Compositions and methods for improved cancer treatment

Abstract

The present disclosure relates generally to compositions and methods for treating cancer. More specifically, the disclosure relates to compositions and their use in altering one of epithelial-to-mesenchymal or mesenchymal-epithelial transition in tumor cells. The method includes 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 (CSCs), wherein the immunotherapy is either an immune checkpoint antagonist or a PARP inhibitor.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates generally to compositions and their use in the treatment of cancer. More specifically, the present invention relates to compositions and their use in altering one of epithelial-mesenchymal or mesenchymal-epithelial transition of tumor cells. [Background technology]

[0002] Background of the Invention

[0002] Reference in this specification to any prior publication (or information derived therefrom) or any known matter is not, and should not be considered as, an acknowledgement or admission, or any form of suggestion, that the prior publication (or information derived therefrom) or known matter forms part of the common general knowledge in the field of endeavor to which this specification pertains.

[0003] Phosphatidylinositol is one of several phospholipids found in cell membranes that plays an important role in intracellular signal transduction. Cell signaling via 3'-phosphorylated phosphoinositides is involved in various cellular processes, such as 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 known as PI3 kinase or PI3K), was originally identified as an activity associated with viral oncoproteins and growth factor receptor tyrosine kinases, which phosphorylate phosphatidylinositol (PI) and its phosphorylated derivatives at the 3'-hydroxyl position of the inositol ring (Panayotou et al. (1992) Trends Cell Biol 2: 358-60).

[0004] Phosphoinositide 3-kinase (PI3K) is a lipid kinase that phosphorylates lipids at the 3-hydroxyl residue of the inositol ring (Whitman et al. (1988) Nature, 332:664). The 3-phosphorylated phospholipid (PIP3) generated by PI3 kinase acts as a second messenger to recruit kinases with lipid-binding domains (including pleckstrin homology (PH) regions), such as Akt and phosphoinositide-dependent kinase-1 (PDK1). Binding of Akt to membrane PIP3 triggers its translocation to the plasma membrane, bringing Akt into contact with PDK1, which activates Akt. The tumor 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).

[0005] The predominant PI3K isoform in cancer is class I PI3 kinase, p110α (alpha) (as described in U.S. Patent Nos. 5,824,492, 5,846,824, and 6,274,327). Other isoforms are involved in cardiovascular and immune-mediated inflammatory diseases (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 WJ, Lane MD eds) Elsevier Academic Press). The PI3K / Akt / PTEN pathway is an attractive target for cancer drug development because such modulators or inhibitors are expected to inhibit proliferation, reverse the suppression of apoptosis, and overcome resistance to cytotoxic agents in cancer cells (Folkes et al. (2008) J. Med. Chem. 51: 5522-5532, Yamaguchi et al. (2006) Jour, of the Nat. Cancer Inst. 98(8):545-556).

[0006] However, despite numerous clinical trials investigating the use of PI3K inhibitors for the treatment of many solid tumors, no candidates have yet been successfully introduced into clinical practice. Therefore, further research is needed to successfully utilize these promising drug candidates to improve efficacy and reduce toxicity for use in these challenging diseases. Summary of the Invention [Means for solving the problem]

[0007] Summary of the Invention

[0007] The present invention is based, in part, on the discovery that PI3K inhibitors have significant activity in inhibiting EMT, inhibiting the formation and maintenance of cancer stem cells (CSCs) and inducing mesenchymal-epithelial transition (MET), making them useful for treating a wide range of cancers (e.g., solid tumors), including recurrent cancers.

[0008]

[0008] In one aspect of the present invention, there is provided a method for modifying epithelial-mesenchymal or mesenchymal-epithelial transition of PI3K-overexpressing cells, the method comprising contacting the PI3K-overexpressing cells with a composition comprising a PI3K inhibitor and an immunotherapy that does not target cancer stem cells (CSCs).

[0009] In another aspect, the present invention provides a method of treating or preventing 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 (CSCs). 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 tumor cell.

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

[0012]

[0012] The epithelial cells are preferably characterized by expression of one or both of EpCAM and MDA5. Further, 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 cells express an epithelial cell signature that includes E-cadherin.

[0013] In some embodiments, the immunotherapy targeting CSCs is an immune checkpoint molecule (ICM) antagonist. For example, the ICM antagonist can 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]

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

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

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

[0018]

[0018] Another aspect of the present invention provides the use of a PI3K inhibitor in combination with an immunotherapy that does not target cancer stem cells (CSCs) to treat a T cell dysfunction disorder or enhance immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, treat or delay the progression of cancer, or treat the recurrence of cancer.

[0019]

[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 (CSCs) in the manufacture of a medicament for treating a T cell dysfunction disorder or enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, treating or delaying the progression of cancer, or treating the recurrence of cancer.

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

[0021]

[0021] In yet another aspect, the present invention provides the use of a PI3K inhibitor, a non-cancer stem cell (CSC)-targeted immunotherapy, and an adjuvant (e.g., a chemotherapeutic agent) to treat or assist in the treatment of a T cell dysfunction disorder, or to enhance immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, to treat or delay the progression of cancer, or to treat the recurrence of cancer.

[0022]

[0022] In another aspect, the present invention provides the use of a PI3K inhibitor, a non-cancer stem cell (CSC)-targeted immunotherapy, and an adjuvant (e.g., a chemotherapeutic agent) in the manufacture of a medicament for treating or assisting in the treatment of a T cell dysfunction disorder, or enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, or treating or delaying the progression of cancer, or treating the recurrence of cancer.

[0023] In some preferred embodiments, the PI3K inhibitor, immunotherapy and adjunctive agent (eg, chemotherapeutic agent) are formulated for combined administration.

[0024]

[0024] Preferably, the use may further comprise a step of detecting elevated levels of one or more of TIM3, TIGIT, PD1, TOX1 and EOMES in T cells (e.g., relative to the levels of TIM3, TIGIT, PD1, TOX1 and EOMES in activated T cells) in a sample obtained from the subject prior to combined administration.

[0025]

[0025] In yet another embodiment, the present invention provides a kit comprising a drug comprising a PI3K inhibitor and an optional pharmaceutically acceptable carrier for treating a T cell dysfunction disorder or enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, treating or delaying the progression of cancer, or treating recurrent cancer in an individual, and a package insert containing instructions for administering the drug in combination with another drug comprising an immunotherapy that does not target cancer stem cells (CSCs) and an optional pharmaceutically acceptable carrier.

[0026]

[0026] In yet another embodiment, the present invention provides a kit comprising a pharmaceutical agent comprising a non-cancer stem cell (CSC)-targeted immunotherapy and an optional pharmaceutically acceptable carrier for treating a T cell dysfunction disorder or enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, treating or delaying the progression of cancer, or treating the recurrence of cancer in an individual, and a package insert containing instructions for administering the pharmaceutical agent in combination with another pharmaceutical agent comprising a PI3K inhibitor and an optional pharmaceutically acceptable carrier.

[0027]

[0027] In another aspect of the present invention, there is provided a kit comprising a first agent comprising a PI3K inhibitor and optionally a pharmaceutically acceptable carrier for treating a T cell dysfunction disorder, or enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, or treating or delaying the progression of cancer, or treating recurrence of cancer in an individual, and a second agent comprising an immunotherapy that does not target cancer stem cells (CSCs) and optionally a pharmaceutically acceptable carrier. Preferably, the kit further comprises a package insert containing instructions for administering the first agent and the second agent in combination to treat a T cell dysfunction disorder, or enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, or treating or delaying the progression of cancer, or treating recurrence of cancer in an individual.

[0028]

[0028] In another aspect, the present invention provides a pharmaceutical composition for treating cancer in a subject, 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 administration of about 11.25 mg / kg or less to a subject. Preferably, the PI3K inhibitor can be GDC-0084, and the unit dose corresponds to administration of about 7.5 mg / kg or less to a subject. A PI3K inhibitor that is GDC-0084 is also contemplated, and the unit dose corresponds to administration of about 4 mg / kg or less to a subject.

[0030]

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

[0031] In some embodiments, the immunotherapy is selected from an ICM antagonist (eg, a PD1 antagonist, a PDL1 antagonist, a CTLA4 antagonist, etc.) or a PARP inhibitor.

[0032] Brief description of the diagram The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present 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. [Brief explanation of the drawings]

[0033] [Figure 1]

[0033] Provides a KM survival plot for PI3K protein expression in stage IV metastatic cancer survival. [Figure 2]

[0034] Using our optimized laboratory protocol, we processed liquid biopsies from cancer patients in the IO-resistant or IO-responsive cohort, isolated PBMCs from the liquid biopsy samples, and then stained the samples for CSV, PI3KCA, and ABCB5. [Figure 3]

[0035] CSV, ABCB5, EpCAM, PI3KCA, and AKT1 (AKT1 plays a role in promoting invasion and metastasis) fluorescence intensity (FI) and population dynamics (% cell population) were determined in CTCs from liquid biopsies from patients with stage IV metastatic solid tumors using ASI Digital Pathology. CTCs were treated with GDC-0084 or vehicle control. (A) Depicts two mesenchymal markers: CSV, ABCB5, and PI3KCA / AKT1. (B) Percentage population of CSV+ABCB5+ cells or EpCAM+ cells or % population with PI3KCA / AKT1. [Figure 4]

[0036] Using our optimized laboratory protocol, we processed liquid biopsies from patients with stage IV metastatic solid tumor cancer and isolated PBMCs from the liquid biopsy samples. The samples were then stimulated (PMA / CaI stimulation for 4 hours in the presence of ST-brefeldin A) or unstimulated (NS) and / or treated with the GDC-0084 inhibitor. The samples were then stained for markers from three separate panels: exhaustion signature panel: EOMES, PD1, and CD8; checkpoint panel: TIGIT, TIM3, and CD8; and effector signature panel: perforin, GZNb, and CD8. [Figure 5]

[0037] For the PI3KCA signature, (A) MCF7 (epithelial breast cancer), MDAMB231 (mesenchymal / CSC breast cancer), MDAMB231-Br (a brain cancer version of MDA-MB-231), and 4T1 (a highly aggressive, IO-resistant murine breast cancer) cell lines, and (B) H1299 (epithelial-like lung cancer), CT26 (epithelial-like highly immunogenic murine colon tumor), and (LLC) (highly treatment-resistant Lewis lung carcinoma) cell lines were stained. Samples were permeabilized with Triton X-100 and stained with primary mouse antibodies against CSV and primary rabbit antibodies against PI3KCA, followed by detection with donkey AF anti-mouse 568 and anti-rabbit 647. [Figure 6]

[0038] (C) Graphical representation of the relative wound density of MDA-MB-231 cells treated with PI3K inhibitors. Black bars: DMSO control; light gray bars: IC25 concentration 1.25 μM; dark gray bars: IC50 concentration 2.5 μM. Error bars correspond to the mean ± SD from eight replicates. P values ​​were calculated using two-way ANOVA analysis with Tukey's multiple comparison test, where 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. [Figure 7]

[0039] The IC50 of GDC-0084 against different cancer cell lines is demonstrated. WST-1 cell proliferation reagent was added to CT26, 4T1, and MCF7 cells pretreated with PI3K inhibitors for 72 hours in a dose-response design. Cell proliferation was measured indirectly by the formation of formazan and absorbance recorded at 450 nm. Percent (%) proliferation was determined, and the EC50 was calculated using Graphpad Prism software as described in the methods. [Figure 8]

[0040] Figure 1 shows that treatment with olaparib upregulates the resistance mesenchymal marker ABCB5. MDA-MB-231 cells were treated with either vehicle ("CTRL") or olaparib ("Ola."). Expression of the resistance cancer stem cell-like marker ABCB5 was analyzed by immunofluorescence staining quantification. Images show IF staining of (A) ABCB5 and (B) ALDH1A1, demonstrating upregulation of ABCB5 and ALDH1A1 expression. ***p≦0.001. This figure demonstrates that PARP inhibition can upregulate markers of the cancer stem cell resistance signature. [Figure 9]

[0041] Figure 1: Olaparib treatment failed to inhibit CSCs or cell proliferation. MDA-MB-231 cells were treated with olaparib for 24 hours. Cells were harvested, stained with APC / CD44 and PE / CD24, and then subjected to flow cytometry. (A) Percent inhibition of CSCs compared to control is shown, followed by representative flow cytometry images. (B) MDA-MB-231 cells were treated with either olaparib or talazoparib at three different concentrations, and cell proliferation was estimated by WST-1 assay. [Figure 10]

[0042] Graphical and photographic representations of PARP knockdown are provided. PARP1 knockdown was performed using two different concentrations of PARP1 siRNA: 5 nM and 10 nM, including 5 nM, which was previously established as the optimal concentration in our laboratory for 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. PARP1 mRNA expression was significantly depleted at both concentrations (A). Panels (B) and (C) show the nuclear fluorescence intensity of PARP. Graphs represent the mean ± standard error from three independent experiments. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****P < 0.0001, two-way ANOVA. [Figure 11]

[0043] Figure 1 shows that PARP1 knockdown increases resistance marker expression. MDA-MB-231 cells were incubated with 5 nM PARP1 siRNA for 48 hours, followed by RNA extraction and cDNA synthesis, followed by cell fixation for qPCR amplification or immunofluorescence staining. (A, B) mRNA expression; (C, D) immunofluorescence intensity of mesenchymal markers after PARP1 knockdown; (E) PD-L1. Relative fold changes in mRNA expression or changes in total nuclear fluorescence intensity (TNFI) or total cytoplasmic fluorescence intensity (TCFI) are shown. *p≤0.05, **p≤0.01, ***p≤0.001, ****P<0.0001, two-way ANOVA. Wound healing (scratch assay) analysis of the effect of the combination of GDC-0084 and olaparib on cell migration. Images represent MDA-MB-231 cell lines treated with (F) DMSO control, (G) GDC-0084 at an IC25 concentration of 1.25 μM and olaparib at 5 nM, and (H) GDC-0084 at an IC50 concentration of 2.5 μM and olaparib at 250 nM. Error bars correspond to the mean ± SD from eight replicates. P values ​​were calculated using two-way ANOVA analysis with Tukey's multiple comparison test; 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. [Figure 12]

