseline prior to administration of the fusion protein of SEQ ID NO:1 to the patient. Preferably, the increase in circulating NK cells and CD8+ cells is greater than the increase in circulating Treg cells. Preferably, the increase in circulating NK cells and CD8+ cells relative to the increase in circulating Treg cells is greater compared to the increase in circulating NK cells and CD8+ cells relative to the increase in circulating Treg cells in patients receiving high-dose rhIL-2 treatment.

[0010] Preferably, the patient has an improved safety profile compared to a patient receiving high-dose recombinant human IL-2 (rhIL-2) treatment. Preferably, the patient has a lower risk of capillary leak syndrome or cytokine release syndrome. Preferably, a dose-dependent increase in circulating NK cells and CD8+ cells in the patient in the absence of a dose-dependent increase in circulating regulatory T (Treg) cells that results. Preferably, the increase in circulating NK cells and CD8+ cells compared to the increase in circulating regulatory T (Tregs) is greater compared to the increase in circulating NK cells and CD8+ cells compared to the increase in circulating regulatory T (Tregs) in patients receiving high-dose recombinant human IL-2 (rhIL-2) treatment, and the patient has a lower risk of capillary leak syndrome. Preferably, the dose of the fusion protein of SEQ ID NO: 1 is administered by intravenous injection or infusion.

[0011] Preferably, after the fusion protein of SEQ ID NO: 1 is administered by intravenous injection or infusion at a dose of at least about 6 μg / kg to about 15 μg / kg per day for 1 to about 5 consecutive or non-consecutive days, a drug withdrawal period of at least about 9 consecutive days follows. Preferably, the drug withdrawal period is at least about 16 days. Preferably, the fusion protein of SEQ ID NO: 1 is administered in at least 2 courses of treatment. The first course of treatment includes administration by intravenous injection or infusion at a dose of at least about 6 μg / kg to about 15 μg / kg per day for a period of 1 to about 5 consecutive or non-consecutive days, followed by a drug withdrawal period of at least about 9 consecutive days, resulting in a total 14-day course. Subsequently, the second course of treatment includes administering by intravenous injection or infusion at a dose of at least about 6 μg / kg to about 15 μg / kg per day for 1 to about 5 consecutive or non-consecutive days, followed by a drug withdrawal period of at least about 16 consecutive days, resulting in a total 21-day course. Preferably, the second course of treatment is started within about 24 hours or more than about 24 hours after the completion of the first course of treatment. Preferably, a 21-day third course of treatment follows the administration of the second course. Preferably, the third course of treatment is started within about 24 hours or more than about 24 hours after the completion of the second course of treatment. Preferably, a 21-day fourth course of treatment follows the administration of the third course. Preferably, the fourth course of treatment is started within about 24 hours or more than about 24 hours after the completion of the third course of treatment.

[0012] Preferably, the step of administering the fusion protein of SEQ ID NO: 1 further includes the step of co-administering a therapeutically effective amount of a therapeutic agent to the patient. Preferably, the therapeutic agent is an immune checkpoint inhibitor or a PARP inhibitor. Preferably, the therapeutic agent is an immune checkpoint inhibitor. Preferably, the immune checkpoint inhibitor inhibits the interaction between PD-1 and PD-L. Preferably, the immune checkpoint inhibitor is pembrolizumab.

[0013] Preferably, the fusion protein of SEQ ID NO: 1 is administered in at least 2 courses of treatment. The first course of treatment includes intravenous injection or infusion at a dose of at least about 6 μg / kg to about 15 μg / kg per day for 1 to about 5 days, either continuously or discontinuously, followed by a drug-free period of at least about 16 consecutive days, resulting in a total 21-day course. Subsequently, the second course of treatment includes administration by intravenous injection or infusion at a dose of at least about 6 μg / kg to about 15 μg / kg per day for a period of at least 5 consecutive days, followed by a drug-free period of at least about 16 consecutive days, resulting in a total 21-day course. Pembrolizumab is co-administered on the first day of the first course and the first day of the second course. Pembrolizumab is co-administered before, simultaneously with, or after the administration of the fusion protein of SEQ ID NO: 1. Preferably, pembrolizumab is co-administered in a composition separate from the fusion protein of SEQ ID NO: 1. Preferably, pembrolizumab is co-administered by I.V. injection or infusion in an amount of 200 mg.

[0014] Preferably, all methods of the present invention result in a dose-dependent increase in circulating NK cells and CD8+ cells in a patient in the absence of a dose-dependent increase in regulatory T (Treg) cells. Preferably, the patient has an improved safety profile compared to a patient receiving high-dose recombinant human IL-2 (rhIL-2) treatment. Preferably, the patient has a lower risk of cytokine release syndrome compared to a patient receiving high-dose recombinant human IL-2 (rhIL-2) treatment. Preferably, the patient has a lower risk of capillary leak syndrome compared to a patient receiving high-dose recombinant human IL-2 (rhIL-2) treatment.

[0015] The present invention also provides a method for treating cancer in a patient, comprising administering to the patient a fusion protein of SEQ ID NO: 1 at a dose of at least about 6 μg / kg to about 15 μg / kg per day, wherein the dose is administered by intravenous injection or infusion for 1 to about 5 days, either continuously or discontinuously, followed by a drug-free period of at least about 9 consecutive days, resulting in a dose-dependent increase in circulating NK cells and CD8+ cells in the patient in the absence of a dose-dependent increase in regulatory T (Treg) cells, and the increase in circulating NK cells and CD8+ cells is at least 2-fold relative to the baseline prior to administration of the fusion protein of SEQ ID NO: 1 to the patient, and preferably, the increase in circulating NK cells and CD8+ cells relative to the increase in circulating regulatory T (Treg) cells is greater compared to the increase in circulating NK cells and CD8+ cells relative to the increase in circulating regulatory T (Treg) cells in patients treated with high-dose recombinant human IL-2 (rhIL-2).

[0016] The present invention also provides a method for treating cancer in a patient, comprising administering to the patient a fusion protein of SEQ ID NO: 1 at a dose of at least about 6 μg / kg to about 15 μg / kg per day, wherein the dose is administered by intravenous injection or infusion for 1 to about 5 days, either continuously or discontinuously, followed by a drug-free period of at least about 9 consecutive days, resulting in a dose-dependent increase in circulating NK cells and CD8+ cells in the patient in the absence of a dose-dependent increase in regulatory T (Treg) cells, and the patient has an improved safety profile compared to patients treated with high-dose recombinant human IL-2 (rhIL-2), and preferably, the increase in circulating NK cells and CD8+ cells relative to the increase in circulating regulatory T (Treg) cells is greater compared to the increase in circulating NK cells and CD8+ cells relative to the increase in circulating regulatory T (Treg) cells in patients treated with high-dose recombinant human IL-2 (rhIL-2).

[0017] The present invention also provides a method for treating cancer in a patient, comprising administering to the patient a fusion protein of SEQ ID NO:1 at a dose of at least about 6 μg / kg to about 15 μg / kg per day, wherein the dose is administered by intravenous injection or infusion for 1 to about 5 days, either continuously or discontinuously, followed by a drug-free period of at least about 9 consecutive days, resulting in a dose-dependent increase in circulating NK cells and CD8+ cells in the patient in the absence of a dose-dependent increase in regulatory T (Treg) cells, and the patient having a lower risk of cytokine release syndrome compared to a patient receiving high-dose recombinant human IL-2 (rhIL-2) treatment, and preferably, the increase in circulating NK cells and CD8+ cells compared to the increase in circulating regulatory T (Treg) cells is greater compared to the increase in circulating NK cells and CD8+ cells compared to the increase in circulating regulatory T (Treg) cells in a patient receiving high-dose recombinant human IL-2 (rhIL-2) treatment.

[0018] The present invention also provides a method for treating cancer in a patient, comprising administering to the patient a fusion protein of SEQ ID NO:1 at a dose of at least about 6 μg / kg to about 15 μg / kg per day, wherein the dose is administered by intravenous injection or infusion for 1 to about 5 days, either continuously or discontinuously, followed by a drug-free period of at least about 9 consecutive days, resulting in a dose-dependent increase in circulating NK cells and CD8+ cells, as well as a dose-dependent increase in regulatory T (Treg) cells in the patient in the absence of a dose-dependent increase in regulatory T (Treg) cells, and the patient having a lower risk of cytokine release syndrome compared to a patient receiving high-dose recombinant human IL-2 (rhIL-2) treatment, and preferably, the increase in circulating NK cells and CD8+ cells compared to the increase in circulating regulatory T (Treg) cells is greater compared to the increase in circulating NK cells and CD8+ cells compared to the increase in circulating regulatory T (Treg) cells in a patient receiving high-dose recombinant human IL-2 (rhIL-2) treatment.

[0019] The present invention also provides a pharmaceutical composition comprising the fusion protein of SEQ ID NO: 1 at about 6 μg / kg to about 70 μg / kg, preferably about 6 μg / kg to about 15 μg / kg, preferably comprising the fusion protein of SEQ ID NO: 1 at about 8 μg / kg to about 15 μg / kg, preferably comprising the fusion protein of SEQ ID NO: 1 at about 8 μg / kg, preferably comprising the fusion protein of SEQ ID NO: 1 at about 10 μg / kg, preferably comprising the fusion protein of SEQ ID NO: 1 at about 12 μg / kg, preferably the fusion protein comprises the fusion protein of SEQ ID NO: 1 at about 14 μg / kg, preferably the fusion protein comprises the fusion protein of SEQ ID NO: 1 at about 15 μg / kg.

[0020] The present invention also provides a method for treating cancer in a patient, the method comprising administering to the patient a dose of at least about 50 μg / kg to about 60 μg / kg per day or a corresponding fixed daily dose based on an adult human of average 60 - 70 kg (e.g., about 3.0 mg / day to approximately about 3.6 mg / day) of the fusion protein of SEQ ID NO: 1, the dose being administered once a day intravenously or by infusion for one week, resulting in a dose-dependent increase in circulating NK cells and CD8+ cells in the patient in the absence of a dose-dependent increase in regulatory T (Treg) cells, wherein the increase in circulating NK cells and CD8+ cells compared to the increase in circulating regulatory T (Treg) cells is greater compared to the increase in circulating NK cells and CD8+ cells compared to the increase in circulating regulatory T (Treg) cells in patients treated with high-dose recombinant human IL-2 (rhIL-2), and the patient has a lower risk of capillary leak syndrome compared to patients treated with high-dose recombinant human IL-2 (rhIL-2).

[0021] The present invention also provides a pharmaceutical composition comprising the fusion protein of SEQ ID NO: 1 at at least about 6 μg / kg to about 70 μg / kg, preferably at least about 6 μg / kg to about 15 μg / kg, or a corresponding fixed daily dose based on an adult human of average 60 - 70 kg (e.g., about 0.4 mg to approximately about 1.0 mg), or a corresponding fixed dose based on a pediatric patient, e.g., about 12 kg to about 50 kg or older pediatric patients.

[0022] The present invention also provides a pharmaceutical composition comprising the fusion protein of SEQ ID NO: 1 in a corresponding fixed dose (e.g., about 3.0 mg to about 3.6 mg) based on an adult human of about 40 μg / kg to about 70 μg / kg, or, for example, an average of 60 - 70 kg, or a corresponding fixed dose based on a pediatric patient, e.g., about 12 kg to about 50 kg or more, preferably a pharmaceutical composition comprising the fusion protein of SEQ ID NO: 1 at about 40 μg / kg to about 70 μg / kg.

[0023] The present invention also provides a method of treating cancer in a patient, the method comprising administering to the patient a dose of the fusion protein of SEQ ID NO: 1 that is less than 6 μg / kg / day, e.g., doses of about 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, and 5.9 μg / kg / day, or a corresponding fixed dose (e.g., about 0.2 mg / day to about 0.4 mg / day) based on an adult, e.g., 60 - 70 kg, or a corresponding fixed dose based on a pediatric patient, e.g., about 12 kg to about 50 kg or more, which 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 regulatory T (Treg) cells, wherein the increase in circulating NK cells and CD8+ cells is greater than the increase in circulating regulatory T (Treg).

[0024] The foregoing and other objects, features, and advantages of the present invention will become apparent from the following more detailed description of the preferred embodiments of the invention as illustrated in the accompanying drawings. In the drawings, like reference numerals refer to the same parts throughout the various figures. The drawings are not necessarily to scale, but rather emphasis is placed on illustrating the principles of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025]

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DETAILED DESCRIPTION OF THE INVENTION

[0026] Definitions One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited to the foregoing description, but rather is as set forth in the appended claims.

[0027] In the claims, the articles “a,” “an,” and “the” may mean one or more than one unless contrary is indicated or otherwise clear from the context. A claim or description that includes “or” between one or more members of a group is satisfied if one, two or more, or all of the members of the group are present in, used in, or otherwise relevant to a given product or process, unless contrary is indicated or otherwise clear from the context. The invention includes embodiments where exactly one member of the group is present in, used in, or otherwise relevant to a given product or process. The invention includes embodiments where two or more or all of the members of the group are present in, used in, or otherwise relevant to a given product or process.

[0028] It should also be noted that the term “comprising” is intended to be non-limiting and permits the inclusion of additional elements or steps without requiring them. Thus, when the term “comprising” is used herein, the term “consisting of” is also included and disclosed.

[0029] When ranges are given, endpoints are included. Further, unless otherwise indicated or otherwise clear from the context and the understanding of one of ordinary skill in the art, values expressed as ranges are to be understood as including any and all subranges within the stated range, between the lower and upper limits of the range, to the tenth of the unit of the lower limit of the range, unless otherwise indicated or otherwise clear from the context.

[0030] As used herein, the term "about" or "approximately" when applied to one or more target values refers to a value similar to the recited reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the recited reference value, where such numbers are possible values (except where such numbers exceed 100% of the possible values), unless otherwise stated or otherwise apparent from the context.

[0031] As used herein, the term "substantially" refers to a qualitative condition that exhibits all or nearly all of the range or degree of a target feature or characteristic. Those skilled in the art of biology will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or a perfect state, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to account for the potential lack of completeness inherent in many biological and chemical phenomena.

[0032] As used herein, any form of administration or co - administration of "combination", "combined therapy", and / or "combined treatment regimen" refers to at least two therapeutically active agents or compositions that can be administered or co - administered simultaneously, or sequentially at different times separated by minutes, hours, or days, either in separate formulations or in a combined formulation. Generally, each agent is administered at the dosage and / or time schedule determined for that agent.

[0033] As used herein, the term "parenteral" refers to dosage forms intended for administration by injection or infusion, including subcutaneous, intravenous, intra - arterial, intraperitoneal, intracardiac, intrathecal, and intramuscular injections, as well as infusion injections via the intravenous route typically.

[0034] The term "therapeutic agent" encompasses any agent that is administered in addition to or in combination with SEQ ID NO: 1 to treat a symptom or disease in an individual in need of treatment for the symptom or disease. Such additional therapeutic agents may include any active ingredient suitable for the particular indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other.

[0035] The term "chemotherapeutic agent" refers to a compound or derivative thereof that interacts with cancer cells and thereby can inhibit cell proliferation and / or kill cells, for example, by interfering with cell division or DNA synthesis or by damaging DNA, effectively targeting cells that divide rapidly in a short period of time. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents (e.g., cyclophosphamide, ifosfamide), antimetabolites (e.g., methotrexate (MTX), 5-fluorouracil, or their 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 agent trimethotrexate (TMTX), temozolomide, raltitrexed, S-(4-nitrobenzyl)-6-thioinosine (NBMPR), 6-benzylguanidine (6-BG), nitrosoureas, namely nitrosourea (arabinopyranosyl-N-methyl-N-nitrosourea (aranosine), carmustine (BCNU, BiCNU), chlorozotocin, ethylnitrosourea (ENU), fotemustine, lomustine (CCNU), nimustine, N-nitroso-N-methylurea (NMU), ranimustine (MCNU), semustine, streptozocin (streptozotocin)), cytarabine, and camptothecin, or any of their therapeutic derivatives.

[0036] As used herein, a single "course" of treatment, e.g., a first course, a second course, a third course, etc., refers to a treatment regimen in which the fusion protein is administered for a desired time period, e.g., 1 to about 5 days of treatment, either continuously or intermittently, or once a week, followed by a drug withdrawal period of a certain number of consecutive days. Thus, one course of treatment includes a time period of continuous or intermittent administration of the fusion protein to the patient, followed by a drug withdrawal period of a number of consecutive days during which there is no administration of the fusion protein to the patient.

[0037] The term "fusion protein" refers to a protein or peptide that is linked to another protein or peptide by a peptide bond between the respective N-terminal and C-terminal amino acid residues, or between the C-terminal and N-terminal amino acid residues, or by insertion of a protein or peptide by two peptide bonds at the N- and C-termini of the inserted protein or peptide into an internal region of a first 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 in an expression host of a fusion protein gene in which the coding sequence of a first protein or peptide is linked to the coding sequence of a second protein or peptide.

[0038] The term "fusion protein" refers to the fusion protein of SEQ ID NO: 1.

[0039] The present invention also contemplates the use of variants of the fusion protein of SEQ ID NO:1 having an amino acid sequence having at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity over a continuous interval of about 20 amino acids to the full length of SEQ ID NO:1. Variants of SEQ ID NO:1 can have a defined sequence identity when compared to SEQ ID NO:1 over a continuous amino acid of defined length (e.g., a "comparison window"). Methods of aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson and Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computer 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, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds., 1995 Supplement)).

[0040] As an example, a variant of the fusion protein of SEQ ID NO: 1 can include an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% amino acid sequence identity with a continuous segment of SEQ ID NO: 1 that is at least 20 amino acids, preferably about 20 to about 40 amino acids, about 40 to about 60 amino acids, about 60 to about 80 amino acids, about 80 to about 100 amino acids, about 100 to about 120 amino acids, about 120 to about 140 amino acids, about 140 to about 150 amino acids, about 150 to about 155 amino acids, about 155 amino acids to up to the full length of SEQ ID NO: 1.

