Dual PI3k-MTOR inhibitor therapies

Abstract

Disclosed are compositions and their use in treating cancers. More particularly, the invention relates to compositions and their use in altering one of epithelial to mesenchymal cell transition, or mesenchymal to epithelial cell transition of a tumour cell. Also disclosed are methods of screening for candidate agents suitable for treating cancer, or for enhancing patient responsiveness to cancer.

Description

JAWS Ref: 751495PCTTITLE OF THE INVENTION“DUAL PI3K-MTOR INHIBITOR THERAPIES”FIELD OF THE INVENTION

[0001] This invention relates generally to compositions and their use in treating cancers. More particularly, the invention relates to compositions and their use in altering one of epithelial to mesenchymal cell transition, or mesenchymal to epithelial cell transition of a tumour cell.BACKGROUND OF THE INVENTION

[0002] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavor to which this specification relates.

[0003] Phosphatidylinositol is one of a number of phospholipids found in cell membranes which play an important role in intracellular signal transduction. Cell signaling via 3’-phosphorylated phosphoinositides has been implicated in a variety of cellular processes, e.g., malignant transformation, growth factor signaling, inflammation, and immunity (Rameh et al. (1999) J. Biol Chem, 274:8347-8350). The enzyme responsible for generating these phosphorylated signaling products, phosphatidylinositol 3-kinase (also referred to as PI 3-kinase or PI3K), was originally identified as an activity associated with viral oncoproteins and growth factor receptor tyrosine kinases that phosphorylate phosphatidylinositol (PI) and its phosphorylated derivatives at the 3’-hydroxyl of the inositol ring (Panayotou et al. (1992) Trends Cell Biol. 2: 358-60).

[0004] Phosphoinositide 3-kinases (PI3K) are lipid kinases that phosphorylate lipids at the 3-hydroxyl residue of an inositol ring (Whitman et al. (1988) Nature, 332:664). The 3-phosphorylated phospholipids (PIP3s) generated by PI3-kinases act as second messengers recruiting kinases with lipid binding domains (including plekstrin homology (PH) regions), such as Akt and phosphoinositide-dependent kinase-1 (PDK1 ). Binding of Akt to membrane PIP3s causes the translocation of Akt to the plasma membrane, bringing Akt into contact with PDK1, which is responsible for activating Akt. The tumour-suppressor phosphatase, PTEN, dephosphorylates PIP3 and therefore acts as a negative regulator of Akt activation. The PI3-kinases Akt and PDK1 are important in the regulation of many cellular processes including cell cycle regulation, proliferation, survival, apoptosis and motility and are significant components of the molecular mechanisms of diseases such as cancer, diabetes and immune inflammation (Vivanco et al. (2002) Nature Rev. Cancer 2:489; Phillips et al. (1998) Cancer 83:41).JAWS Ref: 751495PCT

[0005] The main PI3K isoform in cancer is the Class I PI3-kinase, p110 a (alpha) (as described in U. S. Pat. Nos. 5,824,492; 5,846,824; 6,274,327). Other isoforms are implicated in cardiovascular and immune-inflammatory disease (Workman P (2004) Biochem Soc Trans 32:393-396; Patel et al. (2004) Proceedings of the American Association of Cancer Research (Abstract LB-247) 95th Annual Meeting, March 27-31, Orlando, Florida, USA; Ahmadi K and Waterfield MD (2004) Encyclopedia of Biological Chemistry (Lennarz W J, Lane M D eds) Elsevier Academic Press). The PI3K / Akt / PTEN pathway is an attractive target for cancer drug development since such modulating or inhibitory agents would be expected to inhibit proliferation, reverse the repression of apoptosis and surmount resistance to cytotoxic agents in cancer cells (Folkes et al. (2008) J. Med. Chem. 51: 5522-5532; Yaguchi et al. (2006) Jour, of the Nat. Cancer Inst. 98(8):545-556).

[0006] However, despite a number of clinical studies investigating the use of PI3K inhibitors for the treatment of many solid tumours, no candidates have yet made it into the clinic. Accordingly, further work is required in order to successfully utilize these promising drug candidates to improve efficacy and reduce toxicity to be used for these challenging diseases.SUMMARY OF THE INVENTION

[0007] The present invention is predicated in part on the discovery that PI3K inhibitors have significant activity in inhibiting EMT, in inhibiting formation and maintenance of cancer stem cells (CSC), and in inducing mesenchymal to epithelial transition (MET), which makes them useful, therefore, in treating a range of cancers (e.g., solid tumours) including recurrent cancers.

[0008] In one aspect of the present invention, there is provided a method of treating cancer in a treatment-resistant subject, the method comprising administering to the subject a composition that comprises an agent that disrupts the PI3K / Akt / mTOR pathway and an anti-cancer treatment (e.g., an immunotherapy or PARP inhibitor), to thereby treat the cancer.

[0009] In some embodiments, the agent directly inhibits both PI3K and mTOR. In some embodiments, the agent is selected from the group comprising paxalisib, PI-103, Pl-103BE, GSK1059615, omipalisib, SN202, NSC765844, dactolisib, samotolisib, (S)-4-fluoro-5-(2-(3-methylmorpholino)-6-(1-(methylsulfonyl)cyclopropyl)pyrimidin-4-yl)pyridine-2-amine, voxtalisib, PF-04691502, apitolisib, GNE-477, (2S,6F?)-2,6-dimethyl-4-(4-mopholino-6-(1 H-pyrazol-5-yl)thieno[3,2-c / |pyrimidin-2-yl)mopholine, 2-(2-aminopyrimidin-5-yl)-A / ’-(4-methoxybenzoyl)-4-morpholinothieno[3,2-c / |pyrimidine-6-carbohydrazide, PKI-402, VS-5584, gedatolisib, bimiralisib, (R)-1 -(2-((4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl)amino)-1 H-benzo[d]imidazole-6-carbonyl)pyrrolidine-2-carboxamide, (2-((4-(4,6-dimopholino-1,3,5-triazin-2-yl)phenyl)amino)-1 / 7-benzo[c / |imidazole-6-yl)(morpholino)methanone, CMG 002, PF-04979064, and MCX-83. In some embodiments, the agent is paxalisib.JAWS Ref: 751495PCT

[0010] In some embodiments, the dose of paxalisib administered to the subject is around 7.5 mg / kg.

[0011] In some embodiments, the cancer is a solid cancer. By way of an example, the cancer may be glioblastoma, breast cancer (e.g., TNBC), or ovarian cancer.

[0012] In some embodiments, the immunotherapy is a checkpoint molecule inhibitor therapy. In some embodiments of this type, the checkpoint molecule is selected from PD1, CTLA-4 and PD-L1. In some alternative embodiments, the inhibitor is a PARP inhibitor.

[0013] In some embodiments, the composition further comprises a chemotherapy drug. In some embodiments, the chemotherapy drug is paclitaxel and / or temozolomide.

[0014] In some embodiments, the gene profile of cells obtained from the cancer express one or more of the genes selected from FOXQ1, NNMT, RelA, and NFE2L2.

[0015] In some embodiments, the subject exhibits reduced leukocyte infiltration into the lungs as compared to the level prior to administration.

[0016] In another aspect, the present invention provides a method of screening for candidate molecules suitable for treating cancer, the method comprising determining whether the candidate molecule inhibits a PI3K function or activity and an mTOR function or activity, and upon determining that both a PI3K function or activity and an mTOR function or activity are inhibited, determining that the candidate molecule is suitable for treating cancer.

[0017] In some embodiments, the method further comprises the step of: exposing a sample obtained from a cancer or tumour to the candidate molecule; and determining the level of nuclear p85p: H3K9Me2and / or p85p: EZH2 in the sample; wherein upon the level of nuclear p85p: H3K9Me2and / or p85p: EZH2 in the sample being below a predetermined threshold, determining that the candidate molecule is suitable for treating cancer.

[0018] Accordingly, a determination of whether a candidate agent would be effective for treating cancer can be made by assessing the level of nuclear p85p: H3K27Me2in a biological sample, such as a serum, plasma or blood sample, and comparing it to the level of the nuclear p85p: H3K27Me2in a corresponding sample from a subject that responds to immunotherapy (e.g., anti-PD1 immunotherapy) or samples from multiple control samples, wherein a decrease indicates that the candidate agent will be effective at treating cancer, In some instances, the level of the nuclear p85p: H3K27Me2is compared to a predetermined level or threshold, wherein a decrease in the level of the nuclear p85p: H3K27Me2compared to the threshold indicates that the candidate agent will be effective at treating cancer. The predetermined threshold may be calculated based on the level of nuclear p85p: H3K27Me2in a corresponding sample from a subject known to respond to cancer therapy or from a group of samples from subjects known to respond to cancer therapy.JAWS Ref: 751495PCTBRIEF DESCRIPTION OF THE FIGURES

[0019] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein.

[0020] Figure 1. Dual PI3K-mTOR blockade inhibits cell proliferation and migration, induces mesenchymal to epithelial transition, and reduces MIC signatures. (A) WST proliferation reagent was added to MDA-MB-468, SUM149PT, and 4T1 cells pre-treated with PI3K and PI3K-mTOR inhibitors for 72 h in a dose-response design. Cell proliferation (%) was measured indirectly by the formation of formazan and the absorbance recorded at 450 nm. (B) WST proliferation reagent was added to MDA-MB-231 cells pre-treated with PI3K and PI3K-mTOR inhibitors for 72 h in a dose-response design. Cell proliferation (%) was measured indirectly by the formation of formazan and the absorbance recorded at 450 nm. (C) Representative images from MDA-MB-231 cells scratched prior to treatment with paxalisib for 6 h, 12 h, and 18 h. Bar graph comparing the relative wound densities (%) observed for PI3K-mTOR inhibition at each time point, *“* p<0.0001, versus vehicle, two-way ANOVA with Bonferroni’s multiple comparisons test, n = 8 / group. (D) Representative images from 4T1 cells scratched prior to treatment with paxalisib for 24 h and comparison of the relative wound densities (%Vehicle) observed for paxalisib at its relative ICso value, *“* p<0.0001 versus vehicle, unpaired t-test. n=10 / group. (E), (F) Representative images and immunofluorescence analysis of (E) vimentin and (F) E-cadherin in 24 h paxalisib-treated MDA-MB-231 cells, “** p<0.0001, “* p<0.001 versus vehicle, unpaired t-test, n > 100 cells / group. (G) Flow cytometry analysis showing CD44: CD24 expression (%Vehicle) in 24 h paxalisib-treated MDA-MB-231 cells. (H) Immunofluorescence analysis of ALDH1, ABCB5, and Snail protein expression in 24 h paxalisib-treated MDA-MB-231 cells. p<0.0001, versus vehicle, unpaired t-test, n > 100 cells / group. (I) Immunofluorescence analysis of nuclear and cytoplasmic NFKB p65, FOXQ1, NRF2, and NNMT protein expression in 24 h paxalisib-treated MDA-MB-231 cells, *, p< 0.05, ** p< 0.01, p<0.001, **“, p<0.0001, versus vehicle, unpaired t-test, n > 100 cells / group. (J) Immunofluorescence analysis of IL-6 protein expression (%Total cells) in paxalisib-treated ABCB5+ / EpCAM+CTOs isolated from TNBC patients * p<0.05, versus vehicle, paired t-test, n = 2 I group. (K) qPCR analysis of viral mimicry-associated gene expression in 24 h paxalisib-treated MCF-7 iEMT cells, *p<0.05, versus vehicle, unpaired t-test, n = 2 / group. (L) Comparison of GBP2 mRNA expression (%Vehicle) following 24 h PI3K and PI3K-mTOR treatment in MCF-7 iEMT cells, versus vehicle, Dunnett’s multiple comparisons test, n = 2 / group.

[0021] Figure 2. Paxalisib reduces primary tumor burden and metastasis with a favorable toxicity profile. (A) Treatment regimens using the BALB / c 4T1 TNBC breast cancer model (paxalisib with + / - anti-PD1, with or without Abraxane). (B) Primary tumor volumesJAWS Ref: 751495PCT(%vehicle) and (F) weights of individual mice from each experimental group prior to harvest, * p<0.05, ** p<0.01, *** p<0.001, versus vehicle, Dunnett’s, n = 4-5 / group. (C) Pipeline for dose de-escalation experiments. In stage I, paxalisib was administered daily at a single dose of 15 mg / kg + / - anti-PD1 (10 mg / kg) at day 0 and day 4. In stage II, a split daily dose of paxalisib (up to 15 mg / kg) was administered 4 h apart in combination anti-PD1 at day 0 and day 4. In the final stage, paxalisib was administered as a single daily dose (up to 7.5 mg / kg) in combination with anti-PD1 at day 0 and day 4. (D) Primary tumor volumes (%Vehicle) of individual mice from each experimental stage at harvest, * p<0.05, ** p<0.01, Tukey’s or Dunnett’s (versus Vehicle) multiple comparison test, n = 4-5 / group. (E) Mouse body weights were monitored throughout the duration of the starting dose, split-dose de-escalation, and single-dose de-escalation experiments. (F) Liver weights were measured at the time of harvest. * p<0.05, Tukey’s multiple comparison test, n = 4-5 / group. (G) (Left) Changes in liver inflammation and hepatocyte damage (metabolic and / or degeneration) were scored 1 = mild, 2 = moderate or 3 = severe for each parameter, *p<0.05, **** p<0.0001, versus vehicle, Dunnett’s, n = 4-5 / group. (Middle) Changes in lung weights and leukocytosis, * p<0.05, “* p<0.001, ****p<0.0001, versus vehicle, Dunnett’s, n = 4-5 / group. (Right) Changes in spleen weights and spleen extramedullary hematopoiesis (EMH), * p<0.05, ** p<0.01, *“ p<0.001, *“* p<0.0001, versus vehicle, Dunnett’s, n = 4-5 / group. (H), (I) For triple therapies, toxicity assessments including total body (H) and liver weights (I) were monitored throughout the experiment. (J) Paxalisib combined with aPD1 experiments using the BALB / c 4T1 i.v. metastasis breast cancer model. (K) Mouse body weights were monitored throughout the experiments. (L) Immunofluorescence images of CD45, vimentin, and SNAIL1 in MDA-MB-231 cells spiked into human healthy PBMCs. (M) CTCs were isolated using ScreenCell Cyto kits from mouse blood collected from the 4T1 i.v. metastasis model, followed by Giemsa staining. (N) Captured CTCs were counted on Cyto IS membranes. (O) Images of Indian ink-stained lungs highlighting metastases, including large protruding and small flat nodules and graph shows number of flat and protruding metastatic lung nodules, ** p<0.01, **** p<0.0001, versus vehicle, two-way ANOVA with Tukey’s multiple comparisons test, n = 2 / group.

[0022] Figure 3. CODEX multiplexed imaging reveals changes in immune cell landscapes when of paxalisib is combined with immunotherapy. (A) Normalized expression of 23 proteins in 14 identified cell types. (B) Percentages of indicated cells types out of total cells in stromal, adaptive immune, innate immune, and tumour populations in aPD1 and paxalisib + aPD1 -treated tumours. (C) Differential location of stromal, tumour, adaptive, and innate cell populations in aPD1 and paxalisib + aPD1 -treated tumour tissues. (D) Representative images of neighborhoods mapped to tumour tissues from aPD1 and paxalisib + aPD1 -treated tumours. (E) Quantification of neighborhood fraction in aPD1 and paxalisib + aPD1 -treated tumours.

[0023] Figure 4. NanoString nCounter analysis highlights distinct gene expression profiles in Paxalisib-treated tumours. Heatmaps of immune gene expressionJAWS Ref: 751495PCTprofiles corresponding to cytotoxicity, T cell co-stimulation, interferon responses, and antigen presentation from NanoString nCounter analysis of paxalisib (GDC)-treated tumours.

[0024] Figure 5. PI3K-mT0R inhibition enhances anti-tumor immune profiles in combination with immunotherapy. (A) NanoString nCounter cell abundance analysis of dendritic cell, cytotoxic cell, NK, and T and B cell immune populations from paxalisib + / - anti-PD1 treated tumors, n = 3 / group. (B) NanoString nCounter cell abundance analysis of TIL populations from paxalisib + / - anti-PD1 -treated tumors, n = 3 / group. (C) NanoString nCounter gene expression levels of cytotoxicity-related cytokines, including IFN-y and Granzyme B (Gzmb), n = 3 / group. (D) NanoString nCounter cell abundance analysis of mast cell immune populations from paxalisib + / - anti-PD1 -treated tumors, n = 3 / group. (E) Toluidine blue staining for mast cell detection in anti-PD1 and paxalisib + anti-PD1 -treated tumors. (F) Genes from the mouse model that were significantly different (p<0.05 and |log2FC|>1) between paxalisib vs Control were matched to human CTC-related and CTC cluster-related genes. Significantly, enriched pathways were identified for these matched genes using the EnrichR portal.

[0025] Figure 6. Nuclear p85p expression in Stage III TNBC patients and paxalisib-treated 4T1 TNBC mice. Immunohistochemistry staining of p85p (PIK3R2) in Stage III TNBC patients. The percentage of nuclear p85p positive tumour cells were scored by an expert pathologist.

[0026] Figure 7. PI3K-mTOR inhibition targets p85p: H3K27Me3 switch. (A) Pipeline of the paxalisib treatment regimen in triple combination experiments using the BALB / c 4T1 TNBC breast cancer model. (B) Immunofluorescence images and Immunofluorescence analysis of p85p nuclear intensity in the 4T1 TNBC breast cancer model, * p<0.05, ** p<0.01, Dunnett’s multiple comparisons test. (C) Immunofluorescence images and analysis of p85p and H3K27me3nuclear intensity in the 4T1 TNBC breast cancer model, * p<0.05, **** p<0.0001, versus vehicle, Dunnett’s multiple comparisons test. (D) Immunofluorescence images of p85p and H3K27Me3in MDA-MB-231 cells. (E) Immunofluorescence analysis of p85p and H3K27Me3nuclear intensity staining in paxalisib-treated MDA-MB-231 cells and MDA-MB-468 cells, * p<0.05, *“* p<0.0001, versus vehicle, unpaired t-test. (F) Representative super resolution Duolink images in PBMCs isolated from TNBC patient blood. White spots indicate p85p: H3K27Me3 interactions within nuclei (blue). (G) Image analysis of p85p: H3K27Me3 interactions pre- and post-chemotherapy. Duolink analysis of the p85p: EZH2 interactions (intensity) in MDA-MB-231 cells treated with paxalisib for 0.5, 2 and 3 hours, p<0.0001, unpaired t-test.

[0027] Figure 8. PI3K-mTOR inhibition targets the dual role of EZH2. (A) qPCR analysis of EZH2 mRNA expression in paxalisib-treated MDA-MB-231 cells *“*, p<0.0001, unpaired t-test. (B) Immunofluorescence analysis of EZH2 nuclear intensity staining in paxalisib-treated MDA-MB-231 cells **“, p<0.0001, unpaired t-test. (C) Duolink analysis of the p85p: EZH2 interactions in paxalisib-treated MDA-MB-231 cells, *“*, p<0.0001, unpaired t-JAWS Ref: 751495PCTtest. (D) Duolink analysis of the NFKB: EZH2 interactions in paxalisib-treated MDA-MB-231 cells, ****, p<0.0001, unpaired t-test. (E) Duolink analysis of the p85p: EZH2 interactions (intensity) in MDA-MB-231 cells treated with paxalisib for 0.5, 2 and 3 hours, p<0.0001, unpaired t-test. (F) Overlap between the EZH2 bound upregulated and downregulated genes from the three comparisons (anti-PD1, paxalisib, and paxalisib + anti-PD1, relative to control). Differentially expressed genes were defined as log2Foldchange > 0.5 and p-value < 0.05. blue * indicated genes were also bound with H3K27me3. (G) Distribution of EZH2-bound genomic locations that are part of the nCounter Tumor 360 Signaling panel. (H) EZH2 and NFKB2 ChlP-seq tracks at IL-6 promoters in MDA-MB-231 cells.

[0028] Figure 9. EZH2 co-localizes with NF-KB to activate pro-oncogenic gene expression in TNBC. EZH2 and NFKB2 ChlP-seq tracks at KRT14, FOXQ1, NNMT, RELA and NFE2L2 promoters in MDA-MB-231 cells.

[0029] Figure 10. Associations between PIK3R2 gene expression and survival probability amongst TNBC breast and ovarian cancer patient cohorts. (A) Distant metastasis-free survival (DMFS) in TNBC / basal-like and HER2+ breast cancer datasets with high and low expression levels for PIK3R2 - from analysis of the GSE158309 dataset. Patients were grouped into PIK3R2-high or PIK3R2-low depending on the expression levels of PIK3R2: high is above 75th percentile (solid lines) and low is below 75th percentile (dotted lines).Kaplan-Meier survival curves for plotted using distant metastasis as the endpoint. Patients with high PIK3R2 (solid lines) showed poorer DMFS in both basal-like / TNBC and HER2+ subtypes. (B) Assessing PIK3R2 mRNA expression in stage IV metastatic triple-negative breast cancer patients post chemotherapy (carboplatin, Nab-paclitaxel) + Pembrolizumab durvalumab+Olaparib in a single-arm phase-2 trial. Patients with higher mRNA expression for PIK3R2 showed poorer overall survival compared to patients with lower PIK3R2 mRNA expression. High = above 75th percentile in PIK3R2 expression; Low = lower than 75th percentile. (C) Assessing PIK3R2 mRNA expression levels in triple-negative breast cancer patients who had residual tumours after treatment with neoadjuvant chemotherapy (NACT). All patients here had residual tumours post NACT, however those who presented with a distant relapse had higher pre-treatment levels of PIK3R2 compared to those who did not present with a distant relapse.

[0030] Figure 11. Impact of dual PI3K-mTOR pathway inhibition in triple negative breast cancer. Mechanism 1. PI3K-mTOR blockade inhibits the resistant cancer cell populations (dormant tumor cells, CSCs, and persister cells), thereby overcoming resistance and reducing inflammation, including key pro-inflammatory cytokines such as IL-6. PI3K-mTOR inhibition also increases immune re-invigoration and activates viral mimicry signatures, making the cancer cells more immune visible. Using this biphasic approach of targeting resistance signatures and enhancing cancer immune visibility, when combined with immunotherapy or PARP inhibitors, PI3K-mTOR inhibition reduces primary tumor burden and metastases.Mechanism 2. PI3K-mTOR blockade inhibits the dual role of EZH2. PI3K-mTOR blockade (e.g.,JAWS Ref: 751495PCTwith paxalisib) acts upstream of EZH2, thereby targeting its dual role in resistance. PI3K-mT0R inhibitors potentially target EZH2 by two mechanisms. First, by inhibiting p85p translocation into the nucleus, which diminishes the p85: EZH2 interaction, thereby inhibiting the catalytic repressive role of the epigenetic enzyme resulting in reduced H3K27 tri-methylation. By targeting upstream of EZH2, at the same time PI3K-mT0R blockade inhibits the non-catalytic inducible role of EZH2, thereby reducing NFKB signaling and downstream targets. Alternatively, PI3K-mT0R inhibition directly impacts EZH2 transcription, which in turn inhibits its dual catalytic and non-catalytic roles.DETAILED DESCRIPTION OF THE INVENTION1. Definitions

[0031] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.

[0032] The articles “a” and “an” are used herein to refer to one or to more than one ( / .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.

[0033] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0034] The “amount” or “level” of a biomarker is a detectable level in a sample. These can be measured by methods known to one skilled in the art and also disclosed herein. The expression level or amount of biomarker assessed can be used to determine the response to treatment.

[0035] As used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).

[0036] Throughout this specification, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term “comprising” and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By “consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase “consisting of’ indicates that the listedJAWS Ref: 751495PCTelements are required or mandatory, and that no other elements may be present. By “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0037] “Chemotherapeutic agent” includes compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca), sunitib (SUTENT®, Pfizer / Sugen), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), finasunate (VATALANIB®, Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin, Rapamycin (Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), Lonafamib (SCH 66336), sorafenib (NEXAVAR®, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), AG1478, alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines (such as benzodopa, carboquone, meturedopa, and uredopa); ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including topotecan and irinotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); adrenocorticosteraids (including prednisone and prednisolone); cyproterone acetate; 5a-reductases including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat dolastatin; aldesleukin, talc duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin y11 and calicheamicin w11 (Angew Chem. Inti. Ed. Engl.199433: 183-186); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin andJAWS Ref: 751495PCTdeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara- C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N. J.), ABRAXANE® (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, III.), and TAXOTERE® (docetaxel, doxetaxel; Sanofi-Aventis); chloranmbucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

[0038] Chemotherapeutic agent also includes (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumours such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; buserelin, tripterelin,JAWS Ref: 751495PCTmedroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all transretionic acid, fenretinide, as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-a, Ralf and H-Ras; (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®;PROLEUKIN®, rlL-2; a topoisomerase 1 inhibitor such as LURTOTECAN®; ABARELIX® rmRH; and (ix) pharmaceutically acceptable salts, acids and derivatives of any of the above.

[0039] Chemotherapeutic agent also includes antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech / Biogen Idee), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

[0040] Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, peefusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and the anti-interleukin-12 (ABT-874 / J695, Wyeth Research and Abbott Laboratories) which is a recombinant exclusively human-sequence, full-length IgGi lamda. antibody genetically modified to recognize interleukin-12 p40 protein.

