Compositions and methods for improved cancer treatment

Abstract

This disclosure relates generally to compositions and methods for treating cancers. More particularly, the disclosure 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. Said methods comprise administering to a subject a composition comprising a PI3K inhibitor, such as paxalisib (GDC-0084), and an immunotherapy that does not target cancer stem cells (CSC), wherein said immunotherapy is either an immune checkpoint antagonist, or a PARP inhibitor.

Description

JAWS Ref: 750406PCT TITLE OF THE INVENTION “COMPOSITIONS AND METHODS FOR IMPROVED CANCER TREATMENT” 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: 750406PCT

[0005] The main PI3K isoform in cancer is the Class I PI3-kinase, p110 α (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 altering epithelial to mesenchymal cell transition or mesenchymal to epithelial cell transition of a PI3K overexpressing cell, comprising contacting the PI3K overexpressing cell with a composition comprising a PI3K inhibitor and an immunotherapy that does not target cancer stem cells (CSC).

[0009] In another aspect, the present invention provides method of treating or preventing a cancer in a subject, wherein the cancer comprises at least one PI3K overexpressing cell, the method comprising administering to the subject a composition comprising a PI3K inhibitor and an immunotherapy that does not target cancer stem cells (CSC). Prior to administering the composition to the subject, the subject may be screened for expression of one or more of the biomarkers CSV, EGRF, ABCB5, SoX9, SNAIL, AKT1, EpCAM, MDA5, TIM3, TIGIT, PD1, TOX1, EOMES, and E-cadherin.

[0010] In some embodiments, the PI3K overexpressing cell is a CSC. In some embodiments, the PI3K overexpressing cell is a CSC tumour cell.JAWS Ref: 750406PCT

[0011] In some embodiments, the PI3K overexpressing cell expresses one or more mesenchymal / cancer stem cell markers selected from the group comprising CSV, EGRF, and ABCB5, SoX9, SNAIL, and AKT1.

[0012] The epithelial cell is suitably characterised by the expression of one or both of EpCAM and MDA5. Furthermore, in some embodiments the subject comprises a CD8+ T cell population that expresses one or more of TIM3, TIGIT, PD1, TOX1, and EOMES. Typically, the epithelial cell expresses an epithelial cell signature that comprises E-cadherin.

[0013] In some embodiments, the CSC-targeting immunotherapy is an immune checkpoint molecule (ICM) antagonist. For example, the ICM antagonist may be selected from a PD1 antagonist, a PD-L1 antagonist, a CTLA4 antagonist, or a PD-L2 antagonist. In some embodiments, the ICM antagonist is an antigen-binding molecule (e.g., an antibody).

[0014] In some alternative embodiments, the immunotherapy is a PARP inhibitor.

[0015] For example, the PARP inhibitor may suitably be selected from the group comprising olaparib, talazoparib, veliparib, niraparib, and rucaparib.

[0016] Typically, the PI3K inhibitor is a catalytic PI3K inhibitor. By way of an illustrative example, the PI3K inhibitor may be selected from the group comprising paxalisib (GDC-0084), idelalisib, LY294002, 3-Methyladenine, alpelisib, quercetin, wortmannin, GNE-490, PI3K-IN-36, 740 Y-P, AZD-7648, buparlisib, inavolisib, dactolisib, copanlisib, eganelisib, pictilisib, SAR405, duvelisib, taselisib, recilisib, YM-201636, omipalisib, PI-103, alpha-linolenic acid, fimepinostat, and isorhamnetin.

[0017] In some preferred embodiments, the PI3K inhibitor is paxalisib (GDC- 0084).

[0018] In another aspect of the invention, it is provided the use of a PI3K inhibitor and an immunotherapy that does not target cancer stem cells (CSC) 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 a recurrence of cancer.

[0019] In yet another aspect, the present invention provides the use of a PI3K inhibitor and an immunotherapy that does not target cancer stem cells (CSC) in the manufacture of a medicament 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 a recurrence of cancer.

[0020] In some embodiments, the PI3K inhibitor and the immunotherapy are formulated for concurrent administration.

[0021] In still yet another aspect, the present invention provides the use of a PI3K inhibitor, an immunotherapy that does not target cancer stem cells (CSC) and an ancillary agentJAWS Ref: 750406PCT (e.g., a chemotherapeutic agent) for treating, or for aiding in the treatment of, 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 a recurrence of cancer.

[0022] In another aspect, the invention provides the use of a PI3K inhibitor, an immunotherapy that does not target cancer stem cells (CSC) and an ancillary agent (e.g., a chemotherapeutic agent) in the manufacture of a medicament for treating, or for aiding in the treatment of, 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 a recurrence of cancer.

[0023] In some preferred embodiments, the PI3K inhibitor, immunotherapy, and ancillary agent (e.g., a chemotherapeutic agent) are formulated for concurrent administration.

[0024] Suitably, the uses may further comprise a step of detecting an elevated level of one or more of TIM3, TIGIT, PD1, TOX1, and EOMES in a T cell (e.g., relative to the level of TIM3, TIGIT, PD1, TOX1, and EOMES in an activated T-cell) in a sample obtained from the subject, prior to the concurrent administration.

[0025] In yet another embodiment, the present invention provides a kit comprising a medicament comprising a PI3K inhibitor and an optional pharmaceutically acceptable carrier, and a package insert comprising instructional material for concurrent administration of the medicament with another medicament comprising an immunotherapy that does not target cancer stem cells (CSC) and an optional pharmaceutically acceptable carrier 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 a recurring cancer in an individual.

[0026] In still yet another embodiment, the invention provides a kit comprising a medicament comprising an immunotherapy that does not target cancer stem cells (CSC) and an optional pharmaceutically acceptable carrier, and a package insert comprising instructional material for concurrent administration of the medicament with another medicament comprising a PI3K inhibitor and an optional pharmaceutically acceptable carrier 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 a recurrence of cancer in an individual.

[0027] In another aspect of the present invention, it is provided a kit comprising a first medicament comprising a PI3K inhibitor and an optional pharmaceutically acceptable carrier, and a second medicament comprising an immunotherapy that does not target cancer stem cells (CSC) and an optional pharmaceutically acceptable carrier 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,JAWS Ref: 750406PCT or for treating a recurrence of cancer in an individual. Suitably, the kit further comprises a package insert comprising instructional material for administering concurrently the first medicament and the second medicament 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 a recurrence of cancer in an individual.

[0028] In another aspect, the present invention provides a pharmaceutical composition for treating cancer in a subject, the composition comprising a unit dose of a PI3K inhibitor, wherein the unit dose of the PI3K inhibitor is less than 75% of the therapeutic dose when administered alone.

[0029] In some embodiments, the PI3K inhibitor is GDC-0084, and the unit dose corresponds to an administration to the subject of about 11.25 mg / kg or less. Suitably, the PI3K inhibitor maybe GDC-0084, and the unit dose corresponds to an administration to the subject of about 7.5 mg / kg or less. Also contemplated is the PI3K inhibitor being GDC-0084, and the unit does corresponding to an administration to the subject of about 4 mg / kg or less.

[0030] In some embodiments, the composition further comprises an immunotherapy that does not target cancer stem cells (CSC).

[0031] In some embodiments, the immunotherapy is selected from an ICM antagonist (e.g., a PD1 antagonist, a PDL1 antagonist, a CTLA4 antagonist, etc.) or a PARP inhibitor. BRIEF DESCRIPTION OF THE FIGURES

[0032] 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.

[0033] Figure 1. Provides KM Survival plots for PI3K protein expression in Stage IV metastatic cancer survival.

[0034] Figure 2. Liquid biopsy from cancer patients from IO-resistant or IO- responsive cohorts was processed to isolate PBMCs from liquid biopsy samples using our optimized lab protocol. Samples were then stained for CSV, PI3KCA, ABCB5.

[0035] Figure 3. CSV, ABCB5, EpCAM, PI3KCA, AKT1 (AKT1 plays a role in promoting invasion and metastasis) fluorescent intensity (FI) and population dynamics (% population of cells) were determined in in CTCs from liquid biopsies from a stage IV metastatic solid tumour patients using ASI digital pathology, CTCs were treated with GDC-0084 or vehicle control. (A) Depicts CSV, ABCB5 two mesenchymal markers and PI3KCA / AKT1 (B) Percentage population of CSV+ABCB5+ or EpCAM+ cells or % population with PI3KCA / AKT1.JAWS Ref: 750406PCT

[0036] Figure 4. Liquid biopsy from stage IV metastatic solid tumour cancer patients was processed to isolate PBMCs from liquid biopsy samples using our optimized lab protocol. Samples were then either stimulated (ST- stimulation with PMA / CaI for 4h in the presence of brefeldin A) or not stimulated (NS) and / or treated with GDC-0084 inhibitor. Sample was then stained for three separate panels of markers. Exhaustion Signature Panel: EOMES; PD1; and CD8. Checkpoint Panel: TIGIT; TIM3; and CD8. Effector Signature Panel: Perforin; GZNb; and CD8.

[0037] Figure 5. (A) MCF7 (epithelial breast cancer), MDAMB231 (mesenchymal / CSC breast cancer), MDAMB231-Br (brain cancer version of MDA-MB-231), and 4T1 (highly aggressive, IO-resistant mouse breast cancer) cell lines; and (B) H1299 (epithelial- like lung cancer), CT26 (epithelial-like, highly immunogenic mouse colon tumour), and (LLC) highly treatment-resistant, Lewis lung cancer) cell lines, were stained for the PI3KCA signature. Samples were permeabilised with Triton X-100 and stained with primary mouse antibodies for CSV, primary rabbit antibodies for PI3KCA, detection was with donkey AF anti-mouse 568, anti- rabbit 647.

[0038] Figure 6 shows the effect of PI3K inhibitor GDC-0084 on MDA-MB-231 and CT26 cell migration. Wound healing (scratch assay) analysis of the impact of GDC-0084 on cell migration, where images represent the different cell lines treated with DMSO control, GDC-0084 at IC25 conc.1.25 ^M, and GDC-0084 at IC50 conc.2.5 ^M. (C) Graphical representation of the relative wound density of MDA-MB-231 treated with PI3K inhibitor. Black bars: DMSO control; light grey bars: IC25 conc.1.25 ^M; dark grey bars: IC50 conc.2.5 ^M. Error bars corresponds to mean ± SD from eight replicates. P-values were calculated using two-way ANOVA analysis using Tukey’s multiple comparisons test wherein ns represents not significant, ns = p > 0.05; * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001; and **** represents P < 0.0001.

[0039] Figure 7 demonstrates the IC50 of GDC-0084 for different cancer cell lines. WST-1 Cell Proliferation Reagent was added to CT26, 4T1 and MCF7 cells that were pre- treated for 72 hours with PI3K inhibitor in a dose-response design. Cell proliferation was measured indirectly by the formation of formazan and the absorbance recorded at 450 nm. Percent (%) proliferation was determined before the EC50 was calculated using Graphpad Prism software as described in the methods.

[0040] Figure 8 shows that treatment with olaparib upregulates some resistance, mesenchymal markers. MDA-MB-231 cells were treated with either vehicle (“CTRL”) or olaparib (“Ola.”). Expression of cancer stem cell like markers of resistance (ABCB5, ALDH1A) were analysed by immunofluorescence staining quantification. Images show IF staining of (A) ABCB5 and (B) ALDH1A1 and show upregulation of the expression of ABCB5, ALDH1A1. *** P ≤ 0.001. This figure demonstrates that PARP inhibition can upregulate markers of a cancer stem cell resistance signature.JAWS Ref: 750406PCT

[0041] Figure 9 shows that treatment with olaparib failed to inhibit CSCs or cellular proliferation: MDA-MB-231 cells were treated with olaparib for 24 hours. Cells were harvested and subjected to flow cytometry after staining with APC / CD44 and PE / CD24. (A) shows percentage inhibition of CSCs compared to control, followed by representative flow cytometry images. (B) MDA-MB-231 cells were treated with either olaparib or talazoparib at three different concentrations, and cellular proliferation was estimated with the WST-1 assay.

[0042] Figure 10 provides graphical and photographical representations of a PARP knockdown. PARP1 knock down was conducted with PARP1 siRNA at two different concentrations, 5 nM and 10 nM, with 5 nM previously established as an optimal concentration in our laboratory in other cell lines. After 48 hours of incubation with siRNA, cells were snap frozen at -80°C. Following RNA extraction and cDNA synthesis, TaqMan qRT-PCR was performed to quantify mRNA expression. Both concentrations significantly depleted PARP1 mRNA expression (A). Figures (B), and (C) show nuclear fluorescence intensity of PARP. The graph represents the means ± standard errors from three independent experiments. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P < 0.0001, two-way ANOVA.

[0043] Figure 11 shows that PARP1 knockdown increases resistance marker expression. MDA-MB-231 cells were incubated with 5 nM PARP1 siRNA for 48 hours followed by either RNA extraction and cDNA synthesis, which were then amplified by qPCR, or fixation of cells for immunofluorescence staining. (A, B) mRNA expression, (C, D) immunofluorescence intensity of mesenchymal markers after PARP1 knockdown, (E) PD-L1. The relative fold changes in mRNA expression or changes in total nuclear fluorescence intensity (TNFI) or total cytoplasmic fluorescence intensity (TCFI) is shown. * P ≤0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P < 0.0001, two-way ANOVA. Wound healing (scratch assay) analysis of the impact of GDC-0084 in combination with olaparib on cell migration. Images represent MDA-MB-231 cell line treated with (F) DMSO control, (G) GDC-0084 at IC25 conc.1.25 µM and olaparib at 5 nM. (H) GDC- 0084 at IC50 conc.2.5 µM and olaparib at 250 nM. Error bars corresponds to mean ± SD from eight replicates (I). P-values were calculated using two-way ANOVA analysis using Tukey’s multiple comparisons test wherein ns represents not significant, ns = p > 0.05; * represents P < 0.05; ** represents P < 0.01; ***represents P < 0.001; and **** represents P < 0.0001.

[0044] Figure 12 shows αPD1 therapy in the CT26 model of colon cancer and 4T1 breast cancer model. Treatment regime using the Balb / c CT26 (A) colon cancer model and 4T1 (B) breast cancer model has been demonstrated. Tumour volumes of mice treated with vehicle (IgG) or αPD1(10 mg / kg) (n=5 / group). * p<0.05, ** p<0.01, Unpaired t-test.

