COMBINATION THERAPIES

SEQUENCE LISTING

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BACKGROUND

The ability of T cells to mediate an immune response against an antigen requires two distinct signaling interactions (Viglietta et al. (2007) Neurotherapeutics 4:666-675; Korman et al. (2007) Adv. Immunol. 90:297-339). First, an antigen that has been arrayed on the surface of antigen-presenting cells (APC) is presented to an antigen-specific naive CD4⁺ T cell. Such presentation delivers a signal via the T cell receptor (TCR) that directs the T cell to initiate an immune response specific to the presented antigen. Second, various co-stimulatory and inhibitory signals mediated through interactions between the APC and distinct T cell surface molecules trigger the activation and proliferation of the T cells and ultimately their inhibition.

The immune system is tightly controlled by a network of costimulatory and co-inhibitory ligands and receptors. These molecules provide the second signal for T cell activation and provide a balanced network of positive and negative signals to maximize immune responses against infection, while limiting immunity to self (Wang et al. (2011) J. Exp. Med. 208(3):577-92; Lepenies et al. (2008) Endocrine, Metabolic & Immune Disorders—Drug Targets 8:279-288). Examples of costimulatory signals include the binding between the B7.1 (CD80) and B7.2 (CD86) ligands of the APC and the CD28 and CTLA-4 receptors of the CD4⁺ T-lymphocyte (Sharpe et al. (2002) Nature Rev. Immunol. 2:116-126; Lindley et al. (2009) Immunol. Rev. 229:307-321). Binding of B7.1 or B7.2 to CD28 stimulates T cell activation, whereas binding of B7.1 or B7.2 to CTLA-4 inhibits such activation (Dong et al. (2003) Immunolog. Res. 28(1):39-48; Greenwald et al. (2005) Ann. Rev. Immunol. 23:515-548). CD28 is constitutively expressed on the surface of T cells (Gross et al. (1992) J. Immunol. 149:380-388), whereas CTLA4 expression is rapidly up-regulated following T-cell activation (Linsley et al. (1996) Immunity 4:535-543).

Other ligands of the CD28 receptor include a group of related B7 molecules, also known as the “B7 Superfamily” (Coyle et al. (2001) Nature Immunol. 2(3):203-209; Sharpe et al. (2002) Nature Rev. Immunol. 2:116-126; Collins et al. (2005) Genome Biol. 6:223.1-223.7; Korman et al. (2007) Adv. Immunol. 90:297-339). Several members of the B7 Superfamily are known, including B7.1 (CD80), B7.2 (CD86), the inducible co-stimulator ligand (ICOS-L), the programmed death-1 ligand (PD-L1; B7-H1), the programmed death-2 ligand (PD-L2; B7-DC), B7-H3, B7-H4 and B7-H6 (Collins et al. (2005) Genome Biol. 6:223.1-223.7).

The Programmed Death 1 (PD-1) protein is an inhibitory member of the extended CD28/CTLA-4 family of T cell regulators (Okazaki et al. (2002) Curr Opin Immunol 14: 391779-82; Bennett et al. (2003) J. Immunol. 170:711-8). Two ligands for PD-1 have been identified, PD-L1 (B7-H1) and PD-L2 (B7-DC), that have been shown to downregulate T cell activation upon binding to PD-1 (Freeman et al. (2000) J. Exp. Med. 192:1027-34; Carter et al. (2002) Eur. J. Immunol. 32:634-43). PD-L1 is abundant in a variety of human cancers (Dong et al. (2002) Nat. Med. 8:787-9).

PD-1 is known as an immunoinhibitory protein that negatively regulates TCR signals (Ishida, Y. et al. (1992) EMBO J. 11:3887-3895; Blank, C. et al. (Epub 2006 Dec. 29) Immunol. Immunother. 56(5):739-745). The interaction between PD-1 and PD-L1 can act as an immune checkpoint, which can lead to, e.g., a decrease in tumor infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and/or immune evasion by cancerous cells (Dong et al. (2003) J. Mol. Med. 81:281-7; Blank et al. (2005) Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100) Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2; the effect is additive when the interaction of PD-1 with PD-L2 is blocked as well (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66).