[0041] Chemotherapeutic agent also includes “EGFR inhibitors,” which refers to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity, and is alternatively referred to as an “EGFR antagonist.” Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U. S. Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized 225 (C225 or Cetuximab;ERBUTIX®) and reshaped human 225 (H225) (see, WO 96 / 40210, Imclone Systems Inc.); IMC-11 F8, a fully human, EGFR-targeted antibody (Imclone); antibodies that bind type II mutant EGFR (U. S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind EGFR asJAWS Ref: 751495PCTdescribed in U. S. Pat. No. 5,891,996; and human antibodies that bind EGFR, such as ABX-EGF or Panitumumab (see WO98 / 50433, Abgenix / Amgen); EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF-a for EGFR binding (EMD / Merck); human EGFR antibody, HuMax-EGFR (GenMab); fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6. 3 and E7.6. 3 and described in U. S. Pat. No. 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanized mAb 806 (Johns et al., J. Biol. Chem. 279(29): 30375-30384 (2004)). The anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g., EP659439A2, Merck Patent GmbH). EGFR antagonists include small molecules such as compounds described in U. S. Pat. Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, as well as the following PCT publications: WO98 / 14451, WO98 / 50038, WO99 / 09016, and WO99 / 24037. Particular small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech / OSI Pharmaceuticals); PD 183805 (Cl 1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quin-azolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®) 4-(3'-Chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluorophenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1 H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol)-; (R)-6-(4-hydroxyphenyl)-4-[(1 -phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimi- dine); CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(-dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU 5271; Pfizer); dual EGFR / HER2 tyrosine kinase inhibitors such as lapatinib (TYKERB®, GSK572016 or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2- furanyl]-4-quinazolinamine).

[0042] Chemotherapeutic agents also include “tyrosine kinase inhibitors” including the EGFR-targeted drugs noted in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724,714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisense agent ISIS-5132 available from ISIS

[0043] Pharmaceuticals which inhibit Raf-1 signaling; non-HER targeted TK inhibitors such as imatinib mesylate (GLEEVEC®, available from Glaxo SmithKline); multi-JAWS Ref: 751495PCTtargeted tyrosine kinase inhibitors such as sunitinib (SUTENT®, available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787 / ZK222584, available from Novartis / Schering AG); MAPK extracellular regulated kinase I inhibitor Cl -1040 (available from Pharmacia); quinazolines, such as PD 153035, 4-(3-chloroanilino) quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[2,3-d] pyrimidines; curcumin (diferuloyl methane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner-Lamber); antisense molecules (e.g. those that bind to HER-encoding nucleic acid); quinoxalines (U. S. Pat. No. 5,804,396); tryphostins (U. S. Pat. No.5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis / Schering AG); pan-HER inhibitors such as Cl -1033 (Pfizer); Affinitac (ISIS 3521; isis / Lilly); imatinib mesylate (GLEEVEC®); PKI 166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1 C11 (Imclone), rapamycin (sirolimus, RAPAMUNE®); or as described in any of the following patent publications: U. S. Pat. No. 5,804,396; WO 1999 / 09016 (American Cyanamid); WO 1998 / 43960 (American Cyanamid); WO 1997 / 38983 (Warner Lambert); WO 1999 / 06378 (Warner Lambert); WO 1999 / 06396 (Warner Lambert); WO 1996 / 30347 (Pfizer, Inc); WO 1996 / 33978 (Zeneca); WO 1996 / 3397 (Zeneca) and WO 1996 / 33980 (Zeneca).

[0044] Chemotherapeutic agents also include dexamethasone, interferons, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, and pharmaceutically acceptable salts thereof.

[0045] Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate and fluprednidene acetate; immune selective anti-inflammatory peptides (ImSAIDs) such as phenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG) (IMULAN BioTherapeutics, LLC); anti-rheumatic drugs such as azathioprine, ciclosporin (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomideminocycline,JAWS Ref: 751495PCTsulfasalazine, tumour necrosis factor (TNF) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), golimumab (Simponi), Interleukin 1 (IL-1) blockers such as anakinra (Kineret), T-cell costimulation blockers such as abatacept (Orencia), Interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMERA®);Interleukin 13 (IL-13) blockers such as lebrikizumab; Interferon-a (IFN-a) blockers such as Rontalizumab; Beta 7 integrin blockers such as rhuMAb Beta7; IgE pathway blockers such as Anti-M1 prime; Secreted homotrimeric LTa3 and membrane bound heterotrimer LTa1 / p2 blockers such as Anti-lymphotoxin a (LTa); radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212and radioactive isotopes of Lu); miscellaneous investigational agents such as thioplatin, PS-341, phenyl butyrate, ET-18-OCH3, or farnesyl transferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechine gallate, theaflavins, flavanols, procyanidins, betulinic acid and derivatives thereof; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin); podophyllotoxin; tegafur (UFTORAL®); bexarotene (TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine; perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib), proteosome inhibitor (e.g. PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone; farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

[0046] Chemotherapeutic agents also include non-steroidal anti-inflammatory drugs with analgesic, antipyretic and anti-inflammatory effects. NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase. Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, rofecoxib, and valdecoxib. NSAIDs can be indicated for the symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthropathies, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhoea, metastatic boneJAWS Ref: 751495PCTpain, headache and migraine, postoperative pain, mild-to-moderate pain due to inflammation and tissue injury, pyrexia, ileus, and renal colic.

[0047] The terms “correlated” and “associated” are used interchangeably herein to refer to the association between two measurements (or measured entities). The disclosure provides genetic and / or epigenetic variations, the level(s) of which are associated with disease diagnosis and / or prognosis and / or response to treatment.

[0048] The terms “decrease”, “reduced”, “reduction”, “inhibit”, “suppress”, “attenuate” and the like are all used herein to mean a decrease by a statistically significant amount. In some embodiments, these terms typically mean a decrease by at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein “reduction”, “suppression”, and “inhibition” does not necessitate a complete inhibition or reduction as compared to a reference level. “Complete inhibition” and the like is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal (e.g., for an individual without a given disorder).

[0049] As used herein, the term “epithelial to mesenchymal transition" (EMT) refers to the conversion from an epithelial cell to a mesenchymal phenotype, which is a normal process of embryonic development. EMT is also the process whereby injured epithelial cells that function as ion and fluid transporters become matrix remodeling mesenchymal cells, in carcinomas, this transformation typically results in altered cell morphology, the expression of mesenchymal proteins and increased invasiveness. The criteria for defining EMT in vitro involve the loss of epithelial cell polarity, the separation into individual cells and subsequent dispersion after the acquisition of cell motility (refer to Vincent-Salomon and Thiery, Breast Cancer Res. 2003; 5(2): 101 -6). Classes of molecules that change in expression, distribution, and / or function during EMT, and that are causally involved, include growth factors (e.g., transforming growth factor (TGF)-P, wnts), transcription factors (e.g., SNAI, SMAD, LEF and nuclear p-catenin), molecules of the cell-to-cell adhesion axis (cadherins, catenins), cytoskeletal modulators (Rho family) and extracellular proteases (matrix metalloproteinases, plasminogen activators) (refer to Thompson and Newgreen, Cancer Res. 2005; 65(14): 5991-5).

[0050] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statistically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level (e.g., the absence of a given treatment or agent) and can include, for example, of at least about 10% as compared to a reference level, for example an increase ofJAWS Ref: 751495PCTat least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or up to and including a 100% increase or any increase between 10-100% as compared to a reference level or at least about a 2-fold, or at least about a 3-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold, or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, an “increase” is a statistically significant increase in such level.

[0051] “Measuring" or “measurement” means assessing the presence, absence, quantity or amount (which can be an effective amount) of a given substance within a sample, including the derivation of qualitative or quantitative concentration levels of such substances, or otherwise evaluating the values or categorization of a subject's clinical parameters.Alternatively, the term “assaying,” “detecting" or “detection” may be used to refer to all measuring or measurement as described in this specification.

[0052] As used herein, the term “mesenchymal-to-epithelial transition” (MET) is a reversible biological process that involves the transition from motile, multipolar or spindle- shaped mesenchymal cells to planar arrays of polarized cells called epithelia. MET is the reverse process of EMT. METs occur in normal development, cancer metastasis and induced pluripotent stem cell reprogramming.

[0053] As used herein, the terms “overexpress,” “overexpression,” “overexpressing” or “overexpressed” interchangeably refer to a gene (e.g., PI3KCA gene) that is transcribed or translated at a detectably greater level, usually in a cancer ceil, in comparison to a normal ceil. Overexpression, therefore, refers to both overexpression of protein and RNA (due to increased transcription, post transcriptional processing, translation, posttranslational processing, altered stability and altered protein degradation), as well as local overexpression due to altered protein traffic patterns and augmented functional activity, for example, as in an increased enzyme hydrolysis of substrate. Overexpression can also be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a normal cell or comparison cell (e.g., a breast cell).

[0054] The term “PI3K inhibitor” and grammatical variants thereof are used herein to refer to a molecule that decreases or inhibits at least one function or biological activity of PI3K. For example, PI3K inhibitors may inhibit or reduce the enzymatic activity of PI3K and / or may inhibit or reduce the expression of PI3K.

[0055] As used herein, the term “PI3K overexpressing cell” refers to a vertebrate cell, particularly a mammalian ceil, that expresses PI3K at a detectably greater level than a normal ceil. The cell may be a vertebrate ceil, such as a primate cell; an avian ceil: a livestock animal ceil (such as a sheep ceil, cow cell, horse cell, deer ceil, donkey cell and pig cell); aJAWS Ref: 751495PCTlaboratory test animal cell (such as a rabbit cell, mouse cell, rat cell, guinea pig cell and hamster cell); a companion animal cell (such as a cat cell and dog cell); and a captive wild animal cell (such as a fox ceil, deer ceil and dingo cell). In particular embodiments, the PI3K overexpressing cell is a human cell. In specific embodiments, the PI3K overexpressing ceil is a cancer stem cell or a non-cancer stem cell tumour cell; preferably a cancer stem cell tumour cell. Overexpression can also be by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to a normal ceil or comparison cell (e.g. a breast ceil).

[0056] The term “selective” and grammatical variants thereof are used herein to refer to molecules that inhibit PI3K without substantially inhibiting the function of one or more other PI3K enzyme or isoform. Generally, a molecule that is selective for PI3K exhibits PI3K selectivity of greater than about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or greater than about 100-fold with respect to inhibition of one or more other PI3K enzyme. In other embodiments, selective molecules display at least 50-fold greater inhibition towards PI3K than towards one or more other PI3K enzyme. In further embodiments, selective molecules display at least 100-fold greater inhibition towards PI3K than towards one or more other PI3K enzyme. In still further embodiments, selective molecules display at least 500-fold greater inhibition towards PI3K than towards one or more other PI3K enzyme. In yet further embodiments, selective molecules display at least 100-fold greater inhibition towards PI3K than towards one or more other PI3K enzyme.

[0057] As used herein, the term “predetermined threshold” refers to a value, above or below which, indicates the characteristic of the candidate molecule being tested, such as a suitability for treating cancer. For example, for the purposes of the present invention, a predetermined threshold may represent the level of nuclear p85p: H3K27Me2in a sample from an appropriate control subject, such as a healthy subject or a subject that is not responsive to a cancer therapy (e.g., an immunotherapy), or in pooled samples from multiple control subjects or medians or averages of multiple control subjects. Thus, a level above or below the threshold indicates the effectiveness of the candidate agent of treating cancer, as taught herein. In other examples, a predetermined threshold may represent a value larger or smaller than the level determined for a control subject so as to incorporate a further degree of confidence that a level above or below the predetermined threshold is indicative of the candidate agent being suitable for treating cancer. For example, the predetermined threshold may represent the average or median level of a glycospecies in a group of control subjects, plus or minus 1, 2, 3 or more standard deviations. Those skilled in the art can readily determine an appropriate predetermined threshold based on analysis of biological samples from appropriate control subjects.

[0058] As used herein, a “subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (e.g., Rhesus). Rodents include mice, rates, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animalsJAWS Ref: 751495PCTinclude cows, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cat), canine species (e.g., dog, fox, wolf), avian species (e.g., chicken, emu, ostrich), and fish (e.g., trout, catfish, and salmon). In some embodiments the subject is a mammal (e.g., a primate (e.g., a human)). The terms “individual”, “patient” and “subject” are used interchangeably herein.

[0059] As used herein, the terms “treat”, “treatment”, “treating” and the like, refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder (e.g., cancer or tumour). The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with cancer or tumour. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration, or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term “treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment). A treatment need not cure a disorder (i.e., complete reversal or absence of disease) to be considered effective.

[0060] As used herein, the term “tumour” refers to any neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer” and “cancerous” referto or describe the physiological condition in mammals that is typically characterized in part by unregulated cell growth. As used herein, the term “cancer” refers to non-metastatic and metastatic cancers, including early stage and late stage cancers. The term “precancerous” refers to a condition or a growth that typically precedes or develops into a cancer. The term “non-metastatic” refers to a cancer that is benign or that remains at the primary site and has not penetrated into the lymphatic or blood vessel system or to tissues other than the primary site. Generally, a non -metastatic cancer is any cancer that is a stage 0, I or II cancer. By “early stage cancer” is meant a cancerthat is not invasive or metastatic or is classified as a stage 0, 1 or II cancer. The term “late stage cancer” generally refers to a stage III or IV cancer, but can also refer to a stage II cancer or a sub stage of a stage II cancer. One skilled in the art will appreciate that the classification of a stage II cancer as either an early stage cancer or a late stage cancer depends on the particular type of cancer. Illustrative examples of cancer include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, pancreatic cancer, colorectal cancer, lung cancer, hepatocellular cancer, gastric cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, brain cancer, non-small cell lung cancer, squamous cell cancer of the head and neck, endometrial cancer, multiple myeloma,JAWS Ref: 751495PCTmesothelioma, rectal cancer and esophageal cancer. In an exemplary embodiment, the cancer is breast cancer.

[0061] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise.2. Compositions

[0062] The present invention is predicated at least in part on the realization that dual targeting of PI3K and mTOR, but not PI3K alone, inhibits cancer cell proliferation and migration. Such dual targeting not only promotes a favourable mesenchymal to epithelial phenotype but also inhibits signatures associated with MICs, including the highly aggressive CSC phenotype, persister cancer cell phenotype, and a cancer drug resistance signature.

[0063] Thus, in accordance with the present invention, compositions are provided that comprise an agent that inhibit both PI3K and mTOR, and an anti-cancer therapy (e.g., an immunotherapy and / or a PARP inhibitor therapy). In some embodiments, the anti-cancer therapy does not target CSC.2.1 PI3K - mTOR inhibitors.

[0064] The compositions of the invention comprise an agent that is a inhibitor, which includes and encompasses any active agent that reduces the accumulation, function (e.g., enzymatic activity, localization, etc.), or stability of PI3K; or decrease expression of a PI3KCA gene, and such inhibitors include without limitation, small molecules and macromolecules such as nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, polysaccharides, lipopolysaccharides, lipids or other organic (carbon containing) or inorganic molecules.

[0065] Representative transcripts of this PI3K include nucleotide sequences corresponding to any one the following sequences: (1 ) human PI3K nucleotide sequences as set forth for example in GenBank Accession Nos. NM 006219.3, NM 001256045, NM 005026, NM 001350234, and NM 001350235, (2) nucleotide sequences that share at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with any one of the sequences referred to in (1); (3) nucleotide sequences that hybridize under at least low, medium or high stringency conditions to the sequences referred to in (1 ); (4) nucleotide sequences that encode any one of the following amino acid sequences: human PI3K amino acid sequences as set forth for example in UniProt Accession Nos. 000459, P42336, Q8NEB9, 000750, 075747, P48736 and P42338; (5) nucleotide sequences that encode an amino acid sequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity with any one of the sequences referred to in (4); and nucleotide sequences that encode an amino acid sequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with any one of the sequences referred to in (4).JAWS Ref: 751495PCT

[0066] Suitably, the agent is also an mTOR inhibitor, which includes and encompasses any active agent that reduces the accumulation, function (e.g., enzymatic activity, localization, etc.), or stability of mTOR. Representative transcripts of mTOR include nucleotide sequences corresponding to any one the following sequences: (1 ) human mTOR nucleotide sequences as set forth for example in GenBank Accession Nos. NM_004958.2 andNM 001386501.1; (2) nucleotide sequences that share at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with any one of the sequences referred to in (1 ); (3) nucleotide sequences that hybridize under at least low, medium or high stringency conditions to the sequences referred to in (1); (4) nucleotide sequences that encode any one of the following amino acid sequences: human PI3K amino acid sequences as set forth for example in UniProt Accession No. P42345; (5) nucleotide sequences that encode an amino acid sequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity with any one of the sequences referred to in (4); and nucleotide sequences that encode an amino acid sequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with any one of the sequences referred to in (4).

[0067] Antagonist nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, antagonist nucleic acid molecules can interact with PI3K and mTOR mRNA or the genomic DNA of a PIK3 gene (e.g., PIK3CA) or mTOR gene, or they can interact with a PI3K polypeptide and mTOR polypeptide. Often antagonist nucleic acid molecules are designed to interact with other nucleic acids based on sequence homology between the target molecule and the antagonist nucleic acid molecule. In other situations, the specific recognition between the antagonist nucleic acid molecule and the target molecule is not based on sequence homology between the antagonist nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place.

[0100] The present invention further contemplates peptide or polypeptide-based inhibitor compounds. PI3K inhibitory peptides, as described for example above may be modified by being part of a fusion protein. The fusion protein may also include an mTOR inhibitory peptide, in addition to a transport protein or peptide that functions to increase the cellular uptake of the peptide inhibitors, has another desired biological effect, such as a therapeutic effect, or may have both of these functions. The fusion protein may be produced by methods known to the skilled artisan. The inhibitor peptide may be bound, or otherwise conjugated, to another peptide in a variety of ways known to the art. For example, the inhibitor peptide may be bound to a carrier peptide or other peptide described herein via cross-linking wherein both peptides of the fusion protein retain their activity. As a further example, the peptides may be linked or otherwise conjugated to each other by an amide bond from the C-terminal of one peptide to the N-terminal of the other peptide. The linkage between the inhibitor peptide and the other memberJAWS Ref: 751495PCTof the fusion protein may be non-cleavable, with a peptide bond, or cleavable with, for example, an ester or other cleavable bond known to the art.

[0101] In some embodiments, the transport protein or peptide may be, for example, a Drosophila Antennapedia homeodomain-derived sequence comprising the amino acid sequence CRQIKIWFQNRRMKWKK [SEQ ID NO:1], and may be attached to the inhibitor by cross-linking via an N-terminal Cys-Cys bond (as discussed, for example, in Theodore et al., 1995. J. Neurosci. 15:7158-7167; Johnson et al., 1996. Circ. Res 79: 1086). Alternatively, the inhibitor may be modified by a transactivating regulatory protein (Tat)-derived transport polypeptide (such as from amino acids 47-57 of Tat shown in SEQ ID NO:2; YGRKKRRQRRR) from the human immunodeficiency virus, Type 1, as described in Vives et al., 1997. J. Biol. Chem, 272:16010-16017, U. S. Pat. No. 5,804,604 and GenBank Accession No. AAT48070; or with polyarginine as described in Mitchell et al., 2000. J. Peptide Res. 56:318-325 and Rolhbard et al., 2000. Nature Med. 6: 1253-1257). The inhibitors may be modified by other methods known to the skilled artisan in order to increase the cellular uptake of the inhibitors.

[0102] A PI3K / mTOR inhibitory peptide can also be introduced into a cell by introducing into the cell a nucleic acid comprising a nucleotide sequence that encodes a PI3K inhibitory peptide. The nucleic acid can be in the form of a recombinant expression vector. The PI3K inhibitory peptide-encoding sequence can be operably linked to a transcriptional control element(s), e.g., a promoter, in the expression vector. Suitable vectors include, e.g., recombinant retroviruses, lentiviruses, and adenoviruses; retroviral expression vectors, lentiviral expression vectors, nucleic acid expression vectors, and plasmid expression vectors. In some cases, the expression vector is integrated into the genome of a cell. In other cases, the expression vector persists in an episomal state in a cell.

[0103] Suitable expression vectors include, but are not limited to, viral vectors (e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:25432549, 1994; Borras et al., Gene Ther 6:515524, 1999; Li and Davidson, PNAS 92:77007704, 1995; Sakamoto et al., H Gene Ther 5: 1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Hum Gene Ther 9:8186, 1998, Flannery et al., PNAS 94:69166921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863, 1997;Jomary et al., Gene Ther 4:683690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet. 5:591 594, 1996; Srivastava in WO 93 / 09239, Samulski et al., J. Vir. 63:3822-3828, 1989; Mendelson et al., Virol. 166: 154-165, 1988; and Flotte et al., PNAS (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:1031923, 1997; Takahashi et al., J Virol 73:78127816, 1999); a retroviral vector (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leucosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumour virus); and the like.JAWS Ref: 751495PCT

[0104] The present invention also contemplates small molecule agents that reduce the functional activity of PI3K and mTOR. Small molecule agents that reduce functional activity of PI3K that are suitable for use in the present invention include, for example, compounds selected from formula I:Nand stereoisomers, geometric isomers, tautomers, and pharmaceutically acceptable salts thereof, wherein:the dashed lines indicate an optional double bond, and at least one dashed line is a double bond;X1is S, O, N, NRa, CR1, C(R1)2, or -C(R1)2O-;X2is C, CR2or N;X3is C, CR3or N;A is a 5, 6, or 7-membered carbocyclyl or heterocyclyl ring fused to X2and X3, optionally substituted with one or more R5groups;Rais s H, Ci-Ci2alkyl, C2-Cs alkenyl, C2-Cs alkynyl, -(C1-C12 alkylene)-(C3-Ci2carbocyclyl), -(Ci-Ci2alkylene)-(heterocyclyl having 3-20 ring atoms), -(Ci-Ci2alkylene)- C(=O)-(heterocyclyl having 3-20 ring atoms), -(Ci-Ci2alkylene)-(C6-C2o aryl), and -(Ci-Ci2alkylene)-(heteroaryl having 5-20 ring atoms), where alkyl, alkenyl, alkynyl, alkylene, carbocyclyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with one or more groups independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -C(CH3)3, -CH2OH, - CH2CH2OH, -C(CH3)2OH, -CH2OCH3, -CN, -CH2F, -CHF2, -CF3, -CO2H, -COCH3, - COC(CH3)3, -CO2CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NO2, -NH2, - NHCH3, -N(CH3)2, -NHCOCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, - N(CH3)CH2CH2S(O)2CH3, =0, -OH, -OCH3, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, cyclopropyl, cyclobutyl, oxetanyl, morpholino, and 1,1-dioxo-thiopyran-4-yl;R1, R2and R3are independently selected from H, F, Cl, Br, I, -CH3, -CH2CH3, - C(CH3)3, -CH2OH, -CH2CH2OH, -C(CH3)2OH, -CH2OCH3, -CN, -CF3, -CO2H, -COCH3, - COC(CH3)3, -CO2CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NO2, -NH2, - NHCH3, -N(CH3)2, -NHCOCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, - N(CH3)CH2CH2S(O)2CH3, =0, -OH, -OCH3, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, cyclopropyl, cyclobutyl, oxetanyl, morpholino, and 1,1-dioxo-thiopyran-4-yl;JAWS Ref: 751495PCTR4is selected from is selected from C6-C20 aryl, heterocyclyl having 3-20 ring atoms and heteroaryl having 5-20 ring atoms, each of which are optionally substituted with one or more R6groups independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2CH3, -CH2CN, -CN, -CF3, -CH2OH, -CO2H, -CONH2, -CONH(CH3), -CON(CH3)2, -NO2, -NH2, -NHCH3, -NHCOCH3, -OH, -OCH3, -OCH2CH3, -OCH(CH3)2, -SH, -NHC(=O)NHCH3, -NHC(=O)NHCH2CH3, -NHC(=O)NHCH(CH3)2, -NHS(O)2CH3, -N(CH3)C(=O)OC(CH3)3, -S(O)2CH3, benzyl, benzyloxy, morpholinyl, morpholinomethyl, and 4-methylpiperazin-1-yl; andR5is independently selected from C1-C12 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -(C1-C12 alkylene)-(Cs-Ci2 carbocyclyl), -(C1-C12 alkylene)-(heterocyclyl having 3-20 ring atoms), - (C1-C12 alkylene)-C(=O)-(heterocyclyl having 3-20 ring atoms), -(C1-C12 alkylene)-(C6-C2o aryl), and -(C1-C12 alkylene)-(heteroaryl having 5-20 ring atoms); or two geminal R5groups form a 3, 4, 5, or 6-membered carbocyclyl or heterocyclyl ring, where alkyl, alkenyl, alkynyl, alkylene, carbocyclyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with one or more groups independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -C(CH3)3, -CH2OH, - CH2CH2OH, -C(CH3)2OH, -CH2OCH3, -CN, -CH2F, -CHF2, -CF3, -CO2H, -COCH3, -COC(CH3)3, -CO2CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NO2, -NH2, -NHCH3, -N(CH3)2, -NHCOCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, - N(CH3)CH2CH2S(O)2CH3, =0, -OH, -0CH3, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, cyclopropyl, cyclobutyl, oxetanyl, morpholino, and 1, 1-dioxo-thiopyran-4-yl;mor is selected from:optionally substituted with one or more R7groups independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -C(CH3)3, -CH2OCH3, -CHF2, -CN, -CF3, - CH2OH, -CH2OCH3, -CH2CH2OH, -CH2C(CH3)2OH, -CH(CH3)OH, -CH(CH2CH3)OH, -CH2CH(OH)CH3, -C(CH3)2OH, -C(CH3)2OCH3, -CH(CH3)F, -C(CH3)F2, -CH(CH2CH3)F, -C(CH2CH3)2F, -CO2H, -CONH2, -CON(CH2CH3)2, -COCH3, -CON(CH3)2, -NO2, -NH2, -NHCH3, -N(CH3)2, -NHCH2CH3, -NHCH(CH3)2, -NHCH2CH2OH, -NHCH2CH2OCH3, - NHCOCH3, -NHCOCH2CH3, -NHCOCH2OH, -NHS(O)2CH3, -N(CH3)S(O)2CH3, =0, -OH, -OCH3, -OCH2CH3, -OCH(CH3)2, -SH, -NHC(=O)NHCH3, -JAWS Ref: 751495PCTNHC(=O)NHCH2CH3, -S(O)CH3, -S(O)CH2CH3, -S(O)2CH3, -S(O)2NH2, -S(O)2NHCH3, - S(O)2N(CH3)2, and -CH2S(O)2CH3.