[0044] Figure 1 shows αPD1 therapy in CT26 colon cancer model and 4T1 breast cancer model. Treatment regimes were demonstrated using Balb / c CT26 (A) colon cancer model and 4T1 (B) breast cancer model. Tumor volumes (n=5 / group) in mice treated with vehicle (IgG) or αPD1 (10 mg / kg). *p<0.05, **p<0.01, unpaired t-test. [Figure 13]

[0045] PI3K inhibition targets and inhibits the expression of mesenchymal resistance markers. 4T1 (A) or CT26 (B) cancer cell lines were treated with control or two low concentrations of GDC-0084 (0.2 or 0.3 μM). Samples were then permeabilized with Triton-X100 and stained with primary mouse antibodies against CSV or EpCAM, primary rabbit antibodies against EGFR or FOXN2, or primary goat antibodies against ABCB5, followed by detection with donkey AF anti-mouse 488, anti-rabbit 568, or anti-goat 647 secondary antibodies. Protein targets were imaged using an ASI Digital Pathology System with a 100x objective. Example image areas with scale bars (orange) are depicted above. Analysis was performed by comparing fluorescence intensity for EGFR, cytoplasmic fluorescence intensity (CFI) for CSV, or total fluorescence intensity for ABCB5. Data were plotted in PRISM and analyzed using a one-way ANOVA nonparametric test with Kruskal-Wallis. Significant differences are plotted. [Figure 14]

[0046] PI3K inhibition induces immunovisible markers. 4T1 (A) or CT26 (B) cancer cell lines were treated with control or GDC-0084 (at concentrations of 0.2 or 0.3 mM). Samples were then permeabilized with Triton-X100 and stained with primary mouse antibodies against CSV or EpCAM, primary rabbit antibodies against MDA5, and detected with donkey AF anti-mouse 488, anti-rabbit 568, or anti-goat 647 secondary antibodies. Protein targets were imaged using an ASI Digital Pathology System with a 100x objective. Example image fields with scale bars (orange) are depicted above. Analysis was performed by comparing fluorescence intensities. Data were plotted in PRISM and analyzed using a one-way ANOVA nonparametric test with Kruskal-Wallis. Significant differences are plotted. [Figure 15]

[0047] PI3K inhibition targets and inhibits the stem-like cancer stem cell-like cancer recurrence signature and induces the epithelial signature. MDA-MB-231TNBC cancer cell lines were treated with control or two different PI3K inhibitors, idelalisib (at two low concentrations: 12.5 μM or 25 μM) or LY294002 (at two low concentrations: 2.5 μM or 5 μM). Samples were then permeabilized with Triton-X100 and stained with primary mouse antibodies against CSV or EpCAM, primary rabbit antibodies against SoX9 (a marker for mesenchymal cancer stem cells and advanced, therapy-resistant cancer cells), or PI3K, or primary goat antibodies against ABCB5 or SNAIL (Figures 11A and 11B). Detection was performed with donkey AF anti-mouse 488, anti-rabbit 568, or anti-goat 647 secondary antibodies. Protein targets were imaged using an ASI Digital Pathology System with a 40x objective. Example image regions with scale bars (orange) are depicted above. Analysis was performed comparing nuclear fluorescence intensity for SoX9, cytoplasmic fluorescence intensity (CFI) for CSV and EpCAM, or total fluorescence intensity for ABCB5 or SNAIL. Data were plotted in PRISM and analyzed with a one-way ANOVA nonparametric test using Kruskal-Wallis. Significant differences are plotted. [Figure 16]

[0048] GDC-0084 administration at 7.5 mg / kg reduces clinical abnormalities in a 4T1 syngeneic tumor model. GDC-0084 therapy in a 4T1 breast cancer model. Treatment regime using a Balb / c 4T1 breast cancer model. GDC-0084 was administered daily at 7.5 mg / kg (A) or 15 mg / kg (B). (C) Percentage of mice exhibiting clinical abnormalities (reduced activity, hunched posture, piloerection, weight loss, and metastases) at endpoint after GDC-0084 monotherapy. [Figure 17]

[0049] Administration of 7.5 mg / kg GDC-0084 inhibits tumor burden. Representative images of tumors harvested from mice administered (A) 7.5 mg / kg GDC-0084 or (C) 15 mg / kg + / - αPD1 in a Balb / c 4T1 breast cancer model. (B, D) Primary tumor volume and final tumor weight in mice treated with (B) 7.5 mg / kg GDC-0084 or (D) 15 mg / kg + / - αPD1 (10 mg / kg) once daily. Data presented as mean ± SEM. One-way ANOVA with Tukey post-hoc analysis, ***p<0.001, ****p<0.0001 (n=5 / group). [Figure 18]

[0050] Administration of 7.5 mg / kg GDC-0084 reduces tumor inflammation, including monocyte and neutrophil infiltration. Representative images of H&E-stained tumors and pathology scores for tumor inflammation in mice treated with (A) 7.5 mg / kg or (B) 15 mg / kg GDC-0084 + / - αPD1 once daily in a 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). [Figure 19]

[0051] GDC-0084 administration at 7.5 mg / kg reduces splenomegaly in a 4T1 syngeneic tumor model. Representative images of spleens harvested from mice treated with (A) 7.5 mg / kg or (B) 15 mg / kg GDC-0084 + / - αPD1 once daily in a 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). [Figure 20]

[0052] Administration of 7.5 mg / kg of GDC-0084 reduced SOX9 expression. Qupath analysis of captured images was used to plot bar graphs for: (A) CSC signature (SOX9, PDL1, panCK), n>500,000 cells; (B) CD8 dysfunction signature (CD8, TOX1, PD1), n>10,000 cells; (C) CSC signature (SOX9, PDL1, panCK), n>100,000 cells; (D) CD8 dysfunction signature (CD8, TOX1, PD1), n>10,000 cells. Qupath analysis was used to select DAPI-positive cells, followed by PDL1- and panCK-positive CSC signature cells, and score them based on SOX9 fluorescence intensity. For the CD8 dysfunction signature, DAPI-positive cells were then selected for CD8+ and PD1+ cells and scored based on TOX1 fluorescence intensity. Images were extracted from an Aperio FL fluorescent slide scanner of stained FFPE tumor sections from GDC mouse models treated with either control, anti-PD1 immunotherapy, GDC-0084 (high dose in C / D and low dose in A / B), or the combination. FFPE sections of lung tissue were processed as described above and stained for SOX9, PDL1, panCK, or CD8, TOX1, and PD1, and DAPI was used to stain nuclei. Data are plotted as mean ± SD and represent SOX9 or TOX1 fluorescence intensity. Tukey's post-hoc test, ns = not significant. ***p<0.001, ****p<0.0001. [Figure 21]

[0053] PI3K inhibition alone or in combination induces an epithelial phenotype by targeting the mesenchymal signature in IO-resistant mouse models. FFPE primary tumor samples processed on BONDRX with Opal staining kits targeting PI3KCA, CSV, E-cadherin, or EGFR. (A)-(D) Quantitation of fluorescence intensity (FI) for PI3KCA, CSV, E-cadherin, or EGFR was determined using an ASI Digital Pathology system. [Figure 22]

[0054] PI3K alone or in combination induces an epithelial phenotype in an IO-resistant mouse model. FFPE primary tumor samples were processed on BONDRX with Opal staining kits targeting PI3KCA, CSV, E-cadherin, or EGFR. % population dynamics were determined for % positive cells for E-cadherin expression using the ASI Digital Pathology Platform. [Figure 23]

[0055] PI3K monotherapy or combination therapy induces effector and TRM signatures. Quantification of CD8+ T cell-expressed markers (CD103, CD69, CD44, and CD8) on CD8+ IFNγ+ T cell-positive cells (A) or tissue-resident memory cells was also performed via staining of FFPE tumor samples with the Opal staining kit and image analysis using ASI Digital Pathology. Significant differences are shown as calculated by the Kruskal-Wallis nonparametric test. [Figure 24]

[0056] PI3K alone or in combination inhibits the checkpoint exhaustion signature in CD8+ T cells. FFPE primary tumor samples were processed using a tyramide staining kit targeting CD8, LAG3, and TIM3 checkpoint markers. % population dynamics were determined for % positive cells for each marker using the ASI Digital Pathology Platform. % expression of TIM3-positive and LAG3-positive CD8+ T cells out of total CD8+ T cells was calculated. Graphs represent n=3 mice / group using Kruskal-Wallis test to calculate significance. ns=not significant, *p=<0.02. [Figure 25]

[0057] Combining PI3K inhibition with immunotherapy abrogates metastatic spread in the 4T1 IO-resistant model. GDC-0084 + / - αPD1 therapy in the 4T1 model of metastatic breast cancer. (A) Treatment regime using a Balb / c 4T1 breast cancer model. (B) Number of metastatic tumor nodules present in the lungs of individual mice at day 20 post-inoculation; **p<0.01, Tukey's post-hoc test. [Figure 26]

[0058] Optimal therapeutic dose of GDC-0084 established in a 4T1 TNBC model. (A) Treatment regime using a Balb / c 4T1 TNBC breast cancer model. (B) Pipeline for dose de-escalation experiments. In stage I, GDC-0084 was administered at a dose of 15 mg / kg + / - αPD1 (10 mg / kg) daily on days 0 and 4. In stage II, split daily doses of GDC-0084 (up to 15 mg / kg) were administered every 4 hours in combination with αPD1 on days 0 and 4. In the final stage, GDC-0084 was administered as a single daily dose (up to 7.5 mg / kg) in combination with αPD1 on days 0 and 4. (C) Tumor volumes (% vehicle) of individual mice from each experimental stage before harvest, *p<0.05, **p<0.01, ***p<0.001, ns not significant, unpaired t test or Dunnett's post hoc test, n=4–5 / group. [Figure 27]

[0059] Dose reduction of GDC-0084 in mice improves safety. (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 death. (B) Liver weights were measured at the time of harvest. *p<0.05, unpaired t-test, n=4-5 / group. [Figure 28]

[0060] No hepatotoxicity was observed with the optimal therapeutic dose of GDC-0084. H&E-stained FFPE livers from the split-dose de-escalation and single-dose de-escalation studies were scored by an expert pathologist. (A) Liver inflammation, (B) extramedullary hematopoiesis (EMH), and (C) hepatocellular changes (metabolic and / or degenerative) were scored as 1 = mild, 2 = moderate, or 3 = severe for each parameter. *p<0.05, ***p<0.001, ****p<0.0001 vs. vehicle, Dunnett's post-hoc test, n=4-5 / group. [Figure 29]

[0061] PI3K-mTOR inhibition reduces splenomegaly and extramedullary hematopoiesis in the spleen. (A) Spleen weights were measured at the time of harvest from the split-dose de-escalation and single-dose de-escalation experiments. *p<0.05, **p<0.01, ****p<0.0001 vs. vehicle, Dunnett's post-hoc test, n=4-5 / group. (B) H&E-stained FFPE spleens were scored by a pathologist. Extramedullary hematopoietic (EMH) changes were given a score of 1=mild, 2=moderate, or 3=severe. *p<0.01, ***p<0.001 vs. vehicle, Dunnett's post-hoc test, n=4-5 / group. [Figure 30]

[0062] PI3K-mTOR inhibition reduces leukocyte infiltration into the lungs. H&E-stained FFPE lungs from the split-dose de-escalation and single-dose de-escalation experiments were scored by an expert pathologist. Leukocytic changes were given a score of 1 = mild, 2 = moderate, or 3 = severe. *p<0.01, ***p<0.001 vs. vehicle, Dunnett's post-hoc test, n=4-5 / group. [Figure 31]

[0063] Combined treatment with GDC-0084 and αPD1 prevents lymph node metastasis. (A) Images from H&E-stained FFPE lymph nodes from αPD1-treated 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 for leukocyte infiltration and / or hemorrhage as follows: 1 = 1 to 3 very small areas (width / length << 0.5 mm), 2 = 1 to 3 areas (at least one of which is 0.5 to 1 mm in diameter), or 3 = 2 to 3 or more areas (all visible to the naked eye). [Figure 32]

[0064] Efficacy of GDC-0084 demonstrated in combination with PARP inhibitors. (A) Pipeline for PARP inhibitor experiments. In Stage I, GDC-0084 was administered at a dose of 7.5 mg / kg daily either before (pre) or 30 minutes after (post) olaparib (50 mg / kg). In Stage II, GDC-0084 was administered at a dose of 7.5 mg / kg daily 30 minutes after olaparib (50 mg / kg). (B) Treatment regime using the Balb / c 4T1 TNBC breast cancer model. (C) Tumor volume (% vehicle or olaparib) of individual mice from each experimental stage before harvest. **p<0.01, ns not significant, Tukey's post-hoc test, n=5 / group. [Figure 33]

[0065] Combination therapy of GDC-0084 and olaparib does not induce toxicity. Mouse body and liver weights were assessed after treatment with GDC-0084 (7.5 mg / kg) post-olaparib treatment. ns not significant, Tukey's post-hoc test, n=5 / group. [Figure 34]

[0066] Reduction of liver and lung inflammation after combination therapy with GDC-0084 and olaparib. H&E-stained FFPE livers and lungs from the GDC-0084 post-olaparib study were scored by an independent, expert pathologist. (A) Changes in liver inflammation and extramedullary hematopoiesis (EMH), and (B) lung leukocytosis were given a score of 1 = mild, 2 = moderate, or 3 = severe for each parameter. *p<0.05, **p<0.001, ****p<0.0001 vs. vehicle, Dunnett's post-hoc test, n=5 / group. [Figure 35]

[0067] The combination of GDC-0084 and olaparib reduces splenomegaly and extramedullary hematopoiesis in the spleen. (A) H&E-stained FFPE spleens from the GDC-0084 post-olaparib study were scored by an expert pathologist. Extramedullary hematopoietic (EMH) changes were scored as 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 vs. vehicle, Dunnett's post-hoc test, n=5 / group. *p<0.01, ***p<0.001 vs. vehicle, Dunnett's post-hoc test, n=5 / group. [Figure 36]

[0068] Comparison of PI3K-mTOR inhibitor combination treatments on cancer cell proliferation. MDA-MB-231 cells pretreated with PI3K inhibitors for 72 hours in a dose-response design were treated with WST proliferation reagent. Cell proliferation (%) was measured indirectly by formazan formation and absorbance recorded at 450 nm. [Figure 37]

[0069] GDC-0084 dose-response curve for wound healing (scratch assay). Pre-stimulated (PMA / TGFβ) MCF-7 cells were wounded and then treated with GDC-0084 (0.078-2.5 μM) for 24 hours. Wound density changes were monitored for 24 hours. (A) Representative images from a scratch assay comparing vehicle-treated and GDC-0084 (1.25 μM and 2.5 μM)-treated MCF-7 cells. (B) Dose-response curve (0.078-2.5 μM) comparing wound density (%) in GDC-0084-treated cells over a 24-hour treatment period. [Figure 38]

[0070] Comparison of PI3K-mTOR inhibitor treatment on cancer cell migration. Pre-stimulated (PMA / TGFβ) MCF-7 cells were wounded and then treated with PI3K-mTOR inhibitors for 24 hours. (A) Dose-response curves (0.078-2.5 μM) comparing the percent wound density of PI3K-mTOR inhibitor-treated cells over the 24-hour treatment period. (B) Comparison of the relative wound density observed for each PI3K-mTOR inhibitor at 24 hours. ****p<0.0001 vs. vehicle, Dunnett's post-hoc test, n=5 / group. *p<0.01, ***p<0.001 vs. vehicle, Dunnett's post-hoc test, n=6 / group. DETAILED DESCRIPTION OF THE INVENTION

[0034] Detailed Description of the Invention 1.Definition

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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, the preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

[0035]

[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.

