WO2013044169A1

WO2013044169A1 - Methods for determining combination therapy with il-2 for the treatment of cancer

Info

Publication number: WO2013044169A1
Application number: PCT/US2012/056753
Authority: WO
Inventors: Hua Gong
Original assignee: Nestec SA
Current assignee: Nestec SA
Priority date: 2011-09-21
Filing date: 2012-09-21
Publication date: 2013-03-28
Anticipated expiration: 2014-03-21

Abstract

The present invention provides methods for determining a combination of IL-2 therapy such as Proleukin® therapy with one or more targeted therapies for the treatment of a cancer such as metastatic melanoma or renal cell carcinoma. With the present invention, it is possible to design individualized treatment strategies for cancers by combining IL-2 therapy such as Proleukin® therapy with one or more targeted therapies such as BRAF, MEK, HH, PI3K/AKT and/or receptor tyrosine kinase inhibitors.

Description

METHODS FOR DETERMINING COMBINATION THERAPY WITH IL-2 FOR THE TREATMENT OF CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 61/537,502, filed September 21, 2011, the disclosure of which is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[0002] Renal cell carcinoma (RCC) is the most common type of kidney cancer found in adults. It is estimated that over 200,000 new cases of RCC are diagnosed each year, resulting in over 100,000 deaths annually (Parkin DM et al. , CA Cancer J Clin. (2005) Mar-

Apr;55(2):74-108). In the US alone, over 50,000 new cases of RCC were diagnosed and more than 13,000 deaths were attributed to RCC in 2008. Unfortunately, the use of traditional radiation and chemotherapeutic regimes, which are effective for the treatment of many types of cancers, generally result in poor outcomes when used to treat RCC. As such, RCC therapy has been limited to surgical removal of localized, non-metastatic RCC tumors, immunotherapy (such as with IL-2 and IFN- ), and more recently with targeted therapies including anti-receptor kinase inhibitors, anti-growth factor inhibitors, and anti-mTOR inhibitors.

[0003] To date, IL-2 and IFN-a based immunotherapies remain the only treatment regimens that have been shown to result in increases in the overall survival of certain patients. IL-2 immunotherapy results in durable tumor remission (>10 years of tumor-free survival) in only 5-10% of patients treated. However, given the small rate of durable remission and the considerable toxicities and expenses associated with this immunotherapy, IL-2 therapy is not regarded as standard therapy for RCC. Instead, IL-2 immunotherapy is only administered to a select number of RCC patients.

[0004] Similarly, metastatic melanoma is a disease with a long-term remission rate of less than 10%. As with renal cell carcinoma, high dose IL-2 immunotherapy results in durable remission in only a small number of patients, making IL-2 immunotherapy impractical as a standard treatment. [0005] Thus, there is a need in the art for improved methods of treating melanoma or RCC (e.g., metastatic or non-metastatic) using combination IL-2 therapy. The present invention satisfies this need and provides related advantages as well.

BRIEF SUMMARY OF THE INVENTION

[0006] The present invention provides methods for determining a combination of IL-2 therapy (e.g., Proleukin^®) with one or more targeted therapies for the treatment of a cancer such as RCC or melanoma. With the present invention, it is possible to design individualized treatment strategies by combining IL-2 therapy (e.g., Proleukin^®) with one or more targeted therapies such as, e.g., BRAF, MEK, PI3K/AKT, receptor tyrosine kinase, and/or HH inhibitors. In some embodiments, the present invention enables the rational design of therapy with IL-2 (e.g., Proleukin^®) in combination with one or more targeted therapies for the treatment of a cancer such as RCC or melanoma. The present invention also provides methods for administering a treatment regimen of IL-2 therapy for individuals with or suspected of having RCC or melanoma. [0007] As such, in one aspect, the present invention provides a method for determining a combination of IL-2 therapy with one or more targeted therapeutic agents for treating cancer in a subject, the method comprising:

(a) determining whether a cancer cell obtained from the subject is positive or negative for MART-1 expression; and

(b) selecting an appropriate therapy for the subject comprising:

(i) a combination of an IL-2 polypeptide and a MEK inhibitor and/or a PI3K/AKT inhibitor if the cell is positive for MART-1 expression; or

(ii) a combination of an IL-2 polypeptide and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor if the cell is negative for MART-1 expression.

[0008] In some embodiments, the cancer is melanoma or metastatic melanoma. In some embodiments, the cancer is renal cell carcinoma or metastatic renal cell carcinoma. In some embodiments, the cancer cell is isolated from a sample (e.g., rumor tissue or whole blood) taken from the subject. [0009] In some embodiments, the MEK inhibitor is selected from the group consisting of PD325901, AZD 6244, trametinib, MEK 162, XL518, and combinations thereof.

[0010] In some embodiments, the PI3K/AKT inhibitor is selected from the group consisting of BEZ235, BKM120, BLY719, IPI-145, and combinations thereof. [0011] In some embodiments, the BRAF inhibitor is selected from the group consisting of PLX-4032, dabrafenib, RAF265, LGX818, and combinations thereof.

[0012] In some embodiments, the receptor tyrosine kinase inhibitor is selected from the group consisting of lapatinib, sunitinib, sorafenib, axitinib, pazopanib, gefitinib, erlotinib, pilitinib, canertinib, CP-654577, CP-724714, ARRY-334543, JNJ-26483327, JNJ-26483327, MP-470, AZD8931, and combinations thereof.

[0013] In some embodiments, the Hedgehog inhibitor is selected from the group consisting of GDC0449, erismodegib, LEQ506, saridegib, IPI-269609, BMS-833932, PF-04449913, TAK-441, and combinations thereof.

[0014] In some embodiments, the IL-2 polypeptide comprises a recombinant IL-2 polypeptide. In some embodiments, the recombinant IL-2 polypeptide is PROLEUKIN^® (aldesleukin). In some embodiments, the IL-2 polypeptide is high dose IL-2. In other embodiments, the IL-2 polypeptide is low dose IL-2. In other embodiments, the IL-2 polypeptide is a subcutaneous IL-2.

[0015] In some embodiments, the MART-1 expression is selected from the group consisting of protein expression, RNA expression, and combinations thereof.

[0016] In another aspect, the present invention provides a method for recommending a combination of IL-2 therapy with one or more targeted therapeutic agents for treating cancer in a subject, the method comprising:

(b) recommending an appropriate therapy for the subject comprising:

[0017] In some embodiments, the method further comprises comparing the subject's symptom profile to that from a control subject.

[0018] In some embodiments, the cancer is melanoma or metastatic melanoma. In some embodiments, the cancer is renal cell carcinoma or metastatic renal cell carcinoma. In some embodiments, the cancer cell is isolated from a sample (e.g., tumor tissue or whole blood) taken from the subject.

[0019] In some embodiments, the MEK inhibitor is selected from the group consisting of PD325901, AZD 6244, trametinib, MEK 162, XL518, and combinations thereof. [0020] In some embodiments, the PI3K/AKT inhibitor is selected from the group consisting of BEZ235, BKM120, BLY719, IPI-145, and combinations thereof.

[0021] In some embodiments, the BRAF inhibitor is selected from the group consisting of PLX-4032, dabrafenib, RAF265, LGX818, and combinations thereof.

[0022] In some embodiments, the receptor tyrosine kinase inhibitor (TKI) is selected from the group consisting of lapatinib, sunitinib, sorafenib, axitinib, pazopanib, gefitinib, erlotinib, pilitinib, canertinib, CP-654577, CP-724714, ARRY-334543, JNJ-26483327, JNJ-26483327, MP-470, AZD8931, and combinations thereof.

[0023] In some embodiments, the Hedgehog inhibitor is selected from the group consisting of GDC0449, erismodegib, LEQ506, saridegib, IPI-269609, BMS-833932, PF-04449913, TAK-441 , and combinations thereof.

[0024] In some embodiments, the IL-2 polypeptide comprises a recombinant IL-2 polypeptide. In some embodiments, the recombinant IL-2 polypeptide is PROLEUKIN^® (aldesleukin). In some embodiments, the IL-2 polypeptide is high dose IL-2. In other embodiments, the IL-2 polypeptide is low dose IL-2. In other embodiments, the IL-2 polypeptide is a subcutaneous IL-2.

[0025] In some embodiments, the MART-1 expression is selected from the group consisting of protein expression, RNA expression, and combinations thereof.

[0026] In another aspect, the present invention provides a method for administering an appropriate therapy for treating cancer in a subject, the method comprising:

(b) administering an appropriate therapy to the subject comprising:

(ii) a combination of an IL-2 polypeptide and a BRAF inhibitor, a

PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor if the cell is negative for MART-1 expression. [0027] In some embodiments, the method further comprises comparing the subject's symptom profile to that from a control subject.

[0028] In some embodiments, the cancer is melanoma or metastatic melanoma. In some embodiments, the cancer is renal cell carcinoma or metastatic renal cell carcinoma. In some embodiments, the cancer cell is isolated from a sample (e.g., tumor tissue or whole blood) taken from the subject.

[0029] In some embodiments, the MEK inhibitor is selected from the group consisting of PD325901, AZD 6244, trametinib, MEK162, XL518, and combinations thereof.

[0030] In some embodiments, the PI3K/AKT inhibitor is selected from the group consisting of BEZ235, BKM120, BLY719, IPI-145, and combinations thereof.

[0031] In some embodiments, the BRAF inhibitor is selected from the group consisting of PLX-4032, dabrafenib, RAF265, LGX818, and combinations thereof.

[0032] In some embodiments, the receptor tyrosine kinase inhibitor is selected from the group consisting of lapatinib, sunitinib, sorafenib, axitinib, pazopanib, gefitinib, erlotinib, pilitinib, canertinib, CP-654577, CP-724714, ARRY-334543, JNJ-26483327, JNJ-26483327, MP-470, AZD8931, and combinations thereof.

[0033] In some embodiments, the Hedgehog inhibitor is selected from the group consisting of GDC0449, erismodegib, LEQ506, saridegib, IPI-269609, BMS-833932, PF-04449913, TAK-441, and combinations thereof. [0034] In some embodiments, the IL-2 polypeptide comprises a recombinant IL-2 polypeptide. In some embodiments, the recombinant IL-2 polypeptide is PROLEUKIN® (aldesleukin). In some embodiments, the IL-2 polypeptide is high dose IL-2. In other embodiments, the IL-2 polypeptide is low dose IL-2. In other embodiments, the IL-2 polypeptide is a subcutaneous IL-2. [0035] In some embodiments, the MART-1 expression is selected from the group consisting of protein expression, RNA expression, and combinations thereof.

[0036] In another aspect, the present invention provides a method for treating cancer in a subject using a combination of IL-2 therapy with one or more targeted therapeutic agents after determining MART-1 expression in a cancer cell, the method comprising:

(a) administering a combination of an IL-2 polypeptide and a MEK inhibitor and/or a PI3K/AKT inhibitor if the cell is positive for MART-1 expression; or (b) administering a combination of an IL-2 polypeptide and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor if the cell is negative for MART-1 expression, to effectively treat cancer.

[0037] In another aspect, the present invention provides a method for combining IL-2 therapy with one or more targeted therapeutic agents after determining MART-1 expression in a cancer cell, the method comprising:

(a) administering a combination of an IL-2 polypeptide and a MEK inhibitor and/or a PI3K/AKT inhibitor if the cell is positive for MART-1 expression; or

(b) administering a combination of an IL-2 polypeptide and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor if the cell is negative for MART-1 expression.

[0038] In another aspect, the present invention provides a method for treating cancer in a subject using a combination of IL-2 therapy with one or more targeted therapeutic agents based on MART-1 expression in a cancer cell, the method comprising:

(b) administering a combination of an IL-2 polypeptide and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor if the cell is negative for MART-1 expression, to effectively treat cancer. [0039] In another aspect, the present invention provides a method for combining IL-2 therapy with one or more targeted therapeutic agents based on MART-1 expression in a cancer cell, the method comprising:

[0040] In some aspects, the methods of the invention provide information useful for guiding treatment decisions for patients receiving or about to receive combination IL-2 therapy, e.g., by selecting an appropriate targeted therapy to use in addition to IL-2 therapy for initial treatment, by determining when or how to combine a targeted therapy with IL-2 therapy, and/or by determining how or when to change the current course of therapy (e.g., switch to another targeted therapy that acts on a different signal transduction pathway). [0041] In some aspects, the present invention provides methods for monitoring melanoma progression in a subject having melanoma, wherein the method comprises assaying the subject for the appropriate or therapeutic regimen of combination IL-2 therapy.

[0042] In other aspects, the present invention provides methods for monitoring RCC progression in a subject having RCC, wherein the method comprises assaying the subject for the appropriate or therapeutic regimen of combination IL-2 therapy.

[0043] The disclosure of U.S. Application No. 13/048,840, filed March 15, 2011, is hereby incorporated by reference in its entirety for all purposes.

[0044] Other objects, features, and advantages of the present invention will be apparent to one of skill in the art from the following detailed description and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 shows the dose dependent stimulation of Thl/Th2 cytokines in IL-2 treated PBMCs. Figure 1A shows IL-lb, IFN-g, IL-13 and IL-12. Figure IB shows IL-10, IL-4, IL- 5 andIL-8. Figure 1C shows TNF -alpha. [0046] Figure 2 shows the effect of TKI treatment on melanoma tumor antigen expression. The expression of the following 10 genes was measured by RT-qPCR: MART-1, tyrosinase, MITF, gplOO, c-myc, Perforin, FasL, granzyme B, IRF-1, and OSM. Among these, gplOO, MART-1, tyrosinase, and MITF were found to be regulated by TKI treatment. Figure 2 A represents tumor cells co-cultured with untreated PBMCs. Figure 2B represents tumor cells co-cultured with IL-2 treated PBMCs.

[0047] Figure 3 shows the morphological changes in TKI treated melanoma cells before and after addition of IL-2 treated PBMCs. Figure 3 A shows the morphological changes of MART-1 positive (D10) and MART-1 negative (NA8) cells 72 hours after TKI treatment. Figure 3B shows the morphological changes after incubating the cells with IL-2 treated PBMCs for 48 hours.

[0048] Figure 4 shows the dose dependent growth inhibition of MART-1 positive melanoma cells (D10) and MART-1 negative melanoma cells (NA8) by TKIs. Figure 4A shows tumor cells treated with TKI only. Figure 4B shows cells treated with TKIs in the presence of untreated PBMCs. Figure 4C cells treated with TKIs in the presence of IL-2 treated PBMCs.

[0049] Figure 5 shows the modulation of immune cell functions by targeted therapy treatment in MART-1 positive (D10) and MART-1 negative (NA8) tumor cells. Thl/Th2 cytokines were measured in TKI treated tumor/immune cell co-culture systems. Figure 5A-F shows levels of IFN-gamma, IL-13, IL-5, IL-10, IL-lb and TNF-a, respectively.

DETAILED DESCRD7TION OF THE INVENTION

I. Introduction

[0050] High dose IL-2 immunotherapy has been used to treat patients with renal cell carcinoma and patients with melanoma, yet only a small number of patients have durable remission. The present invention is advantageous because it overcomes current limitations associated with the administration of IL-2 immunotherapy.

[0051] Accordingly, the present invention provides methods for determining a combination of IL-2 therapy (e.g., Proleukin®) with one or more targeted therapies, such as, e.g., BRAF, MEK, PI3K/AKT, receptor tyrosine kinase, and/or HH inhibitors, for the treatment of a cancer such as renal cell carcinoma or melanoma (e.g., metastatic or non-metastatic) in an individual. The methods include determining whether the individual is positive or negative for MART-1 expression. It has been determined as described herein that MART-1 is predictive of an individual's responsiveness to one or more targeted therapies when combined with IL-2.

[0052] With the present invention, it is possible to design individualized treatment strategies by combining IL-2 therapy (e.g., Proleukin^®) with one or more targeted therapies. The present invention also provides methods for selecting a combination of IL-2 therapy (e.g., Proleukin^®) with one or more targeted therapies for an individual having RCC or melanoma based on the expression of MART-1 in the individual. In some embodiments, the present invention enables the rational design of therapy with a high dose (HD) of IL-2 (e.g., Proleukin^®) in combination with one or more targeted therapies for the treatment of a cancer such as RCC or melanoma. [0053] The present invention also provides methods for administering a treatment regimen of IL-2 therapy for an individual having RCC or melanoma based on whether the individual is positive or negative for MART-1 expression.

II. Definitions

[0054] As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0055] The term "cancer" is intended to include any member of a class of diseases characterized by the uncontrolled growth of aberrant cells. The term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue, or solid, and cancers of all stages and grades including pre- and post-metastatic cancers.

Examples of different types of cancer include, but are not limited to, digestive and gastrointestinal cancers such as gastric cancer (e.g., stomach cancer), colorectal cancer, gastrointestinal stromal tumors, gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, and esophageal cancer; breast cancer; lung cancer; gallbladder cancer; liver cancer; pancreatic cancer; appendix cancer; prostate cancer, ovarian cancer; renal cancer (e.g., renal cell carcinoma); cancer of the central nervous system; skin cancer (e.g., melanoma); lymphomas; gliomas; choriocarcinomas; head and neck cancers; osteogenic sarcomas; and blood cancers. As used herein, a "tumor" comprises one or more cancerous cells. In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the cancer is melanoma.

[0056] The term "renal cell carcinoma" ("RCC") includes a disease in which malignant cancer cells form in the lining of the tubules of the kidney. In some embodiments, the renal cell carcinoma is non-metastatic. In some embodiments, the renal cell carcinoma is metastatic.

[0057] The term "melanoma" includes a disease in which malignant cancer cells arise from melanocytes. Melanomas can arise in tissues including, but not limited to, skin, eye, oral mucosa, and genital mucosa. In some embodiments, the melanoma is non-metastatic. In some embodiments, the melanoma is metastatic.

[0058] The term "metastasis" or "metastatic" refers to spread of a cancer from the primary tumor or origin (e.g., kidney for renal cell carcinoma) to other tissues and parts of the body, such as lymph node, lung, adrenal gland, liver, brain, and/or bone.