Glucocorticoid-induced TNFR-related protein (GITR) is a member of the Tumor Necrosis Factor Superfamily (TNFRSF). GITR expression is detected constitutively on murine and human CD4+CD25+ regulatory T cells which can be further increased upon activation. In contrast, effector CD4+CD25− T cells and CD8+CD25− T cells express low to undetectable levels of GITR, which is rapidly upregulated following T cell receptor activation. Expression of GITR has also been detected on activated NK cells, dendritic cells, and macrophages. Signal transduction pathway downstream of GITR has been shown to involve MAPK and the canonical NFκB pathways. Various TRAF family members have been implicated as signaling intermediates downstream of GITR (Nocentini et al. (2005) Eur. J. Immunol. 35:1016-1022).

Cellular activation through GITR is believed to serve several functions depending on the cell type and microenvironment including, but not limited to, costimulation to augment proliferation and effector function, inhibition of suppression by regulatory T cells, and protection from activation-induced cell death (Shevach and Stephens (2006) Nat. Rev. Immunol. 6:613-618). An agonistic monoclonal antibody against mouse GITR effectively induced tumor-specific immunity and eradicated established tumors in a mouse syngeneic tumor model (Ko et al. (2005) J. Exp. Med. 202:885-891).

Given the importance of immune checkpoint pathways in regulating an immune response, the need exists for developing novel combination therapies that activate the immune system.

SUMMARY

Disclosed herein, inter alia, are methods and compositions comprising combination therapies, e.g., a combination comprising two or more (e.g., two, three, four, five, six, or more) therapeutic agents disclosed herein. The therapeutic agents can be chosen from one or more of: an inhibitor of an inhibitory molecule (e.g., an inhibitor of a checkpoint inhibitor), an activator of a costimulatory molecule, a chemotherapeutic agent, a targeted anti-cancer therapy, an oncolytic drug, a cytotoxic agent, or any of the therapeutic agents disclosed herein. In some embodiments, the therapeutic agent can be chosen from: a PD-1 inhibitor, a LAG-3 inhibitor, a TIM-3 inhibitor, a GITR agonist, a SERD, a CDK4/6 inhibitor, a CXCR2 inhibitor, a CSF-1/1R binding agent, a c-MET inhibitor, a TGF-β inhibitor, an A2aR antagonist, an IDO inhibitor, a STING agonist, a Galectin inhibitor, a MEK inhibitor, an IL-15/IL-15RA complex, an IL-1β inhibitor, an MDM2 inhibitor, or any combination thereof.

The combinations described herein can provide a beneficial effect, e.g., in the treatment of a cancer, such as an enhanced anti-cancer effect, reduced toxicity, and/or reduced side effects. For example, a first therapeutic agent, e.g., any of the therapeutic agents disclosed herein, and a second therapeutic agent, e.g., the one or more additional therapeutic agents, or all, can be administered at a lower dosage than would be required to achieve the same therapeutic effect compared to a monotherapy dose. Thus, compositions and methods for treating proliferative disorders, including cancer, using the aforesaid combination therapies are disclosed.

Accordingly, in one aspect, the disclosure features a method of treating (e.g., inhibiting, reducing, ameliorating, or preventing) a disorder, e.g., a hyperproliferative condition or disorder (e.g., a cancer) in a subject. The method includes administering to the subject a combination comprising three or more (e.g., four, five, six, seven, eight, or more) therapeutic agents disclosed herein. In some embodiments, the therapeutic agent is chosen from: a PD-1 inhibitor, a LAG-3 inhibitor, a TIM-3 inhibitor, a GITR agonist, a SERD, a CDK4/6 inhibitor, a CXCR2 inhibitor, a CSF-1/1R binding agent, a c-MET inhibitor, a TGF-β inhibitor, an A2aR antagonist, an IDO inhibitor, a STING agonist, a Galectin inhibitor, a MEK inhibitor, an IL-15/IL-15RA complex, an IL-1β inhibitor, an MDM2 inhibitor, or any combination thereof. In some embodiments, the cancer is chosen from a breast cancer (e.g., a triple negative breast cancer), a pancreatic cancer, a colorectal cancer (e.g., a microsatellite stable colorectal cancer (MSS CRC)), a skin cancer, a gastric cancer, a gastroesophageal cancer, or an ER+ cancer. In some embodiments, the skin cancer is a melanoma (e.g., a refractory melanoma). In some embodiments, the ER+ cancer is an ER+ breast cancer.