[0036]

[0073] As used herein, the term "about" refers to a normal range of error for each value, which is readily apparent to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments that are directed to that value or parameter itself.

[0037]

[0074] The "amount" or "level" of a biomarker is the level that can be detected in a sample. These can be measured by methods known to those of skill in the art and also disclosed herein. The expression level or amount of the biomarker being assessed can be used to determine response to treatment.

[0038]

[0075] As used herein, "and / or" refers to and includes all possible combinations of one or more of the associated listed items and the lack of combinations when interpreted in the alternative (or).

[0039]

[0076] Throughout this specification, unless otherwise required by context, the words "comprise," "comprises," and "comprising" should be understood to mean the inclusion of the specified step or element or group of steps or elements, but not the exclusion of other steps or elements or group of steps or elements. Thus, the use of terms such as "comprise" suggests that the recited elements are required or essential, but that other elements are optional and may or may not be present. "Consisting of" means including and limited to, regardless of what follows the phrase "consisting of." Thus, the phrase "consisting of" suggests that the recited elements are required or essential, and that other elements may not be present. "Consisting essentially of" means including any elements listed after this phrase, and limited to other elements that do not interfere with or contribute to the activity or function specifically set forth in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or function of the recited elements.

[0040]

[0077] "Chemotherapeutic agents" include chemical 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), sunitinib (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® cyclophosphamide, alkylsulfonates such as busulfan, improsulfan and piposulfan, aziridines (e.g., benzodopa, carboquone, meturedopa and uredopa), ethylenimines and methylameramines (including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine), acetogenins (especially bullatacin and bullatacinone), camptothecins (including topotecan and irinotecan), bryostatin, kallistatin, CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs), cryptophycins (especially cryptophycin 1 and cryptophycin 8), adrenocorticosterides (including prednisone and prednisolone),Cyproterone acetate, 5α-reductase inhibitors (including finasteride and dutasteride), vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat, dolastatins, aldesleukin, talc, duocarmycins (including synthetic analogs KW-2189 and CB1-TM1), eleutherobin, pancratistatin, sarcodictiin, spongistatins, nitrogen mustards such as chlorambucil, chromafadine, cyclophosphamide methadone, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novoenbiquin, fenesterine, prednimustine, trofosfamide, uracil mustard, carmustine, e.g., nitrosoureas, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine, antibiotics, e.g., enediyne antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ω1I (Angew Chem. Intl. Ed. Engl. 1994 33: 183-186), dynemicins (including dynemicin A), bisphosphonates such as clodronate, esperamicin, and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYC1N® (doxorubicin), morpholinodoxorubicin, Cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicinone, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin C such as mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU), folic acid analogues such as denopterin, methotrexate,Pteropterin, trimetrexate, purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, antiadrenal agents such as aminoglutethimide, mitotane, trilostane, folic acid replenishers such as furoic acid, aceglatone, aldophosphamide glycosides cid, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatraxate, defofamine, demecolcine, diaziquone, elfomitin, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidynin, maytansinoids such as maytansine and ansamitocin, mitoguazone, mitoxantrone, mopidamol, nitraelin, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine, PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.), razoxane, rhizoxin, schizofuran, spirogermanium, tenuazonic acid, triaziconazole, 2,2',2"-trichlorotriethylamine, trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine), urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside ("Ara-C"), cyclophosphamide, thiotepa, taxoids such as TAXOL (paclitaxel, Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® (Cremophor Free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.) and TAXOTERE® (docetaxel, doxetaxel, Sanofi-Aventis), chlorambucil,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, the topoisomerase inhibitor RFS2000, difluoromethylornithine (DMFO), retinoids such as retinoic acid, and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0041]

[0078] Chemotherapeutic agents include (i) antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs) (e.g., tamoxifen (including NOLVADEX®, tamoxifen tosylate), raloxifene, droloxifene, iodoxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate)); (ii) enzyme aromatase inhibitors; aromatase inhibitors, which inhibit the enzyme and regulate estrogen production in the adrenal gland, such as 4(5)-imidazole, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane, Pfizer), formestane, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole, Novartis), and ARIMIDEX® (anastrozole, AstraZeneca); (iii) antiandrogens, such as flutamide, (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes involved in signal transduction pathways involved in abnormal cell growth, e.g., PKC-α, Ralf, H -Ras, etc., (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®, VAXID®, PROLEUKIN®, rIL-2, topoisomerase 1 inhibitors, for example, LURTOTECAN®, ABARELIX® rmRH, and (ix) pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0042]

[0079] Chemotherapeutic agents also include 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).

[0043]

[0080] Additional humanized monoclonal antibodies having therapeutic potential as agents in combination with the compounds of the invention include apolizumab, acelizumab, atlizumab, bapinoyumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidutuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, natalizumab, nimotuzumab, and the like. Mab, norobizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pertuzumab, pexelizumab, palivizumab, ranibizumab, reslizumab, reslizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tuxituuzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and anti-interleukin-12 (ABT-874 / J695, Wyeth Research and Abbott Laboratories), which is a recombinant exclusively human sequence full-length IgG1 lambda antibody genetically modified to recognize the interleukin-12p40 protein.

[0044]

[0081] Chemotherapeutic agents also include "EGFR inhibitors," alternatively referred to as "EGFR antagonists," which refer to compounds that bind to or otherwise directly interact with EGFR and prevent or reduce its signaling activity. Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb579 (ATCC PTA HB8506), MAb455 (ATCC PTA HB8507), MAb225 (ATCC PTA8508), MAb528 (ATCC PTA8509) (see U.S. Pat. No. 4,943,533, Mendelsohn et al.) and their variants, such as chimerized 225 (C225 or cetuximab, ERBUTIX®) and reshaped human 225 (H225) (WO 96 / 40210, Imclone Systems, Inc.). Inc.), IMC-11F8 fully human EGFR-targeting antibody (Imclone), antibodies that bind to type II mutant EGFR (U.S. Pat. No. 5,212,290), humanized and chimeric antibodies that bind to EGFR as described in U.S. Pat. No. 5,891,996, and human antibodies that bind to EGFR, such as ABX-EGF or panitumumab (WO 98 / 50433, Abgenix / Amgen), EMD55900 (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), the human EGFR antibody HuMaxEGFR (GenMab), the fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E7.6.3 described in U.S. Pat. No. 6,235,883, MDX-447 (Medarex Inc), and mAb806 or humanized mAb806 (Johns et al., J. Biol. Chem. 279(29): 30375-30384 (2004)).Anti-EGFR antibodies can be conjugated to cytotoxic agents to form immunoconjugates (see, e.g., European Patent Application Publication No. 659439A2, Merck Patent GmbH). EGFR antagonists include those described in U.S. Patent 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, and 5,866,577. Nos. 2, 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, and the following PCT publications: WO 98 / 14451, WO 98 / 50038, WO 99 / 09016, and WO 99 / 24037.Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech / OSI Pharmaceuticals), PD183805 (Cl1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.), ZD1839, gefitinib (IRESSA®) 4-(3'-chloro-4'-fluoroanilino)-7-methoxy 6-(3-morpholinopropoxy)quinazoline, AstraZeneca), ZM105180 (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), 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]pyrimidine), CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide), EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano and N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[2-methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine (TYKERB®, GSK572016, or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[2-methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine.

[0045]

[0082] Chemotherapeutic agents also include "tyrosine kinase inhibitors," including the EGFR-targeted agents described in the paragraph above; small molecule HER2 tyrosine kinase inhibitors, such as TAK165 available from Takeda; oral selective inhibitors of ErbB2 receptor tyrosine kinase CP-724, 714 (Pfizer and OSI); dual HER inhibitors, such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits both HER2 and EGFR overexpressing cells; oral HER2 and EGFR tyrosine kinase inhibitors lapatinib (GSK572016 available from Glaxo-SmithKline); PKI-166 (available from Novartis); pan-HER inhibitors, such as canertinib (Cl-1033, Pharmacia); and Raf-1 inhibitors, such as the antisense agent ISIS-5132 available from ISIS.

[0046]

[0083] Drugs that inhibit Raf-1 signaling, non-HER-targeted TK inhibitors such as imatinib mesylate (GLEEVEC® available from GlaxoSmithKline), 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 PD153035, 4-(3-chloroanilino)quinazoline, pyridopyrimidines, birimitobirimidines, pyrrolopyrimidines such as CGP59326, CGP60261 and CGP62706, pyrazolopyrimidines, 4-(phenylamino)-7H-pyrrolo[ 2,3-d]pyrimidines, curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide), tyrphostins containing a nitrothiophene moiety, PD-0183805 (Warner-Lambert), antisense molecules (e.g., those that bind to HER-encoding nucleic acids), 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 (ISIS3521, isis / Lilly), imatinib mesylate (GLEEVEC®), PKI166 (Novartis), GW2016 (GlaxoSmithKline), Cl-1033 (Pfizer), EKB-569 (Wyeth), semaxinib (Pfizer), ZD6474 (AstraZeneca), PTK-787 (Novartis / Schering AG), INC-1C11 (Imclone), rapamycin (sirolimus, RAPAMUNE®) or the following patent publications: U.S. Pat.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).

[0047]

[0084] Chemotherapeutic agents include dexamethasone, interferon, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, live BCG, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, erlotinib, filgrastim, histrelin acetate, ibritumomab, interferon alpha-2a, and interferon alfa. Also included are a-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.

[0048]

[0085] Chemotherapeutic agents 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, fluorocortolone, hydrocortisone 17-butyrate, and hydrocortisone. -17-valerate, alclomethasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate, and fluprednidene acetate, immunoselective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG) (IMULAN BioTherapeutics, LLC), antirheumatic drugs such as azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide, minocycline, sulfasalazine, tumor necrosis factor (TNF) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), golimumab (Simponi), interleukins 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 lontalizumab, beta 7 integrin blockers such as rhuMAb Beta7, IgE pathway blockers such as anti-M1 prime, secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa1 / β2 blockers such as anti-lymphotoxin α (LTa), radioisotopes (e.g., At 211 , I 131 , I 125、Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 、P 32 、Pb 212and radioactive isotopes of Lu), various investigational agents such as thioplatinum, PS-341, phenylbutyrate, ET18-OCH3 or farnesyltransferase inhibitors (L-739749, L-744832), polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavin, flavanols, procyanidins, betulinic acid and their derivatives, autophagy inhibitors such as chloroquine, delta-9-tetrahydrocannabinol (dronabinol, MARINOL®), beta-glucan, benzophenone, benzocaine, benzophenone-1, benzocaine ... Tarapachone, lapachol, colchicine, betulinic acid, acetylcamptothecin, scopolectin and 9-aminocamptothecin, podophyllotoxin, tegafur (UFTORAL®), bexarotene (TARGRETIN®), clodronate (e.g., BONEFOS® or OSTAC®), bisphosphonates such as etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), (R)), pamidronate (AREDIA®), tiludronate (SKELID®) or risedronate (ACTONEL®) and epidermal growth factor receptor (EGF-R), vaccines such as the THERATOPE® vaccine, perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341), CCl-779, tipifarnib (R11577), orafenib, ABT510, Bcl-2 inhibitors such as oblimersen sodium ( GENASENSE®), pixantrone, farnesyltransferase inhibitors such as lonafarnib (SCH6636, SARASAR™), and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing, as well as combinations of two or more of the foregoing, such as CHOP (an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone) and FOLFOX (an abbreviation for the treatment regimen combining oxaliplatin (ELOXATIN™) with 5-FU and leucovorin).

[0049]

[0086] Chemotherapeutic agents also include nonsteroidal anti-inflammatory drugs (NSAIDs), which have analgesic, antipyretic, and anti-inflammatory effects. NSAIDs include nonselective 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, and diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, and isoxicam, fenamic acid derivatives such as meclofenamic acid, flufenamic acid, and tolfenamic acid, including mefenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, and valdecoxib. NSAIDs may be indicated for the symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthropathy, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headache and migraine, postoperative pain, mild to moderate pain due to inflammation and tissue injury, fever, ileus, and renal colic.

[0050]

[0087] The terms "correlation" and "association" are used interchangeably herein to refer to an association between two measurements (or measurement entities). The present disclosure provides genetic and / or epigenetic variations, the levels of which are associated with disease diagnosis and / or prognosis and / or response to treatment.

[0051]

[0088] The terms "reduce," "reduced," "reduction," "inhibit," "suppress," "attenuate," and the like are all used herein to refer to a statistically significant amount of reduction. In some embodiments, these terms typically refer to a reduction of at least 10% compared to a reference level (e.g., the absence of a given treatment or agent), and can include, for example, a reduction of 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, "reduce," "suppress," and "inhibition" do not necessarily require complete inhibition or reduction compared to a reference level. "Complete inhibition," etc., is 100% inhibition compared to a reference level. The decrease may preferably be to a level that is considered to be within the normal range (eg, in an individual without a given disorder).