[0045] Figure 13: PI3K inhibition targets and inhibits the expression of mesenchymal resistant markers.4T1 (A) or CT26 (B) cancer cell lines were treated with control or GDC-0084 (at two low concentrations of 0.2 or 0.3 uM). Samples were then permeablized with Triton-X 100 and stained with primary mouse antibodies for CSV or EpCAM, primary rabbit antibodies for EGFR or FOXN2 or primary goat antibodies for ABCB5, detectionJAWS Ref: 750406PCT was with donkey AF anti-mouse 488, anti-rabbit 568 or anti-goat 647 secondary antibodies. The ASI Digital pathology system at 100x objective was used to image protein targets. Example image fields with scale bar (in orange) are depicted above). Analysis was carried out comparing the florescent intensity for EGFR, cytoplasmic florescent intensity (CFI) for CSV or overall florescent intensity for ABCB5. Data was plotted with PRISM and analysis carried out with one- way ANOVA non-parametric test with Kruskal-Wallis. Significant differences are plotted.

[0046] Figure 14 PI3K inhibiton induces marker of immune visibility.4T1 (A) or CT26 (B) cancer cell lines were treated with control or GDC-0084 (at concentration of 0.2 or 0.3 mM). Samples were then permeablized with Triton-X 100 and stained with primary mouse antibodies for CSV or EpCAM, primary rabbit antibodies for MDA5, detection was with donkey AF anti-mouse 488, anti-rabbit 568 or anti-goat 647 secondary antibodies. The ASI Digital pathology system at 100x objective was used to image protein targets. Example image fields with scale bar (in orange) are depicted above). Analysis was carried out comparing the florescent intensity. Data was plotted with PRISM and analysis carried out with one-way ANOVA non-parametric test with Kruskal-Wallis. Significant differences are plotted.

[0047] Figure 15 PI3K inhibition targets and inhibits the Stem-Like Cancer Stem Cell cancer recurrence signature and induces an epithelial signature. MDA-MB-231 TNBC cancer cell line were treated with control or two different PI3K inhibitors, Idelalisib (at two low concentrations of 12.5 µM or 25 µM) or LY294002 (at two low concentrations of 2.5 µM or 5 µM). Samples were then permeablized with Triton-X 100 and stained with primary mouse antibodies for CSV or EpCAM, primary rabbit antibodies for SoX9 (SoX9 is a marker for mesenchymal cancer stem cells and progressive, therapeutic resistance cancer cells) or PI3K or primary goat antibodies for ABCB5 or SNAIL (11A and 11B), detection was with donkey AF anti-mouse 488, anti-rabbit 568 or anti-goat 647 secondary antibodies. The ASI Digital pathology system at 40x objective was used to image protein targets. Example image fields with scale bar (in orange) are depicted above. Analysis was carried out comparing the nuclear florescent intensity for SoX9, cytoplasmic florescent intensity (CFI) for CSV and EpCAM or overall florescent intensity for ABCB5 or SNAIL. Data was plotted with PRISM and analysis carried out with one-way ANOVA non-parametric test with Kruskal-Wallis. Significant differences are plotted.

[0048] Figure 16. GDC-0084 administration at 7.5 mg / kg reduces clinical abnormalities in the 4T1 syngeneic tumour model. GDC-0084 therapy in the 4T1 breast cancer model. Treatment regime using the Balb / c 4T1 breast cancer model. GDC-0084 was administered at 7.5 mg / kg (A) or 15 mg / kg daily (B). (C) % of mice displaying clinical abnormalities (reduced activity, hunched posture, piloerection, weight loss and metastasis) following GDC-0084 monotherapy at endpoint.

[0049] Figure 17. GDC-0084 administration at 7.5 mg / kg abrogates tumour burden. Representative images of tumours harvested from mice administered with (A) 7.5JAWS Ref: 750406PCT mg / kg GDC-0084 (C) 15 mg / kg + / - αPD1 in the Balb / c 4T1 breast cancer model. (B, D) Primary tumour volumes and final tumour weights of mice treated with (B) 7.5 mg / kg GDC-0084 (D) 15 mg / kg once daily + / - αPD1 (10 mg / kg). Data presented as mean ± SEM. One-way ANOVA with Tukey post hoc analysis, ***p < 0.001, **** p<0.0001 (n=5 / group).

[0050] Figure 18. GDC-0084 administration at 7.5 mg / kg reduces tumour inflammation including monocyte and neutrophil infiltration. Representative images of H&E stained tumours and pathology scores for tumour inflammation in mice administered with (A) 7.5 mg / kg GDC-0084 or (B) 15 mg / kg once daily + / - αPD1 in the Balb / c 4T1 breast cancer model. Data presented as mean ± SEM. One-way ANOVA with Tukey post hoc analysis, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant (n = 5 / group).

[0051] Figure 19. GDC-0084 administration at 7.5 mg / kg reduces splenomegaly in the 4T1 syngeneic tumour model. Representative images of spleens harvested from mice administered with (A) 7.5 mg / kg GDC-0084 or (B) 15 mg / kg once daily + / - αPD1 in the Balb / c 4T1 breast cancer model. Spleen weights are presented as mean ± SEM. One-way ANOVA with Tukey post hoc analysis, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p<0.0001, ns = not significant (n = 5 / group).

[0052] Figure 20. GDC-0084 administration at 7.5 mg / kg reduces SOX9 and TOX1 expression. Depicts bar-graphs using Qupath analysis of captured images for: A) CSC signature (SOX9, PDL1, panCK), n > 500,000 cells analyzed. B) CD8 Dysfunctional signature (CD8, TOX1, PD1), n > 10,000 cells analyzed. C) CSC signature (SOX9, PDL1, panCK) for C, n > 100,000 cells analyzed. D) CD8 Dysfunctional signature (CD8, TOX1, PD1), n > 10,000 cells analyzed. Qupath analysis was used to select cells positive for DAPI, then for the CSC signature cells positive for PDL1 and panCK were scored based on SOX9 fluorescent intensity. For the CD8 dysfunctional signature, cells were selected for been DAPI positive, then CD8 and PD1+ and were scored based on TOX1 fluorescent intensity. Images were taken from an Aperio FL Fluorescence slide Scanner of stained FFPE tumour sections from GDC Mouse model treated with either control, anti-PD1 immunotherapy, GDC-0084 (high dose in C / D and low dose in A / B) or combination. FFPE sections of lung tissue were processed as described in previously and stained for SOX9, PDL1, panCK or CD8, TOX1, PD1; DAPI was used to stain nuclei. Data are plotted as means ± SD and represent the fluorescent intensity of SOX9 or TOX1. Tukey’s post hoc test, ns = non-significant, *** p < 0.001, **** p < 0.0001.

[0053] Figure 21. PI3K mono or combination inhibition targets mesenchymal signature and induces epithelial phenotype in IO resistant mouse model. FFPE primary tumour samples which were processed on the BONDRX with the Opal staining kit targeting PI3KCA, CSV, E-Cadherin or EGFR. (A)-(D) Quantification of PI3KCA, CSV, E-Cadherin or EGFR fluorescence intensity (FI) was determined using ASI Digital Pathology.JAWS Ref: 750406PCT

[0054] Figure 22. PI3K mono or combination induces epithelial phenotype in IO resistant mouse model. FFPE primary tumour samples which were processed on the BONDRX with the Opal staining kit targeting PI3KCA, CSV, E-Cadherin or EGFR. % Population dynamics was determined using the ASI Digital pathology platform for the % positive cells for E-Cadherin expression.

[0055] Figure 23. PI3K mono or combination therapy induces a effector and TRM signature. Quantification of CD8+IFNγ+ T cells positive cells (A) or CD8+ T cells expression markers for Tissue Resident Memory cells (CD103, CD69, CD44 and CD8) (B) was also carried via staining FFPE tumour samples with Opal staining Kit and analyzing images with ASI Digital Pathology. Significant differences are indicated calculated by Kruskal-Wallis non- parametric test.

[0056] Figure 24. PI3K mono or combination inhibits checkpoint exhaustion signature in CD8+ T cells. FFPE primary tumour samples which were processed with tyramide staining kit targeting CD8, LAG3 and TIM3 checkpoint markers. % Population dynamics was determined using the ASI Digital pathology platform for the % positive cells for each marker. % expression of TIM3 and LAG3 positive CD8+ T cells out of total CD8+ T cells was calculated. Graph represents n = 3 mice per group, with Kruskal-Wallis test used to calculate significant differences. ns = no significance, * p = <0.02.

[0057] Figure 25. Combination with PI3K inhibition and immunotherapy abrogates metastatic spread in 4T1 IO resistant model. GDC-0084 + / - αPD1 therapy in the 4T1 model of metastatic breast cancer. (A) Treatment regime using the Balb / c 4T1 breast cancer model. (B) Metastatic tumour nodules present in the lungs of individual mice at day 20 post inoculation, ** p < 0.01, Tukey’s post test.

[0058] Figure 26. Optimal therapeutic dose of GDC-0084 established in the 4T1 TNBC model (A) Treatment regime using the Balb / c 4T1 TNBC breast cancer model. (B) Pipeline for dose de-escalation experiments. In stage I, GDC-0084 was administered daily at a dose of 15 mg / kg + / - αPD1 (10 mg / kg) at day 0 and day 4. In stage II, a split daily dose of GDC-0084 (up to 15 mg / kg) was administered four hours apart in combination αPD1 at day 0 and day 4. In the final stage, GDC-0084 was administered as a single daily dose (up to 7.5 mg / kg) in combination with αPD1 at day 0 and day 4. (C) Tumour volumes (% vehicle) of individual mice from each experimental stage prior to harvest, * p < 0.05, ** p < 0.01, *** p < 0.001, ns not significant, unpaired t test or Dunnett’s post-test, n = 4-5 / group.

[0059] Figure 27. Lowering the dose of GDC-0084 improves safety in mice. (A) Mouse body weights were monitored throughout the duration of the starting dose, split-dose de-escalation and single-dose de-escalation experiments. #, indicates mouse fatalities. (B) Liver weights were measured at the time of harvest. * p < 0.05, unpaired t test, n = 4-5 / group.

[0060] Figure 28. No liver toxicity observed using the optimal therapeutic dose of GDC-0084. H&E stained FFPE livers from split-dose de-escalation and single-dose de-JAWS Ref: 750406PCT escalation experiments were scored by an expert pathologist. Changes in (A) liver inflammation, (B) extramedullary hematopoiesis (EMH), and (C) hepatocyte changes (metabolic & / or degeneration) were given a score of 1 = mild, 2 = moderate, or 3 = severe for each parameter. * p < 0.05, *** p < 0.001, ****p < 0.0001, versus vehicle, Dunnett’s post-test, n = 4-5 / group.

[0061] Figure 29. PI3K-mTOR inhibition reduces splenomegaly and extramedullary hematopoiesis in the spleen. (A) Spleen weights were measured at the time of harvest from split-dose de-escalation and single-dose de-escalation experiments. * p < 0.05, ** p < 0.01, **** p < 0.0001, versus vehicle, Dunnett’s post-test, n = 4-5 / group. (B) H&E stained FFPE spleens were scored by a pathologist. Changes in extramedullary hematopoiesis (EMH) were given a score of 1 = mild, 2 = moderate, or 3 = severe. * p < 0.01, *** p < 0.001, versus vehicle, Dunnett’s post-test, n = 4-5 / group.

[0062] Figure 30. PI3K-mTOR inhibition diminishes leukocyte infiltration into the lungs. H&E stained FFPE lungs from split-dose de-escalation and single-dose de- escalation experiments were scored by an expert pathologist. Changes in leukocytosis were given a score of 1 = mild, 2 = moderate or 3 = severe, * p < 0.01, *** p < 0.001, versus vehicle, Dunnett’s post-test, n = 4-5 / group.

[0063] Figure 31. GDC-0084 and αPD1 combination treatment prevents lymph node metastases. (A) Images from H&E stained FFPE lymph nodes from αPD1 and GDC-0084 (7.5 mg / kg) and PD1 treated 4T1 mice. (B) Lymph node sections were examined by an independent expert pathologist and given a score of 1 = 1-3 tiny sites (<< 0.5 mm in width / length), 2 = 1-3 of which at least one is 0.5-1 mm in diameter or 3 = 2-3 or more sites, all grossly visible, with leukocyte infiltration and / or haemorrhage.

[0064] Figure 32. GDC-0084 efficacy demonstrated in combination with PARP inhibitor. (A) Pipeline for PARP inhibitor experiments. In stage I, GDC-0084 was administered daily at a dose of 7.5 mg / kg either before (pre) or 30 minutes after olaparib (50 mg / kg) (post). In stage II, GDC-0084 was administered daily at a dose of 7.5 mg / kg, 30 min after olaparib (50 mg / kg). (B) Treatment regime using the Balb / c 4T1 TNBC breast cancer model. (C) Tumour volumes (% vehicle or olaparib) of individual mice from each experimental stage prior to harvest. ** p < 0.01, ns not significant, Tukey’s post-test, n = 5 / group.

[0065] Figure 33. GDC-0084 and olaparib combination therapy does not induce toxicity. Mouse body weights and liver weights were assessed following treatment with GDC-0084 (7.5 mg / kg) post olaparib treatment. ns, not significant, Tukey’s post-test, n = 5 / group.

[0066] Figure 34. Reduced liver and lung inflammation following GDC-0084 and olaparib combination therapy. H&E stained FFPE livers and lungs from GDC-0084 post olaparib experiments were scored by an independent expert pathologist. (A) Changes in liver inflammation and extramedullary hematopoiesis (EMH) and (B) lung leukocytosis were given aJAWS Ref: 750406PCT score of 1 = mild, 2 = moderate, or 3 = severe for each parameter. * p < 0.05, ** p < 0.001, **** p < 0.0001, versus vehicle, Dunnett’s post-test, n = 5 / group.

[0067] Figure 35. GDC-0084 and olaparib combination reduces splenomegaly and extramedullary hematopoiesis in the spleen. (A) H&E stained FFPE spleens from GDC-0084 post olaparib experiments were scored by an expert pathologist. Changes in extramedullary hematopoiesis (EMH) were given a score of 1 = mild, 2 = moderate or 3 = severe. (B) Spleen weights were measured at the time of harvest * p < 0.05, *** p < 0.001, **** p < 0.0001, versus vehicle, Dunnett’s post-test, n = 5 / group. * p < 0.01, *** p < 0.001, versus vehicle, Dunnett’s post-test, n = 5 / group.