[0059] The term "clear cell carcinoma" refers to a cancer wherein a majority of the carcinogenic cells become vacuolated and/or become filled with glycogen. Generally these cells appear clear when viewed through a microscope. Clear cell carcinoma can occur in many types of cancers, including but not limited to, renal cell carcinoma, ovarian epithelial neoplasm, ovarian carcinoma, hepatocellular carcinoma, uterine carcinoma, breast neoplasm, and lung carcinoma. Clear cells may be characterized by several histological features including, without limitation, sarcomatoid, rhabdoid, papillary, granular/eosinophilic, tubular, alveolar, cystic, and solid. [0060] The term "(metastatic)", when used before "melanoma", includes metastatic or non- metastatic melanoma, or both. Similarly, the term "(metastatic)", when used before "renal cell carcinoma" or "RCC", includes metastatic or non-metastatic RCC, or both.

[0061] The term "classifying" includes "to associate" or "to categorize" a sample with a prognosis, for example, a likelihood of responding to a particular therapy (e.g., IL-2 therapy in combination with one or more other therapies). In certain instances, "classifying" is based on statistical evidence, empirical evidence, or both. In certain embodiments, the methods and systems of classifying use a so-called training set of samples having known responses to a particular therapy or known disease states. Once established, the training data set serves as a basis, model, or template against which the features of an unknown sample are compared, in order to classify the unknown prognosis or disease state of the sample. In certain instances, classifying the sample is akin to providing a prognosis for or diagnosing the disease state of the sample. In certain other instances, classifying the sample is akin to differentiating the prognosis or disease state of the sample from another prognosis or disease state. [0062] The term "interleukin 2" or "IL-2" includes any purified or recombinant IL-2 molecule that possesses an immune stimulatory effect (e.g., enhancement of lymphocyte mitogenesis, stimulation of long-term growth of hIL-2 dependent cells, enhancement of lymphocyte cytotoxicity, induction of lymphokine-activated killer cell (LAK) and/or natural killer cell ( K) activity, induction of interferon-gamma (IFN-γ) production, and the like) useful for the treatment of (metastatic) renal cell carcinoma and/or (metastatic) melanoma, including modified native IL-2 molecules, truncated IL-2 molecules, variant IL-2 molecules, and covalently modified IL-2 molecules (e.g., glycosylated). In one embodiment, IL-2 refers to a human polypeptide encoded by the IL-2 gene (Entrez GenelD: 3558; NM 000586), which is produced after processing of the IL-2 precursor polypeptide (NP 000577), from which the first 20, 21, or 22 amino acids are removed, and variants thereof. In a preferred embodiment, the IL-2 molecule comprises des-alanyl-1, serine-125 human interleukin-2 (PROLEUKIN® (aldesleukin)). Exemplary IL-2 variants, methods of purification thereof, and methods of formulation thereof, can be found, for example, in U.S. Patent Nos.

4,530,787, 4,569,790, 4,572,798, 4,604,377, 4,748,234, 4,853,332, 4,959,314, 5,464,939, RE33,653, 5,229,109, 7,514,073, and 7,569,215, each of which is herein incorporated by reference in their entirety for all purposes.

[0063] The term "IL-2 immunotherapy" or "IL-2 therapy" includes administration of IL-2 to a subject in need thereof via any known route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, inhalation, and other known routes. Non-limiting examples of IL-2 immunotherapy can be found, for example, in Rosenberg SA et al, J Natl Cancer Inst. 1993 Apr 21;85(8):622-32, Guirguis LM et al, J Immunother 2002 Jan-Feb;25(l):82-7, Griffiths and Mellon, Postgrad Med J. 2004 Jun;80(944):320-7, Yang JC et al, J Clin Oncol. 2003 Aug 15;21(16):3127-32, McDermott DF et al, J Clin Oncol. 2005 Jan 1;23(1): 133-41, Negrier S et al, Cancer 2007 Dec l;110(l l):2468-77, McDermott DF, Med Oncol. 2009;26 Suppl 1 :13-7, McDermott DF, Med Oncol. 2009; 26 Suppl 1: 13-7, US Patent Nos. 5,419,900, 5,696,079, 6,045,788, and 6,548,055, each of which is herein incorporated by reference in their entirety for all purposes.

[0064] As used herein, the terms "targeted therapy" and "targeted therapeutic agent" include a small molecule inhibitor of a signaling transduction pathway {e.g., BRAF, MEK, PI3K/AKT, HER2, and/or HH pathway) that is activated in cancer. In some embodiments, IL-2 therapy further comprises administration of at least a second, third, fourth, or fifth therapeutic agent, such as one or more targeted therapeutic agents {e.g., one or more BRAF, MEK, PI3K/AKT, HER2, and/or HH inhibitors), lymphokine activated killer (LAK) cells (U.S. Patent No. 4,690,915), IFN-a (Atzpodien J et al., JClin Oncol. 1995 Feb;13(2):497- 501, Kruit WH et al, Br J Cancer. 1995 Jun;71(6):1319-21, Kruit WH et al, Br J Cancer. 1996 Sep;74(6):951-5, Henriksson R et al, Br J Cancer. 1998 Apr;77(8):1311-7, and US 6,605,273, each of which is herein incorporated by reference in their entirety for all purposes), TNF (US Patent No. 5,098,702), and the like. In a preferred embodiment, IL-2 immunotherapy includes high dose (HD) intravenous administration of des-alanyl-1, serine- 125 human interleukin-2 (PROLEUKIN^® (aldesleukin)) in combination with one or more targeted therapeutic agents {e.g., one or more BRAF, MEK, PI3K/AKT, HER2, and/or HH inhibitors).

[0065] As used herein, the term "BRAF inhibitor" includes any small molecule compound that inhibits {e.g., blocks, disrupts, or inactivates) the activity of the protein kinase BRAF. For instance, the inhibitor can directly bind {e.g., complex) to BRAF, and interrupt {e.g., block) the signal transduction pathway lying downstream of BRAF. Non-limiting examples of BRAF inhibitors include vemurafenib/PLX4032 (Plexxikon/Hoffman-LaRoche), dabrafenib (GlaxoSmithKline), RAF265 (Novartis), RAF265 (Novartis), and combinations thereof.

[0066] As used herein, the term "MEK inhibitor" includes any small molecule compound that inhibits {e.g., blocks, disrupts, or inactivates) the activity of the protein kinase MEK. For instance, the inhibitor can directly bind {e.g., complex) to MEK, and interrupt {e.g., block) signal transduction pathways downstream of MEK. Non-limiting examples of MEK inhibitors include trametinib, MEK 162 (Novartis), XL518 (Genentech), PD-325901 (Pfizer), and combinations thereof.

[0067] As used herein, the term "PI3K/AKT inhibitor" includes includes any small molecule compound that inhibits (e.g., blocks, disrupts, or inactivates) the activity of the protein kinase PI3K and/or AKT. For instance, the inhibitor can directly bind (e.g., complex) to either PI3K or AKT, and interrupt (e.g., block) the signal transduction pathways downstream of either PI3K or AKT. Non-limiting examples of PI3K/AKT inhibitors include BEZ235 (Novartis), BKM120 (Novartis), BLY719 (Novartis), IPI-145 (Infinity

Pharmaceuticals), and combinations thereof.

[0068] As used herein, the term "HH inhibitor" or "Hedgehog inhibitor" includes any small molecule compound that blocks (e.g., disrupts, inactivates, interferes, inhibits) the signaling transduction of the Hedgehog signaling pathway. For example, a HH inhibitor includes a small molecule that binds the G-protein receptor Smoothened (Smo) and blocks its activity. Non-limiting examples of Hedgehog inhibitors include erismodegib LDE-225 (Novartis), LEQ506 (Novartis), vismodegib/GDC-0449 (Genentech/ Hoffman-LaRoche), saridegib/IPI- 926 (Infinity Pharmaceuticals), IPI-269609 (Infinity Pharmaceuticals), BMS-833932/XL-139 (Bristol-Myers Squibb/Exelixis), PF-04449913 (Pfizer), TAK-441 (Millenium), and combinations thereof. [0069] As used herein, the term "receptor tyrosine kinase inhibitor'Or "TKF'includes any small molecule compound that blocks (e.g., disrupts, inactivates, interferes, inhibits) the activity (e.g., phosphorylation) of a receptor tyrosine kinase (RTK). The RTK inhibitor can directly bind to the RTK. Non-limiting examples of RTK inhibitors include pazopanib (for VEGFR-1, VEGFR-2, VEGFR-3, PDGFR, and c-KIT), sorafenib (for CRAF, BRAF, KIT, FLT-3, VEGFR-2, VEGFR-3, and PDGFR-β), sunitinib (for VEGFR-1, VEGFR-2, PDGFR, and c-Kit), axitinib (for VEGFR-1, VEGFR-2, and VEGFR-3), and combinations thereof. RTK inhibitors include HER2 inhibitors.

[0070] As used herein, the term "HER2 inhibitor" includes any small molecule compound that inhibits (e.g., blocks, disrupts, or inactivates) the activity (e.g., phosphorylation) of the receptor tyrosine kinase HER2. For instance, an inhibitor can directly bind to the

extracellular domain of HER2, interrupt (e.g., block) HER2 dimerization, and/or interfere with downstream signal transduction pathways. Non-limiting examples of HER2 inhibitors include gefitinib (AstraZeneca), erlotinib (Hoffman-LaRoche), lapatinib (GlaxoSmithKline), CP-724714 (Pfizer), CP-654577 (Pfizer), AG1478, Arry-380 (Array Biopharma), Arry- 334543 (Array Biopharma), canertinib/CI-1033/PD183805 (Parke-Davis/Pfizer), CL- 387785/EKI-785 (Wyeth-Ayerst), BIBW-2992 (Boehringer Ingelheim), AV-412/MP-412 (AVEO Pharmaceuticals), AE788 (Novartis), CGP-59326A (Novartis), PKI-166/CGP-75166 (Novartis), pelitinib (Wyeth), neratinib (Wyeth), HKI-357 (Wyeth), AC-480/BMS599626 (Mristal Myers Squibb), ZD 1839, Bay846, D69491 (Sugen), DXL-702 (InNexus

Biotechnology), E-75, RB-200h (Receptor BioLogix), and combinations thereof.

[0071] As used herein, the terms "treatment," "treating," and the like, include administering an agent (e.g., IL-2 alone or in combination with targeted therapeutic agents), or carrying out a procedure (e.g., a nephrectomy), for the purposes of obtaining an effect. As such, treatment includes any therapeutic approach taken to relieve or prevent one or more symptoms (e.g., clinical factors) associated with (metastatic) renal cell carcinoma or (metastatic) melanoma (e.g., tumor burden) or improve the health of an individual with (metastatic) renal cell carcinoma or (metastatic) melanoma (e.g., increase progression-free survival, increase overall survival time, and the like). The term encompasses administering any compound, drug, procedure, and/or regimen useful for improving the health of a subject with (metastatic) renal cell carcinoma or (metastatic) melanoma and includes any of the therapeutic agents described herein as well as surgery. One skilled in the art will appreciate that either the course of therapy or the dose of the current course of therapy can be changed, e.g., based upon the results obtained using the methods of the present invention.

[0072] As used herein, the term "circulating cells" comprises extratumoral cells that have either metastasized or micrometastasized from a solid tumor. Examples of circulating cells include, but are not limited to, circulating tumor cells, cancer stem cells, and/or cells that are migrating to the tumor (e.g., circulating endothelial progenitor cells, circulating endothelial cells, circulating pro-angiogenic myeloid cells, circulating dendritic cells, etc.). Patient samples containing circulating cells can be obtained from any accessible biological fluid (e.g., whole blood, serum, plasma, sputum, bronchial lavage fluid, urine, nipple aspirate, lymph, saliva, fine needle aspirate, etc.). In certain instances, the whole blood sample is separated into a plasma or serum fraction and a cellular fraction (i.e., cell pellet). The cellular fraction typically contains red blood cells, white blood cells, and/or circulating cells of a solid tumor such as circulating tumor cells (CTCs), circulating endothelial cells (CECs), circulating endothelial progenitor cells (CEPCs), cancer stem cells (CSCs), disseminated tumor cells of the lymph node, and combinations thereof. The plasma or serum fraction usually contains, inter alia, nucleic acids {e.g., DNA, R A) and proteins that are released by circulating cells of a solid tumor.

[0073] Circulating cells are typically isolated from a patient sample using one or more separation methods including, for example, immunomagnetic separation {see, e.g., Racila et al, Proc. Natl. Acad. Sci. USA, 95:4589-4594 (1998); Bilkenroth et al, Int. J. Cancer,

92:577-582 (2001)), the CellTracks^® System by Immunicon (Huntingdon Valley, PA), microfluidic separation {see, e.g., Mohamed et al, IEEE Trans. Nanobiosc , 3:251-256 (2004); Lin et al, Abstract No. 5147, 97th AACR Annual Meeting, Washington, D.C.

(2006)), FACS {see, e.g., M cuso et al., Blood, 97:3658-3661 (2001)), density gradient centrifugation {see, e.g., Baker et al., Clin. Cancer Res., 13:4865-4871 (2003)), and depletion methods {see, e.g., Meye et al, Int. J. Oncol, 21 :521-530 (2002)).

[0074] The term "sample" includes any biological specimen obtained from an individual. Suitable samples for use in the present invention include, without limitation whole blood, plasma, serum, red blood cells, white blood cells {e.g., peripheral blood mononuclear cells), ductal lavage fluid, nipple aspirate, lymph {e.g., disseminated tumor cells of the lymph node), bone marrow aspirate, saliva, urine, stool {i.e., feces), sputum, bronchial lavage fluid, tears, fine needle aspirate {e.g., harvested by random periareolar fine needle aspiration), any other bodily fluid, a tissue sample {e.g., tumor tissue) such as a biopsy of a tumor {e.g., needle biopsy) or a lymph node {e.g., sentinel lymph node biopsy), a tissue sample {e.g., tumor tissue) such as a surgical resection of a tumor, and cellular extracts thereof {e.g., tumor tissue cellular extract). In some embodiments, the sample is whole blood or a fractional component thereof such as plasma, serum, or a cell pellet. In preferred embodiments, the sample is obtained by isolating circulating cells of a solid tumor from whole blood or a cellular fraction thereof using any technique known in the art. In other embodiments, the sample is a formalin fixed paraffin embedded (FFPE) tumor tissue sample, e.g., from a solid tumor of the skin or kidney or other portion of the body. The use of samples such as serum, saliva, and urine is well known in the art {see, e.g., Hashida et al., J. Clin. Lab. Anal, 11:267-86 (1997)). One skilled in the art will appreciate that samples such as serum, plasma, and cellular extract samples can be diluted prior to the analysis of marker levels. In another embodiment, the sample is a biopsy.

[0075] A "biopsy" refers to the process of removing a tissue sample for diagnostic or prognostic evaluation, and to the tissue specimen itself. Any biopsy technique known in the art can be applied to the methods and compositions of the present invention. The biopsy technique applied will generally depend on the tissue type to be evaluated and the size and type of the tumor (i.e. , solid or suspended (i.e., blood or ascites)), among other factors. Representative biopsy techniques include excisional biopsy, incisional biopsy, needle biopsy (e.g., core needle biopsy, fine-needle aspiration biopsy, etc.), surgical biopsy, and bone marrow biopsy. Biopsy techniques are discussed, for example, in Harriso 's Principles of Internal Medicine, Kasper, et al, eds., 16th ed., 2005, Chapter 70, and throughout Part V. One skilled in the art will appreciate that biopsy techniques can be performed to identify cancerous and/or precancerous cells in a given tissue sample.

[0076] The term "individual," "subject," or "patient" typically refers to humans, but also to other animals including, e.g., other primates, rodents, canines, felines, equines, ovines, porcines, and the like.

[0077] The terms "marker" and "biomarker" include any biochemical marker, serological marker, genetic marker, or other clinical or echographic characteristic that can be measured in a sample. In certain embodiments, a marker of the invention can be used to classify a sample from an individual with (metastatic) RCC or (metastatic) melanoma. Non-limiting examples of suitable markers include cytokines such as Thl/Th2 cytokines (e.g., IFN-γ, IL- 10, IL-12, IL-13, IL-Ιβ, IL-4, TNF-a, IL-5, IL-8, etc.), gene expression markers (e.g.,

MART-1, tyrosinase, MITF, gplOO, c-myc, Perforin, FasL, granzyme B, IRF-1, OSM, etc.), and combinations thereof. Other examples of cytokines include, but are not limited to, TNF- related weak inducer of apoptosis (TWEAK), osteoprotegerin (OPG), IFN-a, IFN-β, IL-1 a, IL-1 receptor antagonist (IL-IRA), IL-2, IL-6, soluble IL-6 receptor (sIL-6R), IL-7, IL-9, IL- 15, IL-17, IL-23, IL-27, and combinations thereof (e.g., combined with one or more Thl/Th2 cytokines). Those skilled in the art will know of additional markers suitable for use in the present invention.