[0052]

[0089] As used herein, the term "epithelial-mesenchymal transition" (EMT) refers to the conversion of epithelial cells to a mesenchymal phenotype, which is a normal process of embryonic development. EMT is also the process by which injured epithelial cells that function as ion and fluid transporters become matrix-remodeling mesenchymal cells; in carcinomas, this conversion typically results in altered cell morphology, expression of mesenchymal proteins, and increased invasiveness. Criteria that define EMT in vitro involve the loss of epithelial cell polarity, separation into individual cells, and subsequent dispersal after the acquisition of cell motility (see Vincent-Salomon and Thiery, Breast Cancer Res. 2003; 5(2): 101-6). Molecular classes whose expression, distribution, and / or function change during EMT and are causally involved include growth factors (e.g., transforming growth factor (TGF)-P, wnt), transcription factors (e.g., SNAI, SMAD, LEF, and nuclear β-catenin), cell-cell adhesion axis molecules (cadherins, catenins), cytoskeletal modulators (Rho family), and extracellular proteases (matrix metalloproteinases, plasminogen activators) (see Thompson and Newgreen, Cancer Res. 2005; 65(14): 5991-5).

[0053]

[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 refer to an increase of at least 10% as compared to a reference level (e.g., in the absence of a given treatment or agent), and can include, for example, at least about 10% as compared to a reference level, e.g., 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% increase as compared to a reference level, or up to a 100% increase, or any increase between 10-100%, or at least about 2-fold, or at least about 3-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold increase as compared to a reference level. With respect to a marker or symptom, an "increase" is a statistically significant increase in such level.

[0054]

[0091] "Measuring" or "measurement" means assessing the presence, absence, quantity, or amount (which may be an effective amount) of a given substance in a sample, including deriving a qualitative or quantitative concentration level of such substance or otherwise assessing or categorizing the value of a clinical parameter of interest. Alternatively, the terms "assaying," "detecting," or "detection" may be used to refer to all of the acts of measuring or determination described herein.

[0055]

[0092] As used herein, the term "mesenchymal-epithelial transition" (MET) is a reversible biological process involving the conversion of motile, multipolar, or spindle-shaped mesenchymal cells to a planar array of polarized cells called epithelium. MET is the reverse process of EMT. MET occurs in normal development, cancer metastasis, and induced pluripotent stem cell reprogramming.

[0056]

[0093] As used herein, the terms "overexpress," "overexpression," "overexpressing," or "overexpressed" interchangeably refer to the transcription or translation of a gene (e.g., the PI3KCA gene) at levels typically detectably greater in cancer cells than in normal cells. Overexpression thus refers to the overexpression of both protein and RNA (due to increased transcription, post-transcriptional processing, translation, post-translational processing, stability modifications, and protein degradation modifications), as well as localized overexpression due to altered protein trafficking patterns and expanded functional activity, e.g., increased enzymatic hydrolysis of a substrate. Overexpression can also be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to normal cells or comparison cells (e.g., breast cells).

[0057]

[0094] The term "PI3K inhibitor" and grammatical variations thereof are used herein to refer to a molecule that reduces or inhibits at least one function or biological activity of PI3K. For example, a PI3K inhibitor may inhibit or reduce the enzymatic activity of PI3K and / or may inhibit or reduce the expression of PI3K.

[0058]

[0095] As used herein, the term "PI3K-overexpressing cells" refers to vertebrate cells, particularly mammalian cells, that express PI3K at levels detectably greater than normal cells. The cells can be vertebrate cells, such as primate cells, avian cells, livestock animal cells (e.g., sheep cells, bovine cells, equine cells, deer cells, donkey cells, and porcine cells), laboratory test animal cells (e.g., rabbit cells, mouse cells, rat cells, guinea pig cells, and hamster cells), companion animal cells (e.g., feline cells and canine cells), and captive wild animal cells (e.g., fox cells, deer cells, and dingo cells). In certain embodiments, the PI3K-overexpressing cells are human cells. In specific embodiments, the PI3K-overexpressing cells are cancer stem cells or non-cancer stem cell tumor cells, preferably cancer stem cell tumor cells. Overexpression can also be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to normal cells or comparison cells (e.g., breast cells).

[0059]

[0096] The term "selective" and grammatical variations thereof are used herein to refer to a molecule that inhibits PI3K without substantially inhibiting the function of one or more other PI3K enzymes or isoforms. Generally, a molecule that is selective for PI3K exhibits about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or about 100-fold greater PI3K selectivity relative to the inhibition of one or more other PI3K enzymes. In other embodiments, a selective molecule exhibits at least 50-fold greater inhibition of PI3K than one or more other PI3K enzymes. In further embodiments, a selective molecule exhibits at least 100-fold greater inhibition of PI3K than one or more other PI3K enzymes. In yet further embodiments, a selective molecule exhibits at least 500-fold greater inhibition of PI3K than one or more other PI3K enzymes. In yet further embodiments, a selective molecule exhibits at least 100-fold greater inhibition of PI3K than one or more other PI3K enzymes.

[0060]

[0097] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate, such as a primate, a rodent, a domestic animal, or a game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (e.g., rhesus monkeys). Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domestic and game animals include cattle, horses, pigs, deer, bison, buffalo, felines (e.g., domestic cats), canines (e.g., dogs, foxes, wolves), avian species (e.g., chickens, emus, ostriches), 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.

[0061]

[0098] As used herein, the terms "treat," "treatment," "treating," and the like refer to therapeutic treatment aimed at reversing, alleviating, ameliorating, inhibiting, slowing, or halting the progression or severity of a condition associated with a disease or disorder (e.g., cancer or tumor). The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder associated with a cancer or tumor. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of the disease is reduced or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the cessation or at least slowing of the progression 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, alleviating one or more symptoms, whether detectable or not, reducing the extent of the disease, stabilizing (i.e., not worsening) the disease, delaying or slowing the progression of the disease, ameliorating or alleviating the disease state, remission (whether partial or total), and / or reducing mortality. The term "treatment" of a disease also includes providing relief from the symptoms or side effects of the disease (including palliative care). Treatment does not require a cure of the disorder (i.e., complete reversal or absence of the disease) to be considered effective.

[0062]

[0099] As used herein, the term "tumor" 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 a physiological condition in a mammal typically characterized by partially unregulated cell growth. As used herein, the term "cancer" refers to early-stage and late-stage cancers, including non-metastatic and metastatic cancers. The term "pre-cancerous" typically refers to a condition or growth that precedes or progresses to cancer. The term "non-metastatic" refers to a cancer that is benign or remains at the primary site and does not penetrate the lymphatic system, vascular system, or tissues outside the primary site. Generally, a non-metastatic cancer is any cancer that is a stage 0, I, or II cancer. "Early-stage cancer" refers to a cancer that is not invasive or metastatic, or is classified as either a stage 0, 1, or II cancer. The term "late-stage cancer" generally refers to stage III or IV cancer, but can also refer to stage II cancer or substages of stage II cancer. Those skilled in the art will appreciate that the classification of stage II cancer as either an early-stage cancer or a late-stage cancer depends on the particular type of cancer. Illustrative examples of cancer include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, pancreatic cancer, colorectal cancer, lung cancer, hepatocellular carcinoma, gastric cancer, liver cancer, bladder cancer, urinary tract cancer, thyroid cancer, kidney cancer, carcinoma, melanoma, brain cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, multiple myeloma, mesothelioma, rectal cancer, and esophageal cancer. In an exemplary embodiment, the cancer is breast cancer.

[0063]

[0100] Each embodiment described herein should be applied mutatis mutandis to each of the other embodiments unless otherwise stated.

[0064] 2. Composition

[0101] The present invention is based, in part, on the determination that exposure of functionally suppressed T cells of a mesenchymal phenotype to a PI3K inhibitor results in epigenetic reprogramming of T cells by de-repression of immune effector function, including increased 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), and decreased expression of biomarkers of T cell exhaustion (e.g., PD-1 and EOMES), and increased expression of the transcription factor TBET, which increases IFN-γ production in cells of the adaptive and innate immune systems. The inventors have also found that PI3K inhibitor-mediated phosphorylation reprogramming confers enhanced susceptibility of exhausted T cells to reactivation by ICM-binding antagonists.

[0065]

[0102] Thus, the present invention provides compositions and methods that utilize PI3K inhibitors (e.g., inhibitors of PI3K activity) and ICM-binding antagonists to enhance immune effector function and / or enhance T cell (e.g., CD8+ T cell) function, including increasing T cell activation and increasing the susceptibility of exhausted T cells to reactivation by ICM-binding antagonists. The methods and compositions of the present invention are therefore particularly useful for treating disorders of T cell dysfunction, including cancer and infectious diseases.

[0066]

[0103] The present invention is based, at least in part, on the identification of compositions that inhibit EMT and induce MET in CSC tumor cells, which led the inventors to envision that the compositions of the present invention may be used to treat or prevent cancer and / or improve the responsiveness of cancers or tumors to immunotherapies that do not target CSCs.

[0067]

[0104] Therefore, in one aspect of the present invention, a composition comprising an inhibitor of PI3K and an immunotherapy that does not target CSCs is provided.

[0068] 2.1 PI3K inhibitors

[0105] The compositions of the present invention include inhibitors that contain and include active agents that either reduce the accumulation, function (e.g., enzymatic activity, localization, etc.) or stability of PI3K or reduce the expression of the PI3KCA gene, and such inhibitors include, but are not limited to, small molecules and macromolecules, such as nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, polysaccharides, lipopolysaccharides, lipids or other organic (carbon-containing) or inorganic molecules.

[0069]

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

[0070]

[0107] Representative transcripts of this type include the following sequences: (1) the human PI3K nucleotide sequence set forth, for example, in GenBank accession numbers NM_006219.3, NM_001256045, NM_005026, NM_001350234, and NM_001350235; (2) sequences that are 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, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity; (3) a nucleotide sequence that hybridizes to the sequence referenced in (1) under at least low, medium, or high stringency conditions; (4) the following amino acid sequences: e.g., UniProt accession numbers P42336, Q8NEB9, O00750, O757 47, P48736 and P42338, (5) a nucleotide sequence encoding any one of the human PI3K amino acid 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 similarity to any one of the sequences referenced in (4). (4) includes a nucleotide sequence encoding an amino acid sequence and a nucleotide sequence corresponding to any one of the nucleotide sequences encoding 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, or 99% sequence identity to any one of the sequences referenced in (4).

[0071]

[0108] Exemplary antagonist nucleic acid molecules include antisense molecules, aptamers, ribozymes and triplex-forming molecules, RNAi and external guide sequences. Nucleic acid molecules can act as effectors, inhibitors, modulators and stimulators of specific activities possessed by target molecules, and functional nucleic acid molecules can possess de novo activity that is independent of any other molecule.

[0072]

[0109] Antagonist nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptide, or carbohydrate chain. Thus, antagonist nucleic acid molecules can interact with PI3K mRNA or genomic DNA of a PIK3 gene (e.g., PIK3CA), or can interact with a PI3K polypeptide. In many cases, 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, specific recognition between an antagonist nucleic acid molecule and a target molecule is not based on sequence homology between the antagonist nucleic acid molecule and the target molecule, but rather on the formation of a tertiary structure that allows specific recognition to occur.

[0073]

[0110] In some embodiments, antisense RNA or DNA molecules are used to directly block PI3K translation by binding to target mRNA and preventing protein translation. Antisense molecules are designed to interact with target nucleic acid molecules through either canonical or non-canonical base pairing. The interaction between the antisense molecule and the target molecule can be designed to promote the destruction of the target molecule, for example, through RNAse H-mediated RNA-DNA hybrid degradation. Alternatively, antisense molecules can be designed to disrupt processing functions, such as transcription or replication, that would normally occur on the target molecule. Antisense molecules can be designed based on the sequence of the target molecule. There are many ways to optimize antisense efficiency by finding the most accessible region of the target molecule. Non-limiting methods include in vitro selection experiments and DNA modification tests using DMS and DEPC. In a specific example, antisense molecules are designed to disrupt the target molecule by 10 -6 , 10 -8 , 10 -10 or 10 -12 The following dissociation constants (K d ) to bind to the target molecule. In a specific embodiment, an antisense oligodeoxyribonucleotide derived from the translation initiation site, for example, the -10 to +10 region, is utilized.

[0074]

[0111] Aptamers are molecules that interact favorably with target molecules in a specific manner. Aptamers are generally small nucleic acids, ranging from 15 to 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, and large molecules, such as reverse transcriptase and thrombin. Aptamers can bind to 10 -12 Aptamers can bind very tightly with a Kd from the target molecule of less than 10 M. -6 , 10 -8 , 10 -10 or 10 -12 Less than K d Aptamers bind to target molecules with a high degree of specificity. For example, aptamers have been isolated that exhibit a greater than 10,000-fold difference in binding affinity between the target molecule and another molecule that differs at only a single position on the molecule. Aptamers have a Kd with the target molecule that is at most 1 / 10, 1 / 100, 1 / 1000, 1 / 10,000, or 1 / 100,000 of the Kd with background binding molecules. d It is desirable to have a method for generating aptamers to a target of interest (e.g., PI3K) that is suitable for use in a nucleic acid synthesis system. A preferred method for generating aptamers to a target of interest (e.g., PI3K) is "Systematic Evolution of Ligands by Exponential Enrichment" (SELEX™). The SELEX™ method is described in U.S. Pat. Nos. 5,475,096 and 5,270,163 (see also WO 91 / 19813). Briefly, a mixture of nucleic acids is contacted with a target molecule under conditions that favor binding. Unbound nucleic acids are separated from bound nucleic acids, and the nucleic acid-target complex is dissociated. The dissociated nucleic acids are then amplified to generate a ligand-enriched mixture of nucleic acids, which is optionally subjected to repeated cycles of binding, dissociation, dissociation, and amplification to generate highly specific, high-affinity nucleic acid ligands to the target molecule.

