MODULATING BIOMARKERS TO INCREASE TUMOR IMMUNITY AND IMPROVE THE EFFICACY OF CANCER IMMUNOTHERAPY

Cross-Reference to Related Applications

This application claims the benefit of U.S. Provisional Application No. 62/456,432, filed on 8 February 2017, and U.S. Provisional Application No. 62/532,587, filed on 14 July 2017; the entire contents of each of said applications are incorporated herein in their entirety by this reference. Background of the Invention

The striking clinical success of cancer immunotherapy with checkpoint blockade suggests it is likely to form the foundation of curative therapy for many malignancies (Reck et al. (2016) N. Engl. J. Med. 375:1823-1833; Hodi et al. (2010) JV. Engl. J. Med. 363:711- 723; Postow et al. (2015) N. Engl. J. Med. 372:2006-2017; Wolchok et al. (2013) N. Engl. J. Med. 369:122-133; Ferris et al. (2016) N. Engl. J. Med. 375:1856-1867; Brahmer et al. (2012) N. Engl. J. Med. 366:2455-2465; Nghiem et al. (2016) N. Engl. J. Med. 374:2542- 2552; Topalian et al. (2012) N. Engl. J. Med. 366:2443-2454); Motzer et al. (2015) N. Engl. J. Med. 373:1803-1813). However, despite these successes, checkpoint blockade does not achieve sustained clinical response in most patients (Tumeh et al. (2014) Nature 515:568- 571; Kelderman et al. (2014) Mol. Oncol. 8:1132-1139; Zaretsky et al. (2016) N. Engl. J. Med. 375:819-829). Additional therapeutic strategies are therefore needed to increase the clinical efficacy of immunotherapy. Moreover, the optimal strategy for combining emerging cancer immunotherapies with checkpoint blockade remains uncertain.

A relatively small number of genes, such as PD-L1, that enable tumors to evade the immune system have been discovered and most of these are already the focus of intense efforts to develop new immunotherapies (Freeman et al. (2000) J. Exp. Med. 192:1027- 1034; Hirano et al. (2005) Cancer Res. 65:1089-1096; Dong et al. (2002) Nat. Med. 8:793- 800; Balachandran et al. (2011) Nat. Med.17:1094-1100; Spranger et al. (2013) Sci Transl Med. 5:200ral 16; Holmgaard et al. (2013) J. Exp. Med. 210:1389-1402; Sockolosky et al. (2016) Proc. Natl. Acad. Sci. U.S.A. 113:E2646-654; Liu et al. (2015) Nat. Med. 21:1209- 1215; Weiskopfet fl/. (2016) J Clin. Invest. 126:2610-2620; Tseng et al. (2013) Proc. Natl. Acad. Sci. U.S.A. 110: 11103-11108; Sica et al. (2003) Immunity 18:849-861; Zang et al. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 19458-19463). Although cancer cells could, in theory, express many more genes that regulate their response or resistance to tumor immunity, strategies to systematically discover such genes are lacking.

Loss-of-function genetic screens have been increasingly used to study the functional consequences of gene deletion on tumor cells (Howard et al. (2016) Functional Genomic Characterization of Cancer Genomes. Cold Spring Harb. Symp. Quant. Biol. (2016); Ebert et al. (2008) Nature 451:335-339; Cowley et al. (2014) Scientific Data l:article number 140035). These approaches include pooled genetic screens using CRISPR-Cas9-mediated genome editing that simultaneously test the role of a large number of genes on tumor cell growth, viability or drug resistance (Wang et al. (2014) Science 343:80-84; Shalem et al. (2014) Science 343:84-87). However, these screens have generally been conducted in vitro, where the contribution of the immune system is absent, or have studied phenotypes such as metastasis that do not directly evaluate the role of tumor immunity (Hart et al. (2015) Cell 163:1515-1526; Yu et al. (2016) Nat. Biotechnol. 34:419-423; Chen et al. (2015) Cell 160:1246-1260).

Despite the dramatic clinical success of cancer immunotherapy with PD-1 checkpoint blockade, most patients do not experience sustained clinical benefit from treatment. Accordingly a great need in the art exists for additional therapeutic strategies.

Summary of the Invention

The present invention is based, at least in part, on the discovery that modifying (e.g. , inhibiting/blocking or promoting one or more biomarkers listed in Table 1, such as one or more kinase signaling inhibitors (e.g., PTPN2 and SOCS1)), in combination with an immunotherapy, results in a synergistic therapeutic benefit for treating cancers that is unexpected given the lack of such benefit observed for the immunotherapy alone.

In one aspect, a method of treating a subject afflicted with a cancer comprising administering to the subject a therapeutically effective amount of an agent that modifies the copy number, the expression level, and/or the activity of one or more biomarkers listed in Table 1 or a fragment thereof, in combination with an immunotherapy, optionally wheren in the agent inhibits the copy number, the expression level, and/or the activity of one or more biomarkers listed in Table 1 or a fragment thereof, is provided.

Numerous embodiments are further provided that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in one embodiment, the agent described herein decreases the copy number, the expression level, and/or the activity of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors , such as tyrosine-protein phosphatase non-receptor type 2 (Ptpn2) and/or Suppressor Of Cytokine Signaling 1 (SOCSl)). In another embodiment, the agent selectively decreases the activity of one or more biomarkers in Table 1, such as decreasing the phosphatase activity and/or the substrate binding activity of one or more kinase signaling inhibitors (e.g., Ptpn2 and/or SOCSl). In still another embodiment, the agent described herein is a small molecule inhibitor, CRISPR single-guide RNA (sgRNA), RNA interfering agent, antisense oligonucleotide, peptide or peptidomimetic inhibitor, aptamer, or intrabody. In another embodiment, the RNA interfering agent is a small interfering RNA (siRNA), CRISPR RNA (crRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwi-interacting RNA (piRNA). In still another embodiment, the RNA interfering agent is a CRISPR single-guide RNA (sgRNA). In yet another embodiment, the sgRNA comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequence listed in Table 2. In one embodiment, the agent described herein comprises an intrabody, or an antigen binding fragment thereof, which specifically binds to the one or more biomarkers in Table 1 and/or a substrate of the one or more biomarkers in Table 1. In another embodiment, the intrabody, or antigen binding fragment thereof, is murine, chimeric, humanized, composite, or human. In still another embodiment, the intrabody, or antigen binding fragment thereof, is detectably labeled, comprises an effector domain, comprises an Fc domain, and/or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabodies fragments. In yet another embodiment, the intrabody, or antigen binding fragment thereof, is conjugated to a cytotoxic agent. In one embodiment, the cytotoxic agent is selected from the group consisting of a chemotherapeutic agent, a biologic agent, a toxin, and a radioactive isotope. In still another embodiment, the agent described herein increases the sensitivity of the cancer cells to an immunotherapy. In another embodiment, the immunotherapy and/or a cancer therapy is administered before, after, or concurrently with the agent. In still another embodiment, the immunotherapy comprises an anti-cancer vaccine and/or virus. In yet embodiment, the immunotherapy is cell-based. In one embodiment, immunotherapy inhibits an immune checkpoint. In another embodiment, the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7- H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP, CD47, CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, IDO, CD39, CD73 and A2aR. In still another embodiment, the immune checkpoint is selected from the group consisting of PD-1, PD-L1, and PD-L2. In yet another embodiment, the immune checkpoint is PD-1. In another embodiment, the one or more biomarker described herein comprises a nucleic acid sequence having at least 95% identity to a nucleic acid sequence listed in Table 1 and/or encodes an amino acid sequence having at least 95% identity to an amino acid sequence listed in Table 1. In another embodiment, the one or more biomarker is human, mouse, chimeric, or a fusion. In still another embodiment, the agent reduces the number of proliferating cells in the cancer and/or reduces the volume or size of a tumor comprising the cancer cells. In another embodiment, the agent increases the sensitivity of the cancer to the immunotherapy. In still another embodiment, the immunotherapy is T-cell-mediated. In yet another embodiment, the agent increases the amount of CD8+ T cells in a tumor comprising the cancer cells. In another another embodiment, the agent increases the level of MHC-I on the surface of the cancer cells. In one embodiment, the anti-cancer function of the agent is dependent on IFNy signaling. In still another embodiment, the method described herein further comprises administering to the subject at least one additional cancer therapy or regimen. In another embodiment, the at least one additional cancer therapy or regimen is administered before, after, or concurrently with the agent and/or the immunotherapy. In another embodiment, the agent described herein is administered in a pharmaceutically acceptable formulation.

In another aspect, a method of killing cancer cells comprising contacting the cancer cells with an agent that inhibits the copy number, the expression level, and/or the activity of one or more biomarkers listed in Table 1 or a fragment thereof, in combination with an immunotherapy, is provided. As described above, numerous embodiments are further provided that can be applied to any aspect of the present invention and/or combined with any other embodiment described herein. For example, in one embodiment, the agent described herein decreases the copy number, the expression level, and/or the activity of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors , such as PTPN2 and SOCS1). In another embodiment, the agent selectively decreases activity of one or more biomarkers in Table 1, such as decreasing the phosphatase activity and/or the substrate binding activity of one or more kinase signaling inhibitors. In still another embodiment, the agent is a small molecule inhibitor, CRISPR single-guide RNA (sgRNA), RNA interfering agent, antisense oligonucleotide, peptide or peptidomimetic inhibitor, aptamer, or intrabody. In one embodiment, the RNA interfering agent described herein is a small interfering RNA (siRNA), CRISPR RNA (crRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwi-interacting RNA (piRNA). In another embodiment, the RNA interfering agent is a CRISPR single-guide RNA (sgRNA). In still another embodiment, the sgRNA comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequence listed in Table 2. In one embodiment, the agent described herein comprises an intrabody, or an antigen binding fragment thereof, which specifically binds to the one or more biomarkers in Table 1 and/or a substrate of the one or more biomarkers in Table 1. In another embodiment, the intrabody, or antigen binding fragment thereof, is murine, chimeric, humanized, composite, or human. In still another embodiment, the intrabody, or antigen binding fragment thereof, is detectably labeled, comprises an effector domain, comprises an Fc domain, and/or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabodies fragments. In yet another embodiment, the intrabody, or antigen binding fragment thereof, is conjugated to a cytotoxic agent. In one embodiment, the cytotoxic agent is selected from the group consisting of a chemotherapeutic agent, a biologic agent, a toxin, and a radioactive isotope. In one embodiment, the agent described herein increases the sensitivity of the cancer cells to an immunotherapy. In another embodiment, the cancer cells are contacted with an immunotherapy and/or a cancer therapy before, after, or concurrently with the agent. In still another embodiment, the immunotherapy comprises an anti-cancer vaccine and/or virus. In one embodiment, the immunotherapy is cell-based. In another embodiment, the immunotherapy inhibits an immune checkpoint. In still another embodiment, the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD 160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP, CD47, CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, IDO, CD39, CD73 and A2aR. In yet another embodiment, the immune checkpoint is selected from the group consisting of PD-1, PD-L1, and PD-L2. In one embodiment, the immune checkpoint is PD-1. In another embodiment, the biomarker described herein comprises a nucleic acid sequence having at least 95% identity to a nucleic acid sequence listed in Table 1 and/or encodes an amino acid sequence having at least 95% identity to an amino acid sequence listed in Table 1. In still another embodiment, the one or more biomarker is human, mouse, chimeric, or a fusion. In one embodiment, the agent described herein reduces the number of proliferating cells in the cancer and/or reduces the volume or size of a tumor comprising the cancer cells. In another embodiment, the agent increases the sensitivity of the cancer to the immunotherapy. In still another embodiment, the immunotherapy is T-cell-mediated. In yet another embodiment, the agent increases the amount of CD8+ T cells in a tumor comprising the cancer cells. In one embodiment, the agent increases the level of MHC-I on the surface of the cancer cells. In another embodiment, the anti-cancer function of the agent is dependent on IFNy signaling. In one embodiment, the method described herein further comprises administering to the subject at least one additional cancer therapy or regimen. In another embodiment, the at least one additional cancer therapy or regimen is administered before, after, or concurrently with the agent and/or the immunotherapy. In another embodiment, the agent described herein is administered in a pharmaceutically acceptable formulation.

In still another aspect, a method of determining whether a subject afflicted with a cancer or at risk for developing a cancer would benefit from inhibiting the copy number, amount, and/or activity of at least one biomarker listed in Table 1 is provided, the method comprising a) obtaining a biological sample from the subject; b) determining the copy number, amount, and/or activity of at least one biomarker listed in Table 1 ; c) determining the copy number, amount, and/or activity of the at least one biomarker in a control; and d) comparing the copy number, amount, and/or activity of the at least one biomarker detected in steps b) and c); wherein the presence of, or a significant increase in, the copy number, amount, and/or activity of, the at least one biomarker listed in Table 1 in the subject sample relative to the control copy number, amount, and/or activity of the at least one biomarker indicates that the subject afflicted with the cancer or at risk for developing the cancer would benefit from inhibiting the copy number, amount, and/or activity of the at least one biomarker listed in Table 1. In one embodiment, the method described herein further comprises recommending, prescribing, or administering an agent that inhibits the at least one biomarker listed in Table 1 if the cancer is determined to benefit from the agent. In another embodiment, the method described herein further comprises administering at least one additional cancer therapy that is administered before, after, or concurrently with the agent. In still another embodiment, the method described herein further comprises recommending, prescribing, or administering cancer therapy other than an agent that inhibits the at least one biomarker listed in Table 1 if the cancer is determined to not benefit from the agent. In yet another embodiment, the cancer therapy is selected from the group consisting of immunotherapy, targeted therapy, chemotherapy, radiation therapy, hormonal therapy, an anti-cancer vaccine, an anti-cancer virus, and a checkpoint inhibitor. In one embodiment, the control sample is determined from a cancerous or non-cancerous sample from either the patient or a member of the same species to which the patient belongs. In another embodiment, the control sample comprises cells.

In yet another aspect, a method for predicting the clinical outcome of a subject afflicted with a cancer expressing one or more biomarkers listed in Table 1 or a fragment thereof to treatment with an immunotherapy is provided, the method comprising a) determining the copy number, amount, and/or activity of at least one biomarker listed in Table 1 in a subject sample; b) determining the copy number, amount, and/or activity of the at least one biomarker in a control having a good clinical outcome; and c) comparing the copy number, amount, and/or activity of the at least one biomarker in the subject sample and in the control; wherein the presence of, or a significant increase in, the copy number, amount, and/or activity of, the at least one biomarker listed in Table 1 in the subject sample as compared to the copy number, amount and/or activity in the control, is an indication that the subject has a poor clinical outcome.

In another aspect, a method for monitoring the progression of a cancer in a subject, wherein the subject is administered a therapeutically effective amount of an agent that inhibits the copy number, amount, and/or activity of at least one biomarker listed in Table 1 and an immunotherapy is provided, the method comprising a) detecting in a subject sample at a first point in time the copy number, amount, and/or activity of at least one biomarker listed in Table 1; b) repeating step a) at a subsequent point in time; and c) comparing the amount or activity of at least one biomarker listed in Table 1 detected in steps a) and b) to monitor the progression of the cancer in the subject.

In still another aspect, a method of assessing the efficacy of an agent that inhibits the copy number, amount, and/or activity of at least one biomarker listed in Table 1 and an immunotherapy for treating a cancer in a subject is provided, comprising a) detecting in a subject sample at a first point in time the copy number, amount, and/or or activity of at least one biomarker listed in Table 1; b) repeating step a) during at least one subsequent point in time after administration of the agent and the immunotherapy; and c) comparing the copy number, amount, and/or activity detected in steps a) and b), wherein the absence of, or a significant decrease in, the copy number, amount, and/or activity of, the at least one biomarker listed in Table 1, in the subsequent sample as compared to the copy number, amount, and/or activity in the sample at the first point in time, indicates that the agent and immunotherapy treats the cancer in the subject. In one embodiment, between the first point in time and the subsequent point in time, the subject has undergone treatment, completed treatment, and/or is in remission for the cancer. In another embodiment, the cancer treatment is selected from the group consisting of immunotherapy, targeted therapy, chemotherapy, radiation therapy, hormonal therapy, an anti-cancer vaccine, an anti-cancer virus, and a checkpoint inhibitor. In still another embodiment, the first and/or at least one subsequent sample is selected from the group consisting of ex vivo and in vivo samples. In yet another embodiment, the first and/or at least one subsequent sample is a portion of a single sample or pooled samples obtained from the subject.

In one embodiment, the sample described herein comprises cells, serum,

peritumoral tissue, and/or intratumoral tissue obtained from the subject. In one

embodiment, the one or more biomarkers listed in Table 1 comprises Ptpn2 and/or SOCS1. In another embodiment, the cancer is responsive to IFNy. In still another embodiment, the cancer cells are in a parainflamed tumor. In yet another embodiment, the cancer cells produce interferon.

In one embodiment, the cancer described herein is selected from the group consisting of melanoma, colorectal cancer, gliomas, neuroblastoma, prostate cancer, breast cancer, pancreatic ductal carcinoma, epithelial ovarian cancer, B-CLL, leukemia, B cell lymphoma, and renal cell carcinoma. In another embodiment, the cancer is in a subject and the subject is an animal model of the cancer. In still another embodiment, the animal model is a mouse model. In one embodiment, the cancer is in a subject and the subject is a mammal. In another embodiment, the mammal is a mouse or a human. In still another embodiment, the mammal is a human.

In still another aspect, an agent that inhibits one or more kinase signaling inhibitors

(e.g., biomarkers in Table I, such as PTPN2 and SOCS1) is provided for treating a cancer in a subject, in combination with an immunotherapy. Such agent may comprise a small molecule inhibitor, an RNA interfering agent, an antisense oligonucleotide, a peptide or peptidomimetic inhibitor, an aptamer, and/or an intrabody, as described herein.

In yet another aspect, a vector comprising an agent that inhibits one or more kinase signaling inhibitors (e.g., biomarkers in Table 1, such as PTPN2 and SOCS1) for treating a cancer in a subject, in combination with an immunotherapy, is provided. In another aspect, a host cell which comprises an agent that inhibits one or more kinase signaling inhibitors (e.g., biomarkers in Table 1, such as PTPN2 and SOCS1) for treating a cancer in a subject, in combination with an immunotherapy, is provided.

In still another aspect, a host cell which comprises a vector comprising an agent that inhibits one or more kinase signaling inhibitors (e.g., biomarkers in Table 1, such as PTPN2 and SOCS1) for treating a cancer in a subject, in combination with an immunotherapy, is provided.

In yet another aspect, a device or kit comprising the agent that inhibits one or more kinase signaling inhibitors (e.g., biomarkers in Table 1, such as PTPN2 and SOCS1) for treating a cancer in a subject, in combination with an immunotherapy, is provided.

Although the aspects and embodiments described above provide representative embodiments for biomarkers of the present invention, such as those listed in Table 1, for which inhibition in combination with an immunotherapy, results in a synergistic therapeutic benefit for treating cancers that is unexpected given the lack of such benefit observed for the immunotherapy alone, certain biomarkers clearly described herein, especially at Table 1, whose promoted expression rather than inhibition in combination with an

immunotherapy (e.g., identified as being enriched in the sgRNA screen rather than being depleted), results in a synergistic therapeutic benefit for treating cancers, are readily apparent. Thus, any aspect and embodiment described herein and above can use such biomarkers and their promoted expression in diagnostic, prognostic, therapeutic, etc.

applications regarding immunotherapy and cancers. For example, in one aspect, a method of killing cancer cells comprising contacting the cancer cells with an agent that promotes rather than inhibits the copy number, the expression level, and/or the activity of one or more such biomarkers listed in Table 1 or a fragment thereof, in combination with an immunotherapy, is provided. In another representative aspect, a method of determining whether a subject afflicted with a cancer or at risk for developing a cancer would benefit from promoting the copy number, amount, and/or activity of such at least one biomarker listed in Table 1 is provided, the method comprising a) obtaining a biological sample from the subject; b) determining the copy number, amount, and/or activity of at least one biomarker listed in Table 1 ; c) determining the copy number, amount, and/or activity of the at least one biomarker in a control; and d) comparing the copy number, amount, and/or activity of the at least one biomarker detected in steps b) and c); wherein the absence of, or a significant decrease in, the copy number, amount, and/or activity of, the at least one biomarker listed in Table 1 in the subject sample relative to the control copy number, amount, and/or activity of the at least one biomarker indicates that the subject afflicted with the cancer or at risk for developing the cancer would benefit from promoting the copy number, amount, and/or activity of the at least one biomarker listed in Table 1.

Brief Description of the Drawings

Figure 1 includes 6 panels, identified as panels A, B, C, D, E, and F, which show that in vivo pooled loss-of-function screening using CRISPR/Cas9 in tumor cells recovers known mediators of immune evasion. Panel A shows a schematic diagram of the in vivo screening system using the B16 transplantable tumor model. Tumor volumes (in mm ³ ) were compared under each condition, averaged for each group at each time point (Panel B, left) or for individual animals on the day of sacrifice (Panel B, right). Bars represent means, while whiskers represent standard deviation. Enrichment analysis was carried out using a hypergeometric test to show functional classes of genes, (from the Gene Ontology Consortium database (GO)) targeted by sgRNAs, that were enriched or depleted in tumors in animals, including animals treated with irradiated tumor cell vaccine (GVAX) and anti- PD-1 antibody and the TCRa ^"/_ animals (Panel C). Frequency histogram (Panel D, top) and collapsed histograms (Panel D, middle) of enrichment or depletion (normalized as Z scores) are shown for all 9,992 sgRNAs screened. Enrichment/depletion scores are averaged from 10 mice per condition. sgRNAs targeting PD-L1 are indicated by the red lines (Panel D, middle). PD-L1 expression is compared among Cas9-expressing B16 tumor cells transfected with one of the four sgRNAs targeting PD-L1 (red) or a control sgRNA (grey) (Panel D, bottom). Similar to Panel D, Panel E shows the depletion of CD47 by its specific sgRNAs (indicated in red (top and middle) and CD47 expression after CRISPR editing with sgRNAs targeting CD47 (bottom). Panel F compares tumor volumes over time between CD47 null (red) and control (grey) tumors growing in mice treated with GVAX and PD-1 blockade (average and standard error of the mean; n = 10 animals per group). ** p < 0.01 ; *** p < 0.001; **** p < 0.0001.

Figure 2 includes 6 panels, identified as panels A, B, C, D, E, and F, which show the performance analysis of the screening in Figure 1. Panel A shows Western blot of B16 cell lysate for Cas9 and β-ACTIN with or without transduction with a lentiviral vector encoding Cas9. A pie chart shows the fraction of genes targeted in the screening in each of the GO term categories indicated (Panel B). Two-dimensional histograms show the pair- wise distribution of sgR As abundance (averaged for each condition) (Panel C).

Saturation analysis of animal replicates from the three in vivo screening conditions is shown in Panel D. Pearson correlations are calculated for the library distribution in one animal vs. any other animal, then for two animals averaged versus any other two averaged, and so on. Saturation approaches r = 0.95. A matrix of the Pearson correlations of the library distribution from one animal compared to any other animal for B16 Pool 1 is shown (Panel E). Expression of CD47 by B16 cells transfected with either CD47-targeting (red) or control (grey) sgRNA is compared (Panel F).

Figure 3 includes 9 panels, identified as panels A, B, C, D, E, F, G, H, and I, which show that loss of IFNy pathway genes causes resistance to immunotherapy. Panel A shows frequency histogram (top) and collapsed histograms (below) of enrichment or depletion (normalized as z scores) for all sgRNAs in GVAX+ PD-1 blockade-treated mice relative to TCRa ^{_ "} mice, as in Figure 1. Red bars indicate the sgRNAs for the genes listed on the left. Schematic diagram of in vivo competitive growth assay using B16 cells is shown for different mice receiving a mixture of tumor cells containing targeting sgRNA or control sgRNA (Panel B). Representative flow plots show the frequencies of control or Statl null B16 cells for the conditions indicated (Panel C). Specifically, mixtures of Statl null tumors and control tumors (in a 1 :10 ratio) were tested in vitro, in TCRa ^"/_ mice, or GVAX + PD-1 blockade-treated wild-type mice. The B16 cell numbers in immunotherapy-treated wild-type mice (treated with different sgRNAs) were compared relative to those in TCRa ^"A mice, the change in the ratios (log ₂ normalized fold change) is shown in Panel D (mean and standard deviation; n = 8-10 mice per group). Tumor growth in immunotherapy-treated animals challenged with B16 cells lacking the indicated genes is compared (Panel E, each line represents one animal; n = 5 animals per group). Tumor volume averaged for each group at each time point (left) and survival (right) for Braf/Pten melanoma cells lacking the indicated gene (by treatment of the indicated sgRNAs) are compared in mice treated with PD-1 blockade (mean and standard error of the mean; n = 5 animals per group) (Panel F). The increases in cell number of B16 cells from wild-type mice treated with different sgRNAs were compared after an in vitro treatment with cytokines, and the change in the ratios (log ₂ normalized fold change) is shown in Panel G (mean and standard deviation; n = 3 replicates per group). Expression of MHC-I (H2K(b)/H2D(b)) on B16 cells lacking the indicated genes is compared with or without IFNy treatment (Panel H). In an in vitro killing assay, wild-type B16 cells (treated with control sgRNA) and Statl null ovalbumin- expressing B-16 cells (treated with Statl sgR A) were co-cultured with ovalbumin-specific T cells in different ratios as indicated. The B16 cell number was compared for each condition and the change in the ratios (log2 normalized fold change) (mean and standard deviation; n = 3 replicates per group) is shown in Panel I. * p < 0.05; ** p < 0.0l; *** p < 0.001; ****p < 0.0001.

Figure 4 includes 7 panels, identified as panels A, B, C, D, E, and F, which show the deletion of interferon pathway molecules from tumor cells. Panel A shows a Western blot of B16 cell lysate for STAT1 (left) and JAK1 (right) after transfection with the indicated sgRNA and Cas9. β-ACTIN serves as a loading control. The numbers below the lane indicate the percent expression of the target relative to control sgRNA cells. Same blots were also carried out for Braf/Pten melanoma cells (Panel B). Expression of Ifhgrl by B16 cells transfected with the indicated Ifhgr 1 -targeting (red) or control (grey) sgRNA and Cas9 is compared (left) and quantified in mean fluorescence intensity (right) (mean and standard deviation) (Panel C). The same expression was compared for Braf/Pten melanoma cells (Panel D). Expression of Ifhar2 by B16 cells transfected with the indicated Ifnarl- targeting (red) or control (grey) sgRNA and Cas9 is compared (left) and quantified in mean fluorescence intensity (right) (mean and standard deviation) (Panel E). Western blot of Braf/Pten melanoma cell lysate for PTPN2 and β-ACTIN after transfection with either control or Ptprc2-targeting sgRNAs is shown (Panel F, left) and Ptpnl transcript abundance for the same conditions is measured by qPCR (mean and standard deviation) (Panel F, right). ** p < 0.01.

Figure 5 includes 10 panels, identified as panels A, B, C, D, E, F, G, H, I, and J, which show that deletion of Ptpnl sensitizes tumor cells to immunotherapy. Panel A shows frequency histogram (top) and collapsed histogram (below) of enrichment or depletion (normalized as Z scores) for all sgRNAs in GVAX + PD-1 blockade-treated mice relative to TCRa ^{_ "} mice, as in previous figures. sgRNAs are indicated in red. Western blot of B 16 cell lysate for PTPN2 and β-ACTIN after transfections with either control or Ptpn2- targeting sgRNAs is compared (Panel B, left). Corresponding Ptpn2 transcript abundance was measured by qPCR and is shown in the percentage to control level (mean and standard deviation) (Panel B, right). Representative flow plots show frequencies of Ptpn2 null and control B16 tumor cells in mixture in vitro or in vivo (Panel C, right), compared to the mixture of tumor cells treated with either one of two control sgRNAs (Panel C, left). Panel D shows the change in the ratios (log ₂ normalized fold change) of B16 cells lacking the indicated genesxontrol cells in immunotherapy-treated mice compared to TCRa ^"7" mice (mean and standard deviation; n = 3-5 mice per group). The cell number of either Ptpn2 null or control B16 cells grown in GVAX + PD-1 blockade-treated wild-type mice, relative to the cell number of those cells grown in TCRa ^"7" mice, is shown in change in the ratios (log2 normalized fold change) (mean and standard deviation; n = 5 animals per group) (Panel E). Representative flow plots show the frequencies of Ptpn2 null, rescued, or overexpressing B16 cells and control B16 cells for the conditions indicated on the left (Panel F). The cell number of Ptpn2 null, rescued, or overexpressing B16 cells or control B16 cells in immunotherapy-treated mice is compared relative to the input population, and shown in change in the ratios (log2 normalized fold change) (mean and standard deviation; n = 5 animals per group) (Panel G). Tumor volume, averaged for each group at each time point, for Ptpn2 null (red) or control (grey) B16 cells growing in mice treated with GVAX and PD-1 blockade was compared (mean and standard error of the mean; n = 20 animals per group) (Panel H). Survival analysis for such groups is also shown (Panel I). Panel J shows tumor volume, averaged for each group at each time point, for Ptpn2 null (red) or control (grey) Braf/Pten melanoma cells growing in untreated WT mice is compared (mean and standard error of the mean; n = 10 animals per group). * p < 0.05; ** p < 0.01; *** p < 0.001; ****p < 0.0001.

Figure 6 includes 10 panels, identified as panels A, B, C, D, E, F, G, H, I, and J, which show that Ptpn2 null tumor cells do not have a growth disadvantage in vivo in the absence of T cells or immunotherapy. Tumor volume, averaged for each group at each time point, for Ptpn2 null (red) or control (grey) B16 cells growing in wild-type (WT) mice with no treatment is shown (mean and standard error of the mean; n = 10 animals per group) (Panel A). The corresponding survival analysis for the groups is shown in Panel B.

Similarly, tumor volume, averaged for each group at each time point, for Ptpn2 null (red) or control (grey) B16 cells growing in TCRa ^"7" mice with no treatment is shown in Panel C (mean and standard error of the mean; n = 10 animals per group) and the corresponding survival analysis is shown in Panel D. Tumor volume, averaged for each group at each time point, for Ptpn2 null (red) or control (grey) Braf/Pten melanoma cells growing in TCRa ^{" "} mice with no treatment is shown in Panel E (mean and standard error of the mean; n = 5 animals per group). The corresponding survival analysis is shown in Panel F. The tumor volume averaged for each group is compared at each time point for Ptpn2 null (red) or control (grey) MC38 cells growing in WT (Panel G) or TCRa ^"7" (Panel I) mice with no treatment (mean and standard error of the mean; n = 10 animals per group). The corresponding survival analyses are shown in Panel H and Panel J, respectively.

Figure 7 includes 6 panels, identified as panels A, B, C, D, E, and F, which show that Ptpn2 deficient tumor cells present antigen and stimulate T cells better than wild-type tumor cells. The number of different lymphoid cells per milligram of tumor was quantified in either Ptpn2 null (red) or control (grey) B16 tumors (mean and standard deviation; n = 8- 10 animals per group) (Panel A). Panel B shows representative images of

immunohistochemistry staining for CD8a in either control or Ptpn2 null tumors (left) and quantification of the average number of infiltrating CD8+ cells in each group (right) (mean and standard devation; n = 4-5 animals per group). Granzyme B expression by CD8 ⁺ T cells in Ptpn2 null (red) or control (grey) B16 tumors (left) was analyzed by representative histogram (Panel C, left) and quantified in the percentage of Granzyme B ⁺ CD8 ⁺ T cells from each group (Panel C, right) (mean and standard deviation; n = 8-10 animals per group). SIINFEKL-H2K(b) presentation on the surface of Ptpn2 null (red) or control (grey) B16 cells after IFNy stimulation for 72 hours was measured (Panel D, left) and quantified in mean fluorescence intensity (Panel D, right) (mean and standard deviation). Intracellular expression of IFNy and TNF by CD8 ⁺ T cells after re-stimulation with Ptpn2 null (red) or control (grey) B16 cells was measured (Panel E, left) and quantified for indicated populations (Panel E, right) (mean and standard deviation). Ptpn2 null (red) or control (grey) B 16 cells were co-cultured with antigen-specific T cells, in the indicated ratio of T cells:tumor cells. The increased cell number after co-culturing was compared and is shown in change in the ratios (log2 normalized fold change) (Panel F). * p < 0.05; ** p < 0.01; ***p < 0.001.

Figure 8 includes 4 panels, identified as panels A, B, C, and D, which show that Ptpn2 null tumors have increased effector T cell populations but no other significant differences in immune infiltrates compared with control tumors. Panel A contains representative plots showing the gating strategy used in Panel A of Figure 7. Populations of interest are gated in blue. The number of CD45+ cells per mg of tumor for Ptpn2 null (red) and control (grey) B16 tumors is compared (Panel B) (mean and standard deviation).

Populations of interest are gated in blue. Effector T cell populations in either Ptpn2 null (red) or control (grey) B16 tumors were quantified and compared (Panel D) (mean and standard deviation). Data pooled from 2 independent experiments with a minimum of 8 mice per group. The corresponding gating strategy for the quantification in Panel D is shown with representative plots (Panel C).

Figure 9 includes 2 panels, identified as panels A and B, which show that Ptpn2 null tumors show no significant differences in myeloid cell infiltration compared to control tumors. Numbers of myeloid cells per mg of tumor for Ptpn2 null (red) and control (grey) B16 tumors were quantified and compared (Panel B) (mean and standard deviation). Data pooled from 2 independent experiments with a minimum of 8 mice per group. The corresponding gating strategy is shown with representative plots (Panel A) (Broz et al. (2014) Cancer Cell 26: 638-652). Populations of interest are gated in blue.

Figure 10 includes 2 panels, identified as panels A and B, which show that Ptpn2 null tumor cells have increased MHC-I expression after IFNy stimulation and have increased sensitivity to costimulation with TNF and IFNy. Expression of MHC-I

(H2K(b)/H2D(b)) on either Ptpn2 null (red) or control (grey) B16 cells after stimulation with IFNy for 72 hours were compared (left) and quantified in mean fluorescence intensity (right) (Panel A) (mean and standard deviation). Panel B shows gene set enrichment analysis showing enrichment of signatures for IFNy, IFNa, and TNFa signaling via NFKB in Ptpn2 null B16 cells after costimulation with TNF and IFNy relative to Ptpn2 null cells stimulated with IFNy alone.

Figure 11 includes 9 panels, identified as panels A, B, C, D, E, F, G, H, and I, which show that Ptpn2 deficiency renders tumor cells more sensitive to immunotherapy by increasing sensitivity to IFNy. Western blot show levels of phosphorylated STAT1 after IFNy treatment of control, Ptpn2 null, rescued, or overexpressing B16 cells (Panel A). Principle components analysis of gene expression profiles from Ptpn2 null (closed symbols) or control (open symbols) B16 cells was carried out with or without the cytokine treatment indicated (Panel B). Numbered vectors correspond to the correlation of the gene signatures indicated to the principle component vectors shown. Panel C shows heat map of differentially expressed genes in the IFNy signature in either Ptpn2 null or control B16 cells after treatment with the indicated combinations of cytokines. B16 cells transfected with the indicated sgRNAs or control B16 cells were treated with different cytokines. Panel D shows gene set enrichement analysis for IFNy signature genes in Ptpn2 null cells relative to control cells for B16 melanoma (top) and four human cancer cell lines (bottom). The increase of cell numbers before and after cytokine treatment was compared and shown in change in the ratios (log ₂ normalized fold change) (Panel E) (mean and standard deviation; n = 3 replicates per group). Panel F shows the change in the ratios (log2 normalized fold change) of human A375 melanoma cells or HT-29 colon carcinoma cells infected with the indicated sgRNAxontrol cells after cell culture with the cytokines indicated below (mean and standard deviation; n = 3 replicates per group). Panel G shows a diagram of the type I and type II interferon pathway and further shows the targets of Ptpn2. Genetic epistasis in the pathway was tested by transfecting cells with a sgRNA for Ptpn2 (filled red circle) and a sgRNA for a member of the interferon pathway (dashed red line) potentially epistatic to Ptpn2 (Panel G). Representative flow plots show the frequencies of StatllPtpn2 double- null cells and Statl null cells in the conditions indicated (in vitro or in GVAX + PD-1 blockade-treated wild-type mice) (Panel H, right) and the frequencies of Ptpn2 null cells and control cells (Panel H, left). Population 2 (blue) B16 cells were grown in

immunotherapy-treated mice and their increase of cell number were compared and shown in change in the ratios (log ₂ normalized fold change) relative to the input population (Panel I) (mean and standard deviation; n = 5 mice per group). ** p < 0.01; *** p < 0.001.

Figure 12 shows that sensitivity to IFNy can be increased using small molecule inhibitors of PTP1B. Change in the ratios (log2 normalized fold change) of B16 cells transfected with the Ptpn2 sgRNAxontrol cells is compared after either IFNy or IFNy + PTP1B inhibitor treatment (mean and standard deviation; n = 3 replicates per group). * P < 0.05.

Detailed Description of the Invention

It has been determined herein that modulatory regulators of one or more biomarkers in Table I, e.g., inhibitors one or more kinase signaling inhibitors, such as PTPN2 and SOCS1, can be used to augment tumor immunity and immunotherapies, such as by increase interferon sensing by tumor cells. Thus, the instant disclosure provides at least a method of treating cancers, e.g., those cancer types otherwise not responsive or weakly responsive to immunotherapies, with a combination of a biomarker modulator, such as a negative regulator of PTPN2,_and/or SOCS1, and another immunotherapy. Tyrosine-protein phosphatase non-receptor type 2 (PTPN2) is a member of the protein tyrosine phosphatase (PTP) family. PTPN2 dephosphorylates receptor protein tyrosine kinases, including INSR, EGFR, CSFIR, PDGFR, as well as non-receptor protein tyrosine kinases like JAKl, JAK2, JAK3, Src family kinases, STAT1, STAT3, STAT5A, STAT5B and STAT6, either in the nucleus or the cytoplasm (Shuai et al. (2003) Nat. Rev. Immunol. 3:900-911; Wiede et al. (2011) J Clin. Invest. 121:4758-4774; Simoncic et al. (2006) Mol. Cell Biol. 26:4149-4160; Kleppe et al. (2010) Nat. Genet. 42:530-535; Zhou et al. (2014) Nature 506:52-57). Since PTPN2 has numerous pleiotropic activties, such as negatively regulating numerous signaling pathways and biological processes like hematopoiesis, inflammatory response, cell proliferation and differentiation, and glucose homeostasis, the particular heretofore unknown and critical effect that PTPN2 exerts on interferon sensing as a gateway regulator of tumor immunity and immunotherapy is surprising. The results described herein ares all the more unexpected given that analyses of PTPN2 function has heretofore been largely confined to hematopoietic cells and not examined in cancer cells, as well as the fact that modulating sensitivity to interferon signaling is critical for immunotherapy effects rather than simply modulating interferon availability since interferon therapy is known to not significantly augment immunotherapy effects. Accordingly, the present invention provides exemplary RNA interfering agents inhibiting one or more biomarkers in Table I, e.g., one or more kinase signaling inhibitors , such as PTPN2 and SOCS1, which may be used in the combination therapy and other methods described herein, such as agents that inhibit the function of one or more biomarkers in Table 1 and/or their ability to interact/bind to their substrates described herein, or by increasing its degradation and/or stability and/or interaction/binding to their inhibitors. Similarly, methods of screening for inhibitors of one or more biomarkers in Table I, e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1, and methods of diagnosing, prognosing, and monitoring cancer involving such inhibitor/immunotherapy combination therapies are provided. As described above, certain of the one or more biomarkers described herein and especially in Table 1 can be inhibited to promote anti-cancer immunotherapy effects and related cancer diagnostic, prognostic, and monitoring methods and certain other biomarkers described herein and especially in Table 1 can have the opposite modulation to effect similar results (e.g., they can be promoted to promote anti-cancer immunotherapy effects and related cancer diagnostic, prognostic, and monitoring methods).

I. Definitions

The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. The term "altered amount" or "altered level" refers to increased or decreased copy number (e.g., germline and/or somatic) of a biomarker nucleic acid, e.g., increased or decreased expression level in a cancer sample, as compared to the expression level or copy number of the biomarker nucleic acid in a control sample. The term "altered amount" of a biomarker also includes an increased or decreased protein level of a biomarker protein in a sample, e.g., a cancer sample, as compared to the corresponding protein level in a normal, control sample. Furthermore, an altered amount of a biomarker protein may be determined by detecting posttranslational modification such as methylation status of the marker, which may affect the expression or activity of the biomarker protein.

The amount of a biomarker in a subject is "significantly" higher or lower than the normal amount of the biomarker, if the amount of the biomarker is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, and preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or than that amount. Alternately, the amount of the biomarker in the subject can be considered "significantly" higher or lower than the normal amount if the amount is at least about two, and preferably at least about three, four, or five times, higher or lower, respectively, than the normal amount of the biomarker. Such "significance" can also be applied to any other measured parameter described herein, such as for expression, inhibition, cytotoxicity, cell growth, and the like.

The term "altered level of expression" of a biomarker refers to an expression level or copy number of the biomarker in a test sample, e.g., a sample derived from a patient suffering from cancer, that is greater or less than the standard error of the assay employed to assess expression or copy number, and is preferably at least twice, and more preferably three, four, five or ten or more times the expression level or copy number of the biomarker in a control sample (e.g., sample from a healthy subjects not having the associated disease) and preferably, the average expression level or copy number of the biomarker in several control samples. The altered level of expression is greater or less than the standard error of the assay employed to assess expression or copy number, and is preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more times the expression level or copy number of the biomarker in a control sample (e.g., sample from a healthy subjects not having the associated disease) and preferably, the average expression level or copy number of the biomarker in several control samples. In some embodiments, the level of the biomarker refers to the level of the biomarker itself, the level of a modified biomarker (e.g., phosphorylated biomarker), or to the level of a biomarker relative to another measured variable, such as a control (e.g., phosphorylated biomarker relative to an unphosphorylated biomarker).

The term "altered activity" of a biomarker refers to an activity of the biomarker which is increased or decreased in a disease state, e.g., in a cancer sample, as compared to the activity of the biomarker in a normal, control sample. Altered activity of the biomarker may be the result of, for example, altered expression of the biomarker, altered protein level of the biomarker, altered structure of the biomarker, or, e.g., an altered interaction with other proteins involved in the same or different pathway as the biomarker or altered interaction with transcriptional activators or inhibitors.

The term "altered structure" of a biomarker refers to the presence of mutations or allelic variants within a biomarker nucleic acid or protein, e.g., mutations which affect expression or activity of the biomarker nucleic acid or protein, as compared to the normal or wild-type gene or protein. For example, mutations include, but are not limited to substitutions, deletions, or addition mutations. Mutations may be present in the coding or non-coding region of the biomarker nucleic acid.

Unless otherwise specified here within, the terms "antibody" and "antibodies" refers to antigen-binding portions adaptable to be expressed within cells as "intracellular antibodies." (Chen et al. (1994) Human Gene Ther. 5:595-601). Methods are well-known in the art for adapting antibodies to target (e.g., inhibit) intracellular moieties, such as the use of single-chain antibodies (scFvs), modification of immunoglobulin VL domains for hyperstability, modification of antibodies to resist the reducing intracellular environment, generating fusion proteins that increase intracellular stability and/or modulate intracellular localization, and the like. Intracellular antibodies can also be introduced and expressed in one or more cells, tissues or organs of a multicellular organism, for example for

prophylactic and/or therapeutic purposes (e.g., as a gene therapy) (see, at least PCT Pubis. WO 08/020079, WO 94/02610, WO 95/22618, and WO 03/014960; U.S. Pat. No.

7,004,940; Cattaneo and Biocca (1997) Intracellular Antibodies: Development and

Applications (Landes and Springer- Verlag pubis.); Kontermann (2004) Methods 34:163- 170; Cohen et al. (1998) Oncogene 17:2445-2456; Auf der Maur et al. (2001) FEBS Lett. 508:407-412; Shaki-Loewenstein et al. (2005) J Immunol. Meth. 303:19-39). Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g. humanized, chimeric, etc.). Antibodies may also be fully human. Preferably, antibodies of the present invention bind specifically or substantially specifically to a biomarker polypeptide or fragment thereof. The terms "monoclonal antibodies" and "monoclonal antibody composition", as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen, whereas the term "polyclonal antibodies" and "polyclonal antibody composition" refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.

Antibodies may also be "humanized", which is intended to include antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs. The term "humanized antibody", as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

The term "assigned score" refers to the numerical value designated for each of the biomarkers after being measured in a patient sample. The assigned score correlates to the absence, presence or inferred amount of the biomarker in the sample. The assigned score can be generated manually (e.g. , by visual inspection) or with the aid of instrumentation for image acquisition and analysis. In certain embodiments, the assigned score is determined by a qualitative assessment, for example, detection of a fluorescent readout on a graded scale, or quantitative assessment. In one embodiment, an "aggregate score," which refers to the combination of assigned scores from a plurality of measured biomarkers, is determined. In one embodiment the aggregate score is a summation of assigned scores. In another embodiment, combination of assigned scores involves performing mathematical operations on the assigned scores before combining them into an aggregate score. In certain, embodiments, the aggregate score is also referred to herein as the "predictive score."

The term "biomarker" refers to a measurable entity of the present invention that has been determined to be predictive of inhibitors of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors , such as PTPN2 and/or SOCS1) and

immunotherapy (e.g., immune checkpoint inhibitors) combinatorial therapy effects on a cancer. Biomarkers can include, without limitation, nucleic acids and proteins, including those shown in the Tables, the Examples, the Figures, and otherwise described herein. As described herein, any relevant characteristic of a biomarker can be used, such as the copy number, amount, activity, location, modification (e.g., phosphorylation), and the like.

A "blocking" antibody or an antibody "antagonist" is one which inhibits or reduces at least one biological activity of the antigen(s) it binds. In certain embodiments, the blocking antibodies or antagonist antibodies or fragments thereof described herein substantially or completely inhibit a given biological activity of the antigen(s).

The term "body fluid" refers to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g. amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit).