[0078] The term "cytokine" includes any of a variety of polypeptides or proteins secreted by immune cells that regulate a range of immune system functions and encompasses small cytokines such as chemokines. The term "cytokine" also includes adipocytokines, which comprise a group of cytokines secreted by adipocytes that function, for example, in the regulation of body weight, hematopoiesis, angiogenesis, wound healing, insulin resistance, the immune response, and the inflammatory response. [0079] The terms "MART-1" and "melanoma antigen recognized by T-cells 1" include an 18kD transmembrane protein antigen which is also known as protein melan-A, MLANA, or melanocyte antigen. MART-1 is also commonly referred to as a melanocyte differentiation antigen. It is found on the surface of melanocyte lineage cells derived from skin, uvea and retinal, melanocyte cell lines, and both melanotic and amelanotic melanomas. Antitumor cytolytic T lymphocytes (CTLs) and tumor-infiltrating lymphocytes (TILs) recognize MART-1 presented by melanoma tumor cells. MART-1 can be detected using antibodies including Melan-A and A 103. MART-1 expression is detectable in the cell cytoplasm of non-cancerous melanocytes, cells of the adrenal cortex, and neoplasms such as benign adrenocortical adenomas, potentially malignant PEComas, malignant adrenocortical carcinoma, malignant clear cell sarcoma, and malignant pleomorphic liposarcoma In some instances, MART-1 is a cytoplasmic immunohistochemical marker for pheochromocytoma and paraganglioma in that a negative stain indicates the disease state. MART-1 is also an immunohistochemical marker for melanomas, angiomyolipomas, adrenal cortical tumors, sex-cord stromal tumors, compound nevi, lymphangiomyomas, and renal cell carcinoma (RCC). In some instances, primary, malignant melanoma can be negative for Melan-A staining, indicating loss of MART-1 expression. In other instances, MART-1 expression is positive for both benign and malignant melanomas. In some instances, MART-1 staining is negative in RCC and neoplasms of epithelial origin, lymphomas, and mesenchymal tumors. In other instances, it has also been shown that patients with a subtype of renal cell carcinoma (RCC), such as RCC with a t(6;l 1) translocation are positive for MART-1 (Zhan et al, J Clin Oncol, 28(34):e709-e713 (2010)).

[0080] As used herein, the terms "RNA" and "mRNA" are used interchangeably unless specified otherwise.

[0081] As used herein, the term "upregulated" or "upregulation" refer to RNA (e.g., mRNA) or protein expression of a marker of interest in a (metastatic) renal cell carcinoma or (metastatic) melanoma sample that is detectably higher than RNA or protein expression of the marker of interest in a control tissue sample. Upregulation can be due to increased transcription, post transcriptional processing, translation, post translational processing, altered stability, or altered protein degradation, as well as local upregulation due to altered protein traffic patterns (increased nuclear localization), and augmented functional activity, e.g., as a transcription factor. Upregulation can be detected using conventional techniques for detecting RNA (e.g., RT-PCR, PCR, microarray) or proteins (e.g., ELISA, Western blots, flow cytometry, immunofluorescence, immunohistochemistry, DNA binding assay techniques). Upregulation can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more for the marker of interest in the sample in comparison to a control (e.g., non-cancer) tissue. In certain instances, upregulation is 1-fold, 2-fold, 3 -fold, 4-fold or more higher levels of RNA or protein levels for the marker of interest in the sample in comparison to a control (e.g., non-cancer) sample.

[0082] As used herein, the terms "downregulated" or "downregulation" refer to RNA or protein expression of a marker of interest in a (metastatic) renal cell carcinoma or (metastatic) melanoma sample that is detectably lower than RNA or protein expression of the marker of interest in a control sample. Downregulation can be due to decreased transcription, post transcriptional processing, translation, post translational processing, altered stability, or altered protein degradation, as well as local downregulation due to altered protein traffic patterns (increased nuclear localization), and augmented functional activity, e.g., as a transcription factor. Downregulation can be detected using conventional techniques for detecting mRNA (e.g., RT-PCR, PCR, microarray) or proteins (e.g., ELISA, Western blots, flow cytometry, immunofluorescence, immunohistochemistry, DNA binding assay techniques). Downregulation can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more for the marker of interest in the sample in comparison to a control (e.g., non-cancer) tissue. In certain instances, downregulation is 1-fold, 2-fold, 3-fold, 4-fold or more lower levels of RNA or protein levels for the marker of interest in the sample in comparison to a control (e.g., non-cancer) sample.

[0083] The term "positive" for a marker of interest refers to the level of RNA or protein expression of a marker of interest in a sample that is detectably higher than RNA or protein expression of the marker of interest in a negative control sample, wherein the marker of interest is not detectable in the negative control sample. In some embodiments, the term "positive" corresponds to the term "upregulated".

[0084] The term "negative" for a marker of interest refers to the level of RNA or protein expression of a marker of interest in a tissue sample that is substantially equal to the RNA or protein expression of the marker of interest in a negative control sample, wherein the marker of interest is not detectable in the control sample. Likewise, the term "negative" for a marker refers to the level of RNA or protein expression of a marker of interest in a sample that is detectably lower than the RNA or protein expression of the marker of interest in a positive control sample, wherein the marker of interest is detectable in the positive control sample. In some embodiments, the term "negative" corresponds to the term "downregulated".

[0085] The term "prognosis" includes a prediction of the probable course or outcome of a (metastatic) renal cell carcinoma or (metastatic) melanoma or the likelihood of response to a particular treatment for (metastatic) renal cell carcinoma or (metastatic) melanoma, e.g., IL-2 therapy or combination therapy as described herein.

[0086] The term "monitoring the progression or regression" of a cancer such as (metastatic) renal cell carcinoma or (metastatic) melanoma includes the use of the methods of the present invention to determine the disease state (e.g., tumor burden) or the continued likelihood of responding to IL-2 treatment or combination therapy of a subject in need thereof.

[0087] The term "monitoring drug efficacy in a subject receiving a drug useful for treating" a cancer such as (metastatic) renal cell carcinoma or (metastatic) melanoma includes the use of the methods of the present invention to determine the disease state (e.g., tumor burden) of a subject after one or more therapeutic agents for treating the cancer have been administered (e.g., IL-2 therapy or combination therapy as described). As used herein, a drug useful for treating a cancer such as (metastatic) renal cell carcinoma or (metastatic) melanoma is any compound or drug or combination thereof used to improve the health, reduce tumor burden, increase progression-free survival, increase overall survival time, and the like, of the subject. Non-limiting examples of drugs useful for treating a cancer such as renal cell carcinoma or melanoma are described herein and include cytokines (e.g., IL-2, IFN-a, etc.), receptor tyrosine kinase inhibitors (e.g. lapatinib, sunitinib, sorafenib, axitinib, pazopanib, and the like), BRAF inhibitors (e.g., PLX-4032, dabrafenib, RAF265, RAF265, etc.), MEK inhibitors (e.g., AZD 6244, PD325901, XL518, etc.), PI3K/AKT inhibitors (e.g., BEZ235, BKM120, BLY719, PI-145, etc.), Hedgehog (HH) inhibitors (e.g., vismodegib/GDC-0449,

erismodegib/LDE-225, LEQ506, saridegib/IPI-926, IPI-269609, BMS-833932/XL-139, PF- 04449913, TAK-441, etc.), VEGF binding agents (e.g., bevacizumab, etc.), mTOR inhibitors (e.g., temsirolimus, everolimus, etc.), free bases thereof, pharmaceutically acceptable salts thereof, derivatives thereof, analogs thereof, and combinations thereof. [0088] The terms "combination therapy" or "combination treatment" include administering of a plurality of agents and/or carrying out a procedure for the purposes of obtaining a therapeutic effect, such as to relieve or prevent one or more symptoms (e.g., clinical factors) associated with (metastatic) renal cell carcinoma or (metastatic) melanoma (e.g., tumor burden) or improve the health of an individual with (metastatic) renal cell carcinoma or (metastatic) melanoma (e.g., increase progression-free survival, increase overall survival time, and the like). As such, combination therapy includes administering a plurality of compounds, drugs, procedures, regimens, and combinations thereof, wherein administering can be for example, but not limited to, simultaneously, continuously, sequentially, alternately, cyclically, and cyclically with periods of rest. The term includes any of the therapeutic agents described herein as well as surgery. One skilled in the art will appreciate that either the combination therapy or the dose of the current combination therapy can be changed, e.g., based upon the results obtained using the methods of the present invention.

[0089] The terms "high dose IL-2" and "HD IL-2" include a dose of interleukin-2 (IL-2) of about or at least about 600,000 International Units (IU)/kg of body weight (kg)/dose, or about or at least about 720,000 IU/kg/dose. In some embodiments, high dose IL-2 includes a dose of IL-2 of from about 600,000 to about 2,000,000 IU/kg/dose, from about 600,000 to about 1,500,000 IU/kg/dose, from about 600,000 to about 1,000,000 IU/kg/dose, or from about 600,000 to about 720,000 IU/kg/dose. In other embodiments, high dose IL-2 includes a dose of IL-2 of about or at least about 10 million International Units (MIU) daily, e.g., about or at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, or more MIU daily, or from about 10 to about 50, from about 10 to about 25, or from about 15 to about 30 MIU daily. In some instances, 600,000 IU/kg/dose of IL-2 is substantially equivalent to 0.037 mg/kg/dose of IL-2. In yet other embodiments, high dose IL-2 includes a dose of IL-2 of about or at least about 0.037 mg/kg/dose, such as, e.g., about or at least about 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, or 1.0 mg/kg/dose, or from about 0.04 to about 1.0, from about 0.04 to about 0.5, from about 0.04 to about 0.1, or from about 0.04 to about 0.08 mg/kg/dose. In certain instances, high dose IL-2 includes a dose of IL-2 of about 22 MIU of Proleukin^® (about 1.3 mg of Proleukin^®) or about 18 MIU of Proleukin^® (about 1.1 mg of Proleukin^®). [0090] The terms "low dose IL-2" and "LD IL-2" include a dose of interleukin-2 (IL-2) of less than about 600,000 IU/kg of body weight/dose, such as about 60,000 or about 72,000 IU/kg/dose, e.g., from about 60,000 to about 72,000 IU/kg/dose. In some embodiments, LD IL-2 includes a dose of about 125,000 or about 250,000 IU/kg/day, e.g., from about 125,000 to about 250,000 IU/kg/day. In other embodiments, low dose IL-2 includes a dose of IL-2 of less than about 10 million International Units (MIU) daily, e.g., less than about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 MIU daily, or from about 1 to about 10, from about 1 to about 5, or from about 1.5 to about 6 MIU daily.

[0091] The term "subcutaneous dose of IL-2" includes a dose of interleukin-2 (IL-2) that is administered by subcutaneous injection of about or at least about 3 million IU twice/day for 5 days/week for about 6 weeks, corresponding to one immunotherapy cycle. In some instances, a second cycle can be given after at least about 28 days of rest.

[0092] The term "transform" or "transforming" includes a physical or chemical change of the sample, marker, or reagent, for example to extract a marker as defined herein. An extraction, a manipulation, a chemical precipitation, an ELISA, an immuno-extraction, a physical or chemical modification of the sample to measure a marker all constitute a transformation. As long as the sample is not identical before and after the transformation step, the change is a transformation. III. Description of the Embodiments

[0093] The present invention provides methods for determining a combination of IL-2 therapy (e.g., Proleukin^®) with one or more targeted therapies for the treatment of a cancer such as metastatic or non-metastatic melanoma or RCC. The present invention also provides methods for recommending, reporting, and administering a treatment regimen of IL-2 therapy (e.g., Proleukin^®) in combination with one or more targeted therapies for individuals with or suspected of having metastatic or non-metastatic melanoma or RCC.

A. Methods for Determining Combination Therapy For Treating Cancer

[0094] In some embodiments, the present invention provides methods for determining a combination of IL-2 therapy with one or more targeted therapeutic agents for treating cancer in a subject, comprising: (a) determining whether a cancer cell obtained from the subject is positive (e.g., upregulated) or negative (e.g., downregulated) for MART-1 expression; and (b) selecting an appropriate therapy for the subject.

[0095] In some embodiments, the method further comprises obtaining a sample from a subject. The sample may comprise any biological specimen as described herein obtained from the subject and typically contains at least one cancer cell. In particular embodiments, the sample is tumor tissue (e.g., fine needle aspirate sample obtained from a tumor), whole blood (e.g., circulating tumor cells isolated from blood), or a cellular extract thereof. In certain instances, the tumor tissue sample is obtained from a solid tumor of the skin, kidney, and/or other portion of the body. [0096] In some embodiments, the subject has cancer or is suspected of having cancer. In some embodiments, the cancer is melanoma or metastatic melanoma. In some embodiments, the cancer is renal cell carcinoma or metastatic renal cell carcinoma. In some embodiments, the cancer is clear cell carcinoma.

[0097] In some embodiments, step (a) of determining whether a cancer cell obtained from the subject is positive or negative for MART-1 expression comprises measuring (e.g., detecting) the presence or the level of MART-1 expression, wherein MART-1 expression is selected from the group consisting of protein expression, RNA expression, and combinations thereof.

[0098] The presence or level of MART- 1 RNA expression in a sample can be measured or detected using any of a variety of techniques (e.g., quantitative or semi-quantitative RT-PCR, microarray analysis, Northern blot analysis, solution hybridization detection, and the like). See, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989) and Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons (Hoboken, NY 2012).

[0099] The presence or level of MART- 1 protein expression in a sample can be measured or detected using any of a variety of techniques (e.g., immunoassay, proximity dual detection assay (e.g., a Collaborative Enzyme Enhanced Reactive Immunoassay (CEER)), ELISA, radioimmunoassay (RIA), immunoblotting (e.g., Western blotting); flow cytometry, immunohistochemistry, mass spectrometry, and the like). A detailed description of proximity dual detection assays (e.g., CEER) can be found in U.S. Pat. Pub. Nos. 2008/0261829 and 201 1/0275097, the disclosures of which are herein incorporated by reference in their entirety for all purposes. Other specific types of immunoassays include antigen capture/antigen competition, antibody capture/antigen competition, two-antibody sandwiches, antibody capture/antibody excess, and antibody capture/antigen excess.

[0100] In some embodiments, an antibody that recognizes (e.g., binds to, forms a complex with, is specific for) an epitope on the MART-1 protein is useful in the methods of the present invention. Non-limiting examples of such an antibody include melan-A antibodies (e.g., A103). For instance, melan-A antibody (B-10) recognizes an epitope between amino acids 81-1 17 near the C-terminus of MART-1 and melan-A antibody (D-6) recognizes an epitope between 3-31 at the N-terminus of MART-1. Antibodies to MART-1 are

commercially available from, for example, but not limited to, Santa Cruz Biotechnology (Santa Cruz, CA), Novus Biologicals (Littleton, CO), Dako (Carpenteria, CA), Vector Laboratories (Burlingame, CA), and Abgent (San Diego, CA). One skilled in the art recognizes that methods of making antibodies are described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY (1988). [0101] In some embodiments, the method further comprises measuring (e.g., detecting) the presence or level of RNA and/or protein expression of at least one other melanoma biomarker such as, but not limited to, gplOO, MITF, and/or tyrosinase. [0102] In some embodiments, the method further comprises measuring (e.g., detecting) the presence or level of RNA and/or protein expression of at least one other RCC biomarker such as, but not limited to, p53, Ki67, CAIX, VEGF, SAA, IGF-1, NMP22, CXCR3, CXCR4, MMP2, MMP6, EpCAM, vimentin, fascin, livin, survivin, CD70, KIT, and/or KAI-1. [0103] In some embodiments, positive expression of MART- 1 is indicated by comparing the level of MART- 1 expression in the sample from the subject to that of a control, and determining that the level of MART- 1 expression is higher than that of the control, wherein the control does not express MART-1 or does not express detectable levels of MART- 1. In other embodiments, positive expression of MART-1 is indicated by comparing the presence of MART-1 expression in the sample to that of a control (e.g., negative control), wherein MART-1 expression is absent in the control, and determining that the sample is positive for MART-1 expression.

[0104] In some embodiments, the control (e.g., negative control) is a sample from an individual that does not have cancer. In other embodiments, the control is a pooled sample from a plurality of individuals that do not have cancer. In some embodiments, the control is a sample from a non-cancerous cell line. In some embodiments, the control is a control value representing the level of MART-1 expression in a sample that is cancer-free.

[0105] In other embodiments, positive expression of MART-1 is indicated by comparing the level of MART-1 expression in the sample to that of a control, and determining that the level of MART-1 expression is substantially equal or higher than that of the control (e.g., positive control), wherein the control expresses MART-1.

[0106] In some embodiments, the control (e.g., positive control) is a sample from an individual with cancer such as (metastatic) melanoma or (metastatic) RCC. In certain other embodiments, the control is a pooled sample from a plurality of individuals who have cancer. In some embodiments, the control is a sample from a cancer cell line. Non-limiting examples of cancer cell lines useful to the invention include melanoma cell lines such as CHL-1, SK- MEL-2, A375, SK-MEL-28, NA8, D10, and HBL, and RCC cell lines such as 1581 RCC, 1764 RCC, 2194 RCC, SNU-228, SNU-267, SNU-328, SNU-349, and SNU-1272. In some embodiments, the control is a control value representing the level of MART-1 expression in a sample from a cancer cell line.

[0107] In some embodiments, positive expression of MART-1 corresponds to upregulated expression of MART-1. For instance, MART-1 expression is determined to be upregulated when the level of expression (e.g., RNA or protein) is increased in the sample from the subject compared to that of a control (e.g., negative control or positive control).

[0108] In some embodiments, negative expression of MART-1 is indicated by comparing the level of MART-1 expression in the sample from the subject to that of a control (e.g., negative control), and determining that the level of MART-1 expression is substantially equal to that of the control, wherein the control does not express MART- 1 or does not express detectable levels of MART-1.

[0109] In some embodiments, the control (e.g., negative control) is a sample from an individual that does not have cancer. In other embodiments, the control is a pooled sample from a plurality of individuals that do not have cancer. In some embodiments, the control is a control value representing the level of MART-1 expression in a cancer-free sample.

[0110] In some embodiments, negative expression of MART-1 is indicated by comparing the level of MART-1 expression in the sample from the subject to that of a control (e.g., positive control), and determining that the level of MART-1 expression is substantially lower than to that of the control (e.g., positive control), wherein the control expresses MART-1.

[0111] In some embodiments, negative expression of MART-1 corresponds to

downregulated expression of MART-1. For instance, MART-1 expression is determined to be downregulated when the level of expression (e.g., RNA or protein) is decreased in the sample from the subject compared to that of a control (e.g., negative control or positive control).