[0068] Figure 36. Comparison of PI3K-mTOR inhibitor combination treatment on cancer cell proliferation. WST Proliferation Reagent was added to MDA-MB-231 cells that were pre-treated for 72 hours with PI3K inhibitors in a dose-response design. Cell proliferation (%) was measured indirectly by the formation of formazan and the absorbance recorded at 450 nm.

[0069] Figure 37. GDC-0084 dose-response curve for wound healing (scratch assay). Pre-stimulated (PMA / TGFβ) MCF-7 cells were scratched prior to treatment with GDC-0084 (0.078-2.5 ^M) for 24 hours. Changes in wound densities were monitored over a 24 hour period. (A) Representative images from the scratch assay comparing Vehicle and GDC-0084 (1.25 ^M and 2.5 ^M) treated MCF-7 cells. (B) Dose-response curves comparing the wound densities (%) of GDC-0084 treated cells (0.078 - 2.5 ^M) over the 24 hour treatment period.

[0070] Figure 38. Comparison of PI3K-mTOR inhibitor treatment on cancer cell migration. Pre-stimulated (PMA / TGFβ) MCF-7 cells were scratched prior to treatment with PI3K-mTOR inhibitors at the indicated doses for 24 hours. (A) Dose-response curves comparing the wound densities (%) of PI3K-mTOR inhibitor treated cells (0.078-2.5 ^M) over the 24 hour treatment period. (B) Comparison of the relative wound densities observed for each PI3K- mTOR inhibitor at 24 hours. **** p < 0.0001, versus vehicle, Dunnett’s post-test, n = 5 / group. * p < 0.01, *** p < 0.001, versus vehicle, Dunnett’s post-test, n = 6 / group. DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

[0071] 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.JAWS Ref: 750406PCT

[0072] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0073] 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.

[0074] 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.

[0075] 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).

[0076] 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 listed elements 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.

[0077] “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®JAWS Ref: 750406PCT 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; 5α-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 γ1I and calicheamicin ω1I (Angew Chem. Intl. 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, ADRIAMYClN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), 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;JAWS Ref: 750406PCT 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, Ill.), 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.

[0078] 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, medroxyprogesterone 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-α, 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®, rIL-2; a topoisomerase 1 inhibitor such as LURTOTECAN®; ABARELIX® rmRH; and (ix) pharmaceutically acceptable salts, acids and derivatives of any of the above.

[0079] Chemotherapeutic agent also includes antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech / Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

[0080] 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,JAWS Ref: 750406PCT 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, pecfusituzumab, 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 IgG1 lamda. antibody genetically modified to recognize interleukin-12 p40 protein.

[0081] 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- 11F8, 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 as described 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-α 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-JAWS Ref: 750406PCT 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]-1H-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).

[0082] 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 (Cl-1033; Pharmacia); Raf-1 inhibitors such as antisense agent ISIS-5132 available from ISIS

[0083] Pharmaceuticals which inhibit Raf-1 signaling; non-HER targeted TK inhibitors such as imatinib mesylate (GLEEVEC®, available from Glaxo SmithKline); multi- targeted 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); Cl-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (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).JAWS Ref: 750406PCT

[0084] 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.

[0085] 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, sulfasalazine, 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-α (IFN-α) 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 / β2 blockers such as Anti-lymphotoxin α (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®);JAWS Ref: 750406PCT 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); CCl-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.

[0086] 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 bone pain, headache and migraine, postoperative pain, mild-to-moderate pain due to inflammation and tissue injury, pyrexia, ileus, and renal colic.

[0087] 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.

[0088] 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).JAWS Ref: 750406PCT

[0089] 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 β-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).

[0090] 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 of 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 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.

[0091] “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.

[0092] 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.JAWS Ref: 750406PCT

[0093] 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 cell, in comparison to a normal cell. 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).

[0094] 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.

[0095] As used herein, the term “PI3K overexpressing cell” refers to a vertebrate cell, particularly a mammalian cell, that expresses PI3K at a detectably greater level than a normal cell. The cell may be a vertebrate cell, such as a primate cell; an avian cell; a livestock animal cell (such as a sheep cell, cow cell, horse cell, deer cell, donkey cell and pig cell); a laboratory 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 cell, deer cell and dingo cell). In particular embodiments, the PI3K overexpressing cell is a human cell. In specific embodiments, the PI3K overexpressing cell 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 cell or comparison cell (e.g. a breast cell).

[0096] 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.JAWS Ref: 750406PCT

[0097] 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 animals include 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.

[0098] 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.

[0099] 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” refer to 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 cancer that 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,JAWS Ref: 750406PCT 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, mesothelioma, rectal cancer and esophageal cancer. In an exemplary embodiment, the cancer is breast cancer.

[0100] Each embodiment described herein is to be applied mutatis mutandis to each and every embodiment unless specifically stated otherwise. 2. Compositions

[0101] The present invention is based in part of the determination that exposure of functionally repressed T-cells of a mesenchymal phenotype to PI3K inhibitors results in epigenetic reprogramming of the T-cells with de-repression of their immune effector function, including elevated expression of biomarkers of T-cell activation and effector capacity (e.g. , IL-2, IFN-γ, and TNF), decreased expression of biomarkers of T-cell effector inhibition and cancer progression (e.g., ZEB1), as well as decreased expression of biomarkers of T-cell exhaustion (e.g., PD-1 and EOMES) and elevated expression of the transcription factor TBET, which increases production of IFN-γ in cells of the adaptive and innate immune systems. The present inventor has also found that PI3K inhibitor-mediated phosphorylation reprogramming confers enhanced susceptibility of exhausted T-cells to reinvigoration by ICM-binding antagonists.

[0102] Thus, in accordance with the present invention, compositions and methods are provided that take advantage of a PI3K inhibitor (e.g., an inhibitor of PI3K activity) and an ICM-binding antagonist to enhance immune effector function, and / or to enhance T-cell (e.g., CD8+ T-cell) function, including increasing T-cell activation and enhancing susceptibility of exhausted T-cells to reinvigoration by ICM-binding antagonists. The methods and compositions of the present invention are thus particularly useful in the treatment of T-cell dysfunctional disorders including cancers and infections.

[0103] The present invention is based, at least in part, on the identification of compositions that inhibit EMT and induce MET of CSC tumour cells. Thus, the inventors conceived that the compositions of the invention may be used for the treatment or prevention of cancer, and / or improving the responsiveness of a cancer or tumour to an immunotherapy that does not target CSC.

[0104] Accordingly, in one aspect of the present invention, there is provided a composition that comprises an inhibitor of PI3K and an immunotherapy that does not target CSC. 2.1 PI3K inhibitors.

[0105] The compositions of the invention comprise a inhibitor, which includes and encompasses any active agent that reduces the accumulation, function (e.g., enzymatic activity,JAWS Ref: 750406PCT 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.

[0106] In some embodiments, the PI3K inhibitor is an antagonistic nucleic acid molecule that functions to inhibit the transcription or translation of PI3K transcripts.

[0107] Representative transcripts of this type 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. P42336, Q8NEB9, O00750, O75747, 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).

[0108] Illustrative antagonist nucleic acid molecules include antisense molecules, aptamers, ribozymes and triplex forming molecules, RNAi and external guide sequences. The nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.

[0109] 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 mRNA or the genomic DNA of a PIK3 gene (e.g., PIK3CA) or they can interact with a PI3K 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.

[0110] In some embodiments, anti-sense RNA or DNA molecules are used to directly block the translation of PI3K by binding to targeted mRNA and preventing proteinJAWS Ref: 750406PCT translation. Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule may be designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively, the antisense molecule may be designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule exist. Non- limiting methods include in vitro selection experiments and DNA modification studies using DMS and DEPC. In specific examples, the antisense molecules bind the target molecule with a dissociation constant (Kd) less than or equal to 10-6, 10-8, 10-10, or 10-12. In specific embodiments, antisense oligodeoxyribonucleotides derived from the translation initiation site, e.g., between -10 and +10 regions are employed.

[0111] Aptamers are molecules that interact with a target molecule, suitably in a specific way. Aptamers are generally small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind small molecules, such as ATP and theophylline, as well as large molecules, such as reverse transcriptase and thrombin. Aptamers can bind very tightly with Kds from the target molecule of less than 10-12M. Suitably, the aptamers bind the target molecule with a Kdless than 10-6, 10-8, 10-10, or 10-12. Aptamers can bind the target molecule with a very high degree of specificity. For example, aptamers have been isolated that have greater than a 10,000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule. It is desirable that an aptamer have a Kd with the target molecule at least 10-, 100-, 1000-, 10,000-, or 100,000-fold lower than the Kdwith a background-binding molecule. A suitable method for generating an aptamer to a target of interest (e.g., PI3K) is the “Systematic Evolution of Ligands by Exponential Enrichment” (SELEX™). The SELEX™ method is described in U.S. Pat. No.5,475,096 and U.S. Pat. No. 5,270,163 (see also International Pub. No. WO 91 / 19813). Briefly, a mixture of nucleic acids is contacted with the target molecule under conditions favorable for binding. The unbound nucleic acids are partitioned from the bound nucleic acids, and the nucleic acid-target complexes are dissociated. Then the dissociated nucleic acids are amplified to yield a ligand-enriched mixture of nucleic acids, which is subjected to repeated cycles of binding, partitioning, dissociating and amplifying as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.

[0112] In other embodiments, anti-PI3K ribozymes are used for catalyzing the specific cleavage of PI3K RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage. There are several different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions, which are based on ribozymes found in naturalJAWS Ref: 750406PCT systems, such as hammerhead ribozymes, hairpin ribozymes, and tetrahymena ribozymes. There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo. Representative ribozymes cleave RNA or DNA substrates. In some embodiments, ribozymes that cleave RNA substrates are employed. Specific ribozyme cleavage sites within potential RNA targets are initially identified by scanning the target molecule for ribozyme cleavage sites, which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.

[0100] Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependent on both Watson-Crick and Hoogsteen base pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is generally desirable that the triplex forming molecules bind the target molecule with a Kd less than 10-6, 10-8, 10-10, or 10-12.

[0101] External guide sequences (EGSs) are molecules that bind a target nucleic acid molecule forming a complex, and this complex is recognized by RNAse P, which cleaves the target molecule. EGSs can be designed to specifically target a RNA molecule of choice. RNAse P aids in processing transfer RNA (tRNA) within a cell. Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA:EGS complex to mimic the natural tRNA substrate. Similarly, eukaryotic EGS / RNAse P- directed cleavage of RNA can be utilized to cleave desired targets within eukaryotic cells.

[0102] In other embodiments, RNA molecules that mediate RNA interference (RNAi) of a PI3K gene or PI3K transcript can be used to reduce or abrogate gene expression. RNAi refers to interference with or destruction of the product of a target gene by introducing a single-stranded or usually a double-stranded RNA (dsRNA) that is homologous to the transcript of a target gene. RNAi methods, including double-stranded RNA interference (dsRNAi) or small interfering RNA(siRNA), have been extensively documented in a number of organisms, including mammalian cells and the nematode C. elegans (Fire et al., 1998. Nature 391, 806- 811). In mammalian cells, RNAi can be triggered by 21- to 23-nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et al., 2002 Mol. Cell.10:549-561; Elbashir et al., 2001. Nature 411:494-498), or by micro-RNAs (miRNA), functional small-hairpin RNA (shRNA), or other dsRNAs which are expressed in vivo using DNA templates with RNA polymerase III promoters (Zeng et al., 2002. Mol. Cell 9: 1327-1333 ; Paddison et al., 2002. Genes Dev.16:948-958; Lee et al., 2002. Nature Biotechnol.20:500-505; Paul et al., 2002. Nature Biotechnol.20:505-508;JAWS Ref: 750406PCT Tuschl, T., 2002. Nature Biotechnol.20:440-448; Yu et al., 2002. Proc. Natl. Acad. Sci. USA 99(9) :6047-6052; McManus et al., 2002. RNA 8:842-850; Sui et al., 2002. Proc. Natl. Acad. Sci. USA 99(6) :5515-5520).

[0103] In specific embodiments, dsRNA per se and especially dsRNA-producing constructs corresponding to at least a portion of a PIK3 gene are used to reduce or abrogate its expression. RNAi-mediated inhibition of gene expression may be accomplished using any of the techniques reported in the art, for instance by transfecting a nucleic acid construct encoding a stem-loop or hairpin RNA structure into the genome of the target cell, or by expressing a transfected nucleic acid construct having homology for a PIK3 gene from between convergent promoters, or as a head-to-head or tail-to-tail duplication from behind a single promoter. Any similar construct may be used so long as it produces a single RNA having the ability to fold back on itself and produce a dsRNA, or so long as it produces two separate RNA transcripts, which then anneal to form a dsRNA having homology to a target gene.

[0104] Absolute homology is not required for RNAi, with a lower threshold being described at about 85% homology for a dsRNA of about 200 base pairs (Plasterk and Ketting, 2000, Current Opinion in Genetics and Dev.10: 562-67). Therefore, depending on the length of the dsRNA, the RNAi-encoding nucleic acids can vary in the level of homology they contain toward the target gene transcript, i.e., with dsRNAs of 100 to 200 base pairs having at least about 85% homology with the target gene, and longer dsRNAs (i.e., 300 to 100 base pairs), having at least about 75% homology to the target gene. RNA-encoding constructs that express a single RNA transcript designed to anneal to a separately expressed RNA, or single constructs expressing separate transcripts from convergent promoters, are suitably at least about 100 nucleotides in length. RNA-encoding constructs that express a single RNA designed to form a dsRNA via internal folding are usually at least about 200 nucleotides in length.

[0105] The promoter used to express the dsRNA-forming construct may be any type of promoter if the resulting dsRNA is specific for a gene product in the cell lineage targeted for destruction. Alternatively, the promoter may be lineage specific in that it is only expressed in cells of a particular development lineage. This might be advantageous where some overlap in homology is observed with a gene that is expressed in a non-targeted cell lineage. The promoter may also be inducible by externally controlled factors, or by intracellular environmental factors.