[0075]

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

[0076]

[0100] Triplex-forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acids. When a triplex molecule interacts with a target region, a structure called a triplex is formed, where there are three DNA strands that form a complex that relies on both Watson-Crick and Hoogsteen base pairing. Triplex molecules are preferred because they can bind to target regions with high affinity and specificity. Triplex-forming molecules can be used in a variety of applications, including: -6 , 10 -8 , 10 -10 or 10 -12 Less than K d It is generally desirable to bind to the target molecule at

[0077] External guide sequences (EGSs) are molecules that bind to target nucleic acid molecules to form a complex that is recognized by RNase P, which cleaves the target molecule. EGSs can be designed to specifically target RNA molecules of choice. RNase P assists in the processing of transfer RNA (tRNA) within cells. 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 direct cleavage of RNA is available to cleave desired targets within eukaryotic cells.

[0078] In other embodiments, RNA molecules that mediate RNA interference (RNAi) of PI3K genes or PI3K transcripts can be used to reduce or suppress gene expression. RNAi refers to the interference or destruction of target gene products by introducing single-stranded or usually double-stranded RNA (dsRNA) homologous to the target gene transcript. RNAi methods, including double-stranded RNA interference (dsRNAi) or small interfering RNA (siRNA), have been extensively documented in several 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 21-23 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 microRNA (miRNA), functional short hairpin RNA (shRNA), or other dsRNA expressed in vivo using a DNA template with an RNA polymerase III promoter (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, 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).

[0079] In a specific embodiment, the expression of the PIK3 gene is reduced or suppressed using dsRNA itself, particularly a dsRNA-producing construct, corresponding to at least a portion of the gene. RNAi-mediated inhibition of gene expression can be achieved using any of the techniques described in the art, for example, by transfecting a nucleic acid construct encoding a stem-loop or hairpin RNA structure into the genome of a target cell, or by expressing a transfected nucleic acid construct with homology to the PIK3 gene as a head-to-head or tail-to-tail duplication between convergent promoters or behind a single promoter. Any similar construct can be used, as long as it produces a single RNA capable of folding back on itself to produce dsRNA, or it produces two separate RNA transcripts that are then annealed to form dsRNA with homology to the target gene.

[0080] Absolute homology is not required for RNAi, and a lower threshold of about 85% homology has been described for dsRNAs of about 200 base pairs (Plasterk and Ketting, 2000, Current Opinion in Genetics and Dev. 10: 562-67). Thus, depending on the length of the dsRNA, the RNAi-encoding nucleic acid may vary in the level of homology it contains to the target gene transcript; i.e., dsRNAs of 100 to 200 base pairs have at least about 85% homology to the target gene, and longer dsRNAs (i.e., 300 to 100 base pairs) have at least about 75% homology to the target gene. RNA-encoding constructs expressing a single RNA transcript designed to anneal to separately expressed RNAs or single constructs expressing separate transcripts from convergent promoters are preferably at least about 100 nucleotides in length. RNA-coding constructs that express a single RNA designed to form dsRNA through internal folding are usually at least about 200 nucleotides in length.

[0081] The promoter used to express the dsRNA-forming construct can be any type of promoter, as long as the resulting dsRNA is specific to the gene product of the cell lineage that is targeted for destruction. Alternatively, the promoter can be lineage-specific, in that it is only expressed in cells of a specific developmental lineage. This can be advantageous when some overlapping homology is observed with genes expressed in non-target cell lineages. The promoter can also be induced by external regulatory factors or intracellular environmental factors.

[0082] In some embodiments, to mediate RNAi, RNA molecules of about 21 to about 23 nucleotides can be used to direct cleavage of a corresponding specific mRNA, as described, for example, in U.S. Patent Application Publication No. 2002 / 0086356. Such 21-23 nt RNA molecules can contain a 3' hydroxyl group and can be single-stranded or double-stranded (as two 21-23 nt RNAs), and the dsRNA molecules can be blunt-ended or contain overhanging ends (e.g., 5', 3').

[0083] In some embodiments, the antagonist nucleic acid molecule is an siRNA. The siRNA can be prepared by any suitable method. For example, see International Publication No. 02 / 44321 (incorporated herein by reference), which discloses siRNAs capable of sequence-specific degradation of target mRNAs when base-paired with a 3' overhanging end. 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. The siRNAs can be chemically synthesized or in vitro synthesized, or can be the result of short double-stranded hairpin-like RNAs (shRNAs) that are processed into siRNAs within the cell. Synthetic siRNAs are generally designed using algorithms and conventional DNA / RNA synthesizers. 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.

[0084]

[0108] The production of siRNA from vectors is more commonly carried out through the transcription of short hairpin RNA (shRNA). Kits for producing vectors containing shRNA are available, such as Imgenex's GENESUPPRESSOR™ construction kit and Invitrogen's BLOCK-IT™ inducible RNAi plasmid and lentiviral vector. In addition, methods for formulating siRNA and delivering it to subjects are also well known in the art. See, for example, U.S. Patent Application Publication Nos. 2005 / 0282188, 2005 / 0239731, 2005 / 0234232, 2005 / 0176018, 2005 / 0059817, 2005 / 0020525, 2004 / 0192626, 2003 / 0073640, 2002 / 0150936, 2002 / 0142980, and 2002 / 0120129, each of which is incorporated herein by reference.

[0085]

[0109] Exemplary RNAi molecules (e.g., PI3K siRNA and shRNA) have been 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 are commercially available from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), OriGene Technologies, Inc. (Rockville, MD, USA), and Sigma-Aldrich Pty Ltd (Castle Hill, NSW, Australia).

[0086] The present invention further contemplates peptide- or polypeptide-based inhibitor compounds. PI3K inhibitory peptides can be modified, for example, to be part of a fusion protein, as described above. The fusion protein can include a transport protein or peptide that functions to increase the cellular uptake of the peptide inhibitor, have another desired biological effect, such as a therapeutic effect, or have both of these functions. Fusion proteins can be produced by methods known to those skilled in the art. The inhibitor peptide can be linked or otherwise conjugated to another peptide in various ways known in the art. For example, the inhibitor peptide can be linked to a carrier peptide or other peptide described herein via a bridge, and both peptides of the fusion protein retain their activity. As a further example, the peptides can be linked to each other or otherwise conjugated by an amide bond from the C-terminus of one peptide to the N-terminus of the other peptide. The link between the inhibitor peptide and the other members of the fusion protein can be non-cleavable, such as with a peptide bond, or cleavable, such as with an ester bond or other cleavable bond known in the art.

[0087]

[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 (e.g., as discussed in Theodore et al., 1995. J. Neurosci. 15:7158-7167; Johnson et al., 1996. Circ. Res 79: 1086). Alternatively, the inhibitors can be modified with a transport polypeptide derived from the transactivating regulatory protein (Tat) from human immunodeficiency virus type 1 (e.g., from amino acids 47-57 of Tat as shown in SEQ ID NO: 2, YGRKKRRQRRR), as described in Vives et al., 1997, J. Biol. Chem. 272:16010-16017, U.S. Patent 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 can also be modified by other methods known to those skilled in the art to increase cellular uptake of the inhibitors.

[0088]

[0112] A PI3K inhibitory peptide can also be introduced into a cell by introducing a nucleic acid containing a nucleotide sequence encoding the PI3K inhibitory peptide into the cell. The nucleic acid can be in the form of a recombinant expression vector. The PI3K inhibitory peptide coding sequence can be operably linked to a transcriptional control element, such as a promoter in the expression vector. Suitable vectors include, for example, 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 the cell. In other cases, the expression vector remains episomal in the cell.

[0089]

[0113] Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia virus, poliovirus, adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5: 1088 1097, 1999; WO 94 / 12649; WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984; and WO 95 / 00655), adeno-associated virus (see, e.g., Ali et al., al., Hum Gene Ther 9:8186, 1998, Flannery et al., PNAS 94:6916 6921, 1997, Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997, Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al. al., Hum Gene Ther 10:641 648, 1999, Ali et al., Hum Mol Genet. 5:591 594, 1996, Srivastava in WO93 / 09239, Samulski et al., J. Vir. 63:3822-3828, 1989, Mendelson et al., Virol. 154-165, 1988 and Flotte et al., PNAS (1993) 90: 10613-10617), SV40, herpes simplex virus, human immunodeficiency virus (e.g., Miyoshi et al., PNAS 94:10319 23, 1997, Takahashi et al., J Virol 73:7812 7816, 1999), retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0090]

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

[0091]

[0115] Small molecule agents that reduce the functional activity of PI3K suitable for use in the present invention include, for example, compounds represented by Formula I: [ka] and stereoisomers, geometric isomers, tautomers and pharmaceutically acceptable salts thereof, wherein The dashed lines represent optional double bonds, and at least one dashed line is a double bond; X 1 are S, O, N, and NR a , C.R. 1 , C(R 1 )2 or -C(R 1 )2O-, X 2 is C, CR 2 or N, X 3 is C, CR 3 or N, A is a 5-, 6-, or 7-membered carbocyclyl or heterocyclyl ring fused to X2 and X3, optionally substituted with one or more R5 groups; R a H, C1~C 12 Alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -(C1-C 12 Alkylene)-(C3-C 12 carbocyclyl), -(C1-C 12 alkylene)-(heterocyclyl having 3 to 20 ring atoms), -(C1-C 12 alkylene)-C(=O)-(heterocyclyl having 3 to 20 ring atoms), -(C1-C 12 Alkylene)-(C6-C 20 aryl) and -(C1-C 12 alkylene)-(heteroaryl having 5 to 20 ring atoms), and alkyl, alkenyl, alkynyl, alkylene, carbocyclyl, heterocyclyl, aryl and heteroaryl are 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; R 1 , R 2 and R 3is H, F, Cl, Br, I, -CH3, -CH2CH3, -C(CH3)3, -CH2OH, -CH2CH2OH, -C(CH3)2OH, -CH2OCH3, -CN, -CF3, -CO2 H, --COCH3, -COC(CH3)3, -CO2CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NO2, -NH2, -NHCH3, independently selected from -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; R 4 is C6~C 20 and selected from aryl, heterocyclyl having 3 to 20 ring atoms, and heteroaryl having 5 to 20 ring atoms, each of which is 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, -NHC one or more R independently selected from OCH3, -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; 6 is optionally substituted with a group, and R 5 is C1~C 12 Alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -(C1-C 12 Alkylene)-(C3-C 12 carbocyclyl), -(C1-C 12 alkylene)-(heterocyclyl having 3 to 20 ring atoms), -C(=O)-(C1-C12 alkylene)-(heterocyclyl having 3 to 20 ring atoms), -(C1-C 12 Alkylene)-(C6-C 20 aryl) and -(C1-C 12 or two geminal R groups form a 3-, 4-, 5-, or 6-membered carbocyclyl or heterocyclyl ring, wherein alkyl, alkenyl, alkynyl, alkylene, carbocyclyl, heterocyclyl, aryl, and heteroaryl are independently selected from F, Cl, Br, I, -CH, -CHCH, -C(CH), -CHOH, -CHCHOH, -C(CH)OH, -CHOCH, -CN, -CHF, -CHF, -CF, -COH, -COCH, -COC(CH), - optionally substituted with one or more groups independently selected from 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; mor is, [ka] selected from F, Cl, Br, I, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -C(CH3)3, -CH2OCH3, -CHF2, -CN, -CF3, -CH2OH, -CH2OCH3, -CH2CH2OH, -CH2C(CH3)2OH, -CH(CH3)OH, -CH(CH2CH3)OH , -CH2CH(OH)CH3, -C(CH3)2OH, -C(CH3)2OCH3, -CH(CH3)F, -C(CH3)F2, -CH(CH2CH3)F, -C (CH2CH3)2F, -CO2H, -CONH2, -CON(CH2CH3)2, -COCH3, -CON(CH3)2, -NO2, -NH2, -NHCH3, -N and optionally substituted with one or more R groups independently selected from (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.

[0092]

[0116] In some other embodiments, the small molecule PI3K inhibitor is selected from the following group: [ka] is selected from.

[0093]

[0117] The present invention allows a subject to receive a lower dose of PI3K inhibitor therapy, which reduces the toxicity associated with administering the therapy to a subject without reducing the efficacy of the therapy in preventing, managing, treating, or ameliorating cancer (e.g., solid tumor cancer).

[0094]

[0118] In some embodiments, reducing the amount / dose of the PI3K inhibitor reduces or minimizes any unwanted side effects associated with PI3K therapy.

[0095] 2.2 Immunotherapy

[0119] The compositions of the present invention also include anti-cancer therapeutic agents, which are typically immunotherapies. Any immunotherapies that do not target cancer stem cells (CSCs) are generally considered suitable for use in the compositions and methods of the present invention. Checkpoint inhibitor molecule antagonists and PARP inhibitors are generally considered particularly suitable.

[0096] 2.2.1 Checkpoint inhibitor molecule (ICM) antagonists 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.

[0097]

[0114] The ICMs antagonized by the therapeutic combinations of the present invention include any one or more inhibitory ICMs 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.

[0098] In some preferred embodiments, the ICM antagonist included in the therapeutic combination is a PD-1 antagonist. In this context, "PD-1 antagonist" includes any chemical compound or biological molecule that blocks the binding of PD-L1 (e.g., PD-L1 expressed on the surface of a cancer cell) to PD-1 expressed on an immune cell (e.g., a T cell, a B cell, or an NKT cell). Alternative names or synonyms for PD-1 include PDCD1, PD1, CD279, and SLEB2. The representative mature amino acid sequence of human PD-1 (UniProt Accession No. Q15116) is: [ka] As shown in.

[0099] Examples of monoclonal antibodies (mAbs) that bind to human PD-1 and are therefore useful in the present invention are described in U.S. Patent Application Publication Nos. 2003 / 0039653, 2004 / 0213795, 2006 / 0110383, 2007 / 0065427, 2007 / 0122378, 2012 / 237522, and International PCT Publication Nos. WO 2004 / 072286, WO 2006 / 121168, WO 2006 / 133396, WO 2007 / 005874, WO 2008 / 083174, WO 2008 / 156 712, WO 2009 / 024531, WO 2009 / 014708, WO 2009 / 114335, WO 2010 / 027828, WO 2010 / 027423, WO 2010 / 036959, WO 2010 / 029435, WO 2010 / 029434, WO 2010 / 063011, WO 2010 / 089411, WO 2011 / 066342, WO 2011 / 110604, WO 2011 / 110621 and WO 2012 / 145493, the entire contents of which are incorporated herein by reference. Specific mAbs useful for the purposes of the present invention include the anti-PD-1 mAbs nivolumab, pembrolizumab, and pidilizumab, and the humanized anti-PD-1 antibodies h409All, h409A16, and h409A17 described in WO 2008 / 156712.