In some embodiments, the combination comprises:

(i) a PD-1 inhibitor, a SERD, and a CDK4/6 inhibitor, e.g., to treat an ER+ cancer or a breast cancer;

(ii) a PD-1 inhibitor, a CXCR2 inhibitor, a CSF-1/1R binding agent, and optionally, one or more (e.g., two or all) of a TIM-3 inhibitor, a c-MET inhibitor, or an A2aR antagonist, e.g., to treat a pancreatic cancer or a colorectal cancer;

(iii) a PD-1 inhibitor, a CXCR2 inhibitor, and one or more (e.g., two or all) of a TIM-3 inhibitor, a c-MET inhibitor, or an A2aR antagonist, e.g., to treat a pancreatic cancer or a colorectal cancer;

(iv) a PD-1 inhibitor, a GITR agonist, and one or more (e.g., two or all) of a TGF-β inhibitor, an A2aR antagonist, or a c-MET inhibitor, e.g., to treat a pancreatic cancer, a colorectal cancer, or a melanoma;

(v) a PD-1 inhibitor, a LAG-3 inhibitor, a GITR agonist, and one or more (e.g., two or all) of a TGF-β inhibitor, an A2aR antagonist, or a c-MET inhibitor, e.g., to treat a pancreatic cancer, a colorectal cancer, or a melanoma;

(vi) a PD-1 inhibitor, an A2aR antagonist, and one or both of a TGF-β inhibitor or a CSF-1/1R binding agent, e.g., to treat a pancreatic cancer, a colorectal cancer, or a melanoma;

(vii) a PD-1 inhibitor, a c-MET inhibitor, and one or more (e.g., two or all) of a TGF-β inhibitor, an A2aR antagonist, or a c-MET inhibitor, e.g., to treat a pancreatic cancer, a colorectal cancer, a gastric cancer, or a melanoma;

(viii) a PD-1 inhibitor, an IDO inhibitor, and one or more (e.g., two, three, four, or all) of a TGF-β inhibitor, an A2aR antagonist, a CSF-1/1R binding agent, a c-MET inhibitor, or a GITR agonist, e.g., to treat a pancreatic cancer, a colorectal cancer, a gastric cancer, or a melanoma;

(ix) a PD-1 inhibitor, a LAG-3 inhibitor, and one or more (e.g., two three, four, five, six or all) of a TGF-β inhibitor, a TIM-3 inhibitor, a c-MET inhibitor, an IL-1β inhibitor, a MEK inhibitor, a GITR agonist or a CSF-1/1R binding agent, e.g., to treat a breast cancer, e.g., a triple negative breast cancer (TNBC);

(x) a PD-1 inhibitor, a CSF-1/1R binding agent, and one or more of (e.g., two, three, or all) of a TGF-β inhibitor, a TIM-3 inhibitor, a c-MET inhibitor, or an IL-1β inhibitor, e.g., to treat a breast cancer (e.g., a TNBC);

(xi) a PD-1 inhibitor, an A2aR antagonist, and one or more (e.g., two three, four, five, or all) of a TGF-β inhibitor, a TIM-3 inhibitor, a c-MET inhibitor, an IL-1β inhibitor, an IL-15/IL-15RA complex, or a CSF-1/1R binding agent, e.g., to treat a breast cancer (e.g., a TNBC), a colorectal cancer (e.g., a microsatellite stable colorectal cancer (MSS CRC), a gastroesophageal cancer or a pancreatic cancer;

(xii) a PD-1 inhibitor, an IL-1β inhibitor, and one or more of (e.g., two, three, four or more) of a TGF-β inhibitor, an IL-15/IL-15RA complex, a c-MET inhibitor, a CSF-1/1R binding agent, or a TIM-3 inhibitor, e.g., to treat a colorectal cancer (e.g., a microsatellite stable colorectal cancer (MSS CRC), a gastroesophageal cancer or a pancreatic cancer;

(xiii) a PD-1 inhibitor, a MEK inhibitor, and one or more of (e.g., two, three, four or more) of a TGF-β inhibitor, an IL-15/IL-15RA complex, a c-MET inhibitor, a CSF-1/1R binding agent, or a TIM-3 inhibitor, e.g., to treat a a colorectal cancer (e.g., a microsatellite stable colorectal cancer (MSS CRC), a gastroesophageal cancer or a pancreatic cancer;