[0106] In some embodiments, RNA molecules of about 21 to about 23 nucleotides, which direct cleavage of specific mRNA to which they correspond, as for example described in U.S. Patent Pub. No. US 2002 / 0086356, can be utilized for mediating RNAi. Such 21- to 23-nt RNA molecules can comprise a 3’ hydroxyl group, can be single-stranded or double stranded (as two 21-to 23-nt RNAs) wherein the dsRNA molecules can be blunt ended or comprise overhanging ends (e.g., 5’, 3’).JAWS Ref: 750406PCT

[0107] In some embodiments, the antagonist nucleic acid molecule is a siRNA. siRNAs can be prepared by any suitable method. For example, reference may be made to International Publication WO 02 / 44321, which discloses siRNAs capable of sequence-specific degradation of target mRNAs when base-paired with 3' overhanging ends, which is incorporated by reference herein. Sequence specific gene silencing can be achieved in mammalian cells using synthetic, short double-stranded RNAs that mimic the siRNAs produced by the enzyme dicer. siRNA can be chemically or in vitro-synthesized or can be the result of short double- stranded hairpin-like RNAs (shRNAs) that are processed into siRNAs inside the cell. Synthetic siRNAs are generally designed using algorithms and a conventional DNA / RNA synthesizer. Suppliers include Ambion (Austin, Tex.), ChemGenes (Ashland, Mass.), Dharmacon (Lafayette, Colo.), Glen Research (Sterling, Va.), MWB Biotech (Esbersberg, Germany), Proligo (Boulder, Colo.), and Qiagen (Vento, The Netherlands). siRNA can also be synthesized in vitro using kits such as Ambion's SILENCER™ siRNA Construction Kit.

[0108] The production of siRNA from a vector is more commonly done through the transcription of a short hairpin RNAs (shRNAs). Kits for the production of vectors comprising shRNA are available, such as, for example, Imgenex's GENESUPPRESSOR™ Construction Kits and Invitrogen's BLOCK-IT™ inducible RNAi plasmid and lentivirus vectors. In addition, methods for formulation and delivery of siRNAs to a subject are also well known in the art. See, e.g., US 2005 / 0282188; US 2005 / 0239731; US 2005 / 0234232; US 2005 / 0176018; US 2005 / 0059817; US 2005 / 0020525; US 2004 / 0192626; US 2003 / 0073640; US 2002 / 0150936; US 2002 / 0142980; and US 2002 / 0120129, each of which is incorporated herein by reference.

[0109] Illustrative RNAi molecules (e.g., PI3K siRNA and shRNA) are described in the art (e.g., Ma et al., 2013. BMC Biochem.14: 20; and Kim et al., 2013. Immune Netw. 13(2):55-62) or available commercially from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), OriGene Technologies, Inc. (Rockville, MD, USA), Sigma-Aldrich Pty Ltd (Castle Hill, NSW, Australia).

[0110] 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 include 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 member of the fusion protein may be non-cleavable,JAWS Ref: 750406PCT with a peptide bond, or cleavable with, for example, an ester or other cleavable bond known to the art.

[0111] 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.

[0112] A PI3K 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.

[0113] 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: 10881097, 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 10:641648, 1999; Ali et al., Hum Mol Genet.5:591594, 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: 750406PCT

[0114] The present invention also contemplates small molecule agents that reduce the functional activity of PI3K (e.g., reduce PI3K-mediated phosphorylation, inhibit binding of PI3K to the promoter of CD44 or uPAR, reduce binding of PI3K (e.g., active PI3K) to chromatin; reduce PI3K-mediated inhibition of guanine exchange factor, GIV / Girdin, reduce PI3K-mediated inhibition of regulatory T cell function, reduce PI3K-mediated EMT etc).

[0115] 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:and 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, C1-C12 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -(C1-C12 alkylene)-(C3-C12 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-C20 aryl), and -(C1-C12 alkylene)-(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, =O, -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, -JAWS Ref: 750406PCT NHCH3, -N(CH3)2, -NHCOCH3, -NHS(O)2CH3, -N(CH3)C(CH3)2CONH2, - N(CH3)CH2CH2S(O)2CH3, =O, -OH, -OCH3, -S(O)2N(CH3)2, -SCH3, -S(O)2CH3, cyclopropyl, cyclobutyl, oxetanyl, morpholino, and 1,1-dioxo-thiopyran-4-yl; R4is 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; and R5is independently selected from C1-C12 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, - (C1-C12 alkylene)-(C3-C12 carbocyclyl), -(C1-C12 alkylene)-(heterocyclyl having 3-20 ring atoms), - (C1-C12 alkylene)-C(=O)-(heterocyclyl having 3-20 ring atoms), -(C1-C12CH(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, -JAWS Ref: 750406PCT NO2, -NH2, -NHCH3, -N(CH3)2, -NHCH2CH3, -NHCH(CH3)2, -NHCH2CH2OH, - NHCH2CH2OCH3, - NHCOCH3, -NHCOCH2CH3, -NHCOCH2OH, -NHS(O)2CH3, - N(CH3)S(O)2CH3, =O, -OH, -OCH3, -OCH2CH3, -OCH(CH3)2, -SH, -NHC(=O)NHCH3, - NHC(=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.

[0116] In some other embodiments, the small molecule PI3K inhibitor is selected from the following group: Paxalisib 3-Methyladenine (GDC-0084)Idelalisib AlpelisibLY294002 WortmanninQuercetin GNE-490

[0117] The present invention allows for the administration of a lower dosage of the PI3K inhibitor therapy to a subject. The ability to utilize lower dosages of the PI3K therapy reduces the toxicity 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).

[0118] In some embodiments, the lower amount / doses of PI3K inhibitor reduces or minimizes any undesired side-effects associated with PI3K therapy.JAWS Ref: 750406PCT 2.2 Immunotherapies

[0119] 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

[0113] 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 include polypeptides, polynucleotides, carbohydrates, and small molecules. In some preferred embodiments, the ICM antagonist is an antigen-binding molecule.

[0114] 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, OX40, OX40L, CD244, CD160, GITR, GITRL, ICOS, GAL-9, 4-1BBL, 4-1BB, CD27L, CD28, CD80, CD86, SIRP-1, CD47, CD48, CD244, CD40, CD40L, HVEM, TMIGD2, HHLA2, VEGI, TNFRS25 and ICOLG.

[0115] 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].

[0116] 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. WO2004 / 072286, WO2006 / 121168, WO2006 / 133396, WO2007 / 005874, WO2008 / 083174, WO2008 / 156712, WO2009 / 024531, WO2009 / 014708, WO2009 / 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 presentJAWS Ref: 750406PCT invention include the anti-PD-1 mAbs nivolumab, pembrolizumab, and pidilizumab, as well as the humanized anti-PD-1 antibodies h409Al l, h409A16, and h409A17 as described in International Patent Publication No. WO2008 / 156712.

[0117] 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-1 sequence 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).

[0118] In certain embodiments, the anti-PD-1 antigen-binding molecule comprises the fully humanized IgG4 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 antigen- binding 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 5

[0119] 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];JAWS Ref: 750406PCT or an antigen-binding fragment thereof, which comprises, consists or consists essentially of the amino acid sequence: QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSK RYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS [SEQ ID NO: 14].

[0120] In some of the same and other embodiments, the anti-PD-1 antigen- binding molecule may comprise the light chain amino acid sequence of nivolumab as set out for example below: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSST LTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO: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].

[0121] In alternate embodiments, the anti-PD-1 antigen-binding molecule comprises the humanized IgG4 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 6

[0122] In some embodiments, the anti-PD-1 antigen-binding molecule competes with the mAb pembrolizumab for binding to PD-1.

[0123] 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:JAWS Ref: 750406PCT 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: QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGIN PSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYW GQGTTVTVSS [SEQ ID NO:24].

[0124] 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].

[0125] 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 7, comprises a heavy chain amino acid sequence of pidilizumab as set forth below:JAWS Ref: 750406PCT QVQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSG ESTYAEEFKGRFVFSLDTSVNTAYLQITSLTAEDTGMYFCVRVGYDALDYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL 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].

[0127] In some of the same and other embodiments, the anti-PD-1 antigen- binding 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].

[0128] Other suitable mAbs are described in the International Patent Publication No. WO2015 / 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).JAWS Ref: 750406PCT

[0129] 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; and

[0130] 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.

[0131] In yet further exemplary embodiments the anti-PD-1 mAb may comprise the IgG1 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 SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC [SEQ ID NO: 49]JAWS Ref: 750406PCT DIVMTQTPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIYLASYLES GVPDRFSGSGSGTAFTLKISRVEAEDVGLYYCQHSRDLPLTFGQGTKLEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC [SEQ ID NO: 50], or a variant or an antigen-binding fragment thereof.

[0132] In other embodiments, the ICM antagonist is a PD-L1 antagonist. Alternative names or synonyms for PD-L1 include PDCD1L1, 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].

[0133] 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-Ll antibodies are described in International Publication Nos. WO2007 / 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-L1 mAbs are described in International PCT Patent Publication No. WO2016 / 007,235, the entire contents of which is also incorporated herein by reference.

[0134] 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 antigen- binding molecule comprises the fully humanized IgG1 mAb durvalumab (as described with reference to “MEDI4736” in International PCT Publication No. WO2011 / 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- L1 antigen-binding molecule comprises the CDR sequences as set forth in Table 8. TABLE 8JAWS Ref: 750406PCTcomprses t e eavy c an amno ac sequence o urvauma as set out or exampe eow: VQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEK 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].

[0136] In some of the same and other embodiments, the anti-PD-L1 antigen- binding 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].

[0137] Alternatively, the anti-PD-L1antigen-binding molecule competes for binding to PD-L1 with the mAb durvalumab.

[0138] In other embodiments, the anti-PD-L1 antigen-binding molecule comprises the fully humanized IgG1 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 fragmentJAWS Ref: 750406PCT 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 9EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYA 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].

[0140] In some of the same and other embodiments, the anti-PD-L1 antigen- binding 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:JAWS Ref: 750406PCT DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK [SEQ ID NO: 59].

[0120] Alternatively, the anti-PD-L1 antigen-binding molecule competes for binding to PD-L1 with the mAb atezolizumab.

[0121] In other embodiments, the anti-PD-L1 antigen-binding molecule comprises the fully humanized IgG1 mAb avelumab (as described in U.S. Patent No.8,217148, the entire contents 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 10EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGIT 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].

[0141] In some of the same and other embodiments, the anti-PD-Ll antigen- binding molecule comprises the light chain amino acid sequence of avelumab as set out for example below:JAWS Ref: 750406PCT 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: QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPS GVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL [SEQ ID NO: 63].

[0123] Alternatively, the anti-PD-L1 antigen-binding molecule competes for binding to PD-L1 with the mAb avelumab.

[0124] 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].

[0125] 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).

[0126] 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: Kl, A2, M3, E33, R35, Q41, S44, Q45, V46, E48, L91, 193, K95, E97, M99, P102, P103, Y104, Y105, L106, 1108, N110.

[0127] In certain embodiments, the anti-CTLA4 antigen-binding molecule comprises the human IgG1 mAb ipilimumab (as described for example in International PublicationJAWS Ref: 750406PCT WO2014 / 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 11TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV 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].

[0130] In some of the same and other embodiments, the anti-CTLA4 antigen- binding 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:JAWS Ref: 750406PCT EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK [SEQ ID NO: 71].

[0131] In some embodiments, the anti-CTAL4 antigen-binding molecule comprises the human IgG2 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- binding fragment thereof. In representative embodiments of this type, the anti-CTLA4 antigen- binding molecule comprises the CDR sequences as set forth in Table 12. TABLE 12NKYYADSVKGRFTISRDNSKNTLYIQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDV 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].

[0133] In some of the same and other embodiments, the anti-CTLA4 antigen- binding molecule comprises the light chain amino acid sequence of tremelimumab as set out for example below: DIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSJAWS Ref: 750406PCT 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: DIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIK [SEQ ID NO: 75].

[0134] 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].

[0135] 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 13VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPJAWS Ref: 750406PCT APELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLY 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].

[0137] 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].

[0138] In some alternative embodiments, the anti-B7-H3 antigen-binding molecule competes for binding to B7-H3 with the mAb enoblituzumab.

[0139] 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].

[0140] 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-JAWS Ref: 750406PCT ((Aminosulfonyl)amino)ethyl)amino)-N-(3-bromo-4-fluorophenyl)-N’-hydroxy-, (C(Z))-). The molecular structure of each of these molecules is provided, below. Indoximod GDC-0919 Epacadostat

[0141] In some embodiments, the ICM antagonist is a killer-cell immunoglobulinGSFHDSPYEWSKSSDPLLVSVTGNPSNSWPSPTEPSSKTGNPRHLHILIGTSVVIILFILL FFLLHRWCSNKKNAAVMDQESAGNRTANSEDSDEQDPQEVTYTQLNHCVFTQRKITRP SQRPKTPPTDIIVYTELPNAESRSKVVSCP [SEQ ID NO: 82].

[0142] 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 14 ` [SEQ ID NO: 197]

[0143] 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: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYAISWVRQAPGQGLEWMGGFIPIFGAA NYAQKFQGRVTITADESTSTAYMELSSLRSDDTAVYYCARIPSGSYYYDYDMDVWGQG TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPvTVSWNSGALTSGVHTJAWS Ref: 750406PCT FPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAor an antgen-bndng ragment t ereo, a representatve exampe o w c comprses, conssts or consists essentially of the amino acid sequence: QVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYAISWVRQAPGQGLEWMGGFIPIFGAA NYAQKFQGRVTITADESTSTAYMELSSLRSDDTAVYYCARIPSGSYYYDYDMDVWGQG TTVTVSS [SEQ ID NO: 84].

[0144] 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 RFSGSGSGT DFTLTISSLEPEDFAVYYCQQRSNWMYTFGQGTKLEIKRT [SEQ ID NO: 86].

[0145] 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 TPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFT ELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLvTGAFGFHLWRRQWRPRRFSALE QGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPE QL [SEQ ID NO: 87].JAWS Ref: 750406PCT

[0146] In some embodiments, the LAG-3 antagonist is an anti-LAG-3 antigen- binding 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.

[0147] In some embodiments, the anti-LAG-3 antigen-binding molecules comprise the CDR sequences set forth in Table 15. TABLE 15

[0148] 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 TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPATVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK [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].