The terms "cancer" or "tumor" or "hyperproliferative" refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain

characteristic morphological features. Unless otherwise stated, the terms include metaplasias. In some embodiments, such cells exhibit such characteristics in part or in full due to the expression and activity of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) and/or their related signaling pathways (e.g., PTPN2-regulated signaling pathways, including NF-kappaB signaling pathway, MAP kinase pathway, JAK-STAT signaling pathway, or other signaling pathways involving receptor tyrosine kinases, non-receptor tyrosine kinases, Src family kinases, and/or signal transducer and activator of transcription (STAT) proteins and/or SOCS1- regulated signaling pathways, including e.g. activated Toll Like Receptror 4 (TLR4) or Toll Like Receptror 2 (TLR2) signaling, MyD88-Mal cascade initiated on plasma membrane, prolactin signaling pathway, IL-2 pathway, calss I MHC mediated antigen processing and presentation, immune response interferon α/β signaling pathway, etc.). In certain embodiments, the cancer cells are capable of responding to interferon because they express functional proteins of the type I interferon signaling pathway and/or type II interferon signaling pathway, such as those shown in panel E of Figure 11. In some embodiments, the cancer cells described herein are not sensitive to at least one of immunotherapies. Such insensitivity, without limitation, may be related to the inactivation or decreased activation, compared to control cells (e.g., normal and/or wild-type non-cancer cells, and/or cancer cells without this insensitivity to immunotherapies), of interferon signaling (e.g., IFNy signaling) in such cancer cells and/or other surrounding cells and/or cells localized near to such cancer cells. Such inactivation or decreased activation of interferon signaling, without limitation, may be related to the inhibition of interferon signaling by one or more biomarkers in Table I, e.g., one or more kinase signaling inhibitors, such as PTPN2 and/or SOCSl (e.g., by overexpression and/or gain-of-function of the gene, RNA transcript, and/or protein of such one or more biomarkers in Table 1, and/or by reduced expression and/or loss-of-funciton of endogenous negative regulator(s) of such one or more biomarkers in Table 1). In some embodiments, the cancer cells are treatable with an agent capable of antagonizing one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and/or SOCSl), such as inhibiting expression and/or function of one or more biomarkers in Table 1, as described herein. An exemplary agent, without limitation, may relieve the inhibition of interferon (e.g., IFNy) signaling by PTPN2 and/or SOCSl in such cancer cells and/or other cells surrounding or localized near such cancer cells, thus restoring the IFNy signaling and the sensitivity of such cancer cells to

immunotherapies, especially those immunotherapies related to interferon signaling pathways. In some embodiments, the treatment with the agent antagonizing one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCSl) as described herein would increase IFNy signaling in such cancer cells, compared to pre-treatment situations, or would restore IFNy signaling in such cancer cells to at least comparable to the levels in control cells, so that such cancer cells would regain sensitivity to immunotherapies. The term "interferon signaling" or "IFNy signaling" used herein refers to any cell signaling downstream and/or related to the interaction of interferon (e.g., IFNy) and their receptor(s). Some exemplary IFNy cell signaling include, without limitation, the activation of macrophages and/or induction of Class II major histocompatibility complex (MHC) molecule expression, and/or activation of multiple immune effector genes through the Janus kinase (JAK)-STAT signaling pathway {e.g., through STAT1 transcription factor). The receptor specific for IFNy is IFNyR, comprising two chains, namely IFNyRl (also known as the IFNyR alpha chain) and IFNyR2 (also known as the IFNyR beta chain). IFNyRl is the ligand binding receptor and is required but not sufficient for signal transduction, whereas IFNyR2 do not bind IFNy independently but mainly plays a role in IFNy signaling and is generally the limiting factor in IFNy responsiveness. Both IFNyR chains lack intrinsic kinase/phosphatase activity and thus rely on other signaling proteins like Janus-activated kinase 1 (JAK1), JAK2 and signal transducer and activator of transcription 1 (STAT-1) for signal transduction. IFNyR complex in its resting state is a preformed tetramer and upon IFNy association undergoes a conformational change. This conformational change induces the phosphorylation and activation of JAK1, JAK2, and STAT1 which in turn induces genes containing the gamma- interferon activation sequence (GAS) in the promoter. Many IFNy functions are mediated by direct activation of immune effector genes by STAT1, including genes encoding antiviral proteins, microbicidal molecules, phagocytic receptors, chemokines, cytokines, and antigen-presenting molecules. Canonical Jak-STAT signaling mechanisms leading to activation of well-characterized STAT1 target genes have been previously reviewed (Stark (2007) Cytokine Growth Factor Rev., 18:419-423). In addition, activation of other STATs and alternative signaling pathways can contribute to IFNy function in certain cell contexts (reviewed in van Boxel-Dezaire and Stark, 2007 Curr. Top. Microbiol. Immunol., 316:119- 154 and Gough et ah, 2008 Cytokine Growth Factor Rev., 19:383-394). Importantly, many key IFNy functions are mediated by cross-regulation of cellular responses to other cytokines and inflammatory factors, such as, at least, tumor necrosis factor-alpha, interleukin-4, type I IFNs, and lipopolysaccharide. The capacity of IFNy to cross-regulate signaling pathways induced by other endogenous and exogenous factors is less appreciated, and the underlying mechanisms are more recently described. The mechanisms and

(patho)physiological impact of IFNy-mediated cross-regulation of signal transduction is reviewed by Hu and Ivashkiv (2009) Immunity 31 :539-550. For reviews of multiple IFNy responsive genes, see Samarajiwa et al. (2009) Nucl. Acids Res. 37:D852-D857 and Schneider et al. (2014) Annu. Rev. Immunol. 32:513-545. IFNy signaling can at least promote NK cell activity, increase antigen presentation and lysosome activity of macrophages, activate inducible nitric oxide synthase (iNOS), and induce the production of IgG2a and IgG3 from activated plasma B cells. Many IFN-stimulated genes control viral, bacterial, and parasite infection by directly targeting pathways and functions required during pathogen life cycles. Upregulation of chemokines and chemokine receptors enables cell-to-cell communication, whereas negative regulators of signaling help resolve the IFN- induced state and facilitate the return to cellular homeostasis. Additional IFN-stimulated genes encode for proapoptotic proteins, leading to cell death under certain conditions. IFNy signaling, as described herein, include at least activation or inhibition of at least one IFNy responsive genes well known in the art. The detection methods for such activation or inhibitor of IFNy responsive genes are also well known in the art. In some embodiments, the cancer cells described herein have defective or at least reduced IFNy signaling, preferably due to inhibition by one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1). Upon treatment with the antagonizing agent for such one or more biomarkers in Table 1, as described herein, such cancer cells restore IFNy signaling. Such defective, reduced, or restored IFNy signaling can be detected and/or measured through the expression and/or function of IFN-responsive genes, as described herein, using any known method in the art.

Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-tumorigenic cancer cell, such as a leukemia cell. As used herein, the term "cancer" includes premalignant as well as malignant cancers. Cancers include, but are not limited to, B cell cancer, e.g., multiple myeloma, Waldenstrom's macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematologic tissues, and the like. Other non-limiting examples of types of cancers applicable to the methods encompassed by the present invention include human sarcomas and carcinomas, e.g., fibrosarcoma,

myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,

angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, liver cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, bone cancer, brain tumor, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,

hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. In some embodiments, cancers are epithlelial in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer. In other embodiments, the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer. In still other embodiments, the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma. The epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.

In certain embodiments, the cancer encompasses colorectal cancer (e.g., colorectal carcinoma).

The term "colorectal cancer" as used herein, is meant to include cancer of cells of the intestinal tract below the small intestine (e.g., the large intestine (colon), including the cecum, ascending colon, transverse colon, descending colon, and sigmoid colon, and rectum). Additionally, as used herein, the term "colorectal cancer" is meant to further include cancer of cells of the duodenum and small intestine (jejunum and ileum).

Colorectal cancer also includes neoplastic diseases involving proliferation of a single clone of cells of the colon and includes adenocarcinoma and carcinoma of the colon whether in a primary site or metastasized.

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and ranks second in cancer mortality. Extensive genetic and genomic analysis of human CRC has uncovered germline and somatic mutations relevant to CRC biology and malignant transformation (Fearon et al. (1990) Cell 61, 759-767). These mutations have been linked to well-defined disease stages from aberrant crypt proliferation or hyperplasic lesions to benign adenomas, to carcinoma in situ, and finally to invasive and metastatic disease, thereby establishing a genetic paradigm for cancer initiation and progression. Genetic and genomic instability are catalysts for colon carcinogenesis (Lengauer et al. (1998) Nature 396:643-649). CRC can present with two distinct genomic profiles that have been termed (i) chromosomal instability neoplasia (CIN), characterized by rampant structural and numerical chromosomal aberrations driven in part by telomere dysfunction (Artandi et al. (2000) Nature 406:641-645; Fodde et al. (2001) Nat. Rev. Cancer 1:55-67; Maser and DePinho (2002) Science 297:565-569; Rudolph et al. (2001) Nat. Genet. 28:155-159) and mitotic aberrations (Lengauer et al. (1998) Nature 396:643-649) and (ii) microsatellite instability neoplasia (ΜΓΝ), characterized by near diploid karyotypes with alterations at the nucleotide level due to mutations in mismatch repair (MMR) genes (Fishel et al. (1993) Cell 75:1027-1038; Ilyas et al. (1999) Eur. J. Cancer 35:335-351; Modrich (1991) Annu. Rev. Genet. 25:229-253; Parsons et al. (1995) Science 268:738-740; Parsons et al. (1993) Cell 75:1227-1236). Germline MMR mutations are highly penetrant lesions which drive the ΜΓΝ phenotype in hereditary nonpolyposis colorectal cancers, accounting for 1-5% of CRC cases (de la Chapelle (2004) Nat. Rev. Cancer 4:769-780; Lynch and de la Chapelle (1999) J Med. Genet. 36:801-818; Umar et al. (2004) Nat. Rev. Cancer 4:153-158). While CIN and ΜΓΝ are mechanistically distinct, their genomic and genetic consequences emphasize the requirement of dominant mutator mechanisms to drive intestinal epithelial cells towards a threshold of oncogenic changes needed for malignant transformation.

A growing number of genetic mutations have been identified and functionally validated in CRC pathogenesis. Activation of the WNT signaling pathway is an early requisite event for adenoma formation. Somatic alterations are present in APC in greater than 70% of nonfamilial sporadic cases and appear to contribute to genomic instability and induce the expression of c-myc and Cyclin Dl (Fodde et al. (2001) Nat. Rev. Cancer 1:55- 67), while activating β-catenin mutations represent an alternative means of WNT pathway deregulation in CRC (Morin (1997) Science 275:1787-1790). K-Ras mutations occur early in neoplastic progression and are present in approximately 50% of large adenomas (Fearon and Gruber (2001) Molecular abnormalities in colon and rectal cancer, ed. J. Mendelsohm, P.H., M. Israel, and L. Liotta, W.B. Saunders, Philadelphia). The BRAF serine/threonine kinase and PIK3CA lipid kinase are mutated in 5-18% and 28% of sporadic CRCs, respectively (Samuels et al. (2004) Science 304:554; Davies et al. (2002) Nature 417:949- 954; Rajagopalan et al. (2002) Nature 418:934; Yuen et al. (2002) Cancer Res. 62:6451- 6455). BRAF and K-ras mutations are mutually exclusive in CRC, suggesting over-lapping oncogenic activities (Davies et al. (2002) Nature 417:949-954; Rajagopalan et al. (2002) Nature 418:934). Mutations associated with CRC progression, specifically the adenoma-to- carcinoma transition, target the TP53 and the TGF-β pathways (Markowitz et al. (2002) Cancer Cell 1 :233-236). Greater than 50% of CRCs harbor TP53 inactivating mutations (Fearon and Gruber (2001) Molecular abnormalities in colon and rectal cancer, ed. J.

Mendelsohm, P.H., M. Israel, and L. Liotta, W.B. Saunders, Philadelphia) and 30% of cases possess ΤϋΡβ ΙΙ mutations (Markowitz (2000) Biochim. Biophys. Acta 1470:M13-M20; Markowitz et al. (1995) Science 268:1336-1338). MIN cancers consistently inactivate TGFfi-RII by frameshift mutations, whereas CIN cancers target the pathway via inactivating somatic mutations in the TGFP-RII kinase domain (15%) or in the downstream signaling components of the pathway, including SMAD4 (15%) or SMAD2 (5%) transcription factors (Markowitz (2000) Biochim. Biophys. Acta 1470:M13-M20). In some embodiments, the colorectal cancer is micro satellite instable (MSI) colorectal cancer (Llosa et al. (2014) Cancer Disc. CD-14-0863; published online Oct. 30, 2014). MSI represents about 15% of sporadic CRC and about 5-6% of stage IV CRCs. MSI is caused by epigenetic silencing or mutation of DNA mismatch repair genes and typically presents with lower stage disease than microsatellite stable subset (MSS) CRC. MSI highly express immune checkpoints, such as PD-1, PD-L1, CTLA-4, LAG-3, and IDO. In other embodiments, the colorectal cancer is MSS CRC.

In certain embodiments, the cancer encompasses melanoma. The term "melanoma" as used herein, is generally meant to include cancers that develop from the pigment- containing cells, known as melanocytes, in the basal layer of the epidermis. Melanomas typically occur in the skin but may rarely occur in the mouth, intestines, or eye. In women they most commonly occur on the legs, while in men they are most common on the back. Sometimes they develop from a mole with concerning changes including an increase in size, irregular edges, change in color, itchiness, or skin breakdown. Thus, the term

"melanoma" also includes cancers developing from these cells, tissues, and organs.

Melanomas are among the most dangerous forms of skin cancer and develop when unrepaired DNA damage to skin cells (most often caused by ultraviolet radiation from sunshine or tanning beds) triggers gene mutations that lead the skin cells to multiply rapidly and form malignant tumors. The primary cause of melanoma is ultraviolet light (UV) exposure in those with low levels of skin pigment. Melanomas often resemble moles; some develop from moles. Those with many moles, a history of affected family members, and who have poor immune function are at greater risk. A number of rare genetic defects such as xeroderma pigmentosum also increase risk (Azoury and Lange, 2014 Surg Clin North Am. 2014 94:945-962).

Melanoma can be divided into different types, including, at least, lentigo maligna, lentigo maligna melanoma, superficial spreading melanoma, acral lentiginous melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft-tissue melanoma, melanoma with small nevus-like cells, melanoma with features of a Spitz nevus, uveal melanoma, etc. (see James, et al., 2006 Andrews' Diseases of the Skin: clinical Dermatology. Saunders Elsevier, pp. 694-9)

Diagnosis is by biopsy of any concerning skin lesion, including, at least, shave (tangential) biopsy, punch biopsy, incisional and excisional biopsies, "optical" biopsies (e.g. , by reflectance confocal microscopy (RCM)), fine needle aspiration (FNA) biopsy, surgical lymph node biopsy, sentinel lymph node biopsy, etc. In addition, visual inspection may also be used for diagnosis, such as a popular method for the signs and symptoms of melanoma as mnemonic "ABCDE": Asymmetrical skin lesion, Border of the lesion is irregular, Color: melanomas usually have multiple colors, Diameter: moles greater than 6 mm are more likely to be melanomas than smaller moles, and Enlarging: Enlarging or evolving. Another method as the "ugly duckling sign" is also known in the art (Mascara and Mascara, 1998 Arch Dermatol. 134: 1484-1485).

Treatment of melanoma includes surgery, chemotherapy (such as temozolomide, dacarbazine (also termed DTIC), etc.), radiation therapy, oncolytic virotherapy (e.g., see Forbes et ah, 2013 Front. Genet. 4:184), and immunotherapy (e.g., interleukin-2 (IL-2), interferon, etc.). Targeted therapies (e.g., as in Maverakis et ah, 2015 Acta Derm Venereoh 95: 516-524) may include: 1) adoptive cell therapy (ACT) using TILs immune cells (tumor infiltrating lymphocytes) isolated from a person's own melanoma tumor). Cells are grown in large numbers in a laboratory and returned to the patient after a treatment that temporarily reduces normal T cells in the patient's body. TIL therapy following

lymphodepletion can result in durable complete response in a variety of setups (Besser et ah, 2010 Clin. Cancer Res. 16:2646-2655); and 2) adoptive transfer of genetically altered (expressing T cell receptors (TCRs)) autologous lymphocytes into patient's lymphocytes, where the altered lymphocytes recognize and bind to the surface of melanoma cells and kill them. Other therapies include, at least, B-Raf inhibitors (such as vemurafenib, see

Chapman et al., 2011 N. Engl. J. Med. 364:2507-2516) and ipilimumab (alone or in combination with dacarbazine, see, e.g., Robert et al. (2011) N. Engl. J. Med. 364:2517- 2526).

The term "coding region" refers to regions of a nucleotide sequence comprising codons which are translated into amino acid residues, whereas the term "noncoding region" refers to regions of a nucleotide sequence that are not translated into amino acids {e.g., 5' and 3' untranslated regions).

The term "complementary" refers to the broad concept of sequence

complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds ("base pairing") with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

The term "control" refers to any reference standard suitable to provide a comparison to the expression products in the test sample. In one embodiment, the control comprises obtaining a "control sample" from which expression product levels are detected and compared to the expression product levels from the test sample. Such a control sample may comprise any suitable sample, including but not limited to a sample from a control cancer patient (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a normal patient or the cancer patient, cultured primary cells/tissues isolated from a subject such as a normal subject or the cancer patient, adjacent normal cells/tissues obtained from the same organ or body location of the cancer patient, a tissue or cell sample isolated from a normal subject, or a primary cells/tissues obtained from a depository. In another preferred embodiment, the control may comprise a reference standard expression product level from any suitable source, including but not limited to housekeeping genes, an expression product level range from normal tissue (or other previously analyzed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, survival for one, two, three, four years, etc.) or receiving a certain treatment (for example, standard of care cancer therapy). It will be understood by those of skill in the art that such control samples and reference standard expression product levels can be used in combination as controls in the methods of the present invention. In one embodiment, the control may comprise normal or non-cancerous cell/tissue sample. In another preferred embodiment, the control may comprise an expression level for a set of patients, such as a set of cancer patients, or for a set of cancer patients receiving a certain treatment, or for a set of patients with one outcome versus another outcome. In the former case, the specific expression product level of each patient can be assigned to a percentile level of expression, or expressed as either higher or lower than the mean or average of the reference standard expression level. In another preferred embodiment, the control may comprise normal cells, cells from patients treated with combination chemotherapy, and cells from patients having benign cancer. In another embodiment, the control may also comprise a measured value for example, average level of expression of a particular gene in a population compared to the level of expression of a housekeeping gene in the same population. Such a population may comprise normal subjects, cancer patients who have not undergone any treatment (/ ^' . e. , treatment naive), cancer patients undergoing standard of care therapy, or patients having benign cancer. In another preferred embodiment, the control comprises a ratio transformation of expression product levels, including but not limited to determining a ratio of expression product levels of two genes in the test sample and comparing it to any suitable ratio of the same two genes in a reference standard;

determining expression product levels of the two or more genes in the test sample and determining a difference in expression product levels in any suitable control; and determining expression product levels of the two or more genes in the test sample, normalizing their expression to expression of housekeeping genes in the test sample, and comparing to any suitable control. In particularly preferred embodiments, the control comprises a control sample which is of the same lineage and/or type as the test sample. In another embodiment, the control may comprise expression product levels grouped as percentiles within or based on a set of patient samples, such as all patients with cancer. In one embodiment a control expression product level is established wherein higher or lower levels of expression product relative to, for instance, a particular percentile, are used as the basis for predicting outcome. In another preferred embodiment, a control expression product level is established using expression product levels from cancer control patients with a known outcome, and the expression product levels from the test sample are compared to the control expression product level as the basis for predicting outcome. As demonstrated by the data below, the methods of the present invention are not limited to use of a specific cut-point in comparing the level of expression product in the test sample to the control.

The "copy number" of a biomarker nucleic acid refers to the number of DNA sequences in a cell (e.g. , germline and/or somatic) encoding a particular gene product.

Generally, for a given gene, a mammal has two copies of each gene. The copy number can be increased, however, by gene amplification or duplication, or reduced by deletion. For example, germline copy number changes include changes at one or more genomic loci, wherein said one or more genomic loci are not accounted for by the number of copies in the normal complement of germline copies in a control (e.g., the normal copy number in germline DNA for the same species as that from which the specific germline DNA and corresponding copy number were determined). Somatic copy number changes include changes at one or more genomic loci, wherein said one or more genomic loci are not accounted for by the number of copies in germline DNA of a control (e.g., copy number in germline DNA for the same subject as that from which the somatic DNA and corresponding copy number were determined).

The "normal" copy number (e.g., germline and/or somatic) of a biomarker nucleic acid or "normal" level of expression of a biomarker nucleic acid or protein is the activity /level of expression or copy number in a biological sample, e.g., a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow, from a subject, e.g., a human, not afflicted with cancer, or from a corresponding non-cancerous tissue in the same subject who has cancer.

As used herein, the term "costimulate" with reference to activated immune cells includes the ability of a costimulatory molecule to provide a second, non-activating receptor mediated signal (a "costimulatory signal") that induces proliferation or effector function. For example, a costimulatory signal can result in cytokine secretion, e.g., in a T cell that has received a T cell-receptor-mediated signal. Immune cells that have received a cell-receptor mediated signal, e.g., via an activating receptor are referred to herein as "activated immune cells."

The term "determining a suitable treatment regimen for the subject" is taken to mean the determination of a treatment regimen (i.e., a single therapy or a combination of different therapies that are used for the prevention and/or treatment of the cancer in the subject) for a subject that is started, modified and/or ended based or essentially based or at least partially based on the results of the analysis according to the present invention. One example is starting an adjuvant therapy after surgery whose purpose is to decrease the risk of recurrence, another would be to modify the dosage of a particular chemotherapy. The determination can, in addition to the results of the analysis according to the present invention, be based on personal characteristics of the subject to be treated. In most cases, the actual determination of the suitable treatment regimen for the subject will be performed by the attending physician or doctor.

The term "diagnosing cancer" includes the use of the methods, systems, and code of the present invention to determine the presence or absence of a cancer or subtype thereof in an individual. The term also includes methods, systems, and code for assessing the level of disease activity in an individual.

A molecule is "fixed" or "affixed" to a substrate if it is covalently or non-covalently associated with the substrate such that the substrate can be rinsed with a fluid (e.g. standard saline citrate, pH 7.4) without a substantial fraction of the molecule dissociating from the substrate.

The term "expression signature" or "signature" refers to a group of one or more coordinately expressed biomarkers related to a measured phenotype. For example, the genes, proteins, metabolites, and the like making up this signature may be expressed in a specific cell lineage, stage of differentiation, or during a particular biological response. The biomarkers can reflect biological aspects of the tumors in which they are expressed, such as the cell of origin of the cancer, the nature of the non-malignant cells in the biopsy, and the oncogenic mechanisms responsible for the cancer. Expression data and gene expression levels can be stored on computer readable media, e.g., the computer readable medium used in conjunction with a microarray or chip reading device. Such expression data can be manipulated to generate expression signatures.

"Homologous" as used herein, refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue. By way of example, a region having the nucleotide sequence 5'- ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.

The term "immune cell" refers to cells that play a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

The term "immunotherapy" or "immunotherapies" refer to any treatment that uses certain parts of a subject's immune system to fight diseases such as cancer. The subject's own immune system is stimulated (or suppressed), with or without administration of one or more agent for that purpose. Immunotherapies that are designed to elicit or amplify an immune response are referred to as "activation immunotherapies." Immunotherapies that are designed to reduce or suppress an immune response are referred to as "suppression immunotherapies." Any agent believed to have an immune system effect on the genetically modified transplanted cancer cells can be assayed to determine whether the agent is an immunotherapy and the effect that a given genetic modification has on the modulation of immune response. In some embodiments, the immunotherapy is cancer cell-specific. In some embodiments, immunotherapy can be "untargeted," which refers to administration of agents that do not selectively interact with immune system cells, yet modulates immune system function. Representative examples of untargeted therapies include, without limitation, chemotherapy, gene therapy, and radiation therapy.

Immunotherapy is one form of targeted therapy that may comprise, for example, the use of cancer vaccines and/or sensitized antigen presenting cells. For example, an oncolytic virus is a virus that is able to infect and lyse cancer cells, while leaving normal cells unharmed, making them potentially useful in cancer therapy. Replication of oncolytic viruses both facilitates tumor cell destruction and also produces dose amplification at the tumor site. They may also act as vectors for anticancer genes, allowing them to be specifically delivered to the tumor site. The immunotherapy can involve passive immunity for short-term protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen (e.g. , administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen). For example, anti-VEGF and mTOR inhibitors are known to be effective in treating renal cell carcinoma. Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides and the like, can be used to selectively modulate biomolecules that are linked to the initiation, progression, and/or pathology of a tumor or cancer.

Immunotherapy can involve passive immunity for short-term protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen (e.g. , administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen). Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix

polynucleotides and the like, can be used to selectively modulate biomolecules that are linked to the initiation, progression, and/or pathology of a tumor or cancer.

In some embodiments, the immunotherapy described herein comprises at least one immunogenic chemotherapies. The term "immunogenic chemotherapy" refers to any chemotherapy that has been demonstrated to induce immunogenic cell death, a state that is detectable by the release of one or more damage-associated molecular pattern (DAMP) molecules, including, but not limited to, calreticulin, ATP and HMGB1 (Kroemer et al. (2013), Annu. Rev. Immunol., 31:51-72). Specific representative examples of consensus immunogenic chemotherapies include 5'-fluorouracil, anthracyclines, such as doxorubicin, and platinum drugs, such as oxaliplatin, among others.

In some embodiments, immunotherapy comprises inhibitors of one or more immune checkpoints. The term "immune checkpoint" refers to a group of molecules on the cell surface of CD4+ and/or CD8+ T cells that fine-tune immune responses by down- modulating or inhibiting an anti-tumor immune response. Immune checkpoint proteins are well-known in the art and include, without limitation, CTLA-4, PD-1 , VISTA, B7-H2, B7- H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP, CD47, CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, IDO, CD39, CD73 and A2aR (see, for example, WO 2012/177624). The term further

encompasses biologically active protein fragment, as well as nucleic acids encoding full- length immune checkpoint proteins and biologically active protein fragments thereof. In some embodiment, the term further encompasses any fragment according to homology descriptions provided herein. In one embodiment, the immune checkpoint is PD-1.

Immune checkpoints and their sequences are well-known in the art and

representative embodiments are described below. For example, the term "PD-1" refers to a member of the immunoglobulin gene superfamily that functions as a coinhibitory receptor having PD-L1 and PD-L2 as known ligands. PD-1 was previously identified using a subtraction cloning based approach to select for genes upregulated during TCR-induced activated T cell death. PD-1 is a member of the CD28/CTLA-4 family of molecules based on its ability to bind to PD-L1. Like CTLA-4, PD-1 is rapidly induced on the surface of T- cells in response to anti-CD3 (Agata et al. 25 (1996) Int. Immunol. 8:765). In contrast to CTLA-4, however, PD-1 is also induced on the surface of B-cells (in response to anti-IgM). PD-1 is also expressed on a subset of thymocytes and myeloid cells (Agata et al. (1996) supra; Nishimura et al. (1996) Int. Immunol. 8:773).

The nucleic acid and amino acid sequences of a representative human PD-1 biomarker is available to the public at the GenBank database under NM 005018.2 and NP_005009.2 and is shown in Table 1 (see also Ishida et al. (1992) 20 EMBO J 11 :3887; Shinohara et al. (1994) Genomics 23:704; U.S. Patent 5,698,520). PD-1 has an extracellular region containing immunoglobulin superfamily domain, a transmembrane domain, and an intracellular region including an immunoreceptor tyrosine-based inhibitory motif (ITIM) (Ishida et a/. (1992) E BO J. 11:3887; Shinohara et al. (1994) Genomics 23:704; and U.S. Patent 5,698,520) and an immunoreceptor tyrosine-based switch motif (ITSM). These features also define a larger family of polypeptides, called the

immunoinhibitory receptors, which also includes gp49B, PIR-B, and the killer inhibitory receptors (KIRs) (Vivier and Daeron (1997) Immunol. Today 18:286). It is often assumed that the tyrosyl phosphorylated ITIM and ITSM motif of these receptors interacts with SH2-domain containing phosphatases, which leads to inhibitory signals. A subset of these immunoinhibitory receptors bind to MHC polypeptides, for example the KIRs, and CTLA4 binds to B7-1 and B7-2. It has been proposed that there is a phylogenetic relationship between the MHC and B7 genes (Henry et al. (1999) Immunol. Today 20(6):285-8).

Nucleic acid and polypeptide sequences of PD-1 orthologs in organisms other than humans are well-known and include, for example, mouse PD-1 (NM_008798.2 and NP_032824.1), rat PD-1 (NM_001106927.1 and NP_001100397.1), dog PD-1 (XM_543338.3 and

XP_543338.3), cow PD-1 (NM_001083506.1 and NP_001076975.1), and chicken PD-1 (XM_422723.3 and XP_422723.2).

PD-1 polypeptides are inhibitory receptors capable of transmitting an inhibitory signal to an immune cell to thereby inhibit immune cell effector function, or are capable of promoting costimulation {e.g., by competitive inhibition) of immune cells, e.g., when present in soluble, monomeric form. Preferred PD-1 family members share sequence identity with PD-1 and bind to one or more B7 family members, e.g., B7-1, B7-2, PD-1 ligand, and/or other polypeptides on antigen presenting cells.

The term "PD-1 activity," includes the ability of a PD-1 polypeptide to modulate an inhibitory signal in an activated immune cell, e.g., by engaging a natural PD-1 ligand on an antigen presenting cell. Modulation of an inhibitory signal in an immune cell results in modulation of proliferation of, and/or cytokine secretion by, an immune cell. Thus, the term "PD-1 activity" includes the ability of a PD-1 polypeptide to bind its natural ligand(s), the ability to modulate immune cell costimulatory or inhibitory signals, and the ability to modulate the immune response.

The term "PD-1 ligand" refers to binding partners of the PD-1 receptor and includes both PD-L1 (Freeman et al. (2000) J Exp. Med. 192:1027-1034) and PD-L2 (Latchman et al. (2001) Nat. Immunol. 2:261). At least two types of human PD-1 ligand polypeptides exist. PD-1 ligand proteins comprise a signal sequence, and an IgV domain, an IgC domain, a transmembrane domain, and a short cytoplasmic tail. Both PD-Ll (See Freeman et al. (2000) for sequence data) and PD-L2 (See Latchman et al. (2001) Nat. Immunol. 2:261 for sequence data) are members of the B7 family of polypeptides. Both PD-Ll and PD-L2 are expressed in placenta, spleen, lymph nodes, thymus, and heart. Only PD-L2 is expressed in pancreas, lung and liver, while only PD-Ll is expressed in fetal liver. Both PD-1 ligands are upregulated on activated monocytes and dendritic cells, although PD-Ll expression is broader. For example, PD-Ll is known to be constitutively expressed and upregulated to higher levels on murine hematopoietic cells (e.g., T cells, B cells, macrophages, dendritic cells (DCs), and bone marrow-derived mast cells) and non- hematopoietic cells (e.g., endothelial, epithelial, and muscle cells), whereas PD-L2 is inducibly expressed on DCs, macrophages, and bone marrow-derived mast cells (see Butte et al. (2007) Immunity 27: 111).

PD-1 ligands comprise a family of polypeptides having certain conserved structural and functional features. The term "family" when used to refer to proteins or nucleic acid molecules, is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology, as defined herein. Such family members can be naturally or non- naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin. Members of a family may also have common functional characteristics. PD-1 ligands are members of the B7 family of polypeptides. The term "B7 family" or "B7 polypeptides" as used herein includes costimulatory polypeptides that share sequence homology with B7 polypeptides, e.g., with B7-1, B7-2, B7h (Swallow et al. (1999) Immunity 11 :423), and/or PD-1 ligands (e.g., PD-Ll or PD-L2). For example, human B7-1 and B7-2 share approximately 26% amino acid sequence identity when compared using the BLAST program at NCBI with the default parameters (Blosum62 matrix with gap penalties set at existence 11 and extension 1 (See the NCBI website). The term B7 family also includes variants of these polypeptides which are capable of modulating immune cell function. The B7 family of molecules share a number of conserved regions, including signal domains, IgV domains and the IgC domains. IgV domains and the IgC domains are art-recognized Ig superfamily member domains. These domains correspond to structural units that have distinct folding patterns called Ig folds. Ig folds are comprised of a sandwich of two β sheets, each consisting of anti-parallel β strands of 5-10 amino acids with a conserved disulfide bond between the two sheets in most, but not all, IgC domains of Ig, TCR, and MHC molecules share the same types of sequence patterns and are called the CI -set within the Ig superfamily. Other IgC domains fall within other sets. IgV domains also share sequence patterns and are called V set domains. IgV domains are longer than IgC domains and contain an additional pair of β strands.

Preferred B7 polypeptides are capable of providing costimulatory or inhibitory signals to immune cells to thereby promote or inhibit immune cell responses. For example, B7 family members that bind to costimulatory receptors increase T cell activation and proliferation, while B7 family members that bind to inhibitory receptors reduce

costimulation. Moreover, the same B7 family member may increase or decrease T cell costimulation. For example, when bound to a costimulatory receptor, PD-1 ligand can induce costimulation of immune cells or can inhibit immune cell costimulation, e.g., when present in soluble form. When bound to an inhibitory receptor, PD-1 ligand polypeptides can transmit an inhibitory signal to an immune cell. Preferred B7 family members include B7-1, B7-2, B7h, PD-Ll or PD-L2 and soluble fragments or derivatives thereof. In one embodiment, B7 family members bind to one or more receptors on an immune cell, e.g., CTLA4, CD28, ICOS, PD-1 and/or other receptors, and, depending on the receptor, have the ability to transmit an inhibitory signal or a costimulatory signal to an immune cell, preferably a T cell.

Modulation of a costimulatory signal results in modulation of effector function of an immune cell. Thus, the term "PD-1 ligand activity" includes the ability of a PD-1 ligand polypeptide to bind its natural receptor(s) (e.g. PD-1 or B7-1), the ability to modulate immune cell costimulatory or inhibitory signals, and the ability to modulate the immune response.

The term "PD-Ll" refers to a specific PD-1 ligand. Two forms of human PD-Ll molecules have been identified. One form is a naturally occurring PD-Ll soluble polypeptide, i.e., having a short hydrophilic domain and no transmembrane domain, and is referred to herein as PD-Ll S (shown in Table 1 as SEQ ID NO: 4). The second form is a cell-associated polypeptide, i.e., having a transmembrane and cytoplasmic domain, referred to herein as PD-Ll M (shown in SEQ ID NO: 6). The nucleic acid and amino acid sequences of representative human PD-Ll biomarkers regarding PD-Ll M are also available to the public at the GenBank database under NM_014143.3 and NP 054862.1. PD-L1 proteins comprise a signal sequence, and an IgV domain and an IgC domain. The signal sequence of SEQ ID NO: 4 is shown from about amino acid 1 to about amino acid 18. The signal sequence of SEQ ID NO: 6 is shown :from about amino acid 1 to about amino acid 18. The IgV domain of SEQ ID NO: 4 is shown from about amino acid 19 to about amino acid 134 and the IgV domain of SEQ ID NO: 6 is shown from about amino acid 19 to about amino acid 134. The IgC domain of SEQ ID NO: 4 is shown from about amino acid 135 to about amino acid 227 and the IgC domain of SEQ ID NO: 6 is shown from about amino acid 135 to about amino acid 227. The hydrophilic tail of the PD-L1 exemplified in SEQ ID NO: 4 comprises a hydrophilic tail shown from about amino acid 228 to about amino acid 245. The PD-L1 polypeptide exemplified in SEQ ID NO: 6 comprises a

transmembrane domain shown from about amino acids 239 to about amino acid 259 of SEQ ID NO: 6 and a cytoplasmic domain shown from about 30 amino acid 260 to about amino acid 290 of SEQ ID NO: 6. In addition, nucleic acid and polypeptide sequences of PD-L1 orthologs in organisms other than humans are well-known and include, for example, mouse PD-L1 (NM_021893.3 and NP_068693.1), rat PD-L1 (NM_001191954.1 and

NP_001178883.1), dog PD-Ll (XM_541302.3 and XP_541302.3), cow PD-Ll

(NM_001163412.1 and NP_001156884.1), and chicken PD-L1 (XM_424811.3 and XP_424811.3).

The term "PD-L2" refers to another specific PD-1 ligand. PD-L2 is a B7 family member expressed on various APCs, including dendritic cells, macrophages and bone- marrow derived mast cells (Zhong et al. (2007) Eur. J. Immunol. 37:2405). APC-expressed PD-L2 is able to both inhibit T cell activation through ligation of PD-1 and costimulate T cell activation, through a PD-1 independent mechanism (Shin et al. (2005) J. Exp. Med. 201 : 1531 ). In addition, ligation of dendritic cell-expressed PD-L2 results in enhanced dendritic cell cytokine expression and survival (Radhakrishnan et al. (2003) J. Immunol. 37:1827; Nguyen et al. (2002) J. Exp. Med. 196:1393). The nucleic acid and amino acid sequences of representative human PD-L2 biomarkers {e.g., SEQ ID NOs: 7 and 8) are well-known in the art and are also available to the public at the GenBank database under NM_025239.3 and NP_079515.2. PD-L2 proteins are characterized by common structural elements. In some embodiments, PD-L2 proteins include at least one or more of the following domains: a signal peptide domain, a transmembrane domain, an IgV domain, an IgC domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain. For example, amino acids 1-19 of SEQ ID NO: 8 comprises a signal sequence. As used herein, a "signal sequence" or "signal peptide" serves to direct a polypeptide containing such a sequence to a lipid bilayer, and is cleaved in secreted and membrane bound polypeptides and includes a peptide containing about 15 or more amino acids which occurs at the N-terminus of secretory and membrane bound polypeptides and which contains a large number of hydrophobic amino acid residues. For example, a signal sequence contains at least about 10-30 amino acid residues, preferably about 15- 25 amino acid residues, more preferably about 18-20 amino acid residues, and even more preferably about 19 amino acid residues, and has at least about 35-65%, preferably about 38-50%, and more preferably about 40-45% hydrophobic amino acid residues (e.g., valine, leucine, isoleucine or phenylalanine). In another embodiment, amino acid residues 220-243 of the native human PD-L2 polypeptide and amino acid residues 201-243 of the mature polypeptide comprise a transmembrane domain. As used herein, the term "transmembrane domain" includes an amino acid sequence of about 15 amino acid residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 20, 25, 30, 35, 40, or 45 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an alpha-helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, Zagotta, W. N. et al. (1996) Annu. Rev. Neurosci. 19: 235-263. In still another embodiment, amino acid residues 20-120 of the native human PD-L2 polypeptide and amino acid residues 1-101 of the mature polypeptide comprise an IgV domain. Amino acid residues 121- 219 of the native human PD-L2 polypeptide and amino acid residues 102-200 of the mature polypeptide comprise an IgC domain. As used herein, IgV and IgC domains are recognized in the art as Ig superfamily member domains. These domains correspond to structural units that have distinct folding patterns called Ig folds. Ig folds are comprised of a sandwich of two β sheets, each consisting of antiparallel (3 strands of 5-10 amino acids with a conserved disulfide bond between the two sheets in most, but not all, domains. IgC domains of Ig, TCR, and MHC molecules share the same types of sequence patterns and are called the CI set within the Ig superfamily. Other IgC domains fall within other sets. IgV domains also share sequence patterns and are called V set domains. IgV domains are longer than C- domains and form an additional pair of strands. In yet another embodiment, amino acid residues 1-219 of the native human PD-L2 polypeptide and amino acid residues 1-200 of the mature polypeptide comprise an extracellular domain. As used herein, the term

"extracellular domain" represents the N-terminal amino acids which extend as a tail from the surface of a cell. An extracellular domain of the present invention includes an IgV domain and an IgC domain, and may include a signal peptide domain. In still another embodiment, amino acid residues 244-273 of the native human PD-L2 polypeptide and amino acid residues 225-273 of the mature polypeptide comprise a cytoplasmic domain. As used herein, the term "cytoplasmic domain" represents the C-terminal amino acids which extend as a tail into the cytoplasm of a cell. In addition, nucleic acid and polypeptide sequences of PD-L2 orthologs in organisms other than humans are well-known and include, for example, mouse PD-L2 (NM_021396.2 and NP_067371.1), rat PD-L2

(NM_001107582.2 and NP_001101052.2), dog PD-L2 (XM_847012.2 and XP_852105.2), cow PD-L2 (XM_586846.5 and XP_586846.3), and chimpanzee PD-L2 (XM_001140776.2 and XP_001140776.1).

The term "PD-L2 activity," "biological activity of PD-L2," or "functional activity of

PD-L2," refers to an activity exerted by a PD-L2 protein, polypeptide or nucleic acid molecule on a PD-L2 -responsive cell or tissue, or on a PD- L2 polypeptide binding partner, as determined in vivo, or in vitro, according to standard techniques. In one embodiment, a PD-L2 activity is a direct activity, such as an association with a PD-L2 binding partner. As used herein, a "target molecule" or "binding partner" is a molecule with which a PD-L2 polypeptide binds or interacts in nature, such that PD-L2-mediated function is achieved. In an exemplary embodiment, a PD-L2 target molecule is the receptor RGMb. Alternatively, a PD-L2 activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the PD- L2 polypeptide with its natural binding partner (i.e., physiologically relevant interacting macromolecule involved in an immune function or other biologically relevant function), e.g., RGMb. The biological activities of PD-L2 are described herein. For example, the PD-L2 polypeptides of the present invention can have one or more of the following activities: 1) bind to and/or modulate the activity of the receptor RGMb, PD-1, or other PD-L2 natural binding partners, 2) modulate intra-or intercellular signaling, 3) modulate activation of immune cells, e.g. , T lymphocytes, and 4) modulate the immune response of an organism, e.g., a mouse or human organism.

"Anti-immune checkpoint therapy" refers to the use of agents that inhibit immune checkpoint nucleic acids and/or proteins. Inhibition of one or more immune checkpoints can block or otherwise neutralize inhibitory signaling to thereby upregulate an immune response in order to more efficaciously treat cancer. Exemplary agents useful for inhibiting immune checkpoints include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, that can either bind and/or inactivate or inhibit immune checkpoint proteins, or fragments thereof; as well as R A interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and/or activity of immune checkpoint nucleic acids, or fragments thereof. Exemplary agents for upregulating an immune response include antibodies against one or more immune checkpoint proteins block the interaction between the proteins and its natural receptor(s); a non-activating form of one or more immune checkpoint proteins (e.g. , a dominant negative polypeptide); small molecules or peptides that block the interaction between one or more immune checkpoint proteins and its natural receptor(s); fusion proteins (e.g. the

extracellular portion of an immune checkpoint inhibition protein fused to the Fc portion of an antibody or immunoglobulin) that bind to its natural receptor(s); nucleic acid molecules that block immune checkpoint nucleic acid transcription or translation; and the like. Such agents can directly block the interaction between the one or more immune checkpoints and its natural receptor(s) (e.g., antibodies) to prevent inhibitory signaling and upregulate an immune response. Alternatively, agents can indirectly block the interaction between one or more immune checkpoint proteins and its natural receptor(s) to prevent inhibitory signaling and upregulate an immune response. For example, a soluble version of an immune checkpoint protein ligand such as a stabilized extracellular domain can binding to its receptor to indirectly reduce the effective concentration of the receptor to bind to an appropriate ligand. In one embodiment, anti-PD-1 antibodies, anti-PD-Ll antibodies, and/or anti-PD-L2 antibodies, either alone or in combination, are used to inhibit immune checkpoints. These embodiments are also applicable to specific therapy against particular immune checkpoints, such as the PD-1 pathway (e.g., anti-PD-1 pathway therapy, otherwise known as PD-1 pathway inhibitor therapy).

Protein tyrosine phosphatases (PTPs or PTPases) are a group of enzymes that remove phosphate groups from phosphorylated tyrosine residues on proteins (He et al. (2014) Acta Pharmacol. Sin. 35:1227-1246; Barr et al. (2009) Cell 136:352-363). Protein tyrosine (pTyr) phosphorylation is a common post-translational modification that can create novel recognition motifs for protein interactions and cellular localization, affect protein stability, and regulate enzyme activity. As a consequence, maintaining an appropriate level of protein tyrosine phosphorylation is essential for many cellular functions. Tyrosine- specific protein phosphatases (PTPase; EC 3.1.3.48) catalyze the removal of a phosphate group attached to a tyrosine residue, using a cysteinyl-phosphate enzyme intermediate. These enzymes are key regulatory components in signal transduction pathways (such as the MAP kinase pathway) and cell cycle control, and are important in the control of cell growth, proliferation, differentiation, transformation, and synaptic plasticity (Denu and Dixon (1998) Curr. Opin. Chem. Biol. 2:633-641; Lombroso (2003) Cell. Mol. Life Sci. 60:2465-2482). Together with tyrosine kinases, PTPs regulate the phosphorylation state of many important signaling molecules, such as the MAP kinase family. PTPs are

increasingly viewed as integral components of signal transduction cascades. PTPs have been implicated in regulation of many cellular processes, including, but not limited to: cell growth, cellular differentiation, mitotic cycles, oncogenic transformation, receptor endocytosis, etc. The classification of PTPs can be achieved by mechanism or location. By mechanism, PTP activity can be found in four protein families, including: 1) class I PTPs, which is the largest group of PTPs comprising at least 99 members, such as at least 38 classical PTPs (21 receptor tyrosine phosphatase and 17 non-receptor-type PTPs) and 61 VH-l-like or dual-specific (dTyr and dSer/dThr) phosphatases (DSPs) {e.g., 11 MAPK phosphatases (MPKs), 3 Slingshots, 3 PRLs, 4 CDC14s, 19 atypical DSPs, 5 Phosphatase and tensin homologs (PTENs), and 16 Myotubularins); 2) class II PTP, comprising only one member low-molecular-weight phosphotyrosine phosphatase (LMPTP); 3) class III PTPs, comprising at least CDC25 A, B, and C proteins; and 4) Class IV PTPs, comprising at least Eya 1-4 proteins, which are pTyr-specific phosphatases and believed to have evolved separately from the other three classes. By cellular location, PTPs can be classified as receptor-like PTPs and non-receptor (intracellular) PTPs. The former are transmembrane receptors that contain PTPase domains. In terms of structure, all known receptor PTPases are made up of a variable-length extracellular domain, followed by a transmembrane region and a C-terminal catalytic cytoplasmic domain. Some of the receptor PTPases contain fibronectin type III (FN-III) repeats, immunoglobulin-like domains, MAM domains, or carbonic anhydrase-like domains in their extracellular region. In general, the cytoplasmic region contains two copies of the PTPase domain. The first has enzymatic activity, whereas the second is inactive (Sun et al. (2003) Curr Top Med Chem. 3:739-748; Alonso et al. (2004) Cell 117:699-711). All class I, II, and III PTPs carry a highly conserved active site motif C(X) ₅ R (PTP signature motif), employ a common catalytic mechanism, and possess a similar core structure made of a central parallel beta-sheet with flanking alpha-helices containing a beta-loop-alpha-loop that encompasses the PTP signature motif (Barford et al. (1998) Annu. Rev. Biophys. Biomol. Struct. 27:133-164). Functional diversity between PTPases is endowed by regulatory domains and subunits. For most PTPs, the consensus sequence ( I/V)HCXAGXXR(S/T)G (/. e. , the C(X) ₅ R PTP signature motif) contains the catalytically essential Cys and Arg residues. Intracellular PTPs are often modular molecules containing structural motifs such as Src homology 2 (SH2) domains, PEST sequences, and band 4.1 domains on either the N- or C-terminal side of their catalytic domains.

Among non-receptor PTPs, tyrosine-protein phosphatase non-receptor type 2

(PTPN2) is an enzyme that in humans is encoded by the PTPN2 gene (Brown-Shimer et al. (1990) Proc. Natl. Acad. Sci. USA 87:5148-5152). Epidermal growth factor receptor and the adaptor protein She were reported to be substrates of this PTP, which suggests a role in growth factor-mediated cell signaling. Three alternatively spliced variants of this gene, which encode isoforms differing at their extreme C-termini, have been described. The different C-termini are thought to determine the substrate specificity, as well as the cellular localization of the isoforms. Two highly related but distinctly processed pseudogenes that localize to distinct human chromosomes have been reported. The human PTPN2 gene localizes to chromosome 18pl 1.2-pl 1.3, whereas pseudogenes (gene symbol PTPN2P1 and PTPN2P2) are mapped to chromosomes Iq22-q24 and 13ql2-ql3, respectively. A direct comparison of the specificity of genomic and cDNA probes demonstrated that under identical conditions the genomic probes (containing both exon and intron sequences) readily identified a single specific site of hybridization, whereas the cDNA identified sites of both the gene and its pseudogenes (Johnson et al. (1993) Genomics 16:619-629). Human PTPN2 exists as two forms generated by alternative splicing: a 48-kDa endoplasmic reticulum (ER)-associated form (TC48, 415 amino acid) and a 45-kDa nuclear form (TC45). The three-dimensional PDB structure of PTPN2 is also well-known and described in at least the OCA database (protein ID: 1L8K) at the Weizmann Institute of Science (Rehovot, Israel) available on the World Wide Web at oca.weizmann.ac.il/oca- bin/ocashort?id=lL8K. PTPN2 has a protein tryrosine phosphatase catalytic (PTPc) domain, for example, from amino acid residues 5 to 275 of SEQ ID NO: 2. The PTPc domain comprises different motifs for various functions, such as substrate binding (amino acid residues 216-222 of SEQ ID NO: 2), endoplasmic reticulum (ER) location (amino acid residues 346-415 of SEQ ID NO: 2), and STX17 interaction (amino acid residues 376-415 of SEQ ID NO: 2, also see Muppirala et al. (2012) Biochim. Biophys. Acta 1823:2109- 2119).

The nucleic acid and amino acid sequences of a representative human PTPN2 is available to the public at the GenBank database (Gene ID 5771) and is shown in Table 1. Human PTPN2 isoforms include the longest isoform 1 (GenBank database numbers NM_002828.3 and NP_002819.2), and the shorter isoforms 2 (NM_080422.2 and

NP 536347.1, which contains an alternate 3' region including a part of the C-terminal coding region, resulting in a shorter and distinct C-terminus, as compared to isoform 1), 3 (NM_080423.2 and NP_536348.1; which contains an alternate 3' region including a part of the C-terminal coding region, resulting in a shorter and distinct C-terminus, as compared to isoform 1), 4 (NM_001207013.1 and NP_001193942.1; which contains an additional in- frame exon in the middle coding region and an alternate 3' region including a part of the C- terminal coding region, resulting in an additional internal segment and a shorter and distinct C-terminus, as compared to isoform 1), and 5 (NM_001308287.1 and NP_001295216.1; which differs in the 5' UTR by lacking a portion of the 5' coding region and using an alternative start codon to initiates translation, resulting in a shorter and distinct N-terminus, as compared to isoform 1).