[0041] The term "IL-2 therapy" includes the administration of IL-2-based immunotherapy, as well as the biological functions as related immunotherapies thereof, for example, but not limited to, CD4 + maintenance of regulatory T cells and CD4 + differentiation of various subsets of T cells, promotion of cytotoxic activities of CD8 + T cells and NK cells, inhibition of helper T17 (Th17) differentiation while promoting naive CD4 + differentiation into helper T1 (Th1) and helper T2 (Th2) cells, and regulation of antigen-responsive T cell differentiation programs. Accordingly, as used herein, "IL-2 therapy" includes, but is not limited to, immunotherapies using rhIL-2 or variants of rhIL-2, such as the fusion protein of SEQ ID NO: 1.

[0042] The terms "high-dose IL-2" and "HD IL-2" include an interleukin-2 (IL-2) dose of about or at least about 600,000 international units (IU) / kg body weight (kg) / dose, or about or at least about 720,000 IU / kg / dose.

[0043] The terms "low dose IL-2" and "LD IL-2" include interleukin-2 (IL-2) at a dose of less than about 600,000 IU / kg body weight / dose, such as about 60,000 or about 72,000 IU / kg / dose, such as about 60,000 to about 72,000 IU / kg / dose.

[0044] As used herein, the terms "subject" or "patient" refer to any organism to which the compositions of the present disclosure can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. Preferably, a "patient" refers to a human subject who is seeking or thought to need treatment, who needs treatment, who is receiving treatment, or who is scheduled to receive treatment, or a subject under the care of a skilled professional for a particular disease or condition. A "patient" may be a pediatric patient (over 1 year to 17 years old). In yet other embodiments, the patient may be an infant (less than 1 year old). In yet further embodiments, the patient may be a pediatric patient, and the term "pediatric" is used as understood by those of ordinary skill in the art. For example, pediatric patients include infants, children, and adolescents.

[0045] As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment, have a reasonable benefit / risk ratio, and are free of excessive toxicity, irritation, allergic response, or other problems or complications.

[0046] As used herein, the term "preventing" refers to partially or completely delaying the onset of an infectious disease, disorder, and / or condition, partially or completely delaying the onset of one or more symptoms, features, or clinical findings of a particular infectious disease, disorder, and / or condition, partially or completely delaying the onset of one or more symptoms, features, or findings of a particular infectious disease, disorder, and / or condition, partially or completely delaying progression from an infectious disease, particular disorder, and / or condition, and / or reducing the risk of developing a pathological condition associated with an infectious disease, disorder, and / or condition.

[0047] As used herein, the term "protein" or "peptide" refers to at least two or more amino acid residues linked together by peptide bonds. The amino acid sequence of a protein or peptide is shown in standard form, i.e., from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus).

[0048] As used herein, the term "recombinant production" refers to techniques for manipulating two or more DNA sequences and combining them into one, 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" (edited by Ausubel, 2008, John Wiley & Son).

[0049] As used herein, the term "substantially" refers to a qualitative condition that exhibits all or nearly all of the scope or degree of a desired feature or characteristic. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or a perfect state, nor achieve or avoid absolute results. Thus, the term "substantially" is used herein to account for the potential lack of perfection inherent in many biological and chemical phenomena.

[0050] The terms "therapeutically effective amount" or "effective amount" refer to the administration of a drug to a subject, either alone or as part of a pharmaceutical composition and either as a single dose or as part of a series of doses, which amount, when administered to the subject, is capable of producing any detectable beneficial effect on any symptom, aspect, or feature of a disease, disorder, or condition. A therapeutically effective amount can be ascertained by measuring the relevant physiological effects and can be adjusted in relation to, for example, the dosing regimen and the diagnostic analysis of the subject's condition. By way of example, measurement of 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 pathological condition associated with uncontrolled cell division, a therapeutically effective amount is an amount having the effect of (1) reducing the size of a tumor (i.e., tumor shrinkage), (2) inhibiting abnormal cell division, such as cancer cell division (i.e., making it slower to a certain extent, preferably stopping it), (3) preventing or suppressing the metastasis of cancer cells, and / or (4) alleviating (or preferably eliminating) to some extent one or more symptoms associated with a pathological condition related to or in part caused by uncontrolled or abnormal cell division, such as cancer. An "effective amount" is also an amount that results in a desired PD and PK profile and a desired immune cell profiling upon administration of a therapeutically active composition of the invention.

[0051] The term "treating" a disease (or condition or disorder), as used herein, refers to preventing a disease from occurring (preventive treatment) in a human or animal subject who is considered likely to develop the disease but has not yet experienced or manifested symptoms of the disease, inhibiting the disease (slowing or stopping its onset), achieving alleviation of symptoms or side effects of the disease (including palliative treatment), and causing regression of the disease. The terms "treat", "treating", "treatment", "therapeutic", and "therapy" do not necessarily mean complete cure or elimination of the disease or condition. Any degree of reduction of any undesirable sign or symptom of a disease or condition can be considered treatment and / or therapy. Further, treatment can include actions that may worsen the overall well-being or appearance of a patient. With respect to cancer, these terms also mean that the average life expectancy of an individual afflicted with cancer can be increased, or one or more symptoms of the disease can be reduced. With respect to cancer, "treating" also includes enhancing or prolonging an antitumor response in a subject.

[0052] "Progression-free survival (PFS)", as used in the context of cancer described herein, refers to the length of time from the start of and after treatment of cancer until objective tumor progression or death of the patient. 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 blinded central review of images and optionally further confirmed by ORR or blinded independent central review (BICR).

[0053] "Overall survival (OS)" can be evaluated by the Kaplan-Meier method based on OS rates at specific time points (e.g., 1 year and 2 years), and the corresponding 95% CI is derived based on the formula of Greenwood for each study treatment for each tumor type. The OS rate is defined as the proportion of participants surviving at that time point. The OS of a participant is defined as the time from the first dosing date to the date of death from any cause.

[0054] As used herein, "complete response" means the disappearance of all signs of cancer in response to treatment. Complete response may also be referred to herein as "complete remission".

[0055] As used herein, the term "partial response" means a reduction in the size of a tumor or the extent of cancer in the body in response to treatment. Partial response may also be referred to herein as "partial remission".

[0056] As used herein, the term "cancer" shall be given its ordinary meaning as a general term for diseases in which abnormal cells divide without being suppressed.

[0057] The terms "reducing a tumor" or "tumor shrinkage", as used herein, refer 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 proliferation status of cancer cells (the degree to which cancer cells are amplified), and the like.

[0058] The term "enhancing", as used herein, refers to enabling a subject or tumor cell to improve its ability to respond to a treatment disclosed herein. For example, an enhanced response may 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. As used herein, "enhancing" can also refer to enhancing the number of subjects who respond to a treatment, such as a combination therapy including chemotherapy, drug-resistant immune cells, and immune checkpoint inhibitors. For example, an enhanced response may refer to the total percentage of subjects who respond to the treatment, and the percentage 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.

[0059] "Immune checkpoint proteins" control 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 off or inhibit T cell function. Cancer cells utilize this system by driving the high-level expression of immune checkpoint proteins on their surface, resulting in the suppression of T cells expressing immune checkpoint proteins that enter the tumor microenvironment and thus suppressing the anti-cancer immune response. Therefore, inhibition of immune checkpoint proteins by agents referred to herein as "immune checkpoint protein inhibitors" or "immune checkpoint inhibitors" can result in the restoration of T cell function and the immune response 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 that 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, those derived from PD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists, adenosine A2A receptor antagonists, B7-H3 antagonists, B7-H4 antagonists, BTLA antagonists, KIR antagonists, LAG3 antagonists, TIM-3 antagonists, VISTA antagonists, or TIGIT antagonists.

[0060] As used herein, the term "angiogenesis inhibitor" refers to a drug, compound, antibody, or other agent that prevents the formation of new blood vessels. In cancer treatment, angiogenesis inhibitors can prevent the growth of new blood vessels required for tumor growth. Examples of angiogenesis inhibitors include agents that can target one or more signaling pathways associated with receptor tyrosine kinases (RTKs). Examples of RTKs include, but are not limited to, vascular endothelial growth factor receptors type 1, 2, and 3 (VEGFR1-3), platelet-derived growth factor receptors alpha and beta (PDGFRα / β), and fibroblast growth factor receptors (FGFR) type 1, 2, and 3 (FGFR1-3). Preferred angiogenesis inhibitors of the present invention have broad target selectivity and can simultaneously target and inhibit multiple RTKs, and are referred to herein as "multi-receptor tyrosine kinase inhibitors."

[0061] "Pharmaceutically acceptable salt" refers to a salt of a compound of the present disclosure that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic and can be inorganic or organic acid addition salts and base addition salts. Specifically, such salts include (1) those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc., or organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, etc., or (2) salts formed when the acidic proton present in the parent compound is replaced by a metal ion such as an alkali metal ion, alkaline earth ion, or aluminum ion, or coordinated with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, etc. Examples of salts include, but are not limited to, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc. When the compound contains a basic functional group, non-toxic organic or inorganic acid salts such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate, etc. are further included.

[0062] As a whole, such salts can be prepared by reacting these compounds in free acid or base form with a stoichiometric amount of an appropriate base or acid in water, an organic solvent, or a mixture of the two, and as a whole, a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is preferred. A list of suitable salts can be found in Allen, Jr., L. V., ed., Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press, London, UK (2012).

[0063] The fusion protein of SEQ ID NO: 1 The recombinant human IL-2 variant fusion protein described in International Publication No. 2013 / 184942 is a circular permutation (cp) IL-2 variant fused to the extracellular domain of the IL-2Rα portion of the IL-2 receptor, and is referred to herein as the "fusion protein of SEQ ID NO: 1" or "fusion protein", and has the following amino acid sequence: SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQGSGGGSELCDDDPPEIPHATFKAMAYKEGTMLNCECKRGFRRIKSGSLYMLCTGNSSHSSWDNQCQCTSSATRNTTKQVTPQPEEQKERKTTEMQSPMQPVDQASLPGHCREPPPWENEATERIYHFVVGQMVYYQCVQGYRALHRGPAESVCKMTHGKTRWTQPQLICTG (SEQ ID NO: 1) has.

[0064] Fusion proteins closely related to SEQ ID NO: 1, such as fusion proteins having a sequence identity of about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more to the full length of SEQ ID NO: 1, are also contemplated to be suitable for administration according to the methods of the present invention. Fusion proteins closely related to SEQ ID NO: 1, such as fusion proteins having a sequence identity of about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more to a continuous sequence of at least about 20 amino acids to up to the full length of SEQ ID NO: 1, are also contemplated to be suitable for administration according to the methods of the present invention.

[0065] The fusion protein of SEQ ID NO: 1 can be produced using a biological recombinant expression system or any protein synthesis apparatus. Strategies for recombinant protein expression are well known in the art and typically involve the steps of transfecting a cell with a DNA vector containing a template encoding the fusion protein of SEQ ID NO: 1, and then culturing the cell to transcribe and translate the fusion protein. Typically, the cell is then lysed to extract the expressed protein for subsequent purification. Both prokaryotic in vivo protein expression systems and eukaryotic in vivo protein expression systems are widely used. Preferably, the fusion protein of SEQ ID NO: 1 is produced in CHO cells.

[0066] The present invention provides a pharmaceutical composition comprising a fusion protein of SEQ ID NO:1 at a dose of at least about 6 μg / kg to about 70 μg / kg, preferably a fusion protein of SEQ ID NO:1 at least about 6 μg / kg to at least about 15 μg / kg and a pharmaceutically acceptable excipient, or a corresponding fixed dose (e.g., about 0.4 mg to approximately about 1.0 mg) based on an adult human of average 60 - 70 kg, or a corresponding fixed dose of a pharmaceutical composition based on a pediatric, e.g., a pediatric of about 12 kg to about 50 kg or more. The present invention also provides a pharmaceutical composition at a dose of at least about 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, or 15 μg / kg, or a corresponding fixed dose based on an adult, e.g., of average 60 - 70 kg, or based on a pediatric, e.g., a pediatric of about 12 kg to about 50 kg or more.

[0067] The present invention also provides a pharmaceutical composition comprising a fusion protein of SEQ ID NO:1 at a dose of at least about 3 μg / kg to at least about 5.5 μg / kg and a pharmaceutically acceptable excipient, or a corresponding fixed dose (e.g., about 0.2 mg to about 0.4 mg) based on an adult human of average 60 - 70 kg. The present invention also provides a pharmaceutical composition at a dose of at least about 3, 3.5, 4, 4.5, 5, 5.5 μg / kg, or a corresponding fixed dose based on an adult human of average 60 - 70 kg or based on a pediatric, e.g., a pediatric of about 12 kg to about 50 kg or more.

[0068] The present invention also provides a pharmaceutical composition comprising a fusion protein of SEQ ID NO:1 at a dose of at least about 40 μg / kg to at least about 70 μg / kg and a pharmaceutically acceptable excipient, or a corresponding fixed dose (e.g., about 3.0 mg to about 3.6 mg) based on an adult human of average 60 - 70 kg, or a corresponding fixed dose of a pharmaceutical composition based on a pediatric, e.g., a pediatric of about 12 kg to about 50 kg or more.

[0069] As used herein, pharmaceutically acceptable excipients include any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc. that are suitable for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for their preparation.

[0070] The use of any conventional excipient medium is contemplated to be within the scope of the present disclosure, except where it is incompatible with the substance or its derivatives, for example, by producing any undesirable biological effects or otherwise interacting detrimentally with any other component of the pharmaceutical composition.

[0071] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents, and emulsifying agents, for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 - butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters, and mixtures thereof.

[0072] In addition to the inert diluent, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, and / or perfuming agents. In certain embodiments for parenteral administration, the composition is mixed with solubilizing agents such as CREMOPHOR®, alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer, and / or combinations thereof.

[0073] Injectable preparations, such as sterile aqueous or oily suspensions, can be formulated according to known techniques using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations can be, for example, sterile injectable solutions, suspensions, and / or emulsions in a non-toxic parenterally acceptable diluent and / or solvent such as a solution in 1,3 - butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile fixed oils have been conventionally used as solvents or suspending media. For this purpose, any bland fixed oil including synthetic mono - or diglycerides can be used. Fatty acids such as oleic acid can be used in the preparation of injectables.

[0074] Injectable formulations can be used after being sterilized, for example, by filtration through a bacteria - retaining filter and / or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile injectable media. General considerations in the formulation and / or manufacture of pharmaceuticals can be found, for example, in Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference).

[0075] The fusion protein of SEQ ID NO: 1 (Figure 1A) is designed to selectively bind to and activate the intermediate-affinity IL-2R rather than the high-affinity IL-2R. The IL-2Rα domain of the fusion protein of SEQ ID NO: 1 sterically hinders the binding of the fusion protein of SEQ ID NO: 1 to the high-affinity IL-2R but still allows binding to the intermediate-affinity IL-2R.

[0076] In vitro and in vivo non-clinical pharmacodynamic (PD) data support selective signaling through the intermediate-affinity IL-2 receptor by the fusion protein of SEQ ID NO: 1, which causes activation and expansion of effector cells such as NK cells and CD8+ cells while minimizing the activation and expansion of immunosuppressive T reg cells. In addition, in vivo in mice, the fusion protein of SEQ ID NO: 1 shows improved tolerance to rhIL-2 at doses that induce equivalent or greater expansion of effector cells compared to T reg cells.

[0077] Additional non-clinical data demonstrate that intravenous or subcutaneous administration of the fusion protein of SEQ ID NO: 1 results in equivalent tumor growth inhibition in a murine syngeneic tumor model and similar peripheral expansion of NK and CD8 + T cells after administration by either route in cynomolgus monkeys.

[0078] The first of the human clinical data described in Example 1 shows that the fusion protein of SEQ ID NO: 1 dose-dependently activates the expansion of CD8+ cells and NK cells in the absence of dose-dependent activation of Tregs. Thus, the fusion protein of SEQ ID NO: 1 can be dosed to human patients at concentrations comparable to high-dose rhIL-2 to induce equivalent or greater expansion of NK cells and CD8+ cells compared to high-dose rhIL-2, but with a much smaller (less than half) relative expansion of immunosuppressive Tregs compared to high-dose rhIL-2 (Table 2). This result was unexpected.

[0079] Dosing regimen Preferably, the fusion protein of SEQ ID NO: 1 is administered to a cancer patient according to the methods and dosing regimens of the present invention. Preferred routes of administration are intravenous, such as intravenous injection and infusion via a central venous access, etc. Additional routes of administration include subcutaneous, intramuscular, oral, nasal, and pulmonary administration. Preferably, the fusion protein can be administered as part of a pharmaceutical composition containing at least one excipient.

[0080] Preferably, the present invention provides a pharmaceutical composition for intravenous (I.V.) administration comprising a fusion protein of SEQ ID NO: 1 at a dose in μg / kg units, preferably from about 0.1 μg / kg to about 70 μg / kg, about 1 μg / kg to about 70 μg / kg, about 1 μg / kg to about 50 μg / kg, about 1 μg / kg to about 30 μg / kg, about 1 μg / kg to about 25 μg / kg, about 1 μg / kg to about 15 μg / kg, about 1 μg / kg to about 10 μg / kg, about 1 μg / kg to about 5 μg / kg, about 1 μg / kg to about 3 μg / kg, about 6 μg / kg to about 70 μg / kg, about 6 μg / kg to about 50 μg / kg, about 6 μg / kg to about 30 μg / kg, about 6 μg / kg to about 25 μg / kg, about 6 μg / kg to about 15 μg / kg, about 6 μg / kg to about 10 μg / kg, about 6 μg / kg to about 8 μg / kg, about 8 μg / kg to about 70 μg / kg, about 8 μg / kg to about 50 μg / kg, about 8 μg / kg to about 30 μg / kg, about 8 μg / kg to about 25 μg / kg, about 8 μg / kg to about 15 μg / kg, about 8 μg / kg to about 10 μg / kg, about 10 μg / kg to about 70 μg / kg, about 10 μg / kg to about 50 μg / kg, about 10 μg / kg to about 30 μg / kg, about 10 μg / kg to about 25 μg / kg, about 10 μg / kg to about 15 μg / kg, about 12 μg / kg to about 12 μg / kg, about 12 μg / kg to about 50 μg / kg, about 12 μg / kg to about 30 μg / kg, about 12 μg / kg to about 25 μg / kg, about 12 μg / kg to about 15 μg / kg, about 14 μg / kg to about 70 μg / kg, about 14 μg / kg to about 50 μg / kg, about 14 μg / kg to about 30 μg / kg, about 14 μg / kg to about 25 μg / kg, about 30 μg / kg to about 70 μg / kg, about 30 μg / kg to about 50 μg / kg, about 40 μg / kg to about 70 μg / kg, about 40 μg / kg to about 50 μg / kg, about 50 μg / kg to about 70 μg / kg, about 50 μg / kg to about 60 μg / kg, which is preferably used for calculating doses in pediatric patients in many cases but is also useful for calculating doses for adults, or its corresponding fixed dose (e.g., about 1 mg to about 4 mg) based on an adult of, for example, 60 - 70 kg, or a corresponding fixed dose array based on a pediatric patient, for example, a pediatric patient of about 12 kg to about 50 kg or more.