[0105] In some other embodiments, the small molecule PI3K:mTOR dual inhibitor is selected from the following group:Paxalisib PI-103.S. LNPI-103BE GSK1059615LY294002 OmnipalisibSN202 NSC765844Dactolisib Samotolisib(S)-4-fluoro-5-(2- (3- methylmopholino)- 6-(1- Voxtalisib (methylsulfonyl)cyclopropyl)HSWpyrimidin-4-yl)pyridin-2-amineJAWS Ref: 751495PCTPF-04691502 Apitolisib(2S,6R)-2,6- dimethyl-4-(4- morpholino-6- H GNE-477 (1 / 7-pyrazol-5- yl)thieno[3,2-c / |pyrimidin-2-Y yl)morpholine2-(2-aminopyrimidin-5-yl)- / V’-(4-methoxybenzoyl)- PKI-402 4-morpholinothieno[ 3,2-c / |[pyrimidine- 6-carbohydrazideVS-5584 Gedatolisib(R)-1-(2-((4- (4,6- dimorpholino- _..<x 1,3,5-triazin-2-0V Bimiralisib...•■U I yl)phenyl) ■!"fT / amino)-1 H-B benzo[c / |imidazole-6- carbonyl)JAWS Ref: 751495PCTpyrrolidine-2- carboxamide(2-((4-(4,6- dimorpholino- 1,3,5-triazin-2- yl)phenyl)amino)- 1 / 7-benzo[c / | imidazole-6-yl)morpholino) methanonePF-04979064

[0106] In some particularly preferred embodiments, the agent is paxalisib.

[0107] The present invention allows for the administration of a lower dosage of the dual PI3K / mTOR inhibitor therapy to a subject, than would typically be administered in order to achieve an effective biological effect when used alone (i.e., not in combination with an immunotherapy or PARP inhibitor and, optionally, a chemotherapy). The ability to utilize lower dosages of the dual PI3K / mTOR therapy reduces the likely toxicity often associated with the administration of the therapy to a subject, without reducing the efficacy of said therapies in the prevention, management, treatment, or amelioration of cancer (e.g., a solid tumour cancer).

[0108] In some embodiments, the lower amount / doses of the dual PI3K / mTOR inhibitor reduces or minimizes any undesired side-effects associated with PI3K therapy.2.2 Immunotherapies

[0109] The compositions of the invention also comprise an anti-cancer therapy, which is typically an immunotherapy. Any immunotherapy that does not target cancer stem cells (CSC) is generally considered suitable for use in the compositions and methods of the present invention. Checkpoint inhibitor molecule antagonists and PARP inhibitors are generally considered as being particularly suitable.2.2.1 Checkpoint inhibitor molecule (ICM) antagonists

[0068] Any suitable ICM antagonist that can be used in therapy is contemplated for use in the practice of the present invention. For example, suitable ICM antagonists includeJAWS Ref: 751495PCTpolypeptides, polynucleotides, carbohydrates, and small molecules. In some preferred embodiments, the ICM antagonist is an antigen-binding molecule.

[0069] The ICM that is antagonized by the therapeutic combinations of the present invention include any one or more of the inhibitory ICM selected from: PD-1, PD-L1, PD-L2, CTLA-4, A2AR, A2BR, CD276, VTCN 1, BTLA, IDO, KIR, LAG 3, TIM-3, VISTA, CD73, CD96, CD155, DNAM-1, TIGIT, CD112, CRTAM, 0X40, OX40L, CD244, CD160, GITR, GITRL, ICOS, GAL-9, 4-1 BBL, 4-1 BB, CD27L, CD28, CD80, CD86, SIRP-1, CD47, CD48, CD244, CD40, CD40L, HVEM, TMIGD2, HHLA2, VEGI, TNFRS25 and ICOLG.

[0070] In some preferred embodiments, an ICM antagonist included in the therapeutic combination is a PD-1 antagonist. In this regard, a “PD-1 antagonist” includes any chemical compound or biological molecule that blocks binding of PD-L1 (for example, PD-L1 expressed the surface of a cancer cell) to PD-1 that is expressed on an immune cell (for example, a T-cell, B-cell, or NKT cell). Alternative names or synonyms for PD-1 include PDCD1, PD1, CD279, and SLEB2. A representative mature amino acid sequence of human PD-1 (UniProt accession no. Q15116) is set out below:PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDK LAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKE SLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRA ARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFP SGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL [SEQ ID NO: 3],

[0071] Examples of monoclonal antibodies (mAbs) that bind to human PD-1, and therefore of use in the present invention, are described in US Patent Publication Nos.US2003 / 0039653, US2004 / 0213795, US2006 / 0110383, US2007 / 0065427, US2007 / 0122378, US2012 / 237522, and International PCT Publication Nos. W02004 / 072286, W02006 / 121168, W02006 / 133396, W02007 / 005874, W02008 / 083174, WO2008 / 156712, W02009 / 024531, W02009 / 014708, W02009 / 114335, WO2010 / 027828, WO2010 / 027423, WO2010 / 036959, WO2010 / 029435, WO2010 / 029434, WO2010 / 063011, WO2010 / 089411, WO2011 / 066342, WO2011 / 110604, WO2011 / 110621, and WO2012 / 145493 (the entire contents of which are incorporated herein by reference). Specific mAbs that are useful for the purposes of the present invention include the anti-PD-1 mAbs nivolumab, pembrolizumab, and pidilizumab, as well as the humanized anti-PD-1 antibodies h409AI I, h409A16, and h409A17 as described in International Patent Publication No. WO2008 / 156712.

[0072] The anti-PD-1 antigen-binding molecules of the invention preferably bind to a region of the extracellular domain of PD-1. By way of example, the anti-PD-1 antigen-binding molecules may specifically bind to a region of the extracellular domain of human PD-1, which comprises one or both of the amino acid sequences SFVLNWYRMSPSNQTDKLAAFPEDR [SEQ ID NO:4] (i.e., residues 62 to 86 of the native PD-1 sequence set forth in SEQ ID NO: 3) and SGTYLCGAISLAPKAQIKE [SEQ ID NO: 5] (i.e., residues 118 to 136 of the native PD-1JAWS Ref: 751495PCTsequence set forth in SEQ ID NO: 3). In another example, the anti-PD-1 antigen-binding molecule binds to a region of the extracellular domain of human PD-1 that comprises the amino acid sequence NWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV [SEQ ID NO: 6] (i.e., corresponding to residue 66 to 97 of the native human PD-1 sequence set forth in SEQ ID NO: 3).

[0073] In certain embodiments, the anti-PD-1 antigen-binding molecule comprises the fully humanized lgG4 mAb nivolumab (as described in detail in US Patent No. 8,008,449 (referred to as "5C4"), which is incorporated herein by reference in its entirety) or an antigenbinding fragment thereof. In representative examples of this type, the anti-PD-1 antigen-binding molecule comprises the CDR sequences as set forth in Table 5.TABLE 5Heavy Chain Light ChainCDR1 NSGMH [SEQ ID NO: 7] CDR1 RASQSVSSYLA [SEQ ID NO: 10] CDR2 VIWYDGSKRYYADSVKG CDR2 DASNRAT [SEQ ID NO: 11][SEQ ID NO: 8]CDR3 NDDYW [SEQ ID NO: 9] CDR3 QQSSNWPRT [SEQ ID NO: 12]

[0074] In more specific embodiments, the anti-PD-1 antigen-binding molecule comprises a heavy chain amino acid sequence of nivolumab as set out for example below:QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSK RYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSAS TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPvTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR WQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK [SEQ ID NO: 13];or an antigen-binding fragment thereof, which comprises, consists or consists essentially of the amino acid sequence:QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSK RYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS[SEQ ID NO: 14],

[0075] In some of the same and other embodiments, the anti-PD-1 antigenbinding molecule may comprise the light chain amino acid sequence of nivolumab as set out for example below:JAWS Ref: 751495PCTEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID N0:15];or an antigen-binding fragment thereof, which comprises, consists or consists essentially of the amino acid sequence:EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGT DFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK [SEQ ID NO:16],

[0076] In alternate embodiments, the anti-PD-1 antigen-binding molecule comprises the humanized lgG4 mAb pembrolizumab or an antigen-binding fragment thereof. In non-limiting examples of this type, the anti-PD-1 antigen-binding molecule comprises the CDR sequences as set forth in Table 6.TABLE 6Heavy Chain Light ChainCDR1 NYYMY [SEQ ID NO: 17] CDR1 RASKGVSTSGYSYLH[SEQ ID NO: 20]CDR2 GINPSNGGTNFNEKFKN CDR2 LASYLES [SEQ ID NO: 21][SEQ ID NO: 18]CDR3 RDYRFDMGFDY CDR3 QHSRDLPLT [SEQ ID NO: 22][SEQ ID NO: 19]

[0077] In some embodiments, the anti-PD-1 antigen-binding molecule competes with the mAb pembrolizumab for binding to PD-1.

[0078] In additional embodiments, the anti-PD-1 antigen-binding molecule comprises the heavy chain amino acid sequence of pembrolizumab as set out for example below:QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGT TVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPvTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK [SEQ ID NO:23];or an antigen-binding fragment thereof, which comprises, consists or consists essentially of the amino acid sequence:JAWS Ref: 751495PCTQVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGIN PSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYW GQGTTVTVSS [SEQ ID NO:24],

[0079] Similarly, the anti-PD-1 antigen-binding molecule may comprise a light chain amino acid sequence of pembrolizumab as set out for example below:EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 25];or an antigen-binding fragment thereof, which comprises, consists or consists essentially of the amino acid sequence:EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK [SEQ ID NO: 26],

[0080] In yet other embodiments of this type, the anti-PD-1 antigen-binding molecule comprises the mAb pidilizumab or an antigen-binding fragment thereof. In some related embodiments, the anti-PD-1 antigen-binding molecule comprises CDR sequences as set forth in Table 7.TABLE 7Heavy chain Light chain CDR1 NYGMN [SEQ ID NO: 102] CDR1 SARSSVSYMH [SEQ ID NO: 105] CDR2 WINTDSGESTYAEEFKG CDR2 RTSNLAS [SEQ ID NO:[SEQ ID NO: 103] 106] CDR3 VGYDALDY [SEQ ID NO: 104] CDR3 QQRSSFPLT [SEQ ID NO: 107]

[0081] In more specific embodiments, the anti-PD-1 antigen-binding molecule comprises a heavy chain amino acid sequence of pidilizumab as set forth below:QVQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSG ESTYAEEFKGRFVFSLDTSVNTAYLQITSLTAEDTGMYFCVRVGYDALDYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLJAWS Ref: 751495PCTPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 27];or an antigen-binding fragment thereof, which comprises, consists or consists essentially of the amino acid sequence:QVQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSG ESTYAEEFKGRFVFSLDTSVNTAYLQITSLTAEDTGMYFCVRVGYDALDYWGQGTLVTV SS [SEQ ID NO: 28],

[0082] In some of the same and other embodiments, the anti-PD-1 antigenbinding molecule comprises the light chain amino acid sequence of pidilizumab as shown below:EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPS RFSGSGSGTSYCLTINSLQPEDFATYYCQQRSSFPLTFGGGTKLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 29],or an antigen-binding fragment thereof, which comprises, consists or consists essentially of the amino acid sequence:EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPS RFSGSGSGTSYCLTINSLQPEDFATYYCQQRSSFPLTFGGGTKLEIK [SEQ ID NO: 30],

[0083] Other suitable mAbs are described in the International Patent Publication No. WQ2015 / 026634, which is hereby incorporated by reference herein in its entirety. These include mAbs, or antigen-binding fragments thereof, which comprise: (a) light chain CDRs with amino acid sequences: RASKSVSTSGFSYLH [SEQ ID NO: 31], LASNLES [SEQ ID NO: 32], and QHSWELPLT [SEQ ID NO: 33] (CDR1, CDR2, and CDR3, respectively) and heavy chain CDRs with amino acid sequences SYYLY [SEQ ID NO: 34], GVNPSNGGTNFSEKFKS [SEQ ID NO: 35] and RDSNYDGGFDY [SEQ ID NO: 36] (CDR1, CDR2, and CDR3, respectively); or (b) light chain CDRs with amino acid sequence RASKGVSTSGYSYLH [SEQ ID NO: 37], LASYLES [SEQ ID NO: 38], and QHSRDLPLT [SEQ ID NO: 39] (CDR1, CDR2, and CDR3, respectively), and heavy chain CDRs with amino acid sequence NYYMY [SEQ ID NO: 40], GINPSNGGTNFN EKFKN [SEQ ID NO: 41], and RDYRFDMGFDY [SEQ ID NO: 42] (CDR1, CDR2, and CDR3, respectively).

[0084] By way of an illustration, such mAbs may comprise (a) a heavy chain variable region comprising:QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGT TVTVSS [SEQ ID NO: 43],or a variant or antigen-binding fragment thereof; andJAWS Ref: 751495PCT

[0085] a light chain variable region comprising an amino acid sequence selected from:EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSG SGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK [SEQ ID NO: 44], IVLTQSPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIYLASYLESG VPDRFSGSGS GTDFTLKISRVEAEDVGVYYCQHSRDLPLTFGQGTKLEIK [SEQ ID NO: 45], or DIVMTQTPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIYLASYLES GVPDRFSGS GSGTAFTLKISRVEAEDVGLYYCQHSRDLPLTFGQGTKLEIK [SEQ ID NO: 46], or a variant or antigen-binding fragment thereof.

[0086] In yet further exemplary embodiments, the anti-PD-1 mAb may comprise the lgG1 heavy chain comprising:QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNG GTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGT TVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKP REEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYT LPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK [SEQ ID NO: 47]or a variant or antigen-binding fragment thereof; and a light chain comprising any one of:EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLES GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 48], EIVLTQSPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIYLASYLES GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSRDLPLTFGQGTKLEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 49] DIVMTQTPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIYLASYLES GVPDRFSGSGSGTAFTLKISRVEAEDVGLYYCQHSRDLPLTFGQGTKLEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 50], or a variant or an antigen-binding fragment thereof.JAWS Ref: 751495PCT

[0087] In other embodiments, the ICM antagonist is a PD-L1 antagonist.Alternative names or synonyms for PD-L1 include PDCD1 L1, PDL1, B7H1, B7-4, CD274, and B7-H. Generally, the PD-L1 antagonists specifically bind to the native amino acid sequence of human PD-L1 (UniProt accession no. Q9NZQ7) as set out below:MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEME DKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYG GADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKT TTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHL VILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET [SEQ ID NO: 51],

[0088] Suitably, the PD-L1 antagonist is an anti-PD-L1 antigen-binding molecule. By way of example, anti-PD-L1 antigen-binding molecules that are suitable for use with the present invention include the anti-PD-L1 mAbs durvalumab (MEDI4736), atezolizumab (Tecentriq), BMS-936559 / MDX-1105, MSB0010718C, LY3300054, CA-170, GNS-1480, MPDL3280A, and avelumab. These and other anti-PD-LI antibodies are described in International Publication Nos. W02007 / 005874 and WO2010 / 077634, and U. S. Patent Nos. 8,217,149, and 8,779,108, the entirety of each is incorporated herein by reference. Further anti-PD-LI mAbs are described in International PCT Patent Publication No. WO2016 / 007,235, the entire contents of which is also incorporated herein by reference.

[0089] The anti-PD-L1 antigen-binding molecules suitably bind to a region of the extracellular domain of PD-L1. By way of illustration, the anti-PD-L1 antigen-binding molecules may specifically bind to a region of the extracellular domain of human PD-L1 that comprises the amino acid sequence SKKQSDTHLEET [SEQ ID NO: 13] (i.e., residues 279 to 290 of the native PD-L1 sequence set forth in SEQ ID NO: 14). In certain embodiments, the anti-PD-L1 antigenbinding molecule comprises the fully humanized IgG 1 mAb durvalumab (as described with reference to “MEDI4736” in International PCT Publication No. WQ2011 / 066389, and U. S.Patent Publication No 2013 / 034559, which are incorporated herein by reference in their entirety) or an antigen-binding fragment thereof. In representative embodiments of this type, the anti-PD-LI antigen-binding molecule comprises the CDR sequences as set forth in Table 8.TABLE 8Heavy chain Light chainCDR1 RYWMS CDR1 RASQRVSSSYLA[SEQ ID NO: 132] [SEQ ID NO: 135]CDR2 NIKQDGSEKYYVDSVK CDR2 DASSRATGIPD[SEQ ID NO: 133] [SEQ ID NO: 136]CDR3 EGGWFGELAFDY CDR3 QQYGSLPWT[SEQ ID NO: 134] [SEQ ID NO: 137]

[0090] In more specific embodiments, the anti-PD-L1 antigen-binding molecule comprises the heavy chain amino acid sequence of durvalumab as set out for example below:JAWS Ref: 751495PCTVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEK YYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG [SEQ ID NO: 52], or an antigen-binding fragment thereof, which comprises, consists or consists essentially of the amino acid sequence:VQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEK YYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTL VTVSS [SEQ ID NO: 53],

[0091] In some of the same and other embodiments, the anti-PD-L1 antigenbinding molecule may comprise the light chain amino acid sequence:EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 54],or an antigen-binding fragment thereof, which comprises, consists or consists essentially of the amino acid sequence:EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIK [SEQ ID NO: 55],

[0092] Alternatively, the anti-PD-L1 antigen-binding molecule competes for binding to PD-L1 with the mAb durvalumab.

[0093] In other embodiments, the anti-PD-L1 antigen-binding molecule comprises the fully humanized IgG 1 mAb atezolizumab (as described in U. S. Patent No. 8,217148, the entire content of which is incorporated herein by reference) or an antigen-binding fragment thereof. In representative embodiments of this type, the anti-PD-L1 antigen-binding molecule comprises the CDR sequences as set forth in Table 9.TABLE 9Heavy chain Light chain CDR1 GFTFSXiSWIH [SEQ ID NO: 142] CDR1 RASQX4X5X6TX7X8A [SEQ ID NO: 145] CDR2 AWIX2PYGGSX3YYADSVKG CDR2 SASX9LX10S[SEQ ID NO: 143] [SEQ ID NO: 146]JAWS Ref: 751495PCTCDR3 RHWPGGFDY [SEQ ID NO: 144] CDR3 QQX11X12X13X14PX15T [SEQ ID NO: 147] wherein Xi is D or G; X2 is S or L; X3 is T or S; X4 is D or V; Xs is V or I; Xe is S or N; X7 is A or F; X8is V or L; X9is F or T; X10 is Y or A; Xu is Y, G, or F; X12 is L, Y or F; X13 is Y, N, T,G, F or I; X14 is H, V, P, T, or I; and X15 is A, W, R, P, or T.

[0094] In more specific embodiments, the anti-PD-L1 antigen-binding molecule comprises the heavy chain amino acid sequence of atezolizumab as set forth for example below:EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYA DSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKD TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK [SEQ ID NO: 56],or an antigen-binding fragment thereof, which comprises, consists or consists essentially of the amino acid sequence:EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGS TYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVT VSS [SEQ ID NO: 57],

[0095] In some of the same and other embodiments, the anti-PD-L1 antigenbinding molecule comprises the light chain amino acid sequence of atezolizumab as provided for example below:DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO:58],or an antigen-binding fragment thereof, which comprises, consists or consists essentially of the amino acid sequence:DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK [SEQ ID NO: 59],

[0110] Alternatively, the anti-PD-L1 antigen-binding molecule competes for binding to PD-L1 with the mAb atezolizumab.

[0111] In other embodiments, the anti-PD-L1 antigen-binding molecule comprises the fully humanized lgG1 mAb avelumab (as described in U. S. Patent No. 8,217148, the entireJAWS Ref: 751495PCTcontents of which is incorporated herein by reference) or an antigen-binding fragment thereof. In representative embodiments of this type, the anti-PD-L1 antigen-binding molecule comprises the CDR sequences as set forth in Table 10.TABLE 10Heavy chain Light chain CDR1 X1YX2MX3 [SEQ ID NO: 152] CDR1 TGTX7XsDVGX9YNYVS [SEQ ID NO: 155] CDR2 SIYPSGGX4TFYADX5VKG CDR2 X10VX11X12RPS [SEQ ID NO: 153] [SEQ ID NO: 156] CDR3 IKLGTVTTVXeY CDR3 SSX13X14X15X16X17RV [SEQ ID NO: 154] [SEQ ID NO: 157] wherein Xi is M, I, or S; X2 is R, K, L, M, or I; X3 is F or M; X4 is F or I; Xs is S or T; Xe is E or D; X7 is N or S; Xs is T, R, or S; X9 is A or G; X10 is E or D; Xn is I, N, or S; X12 is D, H, or N;X13 is F or Y; X14 is N or S; X15 is R, T, or S; X16 is G or S; and X17 is I or T.

[0112] In specific embodiments, the anti-PD-L1 antigen-binding molecule comprises the heavy chain amino acid sequence of avelumab as provided for example below:EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGIT FYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 60], or an antigen-binding fragment thereof, which comprises, consists or consists essentially of the amino acid sequence:EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGIT FYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVT VSS [SEQ ID NO: 61],

[0096] In some of the same and other embodiments, the anti-PD-LI antigenbinding molecule comprises the light chain amino acid sequence of avelumab as set out for example below:QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPS GVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANP TVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKY AASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS [SEQ ID NO: 62], or an antigen-binding fragment thereof, which comprises, consists or consists essentially of the amino acid sequence:JAWS Ref: 751495PCTQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPS GVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL [SEQ ID NO: 63],

[0113] Alternatively, the anti-PD-L1 antigen-binding molecule competes for binding to PD-L1 with the mAb avelumab.

[0114] In some embodiments, the ICM antagonist is an antagonist of CTLA4. Alternative names or synonyms for CTLA4 include ALPS5, CD, CD152, CELIAC3, CTLA-4, GRD4, GSE, IDDM 12. Generally, the CTLA4 antagonists bind specifically to the mature amino acid sequence of human CTLA4 (UniProt accession no. P16410) as set out for example below:KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGN ELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDP EPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQ FQPYFIPIN [SEQ ID NO: 64],

[0115] Suitably, the CTLA4 antagonist is an anti-CTLA4 antigen-binding molecule. By way of example, anti-CTLA4 antigen-binding molecules that are suitable for use with the present invention include the anti-CTLA4 mAbs ipilimumab (BMS-734016, MDX-010, MDX-101) and tremelimumab (ticilimumab, CP-675,206).

[0116] The anti-CTLA4 antigen-binding molecules suitably bind to a region of the extracellular domain of CTLA4. By way of illustration, the anti-CTLA4 antigen-binding molecules may specifically bind to a region of the extracellular domain of human CTLA4 that comprises any one or more of the amino acid sequences YASPGKATEVRVTVLRQA [SEQ ID NO: 65] (i.e., residues 26 to 42 of the native CTLA4 sequence set forth in SEQ ID NO: 64), DSQVTEVCAATYMMGNELTFLDD [SEQ ID NO: 66] (i.e., residues 43 to 65 of the native CTLA4 sequence set forth in SEQ ID NO: 64), and VELMYPPPYYLGIG [SEQ ID NO: 67] (i.e., residues 96 to 109 of the native CTLA4 sequence set forth in SEQ ID NO: 64). Alternatively or in addition, the anti-CTLA4 antigen-binding molecules may specifically bind to a region of the extracellular domain of human CTLA4 that comprises any one or more and preferably all of the following residues of the mature form of CTLA4: KI, A2, M3, E33, R35, Q41, S44, Q45, V46, E48, L91, 193, K95, E97, M99, P102, P103, Y104, Y105, L106, 1108, N110.