[0112] In some embodiments, if the cancer cell is positive for MART-1 expression (e.g., RNA expression or protein expression), then a combination of an IL-2 polypeptide (e.g., IL-2 immunotherapy) and a MEK inhibitor and/or a PI3K/AKT inhibitor is selected as appropriate therapy for the subject. In some embodiments, if MART-1 expression (e.g., RNA expression or protein expression) is upregulated in the cancer cell, then a combination of an IL-2 polypeptide and a MEK inhibitor and/or a PI3K/AKT inhibitor is selected as appropriate therapy for the subject.

[0113] In some instances, a combination of an IL-2 polypeptide and a MEK inhibitor is selected as the appropriate therapy for the subject if the cancer cell is positive and/or upregulated for MART-1 expression. In other instances, a combination of an IL-2 polypeptide and a PI3K/AKT inhibitor is selected as the appropriate therapy for the subject if the cancer cell is positive and/or upregulated for MART-1 expression. In yet other instances, a combination of an IL-2 polypeptide, a MEK inhibitor and a PI3K/AKT inhibitor is selected as the appropriate therapy for the subject if the cancer cell is positive and/or upregulated for MART-1 expression.

[0114] In some embodiments, if the cancer cell is negative for MART-1 expression (e.g., RNA expression or protein expression), then a combination of an IL-2 polypeptide (e.g., IL-2 immunotherapy) and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor is selected as appropriate therapy for the subject. In some embodiments, if MART-1 expression (e.g., RNA expression or protein expression) is downregulated in the cancer cell, then a combination of an IL-2 polypeptide and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor is selected as appropriate therapy for the subject.

[0115] In some instances, a combination of an IL-2 polypeptide and a BRAF inhibitor is selected as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In other instances, a combination of an IL-2 polypeptide and a PI3K/AKT inhibitor is selected as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In yet other instances, a combination of an IL-2 polypeptide and a receptor tyrosine kinase inhibitor is selected as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In still yet other instances, a combination of an IL-2 polypeptide and a Hedgehog inhibitor is selected as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression.

[0116] In further instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, and a PI3K/AKT inhibitor is selected as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In other instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, and a receptor tyrosine kinase inhibitor is selected as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In yet other instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, and a Hedgehog inhibitor is selected as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In still yet other instances, a combination of an IL-2 polypeptide, a PI3K/AKT inhibitor, and a receptor tyrosine kinase inhibitor is selected as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In further instances, a

combination of an IL-2 polypeptide, a PI3K/AKT inhibitor, and a Hedgehog inhibitor is selected as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In other instances, a combination of an IL-2 polypeptide, a receptor tyrosine kinase inhibitor, and a Hedgehog inhibitor is selected as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression.

[0117] In yet other instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, a PI3K/AKT inhibitor, and a receptor tyrosine kinase inhibitor is selected as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In still yet other instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, a PI3K/AKT inhibitor, and a Hedgehog inhibitor is selected as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In further instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, a receptor tyrosine kinase inhibitor, and a Hedgehog inhibitor is selected as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In other instances, a combination of an IL-2 polypeptide, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and a Hedgehog inhibitor is selected as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In yet other instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and a Hedgehog inhibitor is selected as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression.

[0118] In some embodiments, the MEK inhibitor is selected from the group consisting of PD325901, AZD 6244, trametinib, MEK162, XL518, and combinations thereof.

[0119] In some embodiments, the PI3 /AKT inhibitor is selected from the group consisting of BEZ235, BKM120, BLY719, IPI-145, and combinations thereof.

[0120] In some embodiments, the BRAF inhibitor is selected from the group consisting of PLX-4032, dabrafenib, RAF265, LGX818, and combinations thereof.

[0121] In some embodiments, the receptor tyrosine kinase inhibitor is selected from the group consisting of lapatinib, sunitinib, sorafenib, axitinib, pazopanib, gefitinib, erlotinib, pilitinib, canertinib, CP-654577, CP-724714, ARRY-334543, JNJ-26483327, JNJ-26483327, MP-470, AZD8931, and combinations thereof. [0122] In some embodiments, the HH inhibitor is selected from the group consisting of GDC0449, erismodegib, LEQ506, saridegib, 1PI-269609, BMS-833932, PF-04449913, TAK- 441, and combinations thereof.

[0123] In some embodiments, the IL-2 polypeptide (e.g., IL-2 immunotherapy) comprises a recombinant IL-2 polypeptide. In some embodiments, the recombinant IL-2 polypeptide is PROLEUKIN^® (aldesleukin). In some embodiments, the IL-2 immunotherapy is high dose (HD) IL-2. In other embodiments, the IL-2 immunotherapy is low dose (LD) IL-2.

[0124] In some embodiments, HD IL-2 immunotherapy comprises at least one dose of IL-2 (e.g., recombinant IL-2 such as Proleukin^®) of about or at least about 600,000 IU/kg of body weight (kg)/dose, or about or at least about 720,000 IU/kg/dose. Examples of suitable ranges and additional types of dosing units (e.g., MIU daily, mg/kg dose, etc.) are described herein.

[0125] In some embodiments, HD IL-2 includes a course of therapy comprising a treatment schedule of about 5 treatment days, about nine days of rest from treatment, and then about 5 additional days of treatment. In certain embodiments, the HD IL-2 course of therapy includes administration of IL-2 via intravenous (i.v.) injection as a bolus dose over about 15 minutes. In certain instances, during each 5 day treatment cycle, a dose is administered about every eight hours for a maximum of about 14 doses. In some instances, (metastatic) RCC patients treated with HD IL-2 receive a median of about 20 doses of the about 28 doses during the first course of therapy. In some instances, (metastatic) melanoma patients treated with HD IL-2 receive a median of about 18 doses of the about 28 doses during the first course of therapy.

[0126] In some embodiments, LD IL-2 immunotherapy comprises at least one dose of IL-2 (e.g., recombinant IL-2 such as Proleukin®) of less than about 600,000 IU/kg/dose, such as about 60,000 or about 72,000 IU/kg/dose. Examples of suitable ranges and additional types of dosing units (e.g., MIU daily, etc.) are described herein.

[0127] In certain instances, LD IL-2 includes a course of therapy comprising a treatment schedule of about 5 treatment days, about nine days of rest from treatment, and then about 5 additional days of treatment, wherein the patient receives a bolus dose about every 8 hours for a maximum of about 14 doses during the 5 treatment days, optionally wherein the bolus dose is administered via i.v. injection of about 60,000 to about 72,000 IU/kg/dose over about 15 minutes. In other instances, LD IL-2 includes a treatment schedule of daily subcutaneous (s.c.) injections of IL-2 at about 250,000 IU/kg/day for about 1 week, followed by daily s.c. injections of about 125,000 IU/kg/day for about 5 weeks. [0128] In some embodiments, the IL-2 polypeptide and the targeted therapeutic agent {e.g., the BRAF inhibitor, PI3K/AKT inhibitor, MEK inhibitor, receptor tyrosine kinase inhibitor and/or HH inhibitor) are administered simultaneously during the course of therapy. In some embodiments, the IL-2 polypeptide and the targeted therapeutic agent {e.g., the BRAF inhibitor, PI3K/AKT inhibitor, MEK inhibitor, receptor tyrosine kinase inhibitor and/or HH inhibitor) are administered sequentially during the course of therapy.

[0129] In some embodiments, the present invention provides methods for monitoring drug efficacy in a subject receiving a combination of drugs useful for treating melanoma or RCC {e.g., metastatic or non-metastatic). B. Methods for Recommending Combination IL-2 Therapy

[0130] In another embodiment, the present invention provides methods for recommending (e.g., reporting) a combination of IL-2 therapy with one or more targeted therapeutic agents for treating cancer in a subject, comprising: (a) determining whether a cancer cell obtained from the subject is positive {e.g., upregulated) or negative {e.g., downregulated) for MART-1 expression; and (b) recommending {e.g., reporting) an appropriate therapy for the subject.

[0131] In some embodiments, the method further comprises obtaining a sample from a subject. The sample may comprise any biological specimen as described herein obtained from the subject and typically contains at least one cancer cell. In particular embodiments, the sample is tumor tissue {e.g., fine needle aspirate sample obtained from a tumor), whole blood {e.g., circulating tumor cells isolated from blood), or a cellular extract thereof. In certain instances, the tumor tissue sample is obtained from a solid tumor of the skin, kidney, and/or other portion of the body.

[0132] In some embodiments, the subject has cancer or is suspected of having cancer. In some embodiments, the cancer is melanoma or metastatic melanoma. In some embodiments, the cancer is renal cell carcinoma or metastatic renal cell carcinoma. In some embodiments, the cancer is clear cell carcinoma.

[0133] In some embodiments, step (a) of determining whether a cancer cell obtained from the subject is positive or negative for MART-1 expression comprises measuring {e.g., detecting) the presence or the level of MART-1 expression, wherein MART-1 expression is selected from the group consisting of protein expression, RNA expression, and combinations thereof. [0134] The presence or level of MART- 1 RNA expression in a sample can be measured or detected using any of a variety of techniques (e.g., quantitative or semi-quantitative RT-PCR, microarray analysis, Northern blot analysis, solution hybridization detection, and the like). See, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989) and Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons (Hoboken, NY 2012).

[0135] The presence or level of MART- 1 protein expression in a sample can be measured or detected using any of a variety of techniques (e.g., immunoassay, proximity dual detection assay (e.g., CEER), radioimmunoassay (RIA), ELISA, and immunoblotting (e.g., Western blotting),- flow cytometry, immunohistochemistry, mass spectrometry, and the like). Detailed descriptions of a proximity dual detection assay (e.g., CEER) can be found in U.S. Pat. Pub. Nos. 2008/0261829 and 2011/0275097, the disclosures of which are herein incorporated by reference in their entirety for all purposes. Other specific types of immunoassays include antigen capture/antigen competition, antibody capture/antigen competition, two-antibody sandwiches, antibody capture/antibody excess, and antibody capture/antigen excess.

[0136] In some embodiments, an antibody that recognizes (e.g., is specific for, binds to, forms a complex with) an epitope of MART- 1 protein is useful in the methods of the present invention. Non-limiting examples of such an antibody include Melan-A antibodies (e.g., A 103). For instance, melan-A antibody (B-10) recognizes an epitope between amino acids 81-117 near the C-terminus of MART- 1 and melan-A antibody (D-6) recognizes an epitope between 3-31 at the N-terminus of MART-1. Antibodies to MART-1 are commercially available from, for example, but not limited to, Santa Cruz Biotechnology (Santa Cruz, CA), Novus Biologicals (Littleton, CO), Dako (Carpenteria, CA), Vector Laboratories

(Burlingame, CA), and Abgent (San Diego, CA). One skilled in the art recognizes that methods of making antibodies are described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY 1988).

[0137] In some embodiments, the method further comprises measuring (e.g., detecting) the presence or level of RNA and/or protein expression of at least one other melanoma biomarker such as, but not limited to, gplOO, MITF, and/or tyrosinase. [0138] In some embodiments, the method further comprises measuring (e.g., detecting) the presence or level of RNA and/or protein expression of at least one other RCC biomarker such as, but not limited to, p53, Ki67, CAIX, VEGF, SAA, IGF-1, NMP22, CXCR3, CXCR4, MMP2, MMP6, EpCAM, vimentin, fascin, livin, survivin, CD70, KIT, and/or KAI-1. [0139] In some embodiments, positive expression of MART- 1 is indicated by comparing the level of MART- 1 expression in the sample from the subject to that of a control, and determining that the level of MART- 1 expression is higher than that of the control, wherein the control does not express MART-1 or does not express detectable levels of MART- 1. In other embodiments, positive expression of MART-1 is indicated by comparing the presence of MART-1 expression in the sample to that of a control (e.g., negative control), wherein MART-1 expression is absent in the control, and determining that the sample is positive for MART-1 expression.

[0140] In some embodiments, the control (e.g., negative control) is a sample from an individual that does not have cancer. In other embodiments, the control is a pooled sample from a plurality of individuals that do not have cancer. In some embodiments, the control is a sample from a non-cancerous cell line. In some embodiments, the control is a control value representing the level of MART-1 expression in a sample that is cancer-free.

[0141] In other embodiments, positive expression of MART-1 is indicated by comparing the level of MART-1 expression in the sample to that of a control, and determining that the level of MART-1 expression is substantially equal or higher than that of the control (e.g., positive control), wherein the control expresses MART-1.

[0142] In some embodiments, the control (e.g., positive control) is a sample from an individual with cancer such as melanoma or RCC. In other embodiments, the control is a pooled sample from a plurality of individuals who have cancer. In some embodiments, the control is a sample from a cancer cell line. Non-limiting examples of cancer cell lines useful to the invention include melanoma cell lines such as CHL-1, SK-MEL-2, A375, SK-MEL-28, NA8, D10, and HBL, and RCC cell lines such as 1581 RCC, 1764 RCC, 2194 RCC, SNU- 228, SNU-267, SNU-328, SNU-349, and SNU-1272. In some embodiments, the control is a control value representing the level of MART-1 expression in a sample from a cancer cell line.

[0143] In some embodiments, positive expression of MART-1 corresponds to upregulated expression of MART-1. For instance, MART-1 expression is determined to be upregulated when the level of expression (e.g., RNA or protein) is increased in the sample from the subject compared to that of a control (e.g., negative control or positive control).

[0144] In some embodiments, negative expression of MART-1 is indicated by comparing the level of MART-1 expression in the sample from the subject to that of a control (e.g., negative control), and determining that the level of MART-1 expression is substantially equal to that of the control, wherein the control does not express MART-1 or does not express detectable levels of MART-1.

[0145] In some embodiments, the control (e.g., negative control) is a sample from an individual that does not have cancer. In other embodiments, the control is a pooled sample from a plurality of individuals that do not have cancer. In some embodiments, the control is a control value representing the level of MART-1 expression in a cancer-free sample.

[0146] In some embodiments, negative expression of MART-1 is indicated by comparing the level of MART-1 expression in the sample from the subject to that of a control (e.g., positive control), and determining that the level of MART-1 expression is substantially lower than to that of the control (e.g., positive control), wherein the control expresses MART-1.

[0147] In some embodiments, negative expression of MART-1 corresponds to

[0148] In some embodiments, if the cancer cell is positive for MART-1 expression (e.g., RNA expression or protein expression), then a combination of an IL-2 polypeptide (e.g., IL-2 immunotherapy) and a MEK inhibitor and/or a PI3K/AKT inhibitor is recommended as appropriate therapy for the subject. In some embodiments, if MART-1 expression (e.g., RNA expression or protein expression) is upregulated in the cancer cell, then a combination of an IL-2 polypeptide and a MEK inhibitor and/or a PI3K/AKT inhibitor is recommended as appropriate therapy for the subject.

[0149] In some instances, a combination of an IL-2 polypeptide and a MEK inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is positive and/or upregulated for MART-1 expression. In other instances, a combination of an IL-2 polypeptide and a PI3K/AKT inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is positive and/or upregulated for MART-1 expression. In yet other instances, a combination of an IL-2 polypeptide, a MEK inhibitor and a PI3K/AKT inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is positive and/or upregulated for MART-1 expression.

[0150] In some embodiments, if the cancer cell is negative for MART-1 expression (e.g., RNA expression or protein expression), then a combination of an IL-2 polypeptide (e.g., IL-2 immunotherapy) and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor is recommended as appropriate therapy for the subject. In some embodiments, if MART-1 expression (e.g., RNA expression or protein expression) is downregulated in the cancer cell, then a combination of an IL-2 polypeptide and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor is recommended as appropriate therapy for the subject.

[0151] In some instances, a combination of an IL-2 polypeptide and a BRAF inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In other instances, a combination of an IL-2 polypeptide and a PI3K/A T inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In yet other instances, a combination of an IL-2 polypeptide and a receptor tyrosine kinase inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In still yet other instances, a combination of an IL-2 polypeptide and a Hedgehog inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression.

[0152] In further instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, and a PI3K/AKT inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In other instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, and a receptor tyrosine kinase inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In yet other instances, a

combination of an IL-2 polypeptide, a BRAF inhibitor, and a Hedgehog inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In still yet other instances, a combination of an IL-2 polypeptide, a PI3K/AKT inhibitor, and a receptor tyrosine kinase inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In further instances, a combination of an IL-2 polypeptide, a PI3K/AKT inhibitor, and a Hedgehog inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In other instances, a combination of an IL-2 polypeptide, a receptor tyrosine kinase inhibitor, and a Hedgehog inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. [0153] In yet other instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, a PI3K/AKT inhibitor, and a receptor tyrosine kinase inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In still yet other instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, a PI3K/AKT inhibitor, and a Hedgehog inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In further instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, a receptor tyrosine kinase inhibitor, and a Hedgehog inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In other instances, a combination of an IL-2 polypeptide, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and a Hedgehog inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In yet other instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and a Hedgehog inhibitor is recommended as the appropriate therapy for the subject if the cancer cell is negative and/or downregulated for MART-1 expression.

[0154] In some embodiments, the MEK inhibitor is selected from the group consisting of PD325901, AZD 6244, trametinib, MEK 162, XL518, and combinations thereof.

[0155] In some embodiments, the PI3K/AKT inhibitor is selected from the group consisting of BEZ235, BKM120, BLY719, IPI-145, and combinations thereof.

[0156] In some embodiments, the BRAF inhibitor is selected from the group consisting of PLX-4032, dabrafenib, RAF265, LGX818, and combinations thereof.

[0157] In some embodiments, the receptor tyrosine kinase inhibitor is selected from the group consisting of lapatinib, sunitinib, sorafenib, axitinib, pazopanib, gefitinib, erlotinib, pilitinib, canertinib, CP-654577, CP-724714, ARRY-334543, JNJ-26483327, JNJ-26483327, MP-470, AZD8931, and combinations thereof.