[0081] Preferably, the fusion protein of SEQ ID NO: 1 is administered to a patient by intravenous infusion at a dose of at least about 6 μg / kg / day to at least about 15 μg / kg / day, or a corresponding fixed daily dose based on, for example, an adult human of average 60 - 70 kg (e.g., about 0.4 mg / day to approximately 1.0 mg / day), or a corresponding fixed daily dose based on a pediatric patient, for example, about 12 kg to about 50 kg or more. Preferably, the dose is about 6 μg / kg per day. Preferably, the dose is about 8 μg / kg per day. Preferably, the dose is about 10 μg / kg per day. Preferably, the dose is about 12 μg / kg per day. Preferably, the dose is about 14 μg / kg per day. Preferably, the dose is about 15 μg / kg per day. Preferably, the dose is about 6 μg / kg / day, 6.5 μg / kg / day, 7 μg / kg / day, 7.5 μg / kg / day, 8 μg / kg / day, 8.5 μg / kg / day, 9 μg / kg / day, 9.5 μg / kg / day, 10 μg / kg / day, 10.5 μg / kg / day, 11 μg / kg / day, 11.5 μg / kg / day, 12 μg / kg / day, 12.5 μg / kg / day, 13 μg / kg / day, 13.5 μg / kg / day, 14 μg / kg / day, 14.5 μg / kg / day, 15 μg / kg / day, or a corresponding fixed daily dose based on an adult human of average 60 - 70 kg, or a corresponding fixed daily dose based on a pediatric patient, for example, about 12 kg to about 50 kg or more.

[0082] Higher doses, for example, doses of 16 μg / kg / day, 18 μg / kg / day, 20 μg / kg / day, 22 μg / kg / day, 24 μg / kg / day, 26 μg / kg / day, 28 μg / kg / day, 30 μg / kg / day, 40 μg / kg / day, 50 μg / kg / day, 60 μg / kg / day, 70 μg / kg / day, or a corresponding fixed daily dose based on, for example, an adult human of average 60 - 70 kg (e.g., about 1 mg to about 4 mg), or a corresponding fixed dose based on a pediatric patient, for example, about 12 kg to about 50 kg or more may be contemplated for administration to a patient.

[0083] Preferably, the fusion protein of SEQ ID NO: 1 is administered as a single I.V. infusion per day. The single I.V. infusion may take from 5 minutes to 2 hours.

[0084] Preferably, the dosing regimen for administration of the fusion protein provides one or more treatment courses. A single treatment course can be conducted over a period of days in the range of 1 to 90 days. Preferably, a single treatment course extends over a period of 14 or 21 days. A treatment course can involve multiple consecutive days on which the fusion protein is administered once daily to the patient via I.V. infusion, followed by multiple consecutive days on which the fusion protein is not administered to the patient, herein also referred to as a "drug holiday". It is contemplated that the dosing days may not be consecutive, for example, if the patient experiences discomfort with daily dosing. Preferably, the fusion protein is administered to the patient for at least about 1, 2, 3, 4, or 5 consecutive days. Preferably, administration of the fusion protein need not be continuous and may be conducted over a course of 1, 2, 3, 4, or 5 non - consecutive days. Preferably, the fusion protein is administered once daily to the patient by I.V. infusion for 1 to about 5 consecutive days. Preferably, the fusion protein is administered once daily to the patient for a desired total number of infusion days prior to the drug holiday, for example, by infusing on day 1 and providing 1 or 2 days in between until the next infusion, over a course of 1 to 5 non - consecutive days.

[0085] Preferably, the first course of treatment includes the step of administering the fusion protein once daily by I.V. infusion for five (5) consecutive days during a first treatment course that lasts about 14 days, followed by a 9 - day drug holiday. Preferably, a second course of treatment follows the first course of treatment. The second course of treatment may start at any time after the first course of treatment, but preferably starts within about 24 hours or more than about 24 hours after the first course of treatment has ended.

[0086] Preferably, the treatment of the second course comprises administering the fusion protein of SEQ ID NO: 1 once daily by continuous five (5)-day I.V. infusion during a treatment course lasting about 21 days, followed by a drug-free period of at least about 16 consecutive days. Additional treatment courses, such as the third, fourth, and fifth treatment courses, may follow the second treatment course, preferably starting within about 24 hours after the previous course ends. Preferably, all treatment courses after the first treatment course are treatment courses of at least about 21 days.

[0087] Preferably, the fusion protein is administered with another therapeutic agent and / or anti-cancer agent described below. Preferably, the therapeutic agent is pembrolizumab, an immune checkpoint inhibitor. Preferably, pembrolizumab is administered in a separate composition from the fusion protein, preferably by I.V. infusion before, after, or simultaneously with the infusion of the fusion protein. Preferably, pembrolizumab is administered once daily at a dose of about 200 μg or according to standard prescribing recommendations. Preferably, pembrolizumab is administered with the fusion protein on the first day of each course of treatment. An exemplary treatment regimen is shown in Figure 2. Preferably, when the fusion protein is co-administered with pembrolizumab, the first course of treatment using the fusion protein and all subsequent courses of treatment (e.g., the second, third, fourth, and fifth treatment courses) are, as a whole, about 21-day courses in which, after the fusion protein is administered once daily by continuous five (5)-day I.V. infusion, a drug-free period of at least about 16 consecutive days follows before the next course of administration. As discussed above, the dosing days need not be consecutive and may be carried out over a non-consecutive process of 1, 2, 3, 4, or 5 days.

[0088] The present invention also provides a dosing regimen in which the fusion protein of SEQ ID NO: 1 is administered periodically, for example, only once every about 3 days to once every about 60 days. Preferably, the periodic dosing is once every about 3 days to once every about 21 days. Preferably, the periodic dosing is once every 3 days, once every 4 days, once every 7 days, once every 14 days, or once every 21 days. In this periodic dosing, for example, a once-weekly dosing regimen, the dose of the fusion protein is administered once a week. For example, the dose is about 40 μg / kg to about 70 μg / kg. Preferably, the dose is about 50 μg / kg to about 60 μg / kg, or its corresponding fixed dose based on, for example, an adult of 60 kg to 70 kg or a pediatric patient, for example, a pediatric patient of about 12 kg to about 50 kg or more.

[0089] Preferably, the fusion protein of SEQ ID NO: 1 is administered by I.V. infusion on days 1, 7, 14, and 21 of each treatment cycle. Preferably, the fusion protein of SEQ ID NO: 1 is administered by I.V. infusion on days 1 and 14 of each treatment cycle. Preferably, the fusion protein of SEQ ID NO: 1 is administered by I.V. infusion on days 1 and 21 of each treatment cycle. Preferably, the fusion protein of SEQ ID NO: 1 is administered by I.V. infusion on days 1, 7, and 14 of each treatment cycle. Preferably, the fusion protein of SEQ ID NO: 1 is administered by I.V. infusion on days 1, 7, and 21 of each treatment cycle.

[0090] Preferably, the fusion protein of SEQ ID NO: 1 is administered periodically. For example, the fusion protein of SEQ ID NO: 1 is administered to a patient by I.V. infusion for one or more days during a treatment cycle. Each I.V. administration during the treatment cycle is separated by about 7 days, about 14 days, or about 21 days, or any combination thereof, unless the administration of SEQ ID NO: 1 is carried out daily at a dose of about 6 μg / kg / day to about 15 μg / kg / day, or a dose of about 16 μg / kg / day to about 70 μg / kg / day, or a corresponding fixed daily dose (e.g., about 1 mg to about 4 mg) based on an adult of average 60 - 70 kg, or a corresponding fixed daily dose based on a pediatric patient, e.g., about 12 kg to about 50 kg or more.

[0091] Preferably, the fusion protein of SEQ ID NO: 1 is administered periodically. For example, the fusion protein of SEQ ID NO: 1 is administered to a patient by I.V. infusion for one or more days during a treatment cycle. Each I.V. administration during the treatment cycle is separated by about 7 days, about 14 days, or about 21 days, or any combination thereof, unless the administration of SEQ ID NO: 1 is carried out daily at a dose of preferably about 16 μg / kg / day to about 70 μg / kg / day, preferably about 16 μg / kg / day to about 50 μg / kg / day, preferably about 16 μg / kg / day to about 30 μg / kg / day, preferably about 16 μg / kg / day to about 20 μg / kg / day, preferably about 30 μg / kg / day to about 50 μg / kg / day, or a corresponding fixed daily dose (e.g., about 1 mg to about 4 mg) based on an adult of average 60 - 70 kg, or a corresponding fixed dose based on a pediatric patient, e.g., about 12 kg to about 50 kg or more.

[0092] Preferably, the regular dosing is administered by I.V. infusion. Preferably, a second therapeutic / anticancer agent, such as pembrolizumab, is co-administered with the fusion protein of SEQ ID NO: 1 by I.V. infusion before, after, or at the same time as, or on the same day as, the administration of the fusion protein of SEQ ID NO: 1.

[0093] The method of the present invention also contemplates a dosing regimen in which the fusion protein of SEQ ID NO: 1 is administered once daily at a dose of less than about 6 μg / kg / day, or an equivalent fixed dose based on a 60 - 70 kg human or based on a pediatric of about 12 kg to about 50 kg or more. For example, the patient may be administered a dose of about 3, 3.5, 4, 4.5, 5, 5.5 μg / kg / day, or a corresponding fixed daily dose based on an adult human of average 60 - 70 kg, or a corresponding fixed dose based on a pediatric, for example, about 12 kg to about 50 kg or more. A once-daily dosing regimen of less than about 6 μg / kg / day, or an equivalent fixed dose based on a 60 - 70 kg human, may be administered according to any of the dosing regimens described above instead of being related to a dose between 6 μg / kg / day and 15 μg / kg / day.

[0094] All of the dosing regimens of the present invention described above 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 regulatory T (Treg) cells, preferably resulting in an increase in circulating NK cells and CD8+ cells that is greater than the increase in circulating Treg cells in the patient. When compared to high-dose or low-dose rhIL-2 therapy, all of the dosing regimens of the present invention preferably require fewer dosing times, for example, once-daily dosing of the fusion protein of SEQ ID NO: 1, compared to dosing three times a day with high-dose or low-dose rhIL-2.

[0095] Preferably, the increase in circulating CD8+ T cells resulting from the administration of the fusion protein of SEQ ID NO: 1 is 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. Preferably, the ratio of the increase in circulating CD8+ T cells resulting from the administration of the fusion protein of SEQ ID NO: 1 is greater than the ratio of the increase in circulating regulatory T cells.

[0096] Preferably, the fusion protein of SEQ ID NO: 1 and pharmaceutical compositions thereof, when combined with one or more immune checkpoint inhibitors that treat and / or prevent various diseases, disorders, and conditions (e.g., cancer), are affected by utilizing specific dosing parameters that serve to minimize any adverse effects associated with the administration of the individual therapies alone. 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) enables a reduction in the amount of immune checkpoint inhibitor required to achieve the treatment goal and, thus, may reduce (or even eliminate) severe and life-threatening immune-mediated adverse reactions that prompted the FDA to require a "black box" warning for a particular immune checkpoint inhibitor (e.g., pembrolizumab).

[0097] Generally, the dosing parameters for monotherapy using the fusion protein of SEQ ID NO: 1 or any combination therapy described herein direct that the dosage be less than an amount at which the dosage can irreversibly become toxic to the subject (i.e., the maximum tolerated dose, "MTD") and greater than an amount required to produce a measurable effect on the subject. Such amounts are determined by considering the route of administration and other factors, e.g., by pharmacokinetic parameters and pharmacodynamic parameters associated with ADME.

[0098] An effective dose (ED) is the dose or amount of a drug that produces a therapeutic response or desired effect in a certain percentage of the subjects to which the drug is administered. 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 the drug is administered. The ED50 is commonly used as a basis for a reasonable prediction of the effect of a drug, but is not necessarily a dose that a clinician may consider appropriate when taking into account all relevant factors. Thus, in some situations, the effective amount may be above the calculated ED50, in other situations, the effective amount may be below the calculated ED50, and in still other situations, the effective amount may be the same as the calculated ED50.

[0099] In addition, an effective amount of the fusion protein of SEQ ID NO: 1, when administered to a subject in a single or multiple doses, can be an amount that produces a desired result in a healthy subject. For example, in the case of a subject experiencing a particular disorder, the effective amount is 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% improvement in diagnostic parameters, measurements, markers, etc. of that disorder as defined by diagnostic parameters, measurements, markers, etc. presented by a subject with 100% normality.

[0100] Preferably, if cancer recurs in a patient after the initial treatment, the fusion protein of SEQ ID NO: 1 is administered again to the patient. For example, if a 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.

[0101] Preferably, the fusion protein of SEQ ID NO: 1 is administered to cancer patients according to the methods and dosing regimens of the present invention. Preferred routes of administration are intravenous, such as intravenous injection and intravenous infusion via central venous access, etc. Further preferred routes of administration include subcutaneous, intramuscular, oral, nasal, and pulmonary administration.

[0102] The treatment regimen of the present invention is administered to the patient until the patient is cured or until the patient no longer benefits from the treatment regimen.

[0103] Improved safety profile The toxicity of rhIL-2 in humans and animals has been well established. At the high doses of rhIL-2 used in most cancer trials, tumor responses are only occasional and significant toxicity has been demonstrated. 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 extravascular leakage of fluid and protein, leading to interstitial edema and organ failure. Findings of CLS include fluid retention, weight gain, peripheral edema, pleural and pericardial effusions, ascites, generalized edema, and signs of severe pulmonary and cardiovascular failure. Symptoms are highly variable among patients and the cause is not fully understood.

[0104] The etiology of endothelial cell (EC) injury is complex and can involve activation or injury to ECs and leukocytes, release of cytokines and inflammatory mediators, changes in cell-cell and cell-matrix adhesion, and changes in cytoskeletal function. CLS limits the dose of IL-2 that can be administered to humans and may in some cases require discontinuation of therapy.

[0105] The methods of the present invention maintain the desired therapeutic activity of IL-2 therapy while reducing the risk of side effects often associated with high-dose therapy, such as, but not limited to, CLS, as well as cytokine release syndrome (CRS) and other syndromes associated with immunotherapy using cytokines, often associated with and / or overlapping with CLS.

[0106] Surprisingly, the dose escalation study in the human clinical trials described in the examples showed that administration of the fusion polypeptide of SEQ ID NO: 1 to patients at a concentration equivalent to that of high-dose rh-IL-2 did not result in the frequency and severity of certain side effects, such as capillary leak syndrome, which is often associated with high-dose rhIL-2 therapy. As described in the examples summarizing the human clinical studies, despite exceeding the EC50 values for natural killer cell and CD8+ T cell activation, the dose-limiting toxicity (DLT) has not yet reached doses of 6 μg / kg / day or higher. Thus, the method of the present invention can provide an improved safety profile for patients with cancer in need of IL-2 therapy, particularly as compared to standard rhIL-2 therapy and especially high-dose rhIL-2 therapy.

[0107] As used herein, for example, an "improved safety profile", or "lower risk of side effects", or "reduced frequency or severity of side effects", particularly in comparison to standard rhIL-2 therapy, especially high-dose rhIL-2 therapy, can be evaluated in several ways. The side effects or symptoms of IL-2 therapy can be quantified. The side effects or symptoms of IL-2 therapy can be quantified, for example, on a semi-quantitative scale of 0 to 5, where 0 represents absence, 1 to 4 represent identifiable increases in severity, and 5 represents maximum severity. Clinical trials often use a scale of 1 to 5, where 1 represents a mild adverse event (side effect), 2 represents a moderate adverse event (side effect), 3 represents a severe adverse event (side effect), 4 represents a life-threatening or disabling adverse event (side effect), and 5 represents death related to an adverse event (side effect). Alternatively, the side effects or symptoms of IL-2 therapy can be quantified as binary events, i.e., present or absent, i.e., 0 or 1. Other semi-quantitative scales will be readily apparent to those skilled in the art. In another embodiment, the side effects or symptoms of IL-2 therapy can be quantified on a quantitative scale, such as mass per volume, temperature, duration, ratio, enzyme activity, oxygen saturation, etc., such as the mass of cytokine per volume of tissue fluid. Those skilled in the art will readily understand how to evaluate 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 cytokine concentration in plasma or serum, temperature (fever), heart rate (tachycardia), blood pressure (hypotension), cardiac dysfunction, renal dysfunction, serum or plasma enzyme concentration (liver function), etc. Any quantification of the side effects or symptoms of IL-2 therapy can be compared to a control, such as a healthy control subject not receiving IL-2 therapy, or to other control subjects receiving, for example, standard high-dose rhIL-2 therapy.

[0108] The "reduced risk" of side effects of IL-2 therapy can be, for example, a reduction of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100% in the side effects or symptoms findings of IL-2 therapy as compared to, for example, high-dose rhIL-2 therapy. Alternatively, treating the side effects or symptoms of IL-2 therapy can be a reduction to about one-half, about one-third, about one-fourth, about one-fifth, about one-sixth, about one-seventh, about one-eighth, about one-ninth, about one-tenth, or less of the side effects or symptoms of IL-2 therapy. Thus, "less severe side effects" will refer to such a reduction in the side effects or symptoms of IL-2 therapy.