(xiv) an IL-1β inhibitor, an A2aR antagonist, and one or both of an IL-15/IL-15Ra complex or a TGF-β inhibitor, e.g., to treat a a colorectal cancer (e.g., a microsatellite stable colorectal cancer (MSS CRC), a gastroesophageal cancer or a pancreatic cancer;

(xv) an IL-15/IL-15Ra complex, and a TGF-β inhibitor, and one or more of (e.g., two, three, or more of) an IL-1β inhibitor, a CSF-1/1R binding agent, a c-MET inhibitor, or an A2aR antagonist, e.g., to treat a a colorectal cancer (e.g., a microsatellite stable colorectal cancer (MSS CRC), a gastroesophageal cancer or a pancreatic cancer;

(xvi) a PD-1 inhibitor, and a TIM-3 inhibitor, and one or more of (e.g., both), a STING agonist, or a CSF-1/1R binding agent, e.g., to treat a solid tumor, e.g., a pancreatic cancer, or a colon cancer;

(xvii) a PD-1 inhibitor, a TIM-3 inhibitor and an A2aR antagonist, and one or more of (e.g., both) a CSF-1/1R binding agent or a TGF-β inhibitor, e.g., to treat a solid tumor, e.g., a pancreatic cancer, or a colon cancer;

(xviii) a Galectin inhibitor, e.g., one or more of (e.g., both), a Galectin 1 inhibitor or a Galectin 3 inhibitor, and a PD-1 inhibitor, e.g., to treat a solid tumor or a hematological malignancy; or

(xix) a PD-1 inhibitor and CXCR2 inhibitor, e.g., to treat a solid tumor, e.g., a colorectal cancer (e.g., a microsatellite stable colorectal cancer (MSS CRC)), a lung cancer (e.g., a non-small cell lung cancer (NSCLC)) or a breast cancer (e.g., a TNBC).

In another aspect, the invention features a method of reducing an activity (e.g., growth, survival, or viability, or all), of a hyperproliferative (e.g., a cancer) cell. The method includes contacting the cell with a combination comprising three or more (e.g., four, five, six, seven, eight, or more) therapeutic agents disclosed herein. In some embodiments, the therapeutic agent is chosen from: a PD-1 inhibitor, a LAG-3 inhibitor, a TIM-3 inhibitor, a GITR agonist, a SERD, a CDK4/6 inhibitor, a CXCR2 inhibitor, a CSF-1/1R binding agent, a c-MET inhibitor, a TGF-β inhibitor, an A2aR antagonist, an IDO inhibitor, a STING agonist, a Galectin inhibitor, a MEK inhibitor, an IL-15/IL-15RA complex, an IL-1β inhibitor, an MDM2 inhibitor, or any combination thereof.

In some embodiments, the combination comprises:

(i) a PD-1 inhibitor, a SERD, and a CDK4/6 inhibitor;

(ii) a PD-1 inhibitor, a CXCR2 inhibitor, a CSF-1/1R binding agent, and optionally, one or more (e.g., two or all) of a TIM-3 inhibitor, a c-MET inhibitor, or an A2aR antagonist;

(iii) a PD-1 inhibitor, a CXCR2 inhibitor, and one or more (e.g., two or all) of a TIM-3 inhibitor, a c-MET inhibitor, or an A2aR antagonist;

(iv) a PD-1 inhibitor, a GITR agonist, and one or more (e.g., two or all) of a TGF-β inhibitor, an A2aR antagonist, or a c-MET inhibitor;

(v) a PD-1 inhibitor, a LAG-3 inhibitor, a GITR agonist, and one or more (e.g., two or all) of a TGF-β inhibitor, an A2aR antagonist, or a c-MET inhibitor;

(vi) a PD-1 inhibitor, an A2aR antagonist, and one or both of a TGF-β inhibitor or a CSF-1/1R binding agent;

(vii) a PD-1 inhibitor, a c-MET inhibitor, and one or more (e.g., two or all) of a TGF-β inhibitor, an A2aR antagonist, or a c-MET inhibitor;