[0158] In some embodiments, the HH inhibitor is selected from the group consisting of GDC0449, erismodegib, LEQ506, saridegib, IPI-269609, BMS-833932, PF-04449913, TAK- 441, and combinations thereof. [0159] In some embodiments, the IL-2 polypeptide comprises a recombinant IL-2 polypeptide. In some embodiments, the recombinant IL-2 polypeptide is PROLEUKIN^® (aldesleukin). In some embodiments, the IL-2 polypeptide is high dose (HD) IL-2. In other embodiments, the IL-2 polypeptide is low dose (LD) IL-2.

[0160] In some embodiments, it is recommended that HD IL-2 immunotherapy comprises at least one dose of IL-2 (e.g., recombinant IL-2 such as Proleukin^®) of about or at least about 600,000 IU/kg/dose, or about or at least about 720,000 IU/kg/dose. Examples of suitable ranges and additional types of dosing units (e.g., MIU daily, mg/kg/dose, etc.) are described herein.

[0161] In some embodiments, HD IL-2 includes a course of therapy comprising a treatment schedule of about 5 treatment days, about nine days of rest from treatment, and then about 5 additional days of treatment. In certain embodiments, the HD IL-2 course of therapy includes administration of IL-2 via intravenous (i.v.) injection as a bolus dose over about 15 minutes. In certain instances, during each 5 day treatment cycle, a dose is administered about every eight hours for a maximum of about 14 doses. In some instances, (metastatic) RCC patients treated with HD IL-2 receive a median of about 20 doses of the about 28 doses during the first course of therapy. In some instances, (metastatic) melanoma patients treated with HD IL-2 receive a median of about 18 doses of the about 28 doses during the first course of therapy.

[0162] In some embodiments, it is recommended that LD IL-2 immunotherapy comprises at least one dose of IL-2 (e.g., recombinant IL-2 such as Proleukin^®) of less than about 600,000 IU/kg/dose, such as about 60,000 or about 72,000 IU/kg/dose. Examples of suitable ranges and additional types of dosing units (e.g., MIU daily, etc.) are described herein.

[0163] In certain instances, LD IL-2 includes a course of therapy comprising a treatment schedule of about 5 treatment days, about nine days of rest from treatment, and then about 5 additional days of treatment, wherein the patient receives a bolus dose about every 8 hours for a maximum of about 14 doses during the 5 treatment days, optionally wherein the bolus dose is administered via i.v. injection of about 60,000 to about 72,000 IU/kg/dose over about 15 minutes. In other instances, LD IL-2 includes a treatment schedule of daily subcutaneous (s.c.) injections of IL-2 at about 250,000 lU/kg/day for about 1 week, followed by daily s.c. injections of about 125,000 IU/kg/day for about 5 weeks. [0164] In some embodiments, the IL-2 polypeptide and the targeted therapeutic agent (e.g., the BRAF inhibitor, PI3K AKT inhibitor, MEK inhibitor, receptor tyrosine kinase inhibitor and/or HH inhibitor) are recommended to be administered simultaneously during the course of therapy. In some embodiments, the IL-2 polypeptide and the targeted therapeutic agent (e.g., the BRAF inhibitor, PI3K/AKT inhibitor, MEK inhibitor, receptor tyrosine kinase inhibitor and/or HH inhibitor) are recommended to be administered sequentially during the course of therapy.

[0165] In some embodiments, the method of the present invention further comprises comparing the subject's symptom profile to that from a control subject. In some embodiments, a symptom profile is determined by identifying the presence or severity of at least one symptom in a subject compared to a control subject; and classifying the sample as a melanoma or metastatic melanoma sample. In some embodiments, the control subject is a healthy subject free of cancer. In some embodiments, the control subject is a subject having melanoma or metastatic melanoma. . In other embodiments, a symptom profile is determined by identifying the presence or severity of at least one symptom in a subject compared to a control subject; and classifying the sample as a RCC or metastatic RCC sample. In some embodiments, the control subject is a healthy subject free of cancer. In some embodiments, the control subject is a subject having RCC or metastatic RCC. [0166] In some aspects, the methods of the invention provide information useful for guiding treatment decisions for a patient receiving or about to receive combination IL-2 therapy, e.g., by selecting an appropriate targeted therapy to use in addition to IL-2 therapy for initial treatment, by determining when or how to combine a targeted therapy with IL-2 therapy, and/or by determining how or when to change the current course of therapy (e.g., switch to another targeted therapy that acts on a different signal transduction pathway).

[0167] In some aspects, the present invention provides methods for monitoring melanoma progression in a subject having melanoma, wherein the method comprises assaying the subject to determine the appropriate targeted therapy to combine with IL-2 therapy.

[0168] In other aspects, the present invention provides methods for monitoring RCC progression in a subject having RCC, wherein the method comprises assaying the subject to determine the appropriate targeted therapy to combine with IL-2 therapy.

[0169] In some embodiments, the present invention provides methods for monitoring drug efficacy in a subject receiving a combination of drugs useful for treating melanoma or RCC (e.g., metastatic or non-metastatic). C. Methods for Administering Combination IL-2 Therapy

[0170] In another embodiment, the present invention provides methods for administering an appropriate therapy for treating cancer in a subject, comprising: (a) determining whether a cancer cell obtained from the subject is positive (e.g., upregulated) or negative (e.g., downregulated) for MART-1 expression; and (b) administering an appropriate therapy to the subject.

[0171] In some embodiments, the method further comprises obtaining a sample from a subject. The sample may comprise any biological specimen as described herein obtained from the subject and typically contains at least one cancer cell. In particular embodiments, the sample is tumor tissue (e.g., fine needle aspirate sample obtained from a tumor), whole blood (e.g., circulating tumor cells isolated from blood), or a cellular extract thereof. In certain instances, the tumor tissue sample is obtained from a solid tumor of the skin, kidney, and/or other portion of the body.

[0172] In some embodiments, the subject has cancer or is suspected of having cancer. In some embodiments, the cancer is melanoma or metastatic melanoma. In some embodiments, the cancer is renal cell carcinoma or metastatic renal cell carcinoma. In some embodiments, the cancer is clear cell carcinoma. [0173] In some embodiments, step (a) of determining whether a cancer cell obtained from the subject is positive or negative for MART-1 expression comprises measuring (e.g., detecting) the presence or the level of MART-1 expression, wherein MART-1 expression is selected from the group consisting of protein expression, RNA expression, and combinations thereof. [0174] The presence or level of MART-1 RNA expression in a sample can be measured or detected using any of a variety of techniques (e.g., quantitative or semi-quantitative RT-PCR, microarray analysis, Northern blot analysis, solution hybridization detection, and the like). See, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989) and Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons (Hoboken, NY 2012).

[0175] The presence or level of MART-1 protein expression in a sample can be measured or detected using any of a variety of techniques (e.g., immunoassay, proximity dual detection assay (e.g., CEER), radioimmunoassay (RIA), ELISA, and immunoblotting (e.g., Western blotting); flow cytometry, immunohistochemistry, mass spectrometry, and the like). Detailed descriptions of a proximity dual detection assay (e.g., CEER) can be found in U.S. Pat. Pub. Nos. 2008/0261829 and 2011/0275097, the disclosures of which are herein incorporated by reference in their entirety for all purposes. Other specific types of immunoassays include antigen capture/antigen competition, antibody capture/antigen competition, two-antibody sandwiches, antibody capture/antibody excess, and antibody capture/antigen excess.

[0176] In some embodiments, an antibody that recognizes (e.g., is specific for, binds to, forms a complex with) an epitope of MART- 1 protein is useful in the methods of the present invention. Non-limiting examples of such an antibody include Melan-A antibodies (e.g., A 103). For instance, melan-A antibody (B- 10) recognizes an epitope between amino acids 81-1 17 near the C-terminus of MART- 1 and melan-A antibody (D-6) recognizes an epitope between 3-31 at the N-terminus of MART- 1. Antibodies to MART-1 are commercially available from, for example, but not limited to, Santa Cruz Biotechnology (Santa Cruz, CA), Novus Biologicals (Littleton, CO), Dako (Carpenteria, CA), Vector Laboratories

[0177] In some embodiments, the method further comprises measuring (e.g., detecting) the presence or level of RNA and/or protein expression of at least one other melanoma biomarker such as, but not limited to, gplOO, MITF, and/or tyrosinase.

[0178] In some embodiments, the method further comprises measuring (e.g., detecting) the presence or level of RNA and/or protein expression of at least one other RCC biomarker such as, but not limited to, p53, Ki67, CAIX, VEGF, SAA, IGF-1, NMP22, CXCR3, CXCR4, MMP2, MMP6, EpCAM, vimentin, fascin, livin, survivin, CD70, KIT, and/or KAI-1.

[0179] In some embodiments, positive expression of MART-1 is indicated by comparing the level of MART-1 expression in the sample from the subject to that of a control, and determining that the level of MART-1 expression is higher than that of the control, wherein the control does not express MART-1 or does not express detectable levels of MART-1. In other embodiments, positive expression of MART-1 is indicated by comparing the presence of MART-1 expression in the sample to that of a control (e.g., negative control), wherein MART-1 expression is absent in the control, and determining that the sample is positive for MART-1 expression.

[0180] In some embodiments, the control (e.g., negative control) is a sample from an individual that does not have cancer. In other embodiments, the control is a pooled sample from a plurality of individuals that do not have cancer. In some embodiments, the control is a sample from a non-cancerous cell line. In some embodiments, the control is a control value representing the level of MART-1 expression in a sample that is cancer-free. [0181] In other embodiments, positive expression of MART- 1 is indicated by comparing the level of MART- 1 expression in the sample to that of a control, and determining that the level of MART-1 expression is substantially equal or higher than that of the control (e.g., positive control), wherein the control expresses MART-1. [0182] In some embodiments, the control (e.g., positive control) is a sample from an individual with cancer such as melanoma or RCC. In other embodiments, the control is a pooled sample from a plurality of individuals who have cancer. In some embodiments, the control is a sample from a cancer cell line. Non-limiting examples of cancer cell lines useful to the invention include melanoma cell lines such as CHL-1, SK-MEL-2, A375, SK-MEL-28, NA8, D10, and HBL, and RCC cell lines such as 1581 RCC, 1764 RCC, 2194 RCC, SNU- 228, SNU-267, SNU-328, SNU-349, and SNU-1272. In some embodiments, the control is a control value representing the level of MART-1 expression in a sample from a cancer cell line.

[0183] In some embodiments, positive expression of MART-1 corresponds to upregulated expression of MART-1. For instance, MART-1 expression is determined to be upregulated when the level of expression (e.g., RNA or protein) is increased in the sample from the subject compared to that of a control (e.g., negative control or positive control).

[0184] In some embodiments, negative expression of MART-1 is indicated by comparing the level of MART-1 expression in the sample from the subject to that of a control (e.g., negative control), and determining that the level of MART-1 expression is substantially equal to that of the control, wherein the control does not express MART-1 or does not express detectable levels of MART-1.

[0185] In some embodiments, the control (e.g., negative control) is a sample from an individual that does not have cancer. In other embodiments, the control is a pooled sample from a plurality of individuals that do not have cancer. In some embodiments, the control is a control value representing the level of MART-1 expression in a cancer-free sample.

[0186] In some embodiments, negative expression of MART-1 is indicated by comparing the level of MART-1 expression in the sample from the subject to that of a control (e.g., positive control), and determining that the level of MART-1 expression is substantially lower than to that of the control (e.g., positive control), wherein the control expresses MART-1.

[0187] In some embodiments, negative expression of MART-1 corresponds to

[0188] In some embodiments, IL-2 therapy is combined with one or more (e.g., a plurality of) targeted therapeutic agents.

[0189] In some embodiments, if the cancer cell is positive for MART-1 expression (e.g., RNA expression or protein expression), then a combination of an IL-2 polypeptide (e.g., IL-2 immunotherapy) and a MEK inhibitor and/or a PI3K/AKT inhibitor is administered as the appropriate therapy to the subject. In some embodiments, if MART-1 expression (e.g., RNA expression or protein expression) is upregulated in the cancer cell, then a combination of an IL-2 polypeptide and a MEK inhibitor and/or a PI3K/AKT inhibitor is administered as the , appropriate therapy to the subject.

[0190] In some instances, a combination of an IL-2 polypeptide and a MEK inhibitor is administered as the appropriate therapy to the subject if the cancer cell is positive and/or upregulated for MART-1 expression. In other instances, a combination of an IL-2 polypeptide and a PI3K/AKT inhibitor is administered as the appropriate therapy to the subject if the cancer cell is positive and/or upregulated for MART-1 expression. In yet other instances, a combination of an IL-2 polypeptide, a MEK inhibitor and a PI3K/AKT inhibitor is administered as the appropriate therapy to the subject if the cancer cell is positive and/or upregulated for MART-1 expression.

[0191] In some embodiments, if the cancer cell is negative for MART-1 expression (e.g., RNA expression or protein expression), then a combination of an IL-2 polypeptide (e.g., IL-2 immunotherapy) and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor is administered as the appropriate therapy to the subject. In some embodiments, if MART-1 expression (e.g., RNA expression or protein expression) is downregulated in the cancer cell, then a combination of an IL-2 polypeptide and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor is administered as the appropriate therapy to the subject.

[0192] In some instances, a combination of an IL-2 polypeptide and a BRAF inhibitor is administered as the appropriate therapy to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In other instances, a combination of an IL-2 polypeptide and a PI3K/AKT inhibitor is administered as the appropriate therapy to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In yet other instances, a combination of an IL-2 polypeptide and a receptor tyrosine kinase inhibitor is administered as the appropriate therapy to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In still yet other instances, a combination of an IL-2 polypeptide and a Hedgehog inhibitor is administered as the appropriate therapy to the subject if the cancer cell is negative and/or downregulated for MART-1 expression.

[0193] In further instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, and a PI3K/AKT inhibitor is administered as the appropriate therapy to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In other instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, and a receptor tyrosine kinase inhibitor is administered as the appropriate therapy to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In yet other instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, and a Hedgehog inhibitor is administered as the appropriate therapy to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In still yet other instances, a combination of an IL-2 polypeptide, a PI3K/AKT inhibitor, and a receptor tyrosine kinase inhibitor is administered as the appropriate therapy to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In further instances, a combination of an IL-2 polypeptide, a PI3K/AKT inhibitor, and a Hedgehog inhibitor is administered as the appropriate therapy to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In other instances, a combination of an IL-2 polypeptide, a receptor tyrosine kinase inhibitor, and a Hedgehog inhibitor is administered as the appropriate therapy to the subject if the cancer cell is negative and/or downregulated for MART-1 expression.

[0194] In yet other instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, a PI3K/AKT inhibitor, and a receptor tyrosine kinase inhibitor is administered as the appropriate therapy to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In still yet other instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, a PI3K/AKT inhibitor, and a Hedgehog inhibitor is administered as the appropriate therapy to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In further instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, a receptor tyrosine kinase inhibitor, and a Hedgehog inhibitor is administered as the appropriate therapy to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In other instances, a combination of an IL-2 polypeptide, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and a Hedgehog inhibitor is administered as the appropriate therapy to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In yet other instances, a combination of an IL-2 polypeptide, a BRAF inhibitor, a PI3 /AKT inhibitor, a receptor tyrosine kinase inhibitor, and a Hedgehog inhibitor is administered as the appropriate therapy to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. [0195] In some embodiments, the MEK inhibitor is selected from the group consisting of PD325901, AZD 6244, trametinib, MEK 162, XL518, and combinations thereof.

[0196] In some embodiments, the PI3K/AKT inhibitor is selected from the group consisting of BEZ235, BKM120, BLY719, IPI-145, and combinations thereof.

[0197] In some embodiments, the BRAF inhibitor is selected from the group consisting of PLX-4032, dabrafenib, RAF265, LGX818, and combinations thereof.

[0198] In some embodiments, the receptor tyrosine kinase inhibitor is selected from the group consisting of lapatinib, sunitinib, sorafenib, axitinib, pazopanib, gefitinib, erlotinib, pilitinib, canertinib, CP-654577, CP-724714, ARRY-334543, JNJ-26483327, JNJ-26483327, MP-470, AZD8931, and combinations thereof. [0199] In some embodiments, the HH inhibitor is selected from the group consisting of

GDC0449, erismodegib, LEQ506, saridegib, IPI-269609, BMS-833932, PF-04449913, TAK- 441, and combinations thereof.

[0200] In some embodiments, the IL-2 polypeptide comprises a recombinant IL-2 polypeptide. In some embodiments, the recombinant IL-2 polypeptide is PROLEUKIN^® (aldesleukin). In some embodiments, the IL-2 polypeptide is high dose (HD) IL-2. In other embodiments, the IL-2 polypeptide is low dose (LD) IL-2.

[0201] In some embodiments, HD IL-2 immunotherapy comprises at least one dose of IL-2 (e.g., recombinant IL-2 such as Proleukin^®) of about or at least about 600,000 IU/kg/dose, or about or at least about 720,000 IU/kg/dose. Examples of suitable ranges and additional types of dosing units (e.g., MIU daily, mg/kg/dose, etc.) are described herein.

[0202] In some embodiments, HD IL-2 includes a course of therapy comprising a treatment schedule of about 5 treatment days, about nine days of rest from treatment, and then about 5 additional days of treatment. In certain embodiments, the HD IL-2 course of therapy includes administration of IL-2 via intravenous (i.v.) injection as a bolus dose over about 15 minutes. In certain instances, during each 5 day treatment cycle, a dose is administered about every eight hours for a maximum of about 14 doses. In some instances, (metastatic) RCC patients treated with HD IL-2 receive a median of about 20 doses of the about 28 doses during the first course of therapy. In some instances, (metastatic) melanoma patients treated with HD IL-2 receive a median of about 18 doses of the about 28 doses during the first course of therapy.

[0203] In some embodiments, LD IL-2 immunotherapy comprises at least one dose of IL-2 (e.g., recombinant IL-2 such as Proleukin^®) of less than about 600,000 IU/kg/dose, such as about 60,000 or about 72,000 IU/kg/dose. Examples of suitable ranges and additional types of dosing units (e.g., MIU daily, etc.) are described herein.