[0109] Preferably, the dosing regimen of the fusion protein according to the invention reduces the frequency and severity of capillary leak syndrome (CLS), also referred to herein as vascular leak syndrome (VLS).

[0110] Among the risks of other side effects often associated with rhIL-2 immunotherapy that can also be reduced by the treatment regimen of the present invention, but not limited thereto, is cytokine release syndrome (CRS). CRS is a serious side effect of immunotherapy that can have symptoms clinically overlapping with those of CLS, but can also cause symptoms quite different from CRS. CRS is thought to result from the proliferation of T cells that release large amounts of cytokines including IL-6, IFN-γ, TNF, IL-2, IL-2 receptor a, IL-8, IL-10, and GMCSF. Patients with CRS may experience any one or more of the following: cardiovascular symptoms including elevated body temperature, tachycardia, hypotension, arrhythmia, and decreased cardiac ejection fraction; pulmonary symptoms including edema, hypoxia, dyspnea, and interstitial pneumonia; acute kidney injury usually caused by decreased renal perfusion; liver and gastrointestinal symptoms including elevated serum transaminases and bilirubin, diarrhea, colitis, nausea, and abdominal pain; hematological symptoms including cytopenia, for example grade 3-4 anemia, thrombocytopenia, leukopenia, neutropenia, and lymphopenia; coagulation function abnormalities including prolonged prothrombin time and activated partial thromboplastin time (PTT), elevated D-dimer, hypofibrinogenemia, disseminated intravascular coagulation syndrome, macrophage activation syndrome (MAS), bleeding, B cell hypoplasia, and hypogammaglobulinemia; infectious diseases including bacteremia, salmonellosis, urinary tract infection, influenza, respiratory syncytial virus, and viral infections such as herpes zoster virus; musculoskeletal symptoms including elevated creatine kinase, myalgia, and fatigue; neurological symptoms including delirium, confusion, and seizure attacks.

[0111] Administration of SEQ ID NO: 1 has been shown to induce lower levels of inflammatory cytokines in mice, for example compared to rhIL-2. See Example 4 and Figure 10.

[0112] Administration of SEQ ID NO: 1 has also been shown to induce lower levels of inflammatory cytokines such as IL-6 and at the same time higher levels of desirable cytokines such as IFN in humans. See Example 5 and Figure 15.

[0113] MAS clinically overlaps with CRS in subjects who may experience hepatosplenomegaly, lymphadenopathy, pancytopenia, liver dysfunction, disseminated intravascular coagulation syndrome, hypofibrinogenemia, hyperferritinemia, and hypertriglyceridemia. Similar to CRS, subjects with MAS exhibit elevated levels of cytokines including IFN-γ and GMCSF.

[0114] Another side effect of immunotherapy including IL-2 therapy is tumor lysis syndrome (TLS), which occurs when the contents of cells are released as a result of therapy that causes cell death, most often in lymphoma and leukemia. TLS is characterized by blood ion and metabolite imbalances and symptoms include nausea, vomiting, acute uric acid nephropathy, acute renal failure, seizure, arrhythmia, and death.

[0115] Neurotoxicity may occur as a result of immunotherapy including IL-2 therapy and symptoms can include cerebral edema, delirium, hallucinations, aphasia, mutism, headache, confusion, changes in level of arousal, ataxia, apraxia, facial nerve palsy, tremors, measurement disturbances, and seizure.

[0116] Patients undergoing IL-2 immunotherapy may experience one or more side effects or symptoms, including anemia, aphasia, arrhythmia, arthralgia, back pain, blood and bone marrow disorders, blood and lymphatic system disorders, heart disorders, chills, coagulation disorders, colitis, confusion, general symptoms, cough, decreased appetite, diarrhea, disorientation, dizziness, dyspnea, encephalopathy, fatigue, fever, gastrointestinal disorders, systemic cardiovascular disorders, hemorrhage, liver disorders, hyperglycemia, hypokalemia, hypothyroidism, increased ALT, increased AST, increased C-reactive protein, infectious febrile neutropenia, leukopenia, malaise, abnormal metabolic test results, metabolic and nutritional disorders, mucosal inflammation, musculoskeletal disorders, myalgia, nausea, nervous system disorders, neurological disorders, neutropenia, edema, pain, palmar-plantar erythrodysesthesia, paresthesia, pneumonia, pruritus, pulmonary disorders, rash, renal and urogenital disorders, respiratory disorders, skin and subcutaneous tissue disorders, somnolence, speech disorders, sweating, thoracic and mediastinal disorders, thrombocytopenia, tremors, tumor flare, tumor lysis syndrome, vascular disorders, and vomiting, which are not necessarily caused by CLS, CRS, MAS, or TLS.

[0117] Cancer indications The treatment regimen of the present invention using the fusion protein of SEQ ID NO: 1 is useful for the treatment of many types of cancer. As used herein, the term "cancer" shall be given its ordinary meaning as a general term for diseases in which abnormal cells divide without being suppressed. In particular, in the context of the multiple embodiments of the present invention, cancer refers to angiogenesis-related cancer. Cancer cells can invade nearby tissues and spread to other parts of the body via the bloodstream and lymphatic system. There are several major types of cancer. For example, carcinoma is a cancer that starts in the skin or the tissue that covers the inside or outside of internal organs. Sarcoma is a cancer that starts in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissues. Leukemia is a cancer that starts in blood-forming tissues such as the bone marrow, producing a large number of abnormal blood cells and entering the bloodstream. Lymphoma is a cancer that starts in cells of the immune system.

[0118] Tumors form when normal cells lose their ability to function as a regulated and integrated unit. Overall, solid tumors are abnormal masses of tissue that typically do not contain cysts or liquid components (although some brain tumors have fluid-filled cysts and central necrosis). A single tumor may even have different populations of cells within it because various processes have gone awry. Solid tumors can be either benign (non-cancerous) or malignant (cancerous). Different types of solid tumors are named according to the type of cells that form them. Examples of solid tumors include sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) do not generally form solid tumors.

[0119] Representative cancers include, but are not limited to, among others, acute lymphoblastic leukemia, adult; acute lymphoblastic leukemia, pediatric; acute myelogenous leukemia, adult; adrenocortical carcinoma; adrenocortical carcinoma, pediatric; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; astrocytoma, pediatric cerebellum; astrocytoma, pediatric brain; extrahepatic bile duct cancer; bladder cancer; bladder cancer, pediatric; bone cancer, osteosarcoma / malignant fibrous histiocytoma; glioblastoma, pediatric; glioblastoma, adult; brainstem glioma, pediatric; brain tumor, adult; brain tumor, brainstem glioma, pediatric; brain tumor, cerebellar astrocytoma, pediatric; brain tumor, cerebral astrocytoma / malignant glioma, pediatric; brain tumor, ependymoma, pediatric; brain tumor, medulloblastoma, pediatric; brain tumor, supratentorial primitive neuroectodermal tumor, pediatric; brain tumor, optic pathway and hypothalamic glioma, pediatric; brain tumor, pediatric (others); breast cancer; breast cancer and pregnancy; breast cancer, pediatric; breast cancer, male; bronchial adenoma / carcinoid, pediatric; carcinoid tumor, pediatric; carcinoid tumor, gastrointestinal tract; carcinoma, adrenocortical; carcinoma, islet cell; cancer of unknown primary; central nervous system lymphoma, primary; cerebellar astrocytoma, pediatric; cerebral astrocytoma / malignant glioma, pediatric; cervical cancer; childhood cancer; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; clear cell sarcoma of tendon sheath; colon cancer; colorectal cancer, pediatric; cutaneous T-cell lymphoma; endometrial cancer; ependymoma, pediatric; epithelial cancer, ovary; esophageal cancer; esophageal cancer, pediatric; Ewing family of tumors; extracranial germ cell tumor, pediatric; extragonadal germ cell tumor; extrahepatic bile duct cancer; eye cancer, intraocular melanoma; eye cancer, retinoblastoma; gallbladder cancer; gastric cancer; gastric cancer, pediatric; gastrointestinal carcinoid tumor; extracranial germ cell tumor, pediatric; extragonadal germ cell tumor; germ cell tumor, ovary; gestational trophoblastic tumor; glioma, pediatric brainstem; glioma, pediatric optic pathway and hypothalamus; hairy cell leukemia; head and neck cancer; hepatocellular (liver) cancer, adult (primary); hepatocellular (liver) cancer, pediatric (primary); Hodgkin lymphoma, adult; Hodgkin lymphoma, pediatric; Hodgkin lymphoma during pregnancy; hypopharyngeal cancer; hypothalamic and optic pathway glioma, pediatric; intraocular melanoma; islet cell carcinoma (pancreatic endocrine); Kaposi sarcoma; kidney cancer; pharyngeal cancer; pharyngeal cancer, pediatric; leukemia, acute lymphoblastic, adult; leukemia, acute lymphoblastic, pediatric; leukemia, acute myelogenous, adult; leukemia, acute myelogenous, pediatric; leukemia, chronic lymphocytic; leukemia, chronic myelogenous; leukemia, hairy cell; lip and oral cavity cancer; liver cancer, adult (primary); liver cancer, pediatric (primary); lung cancer, non-small cell;Lung cancer, small cell; Lymphoblastic leukemia, adult acute; Lymphoblastic leukemia, pediatric acute; Lymphocytic leukemia, chronic; Lymphoma, AIDS-related; Lymphoma, central nervous system (primary); Lymphoma, cutaneous T-cell; Lymphoma, Hodgkin, adult; Lymphoma, Hodgkin; Pediatric; Lymphoma, Hodgkin, during pregnancy; Lymphoma, non-Hodgkin, adult; Lymphoma, non-Hodgkin, pediatric; Lymphoma, non-Hodgkin, during pregnancy; Lymphoma, primary central nervous system; Macroglobulinemia, Waldenström; Male breast cancer; Malignant mesothelioma, adult; Malignant mesothelioma, pediatric; Malignant thymoma; Medulloblastoma, pediatric; Melanoma; Melanoma, intraocular; Merkel cell carcinoma; Mesothelioma, malignant; Metastatic squamous neck cancer of unknown primary; Multiple endocrine neoplasia syndrome, pediatric; Multiple myeloma / plasma cell neoplasms; Mycosis fungoides; Myelodysplastic syndrome; Myelogenous leukemia, chronic; Myeloblastic leukemia, pediatric acute; Myeloma, multiple; Myeloproliferative disorders, chronic; Nasal and paranasal cavity cancer; Nasopharyngeal cancer; Nasopharyngeal cancer, pediatric; Neuroblastoma; Neurofibroma; Non-Hodgkin lymphoma, adult; Non-Hodgkin lymphoma, pediatric; Non-Hodgkin lymphoma during pregnancy; Non-small cell lung cancer; Oral cancer, pediatric; Oral and oropharyngeal cancer; Oropharyngeal cancer; Osteosarcoma / malignant fibrous histiocytoma of bone; Ovarian cancer, pediatric; Ovarian epithelial cancer; Ovarian germ cell tumors; Ovarian low malignant potential tumors; Pancreatic cancer; Pancreatic cancer, pediatric; Pancreatic cancer, islet cell; Paranasal cavity and nasal cancer; Parathyroid cancer; Penile cancer; Pheochromocytoma; Pineal and supratentorial primitive neuroectodermal tumors, pediatric; Pituitary tumor; Plasma cell neoplasms / multiple myeloma; Pleuropulmonary blastoma; Pregnancy and breast cancer; Pregnancy and Hodgkin lymphoma; Pregnancy and non-Hodgkin lymphoma; Primary central nervous system lymphoma; Primary liver cancer, adult; Primary liver cancer, pediatric; Prostate cancer; Rectal cancer; Renal cell (kidney) cancer; Renal cell cancer, pediatric; Renal pelvis and ureter, transitional cell cancer; Retinoblastoma; Rhabdomyosarcoma, pediatric; Salivary gland cancer; Salivary gland cancer, pediatric; Sarcoma, Ewing family tumor; Sarcoma, Kaposi; Sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone; Sarcoma, rhabdomyosarcoma, pediatric; Sarcoma, soft tissue, adult; Sarcoma, soft tissue, pediatric; Sézary syndrome; Skin cancer; Skin cancer, pediatric; Skin cancer (melanoma); Skin cancer, Merkel cell; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma, adult; Soft tissue sarcoma, pediatric; Squamous neck cancer of unknown primary, metastatic; Stomach cancer; Stomach cancer, pediatric; Supratentorial primitive neuroectodermal tumors, pediatric; T-cell lymphoma, cutaneous; Testicular cancer; Thymoma, pediatric; Thymoma, malignant; Thyroid cancer;Thyroid cancer, pediatric; Transitional cell carcinoma of the renal pelvis and ureter; Choriocarcinoma, gestational; Cancer of unknown primary site in children; Rare cancers in children; Transitional cell carcinoma of the ureter and renal pelvis; Urethral cancer; Uterine sarcoma; Vaginal cancer; Visual pathway and hypothalamic glioma, pediatric; Vulvar cancer; Waldenström macroglobulinemia; and Wilms tumor;

[0120] Tumors can be classified as malignant or benign. In either case, there is an abnormal collection and proliferation of cells. In the case of malignant tumors, these cells behave more actively and acquire the characteristic of increased invasiveness. Eventually, tumor cells break away from the microscopic environment in which they originated and spread to another area of the body (usually a very different environment that does not normally contribute to their growth), and may even gain the ability to continue rapid growth and division in this new location. This is called metastasis. When malignant cells have metastasized, it becomes more difficult to achieve a cure. Benign tumors tend to be less invasive and are less likely to metastasize.

[0121] The term "reducing a tumor" as used herein 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 proliferation status of cancer cells (the degree to which cancer cells are amplified), and the like.

[0122] The treatment 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, and tumors that have been previously treated with therapeutic therapies but remain resistant to the previous therapies. Preferably, the treatment 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), and advanced solid tumors, and tumors that have been previously treated with anticancer therapies but remain resistant to the previous therapies.

[0123] Complementary immunotherapy and other combination therapies The fusion protein of SEQ ID NO: 1 can be used as a monotherapy in the treatment regimen of the present invention, but in the context of the present invention, combinations of the fusion protein of SEQ ID NO: 1 with other anti-cancer treatments are also contemplated. Other therapeutic treatment regimens include other therapeutic immunotherapies, such as adoptive cell transfer regimens, antigen-specific vaccination, inhibition of DNA repair proteins (e.g., inhibitors of the nuclear enzyme poly(adenosine 5'-diphosphate-ribose) polymerase [“poly(ADP-ribose) polymerase” “PARP inhibitor”]), and blockers of immune checkpoint inhibitor molecules, such as cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and programmed death 1 (PD-1) antibodies.

[0124] Immune checkpoint proteins control 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 off or inhibit T cell function. Cancer cells utilize this system by driving high-level expression of immune checkpoint proteins on their surface, resulting in suppression of T cells expressing immune checkpoint proteins that enter the tumor microenvironment, thus suppressing the anti-cancer immune response. Accordingly, inhibition of immune checkpoint proteins by agents referred to herein as "immune checkpoint protein (ICP) inhibitors" can result in restoration of T cell function and the immune response 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 that 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. 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 combinations thereof. Preferably, the PD1 immune checkpoint inhibitor comprises one or more anti-PD-1 antibodies including nivolumab and pembrolizumab.

[0125] The methods of combination therapy described herein include administering at least one immune checkpoint inhibitor in combination with the 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 a target immune checkpoint protein when administered in an effective amount as a monotherapy or in combination with the fusion protein of SEQ ID NO: 1. In some cases, for example due to a synergistic effect, in the presence of SEQ ID NO: 1, minimal inhibition of the immune checkpoint protein by the immune checkpoint inhibitor may be sufficient. Many immune checkpoint inhibitors are known in the art.

[0126] Exemplary PD-1 / PD-L1-based immune checkpoint inhibitors include antibody-based therapeutic agents. Exemplary treatment methods that utilize PD-1 / PD-L1-based immune checkpoint inhibition are described in U.S. Patent Nos. 8,728,474 and 9,073,994, and European Patent No. 1537878, and include, for example, the use of anti-PD-1 antibodies. Exemplary anti-PD-1 antibodies are described, for example, 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), and 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, and BMS936559 (Bristol Myers Squibb Co.).

[0127] In certain embodiments, the methods or compositions described herein are administered in combination with a CTLA-4 inhibitor. In the CTLA-4 pathway, the interaction of CTLA-4 on T cells with its ligands (e.g., CD80, also known as B7-1, and CD86) on the surface of antigen-presenting cells (not cancer cells) causes 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) Publications Nos. 98 / 42752, 00 / 37504, and 01 / 14424, and European Patent No. 1212422. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.

[0128] Preferably, the methods or compositions of the invention are administered in combination with (i) a PD-1 or PD-L1 inhibitor, such as a PD-1 or PD-L1 inhibitor disclosed herein, and (ii) a CTLA-4 inhibitor, such as a CTLA-4 inhibitor disclosed herein.

[0129] Examples of FDA-approved immune checkpoint protein inhibitors include · ipilimumab (YERVOY®) · pembrolizumab (KEYTRUDA®) · atezolizumab (TECENTRIQ®) · durvalumab (IMFINZI®) · Avelumab (BAVENCIO®) · Nivolumab (OPDIVO®) may be mentioned.

[0130] A preferred treatment regimen of the present invention combines the fusion protein of SEQ ID NO: 1 administered according to the present invention with the immune checkpoint inhibitor pembrolizumab. Preferably, pembrolizumab is administered on the first day of each treatment cycle of the treatment regimen of the present invention. Preferably, 200 mg of pembrolizumab is administered once every 3 weeks or 21 days as a whole according to the manufacturer's recommendations.