[0117] In certain embodiments, the anti-CTLA4 antigen-binding molecule comprises the human IgG 1 mAb ipilimumab (as described for example in International Publication WQ2014 / 209804 and U. S. Patent Publication No 2015 / 0283234, the entire contents of which are incorporated herein by reference) or an antigen-binding fragment thereof. In representative embodiments of this type, the anti-CTA4 antigen-binding molecule comprises the CDR sequences as set forth in Table 11.TABLE 11Heavy chain Light chainJAWS Ref: 751495PCTCDR1 SYTMH [SEQ ID NO: 162] CDR1 RASQSVGSSYLA [SEQ ID NO: 165] CDR2 FISYDGNNKYYADSVKG CDR2 GAFSRAT [SEQ ID NO: 163] [SEQ ID NO: 166] CDR3 TGWLGPFDY [SEQ ID NO: 164] CDR3 QQYGSSPWT[SEQ ID NO: 167]

[0118] In more specific embodiments, the anti-CTLA4 antigen-binding molecule comprises the heavy chain amino acid sequence of ipilimumab as set out for example below:

[0119] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWV TFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 68],or an antigen-binding fragment thereof, a non-limiting example of which comprises, consists or consists essentially of the amino acid sequence:QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGN NKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTL VTVSS [SEQ ID NO: 69],

[0120] In some of the same and other embodiments, the anti-CTLA4 antigenbinding molecule comprises the light chain amino acid sequence of ipilimumab as set out for example below:EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 70],or an antigen-binding fragment thereof, a representative example of which comprises, consists or consists essentially of the amino acid sequence:EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK [SEQ ID NO: 71].

[0121] In some embodiments, the anti-CTAL4 antigen-binding molecule comprises the human lgG2 mAb tremelimumab (as described for example in U. S. Patent Publication No 2009 / 0074787, the entire content of which is incorporated herein by reference) or an antigen-JAWS Ref: 751495PCTbinding fragment thereof. In representative embodiments of this type, the anti-CTLA4 antigenbinding molecule comprises the CDR sequences as set forth in Table 12.TABLE 12Heavy chain Light chain CDR1 GFTFSSYGMH [SEQ ID NO: 172] CDR1 RASQSINSYLD [SEQ ID NO: 175] CDR2 VIWYDGSNKYYADSV CDR2 AASSLQS [SEQ ID NO: 173] [SEQ ID NO: 176] CDR3 D P RG ATLYYYYYG M D V CDR3 QQYYSTPFT[SEQ ID NO: 174] [SEQ ID NO: 177]

[0122] In more specific embodiments, the anti-CTLA4 antigen-binding molecule comprises the heavy chain amino acid sequence of tremelimumab as set out for example below:QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGS NKYYADSVKGRFTISRDNSKNTLYIQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDV WGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVE CPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 172],or an antigen-binding fragment thereof, a non-limiting example of which comprises, consists or consists essentially of the amino acid sequence:QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNN KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVT VSS [SEQ ID NO: 73],

[0123] In some of the same and other embodiments, the anti-CTLA4 antigenbinding molecule comprises the light chain amino acid sequence of tremelimumab as set out for example below:DIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 74],or an antigen-binding fragment thereof, a representative example of which comprises, consists or consists essentially of the amino acid sequence:JAWS Ref: 751495PCTDIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIK [SEQ ID NO: 75],

[0124] In other embodiments, the ICM antagonist is a B7-H3 antagonist. Generally, the B7-H3 antagonists of the invention bind specifically to the native amino acid sequence of human B7-H3 (UniProt accession no. Q5ZPR3) as set out for example below:MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGF SLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADE GSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVF WQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSS VTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLV HSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVS LQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTS QMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEALWV TVGLSVCLIALLVALAFVCWRKIKQSCEEENAGAEDQDGEGEGSKTALQPLKHSDSKED DGQEIA [SEQ ID NO: 76],

[0125] Suitably, the B7-H3 antagonist is an anti-B7-H3 antigen-binding molecule. By way of an example, an anti-B7-H3 antigen-binding molecule suitable for use with the present invention is the mAb enoblituzumab or an antigen-binding fragment thereof. In some embodiments the anti-B7-H3 antigen-binding molecule comprises CDR sequences as set forth in Table 13.TABLE 13Heavy chain Light chain CDR1 FGMH [SEQ ID NO: 183] CDR1 KASQNVDTNVA [SEQ ID NO: 186] CDR2 YISSDSSAIYYADTVK CDR2 SASYRYS [SEQ ID NO: 184] [SEQ ID NO: 187] CDR3 GRENIYYGSRLDY [SEQ ID NO: CDR3 QQYNNYPFT185] [SEQ ID NO: 188]

[0126] In more specific embodiments, the anti-B7-H3 antigen-binding molecule comprises the heavy chain amino acid sequence of enoblituzumab as set out for example below:VQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIY YADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTT VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCP APELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVJAWS Ref: 751495PCTYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 77], or an antigen-binding fragment thereof, a representative example of which comprises, consists or consists essentially of the amino acid sequence:VQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIY YADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTT VTVSS [SEQ ID NO: 78],

[0127] In some of the same and other embodiments, the anti-B7-H3 antigen-binding molecules comprise the light chain amino acid sequence of enoblituzumab as provided for example below.DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKRTVAAPSVFIFP PSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 79],or an antigen-binding fragment thereof, a representative example of which comprises, consists or consists essentially of the amino acid sequence:DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIK [SEQ ID NO: 80],

[0128] In some alternative embodiments, the anti-B7-H3 antigen-binding molecule competes for binding to B7-H3 with the mAb enoblituzumab.

[0129] In other embodiments, the ICM antagonist is an indoleamine 2,3-dioxygenase (IDO) antagonist. The mature amino acid sequence of human IDO (UniProt accession no. P14902) as set out for example below:MAHAMENSWTISKEYHIDEEVGFALPNPQENLPDFYNDWMFIAKHLPDLIESGQLRERV EKLNMLSIDHLTDHKSQRLARLVLGCITMAYVWGKGHGDVRKVLPRNIAVPYCQLSKKL ELPPILVYADCVLANWKKKDPNKPLTYENMDVLFSFRDGDCSKGFFLVSLLVEIAAASAIK VIPTVFKAMQMQERDTLLKALLEIASCLEKALQVFHQIHDHVNPKAFFSVLRIYLSGWKG NPQLSDGLVYEGFWEDPKEFAGGSAGQSSVFQCFDVLLGIQQTAGGGHAAQFLQDMR RYMPPAHRNFLCSLESNPSVREFVLSKGDAGLREAYDACVKALVSLRSYHLQIVTKYILIP AS QQPKENKTSEDPSKLEAKGTGGTDLMNFLKTVRSTTEKSLLKEG [SEQ ID NO: 81],

[0130] Any IDO antagonist is suitable for use in the therapeutic agents of the present invention. Currently, three small molecule IDO inhibitors are undergoing development for clinical use: GDC-0919 (1-cyclohexyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)ethanol), indoximod (1-methyl-D-tryptophan), and epacadostat (1,2,5-Oxadiazole-3-carboximidamide, 4-((2-((Aminosulfonyl)amino)ethyl)amino)-N-(3-bromo-4-fluorophenyl)-N’-hydroxy-, (C(Z))-). The molecular structure of each of these molecules is provided, below.JAWS Ref: 751495PCTIndoximod GDC-0919 Epacadostat

[0131] In some embodiments, the ICM antagonist is a killer-cell immunoglobulin (KIR) antagonist. In preferred embodiments of this type, the KIR antagonist blocks the interaction between KIR2-DL-1, -2, and -3 and their ligands. The mature amino acid sequence of a human KIR, i.e., KIR2-DL1 (UniProt accession no. P43626) is provided, for example, below:HEGVHRKPSLLAHPGPLVKSEETVILQCWSDVMFEHFLLHREGMFNDTLRLIGEHHDGV SKANFSISRMTQDLAGTYRCYGSVTHSPYQVSAPSDPLDIVIIGLYEKPSLSAQPGPTVLA GENVTLSCSSRSSYDMYHLSREGEAHERRLPAGPKVNGTFQADFPLGPATHGGTYRCF GSFHDSPYEWSKSSDPLLVSVTGNPSNSWPSPTEPSSKTGNPRHLHILIGTSVVIILFILL FFLLHRWCSNKKNAAVMDQESAGNRTANSEDSDEQDPQEVTYTQLNHCVFTQRKITRP SQRPKTPPTDIIVYTELPNAESRSKVVSCP [SEQ ID NO: 82],

[0132] Anti-KIR antigen-binding molecules that are suitable for use in the invention can be generated using methods well known in the art. Alternatively, art- recognized KIR antigen-binding molecules can be used. For example, the anti-KIR antigen-binding molecule comprises the fully humanized mAb Lirilumab or an antigen-binding fragment thereof as described for example in International Publication No. WO2014 / 066532, the entire content of which is hereby incorporated herein in its entirety. Suitably, the anti-KIR antigen-binding molecule comprises the CDR regions as set forth in Table 14.TABLE 14Heavy chain Light chainCDR1 FYAIS [SEQ ID NO: 195] CDR1 RASQSVSSYLA [SEQ ID NO: 198] CDR2 GFIPIFGAANYAQKFQ CDR2 DASNRAT[SEQ ID NO: 196] [SEQ ID NO: 199] CDR3 IPSGSYYYDYDMDV[SEQ ID NO: CDR3 QQRSNWMYT [SEQ ID NO:197] 200]

[0133] In representative embodiments of this type, the anti-KIR antigen-binding molecule may comprise the heavy chain variable domain amino acid sequence of Lirilumab, as set out for example below:JAWS Ref: 751495PCTQVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYAISWVRQAPGQGLEWMGGFIPIFGAA NYAQKFQGRVTITADESTSTAYMELSSLRSDDTAVYYCARIPSGSYYYDYDMDVWGQG TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK [SEQ ID NO: 83], or an antigen-binding fragment thereof, a representative example of which comprises, consists or consists essentially of the amino acid sequence:QVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYAISWVRQAPGQGLEWMGGFIPIFGAA NYAQKFQGRVTITADESTSTAYMELSSLRSDDTAVYYCARIPSGSYYYDYDMDVWGQG TTVTVSS [SEQ ID NO: 84],

[0134] In some of the same and other embodiments, the anti-KIR antigen-binding molecule may comprise the light chain variable domain amino acid sequence of Lirilumab, as set out for example below:EIVLTQSPVTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWMYTFGQGTKLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 85],or an antigen-binding fragment thereof, a representative example of which comprises, consists or consists essentially of the amino acid sequence:EIVLTQSPVTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWMYTFGQGTKLEIKRT [SEQ ID NO: 86],

[0135] In alternative embodiments, the ICM antagonist is a LAG-3 antagonist. LAG-3 is a 503 amino acid type I transmembrane protein, with four extracellular Ig -like domains. LAG-3 is expressed on activated T-cells, NK cells, B-cells, and plasmacytoid DCs. The representative mature amino acid sequence of human LAG-3 (UniProt accession no.P18627), is set out below:LQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPL APGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLR PARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPD RPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSI MYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTG DNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEV TPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTJAWS Ref: 751495PCTELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLVTGAFGFHLWRRQWRPRRFSALE QGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPE QL [SEQ ID NO: 87],

[0136] In some embodiments, the LAG-3 antagonist is an anti-LAG-3 antigenbinding molecule. By way of an illustration, a suitable anti-LAG antigen-binding molecule is the anti-LAG3 humanized mAb, BMS-986016. Other anti-LAG-3 antibodies are described in U. S. Patent Publication No. 2011 / 0150892 and International PCT Publication Nos. WO2010 / 019570 and WO2014 / 008218, each of which is incorporated herein by reference in their entirety.

[0137] In some embodiments, the anti-LAG-3 antigen-binding molecules comprise the CDR sequences set forth in Table 15.TABLE 15Heavy chain Light chainCDR1 DYYWN [SEQ ID NO: 206] CDR1 RASQSISSYLA [SEQ ID NO: 209] CDR2 EINHRGSTNSNPSLKS CDR2 DASNRAT[SEQ ID NO: 207] [SEQ ID NO: 210] CDR3 GYSDYEYNWFDP [SEQ ID NO: CDR3 QQRSNWPLT [SEQ ID NO:208] 211]

[0138] The anti-LAG-3 antigen-binding molecules suitably comprise the mAb BMS-986016 or an antigen-binding fragment thereof. More specifically, in some embodiments, the anti-LAG-3 antigen-binding molecule has the heavy chain amino acid sequence of BMS-986016 as set out for example below:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTN SNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLV TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEF LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTL PPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRL TVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK [SEQ ID NO: 88],or an antigen-binding fragment thereof, a representative example of which comprises, consists or consists essentially of the amino acid sequence:QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTN SNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLV TVSS [SEQ ID NO: 89],

[0139] Similarly, the anti-LAG-3 antigen-binding molecules may comprise a light chain amino acid sequence of BMS-986016 as set forth in SEQ ID NO:45 and provided below, of an antigen-binging fragment thereof:JAWS Ref: 751495PCTEIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: 90]or an antigen-binding fragment thereof, a representative example of which comprises, consists or consists essentially of the amino acid sequence:EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIK [SEQ ID NO: 91], 2.2.2 PARP inhibitors

[0140] Poly(ADP-ribose) polymerase (PARP) enzymes are a family of enzymes that cleave NAD+, releasing nicotinamide, and successively add ADP-ribose units to form ADP-ribose polymers. Activation of PARP enzymes can lead to depletion of cellular NAD+ levels (e.g., PARPs as NAD+ consumers) and mediates cellular signalling through ADP-ribosylation of downstream targets. PARP-1 is a zinc-finger DNA-binding enzyme that is activated by binding to DNA double or single strand breaks. It was known that anti-alkylating agents could deplete the NAD+ content of tumour cells, and the discovery of PARPs explained this phenomenon. (PARP Inhibitors and Cancer Therapy. Curtin N. in Poly ADP Ribosylation. ed. Alexander Burke, Lands Bioscience and Springer Bioscience, 2006: 218-233). Anti-alkylating agents induce DNA strand breaks, which activates of PARP-1, which is part of the DNA repair pathway. Poly ADP-ribosylation of nuclear proteins by PARP-1 converts DNA damage into intracellular signals that can either activate DNA repair (e.g., by the base excision repair (BER) pathway); or trigger cell death in the presence of DNA damage that is too extensive and cannot be efficiently repaired.

[0141] PARP-2 contains a catalytic domain and is capable of catalysing a poly(ADP-ribosyl)ation reaction. PARP-2 displays auto-modification properties similar to PARP-1. The protein is localized in the nucleus in vivo and may account for the residual poly(ADP-ribose) synthesis observed in PARP-1 -deficient cells, treated with alkylating agents or hydrogen peroxide. Some agents that inhibit PARP (e.g., agents primarily aimed at inhibiting PARP-1) may also inhibit PARP-2 (e.g., niraparib).

[0142] The role of PARP enzymes in DNA damage response (e.g., repair of DNA in response to genotoxic stress) has led to the compelling suggestion that PARP inhibitors may be useful anti-cancer agents. PARP inhibitors may be particularly effective in treating cancers resulting from germ line or sporadic deficiency in the homologous recombination DNA repair pathway, such as BRCA-1 and / or BRCA-2 deficient cancers.

[0143] Pre-clinical ex vivo and in vivo experiments suggest that PARP inhibitors are selectively cytotoxic for tumours with homozygous inactivation of BRCA-1 and / or BRCA-2 genes, which are known to be important in the homologous recombination (HR) DNA repair pathway. The biological basis for the use of PARP inhibitors as single agents in cancers withJAWS Ref: 751495PCTdefects in BRCA-1 and / or BRCA-2 is the requirement of PARP-1 and PARP-2 for base excision repair (BER) of the damaged DNA. Upon formation of single-strand DNA breaks, PARP-1 and PARP-2 bind at sites of lesions, become activated, and catalyse the addition of long polymers of ADP-ribose (PAR chains) on several proteins associated with chromatin, including histones. This results in chromatin relaxation and fast recruitment of DNA repair factors that access and repair DNA breaks. Normal cells repair up to 10,000 DNA defects daily and single strand breaks are the most common form of DNA damage. Cells with defects in the BER pathway enter S phase with unrepaired single strand breaks. Pre-existing single strand breaks are converted to double strand breaks as the replication machinery passes through the break. Double strand breaks present during S phase are preferentially repaired by the error-free HR pathway. Cells with inactivation of genes required for HR, such as BRCA-1 and / or BRCA-2, accumulate stalled replication forks during S phase and may use error- prone non-homologous end joining (NHEJ) to repair damaged DNA. Both the inability to complete S phase (because of stalled replication forks) and error-prone repair by NHEJ, are thought to contribute to cell death.

[0144] Without wishing to be bound by theory, it is hypothesized that treatment with PARP inhibitors may selectively kill a subset of cancer cells with deficiencies in DNA repair pathways (e.g., inactivation of BRCA-1 and / or BRCA-2). For example, a tumour arising in a patient with a germline BRCA mutation has a defective homologous recombination DNA repair pathway and would be increasingly dependent on BER, a pathway blocked by PARP inhibitors, for maintenance of genomic integrity. This concept of inducing death by use of PARP inhibitors to block one DNA repair pathway in tumours with pre-existing deficiencies in a complementary DNA repair pathways is called synthetic lethality.

[0145] The therapeutic potential of PARP inhibitors is further expanded by the observation that PARP inhibitors not only have monotherapy activity in HR-deficient tumours, but are also effective in preclinical models in combination with other agents such as cisplatin, carboplatin, alkylating and methylating agents, radiation therapy, and topoisomerase I inhibitors. In contrast to the rationale for monotherapy in which PARP inhibition alone is sufficient for cell death in HR-deficient cancers (due to endogenous DNA damage), PARP is required for repair of DNA damage induced by standard cytotoxic chemotherapy. In some cases, the specific role of PARP is not known, but PARP is known to be required to release trapped topoisomerase l / irinotecan complexes from DNA. Temozolomide-induced DNA damage is repaired by the BER pathway, which requires PARP to recruit repair proteins. Combination therapies that enhance or synergize the cancer therapy without significantly increasing toxicity would provide substantial benefit to cancer patients, including ovarian cancer patients.

[0146] In some embodiments, treatment with PARP inhibitors (e.g., PARP-1 / 2 inhibitors) as provided herein for the various methods and kits disclosed herein may selectively kill a subset of cancer cell types by exploiting their deficiencies in DNA repair. Human cancers exhibit genomic instability and an increased mutation rate due to underlying defects in DNA repair. These deficiencies render cancer cells more dependent on the remaining DNA repairJAWS Ref: 751495PCTpathways and targeting these pathways is expected to have an impact on the survival of the tumour cells than on normal cells.

[0147] In some embodiments, a PARP inhibitor is selected from the group comprising ABT-767, AZD 2461, BGB-290, BGP 15, CEP 8983, CEP 9722, DR 2313, E7016, E7449, fluzoparib (SHR 3162), IMP 4297, INO1001, JPI 289, JPI 547, monoclonal antibody B3-LysPE40 conjugate, MP 124, niraparib (ZEJULA), NU 1025, NU 1064, NU 1076, NU1085, olaparib, ONO2231, PD 128763, R 503, R554, rucaparib (RUBRACA), SBP 101, SC 101914, Simmiparib, talazoparib (BMN-673), veliparib (ABT-888), and WW 46, 2-(4-(Trifluoromethyl)phenyl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-ol, or a pharmaceutically acceptable salt thereof.

[0148] In some embodiments, the PARP inhibitor is a small molecule. In some alternative embodiments, the PARP inhibitor is an antibody agent. In some embodiments, an agent that inhibits PARP is a combination of agents.

[0149] In some embodiments, the PARP inhibitor is selected from the group comprising, consisting, or consisting essentially of: olaparib, niraparib, rucaparib, talazoparib, veliparib, or any combination thereof. In some embodiments, the PARP inhibitor is prepared as a pharmaceutically acceptable salt. In some embodiments, the salt form can exist as a solvated or hydrated polymorphic form.

[0150] Accordingly, the PARP inhibitor may be selected from olaparib, niraparib, rucaparib, and talazoparib, or a pharmaceutically acceptable salt thereof.

[0151] In some embodiments, the PARP inhibitor is olaparib, or a pharmaceutically acceptable salt thereof. In some embodiment, the PARP inhibitor is olaparib.

[0152] Olaparib (AZD2281, KU-0059436) is a potent PARP inhibitor (PARP 1, 2, and 3) that is being developed as a monotherapy as well as for combination with chemotherapy, ionizing radiation and other anticancer agents including novel agents and immunotherapy.

[0153] PARP inhibition is a novel approach to targeting tumours with deficiencies in DNA repair mechanisms. PARP enzymes are essential for repairing DNA single strand breaks (SSBs).

[0154] Inhibiting PARPs leads to the persistence of SSBs, which are then converted to the more serious DNA double strand breaks (DSBs) during the process of DNA replication. During the process of cell division, DSBs can be efficiently repaired in normal cells by homologous recombinational repair (HRR). Tumours with homologous recombinational deficiency (HRD), such as ovarian cancers in patients with breast cancer susceptibility gene 1 / 2 (BRCA1 / 2) mutations, cannot accurately repair DNA damage, which may become lethal to cells as DNA abnormalities accumulate. In such tumour types, olaparib may offer a potentially efficacious and less toxic cancer treatment compared with currently available chemotherapyJAWS Ref: 751495PCTregimens. Olaparib traps the inactive form of PARP on DNA at sites of SSBs, thereby preventing their repair.2.3 Ancillary agents

[0155] In some embodiments, the PI3K inhibitor and ICM-binding antagonist are administered concurrently with an ancillary agent for treating, or for aiding in the treatment of, a T-cell dysfunctional disorder. Non-limiting examples of ancillary agents include cytotoxic agents, gene therapy agents, DNA therapy agents, viral therapy agents, RNA therapy agents, immunotherapeutic agents, bone marrow transplantation agents, nanotherapy agents, or a combination of the foregoing. The ancillary agent may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the ancillary agent is a small molecule enzymatic inhibitor or anti-metastatic agent. In some embodiments, the ancillary agent is a side-effect limiting agent (e.g., agents intended to lessen the occurrence and / or severity of side effects of treatment, such as antinausea agents, etc.). In some embodiments, the ancillary agent is a radiotherapy agent. In some embodiments, the ancillary agent is an agent that targets PI3K / AKT / mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and / or chemopreventative agent. In some embodiments, the ancillary agent is an immunotherapeutic, e.g., a blocking antibody, ipilimumab (also known as MDX-010, MDX-101, or Yervoy®), tremelimumab (also known as ticilimumab or CP-675,206), an antagonist directed against B7-H3 (also known as CD276), e.g., a blocking antibody, MGA271, an antagonist directed against a TGF-p, e.g., metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299, aT cell (e.g., a cytotoxic T cell or CTL) expressing a chimeric antigen receptor (CAR), a T cell comprising a dominant-negative TGF-p receptor, e.g., a dominant-negative TGF-p type II receptor, an agonist directed against CD137 (also known as TNFRSF9, 4-1 BB, or ILA), e.g., an activating antibody, urelumab (also known as BMS-663513), an agonist directed against CD40, e.g., an activating antibody, CP-870893, an agonist directed against 0X40 (also known as CD134), e.g., an activating antibody, administered in conjunction with an anti-OX40 antibody (e.g., AgonOX), an agonist directed against CD27, e.g., an activating antibody, CDX-1127, indoleamine-2,3-dioxygenase (IDO), 1-methyl-D-tryptophan (also known as 1-D-MT), an antibody-drug conjugate (in some embodiments, comprising mertansine or monomethyl auristatin E (MMAE)), an anti -NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599), trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine, or KADCYLA®, Genentech), DMUC5754A, an antibody-drug conjugate targeting the endothelin B receptor (EDNBR), e.g., an antibody directed against EDNBR conjugated with MMAE, an angiogenesis inhibitor, an antibody directed against a VEGF, e.g., VEGF-A, bevacizumab (also known as AVASTIN®, Genentech), an antibody directed against angiopoietin 2 (also known as Ang2), MEDI3617, an antineoplastic agent, an agent targeting CSF-IR (also known as M-CSFR or CD115), anti-CSF-IR (also known as IMC-CS4), an interferon, for example IFN-a or IFN-y, Roferon-A, GM-CSF (also known as recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, sargramostim, or Leukine®), IL-2 (also known as aldesleukin or Proleukin®), IL-12, anJAWS Ref: 751495PCTantibody targeting CD20 (in some embodiments, the antibody targeting CD20 is obinutuzumab (also known as GA101 or Gazyva®) or rituximab), an antibody targeting GITR (in some embodiments, the antibody targeting GITR is TRX518), in conjunction with a cancer vaccine (in some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine; in some embodiments the peptide cancer vaccine is a multivalent long peptide, a multi-peptide, a peptide cocktail, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci, 104: 14-21, 2013)), in conjunction with an adjuvant, a TLR agonist, e.g., Poly-ICLC (also known as Hiltonol®), LPS, MPL, or CpG ODN, TNF, IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an HVEM antagonist, an ICOS agonist, e.g., by administration of ICOS-L, or an agonistic antibody directed against ICOS, an agent targeting CX3CL1, an agent targeting CXCL10, an agent targeting CCL5, an LFA-1 or ICAM1 agonist, a Selectin agonist, a targeted therapeutic agent, an inhibitor of B-Raf, vemurafenib (also known as Zelboraf®, dabrafenib (also known as Tafinlar®), erlotinib (also known as Tarceva®), an inhibitor of a MEK, such as MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K2). cobimetinib (also known as GDC-0973 or XL-518), trametinib (also known as Mekinist®), an inhibitor of K-Ras, an inhibitor of c-Met, onartuzumab (also known as MetMAb), an inhibitor of Aik, AF802 (also known as CH5424802 or alectinib), BKM120, idelalisib (also known as GS-1101 or CAL-101 ), perifosine (also known as KRX-0401), an Akt, MK2206, GSK690693, GDC-0941, an inhibitor of mTOR, sirolimus (also known as rapamycin), temsirolimus (also known as CCI-779 or Torisel®), everolimus (also known as RAD001), ridaforolimus (also known as AP-23573, MK-8669, or deforolimus), OSI-027, AZD8055, INK128, a dual PI3K / mTOR inhibitor, XL765, GDC-0980, BEZ235 (also known as NVP-BEZ235), BGT226, GSK2126458, PF-04691502, PF-05212384 (also known as PKI-587). The ancillary agent may be one or more of the cytotoxic or chemotherapeutic agents described herein.