[0100] The anti-PD-1 antigen-binding molecules of the invention preferably bind to a region of the extracellular domain of PD-1. For example, the anti-PD-1 antigen-binding molecule may specifically bind to a region of the extracellular domain of human PD-1 comprising one or both of the amino acid sequences SFVLNWYRMSPSNQTDKLAAFPEDR [SEQ ID NO: 4] (i.e., residues 62-86 of the native PD-1 sequence shown in SEQ ID NO: 3) and SGTYLCGAISLAPKAQIKE [SEQ ID NO: 5] (i.e., residues 118-136 of the native PD-1 sequence shown 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 comprising the amino acid sequence NWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV [SEQ ID NO: 6] (i.e., corresponding to residues 66-97 of the native human PD-1 sequence shown in SEQ ID NO: 3).

[0101] In certain embodiments, the anti-PD-1 antigen-binding molecule comprises the fully humanized IgG4 mAb nivolumab (as described in detail in U.S. Pat. No. 8,008,449, which is incorporated herein by reference in its entirety (referred to as "5C4")) or an antigen-binding fragment thereof. In a representative example of this type, the anti-PD-1 antigen-binding molecule comprises the CDR sequences shown in Table 5.

[0102] [Table 1]

[0103] In more specific embodiments, the anti-PD-1 antigen-binding molecule is, for example, [ka] The heavy chain amino acid sequence of nivolumab or the amino acid sequence shown in: [ka] and antigen-binding fragments thereof comprising, consisting of, or consisting essentially of:

[0104]

[0120] In some of the same and other embodiments, the anti-PD-1 antigen binding molecule may be, for example: [ka] The light chain amino acid sequence of nivolumab or the amino acid sequence shown in: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT DFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK [SEQ ID NO: 16] It may include an antigen-binding fragment thereof comprising, consisting of, or consisting essentially of:

[0105]

[0121] In an alternative embodiment, the anti-PD-1 antigen-binding molecule comprises the humanized IgG4 mAb pembrolizumab, or an antigen-binding fragment thereof. As a non-limiting example of this type, the anti-PD-1 antigen-binding molecule comprises the CDR sequences shown in Table 6.

[0106] [Table 2]

[0107]

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

[0108]

[0123] In additional embodiments, the anti-PD-1 antigen binding molecule is, for example, [ka] The heavy chain amino acid sequence of pembrolizumab or the amino acid sequence shown in: [ka] and antigen-binding fragments thereof comprising, consisting of, or consisting essentially of:

[0109]

[0124] Similarly, anti-PD-1 antigen-binding molecules can be, for example, [ka] The light chain amino acid sequence of pembrolizumab or the amino acid sequence shown in: [ka] It may include an antigen-binding fragment thereof comprising, consisting of, or consisting essentially of:

[0110]

[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 the CDR sequences set forth in Table 7.

[0111] [Table 3]

[0112]

[0126] In a more specific embodiment, the anti-PD-1 antigen binding molecule is [ka] The heavy chain amino acid sequence of pidilizumab or the amino acid sequence shown in: [ka] and antigen-binding fragments thereof comprising, consisting of, or consisting essentially of:

[0113]

[0127] In some of the same and other embodiments, the anti-PD-1 antigen binding molecule is [ka] The light chain amino acid sequence of pidilizumab or the amino acid sequence shown in: EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTSYCLTINSLQPEDFATYYCQQRSSFPLTFGGGTKLEIK [SEQ ID NO: 30] and antigen-binding fragments thereof comprising, consisting of, or consisting essentially of:

[0114]

[0128] Other suitable mAbs are described in WO 2015 / 026634 (incorporated herein by reference in its entirety), including (a) light chain CDRs having the 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 having the 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 having the amino acid sequences: 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 having the amino acid sequences: NYYMY [SEQ ID NO: 40], GINPSNGGTNFN Included are mAbs or antigen-binding fragments thereof comprising heavy chain CDRs having EKFKN [SEQ ID NO: 41] and RDYRFDMGFDY [SEQ ID NO: 42] (CDR1, CDR2, and CDR3, respectively).

[0115]

[0129] Illustratively, such mAbs include: (a) [ka] or a variant or antigen-binding fragment thereof, and

[0130] [ka] [ka] or [ka] The antibody may comprise a light chain variable region comprising an amino acid sequence selected from the group consisting of:

[0116]

[0131] In a still further exemplary embodiment, the anti-PD-1 mAb is [ka] an IgG1 heavy chain or a variant or antigen-binding fragment thereof comprising: [ka] [ka] [ka] The antibody may comprise a light chain comprising any one of the following:

[0117]

[0132] In another embodiment, 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, PD-L1 antagonists are: [ka] It specifically binds to the native amino acid sequence of human PD-L1 (UniProt accession number Q9NZQ7) shown in

[0118]

[0133] Preferably, the PD-L1 antagonist is an anti-PD-L1 antigen binding molecule. By way of example, anti-PD-L1 antigen binding molecules suitable for use in 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-L1 antibodies are described in WO 2007 / 005874 and WO 2010 / 077634, and U.S. Patent Nos. 8,217,149 and 8,779,108, each of which is incorporated by reference in its entirety. Further anti-PD-L1 mAbs are described in WO 2016 / 007,235, the entire contents of which are incorporated herein by reference.

[0119]

[0134] The anti-PD-L1 antigen-binding molecule preferably binds to a region of the extracellular domain of PD-L1. By way of example, the anti-PD-L1 antigen-binding molecule may specifically bind to a region of the extracellular domain of human PD-L1 comprising the amino acid sequence SKKQSDTHLEET [SEQ ID NO: 13] (i.e., residues 279-290 of the sequence of native PD-L1 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 for "MEDI4736" in PCT Publication WO 2011 / 066389 and US Patent Publication No. 2013 / 034559, which are incorporated herein by reference in their entireties) or an antigen-binding fragment thereof. In an exemplary embodiment of this type, the anti-PD-L1 antigen-binding molecule comprises the CDR sequences set forth in Table 8.

[0120] [Table 4]

[0121]

[0135] In more specific embodiments, the anti-PD-L1 antigen binding molecule is, for example, [ka] The heavy chain amino acid sequence of durvalumab or the amino acid sequence shown in: [ka] and antigen-binding fragments thereof comprising, consisting of, or consisting essentially of:

[0122]

[0136] In some of the same and other embodiments, the anti-PD-L1 antigen binding molecule has a light chain amino acid sequence: [ka] or the amino acid sequence: [ka] It may include an antigen-binding fragment thereof comprising, consisting of, or consisting essentially of:

[0123]

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

[0124]

[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. 8,217,148, the entire contents of which are incorporated herein by reference), or an antigen-binding fragment thereof. In an exemplary embodiment of this type, the anti-PD-L1 antigen binding molecule comprises the CDR sequences shown in Table 9.

[0125] [Table 5]

[0126]

[0139] In more specific embodiments, the anti-PD-L1 antigen binding molecule is, for example, [ka] The heavy chain amino acid sequence of atezolizumab or the amino acid sequence shown in: [ka] and antigen-binding fragments thereof comprising, consisting of, or consisting essentially of:

[0127]

[0140] In some of the same and other embodiments, the anti-PD-L1 antigen binding molecule may be, for example: [ka] The atezolizumab light chain amino acid sequence or amino acid sequence provided in DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK [SEQ ID NO: 59] and antigen-binding fragments thereof comprising, consisting of, or consisting essentially of:

[0128] Alternatively, the anti-PD-L1 antigen binding molecule competes with the mAb atezolizumab for binding to PD-L1.

[0129] In other embodiments, the anti-PD-L1 antigen-binding molecule comprises the fully humanized IgG1 mAb avelumab (as described in U.S. Patent No. 8,217,148, the entire contents of which are incorporated herein by reference), or an antigen-binding fragment thereof. In an exemplary embodiment of this type, the anti-PD-L1 antigen-binding molecule comprises the CDR sequences shown in Table 10.

[0130] [Table 6]

[0131] In specific embodiments, the anti-PD-L1 antigen binding molecule is, for example, [ka] The heavy chain amino acid sequence or amino acid sequence of avelumab provided in [ka] and antigen-binding fragments thereof comprising, consisting of, or consisting essentially of:

[0132]

[0141] In some of the same and other embodiments, the anti-PD-L1 antigen binding molecule may be, for example: [ka] The light chain amino acid sequence of avelumab or the amino acid sequence shown in: [ka] and antigen-binding fragments thereof comprising, consisting of, or consisting essentially of:

[0133] Alternatively, the anti-PD-L1 antigen binding molecule competes with the mAb avelumab for binding to PD-L1.

[0134] 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, IDDM12. Generally, CTLA4 antagonists are, for example, [ka] It specifically binds to the mature amino acid sequence of human CTLA4 shown in (UniProt accession number P16410).

[0135]

[0125] Preferably, the CTLA4 antagonist is an anti-CTLA4 antigen binding molecule. By way of example, anti-CTLA4 antigen binding molecules suitable for use in the present invention include the anti-CTLA4 mAbs ipilimumab (BMS-734016, MDX-010, MDX-101) and tremelimumab (ticilimumab, CP-675,206).

[0136]

[0126] The anti-CTLA4 antigen-binding molecule preferably binds to a region of the extracellular domain of CTLA4. For example, the anti-CTLA4 antigen-binding molecule may specifically bind to a region of the extracellular domain of human CTLA4 comprising any one or more of the amino acid sequences YASPGKATEVRVTVLRQA [SEQ ID NO: 65] (i.e., residues 26-42 of the native CTLA4 sequence shown in SEQ ID NO: 64), DSQVTEVCAATYMMGNELTFLDD [SEQ ID NO: 66] (i.e., residues 43-65 of the native CTLA4 sequence shown in SEQ ID NO: 64), and VELMYPPPYYLGIG [SEQ ID NO: 67] (i.e., residues 96-109 of the native CTLA4 sequence shown in SEQ ID NO: 64). Alternatively or additionally, the anti-CTLA4 antigen binding molecule may specifically bind to a region of the extracellular domain of human CTLA4 that includes any one or more, preferably all, of the following residues in the mature form of CTLA4: K1, A2, M3, E33, R35, Q41, S44, Q45, V46, E48, L91, 193, K95, E97, M99, P102, P103, Y104, Y105, L106, 1108, N110.

[0137] In certain embodiments, the anti-CTLA4 antigen-binding molecule comprises the human IgG1 mAb ipilimumab (e.g., as described in WO 2014 / 209804 and U.S. Patent Application Publication No. 2015 / 0283234, the entire contents of which are incorporated herein by reference), or an antigen-binding fragment thereof. In a representative embodiment, the anti-CTLA4 antigen-binding molecule comprises the CDR sequences shown in Table 11.

[0138] [Table 7]

[0139] In more specific embodiments, the anti-CTLA4 antigen binding molecule is, for example,

[0129] [ka] or an antigen-binding fragment thereof, non-limiting examples of which include the heavy chain amino acid sequence of ipilimumab as set forth in [ka] Comprises, consists of, or consists essentially of.

[0140] In some of the same and other embodiments, the anti-CTLA4 antigen binding molecule is, for example, [ka] or an antigen-binding fragment thereof, a representative example of which is the amino acid sequence: [ka] Comprising, consisting of, or consisting essentially of

[0141] In some embodiments, the anti-CTLA4 antigen binding molecule comprises the human IgG2 mAb tremelimumab (e.g., as described in U.S. Patent Application Publication No. 2009 / 0074787, the entire contents of which are incorporated herein by reference), or an antigen-binding fragment thereof. In an exemplary embodiment, the anti-CTLA4 antigen binding molecule comprises the CDR sequences shown in Table 12.

[0142] [Table 8]

[0143] In more specific embodiments, the anti-CTLA4 antigen binding molecule is, for example, [ka] or an antigen-binding fragment thereof, non-limiting examples of which include the heavy chain amino acid sequence of tremelimumab as set forth in [ka] Comprises, consists of, or consists essentially of.

[0144] In some of the same and other embodiments, the anti-CTLA4 antigen binding molecule is, for example, [ka] or an antigen-binding fragment thereof, a representative example of which is the amino acid sequence: DIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIK [SEQ ID NO: 75] Comprising, consisting of, or consisting essentially of

[0145] In other embodiments, the ICM antagonist is a B7-H3 antagonist. Generally, B7-H3 antagonists of the present invention are, for example, [ka] The antibody specifically binds to the native amino acid sequence of human B7-H3 (UniProt accession number Q5ZPR3) shown in

[0146]

[0135] Preferably, the B7-H3 antagonist is an anti-B7-H3 antigen binding molecule. By way of example, an anti-B7-H3 antigen binding molecule suitable for use in the present invention is the mAb enoblituzumab or an antigen-binding fragment thereof. In some embodiments, the anti-B7-H3 antigen binding molecule comprises the CDR sequences shown in Table 13.

[0147] [Table 9]

[0148] In more specific embodiments, the anti-B7-H3 antigen binding molecule is, for example, [ka] or an antigen-binding fragment thereof, a representative example of which is the heavy chain amino acid sequence of enoblituzumab shown in [ka] Comprising, consisting of, or consisting essentially of

[0149] In some of the same and other embodiments, the anti-B7-H3 antigen binding molecule is, for example, [ka] or an antigen-binding fragment thereof, a representative example of which is the amino acid sequence: DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIK [SEQ ID NO: 80] Comprising, consisting of, or consisting essentially of

[0150] In some alternative embodiments, the anti-B7-H3 antigen binding molecule competes with mAb enoblituzumab for binding to B7-H3.