[0131] The treatment regimen using the fusion protein of SEQ ID NO: 1 of the present invention may also be combined with other therapeutic agents and / or anticancer agents in addition to or instead of immune checkpoint inhibitors. Preferably, the therapeutic agent and / or anticancer agent is an antibody. Preferably, the therapeutic agent is a therapeutic protein. Preferably, the therapeutic agent is a small molecule. Preferably, the anticancer agent is an antigen. Preferably, the therapeutic agent is a population of cells. Preferably, the therapeutic agent is a therapeutic antibody. Preferably, the therapeutic agent is another cytotoxic agent and / or chemotherapeutic agent. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. "Chemotherapeutic agents" include chemical compounds useful for the treatment of cancer.

[0132] antibody Preferably, administration of SEQ ID NO: 1 may be in combination with a therapeutic antibody. Methods for making antibodies and antigen-binding fragments thereof are well known in the art and are disclosed, for example, in U.S. Patent No. 7,247,301, U.S. Patent Application Publication No. 2008 / 0138336, and U.S. Patent No. 7,923,221, all of which are hereby incorporated 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 therapeutic antibody recognized in the art that is approved for use, in clinical trials, or being developed for clinical use. In some embodiments, two or more therapeutic antibodies may be included in the combination therapy of the present invention.

[0133] Non-limiting examples of therapeutic antibodies include, but are not limited to, the following: · Trastuzumab (HERCEPTIN™ by Genentech, South San Francisco, Calif.) used to treat HER-2 / neu positive breast cancer 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 carcinoma, · Rituximab (RITUXAN™ by Genentech) used to treat non-Hodgkin 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, N.Y.) that 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 for treating colorectal cancer, head and neck cancer, and other potential cancer targets. · Tositumomab and tositumomab and iodine I for treating non-Hodgkin lymphoma that may be CD20-positive follicular non-Hodgkin lymphoma with or without transformation, where the disease is rituximab-resistant and has relapsed after chemotherapy. 131 (BEXXAR (trademark) by Corixa Corporation, Seattle, Wash.) · Inotuzumab ozogamicin, Y for treating lymphoma or non-Hodgkin lymphoma that can include relapsed follicular lymphoma, relapsed or resistant low-grade or follicular non-Hodgkin lymphoma, or transformed B-cell non-Hodgkin lymphoma. 111 Inotuzumab ozogamicin, Y 90 Inotuzumab ozogamicin, I 111 Inotuzumab ozogamicin, and Y 90 Inotuzumab ozogamicin (ZEVALIN (trademark) by Biogen Idec, Cambridge, Mass.) · EMD7200 (EMD Pharmaceuticals, Durham, N.C.) used for treating non-small cell lung cancer or cervical cancer. · SGN-30 (a genetically engineered monoclonal antibody targeting the CD30 antigen by Seattle Genetics, Bothell, Wash.) used for treating Hodgkin lymphoma or non-Hodgkin lymphoma. · SGN-15 (a genetically engineered monoclonal antibody targeting the Lewis y-related antigen conjugated to doxorubicin by Seattle Genetics) used for treating non-small cell lung cancer. · SGN-33 (a humanized antibody targeting the CD33 antigen by Seattle Genetics) used for treating acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). · SGN-40 (a humanized monoclonal antibody targeting the CD40 antigen by Seattle Genetics), used for treating multiple myeloma or non-Hodgkin lymphoma, · SGN-35 (a genetically engineered monoclonal antibody targeting the CD30 antigen conjugated with auristatin E by Seattle Genetics), used for treating non-Hodgkin lymphoma, · SGN-70 (a humanized antibody targeting the CD70 antigen by Seattle Genetics), used for treating renal cancer and nasopharyngeal cancer, · SGN-75 (a conjugate composed of the SGN70 antibody and an auristatin derivative by Seattle Genetics), and · SGN-17 / 19 (a fusion protein containing an antibody and an enzyme conjugated with melphalan prodrug by Seattle Genetics), used for treating melanoma or metastatic melanoma.

[0134] The therapeutic antibodies used in the methods of the present invention are not limited to those described herein. For example, the following approved therapeutic antibodies can also be used in the methods of the present invention: brentuximab vedotin (ADCETRIS™) for anaplastic large cell lymphoma and Hodgkin 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.

[0135] Antibodies for use in the present invention can also target molecules expressed by immune cells. For example, but not limited to these, tremelimumab (CP-675,206) and ipilimumab (MDX-010) target CTLA4 and have effects of tumor rejection, protection from re-challenge, and enhanced tumor-specific T cell responses; OX86 targets OX40, increases antigen-specific CD8+ T cells at the tumor site, and enhances tumor rejection; CT-011 targets PD1 and has effects of maintaining and expanding tumor-specific memory T cells, activating NK cells; BMS-663513 targets CD137 and causes regression of established tumors and increase and maintenance of CD8+ T cells; daclizumab (ZENAPAX™) targets CD25, causes transient depletion of CD4+CD25+FOXP3+ Tregs, enhances tumor shrinkage, and increases 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.

[0136] Preferably, the antibody is an inflammation-inducing and / or tumorigenesis-promoting cytokine-targeting antibody, including but not limited to anti-TNF antibodies, anti-IL-1Ra receptor-targeting antibodies, anti-IL-1 antibodies, anti-IL-6 receptor antibodies, and anti-IL-6 antibodies. Preferably, the antibody includes an antibody that targets inflammation-inducing helper T cell type 17 (TH17).

[0137] The therapeutic antibody may be a fragment of the antibody, a complex containing the antibody, or a conjugate containing the antibody. The antibody may optionally be a chimeric antibody, a humanized antibody, or a fully human antibody.

[0138] Therapeutic proteins and polypeptides Preferably, the method of the invention comprises administration of the fusion protein of SEQ ID NO: 1 according to the treatment regimen of the invention in combination with a therapeutic protein or peptide. Therapeutic proteins effective for treating cancer are well known in the art. Preferably, the therapeutic polypeptide or protein is a "suicide protein" that causes cell death either alone or in the presence of other compounds.

[0139] A representative example of such a suicide protein is herpes simplex virus thymidine kinase. Additional examples include varicella zoster virus thymidine kinase, the bacterial gene cytosine deaminase (which converts 5-fluorocytosine to the highly toxic compound 5-fluorouracil), p450 oxidoreductase, carboxypeptidase G2, beta-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, beta-lactamase, nitroreductase, carboxypeptidase A, linamarase (also referred to as beta-glucosidase), the E. coli gpt gene, and the E. coli Deo gene, among others known in the art. In some embodiments, the suicide protein converts a prodrug to a toxic compound.

[0140] As used herein, "prodrug" means any compound useful in the methods of the present invention that is toxic, i.e., can be converted to a toxic product, to tumor cells. The prodrug is converted to 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-iodouracil) for thymidine kinase; ifosfamide for oxidoreductase; 6-methoxypurine arabinoside for VZV-TK; 5-fluorocytosine for cytosine deaminase; doxorubicin for beta-glucuronidase; CB1954 and nitrofurazone for nitroreductase; and N-(cyanoacetyl)-L-phenylalanine or N-(3-chloropropionyl)-L-phenylalanine for carboxypeptidase A. Prodrugs can be readily administered by those skilled in the art. Those skilled in the art will be able to readily determine the most appropriate dosage and route for the administration of the prodrug.

[0141] Preferably, the therapeutic protein or polypeptide is a tumor suppressor, such as p53 or Rb, or a nucleic acid encoding such a protein or polypeptide. Those skilled in the art understand a wide variety of such tumor suppressors, as well as methods for obtaining them and / or the nucleic acids encoding them.

[0142] Other examples of anti-cancer / therapeutic proteins or polypeptides include apoptosis-promoting therapeutic proteins and polypeptides, such as p15, p16, or p21 WAF-1 are included.

[0143] Cytokines and nucleic acids encoding cytokines can also be used as therapeutic proteins and polypeptides. Examples include, but are not limited to, GM-CSF (granulocyte macrophage colony-stimulating factor); TNF-alpha (tumor necrosis factor alpha); interferons including IFN-alpha and IFN-gamma; and interleukins such as 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 (IL-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), interleukin-24 (IL-24), although other embodiments are known in the art.

[0144] Additional examples of cell-destroying genes include, but are not limited to, the mutated cyclin G1 gene. By way of example, the cell-destroying gene can be a dominant negative mutation of the cyclin G1 protein (e.g., WO 01 / 64870).

[0145] Vaccine Preferably, the treatment regimen of the present invention comprises administration of the fusion protein of SEQ ID NO: 1 in combination with administration of a cancer vaccine to stimulate a cancer-specific immune response, such as natural and acquired immune responses, 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 particular type of vaccine, the vaccine composition may include one or more suitable adjuvants known to enhance the immune response to the vaccine in the subject.

[0146] The vaccine may be made, for example, cell-based, i.e., using cells derived from the patient's own cancer cells to identify and obtain the antigen. Exemplary vaccines include tumor cell-based and dendritic cell-based vaccines, where activated immune cells from the subject are delivered back to the same subject together with other proteins to further facilitate the immunological activation of these immune cells primed by tumor antigens. Tumor cell-based vaccines include whole tumor cells and genetically modified tumor cells. Whole tumor cell vaccines may optionally be treated, for example, by irradiation of either the tumor cells or tumor lysates, to enhance antigen presentation. Vaccine administration may also be accompanied by an adjuvant such as Bacillus Calmette-Guerin (BCG) or keyhole limpet hemocyanin (KLH), depending on the type of vaccine used. Plasmid DNA vaccines may also be used and can be administered via direct injection or by a gene gun. Peptide vaccines, virus gene transfer vector vaccines, and antigen-modified dendritic cells (DCs) are also contemplated for use.

[0147] Preferably, the vaccine is a therapeutic cancer peptide-based vaccine. The peptide vaccine can be prepared using a known sequence or from an antigen isolated from the subject's own tumor, and can include neoantigens and modified antigens. Exemplary antigen-based vaccines include those in which the antigen is a tumor-specific antigen. For example, the tumor-specific antigen can be selected, inter alia, from cancer testis antigens, differentiation antigens, and widely expressed overexpressed tumor-associated antigens. Recombinant peptide vaccines based on peptides derived from tumor-associated antigens can be administered or formulated with an adjuvant or immunomodulatory agent when used in the present method. Exemplary antigens for use in peptide-based vaccines include, but are not limited to, the following list as it is intended to be purely exemplary. For example, the peptide vaccine can include cancer testis antigens encoded by genes that are normally silenced in adult tissues but transcriptionally reactivated in tumor cells, such as MAGE, BAGE, NY-ESO-1, and SSX-2. Alternatively, the peptide vaccine can include tissue differentiation-related antigens, i.e., antigens of normal tissue origin that are shared by both normal and tumor tissues. For example, the vaccine can include melanoma-associated antigens, such as gp100, Melan-A / Mart-1, MAGE-3, or tyrosinase, or prostate cancer antigens, such as PSA or PAP. The vaccine can include breast cancer-associated antigens, such as mammaglobin A. Other tumor antigens that can be included in the vaccine for use in the present method include, for example, CEA, MUC-1, HER1 / Nue, hTERT, ras, and B-raf. Other suitable antigens that can be used in the vaccine include SOX-2 and OCT-4 associated with cancer stem cells or the EMT process.

[0148] Antigen vaccines include multi-antigen and single-antigen vaccines. Exemplary cancer antigens can include peptides having about 5 to about 30 amino acids, or about 6 to 25 amino acids, or about 8 to 20 amino acids.

[0149] As described above, immune-stimulating adjuvants (different from RSLAIL-2) can be used to assist in generating an effective immune response in vaccines, particularly tumor-associated antigen-based vaccines. For example, the vaccine can incorporate pathogen-associated molecular patterns (PAMPs) that assist in enhancing immunity. Further suitable adjuvants include monophosphoryl lipid A or other lipopolysaccharides; toll-like receptor (TLR) agonists such as imiquimod, resiquimod (R-848), TLR3, IMO-8400, and lintatrimod. Further adjuvants suitable for use include heat shock proteins.

[0150] Viral or plasmid DNA vectors carrying expression cassettes are typically used for gene vaccines. When administered, these vectors transfect somatic cells or dendritic cells as part of an inflammatory response, thereby resulting in cross-priming or direct antigen presentation. Preferably, a gene vaccine is a vaccine that enables the delivery of multiple antigens in a single immunization. Gene vaccines include DNA vaccines, RNA vaccines, and virus-based vaccines.

[0151] A DNA vaccine for use in the present method is a bacterial plasmid constructed to deliver and express a tumor antigen. The DNA vaccine can be administered by any suitable method of administration, such as subcutaneous or intradermal injection, but can also be directly injected into lymph nodes. Further delivery methods include, for example, gene guns, electroporation, ultrasound, lasers, liposomes, microparticles, and nanoparticles.

[0152] Preferably, the vaccine comprises one or more 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 cancer cells. For example, the neoantigen vaccine may comprise a first vector comprising a nucleic acid construct encoding a plurality of immunogenic polypeptide fragments, each of which is a fragment of a protein mutated in cancer cells, where each immunogenic polypeptide fragment comprises one or more mutated amino acids adjacent to various numbers of wild-type amino acids from the original protein, and each polypeptide fragment is joined head-to-tail to form an immunogenic polypeptide. The respective lengths of the immunogenic polypeptide fragments forming the immunogenic polypeptide may differ.

[0153] Viral gene transfer vector vaccines can also be used, in which recombinant viruses, yeasts, bacteria, etc. are used to introduce cancer-specific proteins into the patient's immune cells. In methods based on vectors that may or may not be oncolytic, the vector can enhance the efficiency of the vaccine, for example, due to its intrinsic immunostimulatory properties. Exemplary virus-based vectors include those derived from the family Poxviridae, such as vaccinia, modified vaccinia strain Ankara, and fowlpox virus. Cancer vaccines containing a replicable vaccinia priming vector and a non-replicable fowlpox boosting vector, i.e., PROSTVAC, are also suitable for use. Each vector contains transgenes for PSA as well as three costimulatory molecules, namely CD80, CD54, and CD58, collectively referred to as TRICOM. Other suitable vector-based cancer vaccines include Trovax as well as TG4010 (encoding the MUC1 antigen and IL-2). Further vaccines for use include bacterial and yeast-based vaccines such as recombinant Listeria monocytogenes and Saccharomyces cerevisiae.

[0154] The foregoing vaccines can be combined with and / or formulated with adjuvants and other immune boosters to enhance their effectiveness. Depending on the particular vaccine, administration can be intratumoral or non-intratumoral (i.e., systemic).

[0155] Other cancer antigens that can be used in 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) fetal antigens of 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 material associated with cancer.

[0156] The cancer antigen can be an epithelial cancer antigen (e.g., breast, gastrointestinal, lung), 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, stomach cancer antigen, renal cell carcinoma antigen, pancreatic cancer antigen, liver cancer antigen, esophageal cancer antigen, head and neck cancer antigen, or colorectal cancer antigen.

[0157] In another embodiment, the cancer antigen is a lymphoma antigen (e.g., non-Hodgkin lymphoma or Hodgkin lymphoma), B-cell lymphoma cancer antigen, leukemia antigen, myeloma (i.e., multiple myeloma or plasmacytoma) antigen, acute lymphoblastic leukemia antigen, chronic myelogenous leukemia antigen, or acute myeloid leukemia antigen. The cancer antigens described are merely exemplary, and any cancer antigen can be targeted in the present invention.

[0158] Preferably, the cancer antigen is the mucin-1 protein or peptide (MUC-1) found in all human adenocarcinomas, including multiple myeloma and some B-cell lymphomas, such as pancreatic adenocarcinoma, colorectal adenocarcinoma, breast cancer, ovarian adenocarcinoma, lung adenocarcinoma, prostate adenocarcinoma, and head and neck adenocarcinoma. Patients with inflammatory bowel disease, whether Crohn's disease or ulcerative colitis, have an increased 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, the extracellular portion is exposed in a non-glycosylated form that is recognized by the immune system (Gendler et al., J Biol Chem 1990;265:15286-15293).

[0159] In another embodiment, the cancer antigen is a mutated B-Raf antigen associated with melanoma and colon cancer. The majority of these mutations show a single nucleotide change of T-A at nucleotide 1796, resulting in a change from valine to glutamic acid at residue 599 within the activation segment of B-Raf. The Raf protein is also indirectly associated with cancer as its oncogenic form is an effector of the activated Ras protein that is present in approximately one-third of all human cancers. Normal, non-mutated B-Raf is involved in cell signaling, transmitting signals from the cell membrane to the nucleus. This protein is typically active only when required to transmit signals. In contrast, mutant B-Raf is always active and has been reported to interfere with signal transmission (Mercer and Pritchard, Biochim Biophys Acta (2003) 1653(1):25-40; Sharkey et al., Cancer Res. (2004) 64(5):1595-1599).

[0160] Preferably, the cancer antigen is the human epidermal growth factor receptor-2 (HER-2 / neu) antigen. Cancers having cells that overexpress HER-2 / neu are referred to as HER-2 / neu + cancers. Exemplary HER-2 / neu +Examples of cancers include prostate cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, skin cancer, liver cancer (e.g., hepatocellular adenocarcinoma), intestinal cancer, and bladder cancer.

[0161] 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™ 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™ accession number AAA58637. Based on these sequences, one of ordinary skill in the art could develop a HER-2 / neu antigen using known assays to find appropriate epitopes that generate an effective immune response.

[0162] Exemplary HER-2 / neu antigens include p369-377 (HLA-A2 peptide from HER-2 / neu), 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, HER2[9 754 and the like.

[0163] Preferably, the cancer antigen is an epidermal growth factor receptor (EGFR) antigen. The EGFR antigen can be an EGFR variant 1 antigen, an EGFR variant 2 antigen, an EGFR variant 3 antigen, and / or an EGFR variant 4 antigen. A cancer having cells that overexpress EGFR is referred to as an 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.

[0164] Preferably, the cancer antigen is a vascular endothelial growth factor receptor (VEGFR) antigen. VEGFR is considered to be a regulator of cancer-induced angiogenesis. A cancer having cells that overexpress VEGFR is called a VEGFR + cancer. Exemplary VEGFR + cancers include breast cancer, lung cancer, small cell lung cancer, colon cancer, colorectal cancer, kidney cancer, leukemia, and lymphocytic leukemia.

[0165] Preferably, the cancer antigen is prostate specific antigen (PSA) and / or prostate specific membrane antigen (PSMA) that are widely expressed in androgen-independent prostate cancer.