[0097] In some embodiments, the ancillary agent is an anti-infective drug. The anti-infective drugs are suitably selected from antimicrobials, which include without limitation compounds that kill or inhibit the growth of microorganisms such as viruses, bacteria, yeast, fungi, protozoa, etc. and thus include antibiotics, amebicides, antifungals, antiprotozoals, antimalarials, antituberculotics and antivirals. Anti-infective drugs also include within their scope anthelmintics and nematocides. Illustrative antibiotics include quinolones (e.g., amifloxacin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, lomefloxacin, oxolinic acid, pefloxacin, rosoxacin, temafloxacin, tosufloxacin, sparfloxacin, clinafloxacin, gatifloxacin, moxifloxacin; gemifloxacin; and garenoxacin), tetracyclines, glycylcyclines and oxazolidinones (e.g., chlortetracycline, demeclocycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, tetracycline, tigecycline; linezolide, eperozolid), glycopeptides, aminoglycosides (e.g., amikacin, arbekacin, butirosin, dibekacin, fortimicins, gentamicin, kanamycin, meomycin, netilmicin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin), p-lactams (e.g., imipenem,JAWS Ref: 751495PCTmeropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefpimizole, cefpiramide, cefpodoxime, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephaacetrile, cephalexin, cephaloglycin, cephaloridine, cephalothin, cephapirin, cephradine, cefinetazole, cefoxitin, cefotetan, azthreonam, carumonam, flomoxef, moxalactam, amidinocillin, amoxicillin, ampicillin, azlocillin, carbenicillin, benzylpenicillin, carfecillin, cioxacillin, dicloxacillin, methicillin, mezlocillin, nafcillin, oxacillin, penicillin G, piperacillin, sulbenicillin, temocillin, ticarcillin, cefditoren, SC004, KY-020, cefdinir, ceftibuten, FK-312, S-1090, CP-0467, BK-218, FK-037, DQ-2556, FK-518, cefozopran, ME1228, KP-736, CP-6232, Ro 09-1227, CPC-20000, LY206763), rifamycins, macrolides (e.g., azithromycin, clarithromycin, erythromycin, oleandomycin, rokitamycin, rosaramicin, roxithromycin, troleandomycin), ketolides (e.g., telithromycin, cethromycin), coumermycins, lincosamides (e.g., clindamycin, lincomycin) and chloramphenicol. Illustrative antivirals include abacavir sulfate, acyclovir sodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdine mesylate, didanosine, efavirenz, famciclovir, fomivirsen sodium, foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine, lamivudine / zidovudine, nelfinavir mesylate, nevirapine, oseltamivir phosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir, saquinavir mesylate, stavudine, valacyclovir hydrochloride, zalcitabine, zanamivir, and zidovudine. Non-limiting examples of amebicides or antiprotozoals include atovaquone, chloroquine hydrochloride, chloroquine phosphate, metronidazole, metronidazole hydrochloride, and pentamidine isethionate. Anthelmintics can be at least one selected from mebendazole, pyrantel pamoate, albendazole, ivermectin and thiabendazole. Illustrative antifungals can be selected from amphotericin B, amphotericin B cholesteryl sulfate complex, amphotericin B lipid complex, amphotericin B liposomal, fluconazole, flucytosine, griseofulvin microsize, griseofulvin ultramicrosize, itraconazole, ketoconazole, nystatin, and terbinafine hydrochloride. Non-limiting examples of antimalarials include chloroquine hydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine, and pyrimethamine with sulfadoxine. Antituberculotics include but are not restricted to clofazimine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine, and streptomycin sulfate.3. Pharmaceutical compositions and formulations

[0098] Also provided herein are pharmaceutical compositions and formulations comprising a PI3K inhibitor, an ICM-binding antagonist and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions and formulations further comprise an ancillary agent as described for example herein.

[0099] Pharmaceutical compositions and formulations as described herein can be prepared by mixing the active ingredients (e.g., a small molecule, nucleic acid, or polypeptide) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)).JAWS Ref: 751495PCTPharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral -active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in US Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

[0100] In some embodiments, especially relating to peptide and polypeptide active agents (e.g., antibodies, inhibitory peptides and immunoadhesins), the active agents and optional pharmaceutically acceptable carriers are in the form of lyophilized formulations or aqueous solutions. Exemplary lyophilized antibody formulations are described in U. S. Pat. No.6,267,958. Aqueous antibody formulations include those described in U. S. Pat. No. 6,171,586 and W02006 / 044908, the latter formulations including a histidine-acetate buffer.

[0101] The compositions and formulations herein may also contain further active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0102] Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interracial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0103] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, orJAWS Ref: 751495PCTmicrocapsules. The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

[0104] Depending on the specific conditions being treated, the formulations may be administered systemically or locally. Suitable routes may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Techniques for formulation and administration may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition.4. Therapeutic uses

[0105] The present invention discloses that a PI3K inhibitor and an ICM-binding antagonist (also referred to herein as the “therapeutic combination” or “combination treatment”) are useful for treating a T-cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T-cell function etc.) in an individual having cancer, for treating or delaying the progression of cancer, or for treating infection in an individual. In specific embodiments, the therapeutic combination is disclosed for treating or delaying the progression of cancer, including metastatic cancer, and for preventing cancer recurrence. Any of the PI3K inhibitors and ICM-binding antagonists known in the art or described herein may be used in this regard.

[0106] In some embodiments, the combination therapy further comprises the use or administration of an ancillary agent (e.g., a chemotherapeutic agent), as described for example herein.

[0107] Suitably, the individual to be treated with the combination therapy comprises a T-cell (e.g., a CD8+ T-cell) with a mesenchymal phenotype, for example, a T-cell that expresses CSV, EGRF, and ABCB5, SoX9, SNAIL, and AKT1 in the same T-cell, and / or at a higher level than in an activated T-cell. The T-cell may be a tumour-infiltrating lymphocyte or a circulating lymphocyte. The T-cell suitably exhibits T-cell exhaustion or anergy and in representative examples of this type, the T-cell expresses a higher level of EOMES than TBET and / or has elevated expression of PD-1. In some embodiments, the T-cell has impaired or repressed immune function and suitably expresses biomarkers of reduced T-cell activation (e.g., reduced production and / or secretion of cytokines such as IL-2, IFN-y, and TNF).Accordingly, TBET, PD1 and EOMES (also referred to herein as “T-cell function biomarkers”) can be used to determine the immune function of T cells in a patient for assessing a patient's T-cell immune status, including susceptibility to treatment with ICM-binding antagonists.

[0108] In some embodiments, the individual is a human.JAWS Ref: 751495PCT

[0109] In some embodiments, the individual has been treated with an ICM-binding antagonist before the combination treatment with an ICM-binding antagonist and a PI3K inhibitor.

[0110] In some embodiments, the individual has cancer that is resistant (has been demonstrated to be resistant) to one or more ICM-binding antagonists. In some embodiments, resistance to an ICM-binding antagonist includes recurrence of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer, in the original site or a new site, after treatment. In some embodiments, resistance to an ICM-binding antagonist includes progression of the cancer during treatment with the ICM-binding antagonist. In some embodiments, resistance to an ICM-binding antagonist includes cancer that does not respond to treatment. The cancer may be resistant at the beginning of treatment or it may become resistant during treatment. In some embodiments, the cancer is at early stage or at late stage.

[0111] In some embodiments of any of the methods, assays and / or kits, anyone or more of the T-cell function biomarkers are detected in the sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technique, and FISH, and combinations thereof.

[0112] In some embodiments of any of the methods, assays and / or kits, any one or more of the T-cell function biomarkers are detected in the sample by protein expression. In some embodiments, protein expression is determined by immunohistochemistry (IHC). In some embodiments, any one or more of the T-cell function biomarkers are detected using an antibody that binds specifically to a respective biomarker.

[0113] In some embodiments, the combination therapy of the invention comprises administration of a PI3K inhibitor and an ICM-binding antagonist. The PI3K inhibitor and ICM-binding antagonist may be administered in any suitable manner known in the art. For example, The PI3K inhibitor and ICM-binding antagonist may be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the PI3K inhibitor is in a separate composition as the ICM-binding antagonist. In some embodiments, the PI3K inhibitor is in the same composition as the ICM-binding antagonist. Accordingly, the combination therapy may involve administering the PI3K inhibitor separately, simultaneously or sequentially with ICM-binding antagonist. In some embodiments, this may be achieved by administering a single composition or pharmacological formulation that includes both types of agent, or by administering two separate compositions or formulations at the same time, wherein one composition includes the PI3K inhibitor and the other, ICM-binding antagonist. In other embodiments, the treatment with the PI3K inhibitor may precede or follow the treatment with the ICM-binding antagonist by intervals ranging from minutes to days. In embodiments where theJAWS Ref: 751495PCTPI3K inhibitor is applied separately to the ICM-binding antagonist, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the PI3K inhibitor would still be able to exert an advantageously effect on a functionally repressed T-cell (e.g., a mesenchymal T-cell) as noted above, and in particular, to render the T-cell with enhanced immune function, including susceptibility of the T-cell to reinvigoration by the ICM-binding antagonist. In such instances, it is contemplated that one would administer both modalities within about 1 -12 hours of each other and, more suitably, within about 2-6 hours of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several hours (2, 3, 4, 5, 6 or 7) to several days (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0114] It is conceivable that more than one administration of either the PI3K inhibitor or the ICM-binding antagonist will be desired. Various combinations may be employed, where the PI3K inhibitor is “A” and the ICM-binding antagonist is “B”, as exemplified below:

[0115] A / B / A B / A / B B / B / A A / A / B B / A / A A / B / B B / B / B / A B / B / A / B A / A / B / B A / B / A / B A / B / B / A B / B / A / A B / A / B / A B / A / A / B B / B / B / A A / A / A / B B / A / A / A A / B / A / A A / A / B / A A / B / B / B B / A / B / B B / B / A / B.

[0116] The PI3K inhibitor and ICM-binding antagonist may be administered by the same route of administration or by different routes of administration. In some embodiments, the ICM-binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the PI3K inhibitor is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. An effective amount of the PI3K inhibitor and ICM-binding antagonist may be administered for prevention or treatment of disease. The appropriate dosage of the PI3K inhibitor and ICM-binding antagonist may be determined based on the type of disease to be treated, the type of the PI3K inhibitor and ICM-binding antagonist, the severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician. In some embodiments, combination treatment with PI3K inhibitor (e.g., GDC-0084) and ICM-binding antagonists (e.g., anti-PD-1 antibody) are synergistic, whereby an efficacious dose of an ICM-binding antagonists (e.g., anti-PD-1 antibody) in the combination is reduced relative to efficacious dose of the ICM-binding antagonists (e.g., anti-PD-1 antibody) as a single agent.

[0117] As a general proposition, the therapeutically effective amount of a peptide or polypeptide active agent (e.g., an antibody, peptide inhibitor, immunoadhesin, etc.) administered to a human will be in the range of about 0.01 to about 50 mg / kg of patient body weight whether by one or more administrations. In some embodiments, the antibody used is about 0.01 to about 45 mg / kg, about 0.01 to about 40 mg / kg, about 0.01 to about 35 mg / kg,JAWS Ref: 751495PCTabout 0.01 to about 30 mg / kg, about 0.01 to about 25 mg / kg, about 0.01 to about 20 mg / kg, about 0.01 to about 15 mg / kg, about 0.01 to about 10 mg / kg, about 0.01 to about 5 mg / kg, or about 0.01 to about 1 mg / kg administered daily, for example. In some embodiments, the peptide or polypeptide active agent (e.g., an antibody, peptide inhibitor, immunoadhesin, etc.) is administered at 15 mg / kg. However, other dosage regimens may be useful. In one embodiment, an anti-PDL1 antibody described herein is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of 21 -day cycles. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. The dose of peptide or polypeptide active agent (e.g., an antibody, peptide inhibitor, immunoadhesin, etc.) administered in a combination treatment may be reduced as compared to a single treatment. The progress of this therapy is easily monitored by conventional techniques.

[0118] Small molecule compounds are generally administered at an initial dosage of about 0.0001 mg / kg to about 1000 mg / kg daily. A daily dose range of about 0.01 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 200 mg / kg, or about 1 mg / kg to about 100 mg / kg, or about 10 mg / kg to about 50 mg / kg, can be used. The dosages, however, may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed.

[0119] In any event, dosages can be empirically determined considering the type and stage of disease diagnosed in a particular patient. The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound in a particular patient. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired. Doses can be given daily, or on alternate days, as determined by the treating physician. Doses can also be given on a regular or continuous basis over longer periods of time (weeks, months or years), such as through the use of a subdermal capsule, sachet or depot, or via a patch or pump. In some embodiments, the PI3K inhibitor, ICM-binding antagonist and optionally an ancillary agent (e.g., a chemotherapeutic agent) are administered on a routine schedule. Alternatively, the combination therapy may be administered as symptoms arise.

[0120] A “routine schedule” as used herein, refers to a predetermined designated period of time. The routine schedule may encompass periods of time which are identical or which differ in length, as long as the schedule is predetermined. For instance, the routine schedule may involve administration of the PI3K inhibitor, ICM-binding antagonist and optionalJAWS Ref: 751495PCTancillary agent on a daily basis, every two days, every three days, every four days, every five days, every six days, a weekly basis, a monthly basis or any set number of days or weeks there-between, every two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, etc.Alternatively, the predetermined routine schedule may involve concurrent administration of the PI3K inhibitor, ICM-binding antagonist and optional ancillary agent on a daily basis for the first week, followed by a monthly basis for several months, and then every three months after that. Any particular combination would be covered by the routine schedule as long as it is determined ahead of time that the appropriate schedule involves administration on a certain day.

[0121] In some embodiments, the treatment methods and uses may further comprise an additional therapy. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation.

[0122] The efficacy of any of the methods described herein (e.g., combination treatments including administering an effective amount of a combination of PI3K inhibitor, ICM-binding antagonist and optional ancillary agent may be tested in various models known in the art, such as clinical or pre-clinical models. Suitable pre-clinical models are exemplified herein and further may include without limitation ID8 ovarian cancer, GEM models, B16 melanoma, RENCA renal cell cancer, CT26 colorectal cancer, MC38 colorectal cancer, and Cloudman melanoma models of cancer.

[0123] The efficacy of any of the methods described herein (e.g., combination treatments including administering an effective amount of a combination of PI3K inhibitor, ICM-binding antagonist and optional ancillary agent) may be tested in a GEM model that develops tumours, including without limitation GEM models of non-small-cell lung cancer, pancreatic ductal adenocarcinoma, or melanoma. For example, a mouse expressing KrasG12D in a p53null background after adenoviral recombinase treatment as described in Jackson et al. (2001 Genes Dev. 15(24): 3243-8) (description of KrasG12D) and Lee et al.. (2012 Dis. Model Meeh. 5(3): 397-402) (FRT-mediated p53null allele) may be used as a pre-clinical model for non-small-cell lung cancer. As another example, a mouse expressing KrasG12D in a p16 / p19null background as described in Jackson et al. (2001, supra) (description of KrasG12D) and Aguirre et al. (2003 Genes Dev. 17(24):3112-26) (p16 / p19null allele) may be used as a pre-clinical model for pancreatic ductal adenocarcinoma (PDAC). As a further example, a mouse with melanocytes expressing BrafV600E in a melanocyte-specific PTENnull background after inducible (e.g., 4-OHT treatment) recombinase treatment as described in Dankort et al. (2007 Genes Dev. 21(4):379-84) (description of Braf.sup. V600E) and Trotman et al. (2003 PLoS Biol.JAWS Ref: 751495PCT1 (3): E59) (PTENnull allele) may be used as a pre-clinical model for melanoma. For any of these exemplary models, after developing tumours, mice are randomly recruited into treatment groups receiving combination PI3K inhibitor, ICM-binding antagonist and optional ancillary agent treatment or control treatment. Tumour size (e.g., tumour volume) is measured during the course of treatment, and overall survival rate is also monitored.

[0124] In some embodiments of the methods of the present disclosure, the cancer (in some embodiments, a sample of the patient’s cancer as examined using a diagnostic test, as described for example herein) comprises tumour-infiltrating lymphocytes (TILs), wherein the TILs are within or otherwise associated with the cancer tissue. In these embodiments, the TILs are assessed for expression of any one or more of the T-cell function biomarkers disclosed herein. For example,. TBET, PD-1 and EOMES can be used as biomarkers of T-cell exhaustion, which is characterized for example by high levels of inhibitory co-receptors and lacking the capacity to produce effector cytokines (Wherry, E. J. 2011 Nature Immunology 12: 492-499; Rabinovich et al., 2007 Annual Review of Immunology 25: 267-296).

[0125] In some embodiments of the methods of the present disclosure, the individual has a T-cell dysfunction that manifests in a T-cell dysfunctional disorder. The T-cell dysfunctional disorder may be characterized by T-cell anergy or decreased ability to secrete cytokines, proliferate or execute cytolytic activity. In some embodiments of the methods of the present disclosure, the T-cell dysfunctional disorder is characterized by repressed T-cell immune function. In some embodiments of the methods of the present disclosure, the T-cell dysfunctional disorder is characterized by T-cell of a mesenchymal phenotype. In some embodiments of the methods of the present disclosure, the T-cell dysfunctional disorder is characterized by T-cell exhaustion. In some embodiments of the methods of the present disclosure, the T-cells are CD4+ and / or CD8+ T cells. In accordance with the present invention, PI3K inhibitor treatment may increase expression of biomarkers of T-cell activation and effector capacity (e.g., IL-2, IFN-y and TNF), decrease expression of biomarkers of T-cell effector inhibition and cancer progression (e.g., ZEBI), decrease expression of biomarkers of T-cell exhaustion (e.g., PD-1 and EOMES) and / or increase expression of the transcription factor TBET, which increases production of IFN-y in cells of the adaptive and innate immune systems. Notably, PI3K inhibitor treatment may confer enhanced susceptibility of exhausted T-cells to reinvigoration by ICM-binding antagonists. As such, the combination treatment PI3K inhibitor and an ICM-binding antagonist may increase T-cell (e.g., CD4+ T-cell, CD8+ T-cell, memory T-cell) priming, activation and / or proliferation relative to prior to the administration of the combination. In some embodiments, the T cells are CD4+ and / or CD8+ T cells.

[0126] In some embodiments of the methods of the present disclosure, activated CD4+ and / or CD8+ T-cells in the individual are characterized by IFN-y producing CD4+ and / or CD8+ T cells and / or enhanced cytolytic activity as compared to before the administration of the combination,.gamma. IFN-y may be measured by any means known in the art, including, e.g., intracellular cytokine staining (ICS) involving cell fixation, permeabilization, and staining with anJAWS Ref: 751495PCTantibody against IFN-y. Cytolytic activity may be measured by any means known in the art, e.g., using a cell killing assay with mixed effector and target cells.

[0127] In some embodiments, CD8+ T-cells are characterized, e.g., by presence of CD8b expression (e.g., by RT-PCR using e.g., Fluidigm) (Cd8b is also known as T-cell surface glycoprotein CD8 beta chain; CD8 antigen, alpha polypeptide p3'7; Accession No. is NM_172213). In some embodiments, CD8+ T cells are from peripheral blood. In some embodiments, CD8+ T cells are from tumour.

[0128] In some embodiments, Treg cells are characterized, e.g., by presence of Fox3p expression (e.g., by RT-PCR e.g., using Fluidigm) (Foxp3 is also known as Forkhead box protein P3; scurfin; FOXP3delta7; immunodeficiency, polyendocrinopathy, enteropathy, X-linked; the accession no. is NM_014009). In some embodiments, Treg are from peripheral blood. In some embodiments, Treg cells are from tumour.

[0129] In some embodiments, inflammatory or activated T-cells are characterized, e.g., by presence of TBET and / or CXCR3 expression or by a TBET: EOMES ratio that correlates with inflammatory or activated T-cells (e.g., by RT-PCR using, e.g., Fluidigm). In some embodiments, inflammatory or activated T cells are from peripheral blood. In some embodiments, inflammatory or activated T cells are from tumour.

[0130] In some embodiments of the methods of the present disclosure, CD4+ and / or CD8+ T cells exhibit increased release of cytokines selected from the group consisting of IFN-y, TNF and interleukins such as IL-2. Cytokine release may be measured by any means known in the art, e.g., using Western blot, ELISA, or immunohistochemical assays to detect the presence of released cytokines in a sample containing CD4+ and / or CD8+ T-cells.

[0131] In some embodiments of the methods of the present disclosure, the CD4+ and / or CD8+ T cells are effector memory T cells. In some embodiments of the methods of the present disclosure, the CD4+ and / or CD8+ effector memory T cells are characterized by having the expression of CD44high CD62Llow. Expression of CD44high CD62Llow may be detected by any means known in the art, e.g., by preparing single cell suspensions of tissue (e.g., a cancer tissue) and performing surface staining and flow cytometry using commercial antibodies against CD44 and CD62L. In some embodiments of the methods of the present disclosure, the CD4+ and / or CD8+ effector memory T cells are characterized by having expression of CXCR3 (also known as C-X-C chemokine receptor type 3; Mig receptor; I PIO receptor; G protein-coupled receptor 9; interferon-inducible protein 10 receptor; Accession No. NM_001504). In some embodiments, the CD4+ and / or CD8+ effector memory T cells are from peripheral blood. In some embodiments, the CD4+ and / or CD8+ effector memory T cells are from tumour.

[0132] In some embodiments of the methods of the present disclosure, the administration of an effective amount of a PI3K inhibitor and an ICM-binding antagonist and optionally an ancillary agent to an individual is characterized by increased levels of inflammatory markers (e.g., CXCR3) on CD8+ T cells as compared to before administration of theJAWS Ref: 751495PCTcombination therapy. CXCR3 / CD8+ T cells may be measured by any means known the art. In some embodiments, CXCR3 / CD8+ T cells are from peripheral blood. In some embodiments, CXCR3 / CD8+ T cells are from tumour.

[0133] In some embodiments of the methods of the invention, Treg function is suppressed as compared to before administration of the combination. In some embodiments, T-cell exhaustion is decreased as compared to before administration of the combination.

[0134] In some embodiments, number of Treg is decreased as compared to before administration of the combination. In some embodiments, the levels of plasma IFN-y is increased as compared to before administration of the combination. Treg number may be assessed, e.g., by determining percentage of CD4+Fox3p+CD45+ cells (e.g., by FACS analysis). In some embodiments, absolute number of Treg, e.g., in a sample, is determined. In some embodiments, Treg are from peripheral blood. In some embodiments, Treg are from tumour.

[0135] In some embodiments, T-cell priming, activation and / or proliferation is increased as compared to before administration of the combination. In some embodiments, the T-cells are CD4+ and / or CD8+ T cells. In some embodiments, T-cell proliferation is detected by determining percentage of Ki67+CD8+ T cells (e.g., by FACS analysis). In some embodiments, T-cell proliferation is detected by determining percentage of Ki67+CD4+ T cells (e.g., by FACS analysis). In some embodiments, the T-cells are from peripheral blood. In some embodiments, the T-cells are from tumour.5. Methods of detection and diagnosis

[0136] In accordance with the present invention, PD-1, TBET and EOMES may be used as known in the art to assess T-cell exhaustion. T-cells can be obtained from T-cell containing patient samples which are suitably selected tissue samples such as tumours and fluid samples such as peripheral blood. In some embodiments, the sample is obtained prior to treatment with the therapeutic combination. In some embodiments, the tissue sample is formalin fixed and paraffin embedded, archival, fresh or frozen. In some embodiments, the sample is whole blood. In some embodiments, the whole blood comprises immune cells, circulating tumour cells and any combinations thereof.

[0137] Presence and / or expression levels / amount of a biomarker (e.g., any one or more of TBET and EOMES, also referred to herein collectively as “T-cell function biornarkers”) can be determined qualitatively and / or quantitatively based on any suitable criterion known in the art, including but not limited to DNA, mRNA, cDNA, proteins, protein fragments and / or gene copy number. In certain embodiments, presence and / or expression levels / amount of a biomarker in a first sample is increased or elevated as compared to presence / absence and / or expression levels / amount in a second sample (e.g., before treatment with the therapeutic combination). In certain embodiments, presence / absence and / or expression levels / amount of a biomarker in a first sample is decreased or reduced as compared to presence and / orJAWS Ref: 751495PCTexpression levels / amount in a second sample. In certain embodiments, the second sample is a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. Additional disclosures for determining presence / absence and / or expression levels / amount of a gene are described herein.

[0138] In some embodiments of any of the methods, elevated expression refers to an overall increase of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of biomarker (e.g., protein or nucleic acid (e.g., gene or mRNA)), detected by standard art known methods such as those described herein, as compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, the elevated expression refers to the increase in expression level / amount of a biomarker in the sample wherein the increase is at least about any of 1.5x, 1.75x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 25x, 50x, 75x, or 10Ox the expression level / amount of the respective biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, elevated expression refers to an overall increase of greater than about 1.5-fold, about 1.75-fold, about-2 fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold as compared to a reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).