Nucleic acid and polypeptide sequences of PTPN2 orthologs in organisms other than humans are well-known and include, for example, chimpanzee {Pan troglodytes)

PTPN2 (XM_009433614.2 and XP_009431889.2; XM_009433613.2 and

XP_009431888.2; XM_009433615.2 and XP_009431890.2; XM_003953237.2 and XP_003953286.2; XM 001171536.4 and XP_001171536.2; XM 009433617.2 and XP_009431892.1; XM_016933257.1 and XP_016788746.1; XM_009433619.2 and XP_009431894.2; XM_009433618.2 and XP_009431893.2; XM_016933256.1 and

XP_016788745.1; XM_016933258.1 and XP_016788747.1; and XM_009433620.2 and XP 009431895.2), dog PTPN2 (XM 014115598.1 and XP_013971073.1;

XM_005623101.2 and XP_005623158.1 ; XM_005623100.2 and XP_005623157.1; and XM_005623099.2 and XP_005623156.1), mouse PTPN2 (NM_001127177.1 and

NP_001120649.1, which represent the longer transcript, and NM_008977.3 and

NP_033003.1, which differs in the 3' UTR and has multiple coding region differences, resulting in a distinct C-terminus and is shorter than the isoform encoded by the longer transcript), cattle PTPN2 (NM_001035431.2 and NP_001030508.1), Norway rat (Rattus norvegicus) PTPN2 (NM_053990.1 and NP_446442.1), chicken PTPN2 (NM OOl 199387.1 and NP_001186316.1), tropical clawed frog (Xenopus tropicalis) PTPN2 (XM_004915252.3 and XP_004915309.2; and XM_002936076.4 and

XP_002936122.1); zebrafish (Da io rerio) PTPN2 (NM_200466.2 and NP_956760.2; and NM_212654.1 and NP_997819.1 ); and fruit fly {Drosophila melanogaster) PTPN2

(NM_167874.2 and NP_728600.1; NM_057340.4 and NP_476688.1; NM_001274324.2 and NP_001261253.1; NM 167875.2 and NP_728601.1; and NM_057339.5 and

NP_476687.1).

The term "PTPN2 activity," includes the ability of a PTPN2 polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein) to bind and catalyze the removal of one or more phosphate groups from one or more its substrates in a cell (e.g., a cancer cell, and/or an immune cell), e.g., by engaging a natural PTPN2 substrate (e.g., INSR, EGFR, CSF1R, PDGFR, JAK1, JAK2, JAK3, Src family kinases, STAT1, STAT3, STAT5A, STAT5B, STAT6, etc.) either in the nucleus or the cytoplasm of the cell (Shuai et al. (2003) Nat. Rev. Immunol. 3:900-911; Wiede et a/. (2011) J Clin. Invest. 121:4758- 4774). Thus, the term "ΡΤΡΝ2 activity" includes the ability of a ΡΤΡΝ2 polypeptide to bind its natural substrate(s), the ability to modulate dephosphorylation of such substrate(s), and the ability to modulate the immune response through such substrate(s) in ΡΤΡΝ2- regulated signaling pathways.

The term "ΡΤΡΝ2 substrate(s)" refers to binding partners of a ΡΤΡΝ2 polypeptide

(and its fragments, domains, and/or motifs thereof, discussed herein) from which one or more phosphate groups can be removed by the ΡΤΡΝ2 polypeptide. Such binding partners are usually members in PTPN2-regulated signaling pathways, such as INSR, EGFR, CSF1R, PDGFR, JAK1, JAK2, JAK3, Src family kinases, STAT1, STAT3, STAT5A, STAT5B, STAT6, etc. The term "INSR" refers to a member of gene superfamily that functions as insulin receptors. INSR is also commonly known under the names CD220, HHF5, insulin receptor isoform Long preproprotein, insulin receptor isoform Short preproprotein, and IR. INSR, localizing on 19pl3.2, encodes a long protein that may be cleaved into four parts: two alpha subunits and two beta subunits. These subunits work together as a functioning receptor. The alpha subunits stick out from the surface of the cell, while the beta subunits remain inside the cell. The alpha subunits attach (bind) to insulin, which causes the beta subunits to trigger signaling pathways within the cell that influence many cell functions. The INSR gene mutations have been associated with at least Donohue Syndrome, Rabson-Mendenhall Syndrome, and type A insulin resistance syndrome. The nucleic acid and amino acid sequences of a representative human INSR is available to the public at the GenBank database. Human INSR isoforms include the longer isoform 1 (GenBank database numbers NM_000208.3 and NP_000199.2; a.k.a. insulin receptor isoform Long preproprotein) and the shorter isoform 2 (NM_001079817.2 and

NP 001073285.1; a.k.a. insulin receptor isoform Short preproprotein).

The term "PTPN2-regulated signaling pathway(s)" includes signaling pathways in which PTPN2 (and its fragments, domains, and/or motifs thereof, discussed herein) binds to at least one of its substrate, through which at least one cellular function and/or activity and/or cellular protein profiles is changed. In some embodiments, PTPN2

dephosphorylates at least one of its substates which bind to it. PTPN2-regulated signaling pathways include at least NF-kappaB signaling pathway, MAP kinase signaling pathway, Jak-STAT signaling pathway, cytokine signaling pathway, interferon gamma (IFNy) signaling pathway, etc. In some embodiments, the PTPN2-regulated signaling pathway is a type I interferon signaling pathway and/or type II interferon signaling pathway, which are summarized in at least Platanias (2005) Nat. Rev. Immunol. 5:375-386. The most studied members of the Type I interferons (IFNs) are the multiple IFNa isotypes and IFN . Type I IFNs are responsible for inducing transcription of a large group of genes which play a role in host resistance to viral infections, as well as activating key components of the innate and adaptive immune systems, including antigen presentation and production of cytokines involved in activation of T cells, B cells, and natural killer cells. Type I IFNs are transcriptionally regulated, and are induced following recognition of pathogen components during infection by various host pattern recognition receptors. Virtually all humans cells are able to synthesize IFNa/β, however some cells have a more pronounced ability to produce these cytokines. At least three pathways {i.e., the RIG-I pathway, the TRJF pathway, and TLR7/8/9-IRF7 pathway) the are involved in producing Type I IFN.

Following their production, Type I IFNs trigger antiviral responses by binding to a common receptor (IFNAR). IFNa/β binding to IFNAR stimulates the JAK1-STAT pathway leading to the assembly of the ISGF3 complex which is composed of STAT1-STAT2 dimers and IRF9. ISGF3 binds to IFN-stimulated response elements (ISRE) in the promoters of IFN- stimulated genes to regulate their expression. Among these genes is IRF7 which initiates the transcription of a second wave of Type I IFNs. This autocrine/paracrine feed-back allows Type I IFNs to create an antiviral state in surrounding cells. IFNy is the only type II interferon. While it does not share structural homology or a common receptor with the type I IFNs, it too has antiviral and immunomodulatory properties. The biologically active form of IFNy is a noncovalently-linked homodimer. This homodimer binds to the extracellular domain of two IFNyRl/CDl 19 chains, which interact with IFNyR2 to form the functional IFNy receptor complex. The IFNyRl subunits of the receptor complex are associated with Jakl, while the IFNyR2 subunits are associated with Jak2. Activation of Jakl and Jak2 results in phosphorylation of the receptor and subsequent recruitment and phosphorylation of STAT1. STAT1 phosphorylation leads to its homodimerization and nuclear

translocation. Once in the nucleus, STAT1 homodimers bind to IFNy-activated sequence (GAS) elements in the promoters of target genes to regulate their transcription. Many of the target genes that are induced by IFNy/STATl signaling are transcription factors that then drive the expression of secondary response genes. In addition, IFNy signaling can activate MAPK, PI3K-Akt, and NF-kappa B signaling pathways to regulate the expression of a number of other genes. IFNy signaling plays a key role in host defense by promoting macrophage activation, upregulating the expression of antigen processing and presentation molecules, driving the development and activation of Thl cells, enhancing natural killer cell activity, regulating B cell functions, and inducing the production of chemokines that promote effector cell trafficking to sites of inflammation. While IFNy has historically been known for its cytotoxic, cytostatic, and anti-tumor properties, multiple studies have also suggested that IFNy may also have context-dependent proliferative and pro-tumorigenic effects. For a summary of the Type II interferon signaling pathway, see at least Raza et al. (2008) BMC Sys. Biol. 2:36). IFNy signaling can at least promote NK cell activity, increase antigen presentation and lysosome activity of macrophages, activate inducible nitric oxide synthase (iNOS), and induce the production of IgG2a and IgG3 from activated plasma B cells. Many IFN-stimulated genes control viral, bacterial, and parasite infection by directly targeting pathways and functions required during pathogen life cycles. The detection methods for such activation or inhibitor of IFNy responsive genes are also well known in the art. In some embodiments, the cancer cells described herein have functional IFNy signaling pathway but inactivated or at least reduced activation of IFNy-responsive target genes, probably due to the inhibition by PTPN2. Upon a treatment with the antagonizing agent for PTPN2, as described herein, such cancer cells restore active IFNy signaling. Such restoration of IFNy signaling can be detected and/or measured through the expression and/or function of IFN-responsive genes, as described herein, using any known method in the art.

The term "PTPN2 inhibitor(s)" includes any natural or non-natural agent prepared, synthesized, manufactured, and/or purified by human that is capable of reducing, inhibiting, blocking, preventing, and/or that inhibits the ability of a PTPN2 polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein). In one embodiment, such inhibitors may reduce or inhibit the binding/interaction between PTPN2 and its substrates or other binding partners. In another embodiment, such inhibitors may reduce or inhibit the catalytic function of PTPN2 as a phosphatase. In still another embodiment, such inhibitors may increase or promote the turnover rate, reduce or inhibit the expression and/or the stability (e.g., the half-life), and/or change the cellular localization of PTPN2, resulting in at least a decrease in PTPN2 levels and/or activity. Such inhibitors may be any molecule, including but not limited to small molecule compounds, antibodies or intrabodies, RNA interfereing (R Ai) agents (including at least siRNAs, shR As, microRNAs (miRNAs), piwi, and other well-known agents). Smalle molecule inhibitors of PTPN2 are known in the art and include, for example, 4-(aminosulfonyl)-7-fluoro-2,l,3-benzoxadiazole (ABDF; see Hansen et al. (2005) Biochemistry 44(21):7704-7712), imatinib mesylate (STI571; see Shimizu et al. (2004) Exp Hematol. 32(11):1057-1063), PTP inhibitor V (PHPS1, see Kim and Cho (2013) Bull. Korean Chem. Soc. 34:3874-3876), ethyl-3,4-dephospatin or other PTPN2 inhibitors described in the PCT Publ. No. WO 2015/188228, inhibitors described in Romsicki et al. (2003) Arch Biochem Biophys. 414:40-50, Iversen et al. (2002) J Biol. Chem. 277(22):19982-19990, and Asante-Appiah et al. (2001) J Biol. Chem.

276(28):26036-26043; commercial available PTP and/or PTPN2 inhibitors (e.g., products from EMD Millipore, Billerica, MA, such as anti-PTPN2 antibodies, ursolic acid, sodium orthovanadate, Dephostatin, Phenylarsine Oxide, PTP Inhibitor I, bpV(HOpic), bpV(phen), bpV(bipy), etc.), inhibitory nucleotide-related inhibitors (such as CRISPR products from OriGene (Rockville, MD) (e.g., gRNA vectors KN202161G1 and KN202161G2),

GenScript ^® (Piscataway, NJ), or Santa Cruz Biotechnology (Dallas, Texas) (e.g., TC-PTP CRISPR/Cas9 KO Plasmid (h); sc-403071), miRNA products from ViGene Biosciences, inhibitory RNA products from Origene (e.g., siRNA, shRNA, etc.) and ViGene

Biosciences (e.g., ready-to-package AAV shRNA), etc. Methods for developoing PTPN2- specific inhibitors based on its structure can be found in, e.g., Iversen et al. (2001)

Biochemistry 40(49):14812-14820. An exemplary method, without limitation, of analyzing the activity of the PTPN2 inhibitors described herein is to analyze whether such inhibitors are capable of 1) inhibiting the phosphatase function of Ptpn2; and/or 2) restoring IFNy signaling and/or cellular sensitivity to immunotherapy. Methods for detecting the phosphatase function of Ptpn2 are well-known in the art. For example, the phosphorylation of Ptpn2 targets, including receptor tyrosine kinases {e.g. , INSR, EGFR, CSF1R, PDGFR, etc.), non-receptor tyrosine kinases {e.g., JAK1, JAK2, JAK3, etc.), Src family kinases {e.g., Fyn, Lck, etc.) and STAT family members {e.g., STAT1, STAT3, STAT5A,

STAT5B, STAT6, etc.), can be measured and compared before and after the Ptpn2 inhibitor treatment.

The term "SOCS1," a.k.a., suppressor of cytokine signaling 1, also known as JAB or TIP3, refers to a group of proteins in the family of STAT-induced STAT inhibitor (SSI) proteins, also known as suppressor of cytokine signalling (SOCS). SSI family members are cytokine-inducible negative regulators of cytokine signaling. The expression of this gene can be induced by a subset of cytokines, including IL2, IL3 erythropoietin (EPO), GM- CSF, and interferon-gamma (IFN-γ). SOCS1 functions downstream of cytokine receptors, and takes part in a negative feedback loop to attenuate cytokine signaling. Knockout studies in mice suggested the role of SOCS1 as a modulator of IFN-γ action, which is required for normal postnatal growth and survival. SOCS1 is involved in negative regulation of cytokines that signal through the JAK/STAT3 pathway. Through binding to JAKs, SOCS1 inhibits their kinase activity. SOCS1 also suppresses Tec protein-tyrosine activity at least in vitro. SOCS1 is a major regulator of signaling by interleukin 6 (IL6) and leukemia inhibitory factor (LIF). SOCS1 is believed to regulate interferon-gamma mediated sensory neuron survival. SOCS1 is believed to be the substrate recognition component of an ECS (Elongin BC-CUL2/5-SOCS-box protein) E3 ubiquitin ligase complex, which mediates the ubiquitination and subsequent proteasomal degradation of target proteins. SOCS1 also appears to be a negative regulator in IGF1R signaling pathway. Known interaction partners of SOCS1 include, at least, CD117 (De Sepulveda et al. (1999) EMBOJ. 18:904-915), colony stimulating factor 1 receptor (Bourette et al.

(2001) J Biol. Chem. 276:22133-22139), growth hormone receptor (Ram and Waxman (1999) J Biol. Chem. 274:35553-35561), IRS2 (Rui et al. (2002) J Biol. Chem. 277:42394- 42398), Janus kinase 2 (Yasukawa et al. (1999; EMBO J. 18:1309-1320), and

TEC (Ohya et al. (1997) J. Biol. Chem. 272:27178-27182). Other predicted binding partners for SOCS1 include, e.g., AXL, FGFR3, INSR, TRIM8, COMMD1, ELOC, ERBB2, RELA, and CUL2. S0CS1 is known to be involved in activated Toll-Like Receptor 4 (TLR4) signaling (e.g., MyD88-Mal cascade initiated on plasma membrane, TLRl, TLR2, TLR4, and TLR6 cascades), prolactin signaling pathway, IL-2 pathway (e.g., growth hormone signaling, IL-2, IL-4 signaling pathway, etc.), class I MHC mediated antigen processing and presentation, mmune response interferon α/β signaling pathway, Jak-Stat signaling pathway, NF-κΒ signaling, etc.

The nucleic acid and amino acid sequences of a representative human SOCS1 is available to the public at the GenBank database (Gene ID 8651) and is shown in Table 1. For example, the nucleic acid and amino acid sequences of human SOCS1 polypeptide is well known (GenBank database numbers NM_003745.1 and NP_003736.1). The domain structure of SOCS1 polypeptide is also well known. For example, as shown in UniProt database reference number 015524, human SOCS1 polypeptide has a SH2 domain comprising, e.g., amino acid residue number 79-174 of NP_003736.1, and a SOCS box domain comprising, e.g., amino acid residue number 161-210 of NP_003736.1. Other known regions include a kinase inhibitory region (KIR) comprising, e.g. , amino acid residue number 55-66 of NP 003736.1, an extended SH2 subdomain (ESS) comprising e.g., amino acid residue number 67-78 of NP_003736.1, and a domain capable of interacting with Elongin BC complex comprising e.g., amino acid residue number 173-182 ofNP_003736.1.

Nucleic acid and polypeptide sequences of SOCS1 orthologs in organisms other than humans are well-known and include, for example, chimpanzee (Pan troglodytes) SOCS1 (XM_016929424.1 and XP_016784913.1), Rhesus monkey SOCS1

(XM_001104595.3 and XP_001104595.1), dog SOCS1 (XM_005622079.1 and

XP_005622136.1), mouse SOCS1 (NM_001271603.1 and NP 001258532.1, which represent the longer transcript variant 1 , and NM_009896.2 and NP_034026.1 , which represent the shorter transcript variant 2 which differs in the 5' UTR compared to variant 1 but encodes the same protein), cattle SOCS1 (XM 002697964.4 and XP_002698010.1), Norway rat (Rattus norvegicus) SOCS1 (NM_145879.2 and NP_665886.2), chicken SOCS1 (NM_001137648.1 and NP_001131120.1), tropical clawed frog (Xenopus tropicalis) SOCS1 (NM_001011327.1 and NP_001011327.1); and zebrafish (Danio rerio) SOCS1 (NM_001003467.1 and NP_001003467.1).

The term "SOCS1 activity," includes the ability of a SOCSlpolypeptide (and its fragments, domains, and/or motifs thereof, discussed herein) to bind and exert one or more of its biological functions to one or more its substrates in a cell (e.g., a cancer cell, and/or an immune cell), e.g., by engaging a natural SOCSl substrate (e.g., those cytokine signaling molecues which are negatively regulated by SOCSl) either in the nucleus or the cytoplasm of the cell (Shuai et al. (2003) Nat. Rev. Immunol. 3:900-911; Wiede et al. (2011) J Clin. Invest. 121 :4758-4774). Thus, the term "SOCSl activity" includes the ability of a

SOCSl polypeptide to bind its natural substrate(s), as described herein, the ability to modulate dephosphorylation of such substrate(s), and the ability to modulate the immune response through such substrate(s) in SOCSl -regulated signaling pathways.

The term "SOCSl substrate(s)" refers to binding partners of a SOCSlpolypeptide (and its fragments, domains, and/or motifs thereof, discussed herein) from which one or more phosphate groups can be removed by the SOCSl polypeptide. Such binding partners are usually members in SOCSl -regulated signaling pathways, as described herein.

The term "SOCSl -regulated signaling pathway(s)" includes signaling pathways in which SOCSl (and its fragments, domains, and/or motifs thereof, discussed herein) binds to at least one of its substrate, through which at least one cellular function and/or activity and/or cellular protein profiles is changed. In some embodiments, such signaling pathways include at least those activating SOCSl expression and/or functions after cytokine signaling, downstream of cytokine receptors. In other embodiments, such signaling pathways include at least those utilized by SOCSl in a negative feedback loop to attenuate cytokine signaling.

The term "SOCSl inhibitor(s)" includes any natural or non-natural agent prepared, synthesized, manufactured, and/or purified by human that is capable of reducing, inhibiting, blocking, preventing, and/or that inhibits the ability of a SOCSlpolypeptide (and its fragments, domains, and/or motifs thereof, discussed herein). In one embodiment, such inhibitors may reduce or inhibit the binding/interaction between SOCSl and its substrates or other binding partners, as described herein. In another embodiment, such inhibitors may reduce or inhibit the function of SOCSl as a negtaive regulator of cytokine signaling. In still another embodiment, such inhibitors may increase or promote the turnover rate, reduce or inhibit the expression and/or the stability (e.g., the half-life), and/or change the cellular localization of SOCSl, resulting in at least a decrease in SOCSllevels and/or activity. Such inhibitors may be any molecule, including but not limited to small molecule compounds, antibodies or intrabodies, RNA interfereing (RNAi) agents (including at least siRNAs, shRNAs, microRNAs (miRNAs), piwi, and other well-known agents). Such inhibitors may be specific to SOCS1 or also inhibit at least one of other related proteins (e.g.,

aminopeptidases). RNA interference for SOCS1 polypepitdes are well known and commercially available (e.g., human or mouse shRNA products (Cat. # TG309195, TG513972, TF713151, etc.) and siRNA products (Cat # SR305688, SR405596, SR508208, etc.) and CRISPR knockout products (Cat. # KN220847 and KN316454) from Origene (Rockville, MD), siRNA/shRNA products (Cat. # sc-40996 and sc-40997) and human or mouse gene knockout kit via CRISPR (Cat. # sc- sc-419667) from Santa Cruz

Biotechonology (Dallas, Texas), and Ready-to-package AAV shRNA clones from Vigene Biosciences (Rockville, MD, Cat. # SH833775). Methods for detection, purification, and/or inhibition of SOCS1 (e.g., by anti-SOCSl antibodies) are also well known and commercially available (e.g., multiple anti-SOCSl antibodies from Origene (Cat. #

TA890038, AM26561BT-N, AM26561AF-N, etc.), Novus Biologicals (Littleton, CO, Cat. # NB100-56637, NBP1-54563, 26930002, etc.), and abeam (Cambridge, MA, Cat. # ab9870, ab211288, ab62584, etc.). SOCS1 knockout human cell lines are also well known and available at the Horizon (Cambridge, UK, Cat. # HZGHC8651 , etc.).

Methods of detecting the activation or inhibition of IFNy-responsive genes and/or the corresponding cellular functionality are well-known in the art and taught throughout the instant disclosure. For example, different cell growth rates are associated with cells with normal or defective interferon (e.g., IFNy) signaling (e.g., cells containing gene deletion of Statl, Jakl, Ifngrl, or other IFNy-responsive genes as shown in Figure 1 , panel C and

Figure 11, panels B and C) in response to TNF, IFNy and/or IFN . Thus, cell growth can be measured and compared before and after treatment with inhibitors of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) with well known techniques, such as an in vitro competition assay. While IFNy- pathway deficient cells (e.g. , cancer cells) may have a significant growth advantage over wild type cancer cells or non-cancer cells, when exposed to IFNy or IFNP, an effective inhibitor described herein may ameliorate such growth advantage or cause growth disadvantages, relative to controls. Other readouts for testing the inhibitory function of such inhibitors may include the expression and/or function of IFNy-responsive genes and/or cellular functions. For example, the activation of Statl, Jakl, Ifngrl, or other IFNy- responsive genes as shown in Figure 1, panel C and Figure 11, panels B and C may be detected and such inhibitors may increase or restore the expression of such responsive genes, such as increased expression of Granzyme B in CCD8+ T cells, MHC-I on tumor cell surface, cytokines (e.g., at least Cxcl9, CxcllO, Cxclll, Ccl5, Tapl, Tapbp, B2m, Cdknla, Casp4, Casp8, Ifit2, Ripkl, and Bakl), etc. Other readouts on cellular function for such inhibitors may include, e.g., tumor size, responsiveness to immunotherapies, overall survival, antigen presentation, T cell recogniation of tumors, CD8+ T cell and γδ+ T cell numbers, apoptosis, T cell infiltration into tumors, or other methods taught in the instant disclosure.

The term "immune response" includes T cell mediated and/or B cell mediated immune responses. Exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity. In addition, the term immune response includes immune responses that are indirectly effected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages.

The term "immunotherapeutic agent" can include any molecule, peptide, antibody or other agent which can stimulate a host immune system to generate an immune response to a tumor or cancer in the subject. Various immunotherapeutic agents are useful in the compositions and methods described herein.

The term "inhibit" includes the decrease, limitation, or blockage, of, for example a particular action, function, or interaction. In some embodiments, cancer is "inhibited" if at least one symptom of the cancer is alleviated, terminated, slowed, or prevented. As used herein, cancer is also "inhibited" if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.

The term "interaction", when referring to an interaction between two molecules, refers to the physical contact (e.g., binding) of the molecules with one another. Generally, such an interaction results in an activity (which produces a biological effect) of one or both of said molecules.

An "isolated protein" refers to a protein that is substantially free of other proteins, cellular material, separation medium, and culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide or fusion protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of a biomarker polypeptide or fragment thereof, in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language "substantially free of cellular material" includes preparations of a biomarker protein or fragment thereof, having less than about 30% (by dry weight) of non-biomarker protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-biomarker protein, still more preferably less than about 10% of non-biomarker protein, and most preferably less than about 5% non- biomarker protein. When antibody, polypeptide, peptide or fusion protein or fragment thereof, e.g., a biologically active fragment thereof, is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

As used herein, the term "isotype" refers to the antibody class (e.g., IgM, IgGl, IgG2C, and the like) that is encoded by heavy chain constant region genes.

As used herein, the term "KD" is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction. The binding affinity of antibodies of the disclosed invention may be measured or determined by standard antibody-antigen assays, for example, competitive assays, saturation assays, or standard immunoassays such as ELISA or RIA.

A "kit" is any manufacture (e.g. a package or container) comprising at least one reagent, e.g. a probe or small molecule, for specifically detecting and/or affecting the expression of a marker of the present invention. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. The kit may comprise one or more reagents necessary to express a composition useful in the methods of the present invention. In certain embodiments, the kit may further comprise a reference standard, e.g., a nucleic acid encoding a protein that does not affect or regulate signaling pathways controlling cell growth, division, migration, survival or apoptosis. One skilled in the art can envision many such control proteins, including, but not limited to, common molecular tags (e.g., green fluorescent protein and beta-galactosidase), proteins not classified in any of pathway encompassing cell growth, division, migration, survival or apoptosis by GeneOntology reference, or ubiquitous housekeeping proteins. Reagents in the kit may be provided in individual containers or as mixtures of two or more reagents in a single container. In addition, instructional materials which describe the use of the compositions within the kit can be included. The term "neoadjuvant therapy" refers to a treatment given before the primary treatment. Examples of neoadjuvant therapy can include chemotherapy, radiation therapy, and hormone therapy. For example, in treating breast cancer, neoadjuvant therapy can allows patients with large breast cancer to undergo breast-conserving surgery.

The "normal" level of expression of a biomarker is the level of expression of the biomarker in cells of a subject, e.g., a human patient, not afflicted with a cancer. An "over- expression" or "significantly higher level of expression" of a biomarker refers to an expression level in a test sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more higher than the expression activity or level of the biomarker in a control sample (e.g., sample from a healthy subject not having the biomarker associated disease) and preferably, the average expression level of the biomarker in several control samples. A "significantly lower level of expression" of a biomarker refers to an expression level in a test sample that is at least 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more lower than the expression level of the biomarker in a control sample (e.g., sample from a healthy subject not having the biomarker associated disease) and preferably, the average expression level of the biomarker in several control samples.

An "over-expression" or "significantly higher level of expression" of a biomarker refers to an expression level in a test sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more higher than the expression activity or level of the biomarker in a control sample (e.g., sample from a healthy subject not having the biomarker associated disease) and preferably, the average expression level of the biomarker in several control samples. A "significantly lower level of expression" of a biomarker refers to an expression level in a test sample that is at least 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more lower than the expression level of the biomarker in a control sample (e.g., sample from a healthy subject not having the biomarker associated disease) and preferably, the average expression level of the biomarker in several control samples.

The term "pre-determined" biomarker amount and/or activity measurement(s) may be a biomarker amount and/or activity measurement(s) used to, by way of example only, evaluate a subject that may be selected for a particular treatment, evaluate a response to a treatment such as inhibitors of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCSl)/immunotherapy combination therapy, and/or evaluate the disease state. A pre-determined biomarker amount and/or activity measurement(s) may be determined in populations of patients with or without cancer. The pre-determined biomarker amount and/or activity measurement(s) can be a single number, equally applicable to every patient, or the pre-determined biomarker amount and/or activity measurement(s) can vary according to specific subpopulations of patients. Age, weight, height, and other factors of a subject may affect the pre-determined biomarker amount and/or activity measurement(s) of the individual. Furthermore, the pre-determined biomarker amount and/or activity can be determined for each subject individually. In one embodiment, the amounts determined and/or compared in a method described herein are based on absolute measurements. In another embodiment, the amounts determined and/or compared in a method described herein are based on relative measurements, such as ratios (e.g. , serum biomarker normalized to the expression of housekeeping or otherwise generally constant biomarker). The pre-determined biomarker amount and/or activity

measurement(s) can be any suitable standard. For example, the pre-determined biomarker amount and/or activity measurement(s) can be obtained from the same or a different human for whom a patient selection is being assessed. In one embodiment, the pre-determined biomarker amount and/or activity measurement(s) can be obtained from a previous assessment of the same patient. In such a manner, the progress of the selection of the patient can be monitored over time. In addition, the control can be obtained from an assessment of another human or multiple humans, e.g., selected groups of humans, if the subject is a human. In such a manner, the extent of the selection of the human for whom selection is being assessed can be compared to suitable other humans, e.g., other humans who are in a similar situation to the human of interest, such as those suffering from similar or the same condition(s) and/or of the same ethnic group. The term "predictive" includes the use of a biomarker nucleic acid and/or protein status, e.g., over- or under- activity, emergence, expression, growth, remission, recurrence or resistance of tumors before, during or after therapy, for determining the likelihood of response of a cancer to inhibitors of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCSl)/immunotherapy combination therapy (e.g., treatment with a combination of such an inhibitor and an immunotherapy, such as an immune checkpoint inhibitor). Such predictive use of the biomarker may be confirmed by, e.g., (1) increased or decreased copy number (e.g., by FISH, FISH plus SKY, single-molecule sequencing, e.g., as described in the art at least at J. Biotechnol., 86:289- 301, or qPCR), overexpression or underexpression of a biomarker nucleic acid (e.g., by ISH, Northern Blot, or qPCR), increased or decreased biomarker protein (e.g., by IHC), or increased or decreased activity, e.g., in more than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more of assayed human cancers types or cancer samples; (2) its absolute or relatively modulated presence or absence in a biological sample, e.g., a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, or bone marrow, from a subject, e.g. a human, afflicted with cancer; (3) its absolute or relatively modulated presence or absence in clinical subset of patients with cancer (e.g., those responding to a particular inhibitor of one or more biomarkers in Table 1 /immunotherapy combination therapy or those developing resistance thereto).

The term "pre-malignant lesions" as described herein refers to a lesion that, while not cancerous, has potential for becoming cancerous. It also includes the term "pre- malignant disorders" or "potentially malignant disorders." In particular this refers to a benign, morphologically and/or histologically altered tissue that has a greater than normal risk of malignant transformation, and a disease or a patient's habit that does not necessarily alter the clinical appearance of local tissue but is associated with a greater than normal risk of precancerous lesion or cancer development in that tissue (leukoplakia, erythroplakia, erytroleukoplakia lichen planus (lichenoid reaction) and any lesion or an area which histological examination showed atypia of cells or dysplasia. In one embodiment, a metaplasia is a pre-malignant lesion.

The terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.

The term "probe" refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example, a nucleotide transcript or protein encoded by or corresponding to a biomarker nucleic acid. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

The term "prognosis" includes a prediction of the probable course and outcome of cancer or the likelihood of recovery from the disease. In some embodiments, the use of statistical algorithms provides a prognosis of cancer in an individual. For example, the prognosis can be surgery, development of a clinical subtype of cancer (e.g., solid tumors, such as esophageal cancer and gastric cancer), development of one or more clinical factors, or recovery from the disease.

The term "response to immunotherapy" or "response to inhibitors of one or more biomarkers in Table 1 /immunotherapy combination therapy" relates to any response of the hyperproliferative disorder (e.g., cancer) to an anti-cancer agent, such as an inhibitor of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) and an immunotherapy, preferably to a change in tumor mass and/or volume after initiation of neoadjuvant or adjuvant therapy. Hyperproliferative disorder response may be assessed, for example for efficacy or in a neoadjuvant or adjuvant situation, where the size of a tumor after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation. Responses may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response may be recorded in a quantitative fashion like percentage change in tumor volume or in a qualitative fashion like

"pathological complete response" (pCR), "clinical complete remission" (cCR), "clinical partial remission" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD) or other qualitative criteria. Assessment of hyperproliferative disorder response may be done early after the onset of neoadjuvant or adjuvant therapy, e.g., after a few hours, days, weeks or preferably after a few months. A typical endpoint for response assessment is upon termination of neoadjuvant chemotherapy or upon surgical removal of residual tumor cells and/or the tumor bed. This is typically three months after initiation of neoadjuvant therapy. In some embodiments, clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out from the end of therapy. The shorthand for this formula is CBR=CR+PR+SD over 6 months. In some embodiments, the CBR for a particular cancer therapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Additional criteria for evaluating the response to cancer therapies are related to "survival," which includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); "recurrence- free survival" (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence or metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence. For example, in order to determine appropriate threshold values, a particular cancer therapeutic regimen can be administered to a population of subjects and the outcome can be correlated to biomarker measurements that were determined prior to administration of any cancer therapy. The outcome measurement may be pathologic response to therapy given in the neoadjuvant setting. Alternatively, outcome measures, such as overall survival and disease-free survival can be monitored over a period of time for subjects following cancer therapy for which biomarker measurement values are known. In certain embodiments, the doses administered are standard doses known in the art for cancer therapeutic agents. The period of time for which subjects are monitored can vary. For example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker measurement threshold values that correlate to outcome of a cancer therapy can be determined using well-known methods in the art, such as those described in the Examples section. The term "resistance" refers to an acquired or natural resistance of a cancer sample or a mammal to a cancer therapy ( i.e., being nonresponsive to or having reduced or limited response to the therapeutic treatment), such as having a reduced response to a therapeutic treatment by 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2- fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more. The reduction in response can be measured by comparing with the same cancer sample or mammal before the resistance is acquired, or by comparing with a different cancer sample or a mammal that is known to have no resistance to the therapeutic treatment. A typical acquired resistance to chemotherapy is called "multidrug resistance." The multidrug resistance can be mediated by P-glycoprotein or can be mediated by other mechanisms, or it can occur when a mammal is infected with a multi-drug-resistant microorganism or a combination of microorganisms. The determination of resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician, for example, can be measured by cell proliferative assays and cell death assays as described herein as "sensitizing." In some embodiments, the term "reverses resistance" means that the use of a second agent in combination with a primary cancer therapy {e.g., chemotherapeutic or radiation therapy) is able to produce a significant decrease in tumor volume at a level of statistical significance {e.g., p<0.05) when compared to tumor volume of untreated tumor in the circumstance where the primary cancer therapy {e.g., chemotherapeutic or radiation therapy) alone is unable to produce a statistically significant decrease in tumor volume compared to tumor volume of untreated tumor. This generally applies to tumor volume measurements made at a time when the untreated tumor is growing log rhythmically.

The terms "response" or "responsiveness" refers to an anti-cancer response, e.g. in the sense of reduction of tumor size or inhibiting tumor growth. The terms can also refer to an improved prognosis, for example, as reflected by an increased time to recurrence, which is the period to first recurrence censoring for second primary cancer as a first event or death without evidence of recurrence, or an increased overall survival, which is the period from treatment to death from any cause. To respond or to have a response means there is a beneficial endpoint attained when exposed to a stimulus. Alternatively, a negative or detrimental symptom is minimized, mitigated or attenuated on exposure to a stimulus. It will be appreciated that evaluating the likelihood that a tumor or subject will exhibit a favorable response is equivalent to evaluating the likelihood that the tumor or subject will not exhibit favorable response {i.e., will exhibit a lack of response or be non-responsive). An "R A interfering agent" as used herein, is defined as any agent which interferes with or inhibits expression of a target biomarker gene by RNA interference (RNAi). Such RNA interfering agents include, but are not limited to, nucleic acid molecules including RNA molecules which are homologous to the target biomarker gene of the present invention, or a fragment thereof, short interfering RNA (siRNA), and small molecules which interfere with or inhibit expression of a target biomarker nucleic acid by RNA interference (RNAi).

"RNA interference (RNAi)" is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target biomarker nucleic acid results in the sequence specific degradation or specific post- transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene {see Coburn and Cullen (2002) J Virol. 76:9225), thereby inhibiting expression of the target biomarker nucleic acid. In one embodiment, the RNA is double stranded RNA (dsRNA). This process has been described in plants, invertebrates, and mammalian cells. In nature, RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes processive cleavage of long dsRNA into double-stranded fragments termed siRNAs. siRNAs are incorporated into a protein complex that recognizes and cleaves target mRNAs. RNAi can also be initiated by introducing nucleic acid molecules, e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silence the expression of target biomarker nucleic acids. As used herein, "inhibition of target biomarker nucleic acid expression" or "inhibition of marker gene expression" includes any decrease in expression or protein activity or level of the target biomarker nucleic acid or protein encoded by the target biomarker nucleic acid. The decrease may be of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the expression of a target biomarker nucleic acid or the activity or level of the protein encoded by a target biomarker nucleic acid which has not been targeted by an RNA interfering agent.

The term "sample" used for detecting or determining the presence or level of at least one biomarker is typically brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, stool {e.g., feces), tears, and any other bodily fluid {e.g., as described above under the definition of "body fluids"), or a tissue sample {e.g. , biopsy) such as a small intestine, colon sample, or surgical resection tissue. In certain instances, the method of the present invention further comprises obtaining the sample from the individual prior to detecting or determining the presence or level of at least one marker in the sample. The term "sensitize" means to alter cancer cells or tumor cells in a way that allows for more effective treatment of the associated cancer with a cancer therapy (e.g., anti- immune checkpoint, chemotherapeutic, and/or radiation therapy). In some embodiments, normal cells are not affected to an extent that causes the normal cells to be unduly injured by the therapies. An increased sensitivity or a reduced sensitivity to a therapeutic treatment is measured according to a known method in the art for the particular treatment and methods described herein below, including, but not limited to, cell proliferative assays (Tanigawa N, Kern D H, Kikasa Y, Morton D L, Cancer Res 1982; 42: 2159-2164), cell death assays (Weisenthal L M, Shoemaker R H, Marsden J A, Dill P L, Baker J A, Moran E M, Cancer Res 1984; 94: 161 - 173 ; Weisenthal L M, Lippman M E, Cancer Treat Rep 1985 ; 69: 615-632; Weisenthal L M, In: Kaspers G J L, Pieters R, Twentyman P R, Weisenthal L M, Veerman A J P, eds. Drug Resistance in Leukemia and Lymphoma. Langhorne, P A: Harwood Academic Publishers, 1993: 415-432; Weisenthal L M, Contrib Gynecol Obstet 1994; 19: 82-90). The sensitivity or resistance may also be measured in animal by measuring the tumor size reduction over a period of time, for example, 6 month for human and 4-6 weeks for mouse. A composition or a method sensitizes response to a therapeutic treatment if the increase in treatment sensitivity or the reduction in resistance is 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2-fold, 3-fold, 4-fold, 5- fold, 10-fold, 15-fold, 20-fold or more, compared to treatment sensitivity or resistance in the absence of such composition or method. The determination of sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician. It is to be understood that any method described herein for enhancing the efficacy of a cancer therapy can be equally applied to methods for sensitizing hyperproliferative or otherwise cancerous cells (e.g., resistant cells) to the cancer therapy.

"Short interfering RNA" (siRNA), also referred to herein as "small interfering

RNA" is defined as an agent which functions to inhibit expression of a target biomarker nucleic acid, e.g., by RNAi. An siRNA may be chemically synthesized, may be produced by in vitro transcription, or may be produced within a host cell. In one embodiment, siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides in length, and more preferably about 19, 20, 21, or 22 nucleotides in length, and may contain a 3' and/or 5' overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand. Preferably the siRNA is capable of promoting RNA interference through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA).

In another embodiment, an siRNA is a small hairpin (also called stem loop) RNA

(shRNA). In one embodiment, these shRNAs are composed of a short (e.g., 19-25 nucleotide) antisense strand, followed by a 5-9 nucleotide loop, and the analogous sense strand. Alternatively, the sense strand may precede the nucleotide loop structure and the antisense strand may follow. These shRNAs may be contained in plasmids, retroviruses, and lentiviruses and expressed from, for example, the pol III U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003) RNA Apr;9(4):493-501 incorporated by reference herein).

RNA interfering agents, e.g., siRNA molecules, may be administered to a patient having or at risk for having cancer, to inhibit expression of a biomarker gene which is overexpressed in cancer and thereby treat, prevent, or inhibit cancer in the subject.

The term "small molecule" is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides,

peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds. In a further embodiment, a small molecule is not biosynthetic.

The term "specific binding" refers to antibody binding to a predetermined antigen.

Typically, the antibody binds with an affinity (KD) of approximately less than 10 ^"7 M, such as approximately less than 10 ^"8 M, 10 ^"9 M or 10 ^"10 M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE® assay instrument using an antigen of interest as the analyte and the antibody as the ligand, and binds to the predetermined antigen with an affinity that is at least 1.1 -, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.5-, 3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 6.0-, 7.0-, 8.0-, 9.0-, or 10.0-fold or greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The phrases "an antibody recognizing an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody which binds specifically to an antigen." Selective binding is a relative term refering to the ability of an antibody to discriminate the binding of one antigen over another.

The term "subject" refers to any healthy animal, mammal or human, or any animal, mammal or human afflicted with a cancer, e.g., brain, lung, ovarian, pancreatic, liver, breast, prostate, and/or colorectal cancers, melanoma, multiple myeloma, and the like. The term "subject" is interchangeable with "patient."

The term "survival" includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); "recurrence-free survival" (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point (e.g. time of diagnosis or start of treatment) and end point (e.g. death, recurrence or metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.

The term "synergistic effect" refers to the combined effect of two or more anti- cancer agents (e.g. , inhibitors of one or more biomarkers in Table 1 (e.g. , one or more kinase signaling inhibitors, such as PTPN2 and SOCSl)/immunotherapy combination therapy) can be greater than the sum of the separate effects of the anti-cancer

agents/therapies alone.

The term "T cell" includes CD4 ⁺ T cells and CD8 ⁺ T cells. The term T cell also includes both T helper 1 type T cells and T helper 2 type T cells. The term "antigen presenting cell" includes professional antigen presenting cells (e.g., B lymphocytes, monocytes, dendritic cells, Langerhans cells), as well as other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes).

The term "therapeutic effect" refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. The term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in an animal or human. The phrase "therapeutically- effective amount" means that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit risk ratio applicable to any treatment. In certain embodiments, a therapeutically effective amount of a compound will depend on its therapeutic index, solubility, and the like. For example, certain compounds discovered by the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

The terms "therapeutically-effective amount" and "effective amount" as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment. Toxicity and therapeutic efficacy of subject compounds may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD ₅₀ and the ED ₅ o. Compositions that exhibit large therapeutic indices are preferred. In some embodiments, the LD50 (lethal dosage) can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more reduced for the agent relative to no administration of the agent. Similarly, the ED ₅ o (i.e., the concentration which achieves a half-maximal inhibition of symptoms) can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent. Also, Similarly, the IC ₅₀ (i.e., the concentration which achieves half-maximal cytotoxic or cytostatic effect on cancer cells) can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent. In some embodiments, cancer cell growth in an assay can be inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%. In another embodiment, at least about a 10% , 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% decrease in a solid malignancy can be achieved.

A "transcribed polynucleotide" or "nucleotide transcript" is a polynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA or cDNA) which is complementary to or homologous with all or a portion of a mature mRNA made by transcription of a biomarker nucleic acid and normal post-transcriptional processing {e.g. splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript.

As used herein, the term "unresponsiveness" includes refractivity of cancer cells to therapy or refractivity of therapeutic cells, such as immune cells, to stimulation, e.g., stimulation via an activating receptor or a cytokine. Unresponsiveness can occur, e.g., because of exposure to immunosuppressants or exposure to high doses of antigen. As used herein, the term "anergy" or "tolerance" includes refractivity to activating receptor- mediated stimulation. Such refractivity is generally antigen-specific and persists after exposure to the tolerizing antigen has ceased. For example, anergy in T cells (as opposed to unresponsiveness) is characterized by lack of cytokine production, e.g., IL-2. T cell anergy occurs when T cells are exposed to antigen and receive a first signal (a T cell receptor or CD-3 mediated signal) in the absence of a second signal (a costimulatory signal). Under these conditions, reexposure of the cells to the same antigen (even if reexposure occurs in the presence of a costimulatory polypeptide) results in failure to produce cytokines and, thus, failure to proliferate. Anergic T cells can, however, proliferate if cultured with cytokines {e.g., IL-2). For example, T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes as measured by ELISA or by a proliferation assay using an indicator cell line. Alternatively, a reporter gene construct can be used. For example, anergic T cells fail to initiate IL-2 gene transcription induced by a heterologous promoter under the control of the 5' IL-2 gene enhancer or by a multimer of the API sequence that can be found within the enhancer (Kang et al. (1992) Science 257:1134).

There is a known and definite correspondence between the amino acid sequence of a particular protein and the nucleotide sequences that can code for the protein, as defined by the genetic code (shown below). Likewise, there is a known and definite correspondence between the nucleotide sequence of a particular nucleic acid and the amino acid sequence encoded by that nucleic acid, as defined by the genetic code.

GENETIC CODE

Alanine (Ala, A) GCA, GCC, GCG, GCT

Arginine (Arg, R) AGA, ACG, CGA, CGC, CGG, CGT

Asparagine (Asn, N) AAC, AAT

Aspartic acid (Asp, D) GAC, GAT

Cysteine (Cys, C) TGC, TGT

Glutamic acid (Glu, E) GAA, GAG Glutamine (Gin, Q) CAA, CAG

Glycine (Gly, G) GGA, GGC, GGG, GGT

Histidine (His, H) CAC, CAT

Isoleucine (He, I) ATA, ATC, ATT

Leucine (Leu, L) CTA, CTC, CTG, CTT, TTA, TTG

Lysine (Lys, K) AAA, AAG

Methionine (Met, M) ATG

Phenylalanine (Phe, F) TTC, TTT

Proline (Pro, P) CCA, CCC, CCG, CCT

Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT

Threonine (Thr, T) ACA, ACC, ACG, ACT

Tryptophan (Trp, W) TGG

Tyrosine (Tyr, Y) TAC, TAT

Valine (Val, V) GTA, GTC, GTG, GTT

Termination signal (end) TAA, TAG, TGA

An important and well-known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.

In view of the foregoing, the nucleotide sequence of a DNA or R A encoding a biomarker nucleic acid (or any portion thereof) can be used to derive the polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence. Likewise, for polypeptide amino acid sequence, corresponding nucleotide sequences that can encode the polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence). Thus, description and/or disclosure herein of a nucleotide sequence which encodes a polypeptide should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence. Similarly, description and/or disclosure of a polypeptide amino acid sequence herein should be considered to also include description and/or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.

Finally, nucleic acid and amino acid sequence information for the loci and biomarkers of the present invention (e.g., biomarkers listed in Tables 1 and 2) are well- known in the art and readily available on publicly available databases, such as the National Center for Biotechnology Information (NCBI). For example, exemplary nucleic acid and amino acid sequences derived from publicly available sequence databases are provided below and include, for example, PCT Publ. WO 2014/022759, which is incorporated herein in its entirety by this reference.