[0166] Preferably, the cancer antigen is Gp-100. Glycoprotein 100 (gp100) is a tumor-specific antigen associated with melanoma.

[0167] Preferably, the cancer antigen is a carcinoembryonic (CEA) antigen. A cancer having cells that overexpress CEA is called a CEA + cancer. Exemplary CEA + cancers include colorectal cancer, gastric cancer, and pancreatic cancer. Exemplary CEA antigens include CAP-1 (i.e., CEA aa571-579), CAP1-6D, CAP-2 (i.e., CEA aa555-579), CAP-3 (i.e., CEA aa87-89), CAP-4 (CEA aa1-11), CAP-5 (i.e., CEA aa345-354), CAP-6 (i.e., CEA aa19-28), and CAP-7.

[0168] Preferably, the cancer antigen is carbohydrate antigen 10.9 (CA19.9). CA19.9 is an oligosaccharide related to the Lewis A blood group substance and is associated with colorectal cancer.

[0169] Preferably, the cancer antigen is a melanoma cancer antigen. Melanoma cancer antigens are 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 / Melan A, pMel17, pMel17 / gp100, gp100 (e.g., gp100 peptide 280-288, gp100 peptide 154-162, gp100 peptide 457-467), TRP-1, TRP-2, NY-ESO-1, p16, beta-catenin, mum-1, and the like.

[0170] 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. Mutations in the ras protein typically occur at position 12 (e.g., arginine or valine substituting glycine), position 13 (e.g., asparagine substituting glycine), position 61 (e.g., leucine from glutamine), and / or position 59. The mutant ras peptide can be useful as a lung cancer antigen, a gastrointestinal cancer antigen, a liver cancer antigen, a myeloid cancer antigen (e.g., acute leukemia, myelodysplasia), a skin cancer antigen (e.g., melanoma, basal cell, squamous cell), a bladder cancer antigen, a colon cancer antigen, a colorectal cancer antigen, and a renal cell cancer antigen.

[0171] 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 cancer antigen, a lymphoma cancer antigen, a prostate cancer antigen, a thyroid cancer antigen, a bladder cancer antigen, a pancreatic cancer antigen, and an ovarian cancer antigen.

[0172] A cancer antigen can be a cell, protein, peptide, fusion protein, DNA encoding a peptide or protein, RNA encoding a peptide or protein, glycoprotein, lipoprotein, phosphoprotein, carbohydrate, lipopolysaccharide, lipid, a chemically linked combination of two or more of them, a fusion of two or more of them, or a mixture of two or more of them, or a virus encoding two or more of them, or an oncolytic virus encoding two or more of them. 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.

[0173] Cell therapy Preferably, the method of the invention comprises administration of the fusion protein of SEQ ID NO: 1 in combination with administration of a therapeutic cell therapy. Cell therapies useful for treating cancer are well known and are disclosed, for example, in U.S. Patent No. 7,402,431. In a preferred embodiment, the cell therapy is a T cell transplant. In a preferred method, the T cells are expanded ex vivo with IL-2 prior to transplantation into the subject. Methods for cell therapy are disclosed, for example, in U.S. Patent No. 7,402,431, U.S. Patent Application Publication 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 Application Publication No. 2008 / 0279836.

[0174] Radiation therapy Preferably, the treatment regimen of the present invention comprises administration of the fusion protein of SEQ ID NO: 1 in further combination with radiation therapy. The term "radiation therapy" can be used interchangeably with the term "radiotherapy" and is a type of cancer treatment that uses a beam of powerful energy to kill cancer cells. Radiation therapy most commonly uses X-rays, but gamma rays, electron beams, or protons can also be used. The term "radiation therapy" most commonly refers to external beam radiation therapy. During this type of radiation, a high-energy beam is generated from a machine outside the patient's body that directs the beam at an exact point on the body. Each session is short and painless and lasts about 15 minutes. As used herein, the term "session" or "treatment session" refers to each radiotherapy treatment. A radiation therapy "regimen" or "schedule" typically consists of a specific number of treatments given over a period of time depending on the type and stage of the cancer.

[0175] Small molecule Preferably, the treatment regimen of the present invention comprises administration of the fusion protein of SEQ ID NO: 1 in combination with administration of an anti-cancer small molecule. Small molecules effective for 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 receptor, FGF receptor, EGF receptor, and PDGF receptor.

[0176] Many therapeutic small molecule RTKIs are known in the art, including but not limited to batatinib (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, and 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 for practicing the methods of the present invention. Such inhibitors can target, for example, HSP2, PRL, PTP1B, or Cdc25 phosphatase.

[0177] Small molecules that target Bcl-2 / Bcl-XL, such as those disclosed in US Patent Application Publication No. 2008 / 0058322, are also useful for practicing 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 induce immunogenic cell death, such as anthracyclines (Kepp et al., Cancer and Metastasis Reviews 2011;30:61-9), are thought to be well-suited for synergistic effects with PK extended IL-2.

[0178] Other cytotoxic and chemotherapeutic agents Preferably, the methods of the present invention include administration of the fusion protein of SEQ ID NO: 1 in combination with administration using chemotherapeutic agents including but not limited to alkylating agents, antitumor antibiotics, antimetabolites, other antitumor antibiotics, and plant-derived agents.

[0179] An alkylating agent is a drug that impairs cellular function by forming covalent bonds with amino, carboxyl, sulfhydryl, and phosphate groups in biologically important molecules. The most important alkylation sites are DNA, RNA, and proteins. Alkylating agents are active depending on cell proliferation but are not cell cycle specific. Suitable alkylating agents for use in the present invention include, but are not limited to, bis(chloroethyl)amine (nitrogen mustard, e.g., chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracil mustard), aziridine (e.g., thiotepa), alkyl alkane sulfonate (e.g., busulfan), nitrosoureas (e.g., BCNU, carmustine, lomustine, streptozocin), non-classical alkylating agents (e.g., altretamine, dacarbazine, and procarbazine), and platinum compounds (e.g., carboplatin, oxaliplatin, and cisplatin).

[0180] Antitumor antibiotics such as adriamycin intercalate into DNA in guanine-cytosine and guanine-thymine sequences, leading to the formation of oxygen free radicals that cause spontaneous oxidation and strand breakage. Other antibiotics suitable for use in the present invention include, but are not limited to, anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, and anthracenedione), mitomycin C, bleomycin, dactinomycin, and plicamycin.

[0181] Suitable antimetabolites 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, and gemcitabine.

[0182] Plant-derived agents include taxanes, which are semi-synthetic derivatives of precursors extracted from the needle-like leaves of yew plants. These drugs have a novel 14-membered ring taxane. Unlike vinca alkaloids that cause microtubule depolymerization, taxanes (e.g., paclitaxel) promote microtubule assembly and stability and thus arrest the mitotic cell cycle. Other plant-derived agents include, but are not limited to, vincristine, vinblastine, vindesine, vinzolidine, vinorelbine, etoposide, teniposide, and docetaxel.

[0183] Compositions for combination therapy Preferably, the fusion protein of SEQ ID NO: 1 is administered together (simultaneously or sequentially) with one or more additional therapeutic agents or other therapeutic agents, such as therapeutic antibodies. Preferably, the fusion protein of SEQ ID NO: 1 is administered before the administration of one or more therapeutic agents, such as therapeutic antibodies. Preferably, the fusion protein of SEQ ID NO: 1 is administered simultaneously with the administration of one or more therapeutic agents, such as therapeutic antibodies. Preferably, the fusion protein of SEQ ID NO: 1 is administered after the administration of one or more therapeutic agents, such as therapeutic antibodies. Preferably, SEQ ID NO: 1 and one or more therapeutic agents, such as therapeutic antibodies, are administered simultaneously. In other embodiments, SEQ ID NO: 1 and one or more therapeutic agents, such as therapeutic antibodies, are administered sequentially. Preferably, the fusion protein of SEQ ID NO: 1 and one or more therapeutic agents, such as therapeutic antibodies, are administered within 1, 2, or 3 days of each other.

[0184] One or more therapeutic agents, such as cytokines, chemotherapeutic agents, small molecules, antigens, or therapeutic antibodies, may be therapeutic agents useful as adjuvant therapy for cancer, are well known in the art, and have been discussed above. Further non-limiting examples of additional agents include GM-CSF (which increases monocyte and neutrophil populations), IL-7 (important for the generation and survival of memory T cells), 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, and anti-CD137, PARP inhibitors, antibodies. In some embodiments, the subject receives the fusion protein of SEQ ID NO: 1 and one or more therapeutic agents during the same prophylactic period, during the occurrence of the disorder, and / or during the treatment period.

[0185] Preferably, the present invention provides a separate pharmaceutical composition comprising the fusion protein of SEQ ID NO: 1 together with a pharmaceutically acceptable diluent, carrier, solubilizing agent, emulsifying agent, preservative, and / or adjuvant, and another pharmaceutical composition comprising one or more therapeutic agents, such as a therapeutic antibody, together with a pharmaceutically acceptable diluent, carrier, solubilizing agent, emulsifying agent, preservative, and / or adjuvant.

[0186] Preferably, the present invention provides a pharmaceutical composition comprising the fusion protein of SEQ ID NO: 1 and one or more therapeutic agents or anticancer agents in the same composition together with a pharmaceutically acceptable diluent, carrier, solubilizing agent, emulsifying agent, preservative, and / or adjuvant.

[0187] Kit Also provided is a kit formulated for SC administration, comprising the fusion protein of SEQ ID NO: 1 and optionally any other chemotherapeutic or anti-cancer agent. The kit is in the form of a physical structure that houses various components as described below and can be used, for example, to carry out the methods described above. The kit can include the fusion protein of SEQ ID NO: 1, which can be in the form of a pharmaceutical composition suitable for administration to a subject (e.g., provided in a sterile container). The pharmaceutical composition can be provided in a ready-to-use form or, for example, in a form that requires reconstitution or dilution prior to administration. If the composition is in a form that requires reconstitution by the user, the kit can also include a buffer, a pharmaceutically acceptable excipient, etc., packaged together with or separately from the fusion protein of SEQ ID NO: 1. For combination therapies (e.g., where a fusion protein of SEQ ID NO: 1 and an immune checkpoint inhibitor are contemplated), the kit can contain the several agents separately or they can already be combined in the kit. Similarly, if additional complementary therapies are required (e.g., a fusion protein of SEQ ID NO: 1, an immune checkpoint inhibitor, and an additional complementary therapy or agent), the kit can contain the several agents separately or two or more agents can already be combined in the kit.

[0188] The kits of the invention can be designed to meet the conditions (e.g., refrigeration or freezing) necessary to properly maintain the components contained therein. The kit can contain a label or package insert that includes identifying information for the components in the kit and instructions for use (e.g., dosing parameters, clinical pharmacology of the active ingredient, e.g., mechanism of action, pharmacokinetics and pharmacodynamics, adverse effects, contraindications, etc.).

[0189] Each component of the kit can be enclosed in an individual container, and all of the various containers can be housed in a single package. The label or document can include manufacturer information such as a lot number and an expiration date. The label or attached document may be integrated with, for example, the physical structure containing the components, separately contained in the physical structure, or attached to a component of the kit (e.g., an ampoule, syringe, or vial).

[0190] Examples of the label or document can further include a computer-readable medium such as a disk (e.g., a hard disk, card, memory disk), an optical disk such as a CD or DVD-ROM / RAM, DVD, MP3, magnetic tape, or an electronic storage medium such as RAM and ROM, or a hybrid thereof such as a magnetic / optical storage medium, FLASH media, or a memory-type card, or the label or document can be incorporated therein. In some embodiments, the actual instructions are not present in the kit, and means for obtaining the instructions from a remote source, e.g., via an Internet site, are provided.

[0191] Equivalents and Ranges When ranges are given, the endpoints are included. Further, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values expressed as ranges are to be considered to include any specific value or subrange within the stated range, to the tenth place of the unit of the lower limit of the range, in each different embodiment of the present invention, unless a different indication is clearly given in the context.

[0192] In addition, it should 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 are considered to be known to one of ordinary skill in the art and thus may be excluded even if the exclusion is not explicitly recited herein.

[0193] Any specific embodiment of the compositions, methods of making, or methods of using the present invention may be excluded from any one or more claims for any reason, whether or not related to the existence of prior art. EXAMPLES

[0194] Example 1 A Phase 1 study of a fusion protein of SEQ ID NO:1 administered intravenously as monotherapy and in combination with pembrolizumab to subjects with advanced solid tumors. The fusion protein of SEQ ID NO: 1 selectively activates the intermediate affinity IL-2R consisting of IL-2Rβ and IL-2Rγ, and induces cytotoxic CD8 + It is a fusion of circularly permuted IL-2 and IL-2 receptor alpha (IL-2Rα) designed to activate T cells and NK cells. The intermediate affinity IL-2R is expressed primarily on effector lymphocytes where it plays a key role in driving antitumor immune responses. Wild-type IL-2 binds IL-2Rα, β, and γ c It activates the high-affinity IL-2R and inhibits the immunosuppressive CD4 at concentrations below those at which effector cells with intermediate-affinity IL-2R are activated. + Controllability T(T reg Selective activation of the intermediate affinity IL-2R can enhance tumor killing and has been shown to have improved antitumor activity compared to IL-2 in mouse models.

[0195] method The fusion protein of SEQ ID NO: 1 is tested in a Phase 1 trial in human subjects having advanced refractory solid tumors. The fusion protein is supplied as a sterile, white to off-white, lyophilized powder for IV or SC administration. The excipients included in the fusion protein formulation are citric acid monohydrate, trisodium citrate dihydrate, sucrose, and polysorbate 20. For IV administration, sterile water for injection [United States Pharmacopeia (USP)] is supplied separately for reconstitution. A citrate buffer containing 1% polysorbate 20 (PS20 diluent) is supplied separately for dilution. A saline solution (0.9% sodium chloride injection, USP) is procured separately as needed for further dilution.

[0196] Four different doses (0.1, 0.3, 1, 3 μg / kg / day) of the fusion protein were administered to patients once daily for 5 consecutive days as a 30-minute intravenous infusion, and repeated in treatment cycles of 14 days (first cycle) or 21 days (subsequent cycles). The first part of the study was a dose escalation primarily aimed at investigating the safety and tolerability of the fusion protein and determining the maximum tolerated dose (MTD) and Phase 2 recommended Phase 2 dose (RP2D). The RP2D is equal to or less than the MTD and its associated dosing schedule, and is selected based on safety, PK, pharmacodynamics, and preliminary anti-tumor activity data observed during dose escalation. Figure 2 shows the treatment regimen followed in this study.

[0197] Four different doses (0.1, 0.3, 1, 3 μg / kg / day) of the fusion protein were administered once daily for 5 consecutive days as a 30-minute intravenous infusion, and repeated in treatment cycles of 14 days (first cycle) or 21 days (subsequent cycles). On Day 1 of the first cycle, pembrolizumab, an immune checkpoint inhibitor, was also administered at a dose of 200 mg. Figure 2 shows the treatment regimen followed in this study.

[0198] For details of the high doses (6 μg / kg / day and 8 μg / kg / day) administered as part of this clinical trial protocol, see below (see also Figures 11-15).

[0199] Grade 3 treatment-related adverse events Consistent with other cytokine therapies, fever and chills were the most frequent treatment-emergent adverse events associated with the fusion protein. No overt capillary leak syndrome has been observed. No grade 4 or 5 adverse events have been reported. At the 3 μg / kg dose level, one case each of grade 3 febrile neutropenia and grade 3 hypoalbuminemia met the protocol definition of dose-limiting toxicity (DLT). After discussion with the treating physicians, these were determined not to be DLTs, and the DLT definition was modified to allow continuation of dose escalation.

[0200] Elevated serum cytokine levels In response to treatment with the fusion protein, a dose-dependent increase in the levels of IL-6 and IFN-γ in serum was observed. IL-6 levels peaked at 4 hours after dosing and returned to baseline at 8 - 10 hours after dosing. Fever occurred concurrently with the peak of IL-6 and returned to baseline at 8 - 12 hours after dosing.

[0201] Conclusions from dosing at 0.1, 0.3, 1, 3 μg / kg / day. · A dose-proportional increase in fusion protein systemic exposure in patients treated with the fusion protein (Figure 3). · A dose-dependent increase in circulating NK cells and CD8 + T cells measured in the peripheral blood of patients (Figure 4). · An indeterminate and dose-independent increase in T reg (Figure 4). · A transient increase in serum IL-6 levels related to dose that occurred concurrently with the occurrence of the AE of chills and fever. · No evidence of capillary leak syndrome.

[0202] Administration at 6 μg / kg / day to cohort 5 Eleven patients with progressive solid tumors in cohort 5 of the dose-escalation trial received daily intravenous administration of the fusion protein at 6 μg / kg per day for 5 consecutive days, followed by a treatment hiatus, and this cycle was repeated. In cycle 1, the treatment hiatus was 9 days, so the cycle length was 14 days (2 weeks). For cycle 2 and subsequent cycles, the treatment hiatus was 16 days, so the cycle length was 21 days (3 weeks). In the first two treatment cycles, the fusion protein was administered to the subjects as inpatients in a medical facility where medical support measures and intensive care units were available as needed. If there was no dose-limiting toxicity (DLT), subsequent administration of the fusion protein was performed on the subjects as outpatients.

[0203] Conclusions from the 6 μg / kg / day dosing to cohort 5 · Dose-proportional increase in fusion protein systemic exposure in patients treated with the fusion protein (Figure 3). · Dose-dependent increase in circulating NK cells and CD8+ T cells measured in the peripheral blood of patients (Figure 4). · T reg An indeterminate and dose-independent increase (Figure 4). · Transient increase in serum IL-6 levels related to dose that occurred simultaneously with the occurrence of the AEs of chills and fever. · No evidence of cytokine release syndrome. · No evidence of capillary leak syndrome. · No patients showed dose-limiting toxicity with a 6 μg / kg / day dosing regimen equivalent to high-dose rhIL-2 administration (aldesleukin). · Considering the results from this 6 μg / kg / day dosing regimen, particularly the absence of dose-limiting toxicity, the maximum tolerated dose (MTD) is considered to be higher than the doses of the 8 μg / kg / day dosing regimen, 10 μg / kg / day dosing regimen, and even the 15 μg / kg / day dosing regimen.