[0204] In certain instances, LD IL-2 includes a course of therapy comprising a treatment schedule of about 5 treatment days, about nine days of rest from treatment, and then about 5 additional days of treatment, wherein the patient receives a bolus dose about every 8 hours for a maximum of about 14 doses during the 5 treatment days, optionally wherein the bolus dose is administered via i.v. injection of about 60,000 to about 72,000 IU kg/dose over about 15 minutes. In other instances, LD IL-2 includes a treatment schedule of daily subcutaneous (s.c.) injections of IL-2 at about 250,000 IU/kg/day for about 1 week, followed by daily s.c. injections of about 125,000 IU/kg/day for about 5 weeks.

[0205] In some embodiments, the IL-2 polypeptide and the targeted therapeutic agent (e.g., the BRAF inhibitor, PI3K/AKT inhibitor, MEK inhibitor, receptor tyrosine kinase inhibitor and/or HH inhibitor) are administered simultaneously. In some embodiments, the IL-2 polypeptide and the targeted therapeutic agent (e.g., the BRAF inhibitor, PI3K/AKT inhibitor, MEK inhibitor, receptor tyrosine kinase inhibitor and/or HH inhibitor) are administered sequentially. In some embodiments, the IL-2 polypeptide and the targeted therapeutic agent are administered alternately. In other embodiments, the course of therapy for IL-2 polypeptide and the course of therapy for the targeted therapy are in succession.

[0206] In some embodiments, the method of the present invention further comprises comparing the subject's symptom profile to that from a control subject. In some

embodiments, a symptom profile is determined by identifying the presence or severity of at least one symptom in a subject compared to a control subject; and classifying the sample as a melanoma or metastatic melanoma sample. In some embodiments, the control subject is a healthy subject free of cancer. In some embodiments, the control subject is a subject having melanoma or metastatic melanoma. In other embodiments, a symptom profile is determined by identifying the presence or severity of at least one symptom in a subject compared to a control subject; and classifying the sample as a RCC or metastatic RCC sample. In some embodiments, the control subject is a healthy subject free of cancer. In some embodiments, the control subject is a subject having RCC or metastatic RCC.

[0207] In some aspects, the methods of the invention provide information useful for guiding treatment decisions for a patient receiving or having received combination IL-2 therapy, e.g., by selecting an appropriate targeted therapy to use in addition to IL-2 therapy after initial treatment, by determining when or how to combine a targeted therapy with IL-2 therapy, and/or by determining how or when to change the current course of therapy (e.g., switch to another targeted therapy that acts on a different signal transduction pathway).

[0208] In some aspects, the present invention provides methods for monitoring melanoma progression in a subject having melanoma and receiving therapy, wherein the method comprises assaying the subject to determine the appropriate targeted therapy to combine with IL-2 therapy.

[0209] In other aspects, the present invention provides methods for monitoring RCC progression in a subject having RCC and receiving therapy, wherein the method comprises assaying the subject to determine the appropriate targeted therapy to combine with IL-2 therapy.

D. Methods for Treating Cancer Using Combination IL-2 Therapy

[0210] In some embodiments, the present invention provides methods for treating cancer in a subject using a combination of IL-2 therapy with one or more targeted therapeutic agents after determining MART-1 expression in a cancer cell (e.g., obtained from the subject), comprising:

(a) administering a combination of an IL-2 polypeptide (e.g., recombinant IL-2 such as Proleukin^®) and a MEK inhibitor and/or a PI3K/AKT inhibitor to the subject if the cell is positive (e.g., upregulated) for MART-1 expression; or

(b) administering a combination of an IL-2 polypeptide (e.g., recombinant

IL-2 such as Proleukin^®) and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor to the subject if the cell is negative (e.g., downregulated) for MART-1 expression, to effectively treat cancer.

[0211] In other embodiments, the present invention provides methods for combining IL-2 therapy with one or more targeted therapeutic agents after determining MART-1 expression in a cancer cell obtained from a subject, comprising: (a) administering a combination of an IL-2 polypeptide (e.g., recombinant IL-2 such as Proleukin^®) and a MEK inhibitor and/or a PI3K/AKT inhibitor to the subject if the cell is positive (e.g., upregulated) for MART-1 expression; or

(b) administering a combination of an IL-2 polypeptide (e.g., recombinant IL-2 such as Proleukin®) and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor to the subject if the cell is negative (e.g., downregulated) for MART-1 expression.

[0212] In other embodiments, the present invention provides methods for treating cancer in a subject using a combination of IL-2 therapy with one or more targeted therapeutic agents based on MART-1 expression in a cancer cell (e.g., obtained from the subject), comprising:

(a) administering a combination of an IL-2 polypeptide (e.g., recombinant IL-2 such as Proleukin^®) and a MEK inhibitor and/or a PBK/AKT inhibitor to the subject if the cell is positive (e.g., upregulated) for MART-1 expression; or

(b) administering a combination of an IL-2 polypeptide (e.g., recombinant IL-2 such as Proleukin®) and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor to the subject if the cell is negative (e.g., downregulated) for MART-1 expression, to effectively treat cancer.

[0213] In other embodiments, the present invention provides methods for combining IL-2 therapy with one or more targeted therapeutic agents based on MART-1 expression in a cancer cell obtained from a subject, comprising:

(a) administering a combination of an IL-2 polypeptide (e.g., recombinant IL- 2 such as Proleukin^®) and a MEK inhibitor and/or a PI3K/AKT inhibitor to the subject if the cell is positive (e.g., upregulated) for MART-1 expression; or

(b) administering a combination of an IL-2 polypeptide (e.g., recombinant IL- 2 such as Proleukin^®) and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor to the subject if the cell is negative (e.g., downregulated) for MART-1 expression.

[0214] In some embodiments, the method further comprises obtaining a sample from a subject. The sample may comprise any biological specimen as described herein obtained from the subject and typically contains at least one cancer cell. In particular embodiments, the sample is tumor tissue (e.g., fine needle aspirate sample obtained from a tumor), whole blood (e.g., circulating tumor cells isolated from blood), or a cellular extract thereof. In certain instances, the tumor tissue sample is obtained from a solid tumor of the skin, kidney, and/or other portion of the body. [0215] In some embodiments, the subject has cancer or is suspected of having cancer. In some embodiments, the cancer is melanoma or metastatic melanoma. In some embodiments, the cancer is renal cell carcinoma or metastatic renal cell carcinoma. In some embodiments, the cancer is clear cell carcinoma. [0216] In some embodiments, MART-1 expression in a cancer cell was determined by or based on measuring (e.g., detecting) the presence or the level of MART-1 protein expression, RNA expression, or combinations thereof. Suitable techniques for measuring the presence or level of MART-1 expression are described herein.

[0217] In some embodiments, the methods are further based on the presence or level of RNA and/or protein expression of at least one other melanoma biomarker such as, but not limited to, gplOO, MITF, and/or tyrosinase.

[0218] In other embodiments, the methods are further based on the presence or level of RNA and/or protein expression of at least one other RCC biomarker such as, but not limited to, p53, Ki67, CAIX, VEGF, SAA, IGF-1, NMP22, CXCR3, CXCR4, MMP2, MMP6, EpCAM, vimentin, fascin, livin, survivin, CD70, KIT, and/or KAI-1.

[0219] In some embodiments, positive expression of MART-1 is indicated by comparing the level of MART-1 expression in the sample from the subject to that of a control, and determining that the level of MART-1 expression is higher than that of the control, wherein the control does not express MART-1 or does not express detectable levels of MART-1. In other embodiments, positive expression of MART-1 is indicated by comparing the presence of MART-1 expression in the sample to that of a control (e.g., negative control), wherein MART-1 expression is absent in the control, and determining that the sample is positive for MART-1 expression.

[0220] In some embodiments, the control (e.g., negative control) is a sample from an individual that does not have cancer. In other embodiments, the control is a pooled sample from a plurality of individuals that do not have cancer. In some embodiments, the control is a sample from a non-cancerous cell line. In some embodiments, the control is a control value representing the level of MART-1 expression in a sample that is cancer-free.

[0221] In other embodiments, positive expression of MART-1 is indicated by comparing the level of MART-1 expression in the sample to that of a control, and determining that the level of MART-1 expression is substantially equal or higher than that of the control (e.g., positive control), wherein the control expresses MART-1. [0222] In some embodiments, the control (e.g., positive control) is a sample from an individual with cancer such as melanoma or RCC. In other embodiments, the control is a pooled sample from a plurality of individuals who have cancer. In some embodiments, the control is a sample from a cancer cell line. Non-limiting examples of cancer cell lines useful to the invention include melanoma cell lines such as CHL-1 , SK-MEL-2, A375, SK-MEL-28, NA8, D10, and HBL, and RCC cell lines such as 1581 RCC, 1764 RCC, 2194 RCC, SNU- 228, SNU-267, SNU-328, SNU-349, and SNU-1272. In some embodiments, the control is a control value representing the level of MART- 1 expression in a sample from a cancer cell line.

[0223] In some embodiments, positive expression of MART- 1 corresponds to upregulated expression of MART- 1. For instance, MART-1 expression is determined to be upregulated when the level of expression (e.g., RNA or protein) is increased in the sample from the subject compared to that of a control (e.g., negative control or positive control).

[0224] In some embodiments, negative expression of MART-1 is indicated by comparing the level of MART-1 expression in the sample from the subject to that of a control (e.g., negative control), and determining that the level of MART-1 expression is substantially equal to that of the control, wherein the control does not express MART-1 or does not express detectable levels of MART-1.

[0225] In some embodiments, the control (e.g., negative control) is a sample from an individual that does not have cancer. In other embodiments, the control is a pooled sample from a plurality of individuals that do not have cancer. In some embodiments, the control is a control value representing the level of MART-1 expression in a cancer-free sample.

[0226] In some embodiments, negative expression of MART-1 is indicated by comparing the level of MART-1 expression in the sample from the subject to that of a control (e.g., positive control), and determining that the level of MART-1 expression is substantially lower than to that of the control (e.g., positive control), wherein the control expresses MART-1.

[0227] In some embodiments, negative expression of MART-1 corresponds to

downregulated expression of MART-1. For instance, MART-1 expression is determined to be downregulated when the level of expression (e.g., RNA or protein) is decreased in the sample from the subject compared to that of a control (e.g., negative control or positive control). [0228] In some embodiments, the methods comprise administering a combination of an IL- 2 polypeptide and a MEK inhibitor to the subject if the cancer cell is positive and/or upregulated for MART-1 expression. In other embodiments, the methods comprise administering a combination of an IL-2 polypeptide and a PI3K/AKT inhibitor to the subject if the cancer cell is positive and/or upregulated for MART-1 expression. In yet other embodiments the methods comprise administering a combination of an IL-2 polypeptide, a MEK inhibitor and a PI3K/AKT inhibitor to the subject if the cancer cell is positive and/or upregulated for MART-1 expression.

[0229] In some embodiments, the methods comprise administering a combination of an IL- 2 polypeptide and a BRAF inhibitor to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In other embodiments, the methods comprise administering a combination of an IL-2 polypeptide and a PI3K/AKT inhibitor to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In yet other embodiments, the methods comprise administering a combination of an IL-2 polypeptide and a receptor tyrosine kinase inhibitor to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In still yet other embodiments, the methods comprise administering a combination of an IL-2 polypeptide and a Hedgehog inhibitor to the subject if the cancer cell is negative and/or downregulated for MART-1 expression.

[0230] In further embodiments, the methods comprise administering a combination of an IL-2 polypeptide, a BRAF inhibitor, and a PI3K/AKT inhibitor to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In other embodiments, the methods comprise administering a combination of an IL-2 polypeptide, a BRAF inhibitor, and a receptor tyrosine kinase inhibitor to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In yet other embodiments, the methods comprise administering a combination of an IL-2 polypeptide, a BRAF inhibitor, and a Hedgehog inhibitor to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In still yet other embodiments, the methods comprise administering a combination of an IL-2 polypeptide, a PI3K/AKT inhibitor, and a receptor tyrosine kinase inhibitor to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In further embodiments, the methods comprise administering a combination of an IL-2 polypeptide, a PI3K/AKT inhibitor, and a Hedgehog inhibitor to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In other embodiments, the methods comprise administering a combination of an IL-2 polypeptide, a receptor tyrosine kinase inhibitor, and a Hedgehog inhibitor to the subject if the cancer cell is negative and/or downregulated for MART-1 expression.

[0231] In yet other embodiments, the methods comprise administering a combination of an IL-2 polypeptide, a BRAF inhibitor, a PI3K/AKT inhibitor, and a receptor tyrosine kinase inhibitor to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In still yet other embodiments, the methods comprise administering a combination of an IL-2 polypeptide, a BRAF inhibitor, a PI3K/AKT inhibitor, and a Hedgehog inhibitor to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In further embodiments, the methods comprise administering a combination of an IL-2 polypeptide, a BRAF inhibitor, a receptor tyrosine kinase inhibitor, and a Hedgehog inhibitor to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In other embodiments, the methods comprise administering a combination of an IL-2 polypeptide, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and a Hedgehog inhibitor to the subject if the cancer cell is negative and/or downregulated for MART-1 expression. In yet other embodiments, the methods comprise administering a combination of an IL-2 polypeptide, a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and a Hedgehog inhibitor to the subject if the cancer cell is negative and/or downregulated for MART-1 expression.

[0232] In some embodiments, the MEK inhibitor is selected from the group consisting of PD325901, AZD 6244, trametinib, MEK162, XL518, and combinations thereof.

[0233] In some embodiments, the PI3K/AKT inhibitor is selected from the group consisting of BEZ235, BKM120, BLY719, IPI-145, and combinations thereof.

[0234] In some embodiments, the BRAF inhibitor is selected from the group consisting of PLX-4032, dabrafenib, RAF265, LGX818, and combinations thereof. [0235] In some embodiments, the receptor tyrosine kinase inhibitor is selected from the group consisting of lapatinib, sunitinib, sorafenib, axitinib, pazopanib, gefitinib, erlotinib, pilitinib, canertinib, CP-654577, CP-724714, ARRY-334543, JNJ-26483327, JNJ-26483327, MP-470, AZD8931, and combinations thereof.

[0236] In some embodiments, the HH inhibitor is selected from the group consisting of GDC0449, erismodegib, LEQ506, saridegib, IPI-269609, BMS-833932, PF-04449913, TAK- 441, and combinations thereof. [0237] In some embodiments, the IL-2 polypeptide comprises a recombinant IL-2 polypeptide. In some embodiments, the recombinant IL-2 polypeptide is PROLEUKIN^® (aldesleukin). In some embodiments, the IL-2 polypeptide is high dose (HD) IL-2. In other embodiments, the IL-2 polypeptide is low dose (LD) IL-2.

[0238] In some embodiments, HD IL-2 immunotherapy comprises at least one dose of IL-2 (e.g., recombinant IL-2 such as Proleukin^®) of about or at least about 600,000 IU/kg/dose, or about or at least about 720,000 IU/kg/dose. Examples of suitable ranges and additional types of dosing units (e.g., MIU daily, mg/kg/dose, etc.) are described herein.

[0239] In some embodiments, HD IL-2 includes a course of therapy comprising a treatment schedule of about 5 treatment days, about nine days of rest from treatment, and then about 5 additional days of treatment. In certain embodiments, the HD IL-2 course of therapy includes administration of IL-2 via intravenous (i.v.) injection as a bolus dose over about 15 minutes. In certain instances, during each 5 day treatment cycle, a dose is administered about every eight hours for a maximum of about 14 doses. In some instances, (metastatic) RCC patients treated with HD IL-2 receive a median of about 20 doses of the about 28 doses during the first course of therapy. In some instances, (metastatic) melanoma patients treated with HD IL-2 receive a median of about 18 doses of the about 28 doses during the first course of therapy.

[0240] In some embodiments, LD IL-2 immunotherapy comprises at least one dose of IL-2 (e.g., recombinant IL-2 such as Proleukin^®) of less than about 600,000 IU/kg/dose, such as about 60,000 or about 72,000 IU/kg/dose. Examples of suitable ranges and additional types of dosing units (e.g., MIU daily, etc.) are described herein.

[0241] In certain instances, LD IL-2 includes a course of therapy comprising a treatment schedule of about 5 treatment days, about nine days of rest from treatment, and then about 5 additional days of treatment, wherein the patient receives a bolus dose about every 8 hours for a maximum of about 14 doses during the 5 treatment days, optionally wherein the bolus dose is administered via i.v. injection of about 60,000 to about 72,000 IU/kg/dose over about 15 minutes. In other instances, LD IL-2 includes a treatment schedule of daily subcutaneous (s.c.) injections of IL-2 at about 250,000 IU/kg/day for about 1 week, followed by daily s.c. injections of about 125,000 IU/kg/day for about 5 weeks.

[0242] In some embodiments, the IL-2 polypeptide and the targeted therapeutic agent (e.g., the BRAF inhibitor, PI3K AKT inhibitor, MEK inhibitor, receptor tyrosine kinase inhibitor and/or HH inhibitor) are administered simultaneously. In some embodiments, the IL-2 polypeptide and the targeted therapeutic agent (e.g., the BRAF inhibitor, PI3K/AKT inhibitor, MEK inhibitor, receptor tyrosine kinase inhibitor and/or HH inhibitor) are administered sequentially. In some embodiments, the IL-2 polypeptide and the targeted therapeutic agent are administered alternately. In other embodiments, the course of therapy for IL-2 polypeptide and the course of therapy for the targeted therapy are in succession.

[0243] In some embodiments, the methods of the invention further comprise comparing the subject's symptom profile to that from a control subject. In some embodiments, a symptom profile is determined by identifying the presence or severity of at least one symptom in a subject compared to a control subject; and classifying the sample as a melanoma or metastatic melanoma sample. In some embodiments, the control subject is a healthy subject free of cancer. In some embodiments, the control subject is a subject having melanoma or metastatic melanoma. In other embodiments, a symptom profile is determined by identifying the presence or severity of at least one symptom in a subject compared to a control subject; and classifying the sample as a RCC or metastatic RCC sample. In some embodiments, the control subject is a healthy subject free of cancer. In some embodiments, the control subject is a subject having RCC or metastatic RCC.