Table 1

SEP ID NO: 1 Human PTPN2 isoform 1 cDNA Sequence (NM 002828.3)

1 gctcgggcgc cgagtctgcg cgctgacgtc cgacgctcca ggtactttcc ccacggccga 61 cagggcttgg cgtgggggcg gggcgcggcg cgcagcgcgc atgcgccgca gcgccagcgc 121 tctccccgga tcgtgcgggg cctgagcctc tccgccggcg caggctctgc tcgcgccagc 181 tcgctcccgc agccatgccc accaccatcg agcgggagtt cgaagagttg gatactcagc 241 gtcgctggca gccgctgtac ttggaaattc gaaatgagtc ccatgactat cctcatagag 301 tggccaagtt tccagaaaac agaaatcgaa acagatacag agatgtaagc ccatatgatc 361 acagtcgtgt taaactgcaa aatgctgaga atgattatat taatgccagt ttagttgaca 421 tagaagaggc acaaaggagt tacatcttaa cacagggtcc acttcctaac acatgctgcc 481 atttctggct tatggtttgg cagcagaaga ccaaagcagt tgtcatgctg aaccgcattg 541 tggagaaaga atcggttaaa tgtgcacagt actggccaac agatgaccaa gagatgctgt 601 ttaaagaaac aggattcagt gtgaagctct tgtcagaaga tgtgaagtcg tattatacag 661 tacatctact acaattagaa aatatcaata gtggtgaaac cagaacaata tctcactttc 721 attatactac ctggccagat tttggagtcc ctgaatcacc agcttcattt ctcaatttct 781 tgtttaaagt gagagaatct ggctccttga accctgacca tgggcctgcg gtgatccact 841 gtagtgcagg cattgggcgc tctggcacct tctctctggt agacacttgt cttgttttga 901 tggaaaaagg agatgatatt aacataaaac aagtgttact gaacatgaga aaataccgaa 961 tgggtcttat tcagacccca gatcaactga gattctcata catggctata atagaaggag 1021 caaaatgtat aaagggagat tctagtatac agaaacgatg gaaagaactt tctaaggaag 1081 acttatctcc tgcctttgat cattcaccaa acaaaataat gactgaaaaa tacaatggga 1141 acagaatagg tctagaagaa gaaaaactga caggtgaccg atgtacagga ctttcctcta 1201 aaatgcaaga tacaatggag gagaacagtg agagtgctct acggaaacgt attcgagagg 1261 acagaaaggc caccacagct cagaaggtgc agcagatgaa acagaggcta aatgagaatg 1321 aacgaaaaag aaaaaggtgg ttatattggc aacctattct cactaagatg gggtttatgt 1381 cagtcatttt ggttggcgct tttgttggct ggacactgtt ttttcagcaa aatgccctat 1441 aaacaattaa ttttgcccag caagcttctg cactagtaac tgacagtgct acattaatca 1501 taggggtttg tctgcagcaa acgcctcata tcccaaaaac ggtgcagtag aatagacatc 1561 aaccagataa gtgatattta cagtcacaag cccaacatct caggactctt gactgcaggt 1621 tcctctgaac cccaaactgt aaatggctgt ctaaaataaa gacattcatg tttgttaaaa 1681 actggtaaat tttgcaactg tattcataca tgtcaaacac agtatttcac ctgaccaaca 1741 ttgagatatc ctttatcaca ggatttgttt ttggaggcta tctggatttt aacctgcact 1801 tgatataagc aataaatatt gtggttttat ctacgttatt ggaaagaaaa tgacatttaa 1861 ataatgtgtg taatgtataa tgtactattg acatgggcat caacactttt attcttaagc 1921 atttcagggt aaatatattt tataagtatc tatttaatct tttgtagtta actgtacttt 1981 ttaagagctc aatttgaaaa atctgttact aaaaaaataa attgtatgtc gattgaattg 2041 tactggatac attttccatt tttctaaaga gaagtttgat atgagcagtt agaagttgga 2101 ataagcaatt tctactatat attgcatttc ttttatgttt tacagttttc cccattttaa 2161 aaagaaaagc aaacaaagaa acaaaagttt ttcctaaaaa tatctttgaa ggaaaattct 2221 ccttactggg atagtcaggt aaacagttgg tcaagacttt gtaaagaaat tggtttctgt 2281 aaatcccatt attgatatgt ttatttttca tgaaaatttc aatgtagttg gggtagatta 2341 tgatttagga agcaaaagta agaagcagca ttttatgatt cataatttca gtttactaga 2401 ctgaagtttt gaagtaaaca cttttcagtt tctttctact tcaataaata gtatgattat 24 61 atgcaaacct tacattgtca ttttaactta atgaatattt tttaaagcaa actgtttaat 2521 gaatttaact gctcatttga atgctagctt tcctcagatt tcaacattcc attcagtgtt 2581 taatttgtct tacttaaact tgaaattgtt gttacaaatt taattgctag gaggcatgga 2641 tagcatacat tattatggat agcatacctt atttcagtgg ttttcaaact atgctcattg 2701 gatgtccagg tgggtcaaga ggttactttc aaccacagca tctctgcctt gtctctttat 2761 atgccacata agatttctgc ataaggctta agtattttaa agggggcagt tatcatttaa 2821 aaacagtttg gtcgggcgcg gtggctcatg cctgtaatcc cagcactttg ggaggctgaa 2881 gtgggcagat cacctgaggt caggagttca agaccagcct ggccaacgtg gtgaaacacc 2941 atctctacta aaaatgcaaa aattagctgg gcatggtgga gggcacctgt aatctcagct 3001 actcaggagg ctgaggtagg agaattgctt gaacccagga gatggaggtt gcagtgagct 3061 gagatcacgt cactgcactc cagccagggc gacagagcga gactccatct caaaagaaac 3121 aaacaaaaaa aacagtttgg gccgggtgtg gtggctcacg cttgtaatcc cagcacttcg 3181 gaaggccaag gcgggcggat cacgaggtca agagatggag actgtcctgg ccaacatggt 3241 gaaatccctt ctttactaaa aatacaaaaa ttatctgggc gtggtggtgc atgcctgtag 3301 tcccagctcc ttgggaggct aaggcaggag aatcacttga acccgggagg cagaggttgc 3361 agtgagccga gattgcacca ctgcactcca gcctggcaac agagcaagac ttcgtctc

SEP ID NO: 2 Human PTPN2 isoform 1 Amino Acid Sequence (NP 002819.2)

1 mpttierefe eldtqrrwqp lyleirnesh dyphrvakfp enrnrnryrd vspydhsrvk 61 lqnaendyin aslvdieeaq rsyiltqgpl pntcchfwlm vwqqkt ka v mlnrive kes

121 vkcaqy ptd dqeml fketg fsvkll sedv ksyytvhllq leninsgetr tishfhyttw 181 pdfgvpespa s flnfl fkvr esgslnpdhg pavihcsagi grsgt fslvd tclvlme kgd 241 dini kqvlln mrkyrmgliq tpdqlrfsym aiiegakci k gdssiqkrwk els kedlspa 301 fdhspnkimt ekyngnrigl eeekltgdrc tgl s s kmqdt meensesalr kriredrkat 361 taqkvqqmkq rlnenerkrk rwlywqpilt kmgfmsvilv gafvgwtl f f qqnal

SEP ID NO: 3 Human PTPN2 isoform 2 cDNA Sequence (NM 080422.2)

1 gctcgggcgc cgagtctgcg cgctgacgtc cgacgctcca ggtactttcc ccacggccga 61 cagggcttgg cgtgggggcg gggcgcggcg cgcagcgcgc atgcgccgca gcgccagcgc 121 tctccccgga tcgtgcgggg cctgagcctc tccgccggcg caggctctgc tcgcgccagc 181 tcgctcccgc agccatgccc accaccatcg agcgggagtt cgaagagttg gatactcagc 241 gtcgctggca gccgctgtac ttggaaattc gaaatgagtc ccatgactat cctcatagag 301 tggccaagtt tccagaaaac agaaatcgaa acagatacag agatgtaagc ccatatgatc 361 acagtcgtgt taaactgcaa aatgctgaga atgattatat taatgccagt ttagttgaca 421 tagaagaggc acaaaggagt tacatcttaa cacagggtcc acttcctaac acatgctgcc 481 atttctggct tatggtttgg cagcagaaga ccaaagcagt tgtcatgctg aaccgcattg 541 tggagaaaga atcggttaaa tgtgcacagt actggccaac agatgaccaa gagatgctgt 601 ttaaagaaac aggattcagt gtgaagctct tgtcagaaga tgtgaagtcg tattatacag 661 tacatctact acaattagaa aatatcaata gtggtgaaac cagaacaata tctcactttc 721 attatactac ctggccagat tttggagtcc ctgaatcacc agcttcattt ctcaatttct 781 tgtttaaagt gagagaatct ggctccttga accctgacca tgggcctgcg gtgatccact 841 gtagtgcagg cattgggcgc tctggcacct tctctctggt agacacttgt cttgttttga 901 tggaaaaagg agatgatatt aacataaaac aagtgttact gaacatgaga aaataccgaa 961 tgggtcttat tcagacccca gatcaactga gattctcata catggctata atagaaggag 1021 caaaatgtat aaagggagat tctagtatac agaaacgatg gaaagaactt tctaaggaag 1081 acttatctcc tgcctttgat cattcaccaa acaaaataat gactgaaaaa tacaatggga 1141 acagaatagg tctagaagaa gaaaaactga caggtgaccg atgtacagga ctttcctcta 1201 aaatgcaaga tacaatggag gagaacagtg agagtgctct acggaaacgt attcgagagg 1261 acagaaaggc caccacagct cagaaggtgc agcagatgaa acagaggcta aatgagaatg 1321 aacgaaaaag aaaaaggcca agattgacag acacctaata ttcatgactt gagaatattc 1381 tgcagctata aattttgaac cattgatgtg caaagcaaga cctgaagccc actccggaaa 1441 ctaaagtgag gctcgctaac cctctagatt gcctcacagt tgtttgttta caaagtaaac 1501 tttacatcca ggggatgaag agcacccacc agcagaagac tttgcagaac ctttaattgg 1561 atgtgttaag tgtttttaat gagtgtatga aatgtagaaa gatgtacaag aaataaatta 1621 ggggagatta ctttgtattg tactgccatt cctactgtat ttttatactt tttggcagca 1681 ttaaatattt ttgttaaata gtcaaaaaaa aaaaaaaaaa a

SEP ID NO: 4 Human PTPN2 isoform 2 Amino Acid Sequence (NP 536347.1)

1 mpttierefe eldtqrrwqp lyleirnesh dyphrvakfp enrnrnryrd vspydhsrvk

61 lqnaendyin aslvdieeaq rsyiltqgpl pntcchfwlm vwqqkt ka v mlnrive kes

121 vkcaqywptd dqeml fketg fsvkll sedv ksyytvhllq leninsgetr tishfhyttw

181 pdfgvpespa s flnfl fkvr esgslnpdhg pavihcsagi grsgt fslvd tclvlme kgd

241 dini kqvlln mrkyrmgliq tpdqlrfsym aiiegakci k gdssiqkrwk els kedlspa

301 fdhspnkimt ekyngnrigl eeekltgdrc tgl s s kmqdt meensesalr kriredrkat

361 taqkvqqmkq rlnenerkrk rprltdt

SEP ID NO: 5 Human PTPN2 isoform 3 cDNA Sequence (NM 080423.2)

1 gctcgggcgc cgagtctgcg cgctgacgtc cgacgctcca ggtactttcc ccacggccga 61 cagggcttgg cgtgggggcg gggcgcggcg cgcagcgcgc atgcgccgca gcgccagcgc 121 tctccccgga tcgtgcgggg cctgagcctc tccgccggcg caggctctgc tcgcgccagc 181 tcgctcccgc agccatgccc accaccatcg agcgggagtt cgaagagttg gatactcagc 241 gtcgctggca gccgctgtac ttggaaattc gaaatgagtc ccatgactat cctcatagag 301 tggccaagtt tccagaaaac agaaatcgaa acagatacag agatgtaagc ccatatgatc 361 acagtcgtgt taaactgcaa aatgctgaga atgattatat taatgccagt ttagttgaca 421 tagaagaggc acaaaggagt tacatcttaa cacagggtcc acttcctaac acatgctgcc 481 atttctggct tatggtttgg cagcagaaga ccaaagcagt tgtcatgctg aaccgcattg 541 tggagaaaga atcggttaaa tgtgcacagt actggccaac agatgaccaa gagatgctgt 601 ttaaagaaac aggattcagt gtgaagctct tgtcagaaga tgtgaagtcg tattatacag 661 tacatctact acaattagaa aatatcaata gtggtgaaac cagaacaata tctcactttc 721 attatactac ctggccagat tttggagtcc ctgaatcacc agcttcattt ctcaatttct 781 tgtttaaagt gagagaatct ggctccttga accctgacca tgggcctgcg gtgatccact 841 gtagtgcagg cattgggcgc tctggcacct tctctctggt agacacttgt cttgttttga 901 tggaaaaagg agatgatatt aacataaaac aagtgttact gaacatgaga aaataccgaa 961 tgggtcttat tcagacccca gatcaactga gattctcata catggctata atagaaggag 1021 caaaatgtat aaagggagat tctagtatac agaaacgatg gaaagaactt tctaaggaag 1081 acttatctcc tgcctttgat cattcaccaa acaaaataat gactgaaaaa tacaatggga 1141 acagaatagg tctagaagaa gaaaaactga caggtgaccg atgtacagga ctttcctcta 1201 aaatgcaaga tacaatggag gagaacagtg agaggccaag attgacagac acctaatatt 1261 catgacttga gaatattctg cagctataaa ttttgaacca ttgatgtgca aagcaagacc 1321 tgaagcccac tccggaaact aaagtgaggc tcgctaaccc tctagattgc ctcacagttg 1381 tttgtttaca aagtaaactt tacatccagg ggatgaagag cacccaccag cagaagactt 1441 tgcagaacct ttaattggat gtgttaagtg tttttaatga gtgtatgaaa tgtagaaaga 1501 tgtacaagaa ataaattagg ggagattact ttgtattgta ctgccattcc tactgtattt 1561 ttatactttt tggcagcatt aaatattttt gttaaatagt caaaaaaaaa aaaaaaaaa

SEP ID NO: 6 Human PTPN2 isoform 3 Amino Acid Sequence (NP 536348.1)

1 mpttierefe eldtqrrwqp lyleirnesh dyphrvakfp enrnrnryrd vspydhsrvk

61 lqnaendyin aslvdieeaq rsyiltqgpl pntcchfwlm vwqqkt kavv mlnrive kes

121 vkcaqywptd dqeml fketg fsvkll sedv ksyytvhllq leninsgetr tishfhyttw

181 pdfgvpespa s flnfl fkvr esgslnpdhg pavihcsagi grsgt fslvd tclvlme kgd

241 dini kqvlln mrkyrmgliq tpdqlrfsym aiiegakci k gdssiqkrwk els kedlspa

301 fdhspnkimt ekyngnrigl eeekltgdrc tgl s s kmqdt meenserprl tdt

SEP ID NO: 7 Human PTPN2 isoform 4 cDNA Sequence (NM 001207013.1)

1 gctcgggcgc cgagtctgcg cgctgacgtc cgacgctcca ggtactttcc ccacggccga

61 cagggcttgg cgtgggggcg gggcgcggcg cgcagcgcgc atgcgccgca gcgccagcgc

121 tctccccgga tcgtgcgggg cctgagcctc tccgccggcg caggctctgc tcgcgccagc

181 tcgctcccgc agccatgccc accaccatcg agcgggagtt cgaagagttg gatactcagc

241 gtcgctggca gccgctgtac ttggaaattc gaaatgagtc ccatgactat cctcatagag

301 tggccaagtt tccagaaaac agaaatcgaa acagatacag agatgtaagc ccatatgatc

361 acagtcgtgt taaactgcaa aatgctgaga atgattatat taatgccagt ttagttgaca

421 tagaagaggc acaaaggagt tacatcttaa cacagggtcc acttcctaac acatgctgcc

481 atttctggct tatggtttgg cagcagaaga ccaaagcagt tgtcatgctg aaccgcattg

541 tggagaaaga atcggttaaa tgtgcacagt actggccaac agatgaccaa gagatgctgt 601 ttaaagaaac aggattcagt gtgaagctct tgtcagaaga tgtgaagtcg tattatacag 661 tacatctact acaattagaa aatatcaatt atattgagaa cttgtggatc acactgtatt 721 tgaaattatt aatgctggat gttaaaaggt cactaaaaag tggtgaaacc agaacaatat 781 ctcactttca ttatactacc tggccagatt ttggagtccc tgaatcacca gcttcatttc 841 tcaatttctt gtttaaagtg agagaatctg gctccttgaa ccctgaccat gggcctgcgg 901 tgatccactg tagtgcaggc attgggcgct ctggcacctt ctctctggta gacacttgtc 961 ttgttttgat ggaaaaagga gatgatatta acataaaaca agtgttactg aacatgagaa 1021 aataccgaat gggtcttatt cagaccccag atcaactgag attctcatac atggctataa 1081 tagaaggagc aaaatgtata aagggagatt ctagtataca gaaacgatgg aaagaacttt 1141 ctaaggaaga cttatctcct gcctttgatc attcaccaaa caaaataatg actgaaaaat 1201 acaatgggaa cagaataggt ctagaagaag aaaaactgac aggtgaccga tgtacaggac 1261 tttcctctaa aatgcaagat acaatggagg agaacagtga gagtgctcta cggaaacgta 1321 ttcgagagga cagaaaggcc accacagctc agaaggtgca gcagatgaaa cagaggctaa 1381 atgagaatga acgaaaaaga aaaaggccaa gattgacaga cacctaatat tcatgacttg 1441 agaatattct gcagctataa attttgaacc attgatgtgc aaagcaagac ctgaagccca 1501 ctccggaaac taaagtgagg ctcgctaacc ctctagattg cctcacagtt gtttgtttac 1561 aaagtaaact ttacatccag gggatgaaga gcacccacca gcagaagact ttgcagaacc 1621 tttaattgga tgtgttaagt gtttttaatg agtgtatgaa atgtagaaag atgtacaaga 1681 aataaattag gggagattac tttgtattgt actgccattc ctactgtatt tttatacttt 1741 ttggcagcat taaatatttt tgttaaatag tcaaaaaaaa aaaaaaaaaa

SEP ID NO: 8 Human PTPN2 isoform 4 Amino Acid Sequence

fNP 001193942.1)

1 mpttierefe eldtqrrwqp lyleirnesh dyphrvakfp enrnrnryrd vspydhsrvk 61 lqnaendyin aslvdieeaq rsyiltqgpl pntcchfwlm vwqqkt ka v mlnrive kes

121 vkcaqywptd dqeml fketg fsvkll sedv ksyytvhllq leninyienl witlyl kllm

181 ldvkrsl ksg etrti shfhy ttwpdfgvpe spas flnfl f kvresgslnp dhgpavihcs

241 agigrsgtfs lvdtclvlme kgddini kqv llnmrkyrmg liqtpdqlrf symaiiegak

301 ci kgds siqk rwkel s kedl spafdhspnk imtekyngnr igleee kltg drctgls s km 361 qdtmeenses alrkriredr kattaqkvqq mkqrlnener krkrprltdt

SEP ID NO: 9 Human PTPN2 isoform 5 cDNA Sequence (NM 001308287.1)

1 tattcaatgc agggaacaga ccagttcatc atggaggcat tccatcagag cgtctagtta

61 gagaagatat gtcatggact gcatcggcac agaagtgggg tttatgtgag agaggagttg 121 gaagtcacac ctgagtggag agcaacgtga aaaggtgatg tcagcaagaa tttaggatgt

181 atggaaagga tggtaaaggc accaactgga tggatcaggg agacatggaa tgcagaatgc

241 aggaaataga tgatcacagt cgtgttaaac tgcaaaatgc tgagaatgat tatattaatg

301 ccagtttagt tgacatagaa gaggcacaaa ggagttacat cttaacacag ggtccacttc

361 ctaacacatg ctgccatttc tggcttatgg tttggcagca gaagaccaaa gcagttgtca 421 tgctgaaccg cattgtggag aaagaatcgg ttaaatgtgc acagtactgg ccaacagatg

481 accaagagat gctgtttaaa gaaacaggat tcagtgtgaa gctcttgtca gaagatgtga

541 agtcgtatta tacagtacat ctactacaat tagaaaatat caatagtggt gaaaccagaa

601 caatatctca ctttcattat actacctggc cagattttgg agtccctgaa tcaccagctt

661 catttctcaa tttcttgttt aaagtgagag aatctggctc cttgaaccct gaccatgggc 721 ctgcggtgat ccactgtagt gcaggcattg ggcgctctgg caccttctct ctggtagaca

781 cttgtcttgt tttgatggaa aaaggagatg atattaacat aaaacaagtg ttactgaaca

841 tgagaaaata ccgaatgggt cttattcaga ccccagatca actgagattc tcatacatgg

901 ctataataga aggagcaaaa tgtataaagg gagattctag tatacagaaa cgatggaaag

961 aactttctaa ggaagactta tctcctgcct ttgatcattc accaaacaaa ataatgactg 1021 aaaaatacaa tgggaacaga ataggtctag aagaagaaaa actgacaggt gaccgatgta

1081 caggactttc ctctaaaatg caagatacaa tggaggagaa cagtgagagt gctctacgga

1141 aacgtattcg agaggacaga aaggccacca cagctcagaa ggtgcagcag atgaaacaga

1201 ggctaaatga gaatgaacga aaaagaaaaa ggtggttata ttggcaacct attctcacta

1261 agatggggtt tatgtcagtc attttggttg gcgcttttgt tggctggaca ctgttttttc 1321 agcaaaatgc cctataaaca attaattttg cccagcaagc ttctgcacta gtaactgaca

1381 gtgctacatt aatcataggg gtttgtctgc agcaaacgcc tcatatccca aaaacggtgc

1441 agtagaatag acatcaacca gataagtgat atttacagtc acaagcccaa catctcagga

1501 ctcttgactg caggttcctc tgaaccccaa actgtaaatg gctgtctaaa ataaagacat

1561 tcatgtttgt taaaaactgg taaattttgc aactgtattc atacatgtca aacacagtat 1621 ttcacctgac caacattgag atatccttta tcacaggatt tgtttttgga ggctatctgg 1681 attttaacct gcacttgata taagcaataa atattgtggt tttatctacg ttattggaaa 1741 gaaaatgaca tttaaataat gtgtgtaatg tataatgtac tattgacatg ggcatcaaca 1801 cttttattct taagcatttc agggtaaata tattttataa gtatctattt aatcttttgt 1861 agttaactgt actttttaag agctcaattt gaaaaatctg ttactaaaaa aataaattgt 1921 atgtcgattg aattgtactg gatacatttt ccatttttct aaagagaagt ttgatatgag 1981 cagttagaag ttggaataag caatttctac tatatattgc atttctttta tgttttacag 2041 ttttccccat tttaaaaaga aaagcaaaca aagaaacaaa agtttttcct aaaaatatct 2101 ttgaaggaaa attctcctta ctgggatagt caggtaaaca gttggtcaag actttgtaaa 2161 gaaattggtt tctgtaaatc ccattattga tatgtttatt tttcatgaaa atttcaatgt 2221 agttggggta gattatgatt taggaagcaa aagtaagaag cagcatttta tgattcataa 2281 tttcagttta ctagactgaa gttttgaagt aaacactttt cagtttcttt ctacttcaat 2341 aaatagtatg attatatgca aaccttacat tgtcatttta acttaatgaa tattttttaa 2401 agcaaactgt ttaatgaatt taactgctca tttgaatgct agctttcctc agatttcaac 24 61 attccattca gtgtttaatt tgtcttactt aaacttgaaa ttgttgttac aaatttaatt 2521 gctaggaggc atggatagca tacattatta tggatagcat accttatttc agtggttttc 2581 aaactatgct cattggatgt ccaggtgggt caagaggtta ctttcaacca cagcatctct 2641 gccttgtctc tttatatgcc acataagatt tctgcataag gcttaagtat tttaaagggg 2701 gcagttatca tttaaaaaca gtttggtcgg gcgcggtggc teatgectgt aatcccagca 2761 ctttgggagg ctgaagtggg cagatcacct gaggtcagga gttcaagacc agcctggcca 2821 acgtggtgaa acaccatctc tactaaaaat gcaaaaatta gctgggcatg gtggagggca 2881 cctgtaatct cagctactca ggaggctgag gtaggagaat tgcttgaacc caggagatgg 2941 aggttgcagt gagctgagat cacgtcactg cactccagcc agggcgacag agcgagactc 3001 catctcaaaa gaaacaaaca aaaaaaacag tttgggccgg gtgtggtggc teaegcttgt 3061 aatcccagca cttcggaagg ecaaggeggg eggatcaega ggtcaagaga tggagactgt 3121 cctggccaac atggtgaaat cccttcttta ctaaaaatac aaaaattatc tgggcgtggt 3181 ggtgcatgcc tgtagtccca gctccttggg aggctaaggc aggagaatca cttgaacccg 3241 ggaggcagag gttgcagtga gecgagattg caccactgca ctccagcctg gcaacagagc 3301 aagacttcgt etc

SEP ID NO; 10 Human PTPN2 isoform 5 Amino Acid Sequence

(NP 001295216.1)

1 mygkdgkgtn wmdqgdmecr mqeiddhsrv klqnaendyi naslvdieea qrsyiltqgp

61 lpntcchfwl mvwqqktkav vmlnriveke svkcaqywpt ddqeml f ket gfsvkllsed

121 vksyytvhll qleninsget rti shfhytt wpdfgvpesp as flnfl f kv resgslnpdh 181 gpavihesag igrsgtfslv dtclvlmekg ddini kqvll nmrkyrmgli qtpdqlrfsy

241 maiiegakci kgds siqkrw kel s kedl sp afdhspnkim tekyngnrig leee kltgdr

301 ctgl s s kmqd tmeensesal rkriredrka ttaqkvqqmk qrlnenerkr krwlywqpil 361 tkmgfmsvil vgafvgwtl f fqqnal SEP ID NO; 11 Mouse PTPN2 isoform 1 cDNA Sequence CNM 001127177.1)

1 ggeggggegg ggegeggage gcgcatgcgc cacagtgcca gcgctctccc eggatagage

61 ggggeccgag cctgtccgct gtggtagttc cgctcgcgct gccccgccgc catgteggea

121 accatcgagc gggagttcga ggaactggat gctcagtgtc gctggcagcc gttatacttg

181 gaaattcgaa atgaatccca tgactatcct catagagtgg ccaagtttcc agaaaacaga 241 aaccgaaaca gatacagaga tgtaagecca tatgatcaca gtcgtgttaa actgeaaagt

301 actgaaaatg attatattaa tgecagctta gttgacatag aagaggcaca aagaagttac

361 atcttaacac agggcccact tccgaacaca tgetgecatt tctggctcat ggtgtggcag

421 caaaagacca aagcagttgt catgetaaac cgaactgtag aaaaagaatc ggttaaatgt

481 gcacagtact ggccaacgga tgacagagaa atggtgttta aggaaaeggg attcagtgtg 541 aagctcttat ctgaagatgt aaaatcatat tatacagtac atctactaca gttagaaaat

601 atcaatactg gtgaaaccag aaccatatct cacttccatt ataccacctg gecagatttt

661 ggggttccag agtcaccagc ttcatttcta aacttcttgt ttaaagttag agaatctggt

721 tgtttgaccc ctgaccatgg acctgeagtg atecattgea gtgegggcat cgggcgctct

781 ggcaccttct ctcttgtaga tacctgtctt gttctgatgg aaaaaggaga ggatgttaat 841 gtgaaacaat tattactgaa tatgagaaag tatcgaatgg gacttattca gacaccggac

901 caactcagat tctcctacat ggecataata gaaggagcaa agtacacaaa aggagattca

961 aatatacaga aacggtggaa agaactttct aaagaagatt tatctcctat ttgtgatcat

1021 tcacagaaca gagtgatggt tgagaagtac aatgggaaga gaataggttc agaagatgaa

1081 aagttaacag ggcttccttc taaggtgcag gatactgtgg aggagagcag tgagagcatt 1141 etaeggaaac gtattcgaga ggatagaaag getacgaegg ctcagaaggt gcagcagatg 1201 aaacagaggc taaatgaaac tgaacgaaaa agaaaaaggt ggttatattg gcaacctatt 1261 ctcactaaga tggggtttgt gtcagtcatt ttggttggcg ctttggttgg ctggacactg 1321 ctttttcact aaatgttcta taaattaata gttttaccca gcacctttct gcactagtag 1381 ctgaccgtgg tgcattaatc tcaagggttt gttagcaatg cctcataccc agaaacactg 1441 cgctagagta gacatcagcc agataaggga tattacagtc acaagcccag catctcagga 1501 ctcatcactg caggttcctc tgagacccag actgtcaatg gctcacaata aagacaagca 1561 tgcttgttgg atactgttac ttcttacagc tgcgttcaca ccagtgtatt gagaaatcct 1621 ttatcccaag gattggcttt tggaggcctt ctggatttta acctgcactt gatataagca 1681 ataaacattg tggttttttt ctacattatt aatggaaaga aaatatcctt taaacaatgt 1741 atgtaatatg taatgtactg ttgaaatggg cattacaact ttatataacc attttagggt 1801 aaatatattt tataagtacc tatttaatct tacttttgta gttaaatgta ctttttaaag 1861 gttcaatctg aaagtctgtt atcatagaaa aataaattgt atgttgactc agttgtatac 1921 tgaatacatt ttccctttcc taagcagacg tttgatagag gcagttgaaa ctataagcaa 1981 gctaagacta ctacacattc ttatttcctt tctatttatg ctttatctta ttttaaaaag 2041 aaaaacaaaa attttctaaa catgtcattg aaggaaattg tttttttctg cgatagttaa 2101 gaagtgacag ttggtcaaaa tatagttgaa aacaaacaaa aacttggttt ctgcaggatg 2161 tggtagcaca cacagtgctc aggaagctaa aacaagaggc tcaatggttt gaagccagcc 2221 aaaactacat agcaaggtcc tatctttaaa gataagagaa aaatagaggt ggtggaggag 2281 agatcagaca acaccaagaa taagaaatcg attcttagcc atatttaatg gacaaacctg 2341 tcatctcagc ttttgggaga tagaggcaga aggctcacaa gttcaaggcc agcttcaact 2401 acatagctag ccccagagtt tggggccagt caggactgca agaaacactg tctcagaaac 24 61 tgaagtggtt taaaaacatt ttgatttctg taaagtaaag cccatgcatg actacactgt 2521 taattttttg tgaaaatgta aatgtaatta cccagacggg ataaattatg gttagtaagt 2581 taaaggaacc agtgttttat acttttgatt tcagttcact agactgaaat tttgaagtaa 2641 aaaaaaattt aatttcttta caagttcaat aaatagtaca atggtgtaca aacttacatt 2701 gtcccttacc tttgtaatga gtatttttaa agcataacca ctaattgggt tttggtggtt 2761 tcaaaccctg cttggtggaa aggttccaaa ccattaggac agcattgctg cttcatctct 2821 tttatatatc acgtaaaagt gcgtggtaaa tcttaattag tttaaatgag acagttaatt 2881 tcttaatgca gtttgaaccc cataggtgta gttagaaatt gtgaatggcc ttgaaaagca 2941 tctcacaaag cgtatgatgt atgtgtgtgt cctgactcag catagctgtc ctaaggcttt 3001 gaaatggaga gcaggtaaga aggatgtttc ctcttgtctg tttaatctct gtttaagcgg 3061 aggccttaga attagatggc tatgggtttt gagctttcta acacttactg gtttgttttt 3121 ccaaaatgta gtatgttatc ctactagacc ttattaaaac ttacagtcca agccaataag 3181 gtggcgtaca cctttaatct caacactaag aacaccaaga cagacagatc cctgtgagtt 3241 caaggctagt ctggtccaca taataagttc ccaggcagcc agaaatagac attgagatcc 3301 tgtcttgaaa gaaagcaaac caactgaaga tagcctgagc ttaaacaact tcccacaaga 3361 aaaactgata aggctgagac cagtccttcc ttggacgata tgctttctag agatagcatt 3421 gagcaccact ctttctgcct cttggtgtgt attttatgtt tgtgaggatt cctttggcat 3481 acggaaccct cagtgctcct ccccggagcc cgtctttctc ccctgaacac atctttaagg 3541 atgagtttta acaggagaac ctttaagtca cactgtcatg ttgcttacta aaggtacatg 3601 gcctgtggtg acagtgtcac tggcatcatc ctgagcctgt atgagatgtg ctgtgctgat 3661 gagagaaggg tgctgggcag agaagggata ctagcagttt ctgatgggtt cacggcttta 3721 aacacagtgt gcgtcagtct cggtagcagc ttattttaac taatttagga ataatagttt 3781 gtcttggatc aaattctgtt ttttgtttgt ttgtttgttt tttgtttttg gtgtttggtt 3841 tttttttaat ttggggaaaa aataggcttt ttaaagggga ttattgttta ctggaaagaa 3901 tcctcacttc ctgtttcctc ccaccttgct gtaatgtcag tggtcacaag attcaccagg 3961 tactgtgtta tctcagcctc ctgatttcta tccatgctca aacctaaagt gtaaaagtac 4021 acattccttt ttaaaaatac gcatatgcat catttctacg ttcagcagaa tctacacatt 4081 tgtcaagttt tccacagttc tcagttcttt ttatccattc cgttatgtgt cacctcatgt 4141 atcaaacagt gaacataaaa agatatgaag acctgtatta attagttttt gtccaaacag 4201 ctgtgctctg aagctgcgtc agaggaaagg tcctaatttc tgagctcagc ttccatgcac 4261 tcggctcggc cctttgtctt aaagtaaagc tagtgctgtg agtttagaac tgtggcccac 4321 gtttcaagtt atgacacaga acagccctct ctggttgtca tttcatttcc ttgtttgctt 4381 ttagcaccag tcccagggtg ctggctccca ttttctgcca ggcacagaaa ggctacagct 4441 gactgcttta aaaatagctc tgcgtagatt ctgcagagaa gctggaacct aatggtagta 4501 aaagtacttt tttttggcca ttgtatacaa tctacttaac aagtttacat ttctgtcaag 4561 acattgcaga ctgaagatct acattgcctt aatttgttac ttactgatac aaatctttat 4 621 ttgtagttgt tgttttggat aggtttgtat attctttttt tttttttttt ttttttttgt 4 681 atgtgtgttg agatagtacc ttgccattgc ccaagcctgg ccttaaactc agctcaaacg 4741 actttcctac ctcagcctgt tgagtaacta acaccacagg tacacactgt gcacacagct 4801 ttcaagtata aatcttaaag agattatttt aaaactgtag ataagatttc aggcccttag 4861 tcaagcgtgg tgcatacctt ctctgagtag ggccatctct gggtcctggt gagtagtgtc 4 921 tatgtctgtg ggaaggaagg gctgctcggg gccttcatct ggctgagctc gattcatctg 4 981 ttcatagcat gggacaaaat accaacagaa atgtccattc tatttacatg ccaacaccta 5041 acaaagtctc ttatttttaa aactccttta tatggctttg ccatagaatc ttgtatatac 5101 tttttttttt ttttcaaaat agaaatgatt ttttttctca ttaaatttgt catcttatta 5161 cttgaaacgt gggcctttgt tattggcagt ggcttgctcc cgaggaggcc tgttctgtcc 5221 accctgtccc agaacgcact catttgagtc agatgccaca gttcttcctc acactggtct 5281 ttggtttata ccatgcagca ccatacctag agtcacagct gtctctaatt gtcccctgaa 5341 tatggaatga gagactcagg ctgtgccctc attcactgct gctctgcact ggagcctgtc 5401 cccaatcaga gaacttgcct cgtggccagc agtcttcctt cctgggtcct gagcagcttc 54 61 aagccttctg cattagtgct ttctcttagc cgtggctgtt gggaagaaga cccactgttc 5521 tccacaggtt gggttgtttt tttttttttt cctggctgtc cttgtcccag cacagtgcca 5581 tcagccattg tgagcagtgc ttaaagtgga aagctacacc agcctaagag gctttgtgta 5641 agctgacgtt taggatttaa agagcctgga ccatctgagt tctgactctg aagctctgct 5701 tggttgtaaa gttccagttg attctgagca gtgaggtgtg aggccactgt caccggtagg 5761 gtctgcttgg atgccgcctg ctttacttgg atctgttttg ttggggactg ctgcaaggag 5821 aattgcatgg gaattttctt ctttttcttt acagagactt ataagcatcg agttattctt 5881 tgtagtcact cattaggcat agtttttttt ttttaagacc catgatgctg ttgctattcc 5941 cccccccctt ttttttttgg ttttttgaga cagggtttct ctgtataact ctggctgtcc 6001 tggaactcac tttgtagacc tcaaactcag aaatccccct gcctttgcct cctgagtgct 6061 gggattaaag gcatgcgcca ccacggcccg gctgctgttg atattttaaa tgactatttt 6121 aaaaagtcgt tcagtgtgga aagttgagga gaggaagcct aggtaagttc ctttaaagca 6181 tgcttggctc acctcggtta gtcctgatca atctcagtcg gatgctaatg taaatgtcgt 6241 gtggcaaaac aacttttaat gcagtctgac tttccctcta acacgggcaa ggaagaagac 6301 accagcattt gcctctgcag cacagaggca gcccccagga tacccacgta gctcattgct 6361 tggtttgctc gcccatttta cttttgcctt attaaaaata aaatggtgaa gatccattca 6421 agtgaatata atagaattat ctcaaaagcc atttatctta atagtcttac aaataaagtc 6481 atttcttaga agctattcca ttgatttcct cttattttgc tacccctaaa cactatttga 6541 aaagaagtaa tgagtttcaa aaaccacagc gtgtctgtta aatggcaaat ttattattct 6601 tggtaaatgt gtatttaaca aacactagga aaggatatct cgtgtgtatg tgagagagaa 6661 agagagagtg cttcacaaca ctttaaataa tgccagccat attttcagat aagaaaccca 6721 gtggaggtgt gactcacgcc ttattttcca gcctgtgcag atagagctga gatgcagact 6781 ccaggctgtg gtttcagtcc ctccaaggct caggctcatt gtgctactcc actgtgtatt 6841 tacttaaacc agatgtttaa gcggggaaat agtagacacc ccactagtgg aggggtggaa 6901 tcccttttac aatgcttcac tgactatggc ggaccagaac gtttctgtgc caaagcccca 6961 cttcattcct ttctgttctg ttccacattc tgccagagtc agaaccagcc gtttggtccc 7021 aggtcctgcg acccattgct atctaaagag tatggttccc taatgagaac actgcagaga 7081 atcactgttg ggaaatcaaa caagactttg tagaccacca caggggcttg gtagatctgc 7141 ctgcctatgg agaaagaagc cagtagacag gaagaagctt cattctcatg gttggggagg 7201 agcctaagtg gtggagatct agtgtattgc ctgtttatac agtgataaag tcaagtattt 7261 tcatgggtag agagcgaggg tggaggaagg gaggggctgc gatcggtgca aaaatggaaa 7321 tacctttaat ctcccaaaag ctttgaccac tggcaaacaa ttgaaatatc agcaaagact 7381 actgctctta atggtcacac cctcttgttt aaatggcgtc cccctcccaa gcattaaatt 7441 gcgctgaact atcacagttt tacttagttc tagtagttat aatcattagc attctccttc 7501 aggagaaaat ctaaatgctg gaaatctaat tcagagataa caagccaact ttatgtgcaa 7561 actttatatt taaactgttt ctagcagtgt tacagtgatt gtccaaactg gattagactt 7621 ttgcgttgaa atcaaagtat gggtaagtct agcacatgta ataaaacctt gctgtttctt 7681 gtggctacat tttttttttt aacttgtctg tctcttagcc taccatgtag aggtcatttc 7741 ttgagttaag atgggatggc ctaaaagatt cagtgtgtag ttactgaaga agtaagtccc 7801 ggcgcctcag agcagtctgt ctcacagccc cgcttccatt tggaaacctg ccattctgga 7861 aggaagcact tggtgttctt ggaatgttca tgttggaatg atttttgttg ttgttgttgt 7921 tgttgacttt ttagttcagt cttagttctt ttgtgtttgt atctatctat gtacatctgt 7981 gtgtgtggtg gccatggatt gaatagatga cttcttattt tatgttttag gccaagattg 8041 acagacacct aaatgttcat gacttgagac tattctgcag ctataaaatt tgaacctttg 8101 atgtgcaaag caagacctga agcccactcc ggaaactaaa gtgaggcttg ctaaccctgt 8161 agattgcctc acaagttgtc tgtttacaaa gtaagctttc catccagggg atgaagaacg 8221 ccaccagcag aagacttgca aaccctttaa tttgatgtat tgttttttaa catgtgtatg 8281 aaatgtagaa agatgtaaag gaaataaatt aggagcgact actttgtatt gtactgccat 8341 tcctaatgta tttttatact ttttggcagc attaaatatt tttattaaat agactatgtt 8401 ggttaaaaaa aaaaaaaaaa aaa SEP ID NO: 12 Mouse PTPN2 isoform 1 Amino Acid Sequence (NP 001120649.1)

1 msatierefe eldaqcrwqp lyleirnesh dyphrvakfp enrnrnryrd vspydhsrvk 61 lqstendyin aslvdieeaq rsyiltqgpl pntcchfwlm vwqqkt kavv mlnrtve kes

121 vkcaqywptd dremvfketg fsvkll sedv ksyytvhllq lenintgetr tishfhyttw

181 pdfgvpespa s flnfl fkvr esgcltpdhg pavihcsagi grsgt fslvd tclvlme kge

241 dvnvkqllln mrkyrmgliq tpdqlrfsym aiiegakytk gdsniqkrwk els kedlspi

301 cdhsqnrvmv ekyngkrigs edekltglps kvqdtvees s esilrkrire drkattaqkv 361 qqmkqrlnet erkrkrwlyw qpiltkmgfv svilvgalvg wtll fh

SEP ID NO: 13 Mouse PTPN2 isoform 2 cDNA Sequence (NM 008977.3)

1 ggcggggcgg ggcgcggagc gcgcatgcgc cacagtgcca gcgctctccc cggatagagc 61 ggggcccgag cctgtccgct gtggtagttc cgctcgcgct gccccgccgc catgtcggca 121 accatcgagc gggagttcga ggaactggat gctcagtgtc gctggcagcc gttatacttg 181 gaaattcgaa atgaatccca tgactatcct catagagtgg ccaagtttcc agaaaacaga 241 aaccgaaaca gatacagaga tgtaagccca tatgatcaca gtcgtgttaa actgcaaagt 301 actgaaaatg attatattaa tgccagctta gttgacatag aagaggcaca aagaagttac 361 atcttaacac agggcccact tccgaacaca tgctgccatt tctggctcat ggtgtggcag 421 caaaagacca aagcagttgt catgctaaac cgaactgtag aaaaagaatc ggttaaatgt 481 gcacagtact ggccaacgga tgacagagaa atggtgttta aggaaacggg attcagtgtg 541 aagctcttat ctgaagatgt aaaatcatat tatacagtac atctactaca gttagaaaat 601 atcaatactg gtgaaaccag aaccatatct cacttccatt ataccacctg gccagatttt 661 ggggttccag agtcaccagc ttcatttcta aacttcttgt ttaaagttag agaatctggt 721 tgtttgaccc ctgaccatgg acctgcagtg atccattgca gtgcgggcat cgggcgctct 781 ggcaccttct ctcttgtaga tacctgtctt gttctgatgg aaaaaggaga ggatgttaat 841 gtgaaacaat tattactgaa tatgagaaag tatcgaatgg gacttattca gacaccggac 901 caactcagat tctcctacat ggccataata gaaggagcaa agtacacaaa aggagattca 961 aatatacaga aacggtggaa agaactttct aaagaagatt tatctcctat ttgtgatcat 1021 tcacagaaca gagtgatggt tgagaagtac aatgggaaga gaataggttc agaagatgaa 1081 aagttaacag ggcttccttc taaggtgcag gatactgtgg aggagagcag tgagagcatt 1141 ctacggaaac gtattcgaga ggatagaaag gctacgacgg ctcagaaggt gcagcagatg 1201 aaacagaggc taaatgaaac tgaacgaaaa agaaaaaggc caagattgac agacacctaa 1261 atgttcatga cttgagacta ttctgcagct ataaaatttg aacctttgat gtgcaaagca 1321 agacctgaag cccactccgg aaactaaagt gaggcttgct aaccctgtag attgcctcac 1381 aagttgtctg tttacaaagt aagctttcca tccaggggat gaagaacgcc accagcagaa 1441 gacttgcaaa ccctttaatt tgatgtattg ttttttaaca tgtgtatgaa atgtagaaag 1501 atgtaaagga aataaattag gagcgactac tttgtattgt actgccattc ctaatgtatt 1561 tttatacttt ttggcagcat taaatatttt tattaaatag actatgttgg ttaaaaaaaa 1621 aaaaaaaaaa a

SEP ID NO: 14 Mouse PTPN2 isoform 2 Amino Acid Sequence (NP 033003.1)

1 msatierefe eldaqcrwqp lyleirnesh dyphrvakfp enrnrnryrd vspydhsrvk

61 lqstendyin aslvdieeaq rsyiltqgpl pntcchfwlm vwqqkt kavv mlnrtve kes 121 vkcaqywptd dremvfketg fsvkll sedv ksyytvhllq lenintgetr tishfhyttw

181 pdfgvpespa s flnfl fkvr esgcltpdhg pavihcsagi grsgt fslvd tclvlme kge

241 dvnvkqllln mrkyrmgliq tpdqlrfsym aiiegakytk gdsniqkrwk els kedlspi

301 cdhsqnrvmv ekyngkrigs edekltglps kvqdtvees s esilrkrire drkattaqkv 361 qqmkqrlnet erkrkrprlt dt

SEP ID NO: 15 Human SOCS1 cDNA Sequence (NM 003745.1. CDS regions from 155 to 790)

1 ggcagctgca cggctcctgg ccccggagca tgcgcgagag ccgccccgga gcgccccgga

61 gccccccgcc gtcccgcccg cggcgtcccg cgccccgccg ccagcgcacc cccggacgct 121 atggcccacc cctccggctg gccccttctg taggatggta gcacacaacc aggtggcagc

181 cgacaatgca gtctccacag cagcagagcc ccgacggcgg ccagaacctt cctcctcttc

241 ctcctcctcg cccgcggccc ccgcgcgccc gcggccgtgc cccgcggtcc cggccccggc

301 ccccggcgac acgcacttcc gcacattccg ttcgcacgcc gattaccggc gcatcacgcg 361 cgccagcgcg ctcctggacg cctgcggatt ctactggggg cccctgagcg tgcacggggc 421 gcacgagcgg ctgcgcgccg agcccgtggg caccttcctg gtgcgcgaca gccgccagcg 481 gaactgcttt ttcgccctta gcgtgaagat ggcctcggga cccacgagca tccgcgtgca 541 ctttcaggcc ggccgctttc acctggatgg cagccgcgag agcttcgact gcctcttcga 601 gctgctggag cactacgtgg cggcgccgcg ccgcatgctg ggggccccgc tgcgccagcg 661 ccgcgtgcgg ccgctgcagg agctgtgccg ccagcgcatc gtggccaccg tgggccgcga 721 gaacctggct cgcatccccc tcaaccccgt cctccgcgac tacctgagct ccttcccctt 781 ccagatttga ccggcagcgc ccgccgtgca cgcagcatta actgggatgc cgtgttattt 841 tgttattact tgcctggaac catgtgggta ccctccccgg cctgggttgg agggagcgga 901 tgggtgtagg ggcgaggcgc ctcccgccct cggctggaga cgaggccgca gaccccttct 961 cacctcttga gggggtcctc cccctcctgg tgctccctct gggtccccct ggttgttgta 1021 gcagcttaac tgtatctgga gccaggacct gaactcgcac ctcctacctc ttcatgttta 1081 catataccca gtatctttgc acaaaccagg ggttggggga gggtctctgg ctttattttt 1141 ctgctgtgca gaatcctatt ttatattttt taaagtcagt ttaggtaata aactttatta 1201 tgaaagtttt tttttt

SEP ID NO: 16 Human SOCS1 Amino Acid Sequence (NP 003736.1)

1 mvahnqvaad navstaaepr rrpeps s s s s s spaaparpr pcpavpapap gdthfrt frs 61 hadyrritra salldacgfy wgpl svhgah erlraepvgt flvrdsrqrn cf falsvkma 121 sgptsirvhf qagrfhldgs res fdcl fel lehyvaaprr mlgaplrqrr vrplqelcrq 181 rivatvgren lariplnpvl rdyl s s fpfq i

SEP ID NO: 17 Mouse SOCS1 cDNA Sequence Transcript Varaint 1

(NM 001271603.1, CDS regions from 277 to 915)

1 agcagagaga actgcggccg tggcagcggc acggctccca gccccggagc atgcgcgaca

61 gccgccccgg agcccccagc cgcggctccc cgcgtcctgc cgccagatga gcccaccgag 121 gctcaagctc cgggcggatt ctgcgtgccg ctctcgctcc ttggggtctg ttggccggcc 181 tgtgccaccc ggacgcccgg ctcactgcct ctgtctcccc catcagcgca gccccggacg 241 ctatggccca cccctccagc tggcccctcg agtaggatgg tagcacgcaa ccaggtggca 301 gccgacaatg cgatctcccc ggcagcagag ccccgacggc ggtcagagcc ctcctcgtcc 361 tcgtcttcgt cctcgccagc ggcccccgtg cgtccccggc cctgcccggc ggtcccagcc 421 ccagcccctg gcgacactca cttccgcacc ttccgctccc actccgatta ccggcgcatc 481 acgcggacca gcgcgctcct ggacgcctgc ggcttctatt ggggacccct gagcgtgcac 541 ggggcgcacg agcggctgcg tgccgagccc gtgggcacct tcttggtgcg cgacagtcgc 601 caacggaact gcttcttcgc gctcagcgtg aagatggctt cgggccccac gagcatccgc 661 gtgcacttcc aggccggccg cttccacttg gacggcagcc gcgagacctt cgactgcctt 721 ttcgagctgc tggagcacta cgtggcggcg ccgcgccgca tgttgggggc cccgctgcgc 781 cagcgccgcg tgcggccgct gcaggagctg tgtcgccagc gcatcgtggc cgccgtgggt 841 cgcgagaacc tggcgcgcat ccctcttaac ccggtactcc gtgactacct gagttccttc 901 cccttccaga tctgaccggc tgccgctgtg ccgcagcatt aagtgggggc gccttattat 961 ttcttattat taattattat tatttttctg gaaccacgtg ggagccctcc ccgcctgggt 1021 cggagggagt ggttgtggag ggtgagatgc ctcccacttc tggctggaga cctcatccca 1081 cctctcaggg gtgggggtgc tcccctcctg gtgctccctc cgggtccccc ctggttgtag 1141 cagcttgtgt ctggggccag gacctgaatt ccactcctac ctctccatgt ttacatattc 1201 ccagtatctt tgcacaaacc aggggtcggg gagggtctct ggcttcattt ttctgctgtg 1261 cagaatatcc tattttatat ttttacagcc agtttaggta ataaacttta ttatgaaagt 1321 ttttttttaa aagaaacaaa

SEP ID NO: 18 Mouse SOCS1 cDNA Sequence Transcript Varaint 2 (NM 009896.2, CDS regions from 157 to 795)

1 agcagagaga actgcggccg tggcagcggc acggctccca gccccggagc atgcgcgaca 61 gccgccccgg agcccccagc cgcggctccc cgcgtcctgc cgccagcgca gccccggacg 121 ctatggccca cccctccagc tggcccctcg agtaggatgg tagcacgcaa ccaggtggca 181 gccgacaatg cgatctcccc ggcagcagag ccccgacggc ggtcagagcc ctcctcgtcc 241 tcgtcttcgt cctcgccagc ggcccccgtg cgtccccggc cctgcccggc ggtcccagcc 301 ccagcccctg gcgacactca cttccgcacc ttccgctccc actccgatta ccggcgcatc 361 acgcggacca gcgcgctcct ggacgcctgc ggcttctatt ggggacccct gagcgtgcac 421 ggggcgcacg agcggctgcg tgccgagccc gtgggcacct tcttggtgcg cgacagtcgc 481 caacggaact gcttcttcgc gctcagcgtg aagatggctt cgggccccac gagcatccgc 541 gtgcacttcc aggccggccg cttccacttg gacggcagcc gcgagacctt cgactgcctt 601 ttcgagctgc tggagcacta cgtggcggcg ccgcgccgca tgttgggggc cccgctgcgc 661 cagcgccgcg tgcggccgct gcaggagctg tgtcgccagc gcatcgtggc cgccgtgggt 721 cgcgagaacc tggcgcgcat ccctcttaac ccggtactcc gtgactacct gagttccttc 781 cccttccaga tctgaccggc tgccgctgtg ccgcagcatt aagtgggggc gccttattat 841 ttcttattat taattattat tatttttctg gaaccacgtg ggagccctcc ccgcctgggt 901 cggagggagt ggttgtggag ggtgagatgc ctcccacttc tggctggaga cctcatccca 961 cctctcaggg gtgggggtgc tcccctcctg gtgctccctc cgggtccccc ctggttgtag 1021 cagcttgtgt ctggggccag gacctgaatt ccactcctac ctctccatgt ttacatattc 1081 ccagtatctt tgcacaaacc aggggtcggg gagggtctct ggcttcattt ttctgctgtg 1141 cagaatatcc tattttatat ttttacagcc agtttaggta ataaacttta ttatgaaagt 1201 ttttttttaa aagaaacaaa

SEP ID NO; 19 Mouse SOCS1 Amino Acid Sequence (NP 001258532.1)

1 mvarnqvaad nai spaaepr rrseps s s s s s s spaapvrp rpcpavpapa pgdthfrt fr 61 shsdyrritr tsalldacgf ywgpl svhga herlraepvg tflvrdsrqr ncf falsvkm 121 asgptsirvh fqagrfhldg sretfdcl fe llehyvaapr rmlgaplrqr rvrplqelcr 181 qrivaavgre nlariplnpv lrdyl s s fpf qi

Summary of genes depleted or enriched in KQ mice with or without immunotherapies

Part I Genes depleted in wild type C57BL6 mice treated with both GVAX and anti-PD-1 antibody against the TCRa KO mice without treatment as control

- Ill -

Part II Genes depleted in wild type C57BL6 mice treated with GVAX against the TCRa KO mice without treatment as control

Part III Genes depleted in TCRa KO mice without treatment against the in vitro control p-value=0 means <3.7344e-05, FDR=0 means <0.0006

Part IV Genes enriched in wild type C57BL6 mice treated with both GVAX and anti-PD-1 antibody against the TCRa KO mice without treatment as control

Part V Genes enriched in TCRa KO mice treated with GVAX against the KO mice without treatment control

Part VI Genes enriched in TCRa KO mice without treatment against the in vitro controls p-value=0 means <3.7400e-05, FDR=0 means <0.000522423296101

* The nucleic acid and polypeptide sequences of the biomarkers of the present invention listed in Table 1 have been submitted at GenBank under the unique identifier provided herein and each such uniquely identified sequence submitted at GenBank is hereby incorporated in its entirely by reference.