[0204] Further results - Antitumor activity during dose escalation of monotherapy: Thirty-six patients were administered SEQ ID NO:1 at a maximum dose of 6 μg / kg / day, and the findings were the same as those reported above. The maximum tolerated dose of SEQ ID NO:1 had not yet been reached. Of the 27 patients who had evaluable scans, 14 (52%) had stable disease (Figure 7). One patient with a history of multiple prior pancreatic adenocarcinoma treatments had long-term stable disease and continued to receive 6 μg / kg / day of SEQ ID NO:1 monotherapy for over 6 months.

[0205] Antitumor activity during pembrolizumab combination therapy SEQ ID NO:1 was co-administered with pembrolizumab to 26 patients. Of the 26 patients, evaluable scans were obtained for 18 patients. Twelve of the 18 patients (67%) had stable disease or better throughout the treatment course (Figure 8).

[0206] One ovarian cancer patient showed a confirmed partial response. One triple-negative breast cancer patient showed a reduction of more than 50% in the size of the target lesion (Figure 9).

[0207] (Example 2) Peripheral blood lymphocyte responses in patients with renal cell carcinoma (RCC) treated with high-dose IL-2. Background Recombinant human interleukin-2 (rhIL-2, Aldesleukin) is approved and used for the treatment of metastatic melanoma and renal cell carcinoma. 1-8 However, the use of rhIL-2 is limited to patients with normal heart and lung function due to the associated capillary leak syndrome and resulting hypotension. 9-12 .

[0208] Despite the inadequate tolerance associated with rhIL-2 treatment, it remains one of the few treatment regimens for metastatic melanoma and renal cell carcinoma that result in complete and durable responses in a subset of patients, up to 12% in melanoma and 7% in renal cell carcinoma. 7,8rhIL-2 is hypothesized to preferentially activate immunosuppressive CD4+ Tregs and induce their expansion (13), and high doses of IL-2 are required to induce signaling in the receptor complexes expressed on potential tumor-killing CD8+ T cells and natural killer (NK) cells.

[0209] Published data indicate that immunosuppressive inducible T cell co-stimulator-positive (ICOS+) Treg cells were significantly expanded in a subset of melanoma patients who received high-dose IL-2 therapy. 14 However, data specifically quantifying and comparing the levels of expansion of cytotoxic effectors such as CD8+ T cells and NK cells relative to Treg cells are not readily available. This study was conducted primarily to evaluate the pharmacodynamic effects of high-dose IL-2 on the numbers of circulating CD8+ T cells, NK cells, and Treg cells.

[0210] Methods · This was a single-center open-label study. · Study site: Beth Israel Deaconess Medical Center, Boston, MA. · Study participants: A cohort of renal cell carcinoma patients receiving treatment with high-dose aldesleukin (IL-2). · The study was approved by the Beth Israel Deaconess Medical Center IRB, protocol #06-105. · Aldesleukin at a dose of 600,000 international units / kg was administered up to 14 times by 15-minute intravenous infusion every 8 hours (cycle 1). After a 9-day rest, this schedule was repeated up to 28 times as tolerated (cycle 2). · Whole blood samples for immunophenotyping by flow cytometry were collected for each patient at the following four time points: - Before the first dose of cycle 1 - Within 24 hours of the last dose of cycle 1 - Before the first dose of cycle 2 - Within 24 hours from the last administration of Cycle 2 · CD8+ T cells, NK cells, and Treg cells were quantified by flow cytometry. · Safety and antitumor activity were monitored throughout the study period. · Efficacy was clinically evaluated based on radiological reports, and the best efficacy was recorded.

[0211] Results Baseline demographic characteristics: · Ten patients with renal cell carcinoma were enrolled. · Median age 55 (range 39 - 62) · Male / Female 6 / 4 · ECOG PS of 0 = 9 / 1 = 1 · Median number of prior treatments 2 (range 1 - 3).

[0212] Number of administrations · Cycle 1: Median 11 (range 8 - 13) · Cycle 2: Median 6 (range 0 - 11) · Total (Cycle 1 + Cycle 2): Median 17 (range 11 - 23).

[0213] Best clinical efficacy · Partial response (PR): 5 · Mixed response: 1 · Progressive disease (PD): 4.

[0214] Pharmacodynamic response · Administration of high-dose IL-2 resulted in a robust increase in circulating Tregs, with the mean maximal increase being approximately 4-fold compared to an approximately 2-fold increase in all circulating CD8+ T cells and NK cells. · Little or no change was observed in the ratios of NK cells / Tregs and CD8+ T cells / Tregs in response to high-dose IL-2. · High inter-subject variability in the pharmacodynamic response was observed, and no clear correlation with clinical efficacy and number of administrations was found.

[0215] All of the treatment-emergent adverse events [Table 1] were consistent with the known adverse event profile of high-dose IL-2 15 .

[0216]

Table 1

[0217] Conclusion · The safety profile and clinical efficacy observed in this patient cohort were similar to the data previously published 15 . · In patients treated with high-dose IL-2, an increase in T that was more robust compared to CD8+ T cells and NK cells was observed, consistent with the known biological activity of IL-2 regs . · These results may be useful in evaluating potential differences in immune responses that may occur with novel cytokine therapeutics in the future

[0218] (References) 1. Rotte A, et al. Cancer Metastasis Rev 2015;34:115-128. 2. Fyfe G, et al. J. Clin. Oncol 1995;13:688-696. 3. Brayer J & Fishman M. J. Immunother 2014;37:187-191. 4. Clement JM & McDermott DF. Clin. Genitourin. Cancer 2009;7:E7-E9. 5. Shanafelt AB, et al. Nat. Biotechnol 2000;18:1197-1202. 6. Phan GQ, et al. J. Clin. Oncol. 2001;19:3477-3482. 7. Payne R, et al. J. Immunother. Cancer 2014;2:13. 8. McDermott DF, et al. J Clin. Oncol 2005;23:133-141. 9. McDermott DF & Atkins MB. Expert Opin. Biol. Ther. 2004;4:455-468. 10. Boyman O, Surh CD & Sprent J. Expert Opin. Biol. Ther. 2006;6:1323-1331. 11. Epstein AL, et al. J. Natl. Cancer Inst. 2003;95:741-749. 12. Nakagawa K, et al. Cancer Res. 1996;56:507-510. 13. Malek TR & Bayer AL. Nat. Rev. Immunol 2004;4:665-674. 14. Sim GC, et al. J. Clin. Invest. 2014;124(1):99-110. 15. Marabondo S and Kaufman HL. Expert Opin Drug Saf. 2017;16(12):1347-1357.

[0219] (Example 3) Comparison of the pharmacodynamic responses of SEQ ID NO: 1 to high-dose rhIL-2 For the purpose of comparing the maximum responses to treatment with SEQ ID NO: 1 and high-dose rhIL-2, the data from Examples 1 and 2 were combined. This comparison is shown in Table 2.

[0220] [Table 2]

[0221] Data also show that the increase in circulating NK cells and CD8+ cells compared to the increase in regulatory T cells (Tregs) is greater than the increase in circulating NK cells and CD8+ cells compared to the increase in regulatory T cells (Tregs) in patients treated with high-dose recombinant human IL-2 (rhIL-2). As shown in Table 2, administration of the fusion protein of SEQ ID NO: 1 to patients according to the treatment regimen of the present invention results in a nearly 2-fold increase in circulating immunosuppressive T regs compared to approximately 4-fold increase in circulating immunosuppressive T regs in patients treated with high-dose rhIL-2 therapy.

[0222] (Example 4) Comparison of the pharmacodynamic responses of SEQ ID NO: 1 to high-dose rhIL-2 in mice Treatment with rhIL-2 induced higher levels of systemic inflammation-promoting cytokines in mice. Serum cytokine production at 2, 4, 6, and 24 hours after administration on days 1 and 4 (n = 4 per time point) was assayed in female C57BL / 6 mice treated SC daily for 4 days with either rhIL-2 (20 μg, 50 μg, and 75 μg) or SEQ ID NO: 1 (8 μg, 20 μg, and 30 μg). The data in Figure 10 show cytokine production of IFNγ and IL-6. Elevated levels of TNFα and IL-6 are found in individuals with infections associated with inflammation such as those seen in sepsis or suffering from chronic inflammatory diseases such as rheumatoid arthritis. Antibodies that block the TNFα and IL-6 pathways are prescribed to treat rheumatoid arthritis, and anti-IL-6 drugs are used to mitigate the effects of inflammation that occur during certain types of cancer therapies such as chimeric antigen receptor (CAR) T cell therapy. Thus, the reduction in the induction of inflammation-promoting cytokines as observed in Figure 10 suggests that treatment with SEQ ID NO: 1 is better tolerated and potentially safer compared to treatment with rhIL-2.

[0223] (Example 5) Continuation of clinical trial, Phase 1 / 2, Part A, expansion dose to 8 μg / kg / day List of abbreviations:

[0224]

Table 3A

[0225]

Table 3B

[0226] Overall test design and plan This is an international, multi-site, open-label, sequential cohort Phase 1 / 2 trial, the same as the trial from which the data and information of Examples 1 and 2 were derived.

[0227] This trial has three parts: Part A, i.e., the escalating monotherapy part; Part B, i.e., the expanded monotherapy part; and Part C, i.e., the combination therapy part with pembrolizumab (see also Example 1). The overview of this trial is shown in Table 3 (Table 4).

[0228]

Table 4

[0229] In Part A of the trial, subjects with progressive solid tumors were given daily IV administration of SEQ ID NO: 1 for 5 days, followed by a treatment break period, and this cycle was repeated. In Cycle 1, the treatment break period was 9 days, so the cycle length was 14 days (2 weeks). The treatment break periods for Cycle 2 and subsequent cycles were 16 days, so the cycle length was 21 days (3 weeks). In the first two treatment cycles, subjects were given SEQ ID NO: 1 as inpatients in a medical facility where medical support measures and intensive care unit were available as needed. If there was no DLT, subsequent administrations of SEQ ID NO: 1 were given to subjects continuing the trial as outpatients.

[0230] In the dose escalation, the cohort of this trial used a standard 3+3 trial design in which the sequence number 1 of the dose levels shown in Table 3 (Table 4) was administered to 3 to 6 subjects per cohort. The starting dose of 0.1 μg / kg / day was selected based on the estimated minimum biological effect level. The doses of subsequent cohorts were increased according to Table 3 (Table 4) until stopped due to DLT or reaching the MTD. If the RP2D or MTD was not reached within the proposed dose range, further dose levels were considered.

[0231] In the dose escalation, for each cohort, over-enrollment of 4 to 7 subjects was allowed, and a 3+3 trial design was used to evaluate safety and tolerability, in which at least 3 evaluable subjects per cohort received intravenous administration of sequence number 1 at the specified dose and schedule. If none of the 3 subjects experienced DLT, enrollment at the next dose level was initiated. If 1 out of 3 subjects experienced DLT, 3 more subjects were enrolled at the same dose level. If no further DLT was observed, enrollment at the next dose level was initiated.

[0232] If 2 or more subjects experienced DLT at a certain dose level, no further dose escalation was performed. One or more lower dose levels may be tested to determine the MTD. The MTD is defined as the dose level immediately below the dose level at which 2 or more out of 6 evaluable subjects experienced DLT. Before dose escalation, a teleconference with a Safety Review Committee (SRC) including at least the treating investigators who enrolled the subjects and the medical monitors of the sponsor was held to review the safety data of the current cohort and determine whether dose escalation was justified.

[0233] Dose-limiting toxicity is defined by any of the following events described in Example 1, Table 1 (Table 1).

[0234] After the RP2D was determined as described in Example 1, the second part of the trial (i.e., Part B) was initiated. In this part of the trial, up to 41 subjects with melanoma and up to 41 subjects with RCC were enrolled to receive SEQ ID NO: 1 at the RP2D. Enrollment into these cohorts follows the Simon's two-stage design enrollment for partial response (uncertain). Assessment of response is based on the RECIST guidelines.

[0235] In the third part of the trial (Part C), subjects were administered SEQ ID NO: 1 in combination with pembrolizumab (see Example 1). Part C was conducted independently and concurrently with the monotherapies of Part A and Part B.

[0236] A run-in phase of 3 to 6 subjects was used to evaluate the safety of SEQ ID NO: 1 in combination with pembrolizumab. In the safety run-in phase, subjects were enrolled regardless of tumor type. Roll-over subjects (cohort 4) were deemed ineligible to participate in the safety run-in phase of Part C.

[0237] During the safety run-in phase, the first 3 subjects were administered SEQ ID NO: 1 at a dose level of 1 μg / kg / day (Example 1). As evaluated by the SRC, all 3 subjects tolerated the therapy in the first 21-day cycle, so the trial was advanced to a dose level of 3 μg / kg / day. As evaluated by the SRC, the first 6 subjects tolerated the therapy adequately in the first 21-day cycle, so expansion cohorts C1, C2, C3, and C4 were initiated.

[0238] As described in the incorporation criteria (Example 1), up to 20 subjects are registered in each of cohorts C1, C2, and C3 based on tumor type and treatment history with PD-1 / PD-L1 pathway inhibitors. Subjects with RCC or melanoma were ineligible for registration in cohorts C1, C2, or C3. Subjects who received the SEQ ID NO: 1 monotherapy in Part A or Part B and experienced disease progression after at least 2 cycles or SD after at least 4 cycles and were expected to tolerate treatment with combination therapy were eligible for treatment in Part C, cohort C4. Subjects who showed PR or CR with monotherapy were ineligible for rollover unless they subsequently exhibited disease progression.

[0239] Subjects were administered 200 mg of pembrolizumab every 3 weeks in combination with SEQ ID NO: 1 by daily IV administration for 5 consecutive days, followed by a 16-day treatment hiatus, and thus the cycle length was 21 days (3 weeks) per cycle (Example 1).

[0240] After determining the RP2D of the monotherapy as described in the example, registration in cohorts C5, C6, and C7 was initiated. Since it was shown that monotherapy doses of SEQ ID NO: 1 above 6 μg / kg / day were tolerated, the dose of SEQ ID NO: 1 in the combination arm was also increased. In cohorts C5, C6, and C7, up to 53 subjects with melanoma, up to 42 subjects with NSCLC, and up to 36 subjects with head and neck squamous cell carcinoma can be registered to receive combination administration of pembrolizumab and SEQ ID NO: 1 at the RP2D. Registration in these cohorts follows the Simon's 2-stage design registration for PR (uncertain). Efficacy evaluation is based on the RECIST and iRECIST guidelines. Considering that the RP2D was determined by Example 1 to be 6 μg / kg / day, dose escalation was considered for subjects in cohorts C1, C2, C3, and C4 assigned to dose levels of 1 μg / kg / day or 3 μg / kg / day who tolerated the combination therapy well.

[0241] Tumor Evaluation Antitumor activity was determined by measuring the baseline and the extent of the known disease approximately every 5 - 6 weeks after each even - numbered treatment cycle.

[0242] Appropriate radiological techniques (computed tomography scanning, magnetic resonance imaging, radionuclide imaging) were performed to evaluate the disease area. Superficial skin tumors were measured with calipers, photographed, and evaluated. In parts A, B, and C, determination of response was performed according to the standard RECIST and iRECIST criteria. According to the RECIST guidelines, tumors are evaluated as CR, PR, SD, or PD. According to the iRECIST guidelines, tumors are evaluated as immune - CR (iCR), immune - PR (iPR), immune - SD (iSD), or immune - PD (iPD). For the purposes of this study, this evaluation cannot be determined unless the subject meets the definition of SD / iSD for at least 12 weeks.

[0243] In trials using immunotherapeutic agents, it has been shown that CR, PR, or SD can occur after an increase in the tumor volume characterized as PD according to the RECIST criteria. Conventional response criteria such as RECIST may not fully evaluate the activity of immunotherapeutic agents. Since response to immunotherapy can occur after conventional PD, radiologically evaluated PD may not mean treatment failure. In the case of immunotherapy, the appearance of measurable antitumor activity may take longer than for cytotoxic therapy. When using immunotherapeutic agents, clinically insignificant PD, defined as small new lesions in the presence of other responsive lesions that can occur even if the subject is responsive to immunotherapy, should be tolerated. Stable disease may also represent antitumor activity in iRECIST. Therefore, RECIST and iRECIST were used to ensure a more comprehensive evaluation of the tumor response to SEQ ID NO: 1.

[0244] The ORR / iORR is the number of subjects presenting CR / iCR or PR or iPR divided by the number of subjects evaluable for antitumor activity. The duration of response was also determined. The ORR / iORR was calculated separately for subjects in the dose-escalation part (Part A) of the trial, the dose-expansion part (Part B) of the trial, and the combination therapy part (Part C) of the trial. Tumor images were collected and stored centrally. Using the centralized readings, scans starting from the second stage (N2) of the cohort in Part B and the C5, C6, and C7 cohorts in Part C can be evaluated. Antitumor activity is represented as follows: · ORR based on RECIST · iORR based on iRECIST · DCR by RECIST · iDCR by iRECIST · DOR by RECIST · iDOR by iRECIST · PFS by RECIST · Immune PFS (iPFS) by iRECIST · DRR by RECIST (Part B and Parts C5, C6, C7) · iDRR by iRECIST (Part B and Parts C5, C6, C7).

[0245] Evaluation of Pharmacokinetics, Pharmacodynamics, and Immunogenicity Pharmacokinetics Serum samples for PK evaluation of SEQ ID NO: 1 were obtained from each subject at predetermined time points. SEQ ID NO: 1 in human serum was quantified using a validated electrochemiluminescence method using the Meso Scale Discovery platform. Non-compartmental PK analysis was performed to estimate the PK parameters of SEQ ID NO: 1.

[0246] Immunogenicity Serum samples were obtained from each subject at a predetermined time point to evaluate the induction of antibodies against SEQ ID NO: 1. Anti-drug antibodies against SEQ ID NO: 1 in human serum were detected using a validated electrochemiluminescence method that uses the platform of Meso Scale Discovery. The evaluation of the induction of the immune response for each test subject was based on the comparison of the sample results before and after administration.