[0617] In some embodiments of any of the methods, reduced expression refers to an overall reduction of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of biomarker (e.g., protein or nucleic acid (e.g., gene or mRNA)), detected by standard art known methods such as those described herein, as compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, reduced expression refers to the decrease in expression level / amount of a biomarker in the sample wherein the decrease is at least about any of 0.9x, 0.8x, 0.7x, 0.6x, 0.5x, 0.4x, 0.3x, 0.2x, 0.1 x, 0.05x, or 0.01 x the expression level / amount of the respective biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

[0139] Presence and / or expression level / amount of various biomarkers in a sample can be analyzed by a number of methodologies, many of which are known in the art and understood by the skilled artisan, including, but not limited to, immunohistochemistry (“IHC”), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting (“FACS”), MassARRAY, proteomics, quantitative blood based assays (as for example Serum ELISA), biochemical enzymatic activity assays, in situ hybridization, Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction (“PCR”) including quantitative real time PCR (“qRT-PCR”) and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like), RNA-Seq, FISH, microarray analysis, gene expression profiling, and / or serial analysis of gene expression (“SAGE”), as well as any one of the wide variety of assays that can be performed by protein, gene, and / or tissue array analysis. Typical protocols for evaluating the status of genesJAWS Ref: 751495PCTand gene products are found, for example in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Multiplexed immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery (" MSD") may also be used.

[0140] In some embodiments, presence and / or expression level / amount of a biomarker is determined using a method comprising: (a) performing gene expression profiling, PCR (such as rtPCR or qRT-PCR), RNA-seq, microarray analysis, SAGE, MassARRAY technique, or FISH on a sample (such as a subject cancer sample); and b) determining presence and / or expression level / amount of a biomarker in the sample. In some embodiments, the microarray method comprises the use of a microarray chip having one or more nucleic acid molecules that can hybridize under stringent conditions to a nucleic acid molecule encoding a gene mentioned above or having one or more polypeptides (such as peptides or antibodies) that can bind to one or more of the proteins encoded by the genes mentioned above. In one embodiment, the PCR method is qRT-PCR. In one embodiment, the PCR method is multiplex-PCR. In some embodiments, gene expression is measured by microarray. In some embodiments, gene expression is measured by qRT-PCR. In some embodiments, expression is measured by multiplex-PCR.

[0141] Methods for the evaluation of mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled riboprobes specific for the one or more genes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for one or more of the genes, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like).

[0142] Samples from mammals can be conveniently assayed for mRNAs using Northern, dot blot or PCR analysis. In addition, such methods can include one or more steps that allow one to determine the levels of target mRNA in a biological sample (e.g., by simultaneously examining the levels a comparative control mRNA sequence of a “housekeeping” gene such as an actin family member). Optionally, the sequence of the amplified target cDNA can be determined.

[0143] Optional methods include protocols which examine or detect mRNAs, such as target mRNAs, in a tissue or cell sample by microarray technologies. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support. The array is configured such that the sequence and position of each member of the array is known. For example, a selection of genes whose expression correlates with increased or reduced clinical benefit of anti-angiogenic therapy may be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene.JAWS Ref: 751495PCT

[0144] According to some embodiments, presence and / or expression level / amount is measured by observing protein expression levels of an aforementioned gene. In certain embodiments, the method comprises contacting the biological sample with antibodies to a biomarker (e.g., anti-PD-1 antibodies, anti-PI3K antibodies, anti-TBET antibodies, antibodies, anti-EOMES antibodies) described herein under conditions permissive for binding of the biomarker, and detecting whether a complex is formed between the antibodies and biomarker. Such method may be an in vitro or in vivo method. In some embodiments, one or more antibiomarker antibodies are used to select subjects eligible for combination therapy with a PI3K inhibitor and an ICM-binding antagonist.

[0145] In certain embodiments, the presence and / or expression level / amount of biomarker proteins in a sample is examined using IHC and staining protocols. IHC staining of tissue sections has been shown to be a reliable method of determining or detecting presence of proteins in a sample. In some embodiments, expression of a T-cell function biomarker in a sample from an individual is elevated protein expression and, in further embodiments, is determined using IHC. In one embodiment, expression level of biomarker is determined using a method comprising: (a) performing IHC analysis of a sample (such as a subject cancer sample) with an antibody; and (b) determining expression level of a biomarker in the sample. In some embodiments, IHC staining intensity is determined relative to a reference. In some embodiments, the reference is a reference value. In some embodiments, the reference is a reference sample (e.g., control cell line staining sample or tissue sample from non-cancerous patient).

[0146] In some embodiments, T-cell function biomarker expression is evaluated on a tumour or tumour sample. As used herein, a tumour or tumour sample may encompass part or all of the tumour area occupied by tumour cells. In some embodiments, a tumour or tumour sample may further encompass tumour area occupied by tumour associated intratumoural cells and / or tumour associated stroma (e.g., contiguous peri-tumoural desmoplastic stroma). Tumour associated intratumoural cells and / or tumour associated stroma may include areas of immune infiltrates (e.g., tumour infiltrating immune cells as described herein) immediately adjacent to and / or contiguous with the main tumour mass. In some embodiments, T-cell function biomarker expression is evaluated on tumour cells. In some embodiments, T-cell function biomarker expression is evaluated on immune cells within the tumour area as described above, such as tumour infiltrating immune cells.

[0147] In alternative methods, the sample may be contacted with an antibody specific for said biomarker under conditions sufficient for an antibody-biomarker complex to form, and then detecting said complex. The presence of the biomarker may be detected in a number of ways, such as by Western blotting and ELISA procedures for assaying a wide variety of tissues and samples, including plasma or serum. A wide range of immunoassay techniques using such an assay format are available, see, e.g., U. S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These include both single-site and two-site or “sandwich” assays of the non-JAWS Ref: 751495PCTcompetitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labeled antibody to a target biomarker.

[0148] Presence and / or expression level / amount of a selected T-cell function biomarker in a tissue or cell sample may also be examined by way of functional or activitybased assays. For instance, if the biomarker is an enzyme (e.g., PI3K), one may conduct assays (e.g., kinase assays) known in the art to determine or detect the presence of the given enzymatic activity in the tissue or cell sample.

[0149] In certain embodiments, the samples are normalized for both differences in the amount of the biomarker assayed and variability in the quality of the samples used, and variability between assay runs. Such normalization may be accomplished by detecting and incorporating the expression of certain normalizing biomarkers, including well known housekeeping genes.

[0150] Alternatively, normalization can be based on the mean or median signal of all of the assayed genes or a large subset thereof (global normalization approach). On a gene-by-gene basis, measured normalized amount of a subject tumour mRNA or protein is compared to the amount found in a reference set. Normalized expression levels for each mRNA or protein per tested tumour per subject can be expressed as a percentage of the expression level measured in the reference set. The presence and / or expression level / amount measured in a particular subject sample to be analyzed will fall at some percentile within this range, which can be determined by methods well known in the art.

[0151] In some embodiments, the sample is a clinical sample. In other embodiments, the sample is used in a diagnostic assay. In some embodiments, the sample is obtained from a primary or metastatic tumour. Tissue biopsy is often used to obtain a representative piece of tumour tissue. Alternatively, tumour cells can be obtained indirectly in the form of tissues or fluids that are known or thought to contain the tumour cells of interest. For instance, samples of lung cancer lesions may be obtained by resection, bronchoscopy, fine needle aspiration, bronchial brushings, or from sputum, pleural fluid or blood. Genes or gene products can be detected from cancer or tumour tissue or from other body samples such as urine, sputum, serum or plasma. The same techniques discussed above for detection of target genes or gene products in cancerous samples can be applied to other body samples. Cancer cells may be sloughed off from cancer lesions and appear in such body samples. By screening such body samples, a simple early diagnosis can be achieved for these cancers. In addition, the progress of therapy can be monitored more easily by testing such body samples for target genes or gene products.

[0152] In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or combined multiple samples from the same subject or individual that are obtained at one or more different time points than when the test sample is obtained. For example, a reference sample, reference cell,JAWS Ref: 751495PCTreference tissue, control sample, control cell, or control tissue is obtained at an earlier time point from the same subject or individual than when the test sample is obtained. Such reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be useful if the reference sample is obtained during initial diagnosis of cancer and the test sample is later obtained when the cancer becomes metastatic.

[0153] In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more healthy individuals who are not the subject or individual. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more individuals with a disease or disorder (e.g., cancer) who are not the subject or individual. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is pooled RNA samples from normal tissues or pooled plasma or serum samples from one or more individuals who are not the subject or individual. In certain embodiments, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is pooled RNA samples from tumour tissues or pooled plasma or serum samples from one or more individuals with a disease or disorder (e.g., cancer) who are not the subject or individual.

[0154] In some embodiments, the sample is a tissue sample from the individual. In some embodiments, the tissue sample is a tumour tissue sample (e.g., biopsy tissue). In some embodiments, the tissue sample is lung tissue. In some embodiments, the tissue sample is renal tissue. In some embodiments, the tissue sample is skin tissue. In some embodiments, the tissue sample is pancreatic tissue. In some embodiments, the tissue sample is gastric tissue. In some embodiments, the tissue sample is bladder tissue. In some embodiments, the tissue sample is esophageal tissue. In some embodiments, the tissue sample is mesothelial tissue. In some embodiments, the tissue sample is breast tissue. In some embodiments, the tissue sample is thyroid tissue. In some embodiments, the tissue sample is colorectal tissue. In some embodiments, the tissue sample is head and neck tissue. In some embodiments, the tissue sample is osteosarcoma tissue. In some embodiments, the tissue sample is prostate tissue. In some embodiments, the tissue sample is ovarian tissue, HCC (liver), blood cells, lymph nodes, and / or bone / bone marrow tissue. In some embodiments, the tissue sample is colon tissue. In some embodiments, the tissue sample is endometrial tissue. In some embodiments, the tissue sample is brain tissue (e.g., glioblastoma, neuroblastoma, and so forth).

[0155] In some embodiments, a tumour tissue sample (the term “tumour sample” is used interchangeably herein) may encompass part or all of the tumour area occupied by tumour cells. In some embodiments, a tumour or tumour sample may further encompass tumour area occupied by tumour associated intratumoural cells and / or tumour associated stroma (e.g., contiguous peri-tumoural desmoplastic stroma). Tumour associated intratumoural cells and / or tumour associated stroma may include areas of immune infiltrates (e.g., tumour infiltratingJAWS Ref: 751495PCTimmune cells as described herein) immediately adjacent to and / or contiguous with the main tumour mass.

[0156] In some embodiments, tumour cell staining is expressed as the percent of all tumour cells showing membranous staining of any intensity. Infiltrating immune cell staining may be expressed as the percent of the total tumour area occupied by immune cells that show staining of any intensity. The total tumour area encompasses the malignant cells as well as tumour-associated stroma, including areas of immune infiltrates immediately adjacent to and contiguous with the main tumour mass. In addition, infiltrating immune cell staining may be expressed as the percent of all tumour infiltrating immune cells.

[0157] In some embodiments of any of the methods, the disease or disorder is a tumour. In some embodiments, the tumour is a malignant cancerous tumour (i.e., cancer). In some embodiments, the tumour and / or cancer is a solid tumour.

[0158] A solid tumour includes any cancer of body tissues other than blood, bone marrow, or the lymphatic system. Solid tumours can be further divided into those of epithelial cell origin and those of non-epithelial cell origin. Examples of epithelial cell solid tumours include tumours of the gastrointestinal tract, colon, colorectal (e.g., basaloid colorectal carcinoma), breast, prostate, lung, kidney, liver, pancreas, ovary (e.g., endometrioid ovarian carcinoma), head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gall bladder, labium, nasopharynx, skin, uterus, male genital organ, urinary organs (e.g., urothelium carcinoma, dysplastic urothelium carcinoma, transitional cell carcinoma), bladder, and skin. Solid tumours of non-epithelial origin include sarcomas, brain tumours, and bone tumours. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is second-line or third-line locally advanced or metastatic non-small cell lung cancer. In some embodiments, the cancer is adenocarcinoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast carcinoma (e.g., triple-negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma. In some embodiments, the cancer is a primary tumour. In some embodiments, the cancer is a metastatic tumour at a second site derived from any of the above types of cancer.

[0159] In some embodiments of any of the methods, the cancer displays human effector cells (e.g., is infiltrated by human effector cells). Methods for detecting human effector cells are well known in the art, including, e.g., by IHC. In some embodiments, the cancer displays high levels of human effector cells. In some embodiments, human effector cells are one or more of NK cells, macrophages, monocytes. In some embodiments, the cancer is any cancer described herein. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast carcinoma (e.g., triple-negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.JAWS Ref: 751495PCT

[0160] In some embodiments of any of the methods, the cancer displays cells expressing FcR (e.g., is infiltrated by cells expressing FcR). Methods for detecting FcR are well known in the art, including, e.g., by IHC. In some embodiments, the cancer displays high levels of cells expressing FcR. In some embodiments, FcR is FcyR. In some embodiments, FcR is activating FcyR. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast carcinoma (e.g., triple-negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.

[0161] In some embodiments, the T-cell function biomarker is detected in the sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technique, and FISH, and combinations thereof. In some embodiments, the T-cell function biomarker is detected using FACS analysis. In some embodiments, the T-cell function biomarker is PD-1. In some embodiments, the PD-1 expression is detected in blood samples. In some embodiments, the PD-1 expression is detected on circulating immune cells in blood samples. In some embodiments, the circulating immune cell is a CD3+ / CD8+ T cell. In some embodiments, prior to analysis, the immune cells are isolated from the blood samples. Any suitable method to isolate / enrich such population of cells may be used including, but not limited to, cell sorting. In some embodiments, the PD-1 expression is reduced in samples from individuals that respond to treatment with a PI3K inhibitor and / or ICM-binding antagonist, such as an anti-PD-1 antibody. In some embodiments, the PD-1 expression is elevated on circulating immune cells, such as CD3+ / CD8+ T cells, in blood samples.

[0162] Also provided herein are methods for monitoring pharmacodynamic activity of an ICM-binding antagonist treatment by measuring the expression level of one or more T-cell function biomarkers as described herein in a sample comprising leukocytes obtained from the subject, where the subject has been treated with an ICM-binding antagonist and a PI3K inhibitor, and where the one or more T-cell function biomarkers are selected from TBET, PD-1 and EOMES, and determining the treatment as demonstrating pharmacodynamic activity based on the expression level of the one or more T-cell function biomarkers in the sample obtained from the subject, as compared with a reference, where an increased expression level of the one or more T-cell function biomarkers as compared with the reference indicates pharmacodynamic activity to the PD-1 antagonist treatment. These methods may further comprise measuring the expression level of one or more additional biomarkers of T cell function and / or cellular composition (e.g., percentage of Treg and / or absolute number of Treg; e.g., number of CD8+ effector T cells), wherein the additional biomarkers of T cell function include a cytokine, e.g., IFN-y, a T cell marker, or a memory T cell marker (e.g., a marker of T effector memory cells); and determining the treatment as demonstrating pharmacodynamic activity based on theJAWS Ref: 751495PCTexpression level of the one or more T-cell function biomarkers, the one or more additional biomarkers of T cell function and / or cellular composition in the sample obtained from the subject, as compared with a reference, where an increased expression level of the one or more T-cell function biomarkers, the one or more additional biomarkers of T cell function and / or cellular composition as compared with the reference indicates pharmacodynamic activity to the PD-1 antagonist treatment. Expression level of the biomarker(s) and / or cellular composition may be measured by one or more methods as described herein.

[0163] As used herein, “pharmacodynamic (PD) activity” may refer to an effect of a treatment (e.g., a PI3K inhibitor in combination with an ICM-binding antagonist treatment) to the subject. An example of a PD activity may include modulation of the expression level of one or more genes. Without wishing to be bound to theory, it is thought that monitoring PD activity, such as by measuring expression of one or more T-cell function biomarkers, may be advantageous during a clinical trial examining a PI3K inhibitor and ICM-binding antagonist. Monitoring PD activity may be used, for example, to monitor response to treatment, toxicity, and the like.

[0164] In some embodiments, the expression level of one or more marker genes, proteins and / or cellular composition may be compared to a reference which may include a sample from a subject not receiving a treatment (e.g., a PI3K inhibitor treatment in combination with an ICM-binding antagonist). In some embodiments, a reference may include a sample from the same subject before receiving a treatment (e.g., a PI3K inhibitor treatment in combination with an ICM-binding antagonist). In some embodiments, a reference may include a reference value from one or more samples of other subjects receiving a treatment (e.g., a PI3K inhibitor treatment in combination with an ICM-binding antagonist). For example, a population of patients may be treated, and a mean, average, or median value for expression level of one or more genes may be generated from the population as a whole. A set of samples obtained from cancers having a shared characteristic (e.g., the same cancer type and / or stage, or exposure to a common treatment such as a PI3K inhibitor treatment in combination with an ICM-binding antagonist) may be studied from a population, such as with a clinical outcome study. This set may be used to derive a reference, e.g., a reference number, to which a subject's sample may be compared. Any of the references described herein may be used as a reference for monitoring PD activity.

[0165] Certain aspects of the present disclosure relate to measurement of the expression level of one or more biomarkers (e.g., gene expression products including mRNAs and proteins) in a sample. In some embodiments, a sample may include leukocytes. In some embodiments, the sample may be a peripheral blood sample (e.g., from a patient having a tumour). In some embodiments, the sample is a tumour sample. A tumour sample may include cancer cells, lymphocytes, leukocytes, stroma, blood vessels, connective tissue, basal lamina, and any other cell type in association with the tumour. In some embodiments, the sample is a tumour tissue sample containing tumour-infiltrating leukocytes. In some embodiments, theJAWS Ref: 751495PCTsample may be processed to separate or isolate one or more cell types (e.g., leukocytes). In some embodiments, the sample may be used without separating or isolating cell types.

[0166] A tumour sample may be obtained from a subject by any method known in the art, including without limitation a biopsy, endoscopy, or surgical procedure. In some embodiments, a tumour sample may be prepared by methods such as freezing, fixation (e.g., by using formalin or a similar fixative), and / or embedding in paraffin wax. In some embodiments, a tumour sample may be sectioned. In some embodiments, a fresh tumour sample (i.e., one that has not been prepared by the methods described above) may be used. In some embodiments, a tumour sample may be prepared by incubation in a solution to preserve mRNA and / or protein integrity.

[0167] In some embodiments, the sample may be a peripheral blood sample. A peripheral blood sample may include white blood cells, PBMCs, and the like. Any technique known in the art for isolating leukocytes from a peripheral blood sample may be used. For example, a blood sample may be drawn, red blood cells may be lysed, and a white blood cell pellet may be isolated and used for the sample. In another example, density gradient separation may be used to separate leukocytes (e.g., PBMCs) from red blood cells. In some embodiments, a fresh peripheral blood sample (i.e., one that has not been prepared by the methods described above) may be used. In some embodiments, a peripheral blood sample may be prepared by incubation in a solution to preserve mRNA and / or protein integrity.

[0168] In some embodiments, responsiveness to treatment may refer to any one or more of: extending survival (including overall survival and progression free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer. In some embodiments, responsiveness may refer to improvement of one or more factors according to the published set of RECIST guidelines for determining the status of a tumour in a cancer patient, i.e., responding, stabilizing, or progressing. For a more detailed discussion of these guidelines, see, Eisenhauer et al. (2009 Eur J Cancer 45: 228-47), Topalian et al. (2012 N Engl J Med 366:2443-54), Wolchok et al. (2009 Clin Can Res 15: 7412-20) and Therasse et al. (2000 J. Natl. Cancer Inst. 92:205-16). A responsive subject may refer to a subject whose cancer(s) show improvement, e.g., according to one or more factors based on RECIST criteria. A non-responsive subject may refer to a subject whose cancer(s) do not show improvement, e.g., according to one or more factors based on RECIST criteria.

[0169] Conventional response criteria may not be adequate to characterize the anti-tumour activity of therapeutic agents of the invention, which can produce delayed responses that may be preceded by initial apparent radiological progression, including the appearance of new lesions. Therefore, modified response criteria have been developed that account for the possible appearance of new lesions and allow radiological progression to be confirmed at a subsequent assessment. Accordingly, in some embodiments, responsiveness may refer to improvement of one of more factors according to immune-related response criteriaJAWS Ref: 751495PCT(irRC). See, e.g., Wolchok et al. (2009, supra). In some embodiments, new lesions are added into the defined tumour burden and followed, e.g., for radiological progression at a subsequent assessment. In some embodiments, presence of non-target lesions is included in assessment of complete response and not included in assessment of radiological progression. In some embodiments, radiological progression may be determined only on the basis of measurable disease and / or may be confirmed by a consecutive assessment >4 weeks from the date first documented.

[0170] In some embodiments, responsiveness may include immune activation. In some embodiments, responsiveness may include treatment efficacy. In some embodiments, responsiveness may include immune activation and treatment efficacy.6. Kits

[0171] In other aspects of the invention, therapeutic kits are provided comprising a PI3K inhibitor and an ICM-binding antagonist. In some embodiments, the therapeutic kits further comprise a package insert comprising instructional material for administering concurrently the PI3K inhibitor and the ICM-binding antagonist to treat a T-cell dysfunctional disorder, or to enhance immune function (e.g., immune effector function, T-cell function etc.) in an individual having cancer, or to treat or delay cancer progression, or to treat infection in an individual. Any of PI3K inhibitor and ICM-binding antagonist described herein or known in the art may be included in the kits.

[0172] In some embodiments, the PI3K inhibitor and ICM-binding antagonist are in the same container or separate containers. Suitable containers include, for example, bottles, vials, bags and syringes. The container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation and the label on, or associated with, the container may indicate directions for use. The kits may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructional material for use. In some embodiments, the kits further include one or more of other agents (e.g., a chemotherapeutic agent, and anti-neoplastic agent). Suitable containers for the one or more agent(s) include, for example, bottles, vials, bags and syringes.

[0173] In other embodiments of the invention, diagnostic kits are provided for determining expression of biomarkers, including the T-cell function biomarkers disclosed herein, which include reagents that allow detection and / or quantification of the biomarkers. Such reagents include, for example, compounds or materials, or sets of compounds or materials, which allow quantification of the biomarkers. In specific embodiments, the compounds, materials or sets of compounds or materials permit determining the expression level of a gene (e.g., T-cell function biomarker gene), including without limitation the extraction of RNA material, the determination of the level of a corresponding RNA, etc., primers for the synthesis of aJAWS Ref: 751495PCTcorresponding cDNA, primers for amplification of DNA, and / or probes capable of specifically hybridizing with the RNAs (or the corresponding cDNAs) encoded by the genes, TaqMan probes, proximity assay probes, ligases, antibodies etc.

[0174] The kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, blotting membranes, microtiter plates, dilution buffers and the like. For example, a nucleic acid-based detection kit may include (i) a T-cell function biomarker polynucleotide (which may be used as a positive control), (ii) a primer or probe that specifically hybridizes to a T-cell function biomarker polynucleotide. Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (reverse transcriptase, Tag, Sequenase™, DNA ligase etc. depending on the nucleic acid amplification technique employed), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification. Such kits also generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe. Alternatively, a proteinbased detection kit may include (i) a T-cell function biomarker polypeptide (which may be used as a positive control), (ii) an antibody that binds specifically to a T-cell function biomarker polypeptide. The kit can also feature various devices (e.g., one or more) and reagents (e.g., one or more) for performing one of the assays described herein; and / or printed instructional material for using the kit to quantify the expression of a T-cell function biomarker gene. The reagents described herein, which may be optionally associated with detectable labels, can be presented in the format of a microfluidics card, a chip or chamber, a microarray or a kit adapted for use with the assays described in the examples or below, e.g., RT-PCR or Q PCR techniques described herein.

[0175] Materials suitable for packing the components of the diagnostic kits may include crystal, plastic (polyethylene, polypropylene, polycarbonate and the like), bottles, vials, paper, envelopes and the like. Additionally, the kits of the invention can contain instructional material for the simultaneous, sequential or separate use of the different components contained in the kit. The instructional material can be in the form of printed material or in the form of an electronic support capable of storing instructions such that they can be read by a subject, such as electronic storage media (magnetic disks, tapes and the like), optical media (CD-ROM, DVD) and the like.

[0176] Alternatively or in addition, the media can contain Internet addresses that provide the instructional material.

[0177] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following nonlimiting experimental examples.JAWS Ref: 751495PCTEXPERIMENTALDual PI3K-mT0R blockade with paxalisib inhibits proliferation and migration, induces mesenchymal to epithelial transition, and reduces MIC signatures.

[0156] The present inventors first compared the efficacy of a pan-PI3K, isoform-specific PI3K, and dual targeting PI3K-mTOR inhibition in breast cancer cell lines MDA-MB-231 MDA-MB-468, SUM149PT, and 4T1 cells (a mouse line with a “cold" tumor profile, i.e., unresponsive to immunotherapy) using wortmannin (pan-PI3K inhibitor), idelalisib (p110 b,y inhibitor), alpelisib (p110a inhibitor), and LY294002 (p110a,p,5 inhibitor) and the dual PI3K-mTOR inhibitors omipalisib (p110a / p / b / y,mTORC1 / 2), apitolisib (PI3Ka / p / b / y, mTOR), dactolisib (p110a,p,b,y, mTOR), and paxalisib (p110a,p,b,y, mTOR inhibitor) (Figure 1A, 1 B). The inhibitory effect of dual PI3K-mTOR inhibitors was greater than that of pan- or isoform-specific inhibitors in MDA-MB-231 TNBC cells, with paxalisib, omipalisib, apitolisib, and dactolisib all having ICso values <2.5 pM (Figure 1 A). Dual PI3K-mTOR inhibition with paxalisib and dactolisib was similarly effective in MDA-MB-468 and SUM149PT TNBC cells, and paxalisib had an ICso of 0.33 pM in 4T1 cells (Supplementary Figure 1 A). Furthermore, PI3K / PI3K-mTOR inhibition with paxalisib inhibited cancer cell migration in the MDA-MB-231 (Figure 1C) and 4T1 (Figure 1 D) models. Given the favorable in vitro inhibitory profile of paxalisib, we used this inhibitor in subsequent experiments.