[0244] In some aspects, the methods of the invention provide information useful for guiding treatment decisions for a patient receiving or having received combination IL-2 therapy, e.g., by selecting an appropriate targeted therapy to use in addition to IL-2 therapy after initial treatment, by determining when or how to combine a targeted therapy with IL-2 therapy, and/or by determining how or when to change the current course of therapy (e.g., switch to another targeted therapy that acts on a different signal transduction pathway).

IV. Methods of Administration

[0245] According to the methods of the present invention, anticancer drugs such as IL-2 immunotherapy and targeted therapeutic agents described herein are administered to a subject by any convenient means known in the art. The methods of the present invention can be used to select a suitable anticancer drug or combination of anticancer drugs for the treatment of a tumor, e.g., melanoma or RCC, in a subject. The methods of the present invention can also be used to identify the response of a tumor, e.g., melanoma or RCC, in a subject to treatment with an anticancer drug or combination of anticancer drugs. In addition, the methods of the present invention can be used to predict the response of a subject having a tumor, e.g., melanoma or RCC, to treatment with an anticancer drug or combination of anticancer drugs. One skilled in the art will appreciate that the anticancer drugs described herein can be administered alone or as part of a combined therapeutic approach with conventional chemotherapy, radiotherapy, hormonal therapy, immunotherapy, and/or surgery.

[0246] In certain embodiments, the anticancer drug comprises an anti-signaling agent (i.e., a cytostatic drug or a targeted therapeutic agent) such as small molecule inhibitor that targets a signaling pathway active in cancer; an anti-proliferative agent; a chemotherapeutic agent (i.e., a cytotoxic drug); a hormonal therapeutic agent; a radiotherapeutic agent; a vaccine; and/or any other compound with the ability to reduce or abrogate the uncontrolled growth of aberrant cells such as cancerous cells. In some embodiments, the subject is treated with one or more of these anticancer drugs. [0247] Anticancer drugs can be administered with a suitable pharmaceutical excipient as necessary and can be carried out via any of the accepted modes of administration. Thus, administration can be, for example, oral, buccal, sublingual, gingival, palatal, intravenous, topical, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intravesical, intrathecal, intralesional, intranasal, rectal, vaginal, or by inhalation. By "co-administer" it is meant that an anticancer drug is administered at the same time, just prior to, or just after the administration of a second drug (e.g., another anticancer drug, a drug useful for reducing the side-effects associated with anticancer drug therapy, a radiotherapeutic agent, a hormonal therapeutic agent, an immunotherapeutic agent, etc.). [0248] A therapeutically effective amount of an anticancer drug may be administered repeatedly, e.g., at least 2, 3, 4, 5, 6, 7, 8, or more times, or the dose may be administered by continuous infusion. The dose may take the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, pills, pellets, capsules, powders, solutions, suspensions, emulsions, suppositories, retention enemas, creams, ointments, lotions, gels, aerosols, foams, or the like, preferably in unit dosage forms suitable for simple administration of precise dosages.

[0249] As used herein, the term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of an anticancer drug calculated to produce the desired onset, tolerability, and/or therapeutic effects, in association with a suitable pharmaceutical excipient (e.g., an ampoule). In addition, more concentrated dosage forms may be prepared, from which the more dilute unit dosage forms may then be produced. The more concentrated dosage forms thus will contain substantially more than, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times the amount of the anticancer drug.

[0250] Methods for preparing such dosage forms are known to those skilled in the art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, 18TH ED., Mack Publishing Co., Easton, PA (1990)). The dosage forms typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, adjuvants, diluents, tissue permeation enhancers, solubilizers, and the like. Appropriate excipients can be tailored to the particular dosage form and route of administration by methods well known in the art (see, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra). [0251] Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols, e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc. The dosage forms can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preserving agents such as methyl-, ethyl-, and propyl-hydroxy-benzoates (i.e., the parabens); pH adjusting agents such as inorganic and organic acids and bases; sweetening agents; and flavoring agents. The dosage forms may also comprise biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.

[0252] For oral administration, the therapeutically effective dose can be in the form of tablets, capsules, emulsions, suspensions, solutions, syrups, sprays, lozenges, powders, and sustained-release formulations. Suitable excipients for oral administration include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.

[0253] In some embodiments, the therapeutically effective dose takes the form of a pill, tablet, or capsule, and thus, the dosage form can contain, along with an anticancer drug, any of the following: a diluent such as lactose, sucrose, dicalcium phosphate, and the like; a disintegrant such as starch or derivatives thereof; a lubricant such as magnesium stearate and the like; and a binder such a starch, gum acacia, polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof. An anticancer drug can also be formulated into a suppository disposed, for example, in a polyethylene glycol (PEG) carrier. [0254] Liquid dosage forms can be prepared by dissolving or dispersing an anticancer drug and optionally one or more pharmaceutically acceptable adjuvants in a carrier such as, for example, aqueous saline (e.g., 0.9% w/v sodium chloride), aqueous dextrose, glycerol, ethanol, and the like, to form a solution or suspension, e.g., for oral, topical, or intravenous administration. An anticancer drug can also be formulated into a retention enema.

[0255] For topical administration, the therapeutically effective dose can be in the form of emulsions, lotions, gels, foams, creams, jellies, solutions, suspensions, ointments, and transdermal patches. For administration by inhalation, an anticancer drug can be delivered as a dry powder or in liquid form via a nebulizer. For parenteral administration, the

therapeutically effective dose can be in the form of sterile injectable solutions and sterile packaged powders. Preferably, injectable solutions are formulated at a pH of from about 4.5 to about 7.5.

[0256] The therapeutically effective dose can also be provided in a lyophilized form. Such dosage forms may include a buffer, e.g., bicarbonate, for reconstitution prior to

administration, or the buffer may be included in the lyophilized dosage form for

reconstitution with, e.g., water. The lyophilized dosage form may further comprise a suitable vasoconstrictor, e.g., epinephrine. The lyophilized dosage form can be provided in a syringe, optionally packaged in combination with the buffer for reconstitution, such that the reconstituted dosage form can be immediately administered to a subject.

[0257] A subject can also be monitored at periodic time intervals to assess the efficacy of a certain therapeutic regimen. For example, the presence and/or level of a specific marker may change based on the therapeutic effect of treatment with one or more of the anticancer drugs described herein. The subject can be monitored to assess response and understand the effects of certain drugs or treatments in an individualized approach. Additionally, subjects who initially respond to a specific anticancer drug or combination of anticancer drugs may become refractory to the drug or drug combination, indicating that these subjects have developed acquired drug resistance. These subjects can be discontinued on their current therapy and an alternative treatment prescribed in accordance with the methods of the present invention.

[0258] In certain aspects, the methods described herein can be used in conjunction with panels of gene expression markers that predict the likelihood of cancer prognosis and/or recurrence in various populations. These gene panels can be useful for identifying individuals who are unlikely to experience recurrence and, thus, unlikely to benefit from adjuvant chemotherapy. The expression panels can be used to identify individuals who can safely avoid adjuvant chemotherapy, without negatively affecting disease-free and overall survival outcomes. Suitable systems include, but are not limited to, Oncotype DX™, which is a 21 -gene panel from Genomic Health, Inc.; MammaPrint,^® which is a 70-gene panel from Agendia; and a 76-gene panel from Veridex.

[0259] In addition, in certain other aspects, the methods described herein can be used in conjunction with panels of gene expression markers that identify the original tumors for cancers of unknown primary (CUP). These gene panels can be useful in identifying patients with metastatic cancer who would benefit from therapy consistent with that given to patients diagnosed initially with cancer. Suitable systems include, but are not limited to, the Aviara CancerTYPE ID assay, an RT-PCR-based expression assay that measures 92 genes to identify the primary site of origin for 39 tumor types; and the Pathwork^® Tissue of Origin Test, which measures the expression of more than 1600 genes on a microarray and compares a tumor's gene expression "signature" against those of 15 known tissue types." V. Example

[0260] The present invention will be described in greater detail by way of specific example. The following example is offered for illustrative purposes, and is not intended to limit the invention in any manner. One of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results. Example 1. Determination of Rational Combination of High Dose (HD) IL-2 with New Targeted Therapies.

[0261] This example demonstrates the selection of an optimal drug combination of IL-2 immunotherapy with targeted therapies to cripple melanoma tumor cell growth in an in vitro tumor/immune cell co-culture model and in vivo animal models. In particular, this example provides the rational design of combination therapies in clinical trials to improve the survival durable rates of melanoma cancer patients by a multi-therapeutic modality.

[0262] In certain embodiments, this example provides an understanding of the mechanism of action of combination strategies in preclinical studies that is informative for the design of individualized combination treatment strategies in the clinic. Exemplary Embodiments of the Invention

[0263] In a first aspect, the present example delineates the dose effect of IL-2 on immune cell and tumor cell functions. It has been reported that IL-2 regulates immune system functions in a dose dependent manner. For instance, at high doses (HD), IL-2 stimulates immune cell activation. This has been translated into clinical treatment of metastatic melanoma and renal cell carcinoma (RCC) using recombinant IL-2 (Proleukin^®). In one embodiment, the objective is to establish and validate a tumor and immune cell co-culture model. In another embodiment, the objective is to determine the dose effect of IL-2 on immune cells. In certain instances, tumor and immune cell co-culture are treated with LD and HD IL-2 for several time points. Activation of T lymphocytes as reflected by the change in CD markers and the change in Treg cell population are measured by flow cytometry.

[0264] In a second aspect, the present example elucidates the therapeutic effect of using a combination of HD IL-2 with BRAF and MEK inhibitors in tumor/immune cell co-culture. In one embodiment, the objective is to assess the sensitivity of a panel of tumor cell lines carrying BRAF mutations to IL-2 with a panel of targeted agents for metastatic melanoma that are currently undergoing clinical evaluation. Inhibition of tumor cell growth in single agent and drug combination treatment can be determined by Cell-Titer-Glo. In another embodiment, the objective is to assess the sensitivity of a panel of tumor cell lines carrying wild-type BRAF to IL-2 with the same panel of targeted agents for metastatic melanoma.

[0265] In a third aspect, the present example defines the effect on immune cell functions by combining HD IL-2 with BRAF and MEK inhibitors in a tumor/immune cell co-culture system. In one embodiment, the objective is to explore the role of combination treatment on BRAF wild-type cells. In another embodiment, the objective is to determine the efficacy of combination regimens on tumor cells carrying mutant BRAF genes.

[0266] In a fourth aspect, the present example describes testing drug combinations selected from the tumor/immune cell co-culture study in mammalian (e.g., rodent models). Effective drug combinations as selected by the in vitro tumor/immune cell co-culture are further tested in animal models to verify their efficacy in vivo. The effect of drug sequence on tumor inhibition in vivo can also be investigated.

[0267] In one particular aspect, this example is directed to the rational design of combining high dose (HD) IL-2 with targeted therapies for the treatment of metastatic melanoma.

Introduction

[0268] Melanoma is the cause of about 75% of the deaths related to skin cancer (Jerant AF, Johnson JT, Sheridan CD, Caffrey TJ (2000). "Early detection and treatment of skin cancer". Am Fam Physician 62 (2): 357-68). Worldwide about 160,000 new cases of melanoma are diagnosed yearly, and according to a WHO report, about 48,000 melanoma related deaths occur per year (Lucas, R (2006). Global Burden of Disease of Solar Ultraviolet Radiation, Environmental Burden of Disease Series, No. 13. News release, World Health Organization). Melanoma is the 6th most common malignancy in men and women in the United States. The incidence of melanoma has been increasing worldwide at a rate faster than any other solid malignancy. The overall survival for patients with metastatic melanoma is poor, with a median survival of only 7.5 months after diagnosis. Thus, better forms of treatment are urgently needed.

[0269] One promising area of treatment for patients with melanoma involves the use of immunotherapy. The basis of immunotherapy is to activate a person's own immune system so that it will destroy the melanoma cells within the body. IL-2 (Proleukin^®), a T cell growth factor, is currently FDA-approved for the treatment of stage IV melanoma. However, Proleukin^® treatment only results in an objective response rate of approximately 16% obtained in the treated patients, and a complete response rate of 6%. Nevertheless, complete responses are most often durable. Thus, the longevity of clinical response is well established for this approach. However, the overall percentage of responding patients remains low even with careful selection of patients on clinical grounds. Studies have demonstrated that IL-2 offers the possibility of a complete and long-lasting remission in this disease, although only in a small percentage of patients (Buzaid A (2004). "Management of metastatic cutaneous melanoma". Oncology 18 (11): 1443-50). Another recently approved immunotherapy, Ipilimumab (Yervoy), is a human antibody that binds to and blocks the activity of CTLA-4 (cytotoxic T lymphocyte-associated antigen 4), and thus causes a sustained and active immune response against cancer cells. Patients receiving ipilimumab plus dacarbazine had significantly greater overall survival compared to the group receiving dacarbazine plus placebo (11.2 months vs. 9.1 months). [0270] Increased understanding of the molecular biology behind the development of melanoma has led to the development of novel targeted therapies. Specifically, advancement in knowledge of alterations in signal transduction pathways in melanoma has led to the rapid development of a number of pharmacologic agents that inhibit these pathways. About 60% of melanomas contain a mutation in the B-Raf gene. Various inhibitors of BRAF and the MAPK signaling pathway have been tested in clinical trials in the treatment of melanoma. Treatment with a selective MEK inhibitor yielded modest response rates amongst patients with a known BRAF mutation in a phase II trial involving melanoma (Dummer R, et al. J Clin Oncol 2008;26). Clinical trials suggested that B-Raf inhibitors including Plexxicon's vemurafenib could lead to substantial tumor regression in a majority of patients if their tumor contains the B-Raf mutation (Paul B. Chapman et al (2011). "Improved Survival with Vemurafenib in Melanoma with BRAF V600E Mutation". New England Journal of

Medicine). Preliminary results of a phase I/II trial with a selective BRAF inhibitor suggest that objective responses are seen in the vast majority of patients whose tumors harbor BRAFV600E (Flaherty KT, 2009). However, even the most encouraging of these agents yielded a median duration of response of only 8.5 months.

[0271] It has been reported that the MAPK signal, along with the STAT3 signal, is essential for immune evasion by human melanomas that have constitutively active MAPK signaling and is a potential molecular target for overcoming melanoma cell evasion of the immune system (Liu SQ, Kawai K, Shiraiwa H, Hayashi H, Akaza H, Hashizaki K, Shiba R, Saijou K and Ohno T (1998) High rate of induction of human autologous cytotoxic T lymphocytes against renal cell carcinoma cells cultured with an interleukin cocktail. Jpn J Cancer Res 89: 1 195-1201). Another potential approach involves combining BRAF/MAPK- targeted therapy with immunotherapy. The rationale for combination targeted

therapy/immunotherapy is based on evidence that MAPK pathway inhibition leads to increased expression of melanocyte differentiation antigens (Kono M, et al. Mol Cancer Res 2006 ;4( 10): 779-92). Recognition of melanocyte differentiation antigens is central to melanoma immunotherapy, and may translate into improved response rates to therapy.

[0272] A recent study demonstrated a significant increase in expression of melanocyte differentiation antigens in melanoma cell lines treated with MEK inhibitors and a specific BRAF inhibitor, which was associated with enhanced recognition by antigen-specific T lymphocytes (Wargo, et al, Cancer Research, July 2010). Importantly, BRAF-targeted therapy had no effect on T lymphocyte function whereas MEK inhibition caused significant alterations in T cell reactivity. This study provided the basis for combined targeted therapy and immunotherapy for melanoma, corroborating previous observations that MAPK pathway inhibition leads to an increased expression of melanoma differentiation antigens.

[0273] Other agents including Hedgehog (HH) inhibitors such as GDC-0449 and MEK inhibitors such as GSK-1 120212 and AZD-6044 have been tested in clinical trials in patients with metastatic melanoma and demonstrated clinical benefit. Understanding the underlying biology to devise a rational design of HD IL-2 and target therapy combination may offer high response rates and durable responses in patients with metastatic melanoma. Another possible advantage of combining an immunotherapy such as IL-2 with targeted therapies is the lack of overlapping adverse events (AEs), while a combination of multiple immunotherapies may be associated with overlapping AEs in patients. [0274] The present example describes a preclinical study to evaluate the combination of various new agents with interleukin-2 in a tumor/immune co-culture system. Multicellular tumor spheroid (MCTS) models have been used for studying the effect of

immunotherapeutics (Mueller-Klieser 2006). The 3D model enables long-term co-culture of tumor cells with immune cells to study tumor-immune cell interactions. MCTS co-culture was applied for the evaluation of HD IL-2 in combination with targeted therapies for immune cell-mediated antitumor effects. Importantly, the effects of these agents on T lymphocytes as a basis for selecting which targeted agents to explore in combination with immunotherapy for melanoma is addressed herein. Materials and Methods

[0275] Cell lines: Four human melanoma cell lines are purchased from ATCC, including 2 BRAF wild-type cell lines (CHL-1 and SK-MEL-2) and 2 BRAF mutant cell lines (A375 and SK-MEL-28).

[0276] Generation of CTLs was carried out as described previously (Liu et al., 1998). Briefly, PBMCs from heparinized peripheral blood were separated by conventional Ficoll- Paque gradient centrifugation, and suspended at a concentration of 1 * 10⁶ cell ml^"1 in

RHAM medium supplemented with autologous plasma (5%) or heat-inactivated FBS (5%), IL-1 (Genzyme Co., 167 U ml^"1), IL-2 (Sionogi Co., Ltd., 67 U ml^"1), IL-4 (Genzyme Co., 67 U ml^"1), and IL-6 (Genzyme Co., 134 U ml^"1).