[0151] In other embodiments, the ICM antagonist is an indoleamine 2,3-dioxygenase (IDO) antagonist. For example, [ka] is the mature amino acid sequence of human IDO (UniProt accession number P14902) shown in

[0152] Any IDO antagonist is suitable for use in the therapeutic agents of the present invention. Currently, three small molecule IDO inhibitors are in development for clinical use: GDC-0919 (1-cyclohexyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)ethanol), indoximod (1-methyl-D-tryptophan), and epacadostat (1,2,5-oxadiazole-3-carboximidamide, 4-((2-((aminosulfonyl)amino)ethyl)amino)-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-, (C(Z))-). The molecular structures of each of these molecules are provided below. [ka]

[0153] In some embodiments, the ICM antagonist is a killer cell immunoglobulin (KIR) antagonist. In preferred embodiments of this type, the KIR antagonist blocks the interaction of KIR2-DL-1, 2, and 3 with their ligands. The mature amino acid sequence of human KIR, i.e., KIR2-DL1 (UniProt Accession No. P43626), is, for example: [ka] will be provided to.

[0154] Anti-KIR antigen-binding molecules suitable for use in the present invention can be produced 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 includes the fully humanized mAb lirilumab or an antigen-binding fragment thereof, as described in WO 2014 / 066532 (the entire contents of which are incorporated herein in their entirety). Preferably, the anti-KIR antigen-binding molecule comprises the CDR regions shown in Table 14.

[0155] [Table 10]

[0156] In exemplary embodiments of this type, the anti-KIR antigen binding molecule is, for example, [ka] or an antigen-binding fragment thereof, a representative example of which is the amino acid sequence: [ka] Comprising, consisting of, or consisting essentially of

[0157] In some of the same and other embodiments, the anti-KIR antigen binding molecule is, for example, [ka] or an antigen-binding fragment thereof, a representative example of which is the amino acid sequence: [ka] Comprising, consisting of, or consisting essentially of

[0158] In an alternative embodiment, 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 number P18627) is: [ka] As shown in.

[0159] In some embodiments, the LAG-3 antagonist is an anti-LAG-3 antigen binding molecule. Illustratively, a suitable anti-LAG antigen binding molecule is the anti-LAG-3 humanized mAb BMS-986016. Other anti-LAG-3 antibodies are described in U.S. Patent Application Publication No. 2011 / 0150892 and PCT Publication Nos. WO 2010 / 019570 and WO 2014 / 008218 (each of which is incorporated herein by reference in its entirety).

[0160]

[0147] In some embodiments, the anti-LAG-3 antigen binding molecule comprises the CDR sequences shown in Table 15.

[0161] [Table 11]

[0162] The anti-LAG-3 antigen-binding molecule preferably comprises mAb BMS-986016 or an antigen-binding fragment thereof. More specifically, in some embodiments, the anti-LAG-3 antigen-binding molecule comprises: [ka] or an antigen-binding fragment thereof, and a representative example thereof has the heavy chain amino acid sequence of BMS-986016 shown in [ka] Comprising, consisting of, or consisting essentially of

[0163] Similarly, the anti-LAG-3 antigen-binding molecule has the light chain amino acid sequence of BMS-986016 shown in SEQ ID NO: 45 and provided below: [ka] or an antigen-binding fragment thereof, a representative example of which is the amino acid sequence: EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGT DFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIK [SEQ ID NO: 91] Comprising, consisting of, or consisting essentially of

[0164] 2.2.2 PARP inhibitors Poly(ADP-ribose) polymerase (PARP) enzymes are a family of enzymes that cleave NAD+ to release nicotinamide and sequentially add ADP-ribose units to form ADP-ribose polymers. Activation of PARP enzymes can result in depletion of cellular NAD+ levels (e.g., PARP as an NAD+ consumer) and mediate cell signaling 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. Anti-alkylating agents are known to potentially deplete the NAD+ content of tumor cells, and the discovery of PARP elucidated 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 and activate PARP-1, which is part of the DNA repair pathway. Poly (ADP-ribosylation) of nuclear proteins by PARP-1 converts DNA damage into an intracellular signal that can either activate DNA repair (e.g., via the base excision repair (BER) pathway) or trigger cell death in the presence of DNA damage that is too extensive to allow efficient repair.

[0165] PARP-2 contains a catalytic domain and is capable of catalyzing the poly(ADP-ribosyl)ation reaction. PARP-2 exhibits automodification properties similar to PARP-1. This protein is localized to the nucleus in vivo and can compensate 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 that primarily aim to inhibit PARP-1) can also inhibit PARP-2 (e.g., niraparib).

[0166] The role of PARP enzymes in DNA damage response (e.g., DNA repair in response to genotoxic stress) has led to the compelling proposal that PARP inhibitors may be useful anticancer drugs. PARP inhibitors may be particularly effective in treating cancers arising from the germline or sporadic defects in the homologous recombination DNA repair pathway, such as BRCA-1 and / or BRCA-2 deficient cancers.

[0167] Preclinical ex vivo and in vivo experiments suggest that PARP inhibitors are selectively cytotoxic to tumors with homozygous inactivation of the 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 BRCA-1 and / or BRCA-2 deficiencies lies in the requirement of PARP-1 and PARP-2 for base excision repair (BER) of damaged DNA. Upon formation of a single-strand DNA break, PARP-1 and PARP-2 bind to the lesion site, become activated, and catalyze the addition of long polymers of ADP-ribose (PAR chains) to several proteins associated with chromatin, including histones. This results in chromatin relaxation and the rapid recruitment of DNA repair factors to access and repair the DNA break. Normal cells repair up to 10,000 DNA defects per day, with single-strand breaks being 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 threads 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 repair damaged DNA using error-prone non-homologous end joining (NHEJ). Both the inability to complete S phase (due to stalled replication forks) and error-prone repair by NHEJ are thought to contribute to cell death.

[0168] Without wishing to be bound by theory, it is hypothesized that treatment with PARP inhibitors can selectively kill a subset of cancer cells that have defects in DNA repair pathways (such as inactivation of BRCA-1 and / or BRCA-2).For example, tumors arising in patients with germline BRCA mutations may have defects in homologous recombination DNA repair pathways and become increasingly dependent on BER, a pathway that is blocked by PARP inhibitors, to maintain genome integrity.This concept of using PARP inhibitors to block one DNA repair pathway in tumors with existing defects in complementary DNA repair pathways is called synthetic lethality.

[0169] The therapeutic potential of PARP inhibitors is further expanded by the observation that they not only have monotherapy activity in HR-deficient tumors, but also are effective in preclinical models in combination with other agents, such as cisplatin, carboplatin, alkylating and methylating agents, radiotherapy, and topoisomerase I inhibitors. In contrast to the monotherapy rationale that PARP inhibition alone is sufficient to cause cell death in HR-deficient cancers (due to endogenous DNA damage), PARP is required for the repair of DNA damage induced by standard cytotoxic chemotherapy. While the specific role of PARP in some cases is unknown, it is known that PARP is 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 demonstrate potentiation or synergy in cancer therapy without significantly increasing toxicity would provide substantial benefit to cancer patients, including those with ovarian cancer.

[0170] In some embodiments, treatment with PARP inhibitors (e.g., PARP-1 / 2 inhibitors) provided herein in the various methods and kits disclosed herein can selectively kill a subset of cancer cell types by exploiting DNA repair defects. Human cancers exhibit genomic instability and increased mutation rates due to underlying DNA repair defects. These defects make cancer cells more dependent on remaining DNA repair pathways, and targeting these pathways is expected to affect tumor cell survival relative to normal cells.

[0171] In some embodiments, the PARP inhibitor is selected from the group consisting of ABT-767, AZD 2461, BGB-290, BGP 15, CEP 8983, CEP 9722, DR 2313, E7016, E7449, fluzoparib (SHR3162), IMP 4297, INO1001, JPI 289, JP I547, monoclonal antibody B3-LysPE40 conjugate, MP 124, niraparib (ZEJULA), NU 1025, NU 1064, NU 1076, NU1085, olaparib, ONO2231, PD 128763, R503, R554, rucaparib (RUBRACA), SBP 101, SC 101914, Simmiparib, Talazoparib (BMN-673), Veliparib (ABT-888) and WW46 2-(4-(trifluoromethyl)phenyl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4-ol or a pharmaceutically acceptable salt thereof).

[0172] In some embodiments, the PARP inhibitor is a small molecule. In some alternative embodiments, the PARP inhibitor is an antibody agent. In some embodiments, the agent that inhibits PARP is a combination of agents.

[0173] 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 may exist as a solvated or hydrated polymorphic form.

[0174]

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

[0175] In some embodiments, the PARP inhibitor is olaparib or a pharmaceutically acceptable salt thereof. In certain embodiments, the PARP inhibitor is olaparib.

[0176]

[0162] Olaparib (AZD2281, KU-0059436) is a potent PARP inhibitor (PARP1, 2 and 3) being developed as a monotherapy and in combination with chemotherapy agents, ionizing radiation and other anti-cancer agents (including novel agents and immunotherapies).

[0177] PARP inhibition is a novel approach to target tumors with defective DNA repair mechanisms. The PARP enzyme is essential for the re-pairing of DNA single-strand breaks (SSBs).

[0178] Inhibition of PARP leads to the persistence of SSBs, which are subsequently converted into 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 recombination repair (HRR). Tumors with homologous recombination deficiency (HRD), such as ovarian cancer in patients with breast cancer susceptibility gene 1 / 2 (BRCA1 / 2) mutations, cannot accurately repair DNA damage, and DNA abnormalities can accumulate, leading to cell death. In such tumor types, olaparib may potentially offer a more effective and less toxic cancer treatment than currently available chemotherapy regimens. Olaparib traps the inactive form of PARP on DNA at SSB sites, thereby preventing their repair.

[0179] 2.3 Adjuvants In some embodiments, the PI3K inhibitor and ICM-binding antagonist are co-administered with an adjunct agent to treat or support the treatment of a T-cell dysfunction disorder. Non-limiting examples of adjunct agents include cytotoxic agents, gene therapy agents, DNA therapy agents, viral therapy agents, RNA therapy agents, immunotherapy agents, bone marrow transplant agents, nanotherapy agents, or combinations of the above. The adjunct agent may take the form of an adjuvant or neoadjuvant therapy agent. In some embodiments, the adjunct agent is a small molecule enzymatic inhibitor or an anti-metastatic agent. In some embodiments, the adjunct agent is a side effect limiting agent (e.g., an agent intended to reduce the occurrence and / or severity of side effects of treatment, such as an anti-nausea agent). In some embodiments, the adjunct agent is a radiation therapy agent. In some embodiments, the adjunct agent is an agent targeting the PI3K / AKT / mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. In some embodiments, the adjunctive agent is an immunotherapeutic agent, such as the blocking antibody ipilimumab (also known as MDX-010, MDX-101, or Yervoy®), tremelimumab (also known as ticilimumab or CP-675, 206), a B7-H3 directed antagonist (also known as CD276), such as the blocking antibody MGA271, a TGF-β directed antagonist, such as meterimumab (also known as CAT-192), fresolimumab (also known as GC1008), LY2157299, a chimeric antigen receptor (CAR)-expressing T cells (e.g., T cells comprising a dominant negative TGF-β receptor, such as a dominant negative TGF-β type II receptor, CD137-directed agonists (also known as TNFRSF9, 4-1BB or ILA), such as the activating antibody urelumab (also known as BMS-663513), CD40-directed agonists, such as the activating antibody CP-870893, OX40-directed agonists (also known as CD134), such as an activating antibody administered in conjunction with an anti-OX40 antibody (e.g., AgonOX), CD27-directed agonists,For example, activating antibodies CDX-1127, indoleamine-2,3-dioxygenase (IDO), 1-methyl-D-tryptophan (also known as 1-D-MT), antibody-drug conjugates (in some embodiments, including mertansine or monomethyl auristatin E (MMAE)), anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599), trastuzumab emtansine (T-DM1, ado-trastuzumab emtansine or KADCYLA®, available from Genentec h), DMUC5754A, antibody-drug conjugates targeting endothelin B receptor (EDNBR), e.g., EDNBR directed antibody conjugated to MMAE, angiogenesis inhibitors, antibodies directed against VEGF, e.g., VEGF-A, bevacizumab (also known as AVASTIN® (Genentech)), angiopoietin 2 (also known as Ang2) directed antibody MEDI3617, antineoplastic agents, agents targeting CSF-IR (such as M-CSFR or CD115), also known as IFN-α or IFN-γ, Roferon-A, GM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor, rhuGM-CSF, also known as sargramostim or Leukine®), IL-2 (also known as aldesleukin or Proleukin®), IL-12, antibodies targeting CD20 (in some embodiments, the antibody targeting CD20 is obinutuzumab (also known as GA101 or Gazyva®) or rituximab), antibodies targeting GITR (in some embodiments, the antibody targeting GITR is TRX518), in association with cancer vaccines (in some embodiments, the cancer vaccine is a peptide cancer vaccine, in some embodiments, it is a personalized peptide vaccine, in some embodiments, the peptide cancer vaccine is a multivalent long peptide, multi-peptide, peptide cocktail, hybrid peptide, or peptide-pulsed dendritic cell vaccine (e.g.,see Yamada et al., Cancer Sci, 104: 14-21, 2013), in association with adjuvants, TLR agonists such as poly-ICLC (also known as Hiltonol®), LPS, MPL or CpG ODN, TNF, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, HVEM antagonists, ICOS agonists such as by administration of ICOS-L or agonistic antibodies directed against ICOS, agents targeting CX3CL1, agents targeting CXCL10, agents targeting CCL5, LFA-1 or ICAM1 agonists, selectin agonists, targeted therapeutic agents, inhibitors of B-Raf, Murafenib (also known as Zelboraf®), dabrafenib (also known as Tafinlar®), erlotinib (also known as Tarceva®), inhibitors of 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®), K-R Inhibitors of as, inhibitors of c-Met, onartuzumab (also known as MetMAb), inhibitors 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), inhibitors of Akt, MK2206, GSK690693, GDC-0941, inhibitors 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 and MK-8669 or deforolimus), OSI-027, AZD8055, INK128, 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 adjuvant agent may be one or more of the cytotoxic or chemotherapeutic agents described herein.