[0157] Several cancer cell populations, including cancer stem cells (CSCs), dormant tumour cells, and persister cancer cells, referred to here as metastasisinitiating cells (MICs), are implicated in metastases and drug resistance. Given that PI3K / mTOR signaling is strongly implicated in TNBC tropism, EMT, and sternness, the inventors next examined the effects of paxalisib on EMT and MIC characteristics in cancer cells in vitro. In MDA-MB-231 TNBC cells, paxalisib treatment reduced expression of the mesenchymal marker vimentin and increased protein expression of the epithelial marker E-cadherin (Figure 1 E, 1 F). Furthermore, the inventors determined the impact of paxalisib on the CD44h'9h / CD24lowpopulation, which is associated with the EMT and CSC trait, and PI3K-mTOR inhibition reduced the CD44 / CD24 ratio by 67% in MDA-MB-231 cells (Figure 1G). Paxalisib also reduced protein expression of three biomarkers associated with drug resistance and the CSC phenotype, ABCB5, ALDH1, and Snail (Figure 1 H) and markers associated with persister cancer cells, NFKB p65, FOXQ1, NNMT, and NRF2 (Figure 11). Taken together, these data indicate that PI3K-mTOR blockade reverses the dynamic EMT process and induces a mesenchymal-to-epithelial phenotype less associated with the hallmarks of MICs.

[0158] Given the importance of inflammation in driving metastases, we next determined the role of the PI3K-mTOR inhibition on pro-inflammatory cytokine production and cancer immune visibility. Treatment of CTCs isolated from TNBC patients with paxalisib reduced IL-6 protein expression by 72% in ABCB5+EpCAM+CTCs (Figure 1 J). Finally, induction of a viral mimicry gene signature has been shown to re-invigorate adaptive immune responses,JAWS Ref: 751495PCTthereby improving responses to immunotherapy. To further investigate this signature in TNBC cells, MDA-MB-231 cells were treated with paxalisib for 24 h (Figure 1 K). Exposure to paxalisib significantly increased expression of several viral mimicry genes, most profoundly GBP2 (Figure 1K). Changes in mRNA expression only occurred in response to PI3K-mT0R inhibition (paxalisib and dactolisib; Figure 1 L), further supporting the importance of dual PI3K-mT0R inhibition for the induction of cancer immune visibility.Paxalisib reduces primary tumor burden and metastasis with a favorable toxicity profile

[0159] The present inventors next sought to test the efficacy of paxalisib in a clin ically-relevant model of immunotherapy resistance in TNBC, the spontaneous metastasis 4T1 TNBC model (Figure 2A). As paxalisib alone (and anti-PD1, as expected) did not reduce primary tumour volume (Figure 2B), paxalisib was combined with anti-PD1 and, in a split-dose de-escalation study (Figure 2C), 7.5 mg / kg was confirmed as the optimal dose for maximum efficacy (Figure 2D) with minimal toxicity when compared with higher doses, as measured by body and liver weights (Figure 2E, F). Consistent with liver weight data, the optimal therapeutic dose of 7.5 mg / kg was not hepatotoxic as assessed by histopathological examination, as compared with higher doses (Figure 2G, left). As the addition of immunotherapy to neoadjuvant chemotherapy has significantly improved outcomes for patients with stage Il-Ill TNBC and patients with metastatic TNBC expressing high levels of PD-L1, but some patients experience drug resistance, we also administered paxalisib in combination with immunotherapy (anti-PD1) + / - chemotherapy (paclitaxel, Abraxane) in the 4T1 model (Figure 2B, 2H, I). The triple combination of paxalisib + anti-PD1 + Abraxane showed the greatest reductions in primary tumor weight and volume (Figure 2B), with no increase in toxicity (Figure 2H, I).

[0160] In the 4T1 model, 4T1 mouse breast tumor cells are injected into the mammary gland of host mice and metastases usually then develop in the lung, lymph nodes, liver, bone and other sites. However, mice treated with the paxalisib-anti-PD1 combination regimen did not develop overt metastasis, suggesting an anti-metastatic effect. Lung inflammatory infiltrates, enlarged spleens (splenomegaly), and extramedullary hematopoiesis (EMH) are associated with cancer progression and metastasis in the 4T1 model even in the absence of macroscopic disease. Supporting a systemic anti-cancer effect of combined paxalisib-anti-PD1, histopathological examination of the lungs revealed a significant decrease in leukocyte infiltration into the lungs at all concentrations, including at the optimal therapeutic dose of 7.5 mg / kg (Figure 2G, middle), and there was a significant reduction in EMH following paxalisib treatment at the optimal therapeutic dose of 7.5 mg / kg (Figure 2G, right).

[0161] To further assess the impact of paxalisib on tumour metastasis, we injected 4T1 tumour cells into the systemic circulation prior to treatment (Figure 2J). Mice treated with paxalisib combined with anti-PD1 maintained normal body weights throughout the first 10 days of the experiment (Figure 2K). CD45 VIM+SNAIL+ CTCs (Figure 2L) were isolated from whole blood collected from the submandibular vein using the ScreenCell®Cyto filtration device.JAWS Ref: 751495PCTSignificantly fewer CTCs (<300 cells) were captured from paxalisib + anti-PD1 -treated mice compared with vehicle mice (>1500 cells) (Figure 2M, N). The present inventors also inflated lungs with Indian ink to visualize metastatic lung nodules (Figure 20), and systematic examination revealed two metastatic phenotypes: large and protruding or small and flat (Figure 2I). There was a significant reduction in the number of both types of metastases when mice were administered paxalisib and anti-PD1 (Figure 20). Therefore, paxalisib inhibits CTCs and the development of metastases.Spatial imaging reveals changes in immune cell landscapes when paxalisib is combined with immunotherapy

[0162] To examine the effects of paxalisib on the tissue microenvironment, the inventors next used spatial immune profiling of tumour tissues from the 4T1 TNBC model to investigate 14 innate and adaptive immune populations, stromal cells, and tumor cell proliferation and their interactions following treatment with paxalisib combined with immunotherapy using a 23-marker (immuno)phenotyping panel (Figure 3A, Table 16). The addition of paxalisib to anti-PD1 treatment reduced the proportion of stromal cells (mainly vascular cells, but also fibroblasts and erythrocytes) (Figure 3B), increased adaptive immune cell populations, particularly B cells (Figure 3B), and increased specific populations of innate immune cells (like neutrophils and dendritic cells), while decreasing others (like macrophages) (Figure 3B). The patterns of infiltration also differed between treatment groups, with B cells and neutrophils accumulating in the center of the tumours and others, like APCs and macrophages, observed at the periphery (Figure 3C).TABLE 16THE 23 ANTIBODY CODEX PANELStromal Innate Adaptive OtherCD49f CD11c CD19 Ki67CD71 CD24 CD90.2Ter119 CD38 TCRb, MHCCD31 Ly6G CD169CD11b CD21 / 35CD45 IgM, IgDCD45CD3, CD4, CD8

[0163] To explore potential interactions between immune cell populations, the inventors conducted cellular neighborhood (CN) analysis (Figure 3D). CN analysis identified ten unique cellular neighborhoods, with an increase in B cell and neutrophil populations in the center of paxalisib + anti-PD1 tumours and DCs at the periphery, and a decrease in macrophages (central) and stromal cells (throughout) (Figure 3E). Paxalisib has a profound effect on the tumour microenvironment, affecting both the immune and stromal compartments.JAWS Ref: 751495PCTPI3K-mT0R inhibition enhances anti-tumor immune profiles in combination with immunotherapy

[0164] To further understand immune cell changes in the microenvironment of paxalisib-treated tumours at higher resolution, the inventors conducted NanoString nCounter analysis of tumours harvested from the 4T1 model. Paxalisib + anti-PD1 treatment enhanced anti-tumor immune responses in both paxalisib and combination treatments, such as increases in antigen presentation, TCR signaling, cytotoxicity, and interferon responses (Figure 4). Cell abundance analysis revealed increases in anti-tumor immune cell populations in response to paxalisib and paxalisib + anti-PD1 treatment, including in cytotoxic cells, dendritic cells (DCs), T cells, and B cells (Figure 5A). Supporting a more favorable therapeutic outcome, relative cell abundance analysis also revealed increased total tumor-infiltrating lymphocyte (TIL) counts in both paxalisib and combination treatment groups, representing a shift in lymphocyte populations through an increase in B cells, mirroring the spatial analysis and notable decreases in exhausted and regulatory T cell populations, providing a more granular analysis of the subtype distribution (Figure 5B). Importantly, cytotoxic T cell function was also increased in response to paxalisib, with increases in granzyme B (Gzmb) and IFNy expression (Figure 5C). Paxalisib and combination therapies also inhibited pro-tumour immune cell populations such as mast cells, which contribute to anti-PD1 resistance and immune-suppressive regulatory T cells (Figure 5D). Mast cell differences were further confirmed at the tissue level using toluidine blue (Figure 5E).

[0165] Collectively, these findings demonstrate that paxalisib + anti-PD1 treatment induces anti-tumour immune profiles (cytotoxic T cells), reduces T cell phenotypes associated with immunotherapy resistance (exhausted T cells, Tregs), whilst reducing pro-tumour innate immune populations such as mast cells.PI3K-mT0R inhibition targets p85f3-H3K27Me3 interactions

[0166] Recent studies have shown that p85 regulatory subunit of PI3K can regulate biological processes through nuclear translocation. Nuclear translocation of p85p stabilizes EZH1 / 2, increasing H3K27 tri-methylation and gene silencing. Therefore, the present inventors sought to confirm nuclear p85p expression in human TNBC. Immunohistochemistry was performed on cancer tissues from Stage 3 TNBC patients (Figure 6), and sections were analyzed by an independent histopathologist blinded to the treatment groups. Consistent numbers of nuclear p85p+cells were observed in all patient tissues, thereby confirming nuclear p85p expression in TNBC (Figure 6). This pattern of expression was also confirmed in vivo in mouse tumour cells isolated from mice treated with combined paxalisib, chemotherapy, and / or immunotherapy (Figure 7A, B). Reduced nuclear p85p expression was observed in paxalisib and paxalisib + anti-PD1 -treated mice, suggesting a direct impact of PI3K-mTOR inhibition on the translocation of p85p into the nucleus (Figure 7B).

[0167] Given p85p nuclear translocation leads to increased H3K27 trimethylation, we next determined the impact of paxalisib treatment on p85p and H3K27Me3JAWS Ref: 751495PCTnuclear expression. Paxalisib alone or triple combination-treated 4T1 mice showed a significant decrease in both nuclear p85p and H3K27me3expression in vimentin+cells (Figure 7C).Immunofluorescence analysis revealed a significant decrease in the nuclear intensity of p85p and H3K27Me3(Figure 7D) in response to paxalisib in MDA-MB-231 and MDA-MB-468 cells (Figure 7E). Notably, co-localization of p85p and H3K27Me3was observed in vehicle but not paxalisib-treated samples (Figure 7D). Finally, to determine the relevance of this p85p: H3K27Me3interaction in TNBC patients, PBMCs were isolated from the blood of TNBC patients before and after chemotherapy and subjected to Duolink PLA (Figure 7F). Elevated nuclear p85p: H3K27Me3interactions were observed after chemotherapy in all patients (Figure 7G), suggesting an association with drug resistance.PI3K-mT0R inhibition targets the dual role of EZH2

[0168] EZH2 has been implicated in metastases and recurrence via two mechanisms. First, EZH2 catalyzes H3K27 tri-methylatiob, acting as a transcriptional repressor and causing epigenetic gene silencing. Second, EZH2 has a non-catalytic inducible role as a transcriptional co-activator, interacting with NF-KB p65, resulting in the positive regulation of cancer-related (EMT, cell cycle, DNA repair) and NF-KB inflammatory target genes.

[0169] To investigate the impact of PI3K-mTOR inhibition on EZH2 expression, MDA-MB-231 cells were treated with paxalisib for 24 h prior to immunofluorescence and qPCR analysis. PI3K-mTOR blockade reduced EZH2 mRNA (Figure 8A) and corresponding protein levels within the nucleus (Figure 8B). Furthermore, paxalisib significantly decreased nuclear p85p: EZH2 (Figure 8C) and P65NFKB: EZH2 (Figure 8D) interactions, as observed by proximity ligation assays. Furthermore, time course analysis quantitating the interaction between p85p and EZH2 shows disruption at 30 minutes, 2 hours, and 3 hours post paxalisib treatment in MDA-MB-231 cells (Figure 8E), suggesting the impact of the drug prior to new protein synthesis of EZH2 mRNA. Thus, these data suggest that the paxalisib, in part, mediates its effects through disruption of the protein-protein interaction between p85p and EZH2, and in part independently of its effect on EZH2 mRNA levels.

[0170] To investigate the impact of paxalisib on EZH2-targeted gene expression, the present inventors overlapped EZH2 / H3K27me3ChlP-seq data with our NanoString nCounter differentially-expressed (DE) genes (Figure 8F). EZH2-bound genes (EZH2+) were mainly upregulated by paxalisib (30 DE genes) and paxalisib + anti-PD1 (19 DE genes) treatments, while anti-PD1 alone mediated nine DE genes with EZH2-binding sites. Notably, there were no common EZH2+ DE genes between paxalisib and anti-PD1 treatments, indicating that they have an impact on completely different biological pathways. The present inventors further employed H3K27me3ChlP-seq data to distinguish the chromatin states of EZH2-bound DE genes, showing that half of the EZH2+ population was associated with the repressive mark H3K27me3(EZH2+ / K27me3+). The inventors next determined the distribution of EZH2-bound genomic locations of both up- and downregulated DE genes after paxalisib and anti-PD1 treatmentJAWS Ref: 751495PCT(Figure 8G). There were distinct EZH2-binding patterns between up- and downregulated genes after paxalisib and anti-PD1 treatment. EZH2 mainly bound at distal intergenic (40%) and promoter (TSS + / - 2-3 kb, 5.9%) regions of upregulated genes, while more proximal promoter regions (TSS < 1 kb, 37.2%; and TSS + / - 1-2 kb, 9.3%) were bound by EZH2.

[0171] A recent study revealed that EZH2 co-localizes with NF-KB to activate pro-oncogenic gene expression in TNBC. Consistently, we found that genes associated with inflammation (IL-6), the drug-resistant phenotype (FOXQ1, NNMT, RelA, NFE2L2), and TNBC metastasis (KRT14) were all bound by both EZH2 and NFKB2 in MDA-MB-231 cells (Figure 8H and Figure 9).

[0172] Together, paxalisib and anti-PD1 -mediated EZH2+ / K27me3+genes are potential gene signatures and therapeutic targets in recurrent cancer. Interestingly, three EZH2+ / K27me3+genes were also co-occupied by NFKB2, suggesting that EZH2+ / K27me3+ / NFKB2+may function as repressor complex in TNBC, since NF-KB both activates and represses gene expression.Associations between PIK3R2 gene expression and survival in patients with TNBC and ovarian cancer

[0173] Finally, we analyzed whether expression of PIK3R2, which encodes p85p, was associated with outcomes in TNBC / basal-like breast cancer (GSE158309 dataset).Patients with basal-like / TNBC expressing high levels of PIK3R2 (solid lines) showed poorer distant metastasis-free survival (DMFS) than patients with low expression (Figure 10A).

[0174] Next, the inventors assessed PIK3R2 mRNA expression in stage IV metastatic TNBC post chemotherapy (carboplatin, nab-paclitaxel) + pembrolizumab durvalumab + olaparib in a single-arm phase-2 trial (Figure 10B). Patients with higher mRNA expression of PIK3R2 showed poorer overall survival compared with patients with lower PIK3R2 mRNA expression. In addition, higher pre-treatment levels of PIK3R2were positively associated with distant relapse in TNBC patients who had residual tumors after treatment with neoadjuvant chemotherapy (NACT) (Figure 10C). PIK3R2 could be a useful prognostic and predictive biomarker.Discussion

[0175] Here, the present inventors provide novel insights into the contribution of the PI3K-mTOR pathway to the TNBC phenotypes associated with growth, migration, metastasis, and therapy resistance. Specifically, the present inventors demonstrate that dual targeting of PI3K and mTOR, but not PI3K alone, inhibits cancer cell proliferation and migration in vitro. Using paxalisib to probe dual PI3K-mTOR pathway inhibition, the present inventors found that it not only promotes a favorable mesenchymal to epithelial phenotype but also inhibits signatures associated with MICs, including CSC, persister cancer cell, and drug resistance signatures. In vivo, paxalisib overcomes immunotherapy resistance to reduce primary tumour burden,JAWS Ref: 751495PCTcirculating CTCs, and metastasis with a favorable toxicity profile. Paxalisib also profoundly affects the tumour immune microenvironment, reducing adaptive immune phenotypes associated with immunotherapy resistance (exhausted T cells, Tregs) and pro-tumor innate immune populations such as mast cells. PI3K-mT0R blockade acts upstream of EZH2, impacting both the classical repressive catalytic p85p-EZH2-H27Me3and active EZH2-NFKB pathways. These data suggest that dual targeting of the PI3K-mT0R pathway disrupts both the catalytic and non-catalytic actions of EZH2 to inhibit metastasis and promote an immunogenic tumor immune microenvironment that could increase the utility of immunotherapy in resistant individuals. Furthermore, expression of PIK3R2, which encodes p85p, was associated with poor prognostic and predictive outcomes in patients with TNBC, paving the way for a precision medicine biomarker for an at-risk group that might be most responsive to PI3K-mT0R inhibition.

[0176] These experiments highlight the importance of dual targeting not only PI3K but also mTOR in tumour cells to reduce and / or prevent metastases and create a pro-immunogenic tumor immune microenvironment (Figure 11, mechanism 1 ). MICs are implicated in metastases and are enriched following standard-of-care treatments. CSCs, which express high levels of ABCB5, ALDH1, CD44, and EpCAM, are defined by their tumour re-populating ability, drug resistance, plasticity, and metastatic capacity. Dormant tumour cells, which may remain quiescent for a long time, are found in naive tumors, drug-treated tumours, and pre-metastatic sites. These cells are characterized by slow proliferation and increased expression of sternness, EMT, plasticity, and drug-resistance markers. Persister cancer cells surviving drug treatment are highly plastic cells that express genes associated with EMT (TAGLN and NNMT), sternness, and the tumor-necrosis factor-a (TNF-a)-nuclear factor KB (NFKB) inflammatory pathway. Originating from mutations or epigenetic reprogramming, collectively, MICs contribute to metastases and recurrence. Using paxalisib as an exemplar PI3K / mTOR inhibitor, the present inventors show that this pathway is critical for targeting metastatic processes.Specifically, it is demonstrated that paxalisib: (i) inhibits the CSC phenotype (CD44h'9hCD24l0W) in human TNBC cells; (ii) simultaneously inhibits protein and mRNA expression of MIC-related genes and the IL-6 and NFKB inflammation signature whilst inducing viral mimicry programs in vitro; and (iii) targets CTCs in the blood and significantly inhibits the growth of lung metastases when combined with anti-PD1. As a major mediator of the immune response, high levels of IL-6 are associated with a poor prognosis, drug resistance, and metastases. Together with inhibiting MIC signatures and inflammation, we show that this approach also induces immune reinvigoration and cancer immune visibility (Figure 11, mechanism 1 ). Epigenetic therapies that activate viral mimicry may enhance adaptive immune responses by increasing antigen processing and presentation, resulting in increased tumor immunogenicity, T cell infiltration, and enhanced immunotherapy responses. Our spatial and transcriptomic analyses support that paxalisib may promote these mechanisms, as immunotherapy-resistant tumours showed a reduction in adaptive immune phenotypes associated with immunotherapy resistanceJAWS Ref: 751495PCT(exhausted T cells, Tregs) and pro-tumour innate immune populations such as mast cells after treatment with paxalisib.

[0177] It is also shown that the nuclear p85p subunit is enriched in TNBC human cell lines and after chemotherapy and immunotherapy. Therefore, this subunit could be utilized as a marker of residual disease. There is accumulating evidence that EZH2 is a key factor controlling drug resistance via its dual functionality. A component of polycomb repressive complex 2 (PRC2), EZH2 is overexpressed in many solid cancers and has been associated with cancer progression and poor survival outcomes. EZH2 catalyzes the trimethylation of lysine 27 on histone H3 (H3K27me3), acting as a transcriptional repressor and epigenetic gene silencer. Recent studies have also identified a non-catalytic role for EZH2 independent of H3K27me3as a transcriptional co-activator. EZH2 physically interacts with NF-KB p65 (RelA) in TNBC cells, independent of PRC2, resulting in the positive regulation of cancer-related (EMT, cell cycle, DNA repair) and NF-KB target genes, including the pro-inflammatory cytokine IL-6 (30). These two functions of EZH2 modulate critical signaling pathways that cause drug resistance. Current drugs that solely target the canonical catalytic activity of EZH2 result in incomplete responses or primary resistance. Given that NF-KB signaling also participates in resistance, the identification of nevel therapeutic strategies that target both the catalytic and non-catalytic roles of EZH2 will better overcome resistance mechanisms, consequently improving clinical outcomes (Figure 11, mechanism 2). These results clearly show that nuclear p85p colocalizes with EZH2 in an epigenetic complex. Paxalisib disrupts the nuclear p85p-EZH2 complex in human TNBC cells and patient-derived TNBC liquid biopsies treated ex vivo. EZH2 also interacts with the PI3K / AKT / mTOR pathway. Existing as a heterodimer, PI3K consists of a p110 catalytic subunit (p110a, p110p, and p1105) and a p85 repressive regulatory subunit (p85a, p85p, p55a, p50a, and p55y). p85 proteins can regulate biological processes through nuclear translocation. Specifically, p85p disassociates from p110a in cancer cells, translocating to the nucleus, stabilizing EZH1 / 2, and increasing H3K27 tri-methylation (. Thus, these findings highlight for the first time that PI3K-mTOR inhibition disrupts both the established p110-p85a complex (Table 17) and the nuclear p85p-EZH2 complex. Thus, PI3K-mTOR inhibitors disrupt both the cytoplasmic and nuclear PI3K axes, which is critical for suppressing the complete contribution of the PI3K pathway in cancer. These findings show that paxalisib inhibits EZH2 transcription, contributing to the overall disruption of the nuclear EZH2 pool.TABLE 17INHIBITION OF HUMAN PI3K ISOENZYMES IN VITRO BY PAXALISIBHuman PI3K Isoenzymes Mean Kiapp(nM)Samplep110a / p85p p110p / p85a p110y p1106 / p85a Paxalisib. 1, lot 1 2.1 39 12 2.9 Paxalisib. 1-2 2.4 37 13 3.5JAWS Ref: 751495PCTPaxalisib. 1-3 1.6 35 8.1 2.1 Paxalisib. 1-4 1.6 33 7.9 2.6 Paxalisib. 1-5 1.8 40 7.5 1.3 Paxalisib. 1-6 4.1 77 15 3.5 Paxalisib. 1-9 2.1 34 6.1 3.7 Paxalisib. 1-10 1.8 36 7.7 1.9 Average of all lots 2.2 41.4 9.7 2.7 Standard Deviation 0.82 14.6 3.2 0.9

[0178] More recently, a non-catalytic role for EZH2 has been identified, in which it acts as a transcriptional co-activator. In the context of TNBC, EZH2 interacts with NF-KB p65, inducing EMT and NFKB inflammatory target genes. To date, EZH2 inhibitors have underperformed clinically, with partial responses or primary resistance major limitations (52,53). The present inventions can now extend recent findings by demonstrating the importance of targeting signaling pathways upstream of EZH2. By inhibiting the PI3K-mTOR pathway through p85p nuclear translocation, both the catalytic and non-catalytic axes of EZH2 could be targeted, thereby overcoming resistance mechanisms and reducing metastases (Figure 11, mechanism 2). Future studies are needed to evaluate the precise mechanism(s) by which the PI3K-mTOR pathway inhibits both axes of downstream EZH2. Nevertheless, this study provides critical evidence for the use of PI3K-mTOR inhibitors such as paxalisib in the treatment of cancer.

[0179] In conclusion, this study demonstrates the utility of combining PI3K-mTOR inhibitors with immunotherapy by exploiting interactions between the PI3K-mTOR signalling pathway and the EZH2 epigenetic network. For the first time we show that dual targeting of the PI3K-mTOR pathway, not PI3K alone, has beneficial effects by inhibiting signatures associated with MICs, altering the tumour immune microenvironment, preventing metastases, and overcoming resistance associated with immunotherapy.Materials & Methods

[0180] All materials and reagents used in the synthesis and testing of the compositions described are commercially available, for example, from Sigma-Aldrich Co., Novabiochem, Abeam, and American Type Culture Collection (ATCC) unless otherwise stated.In vivo animal studies.