* Included in Table 1 are RNA nucleic acid molecules (e.g., thymines replaced with uredines), nucleic acid molecules encoding orthologs of the encoded proteins, as well as DNA or RNA nucleic acid sequences comprising a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with the nucleic acid sequence of any SEQ ID NO listed in Table 1, or a portion thereof. Such nucleic acid molecules can have a function of the full-length nucleic acid as described further herein.

* Included in Table 1 are orthologs of the proteins, as well as polypeptide molecules comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or more identity across their full length with an amino acid sequence of any SEQ ID NO listed in Table 1 , or a portion thereof. Such polypeptides can have a function of the full-length polypeptide as described further herein. * Included in Table 1 are any known components of the inhibition of kinase signaling pathway, including PTPN2 and SOCS1, as well as orthologs of the pathway components and nucleic acid and amino acid variants having the recited homology described in the immediately preceding paragraphs and elsewhere herein. * Included in Table 1 are any known components of the activation of kinase signaling pathway. In some embodiments, their agonists are used to activate the kinase signaling pathway as described herein. For example, such agonists include any natural or non-natural agent prepared, synthesized, manufactured, and/or purified by human that is capable of increasing, promoting, enhancing, and/or inducing the biological ability of a biomarker in Table 1, which activates kinase signaling pathway (and its fragments, domains, and/or motifs thereof, discussed herein). In one embodiment, such agonists may increase or enhance the binding/interaction between a biomarker in Table 1 and its substrates or other binding partners. In another embodiment, such agonists may increase or enhance at least one biomarker functions. In still another embodiment, such agonists may decrease or inhibit the turnover rate, increase or enhance the expression and/or the stability (e.g., the half-life), and/or change the cellular localization of at least one biomarker in Table 1 , resulting in at least an increase in the biomarker levels and/or activity. Such agonists may be any molecule, including but not limited to small molecule compounds, antibodies or intrabodies, polypeptides or fusion proteins (which comprise, e.g., full-length biomarker, or biologically active fragments thereof, with or without any mutations or modifications to maintain or enhancing biomarker expression levels or biological functions. Such agonists may be specific to at least one biomarker or also enhance the copy number, amount, and/or activity of at least one of other proteins having a common domain/motif with the at least one biomarker. These embodiments can also be equally applied to other biomarkers of Table 1 that results in an sgRNA enrichment in the sgR A screens described herein.

* The nucleic acid and amino acid sequences corresponding to the reference ID numbers (e.g., Mouse Marker ID, Mouse NCBI RefSeq Transcript ID, Mouse NCBI RefSeq Protein ID, Human NCBI RefSeq Transcript ID, and Human NCBI RefSeq Protein ID) in Table 1 available to the public as of a particular date, e.g., the application filing date, is necessarily and readily confirmed using the public availability date shown in the Gene Locus header information for each ID on the NCBI website available at ncbi.nlm.nih.gov. II. Subjects

In one embodiment, the subject for whom predicted likelihood of efficacy of an inhibitor of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) and an immunotherapy combination treatment is determined, is a mammal (e.g., mouse, rat, primate, non-human mammal, domestic animal, such as a dog, cat, cow, horse, and the like), and is preferably a human. In another embodiment, the subject is an animal model of cancer. For example, the animal model can be an orthotopic xenograft animal model of a human-derived cancer.

In another embodiment of the methods of the present invention, the subject has not undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and/or immunotherapies. In still another embodiment, the subject has undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and/or immunotherapies.

In certain embodiments, the subject has had surgery to remove cancerous or precancerous tissue. In other embodiments, the cancerous tissue has not been removed, e.g., the cancerous tissue may be located in an inoperable region of the body, such as in a tissue that is essential for life, or in a region where a surgical procedure would cause considerable risk of harm to the patient.

The methods of the present invention can be used to determine the responsiveness to inhibitors of one or more biomarkers in Table 1 {e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCSl) and immunotherapy combination treatment of many different cancers in subjects such as those described herein.

III. Sample Collection, Preparation and Separation

In some embodiments, biomarker amount and/or activity measurement(s) in a sample from a subject is compared to a predetermined control (standard) sample. The sample from the subject is typically from a diseased tissue, such as cancer cells or tissues. The control sample can be from the same subject or from a different subject. The control sample is typically a normal, non-diseased sample. However, in some embodiments, such as for staging of disease or for evaluating the efficacy of treatment, the control sample can be from a diseased tissue. The control sample can be a combination of samples from several different subjects. In some embodiments, the biomarker amount and/or activity measurement(s) from a subject is compared to a pre-determined level. This pre-determined level is typically obtained from normal samples. As described herein, a "pre-determined" biomarker amount and/or activity measurement(s) may be a biomarker amount and/or activity measurement(s) used to, by way of example only, evaluate a subject that may be selected for treatment (e.g., based on the number of genomic mutations and/or the number of genomic mutations causing non-functional proteins for DNA repair genes), evaluate a response to an inhibitor of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCSl) and an immunotherapy combination treatment, and/or evaluate a response to such inhibitor and an immunotherapy combination treatment with one or more additional anti-cancer therapies. A pre-determined biomarker amount and/or activity measurement(s) may be determined in populations of patients with or without cancer. The pre-determined biomarker amount and/or activity measurement(s) can be a single number, equally applicable to every patient, or the pre-determined biomarker amount and/or activity measurement(s) can vary according to specific subpopulations of patients. Age, weight, height, and other factors of a subject may affect the pre-determined biomarker amount and/or activity measurement(s) of the individual. Furthermore, the pre-determined biomarker amount and/or activity can be determined for each subject individually. In one embodiment, the amounts determined and/or compared in a method described herein are based on absolute measurements.

In another embodiment, the amounts determined and/or compared in a method described herein are based on relative measurements, such as ratios (e.g., biomarker copy numbers, level, and/or activity before a treatment vs. after a treatment, such biomarker measurements relative to a spiked or man-made control, such biomarker measurements relative to the expression of a housekeeping gene, and the like). For example, the relative analysis can be based on the ratio of pre-treatment biomarker measurement as compared to post-treatment biomarker measurement. Pre-treatment biomarker measurement can be made at any time prior to initiation of anti-cancer therapy. Post-treatment biomarker measurement can be made at any time after initiation of anti-cancer therapy. In some embodiments, post-treatment biomarker measurements are made 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more after initiation of anti-cancer therapy, and even longer toward indefinitely for continued monitoring. Treatment can comprise anti-cancer therapy, such as a therapeutic regimen comprising one or more inhibitor of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) and immunotherapy combination treatment alone or in combination with other anti-cancer agents, such as with immune checkpoint inhibitors.

The pre-determined biomarker amount and/or activity measurement(s) can be any suitable standard. For example, the pre-determined biomarker amount and/or activity measurement(s) can be obtained from the same or a different human for whom a patient selection is being assessed. In one embodiment, the pre-determined biomarker amount and/or activity measurement(s) can be obtained from a previous assessment of the same patient. In such a manner, the progress of the selection of the patient can be monitored over time. In addition, the control can be obtained from an assessment of another human or multiple humans, e.g., selected groups of humans, if the subject is a human. In such a manner, the extent of the selection of the human for whom selection is being assessed can be compared to suitable other humans, e.g., other humans who are in a similar situation to the human of interest, such as those suffering from similar or the same condition(s) and/or of the same ethnic group.

In some embodiments of the present invention the change of biomarker amount and/or activity measurement(s) from the pre-determined level is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0 fold or greater, or any range in between, inclusive. Such cutoff values apply equally when the measurement is based on relative changes, such as based on the ratio of pre-treatment biomarker measurement as compared to post-treatment biomarker measurement.

Biological samples can be collected from a variety of sources from a patient including a body fluid sample, cell sample, or a tissue sample comprising nucleic acids and/or proteins. "Body fluids" refer to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g., amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit). In a preferred embodiment, the subject and/or control sample is selected from the group consisting of cells, cell lines, histological slides, paraffin embedded tissues, biopsies, whole blood, nipple aspirate, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow. In one embodiment, the sample is serum, plasma, or urine. In another

embodiment, the sample is serum.

The samples can be collected from individuals repeatedly over a longitudinal period of time (e.g., once or more on the order of days, weeks, months, annually, biannually, etc.). Obtaining numerous samples from an individual over a period of time can be used to verify results from earlier detections and/or to identify an alteration in biological pattern as a result of, for example, disease progression, drug treatment, etc. For example, subject samples can be taken and monitored every month, every two months, or combinations of one, two, or three month intervals according to the present invention. In addition, the biomarker amount and/or activity measurements of the subject obtained over time can be conveniently compared with each other, as well as with those of normal controls during the monitoring period, thereby providing the subject's own values, as an internal, or personal, control for long-term monitoring.

Sample preparation and separation can involve any of the procedures, depending on the type of sample collected and/or analysis of biomarker measurement(s). Such procedures include, by way of example only, concentration, dilution, adjustment of pH, removal of high abundance polypeptides (e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives and calibrants, addition of protease inhibitors, addition of denaturants, desalting of samples, concentration of sample proteins, extraction and purification of lipids.

The sample preparation can also isolate molecules that are bound in non-covalent complexes to other protein (e.g., carrier proteins). This process may isolate those molecules bound to a specific carrier protein (e.g. , albumin), or use a more general process, such as the release of bound molecules from all carrier proteins via protein denaturation, for example using an acid, followed by removal of the carrier proteins.

Removal of undesired proteins (e.g., high abundance, uninformative, or

undetectable proteins) from a sample can be achieved using high affinity reagents, high molecular weight filters, ultracentrifugation and/or electrodialysis. High affinity reagents include antibodies or other reagents (e.g., aptamers) that selectively bind to high abundance proteins. Sample preparation could also include ion exchange chromatography, metal ion affinity chromatography, gel filtration, hydrophobic chromatography, chromatofocusing, adsorption chromatography, isoelectric focusing and related techniques. Molecular weight filters include membranes that separate molecules on the basis of size and molecular weight. Such filters may further employ reverse osmosis, nanofiltration, ultrafiltration and microfiltration.

Ultracentrifugation is a method for removing undesired polypeptides from a sample.

Ultracentrifugation is the centrifugation of a sample at about 15,000-60,000 rpm while monitoring with an optical system the sedimentation (or lack thereof) of particles.

Electrodialysis is a procedure which uses an electromembrane or semipermable membrane in a process in which ions are transported through semi-permeable membranes from one solution to another under the influence of a potential gradient. Since the membranes used in electrodialysis may have the ability to selectively transport ions having positive or negative charge, reject ions of the opposite charge, or to allow species to migrate through a semipermable membrane based on size and charge, it renders electrodialysis useful for concentration, removal, or separation of electrolytes.

Separation and purification in the present invention may include any procedure known in the art, such as capillary electrophoresis (e.g., in capillary or on-chip) or chromatography (e.g. , in capillary, column or on a chip). Electrophoresis is a method which can be used to separate ionic molecules under the influence of an electric field.

Electrophoresis can be conducted in a gel, capillary, or in a microchannel on a chip.

Examples of gels used for electrophoresis include starch, acrylamide, polyethylene oxides, agarose, or combinations thereof. A gel can be modified by its cross-linking, addition of detergents, or denaturants, immobilization of enzymes or antibodies (affinity

electrophoresis) or substrates (zymography) and incorporation of a pH gradient. Examples of capillaries used for electrophoresis include capillaries that interface with an electrospray.

Capillary electrophoresis (CE) is preferred for separating complex hydrophilic molecules and highly charged solutes. CE technology can also be implemented on microfluidic chips. Depending on the types of capillary and buffers used, CE can be further segmented into separation techniques such as capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), capillary isotachophoresis (cITP) and capillary electrochromatography (CEC). An embodiment to couple CE techniques to electrospray ionization involves the use of volatile solutions, for example, aqueous mixtures containing a volatile acid and/or base and an organic such as an alcohol or acetonitrile.

Capillary isotachophoresis (cITP) is a technique in which the analytes move through the capillary at a constant speed but are nevertheless separated by their respective mobilities. Capillary zone electrophoresis (CZE), also known as free-solution CE (FSCE), is based on differences in the electrophoretic mobility of the species, determined by the charge on the molecule, and the frictional resistance the molecule encounters during migration which is often directly proportional to the size of the molecule. Capillary isoelectric focusing (CIEF) allows weakly-ionizable amphoteric molecules, to be separated by electrophoresis in a pH gradient. CEC is a hybrid technique between traditional high performance liquid chromatography (HPLC) and CE.

Separation and purification techniques used in the present invention include any chromatography procedures known in the art. Chromatography can be based on the differential adsorption and elution of certain analytes or partitioning of analytes between mobile and stationary phases. Different examples of chromatography include, but not limited to, liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (HPLC), etc.

IV. Biomarker Nucleic Acids and Polypeptides

One aspect of the present invention pertains to the use of isolated nucleic acid molecules that correspond to biomarker nucleic acids that encode a biomarker polypeptide or a portion of such a polypeptide. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double- stranded DNA.

An "isolated" nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid molecule.

Preferably, an "isolated" nucleic acid molecule is free of sequences (preferably protein- encoding sequences) which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kB, 4 kB, 3 kB, 2 kB, 1 kB, 0.5 kB or 0.1 kB of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

A biomarker nucleic acid molecule of the present invention can be isolated using standard molecular biology techniques and the sequence information in the database records described herein. Using all or a portion of such nucleic acid sequences, nucleic acid molecules of the present invention can be isolated using standard hybridization and cloning techniques (e.g. , as described in Sambrook et al. , ed., Molecular Cloning: A

Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

A nucleic acid molecule of the present invention can be amplified using cDNA, mRNA, or genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid molecules so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.

Furthermore, oligonucleotides corresponding to all or a portion of a nucleic acid molecule of the present invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

Moreover, a nucleic acid molecule of the present invention can comprise only a portion of a nucleic acid sequence, wherein the full length nucleic acid sequence comprises a marker of the present invention or which encodes a polypeptide corresponding to a marker of the present invention. Such nucleic acid molecules can be used, for example, as a probe or primer. The probe/primer typically is used as one or more substantially purified oligonucleotides. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 7, preferably about 15, more preferably about 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 or more consecutive nucleotides of a biomarker nucleic acid sequence. Probes based on the sequence of a biomarker nucleic acid molecule can be used to detect transcripts or genomic sequences corresponding to one or more markers of the present invention. The probe comprises a label group attached thereto, e.g., a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.

A biomarker nucleic acid molecules that differ, due to degeneracy of the genetic code, from the nucleotide sequence of nucleic acid molecules encoding a protein which corresponds to the biomarker, and thus encode the same protein, are also contemplated.

In addition, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequence can exist within a population (e.g. , the human population). Such genetic polymorphisms can exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes which occur alternatively at a given genetic locus. In addition, it will be appreciated that DNA polymorphisms that affect RNA expression levels can also exist that may affect the overall expression level of that gene (e.g., by affecting regulation or degradation).

The term "allele," which is used interchangeably herein with "allelic variant," refers to alternative forms of a gene or portions thereof. Alleles occupy the same locus or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be homozygous for the gene or allele. When a subject has two different alleles of a gene, the subject is said to be heterozygous for the gene or allele. For example, biomarker alleles can differ from each other in a single nucleotide, or several nucleotides, and can include substitutions, deletions, and insertions of nucleotides. An allele of a gene can also be a form of a gene containing one or more mutations.

The term "allelic variant of a polymorphic region of gene" or "allelic variant", used interchangeably herein, refers to an alternative form of a gene having one of several possible nucleotide sequences found in that region of the gene in the population. As used herein, allelic variant is meant to encompass functional allelic variants, non-functional allelic variants, SNPs, mutations and polymorphisms. The term "single nucleotide polymorphism" (SNP) refers to a polymorphic site occupied by a single nucleotide, which is the site of variation between allelic sequences. The site is usually preceded by and followed by highly conserved sequences of the allele (e.g., sequences that vary in less than 1/100 or 1/1000 members of a population). A SNP usually arises due to substitution of one nucleotide for another at the polymorphic site. SNPs can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele. Typically the polymorphic site is occupied by a base other than the reference base. For example, where the reference allele contains the base "T" (thymidine) at the polymorphic site, the altered allele can contain a "C" (cytidine), "G" (guanine), or "A" (adenine) at the polymorphic site. SNP's may occur in protein-coding nucleic acid sequences, in which case they may give rise to a defective or otherwise variant protein, or genetic disease. Such a SNP may alter the coding sequence of the gene and therefore specify another amino acid (a "missense" SNP) or a SNP may introduce a stop codon (a "nonsense" SNP). When a SNP does not alter the amino acid sequence of a protein, the SNP is called "silent." SNP's may also occur in noncoding regions of the nucleotide sequence. This may result in defective protein expression, e.g., as a result of alternative spicing, or it may have no effect on the function of the protein.

As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame encoding a polypeptide corresponding to a marker of the present invention. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of a given gene. Alternative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. Any and all such nucleotide variations and resulting amino acid

polymorphisms or variations that are the result of natural allelic variation and that do not alter the functional activity are intended to be within the scope of the present invention.

In another embodiment, a biomarker nucleic acid molecule is at least 7, 15, 20, 25, 30, 40, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 550, 650, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2400, 2600, 2800, 3000, 3500, 4000, 4500, or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule corresponding to a marker of the present invention or to a nucleic acid molecule encoding a protein corresponding to a marker of the present invention. As used herein, the term "hybridizes under stringent conditions" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% (65%, 70%, 75%, 80%, preferably 85%) identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in sections 6.3.1-6.3.6 of Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989). A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 50-65°C.

In addition to naturally-occurring allelic variants of a nucleic acid molecule of the present invention that can exist in the population, the skilled artisan will further appreciate that sequence changes can be introduced by mutation thereby leading to changes in the amino acid sequence of the encoded protein, without altering the biological activity of the protein encoded thereby. For example, one can make nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity. For example, amino acid residues that are not conserved or only semi-conserved among homologs of various species may be non-essential for activity and thus would be likely targets for alteration. Alternatively, amino acid residues that are conserved among the homologs of various species (e.g., murine and human) may be essential for activity and thus would not be likely targets for alteration.

Accordingly, another aspect of the present invention pertains to nucleic acid molecules encoding a polypeptide of the present invention that contain changes in amino acid residues that are not essential for activity. Such polypeptides differ in amino acid sequence from the naturally-occurring proteins which correspond to the markers of the present invention, yet retain biological activity. In one embodiment, a biomarker protein has an amino acid sequence that is at least about 40% identical, 50%, 60%, 70%, 75%, 80%, 83%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or identical to the amino acid sequence of a biomarker protein described herein.

An isolated nucleic acid molecule encoding a variant protein can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of nucleic acids of the present invention, such that one or more amino acid residue substitutions, additions, or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

In some embodiments, the present invention further contemplates the use of anti- biomarker antisense nucleic acid molecules, i.e., molecules which are complementary to a sense nucleic acid of the present invention, e.g., complementary to the coding strand of a double-stranded cDNA molecule corresponding to a marker of the present invention or complementary to an mRNA sequence corresponding to a marker of the present invention. Accordingly, an antisense nucleic acid molecule of the present invention can hydrogen bond to (i.e. anneal with) a sense nucleic acid of the present invention. The antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can also be antisense to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding a polypeptide of the present invention. The non- coding regions ("5' and 3' untranslated regions") are the 5' and 3' sequences which flank the coding region and are not translated into amino acids.

An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 or more nucleotides in length. An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-(carboxyhydroxylmethyi) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,

2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5 -methoxy uracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxy acetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-

3- N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been sub-cloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

The antisense nucleic acid molecules of the present invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a polypeptide corresponding to a selected marker of the present invention to thereby inhibit expression of the marker, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. Examples of a route of administration of antisense nucleic acid molecules of the present invention includes direct injection at a tissue site or infusion of the antisense nucleic acid into a blood- or bone marrow-associated body fluid. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

An antisense nucleic acid molecule of the present invention can be an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual a-units, the strands run parallel to each other (Gaultier et al, 1987, Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al, 1987, Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al, 1987, FEBSLett. 215:327-330).

The present invention also encompasses ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes as described in Haselhoff and Gerlach, 1988, Nature 334:585-591) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. A ribozyme having specificity for a nucleic acid molecule encoding a polypeptide corresponding to a marker of the present invention can be designed based upon the nucleotide sequence of a cDNA corresponding to the marker. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved (see Cech et al. U.S. Patent No. 4,987,071; and Cech et al. U.S. Patent No. 5,116,742). Alternatively, an mRNA encoding a polypeptide of the present invention can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (see, e.g., Bartel and Szostak, 1993, Science 261:1411-1418).

The present invention also encompasses nucleic acid molecules which form triple helical structures. For example, expression of a biomarker protein can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the polypeptide (e.g., the promoter and/or enhancer) to form triple helical structures that prevent transcription of the gene in target cells. See generally Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992) Ann. N. Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays 14(12):807-15. In various embodiments, the nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acid molecules (see Hyrup et al., 1996, Bioorganic & Medicinal Chemistry 4(1): 5- 23). As used herein, the terms "peptide nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g. , DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup et al. (1996), supra; Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675.

PNAs can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g. , inducing transcription or translation arrest or inhibiting replication.

PNAs can also be used, e.g., in the analysis of single base pair mutations in a gene by, e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., SI nucleases (Hyrup (1996), supra; or as probes or primers for DNA sequence and hybridization (Hyrup, 1996, supra; Perry-O'Keefe et al, 1996, Proc. Natl. Acad. Sci. USA 93:14670-675).

In another embodiment, PNAs can be modified, e.g., to enhance their stability or cellular uptake, by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras can be generated which can combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, e.g., RNASE H and DNA polymerases, to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup, 1996, supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup (1996), supra, and Finn et al. (1996) Nucleic Acids Res. 24(17):3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs. Compounds such as 5'-(4-methoxytrityl)amino-5'-deoxy- thymidine phosphoramidite can be used as a link between the PNA and the 5' end of DNA (Mag et al, 1989, Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled in a step-wise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al, 1996, Nucleic Acids Res. 24(17):3357-63). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser et al, \975, Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide can include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al, 1987, Proc. Natl Acad. Sci. USA 84:648-652; PCT

Publication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (see, e.g., Krol et al, 1988, Bio/Techniques 6:958-976) or intercalating agents (see, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, the oligonucleotide can be conjugated to another molecule, e.g. , a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

Another aspect of the present invention pertains to the use of biomarker proteins and biologically active portions thereof. In one embodiment, the native polypeptide

corresponding to a marker can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, polypeptides corresponding to a marker of the present invention are produced by

recombinant DNA techniques. Alternative to recombinant expression, a polypeptide corresponding to a marker of the present invention can be synthesized chemically using standard peptide synthesis techniques.

An "isolated" or "purified" protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. The language "substantially free of cellular material" includes preparations of protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of heterologous protein (also referred to herein as a "contaminating protein"). When the protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, 10%, or 5% of the volume of the protein preparation. When the protein is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. Accordingly such preparations of the protein have less than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors or compounds other than the polypeptide of interest.

Biologically active portions of a biomarker polypeptide include polypeptides comprising amino acid sequences sufficiently identical to or derived from a biomarker protein amino acid sequence described herein, but which includes fewer amino acids than the full length protein, and exhibit at least one activity of the corresponding full-length protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the corresponding protein. A biologically active portion of a protein of the present invention can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of the native form of a polypeptide of the present invention.

Preferred polypeptides have an amino acid sequence of a biomarker protein encoded by a nucleic acid molecule described herein. Other useful proteins are substantially identical {e.g., at least about 40%, preferably 50%, 60%, 70%, 75%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to one of these sequences and retain the functional activity of the protein of the corresponding naturally-occurring protein yet differ in amino acid sequence due to natural allelic variation or mutagenesis.

To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = # of identical positions/total # of positions (e.g., overlapping positions) xlOO). In one embodiment the two sequences are the same length.

The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990) J Mol. Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to a protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, (1988) ComputAppl Biosci, 4:11-7. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Yet another useful algorithm for identifying regions of local sequence similarity and alignment is the FASTA algorithm as described in Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444-2448. When using the FASTA algorithm for comparing nucleotide or amino acid sequences, a PAM120 weight residue table can, for example, be used with a &-tuple value of 2.

The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, only exact matches are counted. The present invention also provides chimeric or fusion proteins corresponding to a biomarker protein. As used herein, a "chimeric protein" or "fusion protein" comprises all or part (preferably a biologically active part) of a polypeptide corresponding to a marker of the present invention operably linked to a heterologous polypeptide (i.e., a polypeptide other than the polypeptide corresponding to the marker). Within the fusion protein, the term "operably linked" is intended to indicate that the polypeptide of the present invention and the heterologous polypeptide are fused in-frame to each other. The heterologous polypeptide can be fused to the amino-terminus or the carboxyl-terminus of the polypeptide of the present invention.

One useful fusion protein is a GST fusion protein in which a polypeptide corresponding to a marker of the present invention is fused to the carboxyl terminus of GST sequences. Such fusion proteins can facilitate the purification of a recombinant polypeptide of the present invention.

In another embodiment, the fusion protein contains a heterologous signal sequence, immunoglobulin fusion protein, toxin, or other useful protein sequence. Chimeric and fusion proteins of the present invention can be produced by standard recombinant DNA techniques. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and re-amplified to generate a chimeric gene sequence (see, e.g., Ausubel et al, supra). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A nucleic acid encoding a polypeptide of the present invention can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the polypeptide of the present invention.

A signal sequence can be used to facilitate secretion and isolation of the secreted protein or other proteins of interest. Signal sequences are typically characterized by a core of hydrophobic amino acids which are generally cleaved from the mature protein during secretion in one or more cleavage events. Such signal peptides contain processing sites that allow cleavage of the signal sequence from the mature proteins as they pass through the secretory pathway. Thus, the present invention pertains to the described polypeptides having a signal sequence, as well as to polypeptides from which the signal sequence has been proteolytically cleaved (i.e., the cleavage products). In one embodiment, a nucleic acid sequence encoding a signal sequence can be operably linked in an expression vector to a protein of interest, such as a protein which is ordinarily not secreted or is otherwise difficult to isolate. The signal sequence directs secretion of the protein, such as from a eukaryotic host into which the expression vector is transformed, and the signal sequence is subsequently or concurrently cleaved. The protein can then be readily purified from the extracellular medium by art recognized methods. Alternatively, the signal sequence can be linked to the protein of interest using a sequence which facilitates purification, such as with a GST domain.

The present invention also pertains to variants of the biomarker polypeptides described herein. Such variants have an altered amino acid sequence which can function as either agonists (mimetics) or as antagonists. Variants can be generated by mutagenesis, e.g., discrete point mutation or truncation. An agonist can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of the protein. An antagonist of a protein can inhibit one or more of the activities of the naturally occurring form of the protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the protein of interest. Thus, specific biological effects can be elicited by treatment with a variant of limited function. Treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein can have fewer side effects in a subject relative to treatment with the naturally occurring form of the protein.

Variants of a biomarker protein which function as either agonists (mimetics) or as antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of the protein of the present invention for agonist or antagonist activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential protein sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display). There are a variety of methods which can be used to produce libraries of potential variants of the polypeptides of the present invention from a degenerate oligonucleotide sequence. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, 1983, Tetrahedron 39:3; Itakura et al, 1984, Annu. Rev. Biochem. 53:323; Itakura et al., 1984, Science 198:1056; Ike et al, 1983 Nucleic Acid Res. 11:477).

In addition, libraries of fragments of the coding sequence of a polypeptide corresponding to a marker of the present invention can be used to generate a variegated population of polypeptides for screening and subsequent selection of variants. For example, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of the coding sequence of interest with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes amino terminal and internal fragments of various sizes of the protein of interest.

Several techniques are known in the art for screening gene products of

combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. The most widely used techniques, which are amenable to high throughput analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of a protein of the present invention (Arkin and Yourvan, 1992, Proc. Natl. Acad. Sci. USA 59:7811-7815; Delgrave et al, 1993, Protein Engineering 6(3):327- 331).

An isolated polypeptide or a fragment thereof (or a nucleic acid encoding such a polypeptide) corresponding to one or more biomarkers of the invention, including the biomarkers listed in Table 1 or fragments thereof, can be used as an immunogen to generate antibodies that bind to said immunogen, using standard techniques for polyclonal and monoclonal antibody preparation according to well-known methods in the art. An antigenic peptide comprises at least 8 amino acid residues and encompasses an epitope present in the respective full length molecule such that an antibody raised against the peptide forms a specific immune complex with the respective full length molecule. Preferably, the antigenic peptide comprises at least 10 amino acid residues. In one embodiment such epitopes can be specific for a given polypeptide molecule from one species, such as mouse or human (i.e., an antigenic peptide that spans a region of the polypeptide molecule that is not conserved across species is used as immunogen; such non conserved residues can be determined using an alignment such as that provided herein).

In some embodiments, the immunotherapy utilizes an inhibitor of at least one immune checkpoint, such as an antibody binds substantially specifically to an immune checkpoint, such as PD-1, and inhibits or blocks its immunoinhibitory function, such as by interrupting its interaction with a binding partner of the immune checkpoint, such as PD-Ll and/or PD-L2 binding partners of PD-1. In one embodiment, an antibody, especially an intrbody, binds substantially specifically to one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) and inhibits or blocks its biological function, such as by interrupting its interaction with a substrate like STAT or JAK proteins. In another embodiment, an antibody, especially an intrbody, binds substantially specifically to a binding partner of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1), such as PTPN2 and/or SOCS1 substrates described herein, and inhibits or blocks its biological function, such as by interrupting its interaction to PTPN2 and/or SOCS1.

For example, a polypeptide immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g. , rabbit, goat, mouse or other mammal) with the immunogen. A preferred animal is a mouse deficeint in the desired target antigen. For example, a PD-1 knockout mouse if the desired antibody is an anti-PD-1 antibody, may be used. This results in a wider spectrum of antibody recognition possibilities as antibodies reactive to common mouse and human epitopes are not removed by tolerance mechanisms. An appropriate immunogenic preparation can contain, for example, a recombinantly expressed or chemically synthesized molecule or fragment thereof to which the immune response is to be generated. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.

Immunization of a suitable subject with an immunogenic preparation induces a polyclonal antibody response to the antigenic peptide contained therein.

Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide immunogen. The polypeptide antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, the antibody directed against the antigen can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A chromatography, to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique (originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also Brown et al. (1981) J Immunol. 127:539-46; Brown et al. (1980) J Biol. Chem. 255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. 76:2927-31; Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique (Cole et al. (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well-known (see generally Kenneth, R. H. in

Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York (1980); Lerner, E. A. (1981) Yale J. Biol. Med. 54:387-402; Gefter, M. L. et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds to the polypeptide antigen, preferably specifically. In some

embodiments, the immunization is performed in a cell or animal host that has a knockout of a target antigen of interest (e.g., does not produce the antigen prior to immunization).

Any of the many well-known protocols used for fusing lymphocytes and

immortalized cell lines can be applied for the purpose of generating a monoclonal antibody against one or more biomarkers of the invention, including the biomarkers listed in Table 1, or a fragment thereof (see, e.g., Galfre, G. et al. (1977) Nature 266:55052; Gefter et al. (1977) supra; Lerner (1981) supra; Kenneth (1980) supra). Moreover, the ordinary skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium"). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3- x63-Ag8.653 or Sp2/0-Agl4 myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC), Rockville, MD. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind a given polypeptide, e.g. , using a standard ELIS A assay.

As an alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal specific for one of the above described polypeptides can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with the appropriate polypeptide to thereby isolate immunoglobulin library members that bind the polypeptide. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia

Recombinant Phage Antibody System, Catalog No. 27-9400-01 ; and the Stratagene

SurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening an antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271; Winter et al. International Publication WO 92/20791; Markland et al.

International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al. International Publication No. WO 92/01047; Garrard et al. International Publication No. WO 92/09690; Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Biotechnology (NY) 9:1369-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J Mol. Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576- 3580; Garrard et al. (1991) Biotechnology (NY) 9:1373-1377; Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978- 7982; and McCafferty et al. (1990) Nature 348:552-554. Since it is well-known in the art that antibody heavy and light chain CDR3 domains play a particularly important role in the binding specificity/affinity of an antibody for an antigen, the recombinant monoclonal antibodies of the present invention prepared as set forth above preferably comprise the heavy and light chain CDR3s of variable regions of the antibodies described herein and well-known in the art. Similarly, the antibodies can further comprise the CDR2s of variable regions of said antibodies. The antibodies can further comprise the CDRls of variable regions of said antibodies. In other embodiments, the antibodies can comprise any combinations of the CDRs.

The CDR1, 2, and/or 3 regions of the engineered antibodies described above can comprise the exact amino acid sequence(s) as those of variable regions of the present invention described herein. However, the ordinarily skilled artisan will appreciate that some deviation from the exact CDR sequences may be possible while still retaining the ability of the antibody, especially an introbody, to bind a desired target, such as one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS 1) and/or a binding partner thereof, either alone or in combination with an

immunotherapy, such as PTPN2 and SOCS1, PTPN2/SOCS1 binding partners/substrates, or an immunotherapy effectively (e.g., conservative sequence modifications). Accordingly, in another embodiment, the engineered antibody may be composed of one or more CDRs that are, for example, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical to one or more CDRs of the present invention described herein or otherwise publicly available.

For example, the structural features of non-human or human antibodies (e.g., a rat anti-mouse/anti-human antibody) can be used to create structurally related human antibodies, especially introbodies, that retain at least one functional property of the antibodies of the present invention, such as binding to PTPN2 and/or SOCS1,

PTPN2/SOCS1 binding partners/substrates, and/or an immune checkpoint. Another functional property includes inhibiting binding of the original known, non-human or human antibodies in a competition ELISA assay.

Antibodies, immunoglobulins, and polypeptides of the invention can be used in an isolated (e.g. , purified) form or contained in a vector, such as a membrane or lipid vesicle (e.g. a liposome). Moreover, amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. It is known that when a humanized antibody is produced by simply grafting only CDRs in VH and VL of an antibody derived from a non-human animal in FRs of the VH and VL of a human antibody, the antigen binding activity is reduced in comparison with that of the original antibody derived from a non-human animal. It is considered that several amino acid residues of the VH and VL of the non-human antibody, not only in CDRs but also in FRs, are directly or indirectly associated with the antigen binding activity. Hence, substitution of these amino acid residues with different amino acid residues derived from FRs of the VH and VL of the human antibody would reduce binding activity and can be corrected by replacing the amino acids with amino acid residues of the original antibody derived from a non-human animal.

Similarly, modifications and changes may be made in the structure of the antibodies described herein, and in the DNA sequences encoding them, and still obtain a functional molecule that encodes an antibody and polypeptide with desirable characteristics. For example, antibody glycosylation patterns can be modulated to, for example, increase stability. By "altering" is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody. Glycosylation of antibodies is typically N-linked. "N-linked" refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagines-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked

glycosylation sites). Another type of covalent modification involves chemically or enzymatically coupling glycosides to the antibody. These procedures are advantageous in that they do not require production of the antibody in a host cell that has glycosylation capabilities for N- or O-linked glycosylation. Depending on the coupling mode used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, orhydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. For example, such methods are described in WO87/05330. Similarly, removal of any carbohydrate moieties present on the antibody may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N- acetylglucosamine or N-acetylgalactosamine), while leaving the antibody intact. Chemical deglycosylation is described by Sojahr et al. (1987) and by Edge et al. (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al. (1987).

Other modifications can involve the formation of immunoconjugates. For example, in one type of covalent modification, antibodies or proteins are covalently linked to one of a variety of non proteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

Conjugation of antibodies or other proteins of the present invention with

heterologous agents can be made using a variety of bifunctional protein coupling agents including but not limited to N-succinimidyl (2-pyridyldithio) propionate (SPDP), succinimidyl (N-maleimidomethyl)cyclohexane-l-carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido

compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene

2,6diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2,4- dinitrobenzene). For example, carbon labeled 1-isothiocyanatobenzyl methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody (WO 94/11026).

In another aspect, the present invention features antibodies conjugated to a therapeutic moiety, such as a cytotoxin, a drug, and/or a radioisotope. When conjugated to a cytotoxin, these antibody conjugates are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to {e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). An antibody of the present invention can be conjugated to a radioisotope, e.g., radioactive iodine, to generate cytotoxic radiopharmaceuticals for treating a related disorder, such as a cancer.

Conjugated antibodies, in addition to therapeutic utility, can be useful for diagnostically or prognostically to monitor polypeptide levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (/ ^' e. , physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin (PE); an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include ¹²⁵ 1, ¹³¹ I, ³⁵ S, or ³ H. [0134] As used herein, the term "labeled", with regard to the antibody, is intended to encompass direct labeling of the antibody by coupling (i.e., physically linking) a detectable substance, such as a radioactive agent or a fluorophore (e.g.

fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or Indocyanine (Cy5)) to the antibody, as well as indirect labeling of the antibody by reactivity with a detectable substance.

The antibody conjugates of the present invention can be used to modify a given biological response. The therapeutic moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-.gamma.; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other cytokines or growth factors.

In one embodiment, an antibody for use in the instant invention is a bispecific or multispecific antibody. A bispecific antibody has binding sites for two different antigens within a single antibody polypeptide. Antigen binding may be simultaneous or sequential. Triomas and hybrid hybridomas are two examples of cell lines that can secrete bispecific antibodies. Examples of bispecific antibodies produced by a hybrid hybridoma or a trioma are disclosed in U.S. Patent 4,474,893. Bispecific antibodies have been constructed by chemical means (Staerz et al. (1985) Nature 314:628, and Perez et al. (1985) Nature 316:354) and hybridoma technology (Staerz and Bevan (1986) Proc. Natl. Acad. Set USA, 83:1453, and Staerz and Bevan (1986) Immunol. Today 7:241). Bispecific antibodies are also described in U.S. Patent 5,959,084. Fragments of bispecific antibodies are described in U.S. Patent 5,798,229.

Bispecific agents can also be generated by making heterohybridomas by fusing hybridomas or other cells making different antibodies, followed by identification of clones producing and co-assembling both antibodies. They can also be generated by chemical or genetic conjugation of complete immunoglobulin chains or portions thereof such as Fab and Fv sequences. The antibody component can bind to a polypeptide or a fragment thereof of one or more biomarkers of the invention, including one or more biomarkers listed in Table 1, or a fragment thereof. In one embodiment, the bispecific antibody could specifically bind to both a polypeptide or a fragment thereof and its natural binding partner(s) or a fragment(s) thereof.

Techniques for modulating antibodies, such as humanization, conjugation, recombinant techniques, and the like are well-known in the art.

In another aspect of this invention, peptides or peptide mimetics can be used to antagonize the activity of one or more biomarkers of the invention, including one or more biomarkers listed in Table 1, or a fragment(s) thereof. In one embodiment, variants of one or more biomarkers listed in Table 1 which function as a modulating agent for the respective full length protein, can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, for antagonist activity. In one embodiment, a variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of variants can be produced, for instance, by enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential polypeptide sequences is expressible as individual polypeptides containing the set of polypeptide sequences therein. There are a variety of methods which can be used to produce libraries of polypeptide variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential polypeptide sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.

In addition, libraries of fragments of a polypeptide coding sequence can be used to generate a variegated population of polypeptide fragments for screening and subsequent selection of variants of a given polypeptide. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a polypeptide coding sequence with a nuclease under conditions wherein nicking occurs only about once per polypeptide, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with SI nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the polypeptide.

Several techniques are known in the art for screening gene products of

combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of polypeptides. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify variants of interest (Arkin and Youvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delagrave et al. (1993) Protein Eng. 6(3):327-331). In one embodiment, cell based assays can be exploited to analyze a variegated polypeptide library. For example, a library of expression vectors can be transfected into a cell line which ordinarily synthesizes one or more biomarkers of the invention, including one or more biomarkers listed in Table 1, or a fragment thereof. The transfected cells are then cultured such that the full length polypeptide and a particular mutant polypeptide are produced and the effect of expression of the mutant on the full length polypeptide activity in cell supernatants can be detected, e.g., by any of a number of functional assays. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively,

potentiation of full length polypeptide activity, and the individual clones further

characterized.

Systematic substitution of one or more amino acids of a polypeptide amino acid sequence with a D-amino acid of the same type {e.g., D-lysine in place of L-lysine) can be used to generate more stable peptides. In addition, constrained peptides comprising a polypeptide amino acid sequence of interest or a substantially identical sequence variation can be generated by methods known in the art (Rizo and Gierasch (1992) Annu. Rev.

Biochem. 61:387, incorporated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.

The amino acid sequences described herein will enable those of skill in the art to produce polypeptides corresponding peptide sequences and sequence variants thereof.

Such polypeptides can be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding the peptide sequence, frequently as part of a larger polypeptide. Alternatively, such peptides can be synthesized by chemical methods. Methods for expression of heterologous proteins in recombinant hosts, chemical synthesis of

polypeptides, and in vitro translation are well-known in the art and are described further in Maniatis et al. Molecular Cloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y.; Berger and Kimmel, Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., San Diego, Calif.; Merrifield, J. (1969) J Am. Chem. Soc. 91:501; Chaiken l. M. (1981) CRC Crit. Rev. Biochem. 11: 255; Kaiser et al. (1989) Science 243:187; Merrifield, B. (1986) Science 232:342; Kent, S. B. H. (1988) Annu. Rev. Biochem. 57:957; and Offord, R. E. (1980) Semisynthetic Proteins, Wiley Publishing, which are incorporated herein by reference).

Peptides can be produced, typically by direct chemical synthesis. Peptides can be produced as modified peptides, with nonpeptide moieties attached by covalent linkage to the N-terminus and/or C-terminus. In certain preferred embodiments, either the carboxy- terminus or the amino-terminus, or both, are chemically modified. The most common modifications of the terminal amino and carboxyl groups are acetylation and amidation, respectively. Amino-terminal modifications such as acylation {e.g., acetylation) or alkylation (e.g., methylation) and carboxy-terminal-modifications such as amidation, as well as other terminal modifications, including cyclization, can be incorporated into various embodiments of the invention. Certain amino-terminal and/or carboxy-terminal modifications and/or peptide extensions to the core sequence can provide advantageous physical, chemical, biochemical, and pharmacological properties, such as: enhanced stability, increased potency and/or efficacy, resistance to serum proteases, desirable pharmacokinetic properties, and others. Peptides described herein can be used

therapeutically to treat disease, e.g., by altering costimulation in a patient.

Peptidomimetics (Fauchere (1986) Adv. Drug Res. 15:29; Veber and Freidinger (1985) TINS p.392; and Evans et al. (1987) J Med. Chem. 30:1229, which are

incorporated herein by reference) are usually developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce an equivalent therapeutic or prophylactic effect. Generally, peptidomimetics are structurally similar to a paradigm polypeptide (i.e., a polypeptide that has a biological or pharmacological activity), but have one or more peptide linkages optionally replaced by a linkage selected from the group consisting of: - CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and - CH2SO-, by methods known in the art and further described in the following references: Spatola, A. F. in "Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins" Weinstein, B., ed., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone Modifications" (general review);

Morley, J. S. (1980) Trends Pharm. Sci. pp. 463-468 (general review); Hudson, D. et al. (1979) Int. J. Pept. Prot. Res. 14:177-185 (-CH2NH-, CH2CH2-); Spatola, A. F. et al. (1986) Life Sci. 38:1243-1249 (-CH2-S); Hann, M. M. (1982) J Chem. Soc. Perkin Trans. I. 307-314 (-CH-CH-, cis and trans); Almquist, R. G. et al. (190) J. Med. Chem. 23:1392- 1398 (-COCH2-); Jennings- White, C. et al. (1982) Tetrahedron Lett. 23:2533 (-COCH2-); Szelke, M. et al. European Appln. EP 45665 (1982) CA: 97:39405 (1982)(-CH(OH)CH2- ); Holladay, M. W. et al. (1983) Tetrahedron Lett. (1983) 24:4401-4404 (-C(OH)CH2-); and Hruby, V. J. (1982) Life Sci. (1982) 31:189-199 (-CH2-S-); each of which is incorporated herein by reference. A particularly preferred non-peptide linkage is - CH2NH-. Such peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity {e.g., a broad-spectrum of biological activities), reduced

antigenicity, and others. Labeling of peptidomimetics usually involves covalent attachment of one or more labels, directly or through a spacer {e.g., an amide group), to non-interfering position(s) on the peptidomimetic that are predicted by quantitative structure-activity data and/or molecular modeling. Such non-interfering positions generally are positions that do not form direct contacts with the macropolypeptides(s) to which the peptidomimetic binds to produce the therapeutic effect. Derivatization {e.g., labeling) of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic.

Also encompassed by the present invention are small molecules which can modulate (either enhance or inhibit) interactions, e.g., between biomarkers described herein or listed in Table 1 and their natural binding partners. The small molecules of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one- compound' library method; and synthetic library methods using affinity chromatography selection. (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91 :11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.

33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233. Libraries of compounds can be presented in solution (e.g., Houghten (1992)

Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner USP '409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382); (Felici (1991) J Mol. Biol. 222:301- 310); (Ladner supra.). Compounds can be screened in cell based or non-cell based assays. Compounds can be screened in pools (e.g. multiple compounds in each testing sample) or as individual compounds.