[0149] 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: EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKRTVAAPSVFIFPPJAWS Ref: 750406PCT 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 RFSGSGSGT DFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIK [SEQ ID NO: 91]. 2.2.2 PARP inhibitors

[0150] 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.

[0151] 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).

[0152] 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.

[0153] 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 with defects 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 andJAWS Ref: 750406PCT 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.

[0154] 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.

[0155] 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 I / 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.

[0156] 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 repair pathways and targeting these pathways is expected to have an impact on the survival of the tumour cells than on normal cells.JAWS Ref: 750406PCT

[0157] 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.

[0158] 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.

[0159] In some embodiments, the PARP inhibitor is selected from 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.

[0160] Accordingly, the PARP inhibitor may be selected from olaparib, niraparib, rucaparib, and talazoparib, or a pharmaceutically acceptable salt thereof.

[0161] In some embodiments, the PARP inhibitor is olaparib, or a pharmaceutically acceptable salt thereof. In some embodiment, the PARP inhibitor is olaparib.

[0162] 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.

[0163] 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).

[0164] 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 chemotherapy regimens. Olaparib traps the inactive form of PARP on DNA at sites of SSBs, thereby preventing their repair.JAWS Ref: 750406PCT 2.3 Ancillary agents

[0165] 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-β, e.g., metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299, a T cell (e.g., a cytotoxic T cell or CTL) expressing a chimeric antigen receptor (CAR), a T cell comprising a dominant-negative TGF-β receptor, e.g., a dominant-negative TGF-β type II receptor, an agonist directed against CD137 (also known as TNFRSF9, 4-1BB, 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 OX40 (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- lR (also known as IMC-CS4), an interferon, for example IFN-α or IFN-γ, 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, an antibody targeting CD20 (in some embodiments, the antibody targeting CD20 is obinutuzumab (also known as GA101 or Gazyva®) or rituximab), an antibody targeting GITR (in someJAWS Ref: 750406PCT 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 Alk, 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 CCl-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.

[0142] 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), β-lactams (e.g., imipenem, meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam,JAWS Ref: 750406PCT 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, cloxacillin, 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, OPC-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

[0143] 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.

[0144] 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)). Pharmaceutically 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,JAWS Ref: 750406PCT 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.

[0145] 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 WO2006 / 044908, the latter formulations including a histidine-acetate buffer.

[0146] 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.

[0147] 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).

[0148] 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, or microcapsules. 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.JAWS Ref: 750406PCT

[0149] 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

[0150] 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.

[0151] 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.

[0152] 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-γ, 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.

[0153] In some embodiments, the individual is a human.

[0154] 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.JAWS Ref: 750406PCT

[0155] 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.

[0156] 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 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.

[0157] 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.

[0158] 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 the PI3K 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 withJAWS Ref: 750406PCT 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.

[0159] 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:

[0160] 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.

[0161] 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.

[0162] 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, about 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.) isJAWS Ref: 750406PCT 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.

[0163] 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.

[0164] 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.

[0165] 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 optional ancillary 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.JAWS Ref: 750406PCT 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.

[0166] 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.

[0167] 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.

[0168] 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 Mech.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. 1(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 ancillaryJAWS Ref: 750406PCT 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.

[0169] 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).

[0170] 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-γ and TNF), decrease expression of biomarkers of T-cell effector inhibition and cancer progression (e.g., ZEBl), 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-γ 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.

[0171] In some embodiments of the methods of the present disclosure, activated CD4+ and / or CD8+ T-cells in the individual are characterized by IFN-γ producing CD4+ and / or CD8+ T cells and / or enhanced cytolytic activity as compared to before the administration of the combination, .gamma. IFN-γ may be measured by any means known in the art, including, e.g., intracellular cytokine staining (ICS) involving cell fixation, permeabilization, and staining with an antibody against IFN-γ. 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.JAWS Ref: 750406PCT

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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-γ, 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.

[0176] 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; IPIO 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.

[0177] 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 the combination therapy. CXCR3 / CD8+ T cells may be measured by any means known the art. InJAWS Ref: 750406PCT some embodiments, CXCR3 / CD8+ T cells are from peripheral blood. In some embodiments, CXCR3 / CD8+ T cells are from tumour.

[0178] 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.

[0179] In some embodiments, number of Treg is decreased as compared to before administration of the combination. In some embodiments, the levels of plasma IFN-γ 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.

[0180] 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

[0181] 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.

[0182] 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 / or expression levels / amount in a second sample. In certain embodiments, the second sample is aJAWS Ref: 750406PCT 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.

[0183] 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 100x 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, 0.1x, 0.05x, or 0.01x the expression level / amount of the respective biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

[0184] 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 genes and gene products are found, for example in Ausubel et al., eds., 1995, Current Protocols InJAWS Ref: 750406PCT 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.

[0185] 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.

[0186] 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).

[0187] 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.

[0188] 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: 750406PCT

[0189] 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 anti- biomarker antibodies are used to select subjects eligible for combination therapy with a PI3K inhibitor and an ICM-binding antagonist.

[0190] 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).

[0191] 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.

[0192] 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: 750406PCT competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labeled antibody to a target biomarker.

[0193] 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 activity- based 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.

[0194] 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.

[0195] 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.

[0196] 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.

[0197] 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: 750406PCT reference 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.

[0198] 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.

[0199] 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).

[0200] 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: 750406PCT immune cells as described herein) immediately adjacent to and / or contiguous with the main tumour mass.

[0201] 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.

[0202] 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.

[0203] 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.

[0204] 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: 750406PCT

[0205] 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.

[0206] 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.

[0207] 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-γ, 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: 750406PCT 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 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.

[0208] 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.

[0209] 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.

[0210] 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: 750406PCT sample 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.

[0211] 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.

[0212] 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.

[0213] 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.

[0214] 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: 750406PCT (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.

[0215] 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

[0216] 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.

[0217] 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.

[0218] 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: 750406PCT corresponding 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.

[0219] 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 protein- based 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.

[0220] 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.

[0221] Alternatively or in addition, the media can contain Internet addresses that provide the instructional material.

[0222] 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 non- limiting experimental examples. EXAMPLES Example 1JAWS Ref: 750406PCT PI3K expression is associated with reduced cancer survival.

[0166] PI3KCA is part of the cytoplasmic / plasma membrane PI3KCA / AKT / MTOR signalling pathway (Hassan et al., 2013). PI3KCA mutational burden has been associated with cancer progression via the AKT pathway (which plays a role in promoting invasion and metastasis) (Jiang et al., 2020). Recent studies have shown that the PI3KCA signalling pathway is associated with cancer stem cells (CSC) and EMT (Chen et al., 2020; Xia et al., 2015). PI3KCA has a role in immune evasion by phosphorylating AKT(P) inducing PDL1 expression and mediating immune evasion (Yang et al., 2017).35% of breast cancer patients had mutant positive tumours (Fusco et al., 2021) and in TNBC PI3KCA mutations was present in ~6% of patients (Mosele et al., 2020). This does not account for the majority of patients with recurrence or immunotherapy resistance.

[0167] Using the web tool KM-plotter (Lanczky and Gyorffy, 2021) the present inventors plotted the overall survival probability for low and high protein expression of PI3K (Figure 1). This analysis revealed that in both metastatic breast cancer and non-small cell lung cancer patient cohorts there was significantly reduced survival in the high expression of PI3K cohort. This analysis was undertaken to demonstrate that PI3K enrichment is associated with significantly reduced survival probability in multiple cancer types. Liquid Biopsy based stratification to detect enrichment of PI3K CSC and CD8 T cell signature and inhibition of PI3K targets CSC recurrence signature and exhausted T cell signature in patient liquid biopsies.

[0168] The present inventors identified a dual CSC and T-cell signature in patient liquid biopsies from stage IV solid tumour cohorts (i.e., melanoma, NSCLC, RCC, HCC, breast cancers, GBM). A novel, cancer stem cell, stem-like mesenchymal signature enriched for PI3K in stage IV cancer patients was identified using our novel liquid biopsy platform (Figure 2). These CSC are the key cell type implicated in cancer recurrence and are not targeted by chemotherapy, immunotherapy or radiotherapy, i.e., conventional cancer therapy. However, the present inventors hypothesised that these patients would respond to the mono-therapy and / or a combination therapy approach of the present invention. These CTCs are positive for mesenchymal, CSC markers CSV and ABCB5.

[0169] The present inventors also identified a novel T-cell exhaustion / dysfunctional signature enriched for checkpoint proteins (TIM3, TIGIT, PD1), and exhaustion markers (TOX1, EOMES).

[0170] Analysis revealed that overall expression intensity as measured by immunofluorescent analysis using the digital pathology system for CSV and ABCB5 was significantly reduced in the GDC-0084 treated samples (Figure 3A). In contrast, EpCAM which is a marker of non-mesenchymal, epithelial cells displayed little change in expression (Figure 3A). Population dynamic analysis using the digital pathology system revealed that the population of cells positive for CSV and ABCB5 decreased by approximately 56%, whereasJAWS Ref: 750406PCT EpCAM was increased by 38% (Figure 3B). PI3KCA inhibition with GDC-0084 also inhibited the % positive cells for AKT1 (AKT1 contributes to a mesenchymal metastatic signature) as well as the overall expression of AKT1 (AKT1 is a key tumourgenic marker regulated by PI3KCA). This indicates that targeting PI3KCA also targets critical regulates of both metastasis, mesenchymal transition and the cancer stem cell signature (CSC). Liquid Biopsy based assay from stage IV solid tumour cancer patients demonstrates PI3K inhibition inhibits T-cell dysfunction and induces an effector signature

[0171] Analysis of CD8+ T cells was carried out for overall expression intensity as measured by immunofluorescent analysis using the present inventors’ lab’s Digital Pathology System. Exhaustion Panel demonstrated significant inhibition of EOMES and PD1 expression in both NS and ST samples by GDC-0084 (Figure 4A). The immune checkpoint inhibitor panel demonstrates significant inhibition of TIM3 in both NS and ST CD8+ T cells by GDC-0084 treatment, whereas GDC-0084 only significantly inhibited TIGIT in the NS CD8+ T cells (Figure 4B). Figure 4C shows the effector signature panel, which in contrast to Figure 4A and 4B, demonstrates a significant increase in expression of perforin and GZNb in the GDC-0084 treated CD8+ T cells in both NS and ST groups. PI3K enriched in cancer cell lines with enriched mesenchymal CSC signature.

[0172] Whole cell intensity analysis was undertaken using the ASI Digital pathology system as per normalized settings. Analysis of the expression of PI3KCA in breast cancer cell lines (Figure 5A) revealed a pattern of increasing expression as the cell lines went from a epithelial signature to a more mesenchymal, metastatic, immunotherapy resistant signature (MCF7 is a epithelial breast cancer cell line with low CSC population, MDA-MB-231 is a TNBC cell line with >90% mesenchymal CSCs, MDA-MB-231-BR is a brain metastatic cancer clone of MDA-MB-231 with >90% mesenchymal CSCs and lastly 4T1 is a immunotherapy resistant, metastatic highly aggressive breast cancer model with >90% CSCs).

[0173] Interestingly, a similar pattern is seen for H1299 (epithelial lung cancer), CT26 (IO responsive, epithelial colon cancer) and LLC (highly aggressive, metastatic lung cancer model) (Figure 5B). H1299 and CT26 are both epithelial-like, immunogenic cell lines whereas LLC is highly resistant to immunotherapy and is aggressive. Overall, these data suggest that PI3KCA from a whole cell expression perspective is enriched in metastatic, treatment resistant cancer cell lines. This indicates the importance of targeting PI3KCA to treat metastatic, treatment resistant cancer which is a significant unmet burden. • MCF7 Epithelial breast cancer cell line; • MDAMB231 Mesenchymal / CSC breast cancer cell line; • MDAMB231-Br Brain cancer version of MDA-MB-231; • 4T1 Highly aggressive, IO resistant mouse breast cancer model;JAWS Ref: 750406PCT • H1299: Epithelial like Lung cancer cell line; • CT26: Epithelial like, highly immunogenic mouse colon tumour cell line; and • LLC: Highly treatment resistant, Lewis lung cancer cell line, resistant to immunotherapy (Li et al., 2021). Effect of PI3K inhibitor GDC-0084 on MDA-MB-231 and CT26 cell migration & invasion.

[0174] Interestingly, in up to 70% of breast cancer brain metastasis patients, the PI3K / Akt / mTOR pathway is activated. Upregulation of this pathway has been associated with a worse prognosis; however no approved drugs are available to these patients. GDC-0084, a PI3K inhibitor, is a brain penetrant that has shown promising activity in a preclinical model of glioblastoma. GDC-0084, at both its IC25 (1.25 ^M) and IC50 (2.5 ^M), inhibits breast cancer cell migration, with the IC25 concentration having greater inhibition on MDA-MB231 cell migration (Figure 6). The wound healing analysis demonstrates inhibition of CT26 cell migration upon treatment with GDC-0084 (PI3K inhibitor). DMSO treatment was utilized as a control. These data show that GDC-0084 at 0.3 ^M has a more prominent effect on wound healing as compared to the lower concentration (i.e., 0.2 ^M) (Figure 6C). This suggests that GDC-0084 is capable of inhibiting cancer cell migration at higher concentrations. GDC-0084 shows the greatest inhibitory effect on responsive colon cancer cell line CT26. The present inventors utilized DMSO treatment as a control. Determining the IC50 of GDC-0084 for different cancer cell lines.