[0181] All cancer cell lines (4T1, mouse metastatic TNBC; MDA-MB-231, MDA-MD-468, SUM149PT, human TNBC) were sourced from the ATCC (Gaithersburg, MD) and routinely mycoplasma tested. 4T1, MDA-MB-231, and SUM149PT cells were maintained and cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco; ThermoFisher Scientific, Waltham, MA)JAWS Ref: 751495PCTsupplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin / streptomycin. MDA-MB-468 cells were maintained and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco) supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin / streptomycin. For in vitro and in vivo studies, all PI3K and PI3K-mTOR pathway inhibitors (dactolisib, apitolisib, pmipalisib, LY294002, alpelisib, idelalisib, and wortmannin) were sourced from Selleck Chemicals (Houston, TX), with the exception of paxalisib (Kazia Therapeutics, Sydney, Australia).WST-1 cell viability assay

[0182] 4T1, MDA-MB-231, MDA-MB-468, and SUM149PT cells were seeded at optimized densities in 96-well flat-bottom tissue culture plates in a total volume of 100 pL / well. Cells were left to adhere overnight at 37°C and 5% CO2 prior to treating with PI3K pathway inhibitors at the indicated doses. Cells were incubated at 37°C and 5% CC for 72 hours, after which medium was removed and replaced with 100 pL / well of WST-1 cell proliferation reagent (Sigma-Aldrich) at a 1:10 final dilution in complete cell culture medium. Absorbance was recorded at 450 nm after a one-hour incubation period using a microplate spectrophotometer. Percent proliferation was calculated by subtracting the mean absorbance of the background control (blank) from the sample absorbance. The IC50 value of each inhibitor was determined by log(inhibitor) vs. response - variable slope using Prism 10 software (GraphPad Software, La Jolla, CA).Scratch wound healing assay

[0183] 4T1 and MDA-MD-231 cells were stimulated with PMA / TGF-p as indicated above for 24 hours prior to seeding at optimized cell densities in 96-well IncuCyte Image Lock plates (Sartorius, Gottingen, Germany). Cells were seeded in a total volume of 100 pL / well in low-serum medium (2%) and left to adhere overnight at 37°C and 5% CO2 to ensure 100% confluency. After 24 hours, wounds were created using the 96-pin IncuCyte WoundMaker Tool (Sartorius, Gottingen, Germany). Post wounding, cells were washed to remove non-adherent cells and then treated with PI3K pathway inhibitors in low-serum medium at the established IC50 doses. Plates were then placed into the IncuCyte Zoom live-cell analysis system (Sartorius, Gottingen, Germany), and wound healing was tracked using a scan interval of 4 hours over a 24-hour period. Samples were analyzed using IncuCyte ZOOM 2018A in-built analysis (Sartorius, Gottingen, Germany).Flow cytometry

[0184] Fluorescence-activated cell sorting (FACS) was performed on single-cell suspensions stained with anti-CD44-BV605 (BD Biosciences, San Jose, CA) and anti-CD24-PE antibodies (BD Biosciences), with the LIVE / DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen; Thermo Fisher Scientific, Waltham. MA) to monitor cell viability. The proportion of CD44h'9h / CD24lowcells were analyzed using FlowJo software version 10.8.1 (BD Biosciences).JAWS Ref: 751495PCTRNA extraction and cDNA synthesis

[0185] Cells were lysed in 500 pL TRIzol Reagent (Invitrogen; Thermo Fisher Scientific) before RNA isolation using the Direct-zol RNA Miniprep Kit (Zymo Research, Irvine, CA) and treated with RNase-free DNase I (Qiagen, Hilden, Germany) following the manufacturer’s protocols. The NanoDrop (Thermo Fisher Scientific) instrument was then used to determine RNA concentration and purity (A260 / A280). RNA (1 pg) was reverse transcribed to cDNA using the Superscript VILO IV Master Mix (Thermo Fisher Scientific) following the supplier's protocol. cDNA was diluted 1:20 with RNase-free water for qRT-PCR.gRT-PCR

[0186] Gene transcription was analyzed using TaqMan Gene Expression Master Mix (Thermo Fisher Scientific) on the ABI Viia 7 real-time PCR machine (Applied Biosystems; Thermo Fisher Scientific). Cts were converted to arbitrary copy numbers and normalized to the geomean of the housekeeping genes ACTB and PPIA. The following human TaqMan probes were used: ATR, ATM, RAD51, EXO1, IL6, GBP, IFIT1, IFIT2, IFIT3, IFIH1, IRF1, RSAD2, p50, PDL1, PARP1, and EZH2. No template and no reverse transcriptase controls were included to exclude genomic DNA contamination.PBMC isolation

[0187] Whole blood from TNBC patients was stored in EDTA tubes for CTC identification. Unwanted cells were targeted using glycophorin A on red blood cells and then removed via centrifugation over a buoyant density medium (Lymphoprep™, STEMCELL Technologies, Vancouver, Canada). Purified PBMCs were then extracted as a highly enriched population from the interface between the plasma and the buoyant density medium and harvested in 20% FBS in PBS.Circulating tumor cell enrichment

[0188] Whole blood from patients with TNBC was stored in EDTA tubes for CTC identification. RosetteSep™ Human CD45 Depletion Cocktail (STEMCELL Technologies) was used to enrich CTCs from whole blood by depleting CD45+cells. Unwanted cells were targeted for depletion with tetrameric antibody complexes recognizing CD45, CD66b, and glycophorin A on red blood cells. Unwanted cells were then removed via centrifugation over Lymphoprep™. The purified epithelial tumour cells were then extracted as a highly enriched population from the interface between the plasma and the buoyant density medium and were harvested in 20% FBS in PBS.

[0189] In the 4T1 intravenous (i.v.) metastasis mouse model, 300 pL of whole blood was collected from the submandibular vein and stored in EDTA tubes. ScreenCell®Cyto filtration devices (ScreenCell, Sarcelles, France) were used to capture single and clustered CTCs according to the manufacturer’s instructions. Captured cells were validated as CTCs by immunofluorescence staining with primary antibodies targeting vimentin, Snail-1, and CD45.JAWS Ref: 751495PCTImages were scanned using the Nikon Crest Optics Deep SIM super-resolution microscope (CrestOpics, Rome, Italy) and analyzed using Imaged software as required.Histochemistry and immunohistochemistry

[0190] Modified Giemsa (Sigma-Aldrich) was diluted 1 in 10 with pH 6.8 PBS and syringe-filtered (0.2 pm) to remove precipitates. The isolation support (IS) with microporous (6.5 ± 0.33 pm) membrane filter was submerged in 3 mL of dilute modified Giemsa for 30 minutes in a 6-well plate. Excess Giemsa was removed with 3 x 2 minute washes with 3 mL pH 6.8 PBS. Cells on the whole membrane filter were counted after imaging with the Applied Spectral Imaging (ASI) Digital pathology platform (Carlsbad, CA).

[0191] Formalin-fixed paraffin-embedded (FFPE) mouse tumor tissues were stained with toluidine blue to highlight mast cells and imaged using the Aperio AT Turbo (Leica Biosystems).

[0192] Hematoxylin and eosin (H& E)-stained formalin-fixed, paraffin-embedded (FFPE) livers, lungs, and spleens were subjected to histopathological analysis by an independent expert histopathologist. Changes in inflammation, extramedullary hematopoiesis (EMH), hepatocytes, and lung leukocytosis were scored on a scale of 1 = mild, 2 = moderate, or 3 = severe.

[0193] For human studies, immunohistochemistry was performed on five FFPE biopsy specimens from patients with stage 3 TNBC using an antibody directed against PIK3R2 (HPA069291, Atlas Antibodies; Merck) and imaged using the Aperio AT Turbo (Leica Biosystems). All tissues were analyzed for nuclear PIK3R2 (p85p) expression by a qualified pathologist and expressed as % positivity.Immunofluorescence

[0194] MDA-MB-231 and MDA-MB-468 cells were cultured on glass microscope slides at optimized densities and fixed in 3.7% paraformaldehyde following treatment. CTCs and PBMCs were fixed in 3.7% paraformaldehyde, and cytospinning was used to mount them onto glass microscope slides. Cells were permeabilized by incubating with 0.5% Triton X 100 for 15 min, blocked with 1% BSA in PBS, and probed with the following primary: Primary antibodies targeted vimentin (sc-6260, Santa Cruz Biotechnology, Santa Cruz, CA), E-cadherin (3199S, Cell Signaling Technology, Danvers, MA), Abcb5 (Ab77549, Abeam, Cambridge, UK), Snai-1 (sc-10433, Santa Cruz Biotechnology), ALDH1 (Ab52492, Abeam), p65 (Ab16502, Abeam), FOXQ1 (sc-166266, Santa Cruz Biotechnology), NRF2 (16396-1 -AP, Proteintech, Rosemont, IL), NNMT (Ab119758, Abeam), IL-6 (10C12, Leica Biosystems, Mt Waverley, Victoria, Australia), EpCAM (sc-25308, Santa Cruz Biotechnologies), p85p (Ab180967, Abeam), H3K27Me3 (Ab6002, Abeam), and EZH2 (AB283270, Abeam). Protein expression was visualized with a donkey anti-rabbit Alexa Fluor 488, anti-mouse Alexa Fluor 568, or donkey anti-goat Alexa Fluor 647 (Thermo Fisher Scientific). Cover slips were mounted on glass microscope slides with ProLong Glass Antifade reagent (Thermo Fisher Scientific). ProteinJAWS Ref: 751495PCTtargets were imaged using the Zeiss 780 NLO confocal microscope (Zeiss, Oberkochen, Baden-Wurttemberg, Germany) or the Applied Spectral Imaging (ASI) Digital Pathology platform (Carlsbad, California) and analyzed using Imaris 10.1.1, QuPath, or automated ASI software, as required.Duolink proximity ligation assay

[0195] MDA-MB-231 cells were seeded onto coverslips in 12-well plates at a density of 75,000 cells per well in 1 mL of growth medium (DMEM supplemented with 10% fetal bovine serum, 200 mM L-glutamine, and antibiotics) and incubated overnight at 37°C to allow for cell attachment. The following day, cells were treated with 10 pM Paxalisib or DMSO (vehicle control) for 0.5, 2, 3 or 24 hours. Cells were then fixed with 3.7% formaldehyde and subjected to Duolink PLA according to the manufacturer's protocol. Briefly, cells were fixed and cytospinning was used to mount them onto glass microscope slides. The Duolink proximity ligation assay (MilliporeSigma, Burlington, MA) was performed using antibodies targeting p85p and H3K27Me3, p85p and EZH2, and EZH2 and NFKB, as described above. Digital images were acquired on the Zeiss 780 NLO confocal microscope and analyzed using QuPath 0.4.3 software.Animal studies

[0196] Six-to-eight-week-old female BALB / c mice were procured from the OzGene (Perth, Western Australia) and allowed to acclimatize for one week. For the orthotopic breast tumor model, 1 x 1054T1 cells suspended in phosphate-buffered saline (PBS) were administered into the 4thright mammary fat pad of BALB / c mice. For the i.v. metastatic breast cancer model, all female BALB / c mice were inoculated intravenously with 4T1 cells by tail vein injection with 0.1 mL of 1 x 105cell suspension. In the murine colon adenocarcinoma “hot" tumor model (i.e., responsive to immunotherapy), 5 x 105CT-26 cells were subcutaneously implanted into the right flank of female BALB / c mice. Once primary tumors reached -50-100 mm3or the day after IV inoculation, mice were subjected to daily oral administration of paxalisib at the indicated doses in combination with either anti-PD1 treatment (10 mg / kg) or anti-PD1+Abraxane (nab paclitaxel) (30 mg / kg) administered intraperitoneally at day 0 and day 4. Tumors were measured using calipers, and volumes were calculated using a modified ellipsoidal formula1 / a (a / b2), where a = longest side and b = shortest side. Mice were monitored daily for clinical abnormalities and tumor volumes were measured thrice weekly. Once tumors reached the maximum permitted size (1000 mm3) in the vehicle group, all tumors and associated organs (lung, liver, spleen) were harvested, weighed, imaged, and fixed in 4% paraformaldehyde for subsequent analysis. For metastases studies, lungs were perfused with Indian ink and de-stained in Fekete’s solution. Large protruding and small flat metastatic nodules were manually counted and the ratio of large:small quantified.JAWS Ref: 751495PCTTumor digestion

[0197] 4T1 tumors were harvested in cold DMEM supplemented with 2.5% FCS before being finely cut using surgical scalpels and enzymatically dissociated using collagenase type 4 (Worthington Biochemical Corp., Lakewood, NJ) at a concentration of 1 mg collagenase / g of tumor at 37°C for 1 hour. Dissociated cells were then passed through a 0.2 pm filter before fixation in 3.7% paraformaldehyde and cytospinning onto glass microscope slides for immunofluorescence staining.NanoString nCounter assay

[0198] Total RNA was extracted from FFPE mouse tumor tissue using the RNeasy FFPE kit according to the manufacturer’s protocols (Qiagen, Hilden, Germany). RNA concentration was measured using the Qubit RNA HS assay kit (Thermo Fisher Scientific).

[0199] RNA samples were subjected to hybridization with the multiplexed mouse tumor signaling 360 panel codeset. Then, hybridized samples were loaded onto nCounter prep station chips, and data were acquired using the nCounter Digital Analyzer (NanoString Technologies, Seattle, WA) following the manufacturer's procedures. Data were analyzed with nSolver Analysis Software v4.0 (NanoString Technologies). Counts were normalized according to the expression levels of housekeeping genes and positive / negative-control probes.NanoString nCounter and RNA-seg / ChlP-seg integrated analysis

[0200] ChlP-Seq data (GSE223959) were accessed in scaled BigWig format for both MDA-MB-231 specific Input and EZH2 samples. BigWig files were converted to Wig format using bigWigToWig and further to bed format using wig2bed from the BEDOPS package. EZH2 binding sites (peaks) of significant enrichment relative to input control were identified using MACS2 using the callpeak command by extending in silico the reads to a fragment size of 250 bp. Bound genes were defined as any ChlP-seq peak call that overlapped a gene body + / -3 kb; bound promoters were defined as any ChlP-seq peak call that overlapped a transcription start site (TSS) + / -3 kb. ChlP-Seq peaks were annotated using ChlPseeker R package.

[0201] EZH2 chromatin-binding sites in MDA-MB-231 cells were compared to publicly available MDA-MB-231 ChlP-seq data of the repressive mark H3K27me3 (GSE77772) obtained in BigWig format containing the log-transformed fold-enriched in ChIP relative to Input. bigWigToBedGraph was used to convert to BedGraph format, which was further used for calling peaks (binding sites) using MACS2 bdgpeakcall with a cut-off of 0.5.

[0202] Further, nCounter Tumor 360 Signaling panel genes bound by EZH2 were checked for overlap with genes bound by H3K27me3.

[0203] Differentially expressed genes between Paxalisib vs Control in the mouse model were matched to human CTC and CTC cluster-related genes coming from red, pink, grey, and blue modules from the Gkountela etal. Cell study (Figures 3C and Figure 4B of their paper). Volcano plots showing log2FC and p-values for the overlapping genes were generatedJAWS Ref: 751495PCTusing ggplot2\n R. Significantly enriched pathways from KEGG and Reactome were identified using the EnrichR portal (Kuleshov et al., 2016).STEP spatial immune profiling and analysis

[0204] OCT frozen mouse tumor tissues were immunolabelled with an 18-panel antibody cocktail (CD19, CD21 / 35, CD3, TCRb, CD4, CD90.2, CD11b, CD45, Ly6G, IgM, CD24, CD11c, CD38, CD31, Teri 19, CD49f, CD71, Ki67) and subjected to the Spatial Tissue Exploration Program (STEP) platform (Akoya Biosciences, Marlborough, MA).Multiplexed Image Bioinformatics analysisData pre-processing

[0205] The final QPTIFF files of composite images were opened in QuPath (15), where quality control can be performed by visual assessment of the whole slide. All markers were evaluated individually based on appropriate cellular localization and staining pattern. Artifacts such as debris, tissue folding, and out-of-focus regions were manually cropped and excluded from the downstream analysis.Segmentation and phenotyping

[0206] Nuclear segmentation was first performed using StarDist to the DAPI channel, with the deep learning model retrained by Akoya internally (16). Cytoplasm segmentation was then estimated from nuclear expansion by morphological dilation of 6 pm. The centroid of each cell was determined by the x-y coordinates within the image. Mean fluorescent intensity (MFI) of each marker was extracted for each segmented cell from the nuclear mask and cytoplasm mask according to protein localization, yielding a single-cell protein signals matrix. Cells without any marker expression or abnormally high signal sum were removed. Cell segmentation and filtering left 528,659 cells across two samples.

[0207] Each protein signal was then z-scored across all cells, such that each marker had a mean of zero and a standard deviation of one. Only QC-passed lineage markers were used for phenotyping analysis. The Leiden algorithm and GPU-accelerated methods were used for unsupervised clustering, with cluster visualization using UMAP and t-SNE (17,18). For Leiden clusters, resolutions of 1 -6 were tested, and the optimal resolution of 2 was chosen for sub-clustering and manual annotation. We performed multi-level refinement by additional rounds of sub-clustering of unclear clusters. Clusters were visualized and annotated on the image after each clustering. Cell phenotypes were assigned based on protein expression in the hierarchical clustering heatmaps and their location in the images. Clusters with similar expression patterns were combined. The number of cell types was then calculated, and the percentages were compared across different samples.Cellular neighborhood analysis

[0208] Cellular neighborhood (CN) analysis was performed as previously described (19). Briefly, for each annotated cell, we use the k-nearest neighbor algorithm to enumerate theJAWS Ref: 751495PCTcell types of its 10 nearest spatial neighbors (including the center cell) in Euclidean space. These local neighborhood windows were then clustered based on the composition of cell types within each window using the MiniBatchKMeans algorithm, with k=10. Each cell was then allocated to the CN defined by the containing window. CNs were annotated based on the enrichment of certain cell types. The percentage of each CN was also calculated.Population-based cancer survival analysis

[0209] Human cancer datasets meeting the following criteria were downloaded from Array Express (AE), Gene Expression Omnibus (GEO), and Sequence Read Archive (SRA): (a) at least 20 expression profiles from cancer patient tissues (not cell lines or preclinical models); (b) annotated with clinical outcomes of at least one of i) categorical outcomes after treatment, e.g., progressive disease, stable disease, pathological complete response, response, no response, and distant metastasis; and / or ii) continuous outcome data including survival and event follow-up, e.g., overall, disease-specific, distant metastasis-free, or progression-free survival.

[0210] For cohort analysis, within each cohort, we compared the mRNA expression levels of PIK3R2 by categorizing the samples into high- and low-risk subgroups, e.g., high risk = distant metastasis and low risk = no metastasis. Samples were also categorized into high and low subgroups based on PIK3R2 expression, where high = >75th percentile of PIK3R2 expression and low = <75th percentile. Where mutation or copy-number data were available, samples were categorized into altered and unaltered for the PIK3R2 gene: altered = harbors a mutation or copy number change in PIK3R2; unaltered = otherwise. Box and scatter plots comparing PIK3R2 expression between the above subgroups were plotted using the ggplot package in R version>4.0 (20). Statistical significance between subgroups was compared with the Wilcoxon test. Kaplan-Meier survival curves between high- and low-risk groups were plotted using the ggsurve package and the statistical difference between the curves were computed using log-rank test.Statistical analysis

[0211] Statistical significance was assessed in Prism 10 (GraphPad Software, La Jolla, CA) using either paired or unpaired t-tests or one-way ANOVA (Dunnett’s or Tukey’s multiple comparison test), as indicated. P< 0.05 was considered statistically significant (*, P< 0.05; **, P < 0.01; ***, P < 0.001; P < 0.0001 ).REFERENCESHassan B, Akcakanat A, Holder AM, Meric-Bernstam F. Targeting the PI3-kinase / Akt / mTOR signaling pathway. Surg Oncol Clin N Am. 2013 Oct;22(4):641-64.Jiang, N., Dai, Q., Su, X. et al. Role of PI3K / AKT pathway in cancer: the framework of malignant behavior. Mol Biol Rep 47, 4587-4629 (2020).JAWS Ref: 751495PCTChen X, Cao Y, Sedhom W, Lu L, Liu Y, Wang H, Oka M, Bornstein S, Said S, Song J, Lu SL. Distinct roles of PI3KCA in the enrichment and maintenance of cancer stem cells in head and neck squamous cell carcinoma. Mol Oncol. 2020 Jan;14(1):139-158.Xia P, Xu XY. PI3K / Akt / mTOR signaling pathway in cancer stem cells: from basic research to clinical application. Am J Cancer Res. 2015 Apr 15;5(5):1602-9.Yang L, Huang F, Mei J, Wang X, Zhang Q, Wang H, Xi M, You Z. Posttranscriptional Control of PD-L1 Expression by 17p-Estradiol via PI3K / Akt Signaling Pathway in ERa-Positive Cancer Cell Lines. Int J Gynecol Cancer. 2017 Feb;27(2):196-205.Fusco N, Malapelle U, Fassan M, Marchid C, Buglioni S, Zupo S, Criscitiello C, Vigneri P, Dei Tos AP, Maiorano E, Viale G. PI3KCA Mutations as a Molecular Target for Hormone Receptor-Positive, HER2-Negative Metastatic Breast Cancer. Front Oncol. 2021 Mar 25;11:644737. Mosele F, Stefanovska B, Lusque A, et al. Outcome and molecular landscape of patients with PI3KCA-mutated metastatic breast cancer. Annals of Oncology; Published online 24 January 2020.Lanczky A, Gyorffy B: Web-Based Survival Analysis Tool Tailored for Medical Research (KMplot): Development and Implementation, J Med Internet Res, 2021 Jul 26;23(7):e27633. Kuleshov MV, Jones MR, Rouillard AD, Fernandez NF, Duan Q, Wang Z, Koplev S, Jenkins SL, Jagodnik KM, Lachmann A, McDermott MG, Monteiro CD, Gundersen GW, Ma'ayan A. Enrichr: a comprehensive gene set enrichment analysis web server 2016 update. Nucleic Acids Research. 2016; gkw377.

Claims

JAWS Ref: 751495PCTWHAT IS CLAIMED IS:

1. A method of method of treating cancer in an immunotherapy-resistant subject, the method comprising administering to the subject a composition that comprises an agent that disrupts the PI3K / Akt / mTOR pathway and an immunotherapy (e.g., a checkpoint molecule inhibitor therapy), to thereby treat the cancer.

2. The method of claim 1, wherein the agent inhibits both PI3K and mTOR.

3. The method of claim 1 or claim 2, wherein the agent is selected from the group comprising paxalisib, PI-103, PI-103BE, GSK1059615, omipalisib, SN202, NSC765844, dactolisib, samotolisib, (S)-4-fluoro-5-(2-(3-methylmorpholino)-6-(1 -(methylsulfonyl)cyclopropyl)pyrimidin-4-yl)pyridine-2-amine, voxtalisib, PF-04691502, apitolisib, GNE-477, (2S,6F?)-2,6-dimethyl-4-(4-mopholino-6-(1 / 7-pyrazol-5-yl)thieno[3,2-c / ]pyrimidin-2-yl)mopholine, 2-(2-aminopyrimidin-5-yl)-A / ’-(4-methoxybenzoyl)-4-morpholinothieno[3,2-c / |pyrimidine-6-carbohydrazide, PKI-402, VS-5584, gedatolisib, bimiralisib, (R)-1-(2-((4-(4,6-dimorpholino-1,3,5-triazin-2-yl)phenyl)amino)-1 H-benzo[d]imidazole-6-carbonyl)pyrrolidine-2-carboxamide, (2-((4-(4,6-dimopholino-1,3,5-triazin-2-yl)phenyl)amino)-1 / 7-benzo[c / |imidazole-6-yl)(morpholino)methanone, CMG 002, PF-04979064, and MCX-83.

4. The method of any one of claims 1 to 3, wherein the agent is paxalisib.

5. The method of claim 4, wherein the dose of paxalisib is around 7.5 mg / kg.

6. The method of any one of claims 1 to 5, wherein the cancer is glioblastoma, or breast cancer (e.g., TNBC), ovarian cancer.

7. The method of any one of claims 1 to 6, wherein the immunotherapy is a checkpoint molecule inhibitor therapy.

8. The method of claim 7, wherein the checkpoint molecule is selected from PD1, CTLA-4 and PD-L1.

9. The method of any one of claims 1 to 8, wherein the composition further comprises a chemotherapy drug.

10. The method of claim 9, wherein the chemotherapy drug is paclitaxel and / or temozolomide.

11. The method of any one of claims 1 to 10, wherein the gene profile of cells from the cancer express one or more of the genes selected from FOXQ1, NNMT, RelA, and NFE2L2.JAWS Ref: 751495PCT12. The method of any one of claims 1 to 11, wherein the subject exhibits reduced leukocyte infiltration into the lungs as compared to the level prior to administration.

13. A method of screening for candidate molecules suitable for treating cancer, the method comprising determining whether the candidate molecule inhibits a PI3K function or activity and an mTOR function or activity, and upon determining that both a PI3K function or activity and an mTOR function or activity are inhibited, determining that the candidate molecule is suitable for treating cancer.

14. The method of claim 13, further comprising the step ofexposing a sample obtained from a cancer or tumour to the candidate molecule; and determining the level of nuclear p85p: H3K27Me2in the sample; wherein upon the level of nuclear p85p: H3K27Me2in the sample being below a predetermined threshold, determining that the candidate molecule is suitable for treating cancer.

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