Chimeric or fusion proteins can be prepared for the inhibitors of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) and/or agents for the immunotherapies described herein, such as inhibitors to the biomarkers of the invention, including the biomarkers listed in Table 1 , or fragments thereof. As used herein, a "chimeric protein" or "fusion protein" comprises one or more biomarkers of the invention, including one or more biomarkers listed in Table 1, or a fragment thereof, operatively linked to another polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the respective biomarker. In a preferred embodiment, the fusion protein comprises at least one

biologically active portion of one or more biomarkers of the invention, including one or more biomarkers listed in Table 1 , or fragments thereof. Within the fusion protein, the term "operatively linked" is intended to indicate that the biomarker sequences and the non- biomarker sequences are fused in-frame to each other in such a way as to preserve functions exhibited when expressed independently of the fusion. The "another" sequences can be fused to the N-terminus or C-terminus of the biomarker sequences, respectively.

Such a fusion protein can be produced by recombinant expression of a nucleotide sequence encoding the first peptide and a nucleotide sequence encoding the second peptide. The second peptide may optionally correspond to a moiety that alters the solubility, affinity, stability or valency of the first peptide, for example, an immunoglobulin constant region. In another preferred embodiment, the first peptide consists of a portion of a biologically active molecule (e.g. the extracellular portion of the polypeptide or the ligand binding portion). The second peptide can include an immunoglobulin constant region, for example, a human Cyl domain or Cy4 domain (e.g., the hinge, CH2 and CH3 regions of human IgCy 1, or human IgC/4, see e.g., Capon et al. U.S. Patents 5,116,964; 5,580,756; 5,844,095 and the like, incorporated herein by reference). Such constant regions may retain regions which mediate effector function (e.g. Fc receptor binding) or may be altered to reduce effector function. A resulting fusion protein may have altered solubility, binding affinity, stability and/or valency (i.e., the number of binding sites available per polypeptide) as compared to the independently expressed first peptide, and may increase the efficiency of protein purification. Fusion proteins and peptides produced by recombinant techniques can be secreted and isolated from a mixture of cells and medium containing the protein or peptide. Alternatively, the protein or peptide can be retained cytoplasmically and the cells harvested, lysed and the protein isolated. A cell culture typically includes host cells, media and other byproducts. Suitable media for cell culture are well-known in the art. Protein and peptides can be isolated from cell culture media, host cells, or both using techniques known in the art for purifying proteins and peptides. Techniques for transfecting host cells and purifying proteins and peptides are known in the art.

Preferably, a fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).

The fusion proteins of the invention can be used as immunogens to produce antibodies in a subject. Such antibodies may be used to purify the respective natural polypeptides from which the fusion proteins were generated, or in screening assays to identify polypeptides which inhibit the interactions between one or more biomarkers polypeptide or a fragment thereof and its natural binding partner(s) or a fragment(s) thereof.

Also provided herein are compositions comprising one or more nucleic acids comprising or capable of expressing at least 1, 2, 3, 4, 5, 10, 20 or more small nucleic acids or antisense oligonucleotides or derivatives thereof, wherein said small nucleic acids or antisense oligonucleotides or derivatives thereof in a cell specifically hybridize (e.g., bind) under cellular conditions, with cellular nucleic acids (e.g., small non-coding RNAS such as miRNAs, pre-miRNAs, pri-miRNAs, miRNA*, anti-miRNA, a miRNA binding site, a variant and/or functional variant thereof, cellular mRNAs or a fragments thereof). In one embodiment, expression of the small nucleic acids or antisense oligonucleotides or derivatives thereof in a cell can inhibit expression or biological activity of cellular nucleic acids and/or proteins, e.g., by inhibiting transcription, translation and/or small nucleic acid processing of, for example, one or more biomarkers of the invention, including one or more biomarkers listed in Table 1, or fragment(s) thereof. In one embodiment, the small nucleic acids or antisense oligonucleotides or derivatives thereof are small RNAs (e.g.,

microRNAs) or complements of small RNAs. In another embodiment, the small nucleic acids or antisense oligonucleotides or derivatives thereof can be single or double stranded and are at least six nucleotides in length and are less than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 24, 23, 22, 21,20, 19, 18, 17, 16, 15, or 10 nucleotides in length. In another embodiment, a composition may comprise a library of nucleic acids comprising or capable of expressing small nucleic acids or antisense oligonucleotides or derivatives thereof, or pools of said small nucleic acids or antisense oligonucleotides or derivatives thereof. A pool of nucleic acids may comprise about 2-5, 5- 10, 10-20, 10-30 or more nucleic acids comprising or capable of expressing small nucleic acids or antisense oligonucleotides or derivatives thereof.

In one embodiment, binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix. In general, "antisense" refers to the range of techniques generally employed in the art, and includes any process that relies on specific binding to oligonucleotide sequences.

It is well-known in the art that modifications can be made to the sequence of a miRNA or a pre-miRNA without disrupting miRNA activity. As used herein, the term "functional variant" of a miRNA sequence refers to an oligonucleotide sequence that varies from the natural miRNA sequence, but retains one or more functional characteristics of the miRNA (e.g. cancer cell proliferation inhibition, induction of cancer cell apoptosis, enhancement of cancer cell susceptibility to chemotherapeutic agents, specific miRNA target inhibition). In some embodiments, a functional variant of a miRNA sequence retains all of the functional characteristics of the miRNA. In certain embodiments, a functional variant of a miRNA has a nucleobase sequence that is a least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the miRNA or precursor thereof over a region of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleobases, or that the functional variant hybridizes to the complement of the miRNA or precursor thereof under stringent hybridization conditions. Accordingly, in certain embodiments the nucleobase sequence of a functional variant is capable of hybridizing to one or more target sequences of the miRNA.

miRNAs and their corresponding stem-loop sequences described herein may be found in miRBase, an online searchable database of miRNA sequences and annotation, found on the world wide web at microrna.sanger.ac.uk. Entries in the miRBase Sequence database represent a predicted hairpin portion of a miRNA transcript (the stem-loop), with information on the location and sequence of the mature miRNA sequence. The miRNA stem-loop sequences in the database are not strictly precursor miRNAs (pre-miRNAs), and may in some instances include the pre-miRNA and some flanking sequence from the presumed primary transcript. The miRNA nucleobase sequences described herein encompass any version of the miRNA, including the sequences described in Release 10.0 of the miRBase sequence database and sequences described in any earlier Release of the miRBase sequence database. A sequence database release may result in the re-naming of certain miRNAs. A sequence database release may result in a variation of a mature miRNA sequence.

In some embodiments, miRNA sequences of the invention may be associated with a second RNA sequence that may be located on the same RNA molecule or on a separate RNA molecule as the miRNA sequence. In such cases, the miRNA sequence may be referred to as the active strand, while the second RNA sequence, which is at least partially complementary to the miRNA sequence, may be referred to as the complementary strand. The active and complementary strands are hybridized to create a double-stranded RNA that is similar to a naturally occurring miRNA precursor. The activity of a miRNA may be optimized by maximizing uptake of the active strand and minimizing uptake of the complementary strand by the miRNA protein complex that regulates gene translation. This can be done through modification and/or design of the complementary strand.

In some embodiments, the complementary strand is modified so that a chemical group other than a phosphate or hydroxyl at its 5' terminus. The presence of the 5' modification apparently eliminates uptake of the complementary strand and subsequently favors uptake of the active strand by the miRNA protein complex. The 5' modification can be any of a variety of molecules known in the art, including NH2, NHCOCH3, and biotin.

In another embodiment, the uptake of the complementary strand by the miRNA pathway is reduced by incorporating nucleotides with sugar modifications in the first 2-6 nucleotides of the complementary strand. It should be noted that such sugar modifications can be combined with the 5' terminal modifications described above to further enhance miRNA activities.

In some embodiments, the complementary strand is designed so that nucleotides in the 3' end of the complementary strand are not complementary to the active strand. This results in double-strand hybrid RNAs that are stable at the 3' end of the active strand but relatively unstable at the 5' end of the active strand. This difference in stability enhances the uptake of the active strand by the miRNA pathway, while reducing uptake of the complementary strand, thereby enhancing miRNA activity.

Small nucleic acid and/or antisense constructs of the methods and compositions presented herein can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of cellular nucleic acids (e.g., small RNAs, mRNA, and/or genomic DNA). Alternatively, the small nucleic acid molecules can produce RNA which encodes mRNA, miRNA, pre- miRNA, pri-miRNA, miRNA*, anti-miRNA, or a miRNA binding site, or a variant thereof. For example, selection of plasmids suitable for expressing the miRNAs, methods for inserting nucleic acid sequences into the plasmid, and methods of delivering the

recombinant plasmid to the cells of interest are within the skill in the art. See, for example, Zeng et al. (2002) Mol. Cell 9:1327-1333; Tuschl (2002), Nat. Biotechnol. 20:446-448; Brummelkamp et al. (2002) Science 296:550-553; Miyagishi et al. (2002) Nat. Biotechnol. 20:497-500; Paddison et al. (2002) Genes Dev. 16:948-958; Lee et al. (2002) Nat.

Biotechnol. 20:500-505; and Paul et al. (2002) Nat. Biotechnol. 20:505-508, the entire disclosures of which are herein incorporated by reference.

Alternatively, small nucleic acids and/or antisense constructs are oligonucleotide probes that are generated ex vivo and which, when introduced into the cell, results in hybridization with cellular nucleic acids. Such oligonucleotide probes are preferably modified oligonucleotides that are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, and are therefore stable in vivo. Exemplary nucleic acid molecules for use as small nucleic acids and/or antisense oligonucleotides are phosphoramidate, phosphothioate and methylphosphonate analogs of DNA (see also U.S. Patents 5,176,996; 5,264,564; and 5,256,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van der Krol et al. (1988) BioTechniques 6:958-976; and Stein et al. (1988) Cancer Res 48:2659-2668.

Antisense approaches may involve the design of oligonucleotides (either DNA or RNA) that are complementary to cellular nucleic acids (e.g., complementary to biomarkers listed in Table 1). Absolute complementarity is not required. In the case of double- stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with a nucleic acid (e.g., RNA) it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5' end of the mRNA, e.g., the 5' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3' untranslated sequences of mRNAs have recently been shown to be effective at inhibiting translation of mRNAs as well (Wagner (1994) Nature 372:333). Therefore,

oligonucleotides complementary to either the 5' or 3' untranslated, non-coding regions of genes could be used in an antisense approach to inhibit translation of endogenous mRNAs. Oligonucleotides complementary to the 5' untranslated region of the mRNA may include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could also be used in accordance with the methods and compositions presented herein. Whether designed to hybridize to the 5', 3' or coding region of cellular mRNAs, small nucleic acids and/or antisense nucleic acids should be at least six nucleotides in length, and can be less than about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 40, 30, 25, 24, 23, 22, 21,20, 19, 18, 17, 16, 15, or 10 nucleotides in length.

Regardless of the choice of target sequence, it is preferred that in vitro studies are first performed to quantitate the ability of the antisense oligonucleotide to inhibit gene expression. In one embodiment these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. In another embodiment these studies compare levels of the target nucleic acid or protein with that of an internal control nucleic acid or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide. It is preferred that the control oligonucleotide is of approximately the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.

Small nucleic acids and/or antisense oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded. Small nucleic acids and/or antisense oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc., and may include other appended groups such as peptides (e.g., for targeting host cell receptors), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134), hybridization-triggered cleavage agents. (See, e.g., Krol et al. (1988) BioTech. 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, small nucleic acids and/or antisense oligonucleotides may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization- triggered cleavage agent, etc.

Small nucleic acids and/or antisense oligonucleotides may comprise at least one modified base moiety which is selected from the group including but not limited to 5- fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4- acetylcytosine, 5-(carboxyhydroxytiethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta- D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino- 3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Small nucleic acids and/or antisense oligonucleotides may also comprise at least one modified sugar moiety selected from the group including but not limited to arabinose, 2-fluoroarabinose, xylulose, and hexose.

In certain embodiments, a compound comprises an oligonucleotide (e.g., a miR A or miRNA encoding oligonucleotide) conjugated to one or more moieties which enhance the activity, cellular distribution or cellular uptake of the resulting oligonucleotide. In certain such embodiments, the moiety is a cholesterol moiety (e.g., antagomirs) or a lipid moiety or liposome conjugate. Additional moieties for conjugation include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. In certain embodiments, a conjugate group is attached directly to the oligonucleotide. In certain embodiments, a conjugate group is attached to the oligonucleotide by a linking moiety selected from amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), 8-amino-3,6- dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1- carboxylate (SMCC), 6-aminohexanoic acid (AHEX or AHA), substituted CI -CIO alkyl, substituted or unsubstituted C2-C10 alkenyl, and substituted or unsubstituted C2-C10 alkynyl. In certain such embodiments, a substituent group is selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.

In certain such embodiments, the compound comprises the oligonucleotide having one or more stabilizing groups that are attached to one or both termini of the

oligonucleotide to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the

oligonucleotide from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'-terminus (5'-cap), or at the 3'-terminus (3 - cap), or can be present on both termini. Cap structures include, for example, inverted deoxy abasic caps.

Suitable cap structures include a 4',5 -methylene nucleotide, a l-(beta-D- erythrofuranosyl) nucleotide, a 4'-thio nucleotide, a carbocyclic nucleotide, a 1,5- anhydrohexitol nucleotide, an L-nucleotide, an alpha-nucleotide, a modified base nucleotide, a phosphorodithioate linkage, a threo-pentofuranosyl nucleotide, an acyclic 3',4 -seco nucleotide, an acyclic 3,4-dihydroxybutyl nucleotide, an acyclic 3,5- dihydroxypentyl nucleotide, a 3'-3'-inverted nucleotide moiety, a 3'-3'-inverted abasic moiety, a 3'-2'-inverted nucleotide moiety, a 3'-2'-inverted abasic moiety, a 1,4-butanediol phosphate, a 3'-phosphoramidate, a hexylphosphate, an aminohexyl phosphate, a 3'- phosphate, a 3 '-phosphorothioate, a phosphorodithioate, a bridging methylphosphonate moiety, and a non-bridging methylphosphonate moiety 5'-amino-alkyl phosphate, a 1,3- diamino-2-propyl phosphate, 3-aminopropyl phosphate, a 6-aminohexyl phosphate, a 1,2- aminododecyl phosphate, a hydroxypropyl phosphate, a 5'-5'-inverted nucleotide moiety, a 5'-5'-inverted abasic moiety, a 5'-phosphoramidate, a 5'-phosphorothioate, a 5'-amino, a bridging and/or non-bridging 5'-phosphoramidate, a phosphorothioate, and a 5'-mercapto moiety.

Small nucleic acids and/or antisense oligonucleotides can also contain a neutral peptide-like backbone. Such molecules are termed peptide nucleic acid (PNA)-oligomers and are described, e.g., in Perry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. U.S.A.

93:14670 and in Eglom et al. (1993) Nature 365:566. One advantage of PNA oligomers is their capability to bind to complementary DNA essentially independently from the ionic strength of the medium due to the neutral backbone of the DNA. In yet another embodiment, small nucleic acids and/or antisense oligonucleotides comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In a further embodiment, small nucleic acids and/or antisense oligonucleotides are a-anomeric oligonucleotides. An a-anomeric oligonucleotide forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al. (1987) Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a 2'-0-methylribonucleotide (Inoue et al. (1987) Nucl. Acids Res. 15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett.

215:327-330).

Small nucleic acids and/or antisense oligonucleotides of the methods and compositions presented herein may be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988) Nucl. Acids Res. 16:3209, methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc. For example, an isolated miRNA can be chemically synthesized or recombinantly produced using methods known in the art. In some instances, miRNA are chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional

DNA/RNA synthesizer. Commercial suppliers of synthetic RNA molecules or synthesis reagents include, e.g., Proligo (Hamburg, Germany), Dharmacon Research (Lafayette, Colo., USA), Pierce Chemical (part of Perbio Science, Rockford, 111., USA), Glen Research (Sterling, Va., USA), ChemGenes (Ashland, Mass., USA), Cruachem (Glasgow, UK), and Exiqon (Vedbaek, Denmark).

Small nucleic acids and/or antisense oligonucleotides can be delivered to cells in vivo. A number of methods have been developed for delivering small nucleic acids and/or antisense oligonucleotides DNA or RNA to cells; e.g., antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g. , antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systematically.

In one embodiment, small nucleic acids and/or antisense oligonucleotides may comprise or be generated from double stranded small interfering RNAs (siRNAs), in which sequences fully complementary to cellular nucleic acids (e.g. mRNAs) sequences mediate degradation or in which sequences incompletely complementary to cellular nucleic acids (e.g., mRNAs) mediate translational repression when expressed within cells, or piwiRNAs. In another embodiment, double stranded siRNAs can be processed into single stranded antisense RNAs that bind single stranded cellular RNAs (e.g., microRNAs) and inhibit their expression. RNA interference (RNAi) is the process of sequence-specific, post- transcriptional gene silencing in animals and plants, initiated by double-stranded RNA (dsRNA) that is homologous in sequence to the silenced gene, in vivo, long dsRNA is cleaved by ribonuclease III to generate 21- and 22-nucleotide siRNAs. It has been shown that 21 -nucleotide siRNA duplexes specifically suppress expression of endogenous and heterologous genes in different mammalian cell lines, including human embryonic kidney (293) and HeLa cells (Elbashir et al. (2001) Nature 411 :494-498). Accordingly, translation of a gene in a cell can be inhibited by contacting the cell with short double stranded RNAs having a length of about 15 to 30 nucleotides or of about 18 to 21 nucleotides or of about 19 to 21 nucleotides. Alternatively, a vector encoding for such siRNAs or short hairpin RNAs (shRNAs) that are metabolized into siRNAs can be introduced into a target cell (see, e.g., McManus et al. (2002) RNA 8:842; Xia et al. (2002) Nat. Biotechnol. 20:1006; and Brummelkamp et al. (2002) Science 296:550). Vectors that can be used are commercially available, e.g., from OligoEngine under the name pSuper RNAi System™.

Ribozyme molecules designed to catalytically cleave cellular mRNA transcripts can also be used to prevent translation of cellular mRNAs and expression of cellular

polypeptides, or both (See, e.g., PCT International Publication WO90/11364, published October 4, 1990; Sarver et al. (1990) Science 247:1222-1225 and U.S. Patent No.

5,093,246). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy cellular mRNAs, the use of hammerhead ribozymes is preferred.

Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well-known in the art and is described more fully in Haseloff and Gerlach (1988) Nature 334:585-591. The ribozyme may be engineered so that the cleavage recognition site is located near the 5' end of cellular mRNAs; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

The ribozymes of the methods presented herein also include RNA

endoribonucleases (hereinafter "Cech-type ribozymes") such as the one which occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) and which has been extensively described by Thomas Cech and collaborators (Zaug et al. (1984) Science 224:574-578; Zaug et al. (1986) Science 231 :470-475; Zaug et al. (1986) Nature 324:429-433; WO 88/04300; and Been et al. (1986) Cell 47:207-216). The Cech-type ribozymes have an eight base pair active site which hybridizes to a target RNA sequence whereafter cleavage of the target RNA takes place. The methods and compositions presented herein encompasses those Cech-type ribozymes which target eight base-pair active site sequences that are present in cellular genes.

As in the antisense approach, the ribozymes can be composed of modified oligonucleotides {e.g., for improved stability, targeting, etc.). A preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous cellular messages and inhibit translation. Because ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

Nucleic acid molecules to be used in triple helix formation for the inhibition of transcription of cellular genes are preferably single stranded and composed of

deoxyribonucleotides. The base composition of these oligonucleotides should promote triple helix formation via Hoogsteen base pairing rules, which generally require sizable stretches of either purines or pyrimidines to be present on one strand of a duplex.

Nucleotide sequences may be pyrimidine-based, which will result in TAT and CGC triplets across the three associated strands of the resulting triple helix. The pyrimidine-rich molecules provide base complementarity to a purine-rich region of a single strand of the duplex in a parallel orientation to that strand. In addition, nucleic acid molecules may be chosen that are purine-rich, for example, containing a stretch of G residues. These molecules will form a triple helix with a DNA duplex that is rich in GC pairs, in which the majority of the purine residues are located on a single strand of the targeted duplex, resulting in CGC triplets across the three strands in the triplex.

Alternatively, the potential sequences that can be targeted for triple helix formation may be increased by creating a so called "switchback" nucleic acid molecule. Switchback molecules are synthesized in an alternating 5 '-3', 3 '-5' manner, such that they base pair with first one strand of a duplex and then the other, eliminating the necessity for a sizable stretch of either purines or pyrimidines to be present on one strand of a duplex.

Small nucleic acids (e.g., miRNAs, pre-miRNAs, pri-miRNAs, miRNA*, anti- miRNA, or a miRNA binding site, or a variant thereof), antisense oligonucleotides, ribozymes, and triple helix molecules of the methods and compositions presented herein may be prepared by any method known in the art for the synthesis of DNA and RNA molecules. These include techniques for chemically synthesizing

oligodeoxyribonucleotides and oligoribonucleotides well-known in the art such as for example solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines. Moreover, various well-known modifications to nucleic acid molecules may be introduced as a means of increasing intracellular stability and half-life. Possible

modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone. One of skill in the art will readily understand that polypeptides, small nucleic acids, and antisense oligonucleotides can be further linked to another peptide or polypeptide (e.g., a heterologous peptide), e.g., that serves as a means of protein detection. Non-limiting examples of label peptide or polypeptide moieties useful for detection in the invention include, without limitation, suitable enzymes such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; epitope tags, such as FLAG, MYC, HA, or HIS tags; fluorophores such as green fluorescent protein; dyes; radioisotopes; digoxygenin; biotin; antibodies; polymers; as well as others known in the art, for example, in Principles of Fluorescence Spectroscopy, Joseph R.

Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999).

The modulatory agents described herein (e.g., antibodies, small molecules, peptides, fusion proteins, or small nucleic acids) can be incorporated into pharmaceutical

compositions and administered to a subject in vivo. The compositions may contain a single such molecule or agent or any combination of agents described herein. "Single active agents" described herein can be combined with other pharmacologically active compounds ("second active agents") known in the art according to the methods and compositions provided herein.

The production and use of biomarker nucleic acid and/or biomarker polypeptide molecules described herein can be facilitated by using standard recombinant techniques. In some embodiments, such techniques use vectors, preferably expression vectors, containing a nucleic acid encoding a biomarker polypeptide or a portion of such a polypeptide. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors, namely expression vectors, are capable of directing the expression of genes to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, the present invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the present invention comprise a nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell. This means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in

Goeddel, Methods in Enzymology: Gene Expression Technology vol.185, Academic Press, San Diego, CA (1991). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the present invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.

The recombinant expression vectors for use in the present invention can be designed for expression of a polypeptide corresponding to a marker of the present invention in prokaryotic (e.g., E. coli) or eukaryotic cells (e.g., insect cells {using baculovirus expression vectors}, yeast cells or mammalian cells). Suitable host cells are discussed further in Goeddel, supra. Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988, Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, 1988, Gene 69:301-315) and pET 1 Id (Studier et al, p. 60-89, In Gene

Expression Technology: Methods in Enzymology vol.185, Academic Press, San Diego, CA, 1991). Target biomarker nucleic acid expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target biomarker nucleic acid expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a co-expressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21 (DE3) or HMS174(DE3) from a resident prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacterium with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, p. 119-128, In Gene Expression Technology: Methods in Enzymology vol. 185, Academic Press, San Diego, CA, 1990. Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al, 1992, Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the present invention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expression vector.

Examples of vectors for expression in yeast S. cerevisiae include pYepSecl (Baldari et al, 1987, EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, 1982, Cell 30:933-943), pJRY88 (Schultz et al, 1987, Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and pPicZ (Invitrogen Corp, San Diego, CA).

Alternatively, the expression vector is a baculovirus expression vector. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et al., 1983, Mol. Cell Biol. 3:2156-2165) and the pVL series

(Lucklow and Summers, 1989, Virology 170:31-39).

In yet another embodiment, a nucleic acid of the present invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987, Nature 329:840) and pMT2PC (Kaufman et al, 1987, EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook et al. , supra.

In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue- specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al, 1987, Genes Dev.

1:268-277), lymphoid-specific promoters (Calame and Eaton, 1988, Adv. Immunol. 43:235- 275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989, EMBO J. 8:729-733) and immunoglobulins (Banerji et al, 1983, Cell 33:729-740; Queen and Baltimore, 1983, Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989, Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas- specific promoters (Edlund et al, 1985, Science 230:912-916), and mammary gland- specific promoters (e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss, 1990, Science 249:374-379) and the cc-fetoprotein promoter (Camper and Tilghman, 1989, Genes Dev. 3:537-546).

The present invention further provides a recombinant expression vector comprising a DNA molecule cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operably linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to the mRNA encoding a polypeptide of the present invention. Regulatory sequences operably linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue-specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid, or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes (see Weintraub et al, 1986, Trends in Genetics, Vol. 1(1)).

Another aspect of the present invention pertains to host cells into which a recombinant expression vector of the present invention has been introduced. The terms "host cell" and "recombinant host cell" are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell can be any prokaryotic {e.g., E. coli) or eukaryotic cell {e.g., insect cells, yeast or mammalian cells).

Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride co- precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.

Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. {supra), and other laboratory manuals. For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., for resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

V. Analyzing Biomarker Nucleic Acids and Polypeptides

Biomarker nucleic acids and/or biomarker polypeptides can be analyzed according to the methods described herein and techniques known to the skilled artisan to identify such genetic or expression alterations useful for the present invention including, but not limited to, 1) an alteration in the level of a biomarker transcript or polypeptide, 2) a deletion or addition of one or more nucleotides from a biomarker gene, 4) a substitution of one or more nucleotides of a biomarker gene, 5) aberrant modification of a biomarker gene, such as an expression regulatory region, and the like.

a. Methods for Detection of Copy Number

Methods of evaluating the copy number of a biomarker nucleic acid are well-known to those of skill in the art. The presence or absence of chromosomal gain or loss can be evaluated simply by a determination of copy number of the regions or markers identified herein.

In one embodiment, a biological sample is tested for the presence of copy number changes in genomic loci containing the genomic marker. A copy number of at least 3, 4, 5, 6, 7, 8, 9, or 10 is predictive of poorer outcome of inhibitors of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, e.g. such as PTPN2 and SOCS1) and immunotherapy combination treatments.

Methods of evaluating the copy number of a biomarker locus include, but are not limited to, hybridization-based assays. Hybridization-based assays include, but are not limited to, traditional "direct probe" methods, such as Southern blots, in situ hybridization (e.g., FISH and FISH plus SKY) methods, and "comparative probe" methods, such as comparative genomic hybridization (CGH), e.g., cDNA-based or oligonucleotide-based CGH. The methods can be used in a wide variety of formats including, but not limited to, substrate (e.g. membrane or glass) bound methods or array-based approaches.

In one embodiment, evaluating the biomarker gene copy number in a sample involves a Southern Blot. In a Southern Blot, the genomic DNA (typically fragmented and separated on an electrophoretic gel) is hybridized to a probe specific for the target region. Comparison of the intensity of the hybridization signal from the probe for the target region with control probe signal from analysis of normal genomic DNA (e.g., a non-amplified portion of the same or related cell, tissue, organ, etc.) provides an estimate of the relative copy number of the target nucleic acid. Alternatively, a Northern blot may be utilized for evaluating the copy number of encoding nucleic acid in a sample. In a Northern blot, mRNA is hybridized to a probe specific for the target region. Comparison of the intensity of the hybridization signal from the probe for the target region with control probe signal from analysis of normal RNA (e.g., a non-amplified portion of the same or related cell, tissue, organ, etc.) provides an estimate of the relative copy number of the target nucleic acid. Alternatively, other methods well-known in the art to detect RNA can be used, such that higher or lower expression relative to an appropriate control (e.g., a non-amplified portion of the same or related cell tissue, organ, etc.) provides an estimate of the relative copy number of the target nucleic acid.

An alternative means for determining genomic copy number is in situ hybridization (e.g., Angerer (1987) Meth. Enzymol 152: 649). Generally, in situ hybridization comprises the following steps: (1) fixation of tissue or biological structure to be analyzed; (2) prehybridization treatment of the biological structure to increase accessibility of target DNA, and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological structure or tissue; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization and (5) detection of the hybridized nucleic acid fragments. The reagent used in each of these steps and the conditions for use vary depending on the particular application. In a typical in situ hybridization assay, cells are fixed to a solid support, typically a glass slide. If a nucleic acid is to be probed, the cells are typically denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to the nucleic acid sequence encoding the protein. The targets (e.g., cells) are then typically washed at a predetermined stringency or at an increasing stringency until an appropriate signal to noise ratio is obtained. The probes are typically labeled, e.g., with radioisotopes or fluorescent reporters. In one embodiment, probes are sufficiently long so as to specifically hybridize with the target nucleic acid(s) under stringent conditions. Probes generally range in length from about 200 bases to about 1000 bases. In some applications it is necessary to block the hybridization capacity of repetitive sequences. Thus, in some embodiments, tRNA, human genomic DNA, or Cot-I DNA is used to block non-specific hybridization.

An alternative means for determining genomic copy number is comparative genomic hybridization. In general, genomic DNA is isolated from normal reference cells, as well as from test cells (e.g., tumor cells) and amplified, if necessary. The two nucleic acids are differentially labeled and then hybridized in situ to metaphase chromosomes of a reference cell. The repetitive sequences in both the reference and test DNAs are either removed or their hybridization capacity is reduced by some means, for example by prehybridization with appropriate blocking nucleic acids and/or including such blocking nucleic acid sequences for said repetitive sequences during said hybridization. The bound, labeled DNA sequences are then rendered in a visualizable form, if necessary.

Chromosomal regions in the test cells which are at increased or decreased copy number can be identified by detecting regions where the ratio of signal from the two DNAs is altered. For example, those regions that have decreased in copy number in the test cells will show relatively lower signal from the test DNA than the reference compared to other regions of the genome. Regions that have been increased in copy number in the test cells will show relatively higher signal from the test DNA. Where there are chromosomal deletions or multiplications, differences in the ratio of the signals from the two labels will be detected and the ratio will provide a measure of the copy number. In another embodiment of CGH, array CGH (aCGH), the immobilized chromosome element is replaced with a collection of solid support bound target nucleic acids on an array, allowing for a large or complete percentage of the genome to be represented in the collection of solid support bound targets. Target nucleic acids may comprise cDNAs, genomic DNAs, oligonucleotides (e.g., to detect single nucleotide polymorphisms) and the like. Array-based CGH may also be performed with single-color labeling (as opposed to labeling the control and the possible tumor sample with two different dyes and mixing them prior to hybridization, which will yield a ratio due to competitive hybridization of probes on the arrays). In single color CGH, the control is labeled and hybridized to one array and absolute signals are read, and the possible tumor sample is labeled and hybridized to a second array (with identical content) and absolute signals are read. Copy number difference is calculated based on absolute signals from the two arrays. Methods of preparing immobilized chromosomes or arrays and performing comparative genomic hybridization are well-known in the art (see, e.g., U.S. Pat. Nos: 6,335,167; 6,197,501; 5,830,645; and 5,665,549 and Albertson (1984) EMBO J. 3: 1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No. 430,402; Methods in Molecular Biology, Vol. 33: In situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, N.J. (1994), etc.). In another embodiment, the hybridization protocol of Pinkel, et al. (1998) Nature Genetics 20: 207-211, or of

Kallioniemi (1992) Proc. Natl Acad Sci USA 89:5321-5325 (1992) is used.

In still another embodiment, amplification-based assays can be used to measure copy number. In such amplification-based assays, the nucleic acid sequences act as a template in an amplification reaction {e.g., Polymerase Chain Reaction (PCR). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls, e.g. healthy tissue, provides a measure of the copy number.

Methods of "quantitative" amplification are well-known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided in Innis, et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). Measurement of DNA copy number at microsatellite loci using quantitative PCR analysis is described in Ginzonger, et al. (2000) Cancer Research 60:5405-5409. The known nucleic acid sequence for the genes is sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene. Fluorogenic quantitative PCR may also be used in the methods of the present invention. In fluorogenic quantitative PCR, quantitation is based on amount of fluorescence signals, e.g., TaqMan and SYBR green.

Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren, et al. (1988) Science 241 : 1077, and Barringer et al. (1990) Gene 89: 117), transcription amplification (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication (Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc. Loss of heterozygosity (LOH) and major copy proportion (MCP) mapping (Wang, Z.C., et al. (2004) Cancer Res 64(1):64-71 ; Seymour, A. B., et al. (1994) Cancer Res 54, 2761-4; Hahn, S. A., et al. (1995) Cancer Res 55, 4670-5; Kimura, M., et al. (1996) Genes Chromosomes Cancer 17, 88-93; Li et al, (2008) MBC Bioinform. 9, 204-219) may also be used to identify regions of amplification or deletion.

b. Methods for Detection of Biomarker Nucleic Acid Expression

Biomarker expression may be assessed by any of a wide variety of well-known methods for detecting expression of a transcribed molecule or protein. Non-limiting examples of such methods include immunological methods for detection of secreted, cell- surface, cytoplasmic, or nuclear proteins, protein purification methods, protein function or activity assays, nucleic acid hybridization methods, nucleic acid reverse transcription methods, and nucleic acid amplification methods.

In preferred embodiments, activity of a particular gene is characterized by a measure of gene transcript (e.g. mRNA), by a measure of the quantity of translated protein, or by a measure of gene product activity. Marker expression can be monitored in a variety of ways, including by detecting mRNA levels, protein levels, or protein activity, any of which can be measured using standard techniques. Detection can involve quantification of the level of gene expression (e.g., genomic DNA, cDNA, mRNA, protein, or enzyme activity), or, alternatively, can be a qualitative assessment of the level of gene expression, in particular in comparison with a control level. The type of level being detected will be clear from the context.

In another embodiment, detecting or determining expression levels of a biomarker and functionally similar homologs thereof, including a fragment or genetic alteration thereof (e.g., in regulatory or promoter regions thereof) comprises detecting or determining RNA levels for the marker of interest. In one embodiment, one or more cells from the subject to be tested are obtained and RNA is isolated from the cells. In a preferred embodiment, a sample of breast tissue cells is obtained from the subject.

In one embodiment, RNA is obtained from a single cell. For example, a cell can be isolated from a tissue sample by laser capture microdissection (LCM). Using this technique, a cell can be isolated from a tissue section, including a stained tissue section, thereby assuring that the desired cell is isolated (see, e.g., Bonner et al. (1997) Science 278: 1481; Emmert-Buck et al. (1996) Science 274:998; Fend et al. (1999) Am. J. Path. 154: 61 and Murakami et al. (2000) Kidney Int. 58:1346). For example, Murakami et al, supra, describe isolation of a cell from a previously immunostained tissue section.

It is also be possible to obtain cells from a subject and culture the cells in vitro, such as to obtain a larger population of cells from which RNA can be extracted. Methods for establishing cultures of non-transformed cells, i. e. , primary cell cultures, are known in the art.

When isolating RNA from tissue samples or cells from individuals, it may be important to prevent any further changes in gene expression after the tissue or cells has been removed from the subject. Changes in expression levels are known to change rapidly following perturbations, e.g. , heat shock or activation with lipopolysaccharide (LPS) or other reagents. In addition, the RNA in the tissue and cells may quickly become degraded. Accordingly, in a preferred embodiment, the tissue or cells obtained from a subject is snap frozen as soon as possible.

RNA can be extracted from the tissue sample by a variety of methods, e.g., the guanidium thiocyanate lysis followed by CsCl centrifugation (Chirgwin et al. , 1979, Biochemistry 18:5294-5299). RNA from single cells can be obtained as described in methods for preparing cDNA libraries from single cells, such as those described in Dulac, C. (1998) Curr. Top. Dev. Biol. 36, 245 and Jena et al. (1996) J. Immunol. Methods 190:199. Care to avoid RNA degradation must be taken, e.g., by inclusion of RNAsin.

The RNA sample can then be enriched in particular species. In one embodiment, poly(A)+ RNA is isolated from the RNA sample. In general, such purification takes advantage of the poly -A tails on mRNA. In particular and as noted above, poly-T oligonucleotides may be immobilized within on a solid support to serve as affinity ligands for mRNA. Kits for this purpose are commercially available, e.g., the MessageMaker kit (Life Technologies, Grand Island, NY).

In a preferred embodiment, the RNA population is enriched in marker sequences. Enrichment can be undertaken, e.g., by primer-specific cDNA synthesis, or multiple rounds of linear amplification based on cDNA synthesis and template-directed in vitro

transcription {see, e.g., Wang et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86: 9717; Dulac et al. , supra, and Jena et al. , supra).

The population of RNA, enriched or not in particular species or sequences, can further be amplified. As defined herein, an "amplification process" is designed to strengthen, increase, or augment a molecule within the RNA. For example, where RNA is mRNA, an amplification process such as RT-PCR can be utilized to amplify the mRNA, such that a signal is detectable or detection is enhanced. Such an amplification process is beneficial particularly when the biological, tissue, or tumor sample is of a small size or volume.

Various amplification and detection methods can be used. For example, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770, or reverse transcribe mRNA into cDNA followed by symmetric gap ligase chain reaction (RT-AGLCR) as described by R. L. Marshall, et ah, PCR

Methods and Applications 4: 80-84 (1994). Real time PCR may also be used.

Other known amplification methods which can be utilized herein include but are not limited to the so-called "NASBA" or "3SR" technique described in PNAS USA 87: 1874- 1878 (1990) and also described in Nature 350 (No. 6313): 91-92 (1991); Q-beta

amplification as described in published European Patent Application (EPA) No. 4544610; strand displacement amplification (as described in G. T. Walker et ah, Clin. Chem. 42: 9-13 (1996) and European Patent Application No. 684315; target mediated amplification, as described by PCT Publication W09322461; PCR; ligase chain reaction (LCR) {see, e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et ah, Science 241, 1077 (1988)); self-sustained sequence replication (SSR) {see, e.g., Guatelli et ah, Proc. Nat. Acad. Sci. USA, 87, 1874 (1990)); and transcription amplification {see, e.g., Kwoh et ah, Proc. Natl. Acad. Sci. USA 86, 1173 (1989)).

Many techniques are known in the state of the art for determining absolute and relative levels of gene expression, commonly used techniques suitable for use in the present invention include Northern analysis, RNase protection assays (RPA), microarrays and PCR- based techniques, such as quantitative PCR and differential display PCR. For example, Northern blotting involves running a preparation of RNA on a denaturing agarose gel, and transferring it to a suitable support, such as activated cellulose, nitrocellulose or glass or nylon membranes. Radiolabeled cDNA or RNA is then hybridized to the preparation, washed and analyzed by autoradiography.

In situ hybridization visualization may also be employed, wherein a radioactively labeled antisense RNA probe is hybridized with a thin section of a biopsy sample, washed, cleaved with RNase and exposed to a sensitive emulsion for autoradiography. The samples may be stained with hematoxylin to demonstrate the histological composition of the sample, and dark field imaging with a suitable light filter shows the developed emulsion. Non-radioactive labels such as digoxigenin may also be used.

Alternatively, mRNA expression can be detected on a DNA array, chip or a microarray. Labeled nucleic acids of a test sample obtained from a subject may be hybridized to a solid surface comprising biomarker DNA. Positive hybridization signal is obtained with the sample containing biomarker transcripts. Methods of preparing DNA arrays and their use are well-known in the art (see, e.g., U.S. Pat. Nos: 6,618,6796;

6,379,897; 6,664,377; 6,451,536; 548,257; U.S. 20030157485 and Schena et a/. (1995) Science 20, 467-470; Gerhold et al. (1999) Trends In Biochem. Sci. 24, 168-173; and Lennon et al. (2000) Drug Discovery Today 5, 59-65, which are herein incorporated by reference in their entirety). Serial Analysis of Gene Expression (SAGE) can also be performed (See for example U.S. Patent Application 20030215858).

To monitor mRNA levels, for example, mRNA is extracted from the biological sample to be tested, reverse transcribed, and fluorescently-labeled cDNA probes are generated. The microarrays capable of hybridizing to marker cDNA are then probed with the labeled cDNA probes, the slides scanned and fluorescence intensity measured. This intensity correlates with the hybridization intensity and expression levels.

Types of probes that can be used in the methods described herein include cDNA, riboprobes, synthetic oligonucleotides and genomic probes. The type of probe used will generally be dictated by the particular situation, such as riboprobes for in situ hybridization, and cDNA for Northern blotting, for example. In one embodiment, the probe is directed to nucleotide regions unique to the RNA. The probes may be as short as is required to differentially recognize marker mRNA transcripts, and may be as short as, for example, 15 bases; however, probes of at least 17, 18, 19 or 20 or more bases can be used. In one embodiment, the primers and probes hybridize specifically under stringent conditions to a DNA fragment having the nucleotide sequence corresponding to the marker. As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% identity in nucleotide sequences. In another embodiment, hybridization under "stringent conditions" occurs when there is at least 97% identity between the sequences.

The form of labeling of the probes may be any that is appropriate, such as the use of radioisotopes, for example, ³² P and ³⁵ S. Labeling with radioisotopes may be achieved, whether the probe is synthesized chemically or biologically, by the use of suitably labeled bases. In one embodiment, the biological sample contains polypeptide molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.

In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting marker polypeptide, mRNA, genomic DNA, or fragments thereof, such that the presence of the marker polypeptide, mRNA, genomic DNA, or fragments thereof, is detected in the biological sample, and comparing the presence of the marker polypeptide, mRNA, genomic DNA, or fragments thereof, in the control sample with the presence of the marker polypeptide, mRNA, genomic DNA, or fragments thereof in the test sample,

c. Methods for Detection of Biomarker Protein Expression

The activity or level of a biomarker protein can be detected and/or quantified by detecting or quantifying the expressed polypeptide. The polypeptide can be detected and quantified by any of a number of means well-known to those of skill in the art. Aberrant levels of polypeptide expression of the polypeptides encoded by a biomarker nucleic acid and functionally similar homologs thereof, including a fragment or genetic alteration thereof (e.g., in regulatory or promoter regions thereof) are associated with the likelihood of response of a cancer to inhibitors of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) and immunotherapy combination treatments. Any method known in the art for detecting polypeptides can be used. Such methods include, but are not limited to, immunodiffusion, Immunoelectrophoresis, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),

immunofluorescent assays, Western blotting, binder-ligand assays, immunohistochemical techniques, agglutination, complement assays, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like (e.g., Basic and Clinical Immunology, Sites and Terr, eds., Appleton and Lange, Norwalk, Conn, pp 217-262, 1991 which is incorporated by reference). Preferred are binder-ligand immunoassay methods including reacting antibodies with an epitope or epitopes and competitively displacing a labeled polypeptide or derivative thereof.

For example, ELISA and RIA procedures may be conducted such that a desired biomarker protein standard is labeled (with a radioisotope such as ¹²⁵ I or ³⁵ S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and, together with the unlabeled sample, brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay). Alternatively, the biomarker protein in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled anti-biomarker protein antibody is allowed to react with the system, and radioactivity or the enzyme assayed (ELISA-sandwich assay). Other conventional methods may also be employed as suitable.

The above techniques may be conducted essentially as a "one-step" or "two-step" assay. A "one-step" assay involves contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody. A "two-step" assay involves washing before contacting, the mixture with labeled antibody. Other conventional methods may also be employed as suitable.

In one embodiment, a method for measuring biomarker protein levels comprises the steps of: contacting a biological specimen with an antibody or variant (e.g., fragment) thereof which selectively binds the biomarker protein, and detecting whether said antibody or variant thereof is bound to said sample and thereby measuring the levels of the biomarker protein.

Enzymatic and radiolabeling of biomarker protein and/or the antibodies may be effected by conventional means. Such means will generally include covalent linking of the enzyme to the antigen or the antibody in question, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be effected. Indeed, some techniques for binding enzyme are non-specific (such as using formaldehyde), and will only yield a proportion of active enzyme.

It is usually desirable to immobilize one component of the assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed without laborious and time-consuming labor. It is possible for a second phase to be immobilized away from the first, but one phase is usually sufficient.

It is possible to immobilize the enzyme itself on a support, but if solid-phase enzyme is required, then this is generally best achieved by binding to antibody and affixing the antibody to a support, models and systems for which are well-known in the art. Simple polyethylene may provide a suitable support. Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled antibody with the substrate under controlled conditions well-known in the art.

Other techniques may be used to detect biomarker protein according to a practitioner's preference based upon the present disclosure. One such technique is Western blotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter. Anti-biomarker protein antibodies (unlabeled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-immunoglobulin (suitable labels including ¹²⁵ I, horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.

Immunohistochemistry may be used to detect expression of biomarker protein, e.g., in a biopsy sample. A suitable antibody is brought into contact with, for example, a thin layer of cells, washed, and then contacted with a second, labeled antibody. Labeling may be by fluorescent markers, enzymes, such as peroxidase, avidin, or radiolabeling. The assay is scored visually, using microscopy.

Anti-biomarker protein antibodies, such as intrabodies, may also be used for imaging purposes, for example, to detect the presence of biomarker protein in cells and tissues of a subject. Suitable labels include radioisotopes, iodine ( ¹²⁵ 1, ¹²¹ I), carbon ( ¹⁴ C), sulphur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹² In), and technetium ( ⁹⁹ mTc), fluorescent labels, such as fluorescein and rhodamine, and biotin.

For in vivo imaging purposes, antibodies are not detectable, as such, from outside the body, and so must be labeled, or otherwise modified, to permit detection. Markers for this purpose may be any that do not substantially interfere with the antibody binding, but which allow external detection. Suitable markers may include those that may be detected by X-radiography, NMR or MRI. For X-radiographic techniques, suitable markers include any radioisotope that emits detectable radiation but that is not overtly harmful to the subject, such as barium or cesium, for example. Suitable markers for NMR and MRI generally include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by suitable labeling of nutrients for the relevant hybridoma, for example.

The size of the subject, and the imaging system used, will determine the quantity of imaging moiety needed to produce diagnostic images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of technetium-99. The labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain biomarker protein. The labeled antibody or antibody fragment can then be detected using known techniques.

Antibodies that may be used to detect biomarker protein include any antibody, whether natural or synthetic, full length or a fragment thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to the biomarker protein to be detected. An antibody may have a Kd of at most about 10 ^"6 M, 10 ^"7 M, 10 ^"8 M, 10 ^"9 M, 10- ¹⁰ M, 10 ^"Π Μ, 10 ^{" 12} M. The phrase "specifically binds" refers to binding of, for example, an antibody to an epitope or antigen or antigenic determinant in such a manner that binding can be displaced or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant. An antibody may bind preferentially to the biomarker protein relative to other proteins, such as related proteins.

Antibodies are commercially available or may be prepared according to methods known in the art.

Antibodies and derivatives thereof that may be used encompass polyclonal or monoclonal antibodies, chimeric, human, humanized, primatized (CDR-grafted), veneered or single-chain antibodies as well as functional fragments, i.e., biomarker protein binding fragments, of antibodies. For example, antibody fragments capable of binding to a biomarker protein or portions thereof, including, but not limited to, Fv, Fab, Fab' and F(ab') 2 fragments can be used. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab') 2 fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab') 2 fragments. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab') 2 heavy chain portion can be designed to include DNA sequences encoding the CH, domain and hinge region of the heavy chain.

Synthetic and engineered antibodies are described in, e.g., Cabilly et al, U.S. Pat. No. 4,816,567 Cabilly et al, European Patent No. 0,125,023 Bl; Boss et al, U.S. Pat. No. 4,816,397; Boss et al, European Patent No. 0,120,694 Bl; Neuberger, M. S. et al, WO 86/01533; Neuberger, M. S. et al, European Patent No. 0,194,276 Bl; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400 Bl; Queen et al, European Patent No. 0451216 Bl; and Padlan, E. A. et al, EP 0519596 Al. See also, Newman, R. et al, BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody, and Ladner et al, U.S. Pat. No. 4,946,778 and Bird, R. E. et al, Science, 242: 423-426 (1988)) regarding single-chain antibodies. Antibodies produced from a library, e.g., phage display library, may also be used.

In some embodiments, agents that specifically bind to a biomarker protein other than antibodies are used, such as peptides. Peptides that specifically bind to a biomarker protein can be identified by any means known in the art. For example, specific peptide binders of a biomarker protein can be screened for using peptide phage display libraries, d. Methods for Detection of Biomarker Structural Alterations

The following illustrative methods can be used to identify the presence of a structural alteration in a biomarker nucleic acid and/or biomarker polypeptide molecule in order to, for example, identify PTPN2, SOCS1, or other biomarkers used in the

immunotherapies described herein that are overexpressed, overfunctional, and the like.

In certain embodiments, detection of the alteration involves the use of a

probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al (1994) Proc. Natl. Acad. Sci. USA 91:360-364), the latter of which can be particularly useful for detecting point mutations in a biomarker nucleic acid such as a biomarker gene (see Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a biomarker gene under conditions such that hybridization and amplification of the biomarker gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein. Alternative amplification methods include: self-sustained sequence replication (Guatelli, J. C. et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well-known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

In an alternative embodiment, mutations in a biomarker nucleic acid from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in biomarker nucleic acid can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotide probes (Cronin, M. T. et al. (1996) Hum. Mutat. 7:244-255; Kozal, M. J. et al. (1996) Nat. Med. 2:753-759). For example, biomarker genetic mutations can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin et al. (1996) supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential, overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene. Such biomarker genetic mutations can be identified in a variety of contexts, including, for example, germline and somatic mutations.