(viii) a PD-1 inhibitor, an IDO inhibitor, and one or more (e.g., two, three, four, or all) of a TGF-β inhibitor, an A2aR antagonist, a CSF-1/1R binding agent, a c-MET inhibitor, or a GITR agonist;

(xiii) a PD-1 inhibitor, a MEK inhibitor, and one or more of (e.g., two, three, four or more) of a a TGF-β inhibitor, an IL-15/IL-15RA complex, a c-MET inhibitor, a CSF-1/1R binding agent, or a TIM-3 inhibitor, e.g., to treat a colorectal cancer (e.g., a microsatellite stable colorectal cancer (MSS CRC), a gastroesophageal cancer or a pancreatic cancer;

(xv) an IL-15/IL-15Ra complex, and a TGF-β inhibitor, and one or more of (e.g., two, three, or more) of an IL-1β inhibitor, a CSF-1/1R binding agent, a c-MET inhibitor, or an A2aR antagonist, e.g., to treat a a colorectal cancer (e.g., a microsatellite stable colorectal cancer (MSS CRC), a gastroesophageal cancer or a pancreatic cancer

(xviii) a Galectin inhibitor, e.g., one or more of (e.g., both), a Galectin 1 inhibitor or a Galectin 3 inhibitor, and a PD-1 inhibitor, e.g., to treat a solid tumor or a hematological malignancy; or (xix) a PD-1 inhibitor and CXCR2 inhibitor, e.g., to treat a solid tumor, e.g., a colorectal cancer (e.g., a microsatellite stable colorectal cancer (MSS CRC)), a lung cancer (e.g., a non-small cell lung cancer (NSCLC)) or a breast cancer (e.g., a TNBC).

The method can be performed in a subject, e.g., as part of a therapeutic protocol. The cell can be a cancer cell, e.g., a cell from a cancer described herein, e.g., a breast cancer, a pancreatic cancer, a colorectal cancer (CRC), a skin cancer, a gastric cancer, a gastroesophageal cancer, or an ER+ cancer. In some embodiments, the skin cancer is a melanoma (e.g., a refractory melanoma). In some embodiments, the ER+ cancer is an ER+ breast cancer. In some embodiments, the breast cancer is a TNBC. In some embodiments, the CRC is a MSS CRC.

In some embodiments, a combination described herein is administered to a subject having a cancer, e.g., a cancer described herein. In some embodiments, the cancer has a high mutational burden, e.g., as disclosed in Alexandrov L. B. et al., (2013) Nature 500, 415-421 and Chalmers Z. R., et al., (2017) Genome Medicine 9:34. In some embodiments, the cancer is a breast cancer, a pancreatic cancer, a colorectal cancer (CRC), a skin cancer, a gastric cancer, a gastroesophageal cancer, or an ER+ cancer. In some embodiments, the skin cancer is a melanoma (e.g., a refractory melanoma). In some embodiments, the ER+ cancer is an ER+ breast cancer. In some embodiments, the breast cancer is a TNBC. In some embodiments, the CRC is a MSS CRC.

In certain embodiments of the methods disclosed herein, the method further includes determining one or more biomarkers (e.g., one or more biomarkers disclosed herein) in the subject. In one embodiment, the biomarker is determined in vivo, e.g., non-invasively. In other embodiments, the biomarker is determined in a sample (e.g., a tumor biopsy) acquired from the subject. In embodiments, responsive to a determination of the presence of one or more biomarkers, a combination of the therapeutic agents disclosed herein is administered to the subject.

In another aspect, the invention features a composition (e.g., one or more compositions or dosage forms), that includes a combination comprising three or more (e.g., four, five, six, seven, eight, or more) therapeutic agents disclosed herein. In some embodiments, the therapeutic agent is chosen from: a PD-1 inhibitor, a LAG-3 inhibitor, a TIM-3 inhibitor, a GITR agonist, a SERD, a CDK4/6 inhibitor, a CXCR2 inhibitor, a CSF-1/1R binding agent, a c-MET inhibitor, a TGF-β inhibitor, an A2aR antagonist, an IDO inhibitor, a STING agonist, a Galectin inhibitor, a MEK inhibitor, an IL-15/IL-15RA complex, an IL-1β inhibitor, an MDM2 inhibitor, or any combination thereof.