[0175] Data clearly show the EC50 data for GDC-0084 in the different cell lines. The individual dose response curves for CT26, 4T1, MDA-MB-231, MDA-MB-231-Brm, are MCF7 shown in Figure 7. These data were obtained to demonstrate the efficacy of targeting PI3KCA with GDC-0084 in immunotherapy responder or epithelial cancer cell lines (CT26, MCF7) as compared to immunotherapy resistant or mesenchymal cancer cell lines (4T1, MDA-MB-231 cell lines). These data demonstrate that targeting PI3K with GDC-0084 is able to significantly inhibit proliferation of both epithelial and mesenchymal cancer cell lines. Effect of PARP inhibition on the mesenchymal, CSC signature

[0176] Olaparib and veliparib are PARP inhibitors that have been used to treat some types of breast cancer and ovarian cancer. However, olaparib does not target cancer- stem cells (CSCs) or the mesenchymal signature, and does not impact ABCB5 or ALDH1A (Figure 8). The present inventors next examined the effect of olaparib and veliparib on the CSC signature or proliferation of the mesenchymal TNBC cell line MDA-MB-231, neither of these drugs inhibited the CSC signature or proliferation (Figure 9). Next to further examine the importance of mono-therapeutic targeting of PARP, the present inventors examined the effect of siRNA knock-down of PARP (Figure 10) on the mesenchymal, CSC signature. It was identified that PARP knock-down had no inhibition of these markers of a mesenchymal, CSC signature (Figure 11). The combination treatment of PI3K pathway inhibitor GDC-0084 and PARP inhibitorJAWS Ref: 750406PCT olaparib restricted MDA-MB-231 cell migration (Figure 17). The present inventors demonstrated that the combination of GDC-0084 and olaparib inhibits breast cancer cell migration, with combination “2” (i.e., 1.25 µM GDC-0084 + 250 nM olaparib) having greatest inhibition on MDA- MB-231 migration. This demonstrates that the PARP inhibitors are not effective mono therapeutic agents but may have benefit as a combination with a PI3K inhibitor. αPD1 therapy in the CT26 model of colon cancer and 4T1 breast cancer model.

[0177] The present inventors sought to demonstrate that αPD1 monotherapy causes significant reduction in tumour volumes in the immunotherapy responsive CT26 colon cancer model as compared to the 4T1 breast cancer model (Figure 12). This data establishes that CT26 is an immunotherapy responder line whereas the 4T1 model clearly demonstrates that 4T1 is an immunotherapy-resistant model as demonstrated by the primary tumour burden efficacy in response to αPD1 immunotherapy. PI3K inhibition targets and inhibits the expression of mesenchymal resistant markers

[0178] Next, the inventors demonstrated that treatment with GDC-0084 significantly inhibited CSC / mesenchymal markers, inducing the expression of the epithelial marker EpCAM and also increasing the expression of the viral mimicry / immune visibility marker MDA5 (Figures 13 and 14). This demonstrates that targeting PI3KCA also inhibits metastatic and therapeutic resistance signature as well as making the tumour more immune visible and therefore more responsive to combination with immunotherapy as well.

[0179] Next, the inventors carried out further inhibition analysis with different inhibitors targeting PI3K to confirm the efficacy of targeting PI3K to inhibit the mesenchymal CSC signature. Two different PI3K inhibitors (Figure 15) were used, idelalisib, which has been used to treat certain blood cancers such as chronic lymphocytic leukemia following recurrence and LY294002 which is a strong inhibitor of PI3K currently under trial in patients with recurrent or progressive HNSCC and in patients with mutations in PI3KCA and / or PI3K pathway genes. Treatment with either of the two PI3K inhibitors (Figure 15) at two different low doses significantly inhibited CSC / stem-like cancer recurrence markers CSV, SoX9, ABCB5 and SNAIL The dose of idelalisib at 12.5 µM had the least significant effect. In addition, all idelalisib at 25 µm and both concentrations of LY294002 induced expression of the epithelial marker EpCAM. This data demonstrates that targeting PI3K with alternative inhibitors to GDC-0084 are efficacious at inhibiting the mesenchymal cancer stem cell signature and inducing an epithelial signature. GDC-0084 administration at 7.5 mg / kg reduces clinical abnormalities in the 4T1 syngeneic tumour model.

[0180] Next, the present inventors demonstrated that GDC-0084 monotherapy using 7.5 mg / kg reduces clinical abnormalities observed in the 4T1 breast cancer model. The mouse group receiving the high dose (i.e., 15 mg / kg) suffered from multiple cases of reduced activity and hunched posture as well as significant piloerection (Figure 16). In comparison to 15JAWS Ref: 750406PCT mg / kg, GDC-0084 administration at 7.5 mg / kg reduces clinical abnormalities previously observed at the higher dose. While 40% of mice treated with 15 mg / kg GDC-0084 demonstrate reduced activity, hunched posture, piloerection and weight loss, no clinical abnormalities were present in the 7.5 mg / kg treatment group. These data highlight the reduced toxicity at the lower dose. GDC-0084 administration at 7.5 mg / kg abrogates tumour burden.

[0181] The present inventors next demonstrated that “low dose” GDC-0084 therapy (at 7.5 mg / kg) abrogates primary tumour burden in the 4T1 breast cancer model (see, Figure 17). GDC-0084 administration at 7.5 mg / kg, both as a monotherapy and in combination with αPD1, substantially reduces tumour volume and final tumour weights by more than 50% in the 4T1 breast cancer model. Strikingly, a significant reduction in tumour burden was not observed in mice administered 15 mg / kg GDC-0084 with or without αPD1. GDC-0084 administration at 7.5 mg / kg reduces tumour inflammation including monocyte and neutrophil infiltration.

[0182] The administration of a “low dose” of GDC-0084 administration (i.e., 7.5 mg / kg) reduces tumour inflammation including monocyte and neutrophil infiltration (Figure 18). GDC-0084 administration at 7.5 mg / kg, both as a monotherapy and in combination with αPD1, dramatically reduces tumour inflammation associated with the 4T1 breast cancer model. While tumour inflammation scored “moderate” and “severe” in the control and αPD1 treatment groups, when administered in combination with GDC-0084 at 7.5 mg / kg, only mild inflammation was observed.

[0183] Furthermore, the pathologist noted a substantial reduction in myeloid populations including monocytes and neutrophils in the GDC-0084 administered at 7.5 mg / kg treatment groups. Notably, a significant reduction in inflammation was not observed in mice administered with a daily dose of 15 mg / kg GDC-0084. GDC-0084 administration at 7.5 mg / kg reduces splenomegaly in the 4T1 syngeneic tumour model.

[0184] GDC-0084 administration at 7.5 mg / kg reduces splenomegaly in the 4T1 breast cancer model. In the lower dose of GDC, the spleen is normal sized, appearance in comparison to high doses (Figure 19). GDC administration at 7.5 mg / kg reduces splenomegaly typically associated with the 4T1 breast cancer model.

[0185] While no significant differences in spleen weight were noted between the 15 mg / kg treatment group and the corresponding controls, a dramatic reduction in splenomegaly was observed at the lower dose of GDC-0084 (7.5 mg / kg) both as a monotherapy and in combination with αPD1.JAWS Ref: 750406PCT GDC-0084 administration at 7.5 mg / kg reduces SOX9 and TOX1 expression.

[0186] Combination treatment in both experiments significantly reduced SOX9 (SOX9 is a marker for mesenchymal, therapeutic resistant cancer stem cells) expression, however at 7.5 mg / kg GDC-0084 had a more striking effect in combination, and an exceptional reduction as a monotherapy. Interestingly, at 15 mg / kg GDC-0084 showed induction of TOX1 (TOX1 is marker of T cell exhaustion and dysfunction) in CD8+ T cells whereas at 7.5 mg / kg a significant reduction of TOX1 was observed in CD8+ T cells (Figure 20). These data demonstrate that the “high dose” GDC-0084 induced dysregulation inflammation and contributed to increase T cell dysfunction (higher expression of TOX1) as well as not controlling SOX9, a marker for cancer stem cell like mesenchymal signatures, metastasis and cancer recurrence. In contrast “low dose” GDC-0084 as a monotherapy or combination therapy inhibited SOX9+ expression significantly and significantly reduced expression of TOX1 in CD8+ T cells. Notably, the combination anti-PD1 and “low dose” GDC-0084 (7.5mg) had the strongest effect in abrogating TOX1 expression (Figure 20).

[0187] PI3K mono- or combination inhibition targets mesenchymal signature and induces epithelial phenotype in IO-resistant mouse model.

[0188] Combination treatment with GDC-0084 and anti-PD1 significantly inhibited PI3KCA, mesenchymal markers CSV, and EGFR as well as this combination was also superior induced expression of epithelial marker, E-cadherin (Figure 21).

[0189] Both GDC-0084 monotherapy and the combination treatment comprising GDC-0084 and anti-PD1 significantly induced the population of cells positive for the epithelial marker E-cadherin (see, Figure 22). GDC-0084 with or without αPD1 therapy had no effect on mouse weight in the 4T1 syngeneic tumour model (Figure 17). GDC-0084 combination therapy with αPD1 significantly inhibited tumour burden, but not as a high dose mono-agent, whereas a lose dose mono agent was able to inhibit tumour burden (Figure 17). Notably, this combination also worked in the 4T1 immunotherapy-resistant tumour model. Combination treatment with GDC-0084 and an anti-PD1 inhibitor significantly inhibited PI3KCA and cancer stem cell-like mesenchymal markers CSV and EGFR. Expression of epithelial marker E-cadherin was induced (Figure 21). Strikingly, GDC-0084 monotherapy or combination treatment with GDC- 0084 and an anti-PD1 inhibitor significantly induced the population of cells positive for the epithelial marker E-cadherin (Figure 22). These data demonstrate that a PI3KCA inhibitor in combination with immunotherapy was able to significantly impact tumour burden compared to immunotherapy alone as well as been able to re-program the cancer cell or tumour microenvironment signature to more epithelial signature and therapeutic responsive one. This indicates a viable therapeutic approach for enhancing durable responses for immunotherapy PI3K mono or combination therapy induces an effector and TRM signature.

[0190] Combination treatment is superior to monotherapy and significantly increases infiltration of CD8+ IFN ^+ T cells into the tumour (Figure 23A). Additionally, out ofJAWS Ref: 750406PCT total CD8 T cell population, the proportion of CD8+ T cells expressing TRM markers (i.e., CD44, CD103, CD69) was also significantly increased in the combination group which had superior induction as compared to the PI3K inhibitor monotherapy (Figure 23B). Combination treatment also significantly increased infiltration CD8+ IFN ^+ T cells positive cells into the tumour (Figure 23A). Additionally, out of total CD8 T cell population, the proportion of CD8+ T cells expressing TRM markers (i.e., CD44, CD103, CD69) was also significantly increased in the combination group (Figure 23B). This assay was undertaken to understand if PI3K inhibition monotherapy or combination with immunotherapy re-programmed the immune cell signature in addition to the cancer cell signature. Combination treatment was able to significantly impact and induce the immune effector signature improving anti-tumour immunity. PI3K mono or combination therapy inhibits checkpoint exhaustion signature in CD8+ T cells.

[0191] GDC-0084 and anti-PD1 inhibitor combination treatment significantly reduces the population of CD8+ T cells positive for TIM3 and LAG3 compared to either PD1 or GDC-0084 monotherapy alone (Figure 24). This assay was undertaken to investigate if the signature for dysfunctional immune CD8+ T cells were re-programmed. Analysis revealed that combination therapy was able to significantly reduce the immune dysfunctional signature. Combination of PI3K inhibition and immunotherapy abrogates metastatic spread in 4T1 IO resistant model.

[0192] GDC-0084 combination therapy with αPD1 significantly inhibited metastatic tumour burden but not as a mono agent. This combination was also efficacious in the 4T1 immunotherapy-resistant tumour model (Figure 25). These data demonstrate that a PI3K inhibitor in combination with immunotherapy can significantly impact tumour burden as compared to immunotherapy alone as well as be able to re-program the cancer cell or tumour microenvironment signature to become more therapeutically responsive in an immunotherapy- resistant mouse breast cancer model (4T1) and abrogate metastatic spread of the cancer to the lungs. This indicates a viable therapeutic approach for enhancing durable responses for immunotherapy.’ Discussion

[0193] The inventors have obtained a novel method of both reducing the overall toxicity of PI3K inhibitors (such as GDC-0084) while increasing the overall efficacy of treatment. This was achieved by administering a reduced 7.5 mg of GDC-0084 (the normal dose is a single administration of 15 mg) reduces overall adverse side effects and as well, increases the overall efficacy of the GDC-0084 therapy. In addition, the present inventors identified that the efficacy of PI3K inhibitor combination with immunotherapy is similarly enhanced. The low concentration of therapeutic administration of GDC-0084 in combination with immunotherapy can be further reduced with a similar reduction in the dosage of immunotherapy (such as anti- PD1) resulting in less adverse events and overall enhanced efficacy with a significantly lower dose.JAWS Ref: 750406PCT Summary of efficacy of mono-therapy vs combination therapy

[0194] Mono-therapy at a low dose inhibits checkpoint proteins (Figure 4: TIGIT, Tim3, PD1) and induces effector signature (Figure 4). Re-programs exhaustion signature (EOMES) Figure 4.

[0195] Monotherapy at a lower dose has significantly less clinical abnormalities (Figure 12)

[0196] Monotherapy GDC-0084 at a lower dose was able to significantly inhibit tumour weight (Figure 13)

[0197] However, combination with a low dose displayed features for sustained tumour control. Furthermore, low dose combination was more effective at inhibiting the inflammation (Figure 14 and Figure 15).

[0198] Lower dose in combination was more effective at inhibiting the cancer stem cells (important for recurrence & metastasis) and at re-programing and inhibiting the dysfunctional T cell signature and inducing an effector T cell signature (Figure 16, 19-20).

[0199] The “low dose” in combination with an immunotherapy is more effective at inducing an epithelial signature (Figure 17-18), as compared to a “high dose” or “effective dose”. The combination was also superior at inhibiting metastasis (Figure 21).

[0200] Administration with 15 mg / kg represents a high dose of GDC-0084 (i.e., in the clinic humans are generally administered 20-60 mg), our low dose represents only 50% of this dose. A dose of 25% of the original is also expected to be effective as a single and combination agent. Materials & Methods

[0201] 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.

[0202] Groups of five female BALB / c mice per treatment group were used for xenograft studies.4T1 breast cancer (1 × 105) cells were injected into the right 4th inguinal mammary fat pad of six-week-old BALB / c nude mice. For 4T1 tumour cell injections, 1 × 105cells per mouse were prepared in PBS.