In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence a biomarker gene and detect mutations by comparing the sequence of the sample biomarker with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert (1977) Proc. Natl. Acad. Sci. USA 74:560 or Sanger (1977) Proc. Natl. Acad Sci. USA 74:5463. It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays (Naeve (1995)

Biotechniques 19:448-53), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127- 162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

Other methods for detecting mutations in a biomarker gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242). In general, the art technique of "mismatch cleavage" starts by providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type biomarker sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to base pair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with SI nuclease to enzymatically digest the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397 and Saleeba et al. (1992) Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in biomarker cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662). According to an exemplary embodiment, a probe based on a biomarker sequence, e.g., a wild-type biomarker treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like (e.g., U.S. Pat. No. 5,459,039.)

In other embodiments, alterations in electrophoretic mobility can be used to identify mutations in biomarker genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766; see also Cotton (1993) Mutat. Res. 285:125-144 and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73- 79). Single-stranded DNA fragments of sample and control biomarker nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to ensure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high- melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163; Saiki et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA. Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3' end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11 :238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3' end of the 5' sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification. 3. Anti-Cancer Therapies

The efficacy of inhibitors ofk one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) and immunotherapy combination treatment is predicted according to biomarker amount and/or activity associated with a cancer in a subject according to the methods described herein. In one embodiment, such inhibitors and immunotherapy combination treatments (e.g., one or more such inhibitors and immunotherapy combination treatment in combination with one or more additional anti-cancer therapies, such as another immune checkpoint inhibitor) can be administered, particularly if a subject has first been indicated as being a likely responder to such inhibitors and immunotherapy combination treatment. In another embodiment, such inhibitors and immunotherapy combination treatment can be avoided once a subject is indicated as not being a likely responder to such inhibitors and immunotherapy combination treatment and an alternative treatment regimen, such as targeted and/or untargeted anticancer therapies can be administered. Combination therapies are also contemplated and can comprise, for example, one or more chemotherapeutic agents and radiation, one or more chemotherapeutic agents and immunotherapy, or one or more chemotherapeutic agents, radiation and chemotherapy, each combination of which can be with anti-immune checkpoint therapy. In addition, any representative embodiment of an agent to modulate a particular target can be adapted to any other target described herein by the ordinarily skilled artisn (e.g., direct and indirect PD-1 inhibitors described herein can be applied to other immune checkpoint inhibitors and/or biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1), such as monospecific antibodies, bispecific antibodies, non-activiting forms, small molecules, peptides, interfering nucleic acids, and the like).

The term "targeted therapy" refers to administration of agents that selectively interact with a chosen biomolecule to thereby treat cancer. One example includes immunotherapies such as immune checkpoint inhibitors, which are well-known in the art. For example, anti-PD-1 pathway agents, such as therapeutic monoclonal blocking antibodies, which are well-known in the art and described above, can be used to target tumor microenvironments and cells expressing unwanted components of the PD-1 pathway, such as PD-1, PD-L1, and/or PD-L2.

For example, the term "PD-1 pathway" refers to the PD-1 receptor and its ligands, PD-L1 and PD-L2. "PD-1 pathway inhibitors" block or otherwise reduce the interaction between PD-1 and one or both of its ligands such that the immunoinhibitory signaling otherwise generated by the interaction is blocked or otherwise reduced. Anti-immune checkpoint inhibitors can be direct or indirect. Direct anti-immune checkpoint inhibitors block or otherwise reduce the interaction between an immune checkpoint and at least one of its ligands. For example, PD-1 inhibitors can block PD-1 binding with one or both of its ligands. Direct PD-1 combination inhibitors are well-known in the art, especially since the natural binding partners of PD-1 (e.g., PD-L1 and PD-L2), PD-L1 (e.g., PD-1 and B7-1), and PD-L2 (e.g., PD-1 and RGMb) are known.

For example, agents which directly block the interaction between PD-1 and PD-L1, PD-1 and PD-L2, PD-1 and both PD-L1 and PD-L2, such as a bispecific antibody, can prevent inhibitory signaling and upregulate an immune response (i.e., as a PD-1 pathway inhibitor). Alternatively, agents that indirectly block the interaction between PD-1 and one or both of its ligands can prevent inhibitory signaling and upregulate an immune response. For example, B7-1 or a soluble form thereof, by binding to a PD-L1 polypeptide indirectly reduces the effective concentration of PD-L1 polypeptide available to bind to PD-1.

Exemplary agents include monospecific or bispecific blocking antibodies against PD-1, PD-L1, and/or PD-L2 that block the interaction between the receptor and ligand(s); a non- activating form of PD-1, PD-L1, and/or PD-L2 (e.g., a dominant negative or soluble polypeptide), small molecules or peptides that block the interaction between PD-1, PD-L1, and/or PD-L2; fusion proteins (e.g. the extracellular portion of PD-1, PD-Ll, and/or PD-L2, fused to the Fc portion of an antibody or immunoglobulin) that bind to PD-1, PD-Ll, and/or PD-L2 and inhibit the interaction between the receptor and ligand(s); a non-activating form of a natural PD-1, PD-L2, and/or PD-L2 ligand, and a soluble form of a natural PD-1, PD- L2, and/or PD-L2 ligand.

Indirect anti-immune checkpoint inhibitors block or otherwise reduce the

immunoinhibitory signaling generated by the interaction between the immune checkpoint and at least one of its ligands. For example, an inhibitor can block the interaction between PD-1 and one or both of its ligands without necessarily directly blocking the interaction between PD-1 and one or both of its ligands. For example, indirect inhibitors include intrabodies that bind the intracellular portion of PD-1 and/or PD-Ll required to signal to block or otherwise reduce the immunoinhibitory signaling. Similarly, nucleic acids that reduce the expression of PD-1, PD-Ll, and/or PD-L2 can indirectly inhibit the interaction between PD-1 and one or both of its ligands by removing the availability of components for interaction. Such nucleic acid molecules can block PD-Ll, PD-L2, and/or PD-L2 transcription or translation.

Similary, agents which directly block the interaction between one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCSl)and their binding partners/substrates, and the like, can remove the inhibition to the signaling pathways related to such one or more biomarkers in Table 1 (e.g. , PTPN2- and/or SOCS1 -regulated signaling) and its downstream immune responses, such as increasing sensitivity to interferon signaling. Alternatively, agents that indirectly block the interaction between one or more biomarkers in Table 1 and their binding partners/substrates can remove the inhibition to such biomarkers-related signaling and its downstream immune responses. For example, a truncated or dominant negative form of such one or more biomarkers in Table 1, such as PTPN2 an/or SOCS1 fragments without phosphatase activity, by binding to a substrate of such biomarkers indirectly reduces the effective concentration of such substrate available to bind to such biomarkers in cell. Exemplary agents include monospecific or bispecific blocking antibodies, especially intrbodies, against such biomarkers and/or their substrate(s) that block the interaction between such one or more biomarkers and their substrate(s); a non-active form of such one or more biomarkers and/or their substrate(s) (e.g., a dominant negative polypeptide), small molecules or peptides that block the interaction between such one or more biomarkers and their substrate(s) or the catalytic activity of such biomarkers; and a non-activating form of a natural biomarkers in Table 1 and/or its substrate(s).

Immunotherapies that are designed to elicit or amplify an immune response are referred to as "activation immunotherapies." Immunotherapies that are designed to reduce or suppress an immune response are referred to as "suppression immunotherapies." Any agent believed to have an immune system effect on the genetically modified transplanted cancer cells can be assayed to determine whether the agent is an immunotherapy and the effect that a given genetic modification has on the modulation of immune response. In some embodiments, the immunotherapy is cancer cell-specific. In some embodiments, immunotherapy can be "untargeted," which refers to administration of agents that do not selectively interact with immune system cells, yet modulates immune system function. Representative examples of untargeted therapies include, without limitation, chemotherapy, gene therapy, and radiation therapy.

Immunotherapy can involve passive immunity for short-term protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen (e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen). Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix

polynucleotides and the like, can be used to selectively modulate biomolecules that are linked to the initiation, progression, and/or pathology of a tumor or cancer.

In one embodiment, immunotherapy comprises adoptive cell-based

immunotherapies. Well-known adoptive cell-based immunotherapeutic modalities, including, without limitation, Irradiated autologous or allogeneic tumor cells, tumor lysates or apoptotic tumor cells, antigen-presenting cell-based immunotherapy, dendritic cell-based immunotherapy, adoptive T cell transfer, adoptive CAR T cell therapy, autologous immune enhancement therapy (AIET), cancer vaccines, and/or antigen presenting cells. Such cell- based immunotherapies can be further modified to express one or more gene products to further modulate immune responses, such as expressing cytokines like GM-CSF, and/or to express tumor-associated antigen (TAA) antigens, such as Mage-1, gp-100, patient-specific neoantigen vaccines, and the like.

In another embodiment, immunotherapy comprises non-cell-based

immunotherapies. In one embodiment, compositions comprising antigens with or without vaccine-enhancing adjuvants are used. Such compositions exist in many well-known forms, such as peptide compositions, oncolytic viruses, recombinant antigen comprising fusion proteins, and the like. In still another embodiment, immunomodulatory interleukins, such as IL-2, IL-6, IL-7, IL-12, IL-17, IL-23, and the like, as well as modulators thereof (e.g. , blocking antibodies or more potent or longer lasting forms) are used. In yet another embodiment, immunomodulatory cytokines, such as interferons, G-CSF, imiquimod, TNFalpha, and the like, as well as modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms) are used. In another embodiment, immunomodulatory chemokines, such as CCL3, CCL26, and CXCL7, and the like, as well as modulators thereof (e.g., blocking antibodies or more potent or longer lasting forms) are used. In another embodiment, immunomodulatory molecules targeting immunosuppression, such as STAT3 signaling modulators, NFkappaB signaling modulators, and immune checkpoint modulators, are used. The terms "immune checkpoint" and "anti-immune checkpoint therapy" are described above.

In still another embodiment, immunomodulatory drugs, such as immunocytostatic drugs, glucocorticoids, cytostatics, immunophilins and modulators thereof (e.g., rapamycin, a calcineurin inhibitor, tacrolimus, ciclosporin (cyclosporin), pimecrolimus, abetimus, gusperimus, ridaforolimus, everolimus, temsirolimus, zotarolimus, etc.), hydrocortisone (Cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclometasone, fludrocortisone acetate, deoxycorticosterone acetate (doca) aldosterone, a non-glucocorticoid steroid, a pyrimidine synthesis inhibitor, leflunomide, teriflunomide, a folic acid analog, methotrexate, anti-thymocyte globulin, anti- lymphocyte globulin, thalidomide, lenalidomide, pentoxifylline, bupropion, curcumin, catechin, an opioid, an IMPDH inhibitor, mycophenolic acid, myriocin, fingolimod, an NF- xB inhibitor, raloxifene, drotrecogin alfa, denosumab, an NF-xB signaling cascade inhibitor, disulfiram, olmesartan, dithiocarbamate, a proteasome inhibitor, bortezomib, MG132, Prol, NPI-0052, curcumin, genistein, resveratrol, parthenolide, thalidomide, lenalidomide, flavopiridol, non-steroidal anti-inflammatory drugs (NSAIDs), arsenic trioxide, dehydroxymethylepoxyquinomycin (DHMEQ), I3C(indole-3-carbinol)/DIM(di- indolmethane) (13C DIM), Bay 11 -7082, luteolin, cell permeable peptide SN-50, IKBa.- super repressor overexpression, NFKB decoy oligodeoxynucleotide (ODN), or a derivative or analog of any thereo, are used. In yet another embodiment, immunomodulatory antibodies or protein are used. For example, antibodies that bind to CD40, Toll-like receptor (TLR), OX40, GITR, CD27, or to 4- IBB, T-cell bispecific antibodies, an anti-IL-2 receptor antibody, an anti-CD3 antibody, OKT3 (muromonab), otelixizumab, teplizumab, visilizumab, an anti-CD4 antibody, clenoliximab, keliximab, zanolimumab, an anti-CDl l a antibody, efalizumab, an anti-CD 18 antibody, erlizumab, rovelizumab, an anti-CD20 antibody, afutuzumab, ocrelizumab, ofatumumab, pascolizumab, rituximab, an anti-CD23 antibody, lumiliximab, an anti-CD40 antibody, teneliximab, toralizumab, an anti-CD40L antibody, ruplizumab, an anti-CD62L antibody, aselizumab, an anti-CD80 antibody, galiximab, an anti-CD 147 antibody, gavilimomab, a B-Lymphocyte stimulator (BLyS) inhibiting antibody, belimumab, an CTLA4-Ig fusion protein, abatacept, belatacept, an anti- CTLA4 antibody, ipilimumab, tremelimumab, an anti-eotaxin 1 antibody, bertilimumab, an anti-a4-integrin antibody, natalizumab, an anti-IL-6R antibody, tocilizumab, an anti-LFA-1 antibody, odulimomab, an anti-CD25 antibody, basiliximab, daclizumab, inolimomab, an anti-CD5 antibody, zolimomab, an anti-CD2 antibody, siplizumab, nerelimomab, faralimomab, atlizumab, atorolimumab, cedelizumab, dorlimomab aritox, dorlixizumab, fontolizumab, gantenerumab, gomiliximab, lebrilizumab, maslimomab, morolimumab, pexelizumab, reslizumab, rovelizumab, talizumab, telimomab aritox, vapaliximab, vepalimomab, aflibercept, alefacept, rilonacept, an IL-1 receptor antagonist, anakinra, an anti-IL-5 antibody, mepolizumab, an IgE inhibitor, omalizumab, talizumab, an IL12 inhibitor, an IL23 inhibitor, ustekinumab, and the like.

Nutritional supplements that enhance immune responses, such as vitamin A, vitamin

E, vitamin C, and the like, are well-known in the art (see, for example, U.S. Pat. Nos.

4,981,844 and 5,230,902 and PCT Publ. No. WO 2004/004483) can be used in the methods described herein.

Similarly, agents and therapies other than immunotherapy or in combination thereof can be used with in combination with inhibitors of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS ^/immunotherapies to stimulate an immune response to thereby treat a condition that would benefit therefrom. For example, chemotherapy, radiation, epigenetic modifiers (e.g., histone deacetylase (HDAC) modifiers, methylation modifiers, phosphorylation modifiers, and the like), targeted therapy, and the like are well-known in the art.

The term "untargeted therapy" refers to administration of agents that do not selectively interact with a chosen biomolecule yet treat cancer. Representative examples of untargeted therapies include, without limitation, chemotherapy, gene therapy, and radiation therapy.

In one embodiment, chemotherapy is used. Chemotherapy includes the

administration of a chemotherapeutic agent. Such a chemotherapeutic agent may be, but is not limited to, those selected from among the following groups of compounds: platinum compounds, cytotoxic antibiotics, antimetabolites, anti-mitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof. Exemplary compounds include, but are not limited to, alkylating agents: cisplatin, treosulfan, and trofosfamide; plant alkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomerase inhibitors: teniposide, crisnatol, and mitomycin; anti-folates: methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxifluridine, and cytosine arabinoside; purine analogs:

mercaptopurine and thioguanine; DNA antimetabolites: 2'-deoxy-5-fluorouridine, aphidicolin glycinate, and pyrazoloimidazole; and antimitotic agents: halichondrin, colchicine, and rhizoxin. Compositions comprising one or more chemotherapeutic agents (e.g., FLAG, CHOP) may also be used. FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone. In another embodiments, PARP (e.g., PARP-1 and/or PARP-2) inhibitors are used and such inhibitors are well-known in the art (e.g., Olaparib, ABT-888, BSI-201, BGP-15 (N-Gene Research Laboratories, Inc.); INO-1001 (Inotek Pharmaceuticals Inc.); PJ34 (Soriano et al, 2001; Pacher et al, 2002b); 3-aminobenzamide (Trevigen); 4-amino- 1,8-naphthalimide; (Trevigen); 6(5H)-phenanthridinone (Trevigen); benzamide (U.S. Pat. Re. 36,397); and NU1025 (Bowman et al). The mechanism of action is generally related to the ability of PARP inhibitors to bind PARP and decrease its activity. PARP catalyzes the conversion of .beta. -nicotinamide adenine dinucleotide (NAD+) into nicotinamide and poly-ADP-ribose (PAR). Both poly (ADP-ribose) and PARP have been linked to regulation of transcription, cell proliferation, genomic stability, and carcinogenesis

(Bouchard V. J. etal. Experimental Hematology, Volume 31, Number 6, June 2003, pp. 446-454(9); Herceg Z.; Wang Z.-Q. Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 477, Number 1, 2 Jun. 2001, pp. 97-110(14)).

Poly(ADP-ribose) polymerase 1 (PARP1) is a key molecule in the repair of DNA single- strand breaks (SSBs) (de Murcia J. et al. 1997. Proc Natl Acad Sci USA 94:7303-7307; Schreiber V, Dantzer F, Ame J C, de Murcia G (2006) Nat Rev Mol Cell Biol 7:517-528; Wang Z Q, et al. (1997) Genes Dev 11 :2347-2358). Knockout of SSB repair by inhibition of PARP1 function induces DNA double-strand breaks (DSBs) that can trigger synthetic lethality in cancer cells with defective homology-directed DSB repair (Bryant H E, et al. (2005) Nature 434:913-917; Farmer H, et al. (2005) Nature 434:917-921). The foregoing examples of chemotherapeutic agents are illustrative, and are not intended to be limiting.

In another embodiment, radiation therapy is used. The radiation used in radiation therapy can be ionizing radiation. Radiation therapy can also be gamma rays, X-rays, or proton beams. Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and/or total abdominal and pelvic radiation therapy. For a general overview of radiation therapy, see Hellman, Chapter 16: Principles of Cancer Management: Radiation Therapy, 6th edition, 2001, DeVita et al, eds., J. B. Lippencott Company, Philadelphia. The radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source. The radiation treatment can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass. Also encompassed is the use of photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA), phthalocyanine,

photosensitizer Pc4, demethoxy-hypocrellin A; and 2BA-2-DMHA.

In another embodiment, surgical intervention can occur to physically remove cancerous cells and/or tissues.

In still another embodiment, hormone therapy is used. Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists {e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A derivatives {e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens {e.g., mifepristone, onapristone), or antiandrogens {e.g., cyproterone acetate).

In yet another embodiment, hyperthermia, a procedure in which body tissue is exposed to high temperatures (up to 106°F.) is used. Heat may help shrink tumors by damaging cells or depriving them of substances they need to live. Hyperthermia therapy can be local, regional, and whole-body hyperthermia, using external and internal heating devices. Hyperthermia is almost always used with other forms of therapy (e.g., radiation therapy, chemotherapy, and biological therapy) to try to increase their effectiveness. Local hyperthermia refers to heat that is applied to a very small area, such as a tumor. The area may be heated externally with high-frequency waves aimed at a tumor from a device outside the body. To achieve internal heating, one of several types of sterile probes may be used, including thin, heated wires or hollow tubes filled with warm water; implanted microwave antennae; and radiofrequency electrodes. In regional hyperthermia, an organ or a limb is heated. Magnets and devices that produce high energy are placed over the region to be heated. In another approach, called perfusion, some of the patient's blood is removed, heated, and then pumped (perfused) into the region that is to be heated internally. Whole- body heating is used to treat metastatic cancer that has spread throughout the body. It can be accomplished using warm-water blankets, hot wax, inductive coils (like those in electric blankets), or thermal chambers (similar to large incubators). Hyperthermia does not cause any marked increase in radiation side effects or complications. Heat applied directly to the skin, however, can cause discomfort or even significant local pain in about half the patients treated. It can also cause blisters, which generally heal rapidly.

In still another embodiment, photodynamic therapy (also called PDT, photoradiation therapy, phototherapy, or photochemotherapy) is used for the treatment of some types of cancer. It is based on the discovery that certain chemicals known as photosensitizing agents can kill one-celled organisms when the organisms are exposed to a particular type of light. PDT destroys cancer cells through the use of a fixed-frequency laser light in combination with a photosensitizing agent. In PDT, the photosensitizing agent is injected into the bloodstream and absorbed by cells all over the body. The agent remains in cancer cells for a longer time than it does in normal cells. When the treated cancer cells are exposed to laser light, the photosensitizing agent absorbs the light and produces an active form of oxygen that destroys the treated cancer cells. Light exposure must be timed carefully so that it occurs when most of the photosensitizing agent has left healthy cells but is still present in the cancer cells. The laser light used in PDT can be directed through a fiberoptic (a very thin glass strand). The fiber-optic is placed close to the cancer to deliver the proper amount of light. The fiber-optic can be directed through a bronchoscope into the lungs for the treatment of lung cancer or through an endoscope into the esophagus for the treatment of esophageal cancer. An advantage of PDT is that it causes minimal damage to healthy tissue. However, because the laser light currently in use cannot pass through more than about 3 centimeters of tissue (a little more than one and an eighth inch), PDT is mainly used to treat tumors on or just under the skin or on the lining of internal organs.

Photodynamic therapy makes the skin and eyes sensitive to light for 6 weeks or more after treatment. Patients are advised to avoid direct sunlight and bright indoor light for at least 6 weeks. If patients must go outdoors, they need to wear protective clothing, including sunglasses. Other temporary side effects of PDT are related to the treatment of specific areas and can include coughing, trouble swallowing, abdominal pain, and painful breathing or shortness of breath. In December 1995, the U.S. Food and Drug Administration (FDA) approved a photosensitizing agent called porfimer sodium, or Photofrin®, to relieve symptoms of esophageal cancer that is causing an obstruction and for esophageal cancer that cannot be satisfactorily treated with lasers alone. In January 1998, the FDA approved porfimer sodium for the treatment of early non-small cell lung cancer in patients for whom the usual treatments for lung cancer are not appropriate. The National Cancer Institute and other institutions are supporting clinical trials (research studies) to evaluate the use of photodynamic therapy for several types of cancer, including cancers of the bladder, brain, larynx, and oral cavity.

In yet another embodiment, laser therapy is used to harness high-intensity light to destroy cancer cells. This technique is often used to relieve symptoms of cancer such as bleeding or obstruction, especially when the cancer cannot be cured by other treatments. It may also be used to treat cancer by shrinking or destroying tumors. The term "laser" stands for light amplification by stimulated emission of radiation. Ordinary light, such as that from a light bulb, has many wavelengths and spreads in all directions. Laser light, on the other hand, has a specific wavelength and is focused in a narrow beam. This type of high- intensity light contains a lot of energy. Lasers are very powerful and may be used to cut through steel or to shape diamonds. Lasers also can be used for very precise surgical work, such as repairing a damaged retina in the eye or cutting through tissue (in place of a scalpel). Although there are several different kinds of lasers, only three kinds have gained wide use in medicine: Carbon dioxide (CO2) laser—This type of laser can remove thin layers from the skin's surface without penetrating the deeper layers. This technique is particularly useful in treating tumors that have not spread deep into the skin and certain precancerous conditions. As an alternative to traditional scalpel surgery, the CO2 laser is also able to cut the skin. The laser is used in this way to remove skin cancers.

Neodymium:yttrium-aluminum-garnet (Nd: YAG) laser— Light from this laser can penetrate deeper into tissue than light from the other types of lasers, and it can cause blood to clot quickly. It can be carried through optical fibers to less accessible parts of the body. This type of laser is sometimes used to treat throat cancers. Argon laser—This laser can pass through only superficial layers of tissue and is therefore useful in dermatology and in eye surgery. It also is used with light-sensitive dyes to treat tumors in a procedure known as photodynamic therapy (PDT). Lasers have several advantages over standard surgical tools, including: Lasers are more precise than scalpels. Tissue near an incision is protected, since there is little contact with surrounding skin or other tissue. The heat produced by lasers sterilizes the surgery site, thus reducing the risk of infection. Less operating time may be needed because the precision of the laser allows for a smaller incision. Healing time is often shortened; since laser heat seals blood vessels, there is less bleeding, swelling, or scarring. Laser surgery may be less complicated. For example, with fiber optics, laser light can be directed to parts of the body without making a large incision. More procedures may be done on an outpatient basis. Lasers can be used in two ways to treat cancer: by shrinking or destroying a tumor with heat, or by activating a chemical—known as a photosensitizing agent— that destroys cancer cells. In PDT, a photosensitizing agent is retained in cancer cells and can be stimulated by light to cause a reaction that kills cancer cells. C0 ₂ and Nd:YAG lasers are used to shrink or destroy tumors. They may be used with endoscopes, tubes that allow physicians to see into certain areas of the body, such as the bladder. The light from some lasers can be transmitted through a flexible endoscope fitted with fiber optics. This allows physicians to see and work in parts of the body that could not otherwise be reached except by surgery and therefore allows very precise aiming of the laser beam. Lasers also may be used with low-power microscopes, giving the doctor a clear view of the site being treated. Used with other instruments, laser systems can produce a cutting area as small as 200 microns in diameter— less than the width of a very fine thread. Lasers are used to treat many types of cancer. Laser surgery is a standard treatment for certain stages of glottis (vocal cord), cervical, skin, lung, vaginal, vulvar, and penile cancers. In addition to its use to destroy the cancer, laser surgery is also used to help relieve symptoms caused by cancer (palliative care). For example, lasers may be used to shrink or destroy a tumor that is blocking a patient's trachea (windpipe), making it easier to breathe. It is also sometimes used for palliation in colorectal and anal cancer. Laser- induced interstitial thermotherapy (LITT) is one of the most recent developments in laser therapy. LITT uses the same idea as a cancer treatment called hyperthermia; that heat may help shrink tumors by damaging cells or depriving them of substances they need to live. In this treatment, lasers are directed to interstitial areas (areas between organs) in the body. The laser light then raises the temperature of the tumor, which damages or destroys cancer cells.

The duration and/or dose of treatment with therapies may vary according to the particular therapeutic agent or combination thereof. An appropriate treatment time for a particular cancer therapeutic agent will be appreciated by the skilled artisan. The present invention contemplates the continued assessment of optimal treatment schedules for each cancer therapeutic agent, where the phenotype of the cancer of the subject as determined by the methods of the present invention is a factor in determining optimal treatment doses and schedules.

Any means for the introduction of a polynucleotide into mammals, human or non- human, or cells thereof may be adapted to the practice of this invention for the delivery of the various constructs of the present invention into the intended recipient. In one embodiment of the present invention, the DNA constructs are delivered to cells by transfection, i.e., by delivery of "naked" DNA or in a complex with a colloidal dispersion system. A colloidal system includes macromolecule complexes, nanocapsules,

microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a lipid- complexed or liposome-formulated DNA. In the former approach, prior to formulation of DNA, e.g., with lipid, a plasmid containing a transgene bearing the desired DNA constructs may first be experimentally optimized for expression (e.g., inclusion of an intron in the 5' untranslated region and elimination of unnecessary sequences (Feigner, et al. , Ann NY Acad Sci 126-139, 1995). Formulation of DNA, e.g. with various lipid or liposome materials, may then be effected using known methods and materials and delivered to the recipient mammal. See, e.g., Canonico et al, Am J Respir Cell Mol Biol 10:24-29, 1994; Tsan et al, Am J Physiol 268; Alton et al, Nat Genet. 5:135-142, 1993 and U.S. patent No. 5,679,647 by Carson et al.

The targeting of liposomes can be classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ- specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs, which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.

The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand. Naked DNA or DNA associated with a delivery vehicle, e.g., liposomes, can be administered to several sites in a subject (see below).

Nucleic acids can be delivered in any desired vector. These include viral or non- viral vectors, including adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and plasmid vectors. Exemplary types of viruses include HSV (herpes simplex virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia virus). Nucleic acids can be administered in any desired format that provides sufficiently efficient delivery levels, including in virus particles, in liposomes, in nanoparticles, and complexed to polymers.

The nucleic acids encoding a protein or nucleic acid of interest may be in a plasmid or viral vector, or other vector as is known in the art. Such vectors are well-known and any can be selected for a particular application. In one embodiment of the present invention, the gene delivery vehicle comprises a promoter and a demethylase coding sequence.

Preferred promoters are tissue-specific promoters and promoters which are activated by cellular proliferation, such as the thymidine kinase and thymidylate synthase promoters. Other preferred promoters include promoters which are activatable by infection with a virus, such as the a- and β-interferon promoters, and promoters which are activatable by a hormone, such as estrogen. Other promoters which can be used include the Moloney virus LTR, the CMV promoter, and the mouse albumin promoter. A promoter may be constitutive or inducible. In another embodiment, naked polynucleotide molecules are used as gene delivery vehicles, as described in WO 90/11092 and U.S. Patent 5,580,859. Such gene delivery vehicles can be either growth factor DNA or RNA and, in certain embodiments, are linked to killed adenovirus. Curiel et al, Hum. Gene. Ther. 3:147-154, 1992. Other vehicles which can optionally be used include DNA-ligand (Wu et al. , J. Biol. Chem.

264:16985-16987, 1989), lipid-DNA combinations (Feigner et al, Proc. Natl. Acad. Sci. USA 84:7413 7417, 1989), liposomes (Wang et al, Proc. Natl. Acad. Sci. 84:7851-7855, 1987) and microprojectiles (Williams et al, Proc. Natl. Acad. Sci. 88:2726-2730, 1991).

A gene delivery vehicle can optionally comprise viral sequences such as a viral origin of replication or packaging signal. These viral sequences can be selected from viruses such as astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, retrovirus, togavirus or adenovirus. In a preferred embodiment, the growth factor gene delivery vehicle is a recombinant retroviral vector. Recombinant retroviruses and various uses thereof have been described in numerous references including, for example, Mann et al, Cell 33:153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci. USA 81:6349, 1984, Miller et al, Human Gene Therapy 1:5-14, 1990, U.S. Patent Nos. 4,405,712, 4,861,719, and 4,980,289, and PCT Application Nos. WO 89/02,468, WO 89/05,349, and WO 90/02,806. Numerous retroviral gene delivery vehicles can be utilized in the present invention, including for example those described in EP 0,415,731 ; WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; U.S. Patent No. 5,219,740; WO 9311230; WO 9310218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart, Cancer Res. 53:962-967, 1993; Ram et al, Cancer Res. 53:83-88, 1993; Takamiya et al, J. Neurosci. Res. 33:493-503, 1992; Baba et al, J. Neurosurg.

79:729-735, 1993 (U.S. Patent No. 4,777,127, GB 2,200,651, EP 0,345,242 and

WO91/02805).

Other viral vector systems that can be used to deliver a polynucleotide of the present invention have been derived from herpes virus, e.g., Herpes Simplex Virus (U.S. Patent No. 5,631,236 by Woo et al, issued May 20, 1997 and WO 00/08191 by Neurovex), vaccinia virus (Ridgeway (1988) Ridgeway, "Mammalian expression vectors," In: Rodriguez R L, Denhardt D T, ed. Vectors: A survey of molecular cloning vectors and their uses.

Stoneham: Butterworth,; Baichwal and Sugden (1986) "Vectors for gene transfer derived from animal DNA viruses: Transient and stable expression of transferred genes," In:

Kucherlapati R, ed. Gene transfer. New York: Plenum Press; Coupar et al. (1988) Gene, 68:1-10), and several RNA viruses. Preferred viruses include an alphavirus, a poxivirus, an arena virus, a vaccinia virus, a polio virus, and the like. They offer several attractive features for various mammalian cells (Friedmann (1989) Science, 244:1275-1281;

Ridgeway, 1988, supra; Baichwal and Sugden, 1986, supra; Coupar et ah, 1988; Horwich et ah (1990) J.Virol., 64:642-650).

In other embodiments, target DNA in the genome can be manipulated using well- known methods in the art. For example, the target DNA in the genome can be manipulated by deletion, insertion, and/or mutation are retroviral insertion, artificial chromosome techniques, gene insertion, random insertion with tissue specific promoters, gene targeting, transposable elements and/or any other method for introducing foreign DNA or producing modified DNA/modified nuclear DNA. Other modification techniques include deleting DNA sequences from a genome and/or altering nuclear DNA sequences. Nuclear DNA sequences, for example, may be altered by site-directed mutagenesis.

In other embodiments, recombinant biomarker polypeptides, and fragments thereof, can be administered to subjects. In some embodiments, fusion proteins can be constructed and administered which have enhanced biological properties. In addition, the biomarker polypeptides, and fragment thereof, can be modified according to well-known

pharmacological methods in the art {e.g., pegylation, glycosylation, oligomerization, etc.) in order to further enhance desirable biological activities, such as increased bioavailability and decreased proteolytic degradation.

4. Clincal Efficacy

Clinical efficacy can be measured by any method known in the art. For example, the response to a therapy, such as inhibitors of one or more biomarkers in Table 1 {e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) and immunotherapy combination treatment, relates to any response of the cancer, e.g., a tumor, to the therapy, preferably to a change in tumor mass and/or volume after initiation of neoadjuvant or adjuvant chemotherapy. Tumor response may be assessed in a neoadjuvant or adjuvant situation where the size of a tumor after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation and the cellularity of a tumor can be estimated histologically and compared to the cellularity of a tumor biopsy taken before initiation of treatment. Response may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response may be recorded in a quantitative fashion like percentage change in tumor volume or cellularity or using a semi-quantitative scoring system such as residual cancer burden (Symmans et al., J. Clin. Oncol. (2007) 25:4414-4422) or Miller-Payne score (Ogston et al, (2003) Breast (Edinburgh, Scotland) 12:320-327) in a qualitative fashion like "pathological complete response" (pCR), "clinical complete remission" (cCR), "clinical partial remission" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD) or other qualitative criteria. Assessment of tumor response may be performed early after the onset of neoadjuvant or adjuvant therapy, e.g., after a few hours, days, weeks or preferably after a few months. A typical endpoint for response assessment is upon termination of neoadjuvant chemotherapy or upon surgical removal of residual tumor cells and/or the tumor bed.

In some embodiments, clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR). The clinical benefit rate is measured by determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out from the end of therapy. The shorthand for this formula is CBR=CR+PR+SD over 6 months. In some embodiments, the CBR for a particular anti-immune checkpoint therapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more.

Additional criteria for evaluating the response to immunotherapies, such as anti- immune checkpoint therapies, are related to "survival," which includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); "recurrence-free survival" (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith). The length of said survival may be calculated by reference to a defined start point {e.g., time of diagnosis or start of treatment) and end point {e.g., death, recurrence or metastasis). In addition, criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.

For example, in order to determine appropriate threshold values, a particular anticancer therapeutic regimen can be administered to a population of subjects and the outcome can be correlated to biomarker measurements that were determined prior to administration of any immunotherapy, such as anti-immune checkpoint therapy. The outcome

measurement may be pathologic response to therapy given in the neoadjuvant setting. Alternatively, outcome measures, such as overall survival and disease-free survival can be monitored over a period of time for subjects following immunotherapies for whom biomarker measurement values are known. In certain embodiments, the same doses of immunotherapy agents, if any, are administered to each subject. In related embodiments, the doses administered are standard doses known in the art for those agents used in immunotherapies. The period of time for which subjects are monitored can vary. For example, subjects may be monitored for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months. Biomarker measurement threshold values that correlate to outcome of an immunotherapy can be determined using methods such as those described in the Examples section. 5. Further Uses and Methods of the Present Invention

The compositions described herein can be used in a variety of diagnostic, prognostic, and therapeutic applications. In any method described herein, such as a diagnostic method, prognostic method, therapeutic method, or combination thereof, all steps of the method can be performed by a single actor or, alternatively, by more than one actor. For example, diagnosis can be performed directly by the actor providing therapeutic treatment. Alternatively, a person providing a therapeutic agent can request that a diagnostic assay be performed. The diagnostician and/or the therapeutic interventionist can interpret the diagnostic assay results to determine a therapeutic strategy. Similarly, such alternative processes can apply to other assays, such as prognostic assays.

a. Screening Methods

One aspect of the present invention relates to screening assays, including non-cell based assays and xenograft animal model assays. In one embodiment, the assays provide a method for identifying whether a cancer is likely to respond to inhibitor of one or more biomarker s in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) and immunotherapy combination treatments, such as in a human by using a xenograft animal model assay, and/or whether an agent can inhibit the growth of or kill a cancer cell that is unlikely to respond to such inhibitors and immunotherapy combination treatments. In one embodiment, the present invention relates to assays for screening test agents which bind to, or modulate the biological activity of, at least one biomarker described herein (e.g., in the tables, figures, examples, or otherwise in the specification). In one embodiment, a method for identifying such an agent entails determining the ability of the agent to modulate, e.g. inhibit, the at least one biomarker described herein.

In one embodiment, an assay is a cell-free or cell-based assay, comprising contacting at least one biomarker described herein, with a test agent, and determining the ability of the test agent to modulate (e.g., inhibit) the enzymatic activity of the biomarker, such as by measuring direct binding of substrates or by measuring indirect parameters as described below.

For example, in a direct binding assay, biomarker protein (or their respective target polypeptides or molecules) can be coupled with a radioisotope or enzymatic label such that binding can be determined by detecting the labeled protein or molecule in a complex. For example, the targets can be labeled with ¹²⁵ 1, ³⁵ S, ¹⁴ C, or ³ H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting. Alternatively, the targets can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

Determining the interaction between biomarker and substrate can also be accomplished using standard binding or enzymatic analysis assays. In one or more embodiments of the above described assay methods, it may be desirable to immobilize polypeptides or molecules to facilitate separation of complexed from uncomplexed forms of one or both of the proteins or molecules, as well as to accommodate automation of the assay.

Binding of a test agent to a target can be accomplished in any vessel suitable for containing the reactants. Non-limiting examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. Immobilized forms of the antibodies described herein can also include antibodies bound to a solid phase like a porous, microporous (with an average pore diameter less than about one micron) or macroporous (with an average pore diameter of more than about 10 microns) material, such as a membrane, cellulose, nitrocellulose, or glass fibers; a bead, such as that made of agarose or polyacrylamide or latex; or a surface of a dish, plate, or well, such as one made of polystyrene.

In an alternative embodiment, determining the ability of the agent to modulate the interaction between the biomarker and a substrate or a biomarker and its natural binding partner can be accomplished by determining the ability of the test agent to modulate the activity of a polypeptide or other product that functions downstream or upstream of its position within the signaling pathway (e.g., feedback loops). Such feedback loops are well- known in the art (see, for example, Chen and Guillemin (2009) Int. J. Tryptophan Res. 2:1- 19).

The present invention further pertains to novel agents identified by the above- described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein, such as in an appropriate animal model. For example, an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.

Alternatively, an antibody identified as described herein can be used in an animal model to determine the mechanism of action of such an agent,

b. Predictive Medicine

The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining the amount and/or activity level of a biomarker described herein in the context of a biological sample (e.g., blood, serum, cells, or tissue) to thereby determine whether an individual afflicted with a cancer is likely to respond to inhibitors of one or more biomarkers in Table 1 (e.g. , one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) and immunotherapy combination treatments, such as in a cancer. Such assays can be used for prognostic or predictive purpose alone, or can be coupled with a therapeutic intervention to thereby prophylactically treat an individual prior to the onset or after recurrence of a disorder characterized by or associated with biomarker polypeptide, nucleic acid expression or activity. The skilled artisan will appreciate that any method can use one or more (e.g. , combinations) of biomarkers described herein, such as those in the tables, figures, examples, and otherwise described in the specification.

Another aspect of the present invention pertains to monitoring the influence of agents (e.g., drugs, compounds, and small nucleic acid-based molecules) on the expression or activity of a biomarker described herein. These and other agents are described in further detail in the following sections. The skilled artisan will also appreciated that, in certain embodiments, the methods of the present invention implement a computer program and computer system. For example, a computer program can be used to perform the algorithms described herein. A computer system can also store and manipulate data generated by the methods of the present invention which comprises a plurality of biomarker signal changes/profiles which can be used by a computer system in implementing the methods of this invention. In certain embodiments, a computer system receives biomarker expression data; (ii) stores the data; and (iii) compares the data in any number of ways described herein (e.g., analysis relative to appropriate controls) to determine the state of informative biomarkers from cancerous or pre-cancerous tissue. In other embodiments, a computer system (i) compares the determined expression biomarker level to a threshold value; and (ii) outputs an indication of whether said biomarker level is significantly modulated (e.g., above or below) the threshold value, or a phenotype based on said indication.

In certain embodiments, such computer systems are also considered part of the present invention. Numerous types of computer systems can be used to implement the analytic methods of this invention according to knowledge possessed by a skilled artisan in the bioinformatics and/or computer arts. Several software components can be loaded into memory during operation of such a computer system. The software components can comprise both software components that are standard in the art and components that are special to the present invention (e.g., dCHIP software described in Lin et al. (2004)

Bioinformatics 20, 1233-1240; radial basis machine learning algorithms (RBM) known in the art).

The methods of the present invention can also be programmed or modeled in mathematical software packages that allow symbolic entry of equations and high-level specification of processing, including specific algorithms to be used, thereby freeing a user of the need to procedurally program individual equations and algorithms. Such packages include, e.g., Matlab from Mathworks (Natick, Mass.), Mathematica from Wolfram

Research (Champaign, 111.) or S-Plus from MathSoft (Seattle, Wash.).

In certain embodiments, the computer comprises a database for storage of biomarker data. Such stored profiles can be accessed and used to perform comparisons of interest at a later point in time. For example, biomarker expression profiles of a sample derived from the non-cancerous tissue of a subject and/or profiles generated from population-based distributions of informative loci of interest in relevant populations of the same species can be stored and later compared to that of a sample derived from the cancerous tissue of the subject or tissue suspected of being cancerous of the subject.

In addition to the exemplary program structures and computer systems described herein, other, alternative program structures and computer systems will be readily apparent to the skilled artisan. Such alternative systems, which do not depart from the above described computer system and programs structures either in spirit or in scope, are therefore intended to be comprehended within the accompanying claims.

c. Diagnostic Assays

The present invention provides, in part, methods, systems, and code for accurately classifying whether a biological sample is associated with a cancer that is likely to respond to inhibitors of one or more biomarkers in Table 1 {e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) and immunotherapy combination treatments. In some embodiments, the present invention is useful for classifying a sample {e.g., from a subject) as associated with or at risk for responding to or not responding to such inhibitor and immunotherapy combination treatments using a statistical algorithm and/or empirical data {e.g., the amount or activity of a biomarker described herein, such as in the tables, figures, examples, and otherwise described in the specification).

An exemplary method for detecting the amount or activity of a biomarker described herein, and thus useful for classifying whether a sample is likely or unlikely to respond to inhibitors of one or more biomarkers in Table 1 {e.g. , one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) and immunotherapy combination treatments involves obtaining a biological sample from a test subject and contacting the biological sample with an agent, such as a protein-binding agent like an antibody or antigen-binding fragment thereof, or a nucleic acid-binding agent like an oligonucleotide, capable of detecting the amount or activity of the biomarker in the biological sample. In some embodiments, at least one antibody or antigen-binding fragment thereof is used, wherein two, three, four, five, six, seven, eight, nine, ten, or more such antibodies or antibody fragments can be used in combination {e.g., in sandwich ELISAs) or in serial. In certain instances, the statistical algorithm is a single learning statistical classifier system. For example, a single learning statistical classifier system can be used to classify a sample as a based upon a prediction or probability value and the presence or level of the biomarker. The use of a single learning statistical classifier system typically classifies the sample as, for example, a likely immunotherapy responder or progressor sample with a sensitivity, specificity, positive predictive value, negative predictive value, and/or overall accuracy of at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

Other suitable statistical algorithms are well-known to those of skill in the art. For example, learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex data sets (e.g., panel of markers of interest) and making decisions based upon such data sets. In some embodiments, a single learning statistical classifier system such as a classification tree (e.g., random forest) is used. In other embodiments, a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem. Examples of learning statistical classifier systems include, but are not limited to, those using inductive learning (e.g.,

decision/classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), Probably Approximately Correct (PAC) learning,

connectionist learning (e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons such as multi-layer perceptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc.), reinforcement learning (e.g., passive learning in a known

environment such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement learning, etc.), and genetic algorithms and evolutionary programming. Other learning statistical classifier systems include support vector machines (e.g., Kernel methods), multivariate adaptive regression splines (MARS), Levenberg-Marquardt algorithms, Gauss-Newton algorithms, mixtures of Gaussians, gradient descent algorithms, and learning vector quantization (LVQ). In certain embodiments, the method of the present invention further comprises sending the sample classification results to a clinician, e.g., an oncologist.

In another embodiment, the diagnosis of a subject is followed by administering to the individual a therapeutically effective amount of a defined treatment based upon the diagnosis.

In one embodiment, the methods further involve obtaining a control biological sample (e.g., biological sample from a subject who does not have a cancer or whose cancer is susceptible to inhibitors of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) and immunotherapy combination treatments), a biological sample from the subject during remission, or a biological sample from the subject during treatment for developing a cancer progressing despite such inhibitors and immunotherapy combination treatments,

d. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a cancer that is likely or unlikely to be responsive to inhibitors of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) and immunotherapy combination treatments. The assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with a misregulation of the amount or activity of at least one biomarker described herein, such as in cancer. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disorder associated with a misregulation of the at least one biomarker described herein, such as in cancer. Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with the aberrant biomarker expression or activity,

e. Treatment Methods

The therapeutic compositions described herein, such as the combination of inhibitors of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1) and immunotherapy, can be used in a variety of in vitro and in vivo therapeutic applications using the formulations and/or combinations described herein. In one embodiment, the therapeutic agents can be used to treat cancers determined to be responsive thereto. For example, single or multiple agents that inhibit or block both such inhibitors and a immunotherapy can be used to treat cancers in subjects identified as likely responders thereto.

Modulatory methods of the present invention involve contacting a cell, such as an immune cell with an agent that inhibits or blocks the expression and/or activity of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, e.g. such as

PTPN2 and SOCS1) and an immunotherapy, such as an immune checkpoint inhibitor (e.g., PD-1). Exemplary agents useful in such methods are described above. Such agents can be administered in vitro or ex vivo (e.g., by contacting the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods useful for treating an individual afflicted with a condition that would benefit from an increased immune response, such as an infection or a cancer like colorectal cancer.

Agents that upregulate immune responses can be in the form of enhancing an existing immune response or eliciting an initial immune response. Thus, enhancing an immune response using the subject compositions and methods is useful for treating cancer, but can also be useful for treating an infectious disease (e.g., bacteria, viruses, or parasites), a parasitic infection, and an immunosuppressive disease.

Exemplary infectious disorders include viral skin diseases, such as Herpes or shingles, in which case such an agent can be delivered topically to the skin. In addition, systemic viral diseases, such as influenza, the common cold, and encephalitis might be alleviated by systemic administration of such agents. In one preferred embodiment, agents that upregulate the immune response described herein are useful for modulating the arginase/iNOS balance during Trypanosoma cruzi infection in order to facilitate a protective immune response against the parasite.

Immune responses can also be enhanced in an infected patient through an ex vivo approach, for instance, by removing immune cells from the patient, contacting immune cells in vitro with an agent described herein and reintroducing the in vitro stimulated immune cells into the patient.

In certain instances, it may be desirable to further administer other agents that upregulate immune responses, for example, forms of other B7 family members that transduce signals via costimulatory receptors, in order to further augment the immune response. Such additional agents and therapies are described further below.

Agents that upregulate an immune response can be used prophylactically in vaccines against various polypeptides (e.g., polypeptides derived from pathogens).

Immunity against a pathogen (e.g. , a virus) can be induced by vaccinating with a viral protein along with an agent that upregulates an immune response, in an appropriate adjuvant.

In another embodiment, upregulation or enhancement of an immune response function, as described herein, is useful in the induction of tumor immunity.

In another embodiment, the immune response can be stimulated by the methods described herein, such that preexisting tolerance, clonal deletion, and/or exhaustion (e.g., T cell exhaustion) is overcome. For example, immune responses against antigens to which a subject cannot mount a significant immune response, e.g., to an autologous antigen, such as a tumor specific antigens can be induced by administering appropriate agents described herein that upregulate the immune response. In one embodiment, an autologous antigen, such as a tumor-specific antigen, can be coadministered. In another embodiment, the subject agents can be used as adjuvants to boost responses to foreign antigens in the process of active immunization.

In one embodiment, immune cells are obtained from a subject and cultured ex vivo in the presence of an agent as described herein, to expand the population of immune cells and/or to enhance immune cell activation. In a further embodiment the immune cells are then administered to a subject. Immune cells can be stimulated in vitro by, for example, providing to the immune cells a primary activation signal and a costimulatory signal, as is known in the art. Various agents can also be used to costimulate proliferation of immune cells. In one embodiment immune cells are cultured ex vivo according to the method described in PCT Application No. WO 94/29436. The costimulatory polypeptide can be soluble, attached to a cell membrane, or attached to a solid surface, such as a bead.