[0277] Tumor/Immune cell co-culture: Multicellular tumor spheroids and mixed tumor- immune spheroids were produced in wells of round-bottom 96-well microliter plates

(Greiner) as previously described. Briefly, tumor cells were detached from monolayers in mid-log phase with 0.05% trypsin/0.02% EDTA solution (Boehringer, Mannheim, Germany), washed twice in culture medium and transferred to microtiter plates precoated with 1% type VII agarose (Sigma, Munich, Germany) at 6000 cells/0.2 ml culture medium/well on day 0. The formation of tumor-immune cell spheroids was performed with mixtures of 4,000 tumor cells and 2,000 immune cells, respectively. On day 3, established spheroids were exposed to drug treatment. The concentrations of drugs were determined in previous in vitro experiments and significant tumor growth inhibition was observed at these concentrations after 24-72 hr. Briefly, cryosections of spheroids were incubated with blocking reagent, biotinylated mouse IgG antibody, ABC complex and DAB (Vectastain, Burlingame, CA), and counterstaining was performed with Mayer's hemalaun. CTLs were labeled with the fluorescent vital dyes PKH 67-GL (green) and PKH26-GL (red) (Sigma, Munich, Germany). Coculture of spheroids with labeled PBMC at the effector :target cell ratio of 10: 1, unless stated otherwise, started on day 4. On day 5, spheroids were harvested with a Pasteur pipette, fixed in Zamboni solution, mounted with Tissue-Tek (Sakura Finetek, Torrance, CA) on cork plates, frozen in liquid nitrogen-cooled isopentane, and stored at 28°C until further processing. To evaluate the degree of infiltration, 5 lm sections were cut from each spheroid and fluorescently labeled CTLs were detected using a fluorescent microscope (Olympus, Hamburg, Germany).

Quantification of the degree of CTL infiltration into tumor spheroids was independently performed by two investigators. Since serial sections have confirmed the regularity of CTL penetration throughout the multicellular constructs, the descriptive analysis was confined to sections near the spheroid centre. Five degrees of infiltration were distinguished: negligible (2), weak (11), good (21), very good (31), and extraordinary (41).

[0278] Therapeutic agents: The BRAF inhibitor PLX4032, MEK inhibitor AZD 6244, and FfH inhibitor GDC0449 are tested in combination of high dose (HD) IL-2. Each agent can be used to treat the immune/tumor co-culture individually and in combination with HD IL-2. Therapeutic relevant doses can be used according to the published biological activities of those agents.

Combination Target Drugs

HD IL-2 BRAF PLX-4032

HD IL-2 MEK PD325901

HD IL-2 PI3K/AKT BEZ235

HD IL-2 HER2 Lapatinib

HD IL-2 HH GDC-0449 [0279] Quantification of penetrated leukocytes: Spheroid sections were analyzed with a transmitted light microscope, and photos were taken with a Nikon (D100) camera. For quantification of infiltrated CD45+ cells, a template of concentric rings was constructed with Adobe Photoshop 6.0 in the same pixel size as the microphotographs. Appropriate magnifications and scale bars allow for calibrating the distance between 2 rings, which was 25 μπι at a magnification of 10x and 50 um at 5^χ, respectively. Centers of the image and the template were overlaid in Adobe Photoshop 6.0. With a further feature, it is possible to highlight single rings for an easier manual counting of the total amount of all spheroid cells and CD45+, proliferating, and apoptotic cells. Counted cell numbers of each ring were normalized to the area of the individual ring (mm²).

[0280] Cytokine quantification: Cell-free media supernatants of spheroid cultures were collected every second day. Then, 200 uL were transferred into 1.5-mL reaction tubes. Remaining solid particles were sedimented by centrifugation for 10 min with 400 g at 4°C. The supernatants were stored at -80°C until the samples were analyzed with cytokine bead arrays (CBA, Thl/Th2 Cytokine Kit II, BD, Heidelberg, Germany). Secreted interferon gamma (IFN-γ), IL-4, IL-6, IL- 10, and tumor necrosis factor alpha (TNF-a) level can be quantitated in the supernatant by Mesi Scale assay.

[0281] Flow cytometry analysis: CTLs can undergo flow cytometry (FACS) staining for MART-1 and gp-100 tetramer to assess the frequency of MART- 1+ and gp-100+ T lymphocytes. CD25+Foxp3+ T regulatory cells and natural killer cells can be measured pre- and post-drug treatment.

[0282] Quantitative real-time RT-PCR: Spheroids were treated with or without drug treatment and CTL. After 5 days of co-culture, spheroids were washed 5 times in PBS to remove any attached CTL. Total RNA was extracted using the RNeasy Mini Kit, and a DNase digest was performed (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. Afterwards, RNA concentration was determined and 200 ng of total RNA was reverse transcribed using Reverse-iT RTase Blend (Abgene, Hamburg, Germany) and Anchored Oligo dT primer (Abgene). Concentration of cDNA was determined, and 200 ng were used for measuring mRNA levels of MART-1, gp-100, MITF, TRP-1, and TRP-2 using RT-PCR. Expression levels are shown as fold change over pre-treatment value.

[0283] Inhibition of tumor cell line growth by drug treatment: The tumor cells were seeded into 96-well cell culture plates and maintained in culture for 24 hours. After washing, the cultured cells were incubated in their respective medium containing 5% FBS and various concentrations of the indicated inhibitor for 48 hr. Determination of tumor cell growth inhibition was performed by adding 100 \iL of the combined Cell Titer-Glo® Buffer and Cell Titer-Glo® Substrate Labeling Reagent (Promega) to each well of the plates, followed by incubation at room temperature for 10 min to stabilize the luminescence. The luminescent signal from the cell samples was detected by using an M5 micro-titer plate reader.

[0284] In vivo drug testing in mammalian (e.g., rodent) model: Effective treatment combinations determined in the in vitro co-culture model can be further tested in animal models. Mixed tumor cells and immune cells can be injected into SCID mice, and when the tumor size reaches about 100mm³, IL-2, targeted therapy, or combination treatment is administered to the mice every 3 days per week for 4 weeks. Tumor growth inhibition can be determined as measured by shrinkage of the tumor size. At week 4, mice can be sacrificed, tumor samples resected and tumor antigens measured either by RT-PCR or Flow.

Results

[0285] Modulation of immune cell functions by IL-2 treatment: 10 Thl/Th2 cytokines in IL-2 treated PBMCs were measured. IL-2 stimulated production of IFN-g, IL-10, IL-12, IL-13, IL-lb, IL-4 and TNF-a in PBMCs in a dose dependent manner, while no significant changes in IL-5 and IL-8 cytokines were observed when the PBMCs were treated with IL-2 (Figure 1).

[0286] 10 gene expression markers were measured including MART-1, tyrosinase, MITF, gplOO, c-myc, Perforin, FasL, granzyme B, IRF-1, and OSM. Among these, expression of gplOO, tyrosinase, MART-1, and MITF were up-regulated by BRAF inhibitor, PLX4032, MEK inhibitor, PD-325901, and PI3K/AKT inhibitor, BEZ325. Treatment with Lapatinib and Hedgehog inhibitor, GDC0449, did not affect expression of those tumor antigens. Co- culture of tumor cells with IL-2 treated PBMCs reduced expression of most genes; however, the trend of tumor antigen up-regulation by TKI remained unchanged in the presence of IL-2 treated PBMCs (Figure 2).

[0287] Inhibition of tumor cell growth by IL-2 treatment: Tumor cell growth inhibition was further tested by evaluating selected targeted therapies in the presence/absence of IL-2 treated PBMCs. NA8 and D10 cells were treated with five targeted therapies. Figure 3 shows that growth inhibition by IL-2 in combination with BEZ235 (PI3K inhibitor),

PLX4032 (BRAF inhibitor), Lapatinib (TKI), or GDC0449 (HH inhibitor) was observed in MART-1 negative NA8 cells, while MART-1 expressing D10 cells were sensitive to IL-2 in combination with BEZ235 or PD-325901 (MEK inhibitor). [0288] The effect of TKI treatment on melanoma cell growth was further confirmed using quantitative measurement (ApoTox-Glo, Promega). We measured the mechanism of tumor cell growth inhibition via inhibition of proliferation and/or induction of cytotoxicity and apoptosis. Figure 4 shows that BEZ325 inhibited both D10 and NA8 cell growth

independent of tumor antigen status, while growth of D10 cells was inhibited in a drug concentration dependent manner. Inhibition of cell viability seemed to be the main mechanism of growth inhibition, although mild induction of cytotoxic and apoptotic effect was also observed. BRAF mutation status can be determined by genotyping. TKI resistant cells can be generated to explore the effect of IL-2 on TKI resistant cells. [0289] Modulation of inflammatory cytokines was measured. BEZ 235 and PD325901 were found to inhibit IFN-gamma, IL-lb, TNF-a, and IL-10 production (Figure 5). Although these drugs have anti-tumor activity, they may also inhibit immune cell functions. IL-2 in combination with such TKI may have contraindications, although administering IL-2 may overcome the immunosuppressive function of these drugs in patients. [0290] Inhibition of tumor cell growth by combination treatment: Mammals (e.g., rodents) are treated with TKI alone or in combination with IL-2 to investigate whether combining these drugs has synergistic effects in both in vitro and in vivo models. This information can be used to guide the clinical design of rational combinations of drug treatments. In certain embodiments, the simultaneous drug combination and sequential combination in the model systems can also be evaluated.

Discussion

[0291] In this study, it has been shown that MART-1 negative melanoma cells exhibited tumor growth inhibition when cultured in the presence of a combination of IL-2 and BRAF inhibitor, PI3K/AKT inhibitor, TKI, or HH inhibitor. Growth inhibition was observed in MART-1 positive melanoma cells treated with IL-2 in combination with PI3K/AKT inhibitor or MEK inhibitor.

[0292] Thl/Th2 cytokine imbalance has been reported in the literature. In this study, we measured 9 Thl/Th2 cytokines in IL-2-treated PBMCs in the presence or absence of target agents. We found that most cytokines are upregulated by IL-2 treatment with the exception of IL-5 and IL-8.

[0293] Interferon-γ (IFN-γ) plays a role in the recruitment of leukocytes to the site of infection. IFN-γ is produced by Thl cells and NK cells. It has been reported that IFN- gamma-deficient (IFN-gamma-/-) mice induce potent in vitro immune responses such as anti- alio mixed lymphocyte reaction and CTL responses, whereas they often fail to exhibit in vivo immunity (Nakajima C, Uekusa Y, Iwasaki M, Yamaguchi N, Mukai T, Gao P, Tomura M, Ono S, Tsujimura T, Fujiwara H, Hamaoka T. A role of interferon-gamma (IFN-gamma) in tumor immunity: T cells with the capacity to reject tumor cells are generated but fail to migrate to tumor sites in IFN-gamma-deficient mice. Cancer Res. 2001 ;61(8):3399-405). IFN-gamma was also found to inhibit tumor antigen presentation by freshly prepared epidermal APCs (S Grabbe, S Bruvers, S Beissert and RD Granstein. Interferon-gamma inhibits tumor antigen presentation by epidermal antigen-presenting cells. Journal of Leukocyte Biology, 1994 Vol 55, Issue 6 695-701). [0294] IL-4 plays a paradoxical role in tumor immunity. On the one hand, IL-4 induced the most effective immune response among several cytokines in both prophylactic and therapeutic models. On the other hand, IL-4 is up-regulated in patients with different types of cancers, such as renal cell cancer, non-small lung cancer, prostate cancer, colon cancer, and breast cancer. Onishi et al. found the IL-4 amount at tumor site was associated with the stage and grade of renal cancer. IL-5 is produced by T helper-2 cells and mast cells; it is the key cytokine in eosinophil production, activation and localization. IL-5 is associated with asthma and several related allergic disorders. IL-8 is responsible for the attraction of neutrophils to the vascular endothelium and extravasations into inflamed tissues. It is produced primarily by activated macrophages in response to Toll-like receptor agonists and certain bacterial pathogens.

[0295] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, one of skill in the art will appreciate that certain changes and modifications may be practiced within the scope of the appended claims. In addition, each reference provided herein is incorporated by reference in its entirety to the same extent as if each reference was individually incorporated by reference.

Claims

WHAT IS CLAIMED IS: 1. A method for determining a combination of IL-2 therapy with one or more targeted therapeutic agents for treating cancer in a subject, the method comprising:

(b) selecting an appropriate therapy for the subject comprising:

2. The method of claim 1, wherein the cancer is melanoma or metastatic melanoma.

3. The method of claim 1, wherein the cancer is renal cell carcinoma or metastatic renal cell carcinoma.

4. The method of any one of claims 1 to 3, wherein the cancer cell is from a sample taken from the subject.

5. The method of any one of claims 1 to 4, wherein the MEK inhibitor is selected from the group consisting of PD325901, AZD 6244, trametinib, MEK162, XL518, and combinations thereof.

6. The method of any one of claims 1 to 5, wherein the PI3K/AKT inhibitor is selected from the group consisting of BEZ235, BKM120, BLY719, IPI-145, and combinations thereof.

7. The method of any one of claims 1 to 6, wherein the BRAF inhibitor is selected from the group consisting of PLX-4032, dabrafenib, RAF265, LGX818, and combinations thereof.

8. The method of any one of claims 1 to 7, wherein the receptor tyrosine kinase inhibitor is selected from the group consisting of Iapatinib, sunitinib, sorafenib, axitinib, pazopanib, gefitinib, erlotinib, pilitinib, canertinib, CP-654577, CP-724714, ARRY- 334543, J J-26483327, JNJ-26483327, MP-470, AZD8931, and combinations thereof.

9. The method of any one of claims 1 to 8, wherein the Hedgehog inhibitor is selected from the group consisting of GDC0449, erismodegib, LEQ506, saridegib, IPI-269609, BMS-833932, PF-04449913, TAK-441, and combinations thereof.

10. The method of any one of claims 1 to 9, wherein the IL-2 polypeptide comprises a recombinant IL-2 polypeptide.

11. The method of claim 10, wherein the recombinant IL-2 polypeptide is PROLEUKIN^® (aldesleukin).

12. The method of any one of claims 1 to 11, wherein the IL-2 polypeptide is high dose IL-2.

13. The method of any one of claims 1 to 12, wherein the MART-l expression is selected from the group consisting of protein expression, RNA expression, and combinations thereof.

14. A method for recommending a combination of IL-2 therapy with one or more targeted therapeutic agents for treating cancer in a subject, the method comprising:

(a) determining whether a cancer cell obtained from the subject is positive or negative for MART-l expression; and

(b) recommending an appropriate therapy for the subject comprising:

(i) a combination of an IL-2 polypeptide and a MEK inhibitor and/or a PI3K/AKT inhibitor if the cell is positive for MART-l expression; or

(ii) a combination of an IL-2 polypeptide and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor if the cell is negative for MART-l expression.

15. A method for administering an appropriate therapy for treating cancer in a subject, the method comprising:

(b) administering an appropriate therapy to the subject comprising:

(i) a combination of an IL-2 polypeptide and a MEK inhibitor and/or a PI3K/AKT inhibitor if the cell is positive for MART-l expression; or (ii) a combination of an IL-2 polypeptide and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor if the cell is negative for MART-1 expression.

16. The method of claim 14 or 15, further comprising comparing the subject's symptom profile to that from a control subject.

17. The method of any one of claims 14 to 16, wherein the MART- 1 expression is selected from the group consisting of protein expression, RNA expression, and combinations thereof.

18. The method of any one of claims 14 to 17, wherein the cancer cell is from a sample taken from the subject.

19. The method of any one of claims 14 to 18, wherein the cancer is melanoma or metastatic melanoma.

20. The method of any one of claims 14 to 18, wherein the cancer is renal cell carcinoma or metastatic renal cell carcinoma.

21. The method of any one of claims 14 to 20, wherein the MEK inhibitor is selected from the group consisting of PD325901, AZD 6244, trametinib, MEK162, XL518, and combinations thereof.

22. The method of any one of claims 14 to 21, wherein the PI3K/AKT inhibitor is selected from the group consisting of BEZ235, BKM120, BLY719, IPI-145, and combinations thereof.

23. The method of any one of claims 14 to 22, wherein the BRAF inhibitor is selected from the group consisting of PLX-4032, dabrafenib, RAF265, LGX818, and combinations thereof.

24. The method of any one of claims 14 to 23, wherein the receptor tyrosine kinase inhibitor is selected from the group consisting of lapatinib, sunitinib, sorafenib, axitinib, pazopanib, gefitinib, erlotinib, pilitinib, canertinib, CP-654577, CP- 724714, ARRY-334543, JNJ-26483327, JNJ-26483327, MP-470, AZD8931, and

combinations thereof.

25. The method of any one of claims 14 to 24, wherein the Hedgehog inhibitor is selected from the group consisting of GDC0449, erismodegib, LEQ506, saridegib, IPI-269609, BMS-833932, PF-04449913, TAK-441, and combinations thereof.

26. The method of any one of claims 14 to 25, wherein the IL-2 polypeptide is high dose IL-2.

27. A method for treating cancer in a subject using a combination of IL-2 therapy with one or more targeted therapeutic agents after determining MART-1 expression in a cancer cell, the method comprising:

(b) administering a combination of an IL-2 polypeptide and a BRAF inhibitor, a PI3K/AKT inhibitor, a receptor tyrosine kinase inhibitor, and/or a Hedgehog inhibitor if the cell is negative for MART-1 expression, to effectively treat cancer.

28. A method for combining IL-2 therapy with one or more targeted therapeutic agents after determining MART-1 expression in a cancer cell, the method comprising:

29. A method for treating cancer in a subject using a combination of IL-2 therapy with one or more targeted therapeutic agents based on MART-1 expression in a cancer cell, the method comprising:

30. A method for combining IL-2 therapy with one or more targeted therapeutic agents based on MART-1 expression in a cancer cell, the method comprising: (a) administering a combination of an IL-2 polypeptide and a MEK inhibitor and/or a PI3K/AKT inhibitor if the cell is positive for MART-1 expression; or