[0203] For the CT26 colon carcinoma mouse model, 5 × 105cells were injected via intraperitoneal injection into the abdomens of 6-week-old BALB / c nude mice. Tumour growth was measured thrice weekly by calliper measurements. Mice were treated with 10 mg / kg anti- PD-1 via intraperitoneal injection on days 0 and 5. Tumour volumes (½ (length ×width2)) were measured using a digital calliper and presented as means ± SEM.JAWS Ref: 750406PCT

[0204] Six-week-old female Balb / c mice were purchased from the Animal Resources Centre (ARC) and allowed to acclimatize for 1 week prior to use. All experimental procedures were performed in accordance with the guidelines and regulations approved by the QIMR Berghofer Animal Ethics committee. Mice were shaved at the site of inoculation prior to subcutaneous injection with 1 × 1054T1 cells in 100 µl PBS into the right mammary gland. Treatments were started when tumours reached approximately 100 mm3. Tumours were measured using external callipers and volumes calculated using a modified ellipsoidal formula ½ (a / b2), where a = longest side and b = shortest side. For combination therapy experiments, mice were treated with vehicle (saline) or GDC-0084 (via oral gavage) at indicated doses in combination with anti-PD1 or isotype control (10 mg / kg) twice weekly by intraperitoneal injection. Mice were monitored daily for clinical abnormalities (reduced activity, hunched posture, piloerection, weight loss and metastasis), and tumour volumes and body weights were measured thrice weekly. Once tumours reached the maximal limit (1000 mm3) in the vehicle group, all tumours and associated metastatic organs (lung, liver, spleen) were harvested, weighed, imaged and fixed in 4% paraformaldehyde for associated analysis. WST-1 cell proliferation assay.

[0205] Adherent cell lines CT26, 4T1 and MCF7 were seeded in triplicate at optimized cell densities (100, 100, 3000 and 300 per well respectively) in a 96-well flat bottom tissue culture plate in a total volume of 100 µl / well. The cells were left to adhere overnight at 37°C and 5% CO2. Cells were then treated with a range of different concentrations of inhibitor and left in the incubator at 37°C and 5% CO2 for 72 hours, after which media was removed and replaced with 100 µl / well of WST-1 cell proliferation reagent (Sigma-Aldrich, 11644807001) at a 1:10 final dilution in complete cell culture medium. WST-1 reagent contains water-soluble tetrazolium salts which are cleaved by cellular mitochondrial dehydrogenase enzymes to produce formazan. The amount of formazan produced per reaction is then quantified using a microplate spectrophotometer to measure metabolic activity. The absorbance measured directly correlates to the number of metabolically active cells in culture. Absorbance was recorded at 450 nm at 0.5, 1, and 2 hour incubation periods using a microplate spectrophotometer (following a 30 second mix time). Percent proliferation was calculated by subtracting the mean absorbance of the background control (blank) from the sample absorbance. The EC50 value of each inhibitor was determined by log(inhibitor) vs. response - Variable slope (four parameters) using GraphPad Prism 8 software. Scratch wound healing assay

[0206] Adherent breast cancer cell line MDA-MB-231 was seeded in triplicate at an optimized cell density of 2500 cells per well in a 96-well Incucyte® Image Lock plate in a total volume of 100 ^l / well in low-serum media (2%). The cells were left to adhere overnight at 37°C and 5% CO2. Cells were checked to be at 100% confluence in order to ensure consistent wounds are created. After 24 hours, wounds were created using the 96-pin InCucyte®JAWS Ref: 750406PCT WoundMaker Tool. Post wounding, cells were washed with low serum media to remove non- adherent cells and then treated with a range of different concentrations of inhibitor (IC25 and IC50), prepared in low-serum media. The plate was then placed into the Incucyte® live-cell analysis system and wound healing was tracked using the InCucyte® Zoom live-cell analysis system with scan interval every 6 hours. In vitro metastatic cancer PI3K inhibition signature

[0207] 4T1 or CT26 cancer cell lines were treated with control or GDC-0084 (at concentration of 0.2 µM or 0.3 µM), Idelalisib (at two low concentrations of 12.5 or 25 uM) or LY294002 (at two low concentrations of 2.5 or 5 µM). Samples were then permeablized with Triton X-100 and stained with primary mouse antibodies for CSV or EpCAM, primary rabbit antibodies for EGFR or FOXN2 or MDA5 or primary goat antibodies for ABCB5, detection was with donkey AF anti-mouse 488, anti-rabbit 568 or anti-goat 647 secondary antibodies. The ASI digital pathology system at 100x objective was used to image protein targets. Example image fields with scale bar (in orange) are depicted above. Analysis was carried out comparing the florescent intensity for EGFR, cytoplasmic florescent intensity (CFI) for CSV or overall florescent intensity for ABCB5. Data was plotted with PRISM and analysis carried out with one-way ANOVA non-parametric test with Kruskal-Wallis. Significant differences are plotted. OPAL tissue microscopy.

[0208] The OPAL staining kit for automatic staining use the automatic BONDRX platform was used following the manufacturer’s instructions. Proteins were then mounted and localised. Protein targets were localised by digital pathology laser scanning microscopy. Single 0.5 μm sections were obtained using an ASI Digital pathology microscope using a 100x oil immersion lens running ASI software. The final image was obtained by averaging four sequential images of the same section. Digital images were analyzed using automated ASI software (Applied Spectral Imaging, Carlsbad, CA) to determine the distribution and intensities automatically with automatic thresholding and background correction of the average fluorescence intensity, allowing for the specific targeting of expression of proteins of interest. Isolation of CTCs from Stage IV metastatic cancer patient Liquid biopsies method I.

[0209] A liquid biopsy from a cancer patient was processed for CD45+ cell depletion to enrich for circulating tumour cells (CTCs), or no depletion to isolate PBMCs in the liquid biopsy using our optimized lab protocol. Briefly, our optimized lab protocol is: whole blood was stored in EDTA tubes for circulating tumour cell identification. The RosetteSep™ Human CD45 Depletion Cocktail was used to enrich tumour cells (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 (RBCs). Unwanted cells were then removed via centrifugation over a buoyant density medium Lymphoprep™ (Catalogue #07801). The purified tumour cells were then extracted as a highly enriched population from theJAWS Ref: 750406PCT interface between the plasma and the buoyant density medium and were harvested in 20% FBS in PBS.

[0210] Samples were then stained for CSV (a mesenchymal, metastatic marker for CTCs), ABCB5 (a cancer stem cell marker and marker for chemoresistance) and EpCAM (an epithelial cell marker). Cover slips were mounted on glass microscope slides with ProLong nucblue clear Antifade reagent (Life Technologies). Protein targets were localised by confocal laser scanning microscopy. Single 0.5 μm sections were obtained using an ASI digital pathology system using 100x oil immersion lens running ASI software. The final image was obtained by averaging four sequential images of the same section. Digital images were analysed using both ASI software for population dynamics and ImageJ software (ImageJ, NIH, Bethesda, MD, USA) total cell counts and fluorescent intensity (expression of protein). Isolation of CTCs from Stage IV metastatic cancer patient Liquid biopsies method II

[0211] Liquid biopsy from cancer patient was processed for CD45+ cell depletion to enrich for circulating tumour cells (CTCs) in the liquid biopsy using our optimized lab protocol (as described above under Liquid biopsies method I). Sample was then stained for CSV (a mesenchymal, metastatic marker for CTCs), ABCB5 (a cancer stem cell marker and marker for chemoresistance) and PI3K (a kinase). Cover slips were mounted on glass microscope slides with ProLong nucblue clear Antifade reagent (Life Technologies). Protein targets were localised by confocal laser scanning microscopy. Single 0.5 μm sections were obtained using an ASI digital pathology system using 40x oil immersion lens running ASI software. The final image was obtained by averaging four sequential images of the same section. Digital images were. Example 2 Establishing the optimal therapeutic dose for PI3K-mTOR inhibitor efficacy.

[0212] To establish the optimal therapeutic dose for the GDC-0084 PI3K-mTOR inhibitor the present inventors used the highly aggressive immunotherapy resistant 4T1 breast cancer model. Once tumours were established (approximately 100 mm3), GDC-0084 was administered daily by oral gavage in combination with αPD1 immunotherapy (Figure 26A). Experiments were performed sequentially with initial studies using a high starting dose of GDC of 15 mg / kg + / - αPD1 daily. This was followed by a split-dose de-escalation study whereby daily GDC doses from 1.875 mg / kg to 15 mg / kg were administered as a split dose 4 hours apart in combination with αPD1 and lastly, a single-dose de-escalation study whereby daily GDC doses of 3.75 and 7.5 mg / kg were administered as a single dose in combination with αPD1 (Figure 26B).

[0213] Administration of GDC-0084 at a high daily dose of 15 mg / kg significantly reduced tumour volume by 69% when delivered in combination with immunotherapy (FigureJAWS Ref: 750406PCT 26C, panel 1). Split dose experiments revealed comparable decreases in tumour volume at GDC doses of 15 mg / kg and 7.5 mg / kg in combination with anti-PD1 (Figure 26C, panel 2). In a final single dose de-escalation study, administration of GDC at the lower dose of 7.5 mg / kg in combination with αPD1 reduced primary tumour volumes to the same extent as the high dose (Figure 26C, panel 3), hence 7.5 mg / kg was confirmed as the optimal therapeutic dose. Reducing the dose of GDC-0084 prevents toxicity complications.

[0214] The inventors next sought to further examine toxicity related complications of GDC-0084 treatment by monitoring mouse body and liver weights in the starting dose, split dose, and single dose de-escalation experiments (Figure 27). Mice treated with GDC-0084 at a high daily dose of 15 mg / kg αPD1 experienced weight reductions after day 4 of treatment, ultimately resulting in mouse fatalities in both the starting dose and split dose de-escalation experiments (Figure 27A, panel 1 and 2). However, at the optimal therapeutic dose of 7.5 mg / kg, mice maintained body weights throughout the duration of the experiment and no mouse fatalities occurred (Figure 27A, panel 2 and 3).

[0215] Enlargement of the liver, or hepatomegaly, is associated with organ damage. To determine if GDC-0084 treatment induced liver toxicity, the inventors measured liver weights at the time of harvest (Figure 2B). Administration of GDC-0084 at a high daily dose of 15 mg / kg increased liver weights by approximately 20% by the end of treatment (Figure 27B, panel 1). In contrast, when administered at the lower dose of 7.5 mg / kg, no differences in liver weight were observed (Figure 27B, panels 2 and 3). Taken together, our data confirm that GDC-0084 administration at the optimal therapeutic dose of 7.5 mg / kg has no toxicity-related complications. Reducing the dose of PI3K-mTOR inhibitor decreases liver inflammation.

[0216] To assess changes in liver pathology following GDC treatment, H&E stained liver sections were examined (Figure 28). Using an established scoring criteria, changes in inflammation, extramedullary hematopoiesis and hepatocyte damage were given a score of 1 = mild, 2 = moderate, or 3 = severe for each parameter. Importantly, reducing the dose of GDC- 0084 to 7.5 mg / kg resulted in a significant reduction in liver inflammation (Figure 28A, panel 2). GDC-0084 treatment at the optimal therapeutic dose also reduced extramedullary hematopoiesis which is commonly associated with the 4T1 model (Figure 28B, panel 2). Finally, in support of our liver weight data, GDC-0084 treatment at a high daily dose of 15 mg / kg induced hepatocyte damage confirming liver toxicity (Figure 28C, panel 1). Notably, no significant changes in hepatocytes were observed at the optimal therapeutic dose of GDC-0084 (Figure 28C, panel 2). GDC-0084 αPD1 treatment reduces 4T1-associated splenomegaly and extramedullary hematopoiesis.

[0217] In addition to extramedullary hematopoiesis, enlarged spleens or splenomegaly are commonly associated with the 4T1 model. To examine changes in the spleenJAWS Ref: 750406PCT following GDC-0084 treatment, the inventors measured spleen weights in both the split dose and single dose de-escalation experiments. GDC-0084 administration at the lower dose of 7.5 mg / kg reduced splenomegaly in both dosing regimens (Figure 29A). Furthermore, assessment of spleen pathology also revealed a significant reduction in extramedullary hematopoiesis following GDC-0084 treatment at the optimal therapeutic dose of 7.5 mg / kg (Figure 29B, panel 2). Inhibition of lung leukocyte infiltration following GDC-0084 αPD1 treatment.

[0218] In light of the above data demonstrating that GDC-0084 αPD1 treatment reduces lung metastases, the inventors next sought to assess changes in lung immune cell populations following GDC-0084 treatment. Examination of lung pathology revealed a significant decrease in leukocyte infiltration into the lungs at all concentrations including the optimal therapeutic dose of 7.5 mg / kg (Figure 30). This highlights that GDC-0084 treatment not only impacts the primary tumour and metastases but also inflammation. GDC-0084 αPD1 treatment reduces lymph node metastases.

[0219] In addition to lung metastases, the 4T1 model is also associated with metastases in the lymph node. To assess lymph node metastases following GDC-0084 treatment, H&E stained lymph node sections were examined by an independent expert pathologist and given a score of 1 = 1-3 tiny sites (<< 0.5 mm in width / length), 2 = 1-3 of which at least one is 0.5-1 mm in diameter, or 3 = 2-3 or more sites, all grossly visible, with leukocyte infiltration and / or haemorrhage. In the absence of GDC-0084 treatment, large areas of metastatic tumour growth were observed, however these areas were not visible when immunotherapy was administered in combination with GDC-0084 (7.5 mg / kg) (Figure 31A). Consequently, lymph node metastases scores were substantially lower in GDC-0084 αPD1 treated mice confirming that GDC-0084 not only inhibits lung but also lymph node metastases (Figure 31B). PI3K-mTOR treatment reduces primary tumour volume.

[0220] To determine if GDC-0084 demonstrates efficacy when administered in combination with the PARP inhibitor, Olaparib, the inventors then employed the 4T1 model of TNBC. In initial experiments, GDC-0084 was administered pre or post olaparib and then repeated in a second experiment based upon the optimal dosing regimen (Figure 32A). GDC- 0084 was administered daily by oral gavage, at the optimal therapeutic dose of 7.5 mg / kg in combination with daily intraperitoneal (i.p.) injections of olaparib (Figure 32B). GDC-0084 treatment both pre and post PARP inhibitor, substantially reduced tumour volumes by 53% and 60% respectively (Figure 32C, panel 1). The inventors then confirmed these results in a second experiment using the optimal dosing regimen of GDC-0084 administration 30 minutes post olaparib (Figure 32C panel 2). Taken together, these data demonstrate that in addition to immunotherapy, GDC-0084 also demonstrated efficacy when administered in combination with PARP inhibitor thereby enhancing its applications.JAWS Ref: 750406PCT GDC-0084 and PARP inhibitor combination treatment does not induce toxicity.