6. Administration of Agents

The immune modulating agents of the invention are administered to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo, to enhance immune cell mediated immune responses. By "biologically compatible form suitable for administration in vivo" is meant a form to be administered in which any toxic effects are outweighed by the therapeutic effects. The term "subject" is intended to include living organisms in which an immune response can be elicited, e.g., mammals. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.

Administration of an agent as described herein can be in any pharmacological form including a therapeutically active amount of an agent alone or in combination with a pharmaceutically acceptable carrier.

Administration of a therapeutically active amount of the therapeutic composition of the present invention is defined as an amount effective, at dosages and for periods of time necessary, to achieve the desired result. For example, a therapeutically active amount of an agent may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of peptide to elicit a desired response in the individual. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

Inhibiting or blocking expression and/or activity of one or more biomarkers in Table 1 (e.g., one or more kinase signaling inhibitors, such as PTPN2 and SOCS1), alone or in combination with an immunotherapy, can be accomplished by combination therapy with the modulatory agents described herein. Combination therapy describes a therapy in which one or more biomarkers in Table 1 are inhibited or blocked with an immunotherapy

simultaneously. This may be achieved by administration of the modulatory agent described herein swith the immunotherapy imultaneously (e.g., in a combination dosage form or by simultaneous administration of single agents) or by admimstration of single inhibitory agent for one or more biomarkers in Table 1 and the immunotherapy, according to a schedule that results in effective amounts of each modulatory agent present in the patient at the same time.

The therapeutic agents described herein can be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active compound can be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. For example, for administration of agents, by other than parenteral administration, it may be desirable to coat the agent with, or co-administer the agent with, a material to prevent its inactivation.

An agent can be administered to an individual in an appropriate carrier, diluent or adjuvant, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon. Adjuvants contemplated herein include resorcinols, non- ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluorophosphate (DEEP) and trasylol. Liposomes include water-in-oil-in- water emulsions as well as conventional liposomes (Sterna et al. (1984) J. Neuroimmunol. 7:27).

As described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or

intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.

The phrase "therapeutically-effective amount" as used herein means that amount of an agent that modulates (e.g., inhibits) biomarker expression and/or activity, or expression and/or activity of the complex, or composition comprising an agent that modulates (e.g. , inhibits) biomarker expression and/or activity, or expression and/or activity of the complex, which is effective for producing some desired therapeutic effect, e.g., cancer treatment, at a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable" is employed herein to refer to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and

polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

The term "pharmaceutically-acceptable salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of the agents that modulates (e.g. , inhibits) biomarker expression and/or activity, or expression and/or activity of the complex encompassed by the present invention. These salts can be prepared in situ during the final isolation and purification of the therapeutic agents, or by separately reacting a purified therapeutic agent in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed. Representative salts include the hydrobromide,

hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J Pharm. Sci. 66:1- 19).

In other cases, the agents useful in the methods of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically- acceptable salts with pharmaceutically-acceptable bases. The term "pharmaceutically- acceptable salts" in these instances refers to the relatively non-toxic, inorganic and organic base addition salts of agents that modulates (e.g. , inhibits) biomarker expression and/or activity, or expression and/or activity of the complex. These salts can likewise be prepared in situ during the final isolation and purification of the therapeutic agents, or by separately reacting the purified therapeutic agent in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine. Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et al. , supra).

Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

Formulations useful in the methods of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient, which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.

Methods of preparing these formulations or compositions include the step of bringing into association an agent that modulates (e.g., inhibits) biomarker expression and/or activity, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a therapeutic agent with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a therapeutic agent as an active ingredient. A compound may also be administered as a bolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more

pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the

pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well-known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the

gastrointestinal tract, optionally, in a delayed manner. Examples of embedding

compositions, which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active agent may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more therapeutic agents with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of an agent that modulates (e.g., inhibits) biomarker expression and/or activity include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to a therapeutic agent, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to an agent that modulates (e.g. , inhibits) biomarker expression and/or activity, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as

chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

The agent that modulates (e.g., inhibits) biomarker expression and/or activity, can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound.

Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (T weens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.

Transdermal patches have the added advantage of providing controlled delivery of a therapeutic agent to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the peptidomimetic across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the peptidomimetic in a polymer matrix or gel.

Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.

Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more therapeutic agents in combination with one or more

pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of

microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form.

Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of an agent that modulates (e.g., inhibits) biomarker expression and/or activity, in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.

When the therapeutic agents of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be determined by the methods of the present invention so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.

The nucleic acid molecules of the present invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g. , Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054- 3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

In one embodiment, an agent of the invention is an antibody. As defined herein, a therapeutically effective amount of antibody {i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of an antibody can include a single treatment or, preferably, can include a series of treatments. In a preferred example, a subject is treated with antibody in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of antibody used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result from the results of diagnostic assays. 7. Kits

The present invention also encompasses kits for detecting and/or modulating biomarkers described herein. A kit of the present invention may also include instructional materials disclosing or describing the use of the kit or an antibody of the disclosed invention in a method of the disclosed invention as provided herein. A kit may also include additional components to facilitate the particular application for which the kit is designed. For example, a kit may additionally contain means of detecting the label (e.g., enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a sheep anti-mouse-HRP, etc.) and reagents necessary for controls (e.g., control biological samples or standards). A kit may additionally include buffers and other reagents recognized for use in a method of the disclosed invention. Non-limiting examples include agents to reduce non-specific binding, such as a carrier protein or a detergent.

EXAMPLES

Example 1: Materials and methods for Examples 2-8

a. In vivo CRISPR screening in B16 tumor cells

A Cas9-expressing version of the B16 melanoma cell line was created and confirmed that it could edit DNA efficiently with CRISPR using sgRNAs targeting the PD- Ll gene. For screening the B16-Cas9 cell line, a library of 9,992 optimized sgRNAs was created to target 2,398 genes, selected from the GO term categories: kinase, phosphatase, cell surface, plasma membrane, antigen processing and presentation, immune system process, and chromatin remodeling. The transcript abundance of the genes in these categories were then filtered to include only those that were expressed > RPKM (log ₂ ) = 0.9. These genes were then ranked for expression in the Bl 6 cell line using RNAseq to select for the top 2,398 expressed genes. The library was divided into 4 sub-pools, each containing one sgRNA per gene and 100 non-targeting control sgRNAs. The 4 sub-pools were screened individually and sgRNAs were delivered to B16-Cas9 cells via lentiviral infection at an infection rate of 30%. Transduced B16 cells were purified using a hCD19 reporter by positive magnetic selection (Miltenyi Biotech, Cambridge, MA) and then expanded in vitro before being implanted into animals. For each sub-pool, B16 cells were implanted into 10 TCRa ^"A mice, 10 WT mice treated with GVAX, and 10 WT mice treated with GVAX and PD-1 blockade (see below for treatment protocols). B16 cells transduced with libraries were also grown in vitro at approximately 2000 x library coverage for the same time period as the animal experiment. Mice were sacrificed 12-14 days after tumor implantation tumor genomic DNA was prepared from whole tumor tissue using the Qiagen DNA Blood Midi kit (Qiagen, Hilden, Germany). PCR was used to amplify the sgRNA region and sequencing to determine sgRNA abundance was performed on an Illumina HiSeq system (Illumina, San Diego, CA). Significantly enriched or depleted sgRNAs from any comparison of conditions were identified using the STARS algorithm (Doench et al. (2014) Nat. Biotechnol. 32:1262-1267).

b. Cell lines

B16F10 melanoma and B16-GM-CSF cells were gifts from Dr. Glenn Dranoff. Braf/Pten melanoma cells were a gift from Dr. David Fisher. MC38 colon carcinoma cells were a gift from Dr. Arlene Sharpe. A375 melanoma, A549 lung carcinoma, and HT-29 colon carcinoma were purchased from ATCC. MelJuSo melanoma cells were a gift from the Cancer Cell Line Encyclopedia (Broad Institute). B16, MC38, A549, and HT-29 cells were all grown in DMEM (Gibco) with 10% fetal bovine serum (Gemini biosciences) and antibiotics. A375 and MelJuSo cells were grown in RPMI 1640 (Gibco) with 10% fetal bovine serum and antibiotics. All cell lines were subject to periodic testing for mycoplasma using the LookOut® Mycoplasma PCR detection kit (Sigma).

c. Animal treatment and tumor challenges

The designs of the animal studies and procedures were approved by the Dana Farber Cancer Institute IACUC committee. Dana Farber's specific-pathogen free facility was used for the storage and care of all mice. Seven-week old wild-type female C57BL/6J mice were obtained from Jackson laboratories (Bar Harbor, ME). A colony of B6.129S2- j _cra tmiMonyj (j _cra ) cell-deficient mice were bred on site. Mice were aged-matched to be 7-12 weeks old at the time of tumor inoculation. For screening, 2.0 x 10 ⁶ library- transduced B16-Cas9 cells resuspended in Hanks Balanced Salt Solution (Gibco, Thermo Fisher Scientific, Waltham, MA) were mixed 1 : 1 by volume with Matrigel ^® (Corning, Corning, NY) and subcutaneously injected into the right flank on day 0. Mice were vaccinated with 1.0 x 10 ⁶ GM-CSF-secreting B16 (GVAX) cells that had been irradiated with 3500 Gy on days 1 and 4 to elicit an anti-tumor immune response. Subsequently, mice were treated with 100 μg of rat monoclonal anti-PDl antibody (clone: 29F.1A12) on days 9 and 12 via intraperitoneal injection. For validation assays, 1.0 x 10 ⁶ tumor cells were subcutaneously injected into the right flank without matrigel. Tumors were measured every 3 days beginning on day 6 after challenge until time of death. Measurements were taken manually by collecting the longest dimension (length) and the longest perpendicular dimension (width). Tumor volume was estimated with the formula: (L x W ² )/2. CO2 inhalation was used to euthanize mice on the day of sacrifice,

d. Creation of CRISPR edited tumor cell lines

Transient transfection of Cas9-sgRNA plasmid (pX459, Addgene, Cambridge, MA) was used to edit B 16 and Braf/Pten melanoma cell lines. pX459 was digested with the enzyme Bpil (Thermo Fisher Scientific) as per the manufacturer's instructions and sgRNA oligos were cloned in using standard molecular cloning. For B16 cells, 5 x 10 ⁵ cells were plated in a well of a 6-well plate and were transfected the following day using 2 μg of pX459 plasmid DNA and Turbofect™ (3:1 ratio, Thermo Fisher Scientific). Twenty-four hours after transfection, transfectants were selected in puromycin (6 μg/mL, Thermo Fisher Scientific). For Braf/Pten melanoma cells, 5 x 10 ⁵ cells were plated in a well of a 6-well plate and were transfected the following day using 4 μg of pX459 plasmid DNA and Turbofect™ (3:1 ratio). After selection, cells were grown for 14 days in vitro before being implanted into mice.

e. In vivo competition assays

B16 cells were engineered to express GFP or TdTomato by lentiviral transduction to differentiate populations. Cas9-target sgRNA-transfected cells and Cas9-control sgRNA- transfected cells were mixed and then grown for at least two passages in vitro before implantation into mice. Mixes were analyzed by flow cytometry on the day of tumor inoculation. Mice were euthanized 15-21 days after tumor inoculation for tumor harvest. Tumors were macerated on ice and incubated in collagenase P (2 mg/mL, Sigma, St. Louis, MO) and DNase I (50 μg/mL, Sigma) supplemented DMEM for 10 minutes at 37°C. After incubation, tumor cells were passed through 70 μπι filters to remove undigested tumor. Tumor cells were washed with ice-cold MACS media and stained with Near-IR

LIVE/DEAD ^® (1:1000, BD Biosciences, Franklin Lakes, NJ) for 10 minutes on ice. Tumor cells were then washed and resuspended in ice-cold PBS with 2% FBS. An Accuri™ C6 flow cytometry system (BD Biosiences) was used to analyze final GFP/TdTomato tumor cell ratios.

f. In vitro cytokine stimulations

Differentially labeled B16 cells were plated in 12-well plates in DMEM + 10% FBS containing the indicated combinations of cytokines: TNFa (10 ng/mL, PeproTech, Rocky Hill, NJ), IFNy (100 ng/mL, Cell Signaling Technology, Danvers, MA), and IFN (1000 U/mL, Pbl Assay Science, Piscataway Township, NJ). Cells were passaged every 3 or 4 days. On day 14, tumor cells were analyzed by flow cytometry for changes in the ratio of GFP ⁺ /TdTomato ⁺ cells.

g. In vitro T cell killing assays

Differentially labeled, ovalbumin-expressing B16 cells were plated onto 12 well plates in DMEM + 10% FBS containing IFNp at 50 U/mL. The following day, media was removed and RPMI + 10% FBS was added containing preactivated OT-1 T cells at the indicated ratio. Tumor cells were passaged and fresh OT-1 T cells were added every 2 days until day 6. On day 6, tumor cells were analyzed by flow cytometry for changes in the ratio of GFP7TdTomato ⁺ cells.

h. Analysis tumor-infiltrating lymphocytes by flow cytometry

Mice were injected subcutaneously (s.c.) with 1.0 x 10 ⁶ CRISPR/Cas9-modified B16 cells and treated with GVAX + anti-PD-1 mAb as described above. Tumors were harvested on day 12-13, weighed, mechanically diced, incubated with collagenase P (2 mg/mL, Sigma Aldrich) and DNAse I (50 μg/mL, Sigma Aldrich) for 10 minutes, and pipetted into a single-cell suspension. After filtering through a 70 um filter, cells were blocked with anti-mouse CD 16/32 antibody (BioLegend, San Diego, CA) and stained with indicated antibodies for 30 minutes on ice. Dead cells were excluded using Aqua

Live/Dead ^® (1 :1000, ThermoFisher Scientific) added concurrently with surface antibodies. After washing, cells were fixed with Foxp3/transcription factor staining buffer set

(eBiosciences, San Diego, CA) as per manufacturer's instructions, blocked with mouse and rat serum, then stained with intracellular antibodies. AccuCount Fluorescent particles (Spherotech, Lake Forest, IL) were added for cell quantification prior to analysis on an LSR Fortessa™ cell analyzer (BD Biosciences) using single-color compensation controls and fluorescence-minus-one thresholds to set gate margins. Comparisons between groups were performed using Student's t test.

i. Flow cytometry analysis of B16 tumor cells

B16 cells were trypsinized and washed in PBS + 2% FBS, stained with antibodies for cell surface proteins as per the manufacturer's instructions and then analyzed on an Accuri™ C6 flow cytometry system (BD Biosciences),

j. Restimulation of T cells on B16 tumor cells

OT-I T cells were isolated from the spleens of OT-I T cell receptor transgenic mice using the CD8a ⁺ T cell isolation mouse kit (Miltenyi), as per manufacturer's instructions. Purified OT-I T cells were then stimulated in 24 well plates with plate-bound anti-mouse CD3s antibody (2 μg/ml, BD Biosciences), soluble anti-mouse CD28 antibody (2 μg/ml, BD Biosciences) and recombinant human IL-2 (100 U/ml, DFCI supply center). Fourty- eight hours later, activated OT-I T cells were transferred into fresh media containing recombinant human IL-2 and allowed to expand for 5-7 days. For restimulation, 1 x 10 ⁵ ovalbumin-expressing Ptpn2 null or control-guide transfected B 16 tumor cells were first plated in 24 well plates and stimulated with recombinant mouse IFNy overnight to induce ovalbumin surface expression. 1 x 10 ⁶ pre-activated OT-I T cells were then added to the wells on the next day and co-cultured with the B 16 tumor cells for 2-3 hrs. Subsequently, lx brefeldin (eBiosciences) was added to the cultures for 4 hours to inhibit intracellular protein transport. Afterward, OT-I T cells were harvested from each well, stained with surface markers and then fixed with Foxp3/transcription factor staining buffer set

(eBiosciences) as per the manufacturer's instructions. Intracellular cytokine staining was then performed prior to analysis on an LSR Fortessa™ cell analyzer (BD Biosciences), k. RNAseq analysis of tumor cells

Ptpn2 null or control sgRNA-transfected B 16 cells were stimulated with IFNy (100 ng/mL, Cell Signaling Technology), TNFa (10 ng/mL, Peprotech) or both for 48 hours. RNA was extracted from cell pellets using the Qiagen RNeasy Mini kit according to manufacturer's instructions. First-strand Illumina-barcoded libraries were generated using the NEB RNA Ultra ^™ Directional kit according to manufacturer's instructions, including a 12-cycle PCR enrichment. Libraries were sequenced on an Alumina NextSeq ^™ 500 instrument using paired-end 37 base pair (bp) reads. Data were trimmed for quality using the Trimmomatic pipeline with the following parameters: LEADING: 15 TRAILING: 15 SLIDINGWINDOW:4:15 MINLEN:16. Data were aligned to mouse reference genome mmlO using the Bowtie 2 aligning sequencing tool (available at the World Wide Web website of Johns Hopkins University). HTSeq was used to map aligned reads to genes and to generate a gene count matrix and it is available at the World Wide Web address of www- huber.embl.de/users/anders/HTSeq/doc/overview.htrnl. Normalized counts and differential expression analysis was performed using the DESeq2 R package. The gene set enrichment analysis was performed as described previously in Subramanian et al. (2005) Proc Natl Acad Sci USA 102:15545-15550. Principle Components Analysis (PCA) was performed on the normalized gene counts including all genes that passed a minimal expression filter. Signature scores for the individual samples were generated using FastProject (available at the World Wide Web address of

bmcbioinformatics.biomedcentral.com/articles/10.1186/sl28 59-016-l 176-5) and the Hallmark gene signature collection (Liberzon et al. (2015) Cell Sys. 1:417-425). Pearson correlation coefficients were calculated between the Hallmark gene signatures and PCI and PC2. Selected signatures were plotted on a normalized PCA projection of the dataset.

1. Western blotting

Whole cell ly sates were prepared in lysis buffer (60 mM Tris HC1, 2% SDS, 10% glycerol, complete EDTA-free protease-inhibitor (Roche, Basel, Switzerland), and 500 U/mL benzonase nuclease (Novagen, Merck, Darmstadt, Germany)). Samples were boiled at 100°C and clarified by centrifugation. Protein concentration was measured with a BCA protein assay kit (Pierce, Dallas, Texas). Fifty to one hundred and fifty micrograms of protein was loaded on 4-12% Bolt ^® Bis-Tris Plus gels (Life Technologies, Carlsbad, CA) in MES buffer (Life Technologies). Protein was transferred to 0.45 μηι nitrocellulose membranes (Bio-Rad, Hercules, CA). Membranes were blocked in Tris-buffered saline plus 0.1% Tween 20 (TBS-T) containing 5% non-fat dry milk for 1 hour at room

temperature followed by overnight incubation with primary antibody at 4°C. Membranes were washed with TBS-T and incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. HRP was activated with Supersignal ^® West Dura Extended Duration Substrate (Pierce) and visualized with a chemiluminscent detection system using Fuji ImageQuant ^™ LAS4000 (GE Healthcare Life Sciences, Pittsburgh, PA). Blots were then analyzed using ImageJ and Adobe ^® Photoshop ^® software,

m. Immunohistochemistrv

Whole B16 tumors were fixed for 24 hours in 10% neutral buffered formalin and then permeabilized in 70% ethanol overnight. Fixed tissue was then embedded into paraffin, sectioned, and then mounted onto slides for staining with mouse CD8a

(eBiosciences Cat. #14-0808-82). Slides were imaged on a Leica Scanscope XT and analyzed using Aperio software (Leica Biosy stems, Buffalo Grove, IL).

n. Antibodies

For Western blotting, primary antibodies against STAT1 (Cell Signaling, Cat.

#9172), JAK1 (Cell Signaling, Cat. #3332 and #3344 [clone 6G4]), phospho-STATl (Tyr701) (clone D4A7, Cell Signaling), β-ACTIN (Abeam, Cambridge, UK, Cat. #8227), TC-TPT (PTPN2) (Abeam, Cat. #180764), and FLAG (clone M2, Sigma Aldrich) were used. Peroxidase-conjugated secondaries against Rabbit-IgG (Cat. #111-035-046) and Mouse-IgG (Cat. #115-035-174) were purchased from Jackson Laboratories (Bar Harbor, ME).

For flow cytometry, the following anti-mouse (m) fluorochrome-conjugated antibodies were used: H2K(b)/H2D(b) (clone 28-8-6, BioLegend), CD47 (clone miap301, BioLegend), SIINFEKL-H2K(b) (clone 25-D1.16, BioLegend), Granzyme B (clone GB11, BioLegend), TNF (clone MP6-XT22, BioLegend), IFNy (clone XMG1.2, BioLegend),

CD8a (clone 53-6.7, BioLegend), CD4 (clone RM4-5 or GK15, BioLegend), TCR-β (clone H57-597, BioLegend), PD-1 (clone RPMI-30, BioLegend), Tim-3 (clone TMR3-2.3, BioLegend), CD45 (clone 104 or 30-F11, BioLegend), Ly6C (clone HK1.4, BioLegend), I- A/I-E (clone M5/114.15.2, BioLegend), F4/80 (clone BM8, BioLegend), CDl lc (clone N418, BioLegend), CD24 (clone Ml/69, BioLegend), CD1 lb (clone Ml/70, BioLegend), CD103 (clone 2E7, BioLegend), CD3s (clone 145-2C11, BioLegend), TCRy/δ (clone GL3, BioLegend), NK1.1 (clone PK136, BioLegend), CD44 (clone IM7, BioLegend), Ki-67 (clone B56, BD Biosciences), CD274 (clone MIH5, BD Biosciences), IFNGRl (clone 2E2, Life Technologies), IFNAR2 (R&D Systems; Cat. #FAB1083A), and Foxp3 (clone JFK- 16s, eBioscience).

o. CRISPR sgRNA sequences

CRISPR sgRNA sequences used are shown in Table 2 below:

Table 2

Examples 2-7 disclose the development of a pooled loss-of-function in vivo genetic screening approach that uses CRISPR-Cas9 genome editing to discover genes that increase sensitivity or cause resistance to immunotherapy in a mouse transplantable tumor model. About 2,400 genes expressed by tumor cells were screened in the B16 murine melanoma model to identify those that increase or decrease sensitivity to immunotherapy with tumor vaccination and PD-1 checkpoint blockade. The screen identified known immune evasion molecules PD-L1 and CD47, as tumor cells bearing sgRNAs for these targets were significantly depleted in animals treated with immunotherapy. In contrast, loss of function of any of the genes that sense or signal in response to interferon-γ (IFNy) rendered cells resistant to immunotherapy with PD-1 checkpoint blockade and vaccination, recapitulating resistance mutations identified in melanoma patients (Zaretsky et al. (2016) N. Engl. J. Med. 375:819-829; Gao et al. (2016) Cell 167:397-404.e9). In addition, deletion οϊΡΐρη2, which encodes a protein tyrosine phosphatase, significantly increased sensitivity of tumor cells to immunotherapy by increasing IFNy-mediated effects on antigen presentation and cell growth. These findings reveal that therapeutic strategies, such as Ptpn2 inhibition, that sensitize tumor cells to the effects of IFNy are capable of increasing the efficacy of cancer immunotherapy. Moreover, this screening approach can discover new immunotherapy targets in diverse, unanticipated biologic pathways and prioritize their combination with existing immunotherapies. Example 2: A pooled loss-of-function in vivo genetic screen recovers known immune evasion molecules expressed by tumors

In order to systematically identify new cancer immunotherapy targets and resistance mechanisms, a pooled genetic screening approach was developed to identify genes that increase or decrease the fitness of tumor cells growing in vivo in animals treated with immunotherapy (Figure 1A). First, a B16 melanoma cell line was engineered to express Cas9 (Figure 2 A), confirmed of efficient DNA editing using sgRNAs targeting PD-L1 (Figure ID, bottom). Next, a library of lentiviral vectors was created to encode 9,992 sgRNAs targeting 2,398 genes from relevant functional classes that were expressed at detectable levels in the tumor cell line (Figure 2B). After transduction and in vitro passage to allow gene editing to take place, the tumor cells were transplanted into animals that were then treated with either a GM-CSF-secreting, irradiated tumor cell vaccine (GVAX) or GVAX plus PD-1 blockade using a monoclonal antibody for PD-1, in order to apply immune selective pressure on the tumor cells (Figure IB) (see Dranoff (2003) Oncogene 22:3188-3192; Dranoff et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:3539-3543; and Duraiswamy et al. (2013) Cancer Res. 73:3591-3603; Curran & Allison (2009) Cancer Res 69:7747-7755; Curran et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107:4275-4280). In parallel, the library-transduced tumor cells were transplanted into TCRa ^"7" mice, which lack CD4 ⁺ and CD8 ⁺ T cells and were therefore unable to apply adaptive immune selective pressure on the tumors. This allowed the distinguishing of effects of immune selective pressure on library representation from nonspecific effects on tumor cell viability. After 12-14 days, tumors were harvested (Figure IB), with all sgRNAs recovered from each animal having good inter-animal reproducibility (Figures 2C-2E). The library representation in tumors from immunotherapy-treated wild-type (WT) animals were compared with that found in tumors growing in TCRa ^_/" mice, in which deletion of genes that result in resistance to immunotherapy would be expected to increase tumor sgRNA representation in WT animals, while deletion of genes that result in increased sensitivity of tumors to immunotherapy would decrease sgRNA representation. Analysis of sgRNAs enriched by immune selective pressure revealed those targeting genes involved in cytokine-mediating signaling and immune-system processes (Figure 1C). sgRNAs depleted by immunotherapy included those targeting genes involved in antigen processing, necroptosis, and regulation of immune responses (Figure 1C). These results indicate that the genetic screening approach used here identified genes expressed by tumors cells that play a role in interaction with the immune system.

Inspection of the list of genes targeted by sgRNAs depleted from tumors treated with immunotherapy revealed the known immune evasion molecule PD-L1, indicating that loss of PD-L1 increased the sensitivity of tumor cells to immune attack. sgRNAs targeting PD-L1 were not depleted from tumors in TCRa ^"A mice relative to cells growing in vitro, presumably due to the absence of T cell-mediated selective pressure (Figure ID), but were significantly depleted in WT mice treated with GVAX relative to TCRa ^"7" mice (FDR = 0.004). However, the depletion of PD-L1 -targeting sgRNAs seen in GVAX-treated tumors was not observed in tumors treated with GVAX and anti-PD-1, indicating that loss of PD- LI does not confer a selective disadvantage to tumors when PD-L1 :PD-1 interactions are blocked (Figure ID).

It was also found that sgRNAs targeting CD47, which enables immune evasion by impairing engulfment of tumors cells by phagocytes (as in Liu et al. (2015) Nat. Med. 21:1209-1215; Weiskopf et al. (2016) J Clin. Invest. 126:2610-2620; and Tseng et al. (2013) Proc. Natl. Acad. Sci. U.S.A. 110: 11103-11108), were markedly depleted in tumors treated with either GVAX or with GVAX plus PD-1 blockade (FDR = 0.005, 0.002 respectively) (Figure IE). To confirm that CD47 null tumors were more susceptible to GVAX and PD-1 blockade, CD47 null B16 melanoma cells were generated using transient transfection of a Cas9-sgRNA plasmid (as in Ran et al. (2013) Nat. Protoc. 8:2281-2308) (Figure 2F). It was found that loss of CD47 significantly improved control of tumor growth mediated by GVAX plus anti-PD-1 immunotherapy (Figure l¥,p< 0.01).

Using the pooled loss-of-function in vivo genetic screen for identifying immune evasion molecules expressed by tumors described above, genes that increase or decrease the fitness of MC38 colon cancer cells growing in vivo in animal treated with immunotherapy. Just as with the melanoma cells described above, Ptpn2 was identified among the top hits of approximately 442 genes which, when acquiring a loss-of-function mutation, improved the sensitivity to immunotherapy.

Thus, in vivo genetic screening recovered genes known to confer tumor evasion properties on cancer cells.

Example 3: Loss of IFNy sensing or signaling causes resistance to immunotherapy

Genes that, when deleted, become significantly enriched in immunotherapy treated tumors were analyzed, as these might represent resistance mechanisms. sgRNAs enriched under immune selective pressure included those targeting genes involved in cytokine signaling (Figure 1C). It was observed that sgRNAs targeting five genes required for sensing and signaling through the IFNy pathway (Statl, Jakl, Ifngr2, Ifngrl, and Jak2) were significantly enriched in immunotherapy-treated mice (Figure 3A; FDR<0.09). This finding was confirmed in an in vivo competitive assay that compared the relative growth of mixtures of isogenic Statl null or control B16 cells in animals treated with immunotherapy (Figure 3B). The ratio of Statl null cells to control cells in TCRa ^_/" mice was unchanged relative to cells grown in vitro (Figure 3C), indicating that loss of Statl does not change tumor cell growth or survival in vivo in the absence of T cells. However, in WT mice treated with GVAX plus anti-PD-1 immunotherapy, the relative proportion of Statl null cells increased significantly (Figure 3D; /?<0.01, Student's t test), indicating that Statl null cells have a marked growth advantage over WT tumor cells when under immune attack. Similar results were obtained for Jakl null and Ifngrl null cells (Figure 3D). Consistent with this finding, tumors deficient for Statl or Ifngrl (Figures 4A and 4C) grew

significantly faster than WT tumors when treated with immunotherapy (Figure 3E; /?<0.05, Student's t test). Similar trends were observed for Jakl null tumors (Figure 3E).

To test if this observation was seen in other melanoma models, IFNy sensing genes were deleted from the (Braf/Pten) murine melanoma line (Figures 4B-4D). A variant of the Braf/Pten model that had been subjected to sub-lethal irradiation was used to increase tumor responsiveness to immunotherapy (Lo et ah, submitted). Mice injected with tumor cells deficient in Statl, Jakl, or Ifngrl had significantly larger tumors and shorter survival compared to WT tumors when treated with PD-1 blockade (Figure 3F; p<0.05, Student's t test; p<0.0001, Log-rank test). Thus the efficacy of immunotherapy in both melanoma models depends in part on the sensing of IFNy by tumor cells.

Several mechanisms were tested to determine why IFNy-pathway mutant tumors were resistant to immunotherapy. First, the effect of gene deletion of Statl, Jakl or Ifngrl on cell growth in response to TNF, IFNy and IFNP was tested using an in vitro competition assay. IFNy-pathway deficient B16 tumor cells had a significant growth advantage over WT tumor cells when exposed to IFNy or IFNP (Figure 3G,/?<0.001, Student's t test), and this advantage was even greater with combinations of IFNy and TNF or IFNy and IFNP (Figure 3G,/K0.001, Student's t test). Unlike Statl and Jakl null tumors, it was found that Ifngrl null tumor cells were still sensitive to growth inhibition by IFNP (Figure 3G).

Tumor cells that are insensitive to IFNy could also resist immunotherapy by failing to present antigen. Indeed, it was observed that tumor cells deficient in Statl, Jakl or Ifngrl failed to upregulate MHC-I presentation molecules after stimulation with IFNy (Figure 3H), which might lead to less efficient T cell recognition. To test whether or not T cell recognition of IFNy-insensitive tumors is impaired, WT and Statl null tumor cells that had been engineered to express the model antigen ovalbumin (OVA) as a full-length protein were co-cultured with antigen-specific OT-I CD8 ⁺ T cells in an in vitro killing assay. After six days of co-culture with T cells, a significantly larger fraction of Statl null B16 cells were surviving relative to WT B16 cells (Figure 31; /K0.001, Student's t test), indicating decreased sensitivity of Statl null tumor cells to the effects of cytotoxic T cells. Similar results were obtained for Jakl and Ifngrl null cells. Thus, the screening platform recovered both known immune evasion molecules and resistance mechanisms to immunotherapy.

Example 4: Discovery of novel gene targets to increase the efficacy of

immunotherapy

Deletion of novel candidate immunotherapy targets was found to increase sensitivity of tumor cells to immunotherapy. Analysis of the functional classes of genes targeted by sgRNAs depleted by immunotherapy included antigen processing, necroptosis, and regulation of immune responses (Figure 1C and Table 1 for a summary of results). Thus, genes involved in the interaction of tumor cells with the immune system were enriched in screen hits, and encompassed multiple biological processes.

Among the 50 most depleted genes in immunotherapy treated mice (all with an FDR < 0.08), several prominent biological pathways were identified. In particular, sgRNAs targeting genes involved in inhibition of kinase signaling were determined to be markedly depleted in mice treated with GVAX and PD-1 blockade (Figure 5 A) relative to growth in TCRa ^_/" mice.

Representative genes from the inhibition of kinase signaling pathway were selected to validate based on their highest cumulative score as ranked by the STARS algorithm (Doench et al. (2014) Nat. Biotechnol. 32:1262-1267). These genes included Ptpn2, a phosphatase involved in multiple signaling processes. In vivo competition assays showed that tumor cells deleted for Ptpn2 was strongly selected against in WT animals treated with immunotherapy but grew at equivalent rates to control tumor cells in vitro and in TCR a ^1' mice (Figure 5C and 5D; /K0.01, Student's t test). This indicates that Ptpn2 is synthetically lethal with an anti-tumor immune response, rendering tumor cells more sensitive to immunotherapy but not altering their cell growth or survival in the absence of T cells.

Example 5: Loss of Ptpn2 increases sensitivity of tumors to immunotherapy

In order to identify novel targets for immunotherapy, genes that, when deleted, increased sensitivity of tumor cells to immunotherapy were analyzed. It was found that sgRNAs targeting the pleiotropic protein tyrosine phosphatase, Ptpn2, were markedly depleted in mice treated with GVAX and PD-1 blockade (Figure 5 A, FDR<0.00016). In vivo competition assays showed that tumor cells deleted for Ptpn2 with any of three different sgRNAs (Figure 5B) were strongly selected against in WT animals treated with immunotherapy but grew at equivalent rates to control tumor cells in vitro and in TCRa ^_/" mice (Figures 5C and 5E; /?<0.01, Student's t test). Two additional sgRNAs against Ptpn2 were used in in vivo competition assays to confirm that loss of Ptpn2 sensitizes tumors to immunotherapy (Figures 5B and 5E; /?<0.01, Student's t test; all sgRNAs shown). This indicates that loss of Ptpn2 renders tumor cells more sensitive to immunotherapy but does not affect cell growth or survival in the absence of immune cells.

Consistent with this observation, Ptpn2 null B16 tumors had a significantly better response to immunotherapy measured by tumor size (Figure 5H; /K0.01, Student's t test) and overall survival (Figure 51; /K0.001, Log-rank test). No growth disadvantage was observed in the absence of T cell-mediated immunity or immunotherapy (Figures 6A-6D), again indicating that loss of Ptpn2 did not cause a general impairment of cell growth and survival in vivo. Loss of Ptpn2 increased sensitivity to T cell immunity in two additional tumor models: Braf/Pten melanoma (Figures 4F and 5J; /K0.01, Student's t test) and MC38 colon carcinoma (Figure 6G; /K0.05; Student's t test) in which Ptpn2 deficiency led to a decrease in tumor volume and an increase in survival (Figure 6H; /K0.05, Log-rank test) in immunocompetent animals but not in TCRa ^"7" mice (Figures 6E-6F and 6I-6J). Thus, loss of Ptpn2 sensitizes tumor cells to the effect of immunotherapy.

Enforced expression of Ptpn2 in Ptpn2 null tumor cells (i.e. Ptpn2 rescue) abrogated the sensitivity to immunotherapy in vivo (Figures 5F-5G), making it unlikely that the effect was due to off-target effects of genome editing with Ptp«2-targeting sgRNAs. Moreover, overexpression of Ptpn2 in control B16 tumor cells (i.e. Ptpn2 overexpression) led to an outgrowth of tumor cells in immunotherapy treated mice (Figures 5F-5G; /K0.001, Student's t test), indicating that increased Ptpn2 expression renders tumor cells resistant to the effect of immunotherapy.

Example 6; Loss of Ptpn2 increases antigen presentation and T cell recognition of tumors

To identify the mechanism by which loss of Ptpn2 enhanced the efficacy of immunotherapy, the composition of immune cell subsets in the tumor microenvironment of control and Ptpn2 null tumors was compared using flow cytometry. No difference was observed in the number of total CD45 ⁺ cells, NK cells, CD4 ⁺ T cells, FoxP3 ⁺ regulatory T cells, or cells in the myeloid compartment in Ptpn2 null tumors relative to WT tumors (Figures 7A, 8A-8D and 9A-9B). However, Ptpn2 null tumors contained a significantly greater number of CD8 ⁺ T cells and γδ ⁺ T cells per milligram of tumor (Figure 7A; /K0.05, Student's t test). Confirmation by immunohistochemistry staining for CD8a in Ptpn2 null or control B16 tumors treated with GVAX and PD-1 blockade was performed and a 3 -fold increase in the number of CD8a+ cells, which diffusely infiltrates Ptpn2 deficient tumors, was found (Figure 7B; p<0.0\, Student's t test). In particular, further flow cytometry analysis of infiltrating CD8+ T cells revealed that Ptpn2 null tumors contained a striking increase in the fraction of CD8 ⁺ T cells expressing Granzyme B (Figure 7C, c; /?<0.01, Student's T test). Thus, Ptpn2 loss increased the number of activated, cytotoxic CD8 ⁺ T cell in tumors. The increased number of cytotoxic CD8 ⁺ T cells in Ptpn2 null tumors could be due to better recognition of tumor cells due to increased antigen presentation. To test this, full- length OVA was expressed in Ptpn2 null or WT B16 cells. Staining with a monoclonal antibody specific for the SIINFEKL epitope from OVA in the context of H2K(b) was significantly higher in Ptpn2 null cells treated with IFNy, indicating that loss of Ptpn2 caused increased levels of antigen-loaded MHC-I on the surface of tumor cells (Figure 7D; / O.001, Student's t test). Additionally, Ptpn2 deficient cells increased total MHC-I after treatment with IFNy to a greater degree than did control cells (Figure 10A).

To test whether loss of Ptpn2 made tumor cells more recognizable to T cells, OVA- expressing Ptpn2 null or control cells were cultured with OT-I CD8 ⁺ T cells that recognize the SIINFEKL epitope. T cells cultured with Ptpn2 null OVA-B16 cells were significantly more activated as measured by intracellular IFNy and TNF staining (Figure 7E; / 0.001, Student's t test). To test if Ptpn2 null tumor cells were more sensitive to the presence of T cells, mixed populations of OVA-expressing Ptpn2 null or WT B16 cells were co-cultured with antigen-specific CD8 ⁺ T cells for six days. Ptpn2 deficient tumor cells were preferentially depleted from the co-culture and the extent of depletion increased with a greater ratio of T cells to tumor cells (Figure 7F; /?<0.05, Student's t test). Thus, loss of Ptpn2 in tumor cells increased antigen presentation and sensitivity to cytotoxic CD8 ⁺ T cells.

Example 7; Loss of Ptpn2 increases IFNY sensing by tumor cells

Ptpn2 regulates multiple signaling pathways and has a role in negatively regulating IFNy signaling by dephosphorylating JAK1 and STAT1 (Kleppe et ah (2011) Blood 117:7090-7098; Kleppe et al. (2010) Nat. Genet. 42:530-535; Wiede et ah (2014) J

Autoimmun. 53:105-14; Pike & Tremblay (2016) Cytokine 82: 52-57; Wiede et ah (2011) J Clin. Invest. 121:4758-4774; Doody et al. (2009) Immunol. Rev. 228:325-341). Several approaches were used to determine whether Ptpn2 loss sensitized tumors to immunotherapy by increasing IFNy signaling. First, STAT1 activity was assayed [measured by

accumulation of phosphorylated STAT1 (pSTATl)] in Ptpn2 null cells in response to IFNy stimulation. Ptpn2 null B16 cells had increased pSTATl after treatment with IFNy while overexpression of Ptpn2 repressed this phosphorylation (Figure 11 A).

Transcriptional analysis of the Ptpn2 null and WT B16 tumor cells exposed to IFNy, TNF, or both indicated that loss of Ptpn2 caused a marked change in expression profile in response to cytokine exposure, but did not change gene expression in unstimulated conditions (Figures 1 lB-11C). Co-stimulation with IFNy and TNF together had a synergistic effect on Ptpn2 null cells, as IFNy stimulation appeared to sensitize B16 cells to TNF (Figure 10B). Unsupervised clustering of samples showed cytokine-exposed Ptpn2 null tumor cells clustered separately from WT tumor cells (Figure 1 IB). The gene signatures most strongly correlated with this difference in gene expression included those related to IFNy, IFNa, and TNF signaling. Loss of function of PTPN2 in four human tumor cell lines increased IFNy response gene expression following stimulation with IFNy (Figure 1 ID; FDR<0.001) in a manner similar to loss of function of Ptpn2 in mouse tumor cells. Among the genes upregulated in Ptpn2 null cells (relative to control cells) following cytokine exposure were several T cell chemokines, such as Cxcl9, CxcllO, Cxclll and Ccl5 (Figure 11C), which could act to enhance infiltration of T cells into tumors. Members of the antigen processing and presentation pathway, such as Tapl, Tapbp, and B2m were also upregulated in Ptpn2 null cells (Figure 11C), consistent with our previous observation of enhanced antigen presentation in Ptpn2 deficient cells.

It was also found that cell cycle regulators, such as Cdknla (Abbas & Dutta (2009) Nat. Rev. Cancer 9:400-414), and several genes involved in apoptosis, such as Casp4, Casp8, Ifit2, Ripkl, and Bakl, were significantly upregulated in Ptpn2 null tumor cells treated with cytokines relative to WT tumor cells. Consistent with this finding, exposure to IFNy alone or in combination with IFNP or TNF significantly reduced the growth of Ptpn2 null tumor cells in vitro relative to WT tumor cells (Figure 1 IE; / 0.001, Student's t test), and in PTPN2 null human A375 melanoma cells and HT-29 colon carcinoma cells in vitro (Figure 1 IF; p<0.0\, Student's t test). These results demonstrate that IFNy alone is sufficient to cause a growth disadvantage in both mouse and human tumor cells that lack Ptpn2.

Example 8: Increased sensitivity of Pton2 null tumors to immunotherapy depends on IFNY sensing

To determine whether the mechanism of increased immunotherapy sensitivity of Ptpn2 null tumor cells depends on IFNy sensing and signaling, B16 tumor cell lines that lacked both Ptpn2 and either Statl, Jakl, or Ifngrl were made (Figure 11G). The growth of these double-null cells, relative to tumor cells lacking only Statl, Jakl, or Ifngrl in animals treated with GVAX plus anti-PD-1 immunotherapy, was then tested. Loss of Ptpn2 was associated with a significant growth disadvantage in the presence of immunotherapy as expected (Figures 11H and 1 II). However, loss of function of any of the genes necessary for the sensing of IFNy abolished the growth disadvantage of Ptpn2 null cells (Figures 11H and 1 II; /><0.01, Student's t test). Loss of Ifhar2, a gene that senses IFNP, did not abrogate the sensitivity of Ptpn2 null tumors to GVAX plus anti-PD-1 immunotherapy (Figure 1 II). This genetic epistasis experiment indicates that in the absence of IFNy signaling, Ptpn2 deficiency does not affect tumor cell response to immunotherapy.

The importance of IFNy sensing to tp«2-mediated cancer cell response to immunotherapy was further confirmed using non-genetic methods. For example, sensitivity to IFNy was increased in B16 cells treated with IFNy, as well as a small molecule inhibitor of PTP1B (available on the World Wide Web at fishersci.com/shop/products/ptplb- inhibitor/5397415mg; CAS 765317-72-4), which also inhibits Ptpn2 (Figure 12). Thus, the mechanism by which Ptpn2 deficiency sensitizes tumor cells to immunotherapy is entirely dependent on the sensing of IFNy.

Immunotherapy with checkpoint blockade is rapidly becoming a cornerstone of cancer therapy (Reck et al. (2016) N. Engl. J. Med. 375:1823-1833; Hodi et al. (2010) N N. Engl. J. Med. 363:711-723; Postow et /. (2015) N. Engl. J. Med. 372:2006-2017; Wolchok et al. (2013) N. Engl. J. Med. 369:122-133; Ferris et al. (2016) N. Engl. J. Med. 375:1856- 1867; Brahmer et al. (2012) N. Engl. J. Med. 366:2455-2465; Nghiem et al. (2016) N. Engl. J Med. 374:2542-2552; Topalian et al. (2012) N Engl. J. Med. 366:2443-2454); and Motzer et al. (2015) N. Engl. J. Med. 373:1803-1813). However, incomplete clinical response and the development of resistance limit its efficacy (Tumeh et al. (2014) Nature 515:568-571; Kelderman et al. (2014) Mol. Oncol. 8:1132-1139; Zaretsky et al. (2016) N Engl. J. Med. 375:819-829), indicating that new targets are needed to improve clinical response. It has been demonstrated herein that pooled loss-of-function genetic screens in vivo can identify genes that modulate the efficacy of immunotherapy and therefore represent potential new therapeutic targets. The screening approach identified genes that, when deleted, make cells more sensitive to immunotherapy.

These genetic dependencies included known targets that are currently the focus of intense therapeutic development: PD-L1, which inhibits T cells via PD-1 (Freeman et al. (2000) J Exp.Med. 192:1027-1034; and Dong et al. (2002) Nat. Med. 8:793-800) and CD47, which inhibits tumor cell phagocytosis via SIRPa (Liu et al. (2015) Nat. Med.

21:1209-1215; Weiskopf et al. (2016) J Clin. Invest. 126:2610-2620; Tseng et al. (2013) Proc. Natl. Acad. Sci. U.S.A. 110: 11103-11108). As the number of emerging

immunotherapies, such as blockade of CD47, increases, it is becoming more challenging to select those that should be prioritized for combination therapy with PD-1 checkpoint blockade. Genetic screens can identify genes that cause synthetic lethality with specific immunotherapies, providing a rational means to identify effective combinations.

In addition to known targets, it was also discovered that deletion of new genes (e.g., Ptpn2 and Socsl) involved in diverse biological processes markedly increased the response of tumors to immunotherapy. Ptpn2 encodes a protein tyrosine phosphatase that regulates a range of intracellular processes including IFNy signaling, which it can inhibit by

dephosphorylating STAT1 and JAK1 (Kleppe et al. (2011) Blood 117:7090-7098; Kleppe et al. (2010) Nat. Genet. 42:530-535; Wiede et al. (2014) J Autoimmun. 53:105-114; Pike & Tremblay (2016) Cytokine 82: 52-57; Wiede et al. (2011) J Clin. Invest. 121 :4758-4774; Doody et al. (2009) Immunol. Rev. 228:325-341; Todd et al. (2007) Nat. Genet. 39:857- 864; and The Wellcome Trust Case Control Consortium (2007) Nature 447:661-678). It was found that loss of Ptpn2 in tumor cells increased IFNy signaling and antigen presentation to T cells, and amplified growth arrest in response to cytokines. Genetic epistasis was used to demonstrate that the increased sensitivity of Ptpn2 null tumors to immunotherapy was not only associated with increased IFNy signaling, but required it, because simultaneous deletion of Statl, Jakl and IFNGR (but not loss of IFNAR) abrogated the increased sensitivity of Ptpn2 null tumors to immunotherapy. This study indicates that inhibition of Ptpn2 could augment the efficacy of cancer immunotherapy. Deletion of Ptpn2 has also been reported to increase the function of immune effector cells, providing an additional rationale to develop pharmacologic inhibitors of Ptpn2 (Wiede et al. (2014) J Autoimmun. 53:105-114 and Wiede et al. (2011) J Clin. Invest. 121:4758- 4774). More generally, these findings indicate that sensitizing tumor cells to the effects of inflammatory cytokines might represent a new therapeutic strategy to amplify the efficacy of existing immunotherapies.

The screens identified not only genes that increased sensitivity of tumors to immunotherapy but also those that caused resistance. Loss of function of any gene that sensed or signaled downstream of IFNy conferred a fitness advantage to tumor cells exposed to immune attack. It was noted that this recapitulates the recent findings that loss- of-function mutations in JAK1 and JAK2 emerged in tumors from patients who developed resistance to PD-1 checkpoint blockade (Zaretsky et al. (2016) N. Engl. J. Med. 375:819- 829 and Gao et al. (2016) Cell 167:397-404.e9). Together, these findings indicate that the efficacy of immunotherapy with checkpoint blockade relies in large part on the direct effect of IFNy on tumor cells. Genome editing of mouse transplantable models may provide a useful way to discover and model immunotherapy resistance mechanisms.

One of the central challenges in cancer biology is to identify the genes that underlie the hallmarks of cancer. To date, functional genomic approaches using genome editing have largely focused on identifying genes required by tumor cells for the cancer hallmarks of growth, metastasis and drug resistance (Ebert et al. (2008) Nature 451:335-339; Hart et al. (2015) Cell 163:1515-1526; Yu et al. (2016) Nat. Biotechnol. 34:419-423; Chen et al. (2015) Cell 160:1246-1260). This study extends this approach to the interrogation of the interaction of the tumor cell with the immune system, which can be broadly applied to multiple tumor models and immunotherapy modalities to systematically define genes that govern interactions between cancer cells and the immune system. Incorporation by Reference

All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

Also incorporated by reference in their entirety are any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the world wide web at tigr.org and/or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.