[0247] Pharmacodynamics and Biomarkers In blood and serum samples collected from all subjects of the study, the pharmacodynamic responses of various biomarkers were evaluated. Further biomarker analysis was performed on tumor tissue samples, which was optional for the test subjects.

[0248] Blood-Based Biomarkers The pharmacodynamic effects of SEQ ID NO: 1 were evaluated by measuring circulating CD8+ T cells, T regs , and NK cells in the peripheral blood of each subject at a predetermined time point by flow cytometry. Furthermore, serum samples were obtained from each subject at a predetermined time point. The concentrations of multiple pro-inflammatory cytokines, including interferon γ, tumor necrosis factor α, IL-1β, IL-6, and IL-10, were determined. Circulating tumor DNA (ctDNA) was also measured at a predetermined time point.

[0249] Tumor Tissue Biomarkers Tumor Biopsy Collections of fresh tumor samples by biopsy were collected at baseline from subjects who consented during the study. These samples were analyzed for markers of immune activation by immunohistochemistry and / or immunofluorescence. These were also used for gene expression analysis using methods such as NanoString. To demonstrate the pharmacological effects on the tumor microenvironment, a comparison of the results under treatment and at baseline was used. The analysis of the baseline tumor tissue was used for correlation analysis.

[0250] Overview of Statistical Methodology The statistical analysis methods are described below. Generally, for the variables evaluated, summary statistics (for continuous variables: n, mean, standard deviation, median, minimum, and maximum; for categorical variables: the number and proportion of subjects in each category) were provided. The data were summarized separately for Part A, Part B, and Part C. The baseline was defined separately for each part as the last value before the first administration of the test treatment.

[0251] Overall response rate The evaluation of ORR was based on the review by the study physician of the X-ray images or photographic images, as defined by RECIST 1.1. The overall response rate was defined as the proportion of subjects with objective evidence of CR or PR among the number of subjects evaluable for antitumor activity.

[0252] At the analysis stage, for each subject, the best ORR was assigned as the best response recorded after the start of the test treatment, taking into account all the requirements for confirmation. If applicable, responses recorded after disease progression or the start of a new anticancer treatment were excluded.

[0253] ORR was calculated separately for subjects in the dose-escalation part (Part A) of the study, the dose-expansion part (Part B) of the study, and the combination therapy part (Part C) of the study. The summary of ORR was presented by frequency, proportion, and 95% confidence interval (CI). The CI was obtained using an exact approach given the small sample size. The sum of the diameters of all lesions reported at each visit was graphed using a spider plot (% rate of change over time) and a waterfall plot (% best change). A swimmer plot was used to display the characteristics of the responses in the subjects.

[0254] Immune overall response rate Responses assigned using iRECIST have the prefix "i" (i.e., immune) to distinguish them from responses assigned using RECIST 1.1. The principles used to establish objective tumor responses are mostly the same as those of RECIST 1.1, but the main change in iRECIST is the concept of resetting the criteria if there is tumor shrinkage at the next evaluation following progression on RECIST 1.1. iRECIST defines iUPD (immunologically undefined progressive disease) based on the principles of RECIST 1.1. If the criteria for iUPD have never been met, then the principles of RECIST 1.1 are followed. However, if the criteria for iUPD are met, then the response at the next time point can be iUPD, iSD, iPR, or iCR, or iCPD (immunologically undefined progressive disease). In the case of iRECIST, the best overall response (iBOR) is the response at the best time point recorded between the start and end of the trial treatment, taking into account all the requirements for determination. The immune complete response rate was based on iBOR. iBOR was calculated separately for subjects in the dose escalation part (Part A) of the trial, the dose expansion part (Part B) of the trial, and the combination therapy part (Part C) of the trial. Spider plots, waterfall plots, and swarm plots were used to display the characteristics of responses in the subjects.

[0255] Disease Control Rate The disease control rate is defined as the proportion of subjects having objective evidence of CR, PR, or SD at cycle 4 or later. DCR was calculated separately for subjects in the dose escalation part (Part A) of the trial, the dose expansion part (Part B) of the trial, and the combination therapy part (Part C) of the trial. The summary of DCR was presented by frequency, proportion, and 95% CI. The CI was obtained using an exact approach given the small sample size.

[0256] Immune Disease Control Rate The immune disease control rate is defined as the proportion of subjects having objective evidence of iCR, iPR, or iSD in cycle 4 or later. iDCR was calculated separately for subjects in the dose escalation part (part A) of the trial, the dose expansion part (part B) of the trial, and the combination therapy part (part C) of the trial. The summary of iDCR was presented by frequency, proportion, and 95% CI. The CI was obtained using an exact approach given a small sample size.

[0257] Duration of response (parts B and C) The duration of response is defined as the time from the first record of response (CR or PR) to the first record of objective tumor progression or death regardless of cause. Subjects who are alive and without disease progression at the analysis cut-off date are censored at the time of the last evaluable tumor response assessment before starting a new anti-cancer treatment. Subjects with two or more consecutive missing response evaluations before a visit where death or progression was recorded are censored at the last tumor assessment date when the subject was recorded as without disease progression. Subjects who never achieve CR or PR before starting a new anti-cancer treatment at the lesion site are excluded from the analysis. The response rate was calculated for RECIST responders. In part B, DOR was calculated as follows (in weeks): (date of PD / death in part B - date of first response (CR or PR) in part B + 1) / 7. In part C, DOR was calculated as follows (in weeks): (date of PD / death in part C - date of first response (CR or PR) in part C + 1) / 7. The distribution of DOR for parts B and C was estimated using the Kaplan-Meier methodology. The median point of the estimated DOR, along with the two-sided 95% CI, was determined based on the anti-tumor evaluable population including subjects who experienced CR or PR. Kaplan-Meier curves were created.

[0258] Immune duration of response (parts B and C) The immune response duration is defined as the time from the first record of response (iCR or iPR) to the first record of objective tumor progression or death regardless of cause. Subjects who survived and had no disease progression on the analysis cut-off date were censored at the time of the last evaluable tumor response assessment before starting a new anti-cancer treatment. Subjects with two or more consecutive missing response evaluations before death or progression was recorded were censored at the last tumor assessment date when the subject was recorded as having no disease progression. Subjects who never achieved iCR or iPR before starting a new anti-cancer treatment at the lesion site were excluded from the analysis. The response rate was calculated for iRECIST responders. In Part B, iDOR was calculated as follows (in weeks): (date of iPD / death in Part B - date of first response (iCR or iPR) in Part B + 1) / 7. In Part C, iDOR was calculated as follows (in weeks): (date of iPD / death in Part C - date of first response (iCR or iPR) in Part C + 1) / 7. The distributions of iDOR in Part B and Part C were estimated using the Kaplan-Meier methodology. The median point of the estimated iDOR, along with the two-sided 95% CI, was determined based on the anti-tumor evaluable population including subjects who experienced iCR or iPR. Kaplan-Meier curves were created.

[0259] Durable Response Rate (Parts B and C) The Durable Response Rate is defined as the proportion of subjects in whom an objective response (complete or partial response according to RECIST 1.1) persists continuously for 6 months and starts at any time within 12 months from the start of the investigational drug. DRR was summarized by each tumor type. The summary of DRR was presented by frequency, proportion, and 95% CI. The CI was obtained using an exact approach given the small sample size.

[0260] Immune durability response rate (iDRR) is defined as the proportion of subjects in whom objective response (complete or partial response per iRECIST) persists continuously for 6 months and begins at any time within 12 months from the start of the investigational drug. iDRR was summarized by each tumor type. The summary of iDRR was presented by frequency, proportion, and 95% CI. CI was obtained using an exact approach given the small sample size.

[0261] Progression-free Survival (Parts B and C) Progression-free survival is defined as the time from the first administration of SEQ ID NO: 1 until the first record of objective tumor progression or death regardless of cause. Subjects in whom disease progression was not observed or who did not die were censored at the time when the subject's progression-free status was last confirmed. If a subject started a new anti-cancer treatment (regardless of systemic or local) before the record of progression or death, or if the subject was excluded from the study due to clinically documented disease progression without a record, the subject was censored at the last assessment when the subject was recorded as progression-free before the intervention. Subjects with two or more consecutive missing response evaluations before the visit (or death) at which progression was recorded were censored at the last tumor assessment date when the subject was recorded as progression-free. In Part B, PFS was calculated as follows (in weeks): (date of PD / death in Part B - date of first administration in Part B + 1) / 7. In Part C, PFS was calculated as follows (in weeks): (date of PD / death in Part C - date of first administration in Part C + 1) / 7. The survival distribution of PFS was estimated using the Kaplan-Meier methodology. Based on the anti-tumor evaluable population, the median PFS was determined together with the two-sided 95% CI. Additionally, Kaplan-Meier curves were created. The 6-month and 1-year PFS rates were estimated using the Kaplan-Meier estimates.

[0262] Immune progression-free survival (Parts B and C) The immune progression-free survival period is defined as the time from the first administration to the first record of objective tumor progression or death regardless of cause. Subjects without evidence of disease progression or death were censored at the time when the subject's progression-free status was last confirmed. If a subject started a new anti-cancer treatment (regardless of systemic or local) before the record of progression or death, or if the subject was excluded from the study due to unrecorded clinical disease progression, the subject was censored at the last assessment when the subject was recorded as progression-free before the intervention. Subjects with two or more consecutive missing response evaluations before the visit (or death) with recorded progression were censored at the last tumor assessment date when the subject was recorded as progression-free. In Part B, iPFS was calculated as follows (in weeks): (Date of iPD / death in Part B - Date of first administration in Part B + 1) / 7. In Part C, PFS was calculated as follows (in weeks): (Date of iPD / death in Part C - Date of first administration in Part C + 1) / 7. The distribution of the iPFS survival period was estimated using the Kaplan-Meier methodology. Based on the anti-tumor evaluable population, the iPFS median was determined together with the two-sided 95% CI. Furthermore, Kaplan-Meier curves were created. The 6-month and 1-year iPFS rates were estimated using the Kaplan-Meier estimates.

[0263] Pharmacokinetic analysis Descriptive statistics were used to present and summarize individual serum concentrations and concentration-time data both in graphical and tabular form. Descriptive statistics were used to summarize pharmacokinetic parameters. A subject list of individual PK concentrations was created. Concentration data were summarized according to the nominal (as defined in the protocol) sampling times. Pharmacokinetic parameters were calculated by a non-compartmental analysis method using Phoenix WinNonlin Professional (version 6.1 or later, Pharsight Corporation). The actual elapsed time since administration was used to estimate individual serum PK parameters. Dose proportionality and further PK analysis were performed as needed.

[0264] Pharmacodynamic analysis Pharmacodynamic data were descriptively summarized. If possible, the relationship between the serum PK parameters or concentrations of SEQ ID NO:1 and the pharmacodynamic response was evaluated by correlation analysis or visual inspection.

[0265] Immunogenicity analysis The presence of antibodies against SEQ ID NO:1 was determined and the data were summarized by cohort / dose level.

[0266] Tissue biomarker analysis Baseline values, post-treatment values, and changes in the density of TIL, the ratio of cytotoxic TIL, immunosuppressive TIL, and the density of signals of immune cell-mediated killing were summarized. The correlation between anti-tumor efficacy endpoints (best overall response, progression-free survival) derived from tumor tissue and the baseline status (or value) of the endpoints was estimated. The efficacy endpoints were summarized separately based on the baseline status, or low and high values. The correlation between anti-tumor efficacy endpoints (best overall response, progression-free survival) derived from tumor tissue and the change from the baseline of the endpoints after treatment was estimated.

[0267] Partial and complete responses in patients administered 6 μg / kg dose of SEQ ID NO:1 Initial data from the ongoing Phase 1 / 2 trial showed a partial response in a urethral melanoma patient in the dose escalation cohort of Part B monotherapy receiving 6 μg / kg of SEQ ID NO:1. Prior to participating in the trial, the patient had been treated with nivolumab adjuvant therapy for 1 year following surgical resection of urethral melanoma. During the trial, the patient showed a reduction in serum lactate dehydrogenase (LDH) between Cycle 1 and Cycle 8 of treatment. During the trial, the patient had the following benefits in each cycle of treatment as follows: Cycle 2 - stable disease (SD); 8% increase from baseline in target lesions; Cycle 4 - SD; 17% reduction from baseline in target lesions; Cycle 6 - partial response (PR), 32% reduction from baseline in lesions; Cycle 8 - confirmation of PR by RECIST, 35% reduction from baseline in target lesions.

[0268] Partial and complete responses in patients administered SEQ ID NO: 1 at a dose of 3 μg / kg in combination with pembrolizumab Initial data from an ongoing Phase 1 / 2 trial in patients receiving combination administration of SEQ ID NO: 1 at a dose of 3 μg / kg and pembrolizumab showed at least 1 complete response (CR) and several partial responses (PR) in patients with types of cancer including ovary (CR); ovary (PR); ovary (PR) esophagus (PR); and TNBC (iPR per iRECIST).

[0269] Data from the expanded dose of cohort 6 at 8 μg / kg / day Pharmacokinetics of SEQ ID NO: 1 after IV administration As detailed in Example 1, the RP2D was determined to be a once-daily dose of 6 μg / kg. In accordance with the study design, a new cohort 6 study enrollment was initiated to test at a once-daily dose of 8 μg / kg. Two patients were enrolled and up to 4 additional patients were expected to be enrolled. The following is data obtained from the first 2 patients enrolled.

[0270] The serum concentration-time profile of SEQ ID NO: 1 after the first IV administration (day 1 of cycle 1) of SEQ ID NO: 1 is shown in Figure 11. The mean peak (C max ) and total serum exposure (AUC) of SEQ ID NO: 1 over the dose range evaluated in Part A of Example 1 are shown in Figure 12.

[0271] After the first IV administration of SEQ ID NO: 1, the serum concentration of SEQ ID NO: 1 reached a peak at the end of the 30-minute (0.5-hour) infusion and then decreased exponentially. Systemic exposure to SEQ ID NO: 1 (C max and AUC last ) increased with increasing dose. The increase in C max was approximately dose-proportional over the dose range of 0.1 μg / kg to 8 μg / kg. The increase in AUC last exceeded dose-proportionality in the dose range of 0.1 μg / kg to 3 μg / kg but was approximately dose-proportional from 3 μg / kg to 8 μg / kg.

[0272] A dose of 6 μg / kg was selected as the Phase 2 recommended dose for intravenous administration of SEQ ID NO: 1 (Example 1). Enrollment at 8 μg / kg is ongoing, and two patients are being treated at this dose level.

[0273] Pharmacodynamic effects after intravenous administration of SEQ ID NO: 1 Total NK cells, total CD8 in peripheral blood after the first 2 cycles of treatment with SEQ ID NO: 1 intravenously + T cells, and regulatory T cells (T reg ) The time course of the cell populations is shown in Figure 13. Across the dose range evaluated during the Phase, the fold change from baseline (FCB) of total NK cells, total CD8 + T cells, and T reg at cycle 1 day 8 (C1D8) and cycle 2 day 8 (C2D8) of treatment with SEQ ID NO: 1 intravenously is shown in Figure 14.

[0274] SEQ ID NO: 1 induced a dose-dependent increase in circulating NK cells and CD8 + T cells, and a very slight dose-independent effect on T reg cells, and dose levels of 6 and 8 μg / kg most robustly increased NK cells and CD8 reg T cells without a significant change in the T + profile.

[0275] Furthermore, when evaluating the serum cytokine concentrations of patients treated with various dose levels of SEQ ID NO: 1, a transient increase in the serum concentrations of interferon gamma (IFNγ) and IL-6 was observed in patients administered higher doses (>1 μg / kg) of SEQ ID NO: 1 (Figure 15). Interestingly, the peak of the IL-6 response was observed at a dose level of 3 μg / kg, and the serum IL-6 levels decreased at doses higher than 3 μg / kg such as 6 and 8 μg / kg.

[0276] IL-6 is a pro-inflammatory cytokine that is not thought to contribute to the anti-tumor response but is thought to be associated with the side effects of treatments such as general inflammation and fever. Elevated levels of IL-6 in serum and tumor sites have been demonstrated in several types of cancer. Usually, this elevation is accompanied by poor prognosis and decreased survival rate. Downregulation of IL-6 has been correlated with better efficacy against cancer treatment.

[0277] IFNγ is a very important marker of cytotoxic effector cell function and CD8 + T cells and NK cells produce IFNγ when activated. IFNγ is associated with the anti-tumor immune response, and significant increases were observed at the dose levels of 3, 6, and 8 μg / kg of SEQ ID NO: 1. Notably, the increase in IFNγ was observed at the dose levels of 6 and 8 μg / kg, which were significantly lower than the peak level observed at 3 μg / kg of the IL-6 level. The IFNγ level observed after treatment with SEQ ID NO: 1 at 8 μg / kg was approximately 4-5 times higher than those observed at 3 and 6 μg / kg. This suggests that the dose levels of 6 and 8 μg / kg, and potentially higher dose levels of 10, 12, 15 μg / kg or more, may have certain advantages in terms of anti-tumor immune activity against inflammatory activity compared to the lower dose levels of SEQ ID NO: 1.

[0278] The patents and scientific documents referred to herein establish the knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are hereby incorporated by reference into this specification. All published foreign patents and patent applications cited herein are hereby incorporated by reference into this specification. All other published references, documents, manuscripts, and scientific documents cited herein are hereby incorporated by reference into this specification.

[0279] The present invention has been shown and described in detail with reference to preferred embodiments thereof. However, it will be understood by those skilled in the art that various changes in form and detail can be made without departing from the scope of the present invention as defined in the appended claims. It should also be understood that the embodiments described herein are not mutually exclusive and that the features of various embodiments can be wholly or partly combined in accordance with the present invention.

Claims

**Claim 1** A method for treating cancer in a patient, the method comprising administering to the patient a fusion protein of SEQ ID NO: 1 at a dose of at least about 6 μg / kg / day to about 15 μg / kg / day.

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