[0221] To examine toxicity related complications, the present inventors monitored mouse body and liver weights in PARP inhibitor experiments (Figure 33). At the optimal therapeutic dose of 7.5 mg / kg GDC-0084 does not alter mouse body weights when administered post olaparib (Figure 33A). Similarly, no changes in liver weights were observed confirming no toxicity issues (Figure 33B). Inhibition of inflammation in the lung and liver following GDC-0084 and olaparib combination treatment.

[0222] Given the impact of GDC-0084 and immunotherapy on inflammation, the inventors next sought to examine the liver and lung pathology in PARP inhibitor studies. When administered post olaparib at 7.5 mg / kg, GDC-0084 substantially decreased liver inflammation and extramedullary hematopoiesis (Figure 34A). Examination of lung pathology also highlighted a significant reduction in lung leukocytosis in GDC-0084 and olaparib treated mice (Figure 34B) confirming the impact of GDC-0084 not only on the primary tumour but also inflammation at metastatic sites.

[0223] Finally, assessment of the spleen pathology demonstrated a reduction in extramedullary hematopoiesis in addition to spleen weights following GDC-0084 and olaparib treatment (Figure 35A and 35B). PI3K-mTOR inhibitors reduce cancer cell proliferation.

[0224] The inventors then sought to compare the efficacy of PI3K-mTOR inhibitors such as GDC-0084, and as such conducted proliferation assays using a vast array of inhibitors including the more general PI3K inhibitors: Wortmannin (general PI3K inhibitor), idelalisib (p110 δ,γ inhibitor), Alpelisib (p110α inhibitor), and LY294002 (p110α,β,δ inhibitor), and the PI3K- mTOR inhibitors: omipalisib, apitolisib, dactolisib and GDC-0084 (p110α,β,δ,γ, mTOR inhibitor) (Figure 36). The PI3K-mTOR inhibitors had the most dramatic effect on MDA-MB-231 cell proliferation with omipalisib, apitolisib and dactolisib all having IC50 values below 1.0 μM (Table 16). GDC-0084 in comparison had an IC50 value of 2.5 μM. The more general PI3K inhibitors displayed substantially higher values confirming that inhibition of both PI3K and mTOR is critical in preventing cancer cell proliferation. TABLE 16 Inhibitor IC50(µM) GDC-0084 2.5 Dactolisib 0.4 Apitolisib 0.6 Omipalisib 0.3JAWS Ref: 750406PCT LY294002 25.2 Alpelisib 133.3 Idelalisib 91.6 ND Inhibition of PI3K a

[0225] In order ibition on cancer cell migration the inventors con reast cancer cell line, MCF-7, pre-stimulated with enotype (Figure 37A). In GDC-0084 dose-response was observed at concentrations of 1.25 μM gure 37B).

[0226] Again, t PI3K-mTOR inhibitors, the inventors performed a seco d omipalisib using IC50 concentrations from the proGDC-0084, all PI3K-mTOR inhibitors significantly reduced wound healing density (Figure 38B), confirming that inhibition of both PI3K and mTOR hinders cell migration. Materials & Methods Animal studies

[0227] Female BALB / c mice, 6-8 weeks of age, were procured from the Animal Resources Centre (ARC). Following acquisition, the mice were allowed a one-week acclimatization period within the containment suites at QIMR Berghofer Medical Research Institute (QIMRB). All experimental procedures were performed in accordance with the guidelines and regulations endorsed by the QIMRB Ethics Committee.1 x 1054T1 cells, suspended in phosphate-buffered saline (PBS), was administered into the mammary fat pad of BALB / c mice. Treatments were started when tumours reached approximately 50-100 mm3. In the 4T1 model, mice were subjected to daily oral administration of GDC at the indicated doses in combination with either anti-PD-1 treatment (10 mg / kg) administered intraperitoneally at day 0 and day 4 or olaparib (50 mg / kg) administered daily. Tumours were measured using external callipers and volumes calculated using a modified ellipsoidal formula ½ (a / b2), where a = longest side and b = shortest side. Mice were monitored daily for clinical abnormalities (reduced activity, hunched posture, piloerection, weight loss and metastasis), and tumour volumes were measured thrice weekly. Once tumours reached the maximal limit (1000 mm3) in the vehicle group, all tumours and associated metastatic organs (lung, liver, spleen) were harvested, weighed, imaged and fixed in 4% paraformaldehyde for associated analysis.JAWS Ref: 750406PCT Pathology analysis.

[0228] H&E stained FFPE livers, lungs, lymph nodes and spleens were subjected to pathology analysis by an independent expert pathologist. Changes in inflammation, extramedullary hematopoiesis (EMH), hepatocytes and lung leukocytosis were given a score of 1 = mild, 2 = moderate, or 3 = severe. Lymph node sections were given a score of 1 = 1-3 tiny sites (<< 0.5 mm in width / length), 2 = 1-3 of which at least one is 0.5-1 mm in diameter or 3 = 2- 3 or more sites, all grossly visible, w / leukocyte infiltration & / or haemorrhage. WST-1 cell proliferation assay.

[0229] MDA-MB-231 cells were seeded at optimized densities in a 96-well flat bottom tissue culture plate in a total volume of 100 µL / well. The cells were left to adhere overnight at 37°C and 5% CO2. Cells were then treated with PI3K inhibitors at the indicated doses and left in the incubator at 37°C and 5% CO2 for 72 hours, after which media was removed and replaced with 100 µL / well of WST-1 cell proliferation reagent (Sigma-Aldrich, 11644807001) at a 1:10 final dilution in complete cell culture medium. WST-1 reagent contains water-soluble tetrazolium salts which are cleaved by cellular mitochondrial dehydrogenase enzymes to produce formazan. The amount of formazan produced per reaction is then quantified using a microplate spectrophotometer to measure metabolic activity. The absorbance measured directly correlates to the number of metabolically active cells in culture. Absorbance was recorded at 450 nm at 0.5, 1, and 2 hour incubation periods using a microplate spectrophotometer (following a 30 second mix time). 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 (four parameters) using GraphPad Prism 8 software. Scratch wound healing assay.

[0230] MCF7 cells were stimulated with PMA / TGFβ for 24 hours prior to seeding at optimized cell density in a 96-well Incucyte® Image Lock plate in a total volume of 100 ^L / well in low-serum media (2%). The cells were left to adhere overnight at 37°C and 5% CO2. Cells were checked to be at 100% confluence in order to ensure consistent wounds are created. After 24 hours, wounds were created using the 96-pin Incucyte® WoundMaker Tool. Post wounding, cells were washed with low serum media to remove non-adherent cells and then treated with PI3K inhibitors at the indicated doses prepared in low-serum media. The plate was then placed into the Incucyte® live-cell analysis system and wound healing was tracked using the Incucyte® Zoom live-cell analysis system with scan interval every 4 hours. REFERENCES Hassan 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: 750406PCT Chen 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 17β-Estradiol via PI3K / Akt Signaling Pathway in ERα-Positive Cancer Cell Lines. Int J Gynecol Cancer.2017 Feb;27(2):196-205. Fusco N, Malapelle U, Fassan M, Marchiò 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.

Claims

JAWS Ref: 750406PCT WHAT IS CLAIMED IS:

1. A method of altering epithelial to mesenchymal cell transition or mesenchymal to epithelial cell transition of a PI3K overexpressing cell, comprising contacting the PI3K overexpressing cell with a composition comprising a PI3K inhibitor and an immunotherapy that does not target cancer stem cells (CSC).

2. A method of treating or preventing a cancer in a subject, wherein the cancer comprises at least one PI3K overexpressing cell, the method comprising administering to the subject a composition comprising a PI3K inhibitor and an immunotherapy that does not target cancer stem cells (CSC).

3. The method according to claim 1 or claim 2, wherein the PI3K overexpressing cell is a CSC.

4. The method of claim 3, wherein the PI3K overexpressing cell is a CSC tumour cell.

5. The method of any one of claims 1 to 4, wherein the PI3K overexpressing cell expresses one or more mesenchymal / cancer stem cell markers selected from the group comprising CSV, EGRF, and ABCB5, SoX9, SNAIL, and AKT1.

6. The method of any one of claims 1 to 5, wherein the epithelial cell is characterised by the expression of one or both of EpCAM and MDA5.

7. The method of any one of claims 2 to 6, wherein the subject comprises a CD8+ T cell population that expresses one or more of TIM3, TIGIT, PD1, TOX1, and EOMES.

8. The method of any one of claims 1 to 7, wherein the epithelial cell expresses an epithelial cell signature that comprises E-cadherin.

9. The method of any one of claims 1 to 8, wherein the CSC-targeting immunotherapy is an immune checkpoint molecule (ICM) antagonist.

10. The method of claim 9, wherein the ICM antagonist is a PD1 antagonist, a PD-L1 antagonist, a CTLA4 antagonist, or a PD-L2 antagonist.

11. The method of claim 10, wherein the ICM antagonist is an antigen-binding molecule (e.g., an antibody).

12. The method of any one of claims 1 to 11, wherein the immunotherapy is a PARP inhibitor.

13. The method of any one of claims 1 to 12, wherein the PI3K inhibitor is a catalytic PI3K inhibitor.

14. The method of claim 10, wherein the PARP inhibitor selected from the group comprising olaparib, talazoparib, veliparib, niraparib, and rucaparib.

15. The method of any one of claims 1 to 14, wherein the PI3K inhibitor is selected from the group comprising paxalisib (GDC-0084), idelalisib, LY294002, 3-Methyladenine, alpelisib, quercetin, wortmannin, GNE-490, PI3K-IN-36, 740 Y-P, AZD-7648, buparlisib, inavolisib, dactolisib, copanlisib, eganelisib, pictilisib, SAR405, duvelisib, taselisib, recilisib, YM- 201636, omipalisib, PI-103, alpha-linolenic acid, fimepinostat,and isorhamnetin.JAWS Ref: 750406PCT 16. The method of any one of claims 1 to 15, wherein the PI3K inhibitor is paxalisib (GDC-0084).

17. The method of any one of claims 2-16, wherein the prior to administering the composition to the subject, the subject is screened for expression of one or more of the biomarkers CSV, EGRF, ABCB5, SoX9, SNAIL, AKT1, EpCAM, MDA5, TIM3, TIGIT, PD1, TOX1, EOMES, and E-cadherin.

18. Use of a PI3K inhibitor and an immunotherapy that does not target cancer stem cells (CSC) 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 a recurrence of cancer.

19. Use of a PI3K inhibitor and an immunotherapy that does not target cancer stem cells (CSC) in the manufacture of a medicament 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 a recurrence of cancer.

20. The use of claim 18 or claim 19, wherein the PI3K inhibitor and the immunotherapy are formulated for concurrent administration.

21. Use of a PI3K inhibitor, an immunotherapy that does not target cancer stem cells (CSC) and an ancillary agent (e.g., a chemotherapeutic agent) for treating, or for aiding in the treatment of, 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 a recurrence of cancer.

22. Use of a PI3K inhibitor, an immunotherapy that does not target cancer stem cells (CSC) and an ancillary agent (e.g., a chemotherapeutic agent) in the manufacture of a medicament for treating, or for aiding in the treatment of, 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 a recurrence of cancer.

23. The use of claim 21 or claim 22, wherein the PI3K inhibitor, immunotherapy, and ancillary agent (e.g., a chemotherapeutic agent) are formulated for concurrent administration.

24. The method of any one of claims 18 to 23, further comprising detecting an elevated level of one or more of TIM3, TIGIT, PD1, TOX1, and EOMES in a T cell (e.g., relative to the level of TIM3, TIGIT, PD1, TOX1, and EOMES in an activated T-cell) in a sample obtained from the subject, prior to the concurrent administration.

25. The method of any one of claims 18 to 24, further comprising detecting an elevated level of one or more of TIM3, TIGIT, PD1, TOX1, and EOMES in a T cell (e.g.,JAWS Ref: 750406PCT relative to the level of TIM3, TIGIT, PD1, TOX1, and EOMES in an activated T-cell) in a sample obtained from the subject, prior to the concurrent administration.

26. A kit comprising a medicament comprising a PI3K inhibitor and an optional pharmaceutically acceptable carrier, and a package insert comprising instructional material for concurrent administration of the medicament with another medicament comprising an immunotherapy that does not target cancer stem cells (CSC) and an optional pharmaceutically acceptable carrier 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 a recurring cancer in an individual.

27. A kit comprising a medicament comprising an immunotherapy that does not target cancer stem cells (CSC) and an optional pharmaceutically acceptable carrier, and a package insert comprising instructional material for concurrent administration of the medicament with another medicament comprising a PI3K inhibitor and an optional pharmaceutically acceptable carrier 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 a recurrence of cancer in an individual.

28. A kit comprising a first medicament comprising a PI3K inhibitor and an optional pharmaceutically acceptable carrier, and a second medicament comprising an immunotherapy that does not target cancer stem cells (CSC) and an optional pharmaceutically acceptable carrier 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 a recurrence of cancer in an individual.

29. The kit of claim 28, further comprising a package insert comprising instructional material for administering concurrently the first medicament and the second medicament 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 a recurrence of cancer in an individual.

30. A pharmaceutical composition for treating cancer in a subject, the composition comprising a unit dose of a PI3K inhibitor, wherein the unit dose of the PI3K inhibitor is less than 75% of the therapeutic dose when administered alone.

31. The composition of claim 30, wherein the PI3K inhibitor is GDC-0084, and the unit dose corresponds to an administration to the subject of about 11.25 mg / kg or less.JAWS Ref: 750406PCT 32. The composition of claim 30 or claim 31, wherein the PI3K inhibitor is GDC- 0084, and the unit dose corresponds to an administration to the subject of about 7.5 mg / kg or less.

33. The composition of any one of claims 30 to 32, wherein the PI3K inhibitor is GDC-0084, and the unit does corresponds to an administration to the subject of about 4 mg / kg or less.

34. The composition of any one of claims 30 to 31, wherein the composition further comprises an immunotherapy that does not target cancer stem cells (CSC).

35. The composition of claim 24, wherein the immunotherapy is an ICM antagonist (e.g., a PD1 antagonist, a PDL1 antagonist, a CTLA4 antagonist, etc.) or a PARP inhibitor.

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