[0180] In some embodiments, the adjunct is an anti-infective agent. The anti-infective agent is suitably selected from antimicrobial agents, including, but not limited to, compounds that kill or inhibit the growth of microorganisms such as viruses, bacteria, yeasts, fungi, protozoa, and the like, and thus includes antibiotics, amebicides, antifungals, antiprotozoal agents, antimalarials, antituberculous agents, and antivirals. Anti-infective agents also include within their scope anthelmintics and nematicides. Exemplary antibiotics include quinolones (e.g., amifloxacin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, lomefloxacin, oxolinic acid, pefloxacin, losoxacin, temafloxacin, tosufloxacin, sparfloxacin, clinafloxacin, gatifloxacin, moxifloxacin, gemifloxacin, loxacin and garenoxacin), tetracyclines, glycylcyclines and oxazolidinones (e.g., chlortetracycline, demeclocycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, tetracycline, tigecycline), linezolid, eperezolid, glycopeptides, aminoglycosides (e.g., amikacin, arbekacin, butirosin, dibekacin, fortimycin, 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, cefpimizole, cefpiramide, cefpodoxime, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephaacetril, cephalexin, cephaloglycin, cephaloridine, cephalothin, cephapirin, cephradine, cefinetazole, cefoxitin, cefotetan, aztreonam, 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, FP-736, CP-6232, Ro 09-1227, OPC-20000, LY206763), rifamycins, macrolides (e.g., azithromycin, clarithromycin, erythromycin, oleandomycin, rokitamycin, rosaramycin, roxithromycin, troleandomycin), ketolides (e.g., tetrithromycin, cethromycin), coumermycin, lincosamides (e.g., clindamycin, lincomycin), and chloramphenicol. Exemplary antiviral agents 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 antiprotozoal agents include atovaquone, chloroquine hydrochloride, chloroquine phosphate, metronidazole, metronidazole hydrochloride, and pentamidine isethionate. The anthelmintic agent can be at least one selected from mebendazole, pyrantel pamoate, albendazole, ivermectin, and thiabendazole. Exemplary antifungal agents include amphotericin B, amphotericin B cholesteryl sulfate complex, amphotericin B lipid complex, amphotericin B liposomal, fluconazole,The antimalarial agent may be selected from flucytosine, griseofulvin microsize, griseofulvin ultramicrosize, itraconazole, ketoconazole, nystatin, and terbinafine hydrochloride. Non-limiting examples of antimalarial agents include chloroquine hydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine, sulfadoxine, and pyrimethamine. Antituberculous agents include, but are not limited to, clofazimine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine, and streptomycin sulfate.

[0181] 3. Pharmaceutical Compositions and Formulations 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 adjuvant, as described in the Examples herein.

[0182] Pharmaceutical compositions and formulations described herein can be prepared by mixing an active ingredient (e.g., a small molecule, nucleic acid, or polypeptide) having the desired purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and may include, but are not limited to, buffers such as phosphate, citric acid, and other organic acids, antioxidants (including ascorbic acid and methionine), preservatives (e.g., octadecyldimethylbenzylammonium 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), and / or soluble or soluble ... (minor) 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 counterions 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 dispersions, such as soluble neutral-active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968.In one embodiment, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0183] In some embodiments, particularly those relating to peptide and polypeptide active agents (e.g., antibodies, inhibitory peptides, and immunoadhesins), the active agent and optional pharmaceutically acceptable carrier are in the form of a lyophilized formulation or an aqueous solution. Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulation comprising a histidine-acetate buffer.

[0184]

[0146] The compositions and formulations herein may also contain additional active ingredients as needed 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.

[0185] The active ingredient may be entrapped in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions, in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0186] Sustained-release preparations can be prepared. A suitable example of a sustained-release preparation comprises a semipermeable matrix of solid hydrophobic polymers containing an antibody, the matrix being in the form of a shaped article such as a film or microcapsules. Preparations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0187] Depending on the specific condition to be treated, the formulations may be administered systemically or locally. Suitable routes may include, for example, oral, rectal, transmucosal, or enteral administration, or parenteral delivery, including intramuscular, subcutaneous, intramedullary injection, and intrathecal, direct intraventricular / intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection. Formulation and administration techniques may be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.

[0188] 4. Therapeutic use The present invention discloses that PI3K inhibitors and ICM-binding antagonists (also referred to herein as "therapeutic combinations" or "combination treatments") are useful for treating T-cell dysfunction disorders or enhancing immune function (e.g., immune effector function, T-cell function, etc.) in individuals with cancer, or for treating or slowing the progression of cancer, or for treating infection in individuals. In specific embodiments, therapeutic combinations are disclosed for treating or slowing the progression of cancer, including metastatic cancer, and preventing cancer recurrence. Any PI3K inhibitors and ICM-binding antagonists known in the art or described herein may be used in this regard.

[0189] In some embodiments, the combination therapy further comprises the use or administration of an adjunct agent (eg, a chemotherapeutic agent), as described in the Examples herein.

[0190] Preferably, the individual treated with the combination therapy comprises T cells of a mesenchymal phenotype (e.g., CD8+ T cells), e.g., T cells that express CSV, EGRF, and ABCB5, SoX9, SNAIL, and AKT1 at higher levels than the same T cells and / or activated T cells. The T cells may be tumor-infiltrating lymphocytes or circulating lymphocytes. The T cells preferably exhibit T cell exhaustion or anergy; in representative examples of this type, the T cells express higher levels of EOMES than TBET and / or have elevated expression of PD-1. In some embodiments, the T cells have impaired or suppressed immune function and preferably express biomarkers of reduced T cell activation (e.g., reduced production and / or secretion of cytokines such as IL-2, IFN-γ, TNF, etc.). Therefore, TBET, PD1, and EOMES (also referred to herein as "T cell function biomarkers") can be used to determine T cell immune function in a patient to assess the patient's T cell immune status, including susceptibility to treatment with an ICM-binding antagonist.

[0191]

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

[0192] In some embodiments, the individual has been treated with an ICM binding antagonist prior to combined treatment with an ICM binding antagonist and a PI3K inhibitor.

[0193] In some embodiments, the individual has a cancer that is resistant (demonstrated to be resistant) to one or more ICM-binding antagonists. In some embodiments, resistance to an ICM-binding antagonist includes recurrence of the cancer or a refractory cancer. Recurrence can mean the return of the cancer at the original site or a new site after treatment. In some embodiments, resistance to an ICM-binding antagonist includes the progression of the cancer upon treatment with an ICM-binding antagonist. In some embodiments, resistance to an ICM-binding antagonist includes a cancer that does not respond to treatment. The cancer can be resistant at the start of treatment or can become resistant during treatment. In some embodiments, the cancer is early stage or late stage.

[0194] In some embodiments of any of the present 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 technology and FISH, and combinations thereof.

[0195] In some embodiments of any of the present methods, assays, and / or kits, any one or more of the T cell function biomarkers are detected in a 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 antibodies that specifically bind to the respective biomarkers.

[0196] In some embodiments, the combination therapy of the present invention involves administration of a PI3K inhibitor and an ICM-binding antagonist. The PI3K inhibitor and ICM-binding antagonist can be administered in any suitable manner known in the art. For example, the PI3K inhibitor and ICM-binding antagonist can be administered sequentially (at different times) or concomitantly (simultaneously). In some embodiments, the PI3K inhibitor is in a separate composition from the ICM-binding antagonist. In some embodiments, the PI3K inhibitor is in the same composition as the ICM-binding antagonist. Thus, combination therapy can involve separate, simultaneous, or sequential administration of the PI3K inhibitor and the ICM-binding antagonist. In some embodiments, this can be achieved by administering a single composition or pharmacological formulation containing both types of agents, or by simultaneously administering two separate compositions or formulations, one containing a PI3K inhibitor and the other containing an ICM-binding antagonist. In other embodiments, treatment with the PI3K inhibitor may precede or follow treatment with the ICM-binding antagonist by intervals ranging from minutes to days. In embodiments in which the PI3K inhibitor is administered separately from the ICM-binding antagonist, it will generally be ensured that no significant time period expires between each delivery, so that the PI3K inhibitor can still exert its beneficial effects on functional suppressor T cells (e.g., mesenchymal T cells), as described above, and still provide enhanced immune function to T cells, including specifically, susceptibility of T cells to reactivation by the ICM-binding antagonist. In such cases, administration of both modalities within about 1 to 12 hours of each other, more preferably within about 2 to 6 hours of each other, is contemplated. In some circumstances, it may be desirable to significantly extend the duration of treatment, allowing a lapse of several hours (2, 3, 4, 5, 6, or 7) to several days (1, 2, 3, 4, 5, 6, 7, or 8) between each administration.

[0197] It is contemplated that more than one administration of either the PI3K inhibitor or the ICM-binding antagonist may be desired. Where a PI3K inhibitor is "A" and an ICM-binding antagonist is "B," various combinations may be utilized, as exemplified below.

[0198]

[0160] A / B / AB / A / BB / B / AA / A / BB / A / AA / B / BB / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / AB / A / B / AB / A / A / BB / B / B / AA / A / A / BB / A / A / AA / B / A / AA / A / B / AA / B / B / BB / A / B / BB / B / A / B.

[0199] The PI3K inhibitor and ICM-binding antagonist may be administered by the same or 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. Effective amounts of the PI3K inhibitor and ICM-binding antagonist may be administered for the prevention or treatment of diseases. Appropriate dosages of the PI3K inhibitor and ICM-binding antagonist can be determined based on the type of disease being treated, the type of PI3K inhibitor and ICM-binding antagonist, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician. In some embodiments, combination treatment with a PI3K inhibitor (e.g., GDC-0084) and an ICM-binding antagonist (e.g., an anti-PD-1 antibody) is synergistic, and the efficacious dose of the ICM-binding antagonist (e.g., an anti-PD-1 antibody) in combination is reduced compared to the efficacious dose of the ICM-binding antagonist (e.g., an anti-PD-1 antibody) as a single agent.

[0200] As a general proposition, a therapeutically effective amount of a peptide or polypeptide active agent (e.g., 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 administered in one or more doses. In some embodiments, the antibody used is administered at, for example, 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 per day. In some embodiments, the peptide or polypeptide active agent (e.g., antibody, peptide inhibitor, immunoadhesin, etc.) is administered at 15 mg / kg. However, other dosing regimens may be useful. In one embodiment, the 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 a 21-day cycle. The dose can be administered as a single dose or multiple doses (e.g., two or three doses), e.g., by injection. The dose of a peptide or polypeptide active agent (e.g., an antibody, peptide inhibitor, immunoadhesin, etc.) administered in a combination treatment can be reduced compared to a single treatment. The progress of this therapy is easily monitored by conventional techniques.

[0201] Small molecule compounds are generally administered at an initial dosage of about 0.0001 mg / kg to about 1000 mg / kg daily. Daily dosage ranges 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 may be used. However, dosages may vary depending on the needs of the patient, the severity of the condition being treated, and the compound being utilized.

[0202] In any case, dosage can be empirically determined taking into account the type and stage of disease diagnosed in a particular patient. The dosage administered to a patient, in the context of this invention, should be sufficient to elicit a beneficial therapeutic response in the patient over time. Dosage will also be determined by the existence, nature, and extent of any adverse side effects associated with the administration of a particular compound in a particular patient. Determining the appropriate dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages that are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage can be divided and administered multiple times throughout the day as needed. Doses can be given daily or every other day, as determined by the treating physician. Doses can also be given periodically or continuously over longer periods (weeks, months, years), for example, via the use of subdermal capsules, sachets, or depots, or via patches or pumps. In some embodiments, the PI3K inhibitor, ICM binding antagonist, and optionally ancillary agents (e.g., chemotherapeutic agents) are administered on a routine schedule. Alternatively, the combination therapy can be administered as symptoms arise.

[0203] As used herein, a "routine schedule" refers to a predetermined, designated period of time. A routine schedule can encompass periods of the same or different lengths, so long as the schedule is predetermined. For example, a routine schedule can involve administering the PI3K inhibitor, ICM-binding antagonist, and any adjuvant on a daily, every 2, 3, 4, 5, or 6 day basis, weekly, monthly, or any set number of days or weeks therebetween, such as every 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months. Alternatively, the predetermined routine schedule may involve administering the combination of a PI3K inhibitor, an ICM binding antagonist, and any adjunctive agents on a once-daily basis for an initial week, followed by a monthly basis for several months, and then once every three months thereafter. Any particular combination may be subject to a routine schedule, so long as it is predetermined that administration on specific days will involve the appropriate schedule.

[0204] 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 mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplant, nanotherapy, monoclonal antibody therapy, or a combination of the above. 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.

[0205] The efficacy of any of the methods described herein (e.g., combination treatments comprising administering effective amounts of a combination of a PI3K inhibitor, an ICM binding antagonist, and optional adjuvant agents) can be tested in a variety of models known in the art, such as clinical or preclinical models. Suitable preclinical models are exemplified herein and may further include, but are not limited to, ID8 ovarian cancer, GEM model, B16 melanoma, RENCA renal cell carcinoma, CT26 colorectal cancer, MC38 colorectal cancer, and Cloudman melanoma models of cancer.

[0206] The efficacy of any of the methods described herein (e.g., combination treatments comprising administering effective amounts of a combination of a PI3K inhibitor, an ICM-binding antagonist, and any adjuvant agent) can be tested in tumor-generating GEM models, including, but not limited to, GEM models of non-small cell lung cancer, pancreatic ductal adenocarcinoma, or melanoma. For example, mice expressing KrasG12D on a p53-null background after adenoviral recombinase treatment as described by 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 p53-null allele) can be used as a preclinical model for non-small cell lung cancer. As another example, mice expressing KrasG12D on a p16 / p19 null background as described by Jackson et al. (2001, supra) (description of KrasG12D) and Aguirre et al. (2003 Genes Dev. 17(24):3112-26) (p16 / p19 null allele) can be used as a preclinical model for pancreatic ductal adenocarcinoma (PDAC). As a further example, mice bearing melanocytes expressing BrafV600E in a melanocyte-specific PTEN-null background after inducible (e.g., 4-OHT treatment) recombinase treatment, as described by Dankort et al. (2007 Genes Dev. 21(4):379-84) (Braf.sup.V600E description) and Trotman et al. (2003 PLoS Biol. 1(3):E59) (PTEN-null allele), can be used as a preclinical model for melanoma. In either of these exemplary models, after tumor development, mice are randomly recruited into treatment groups receiving a combination of a PI3K inhibitor, an ICM-binding antagonist, and optional adjuvant treatment, or a control treatment. Tumor size (e.g., tumor volume) is measured during the treatment period, and overall survival is also monitored.

[0207] In some embodiments of the methods of the present disclosure, the cancer (in some embodiments, a patient cancer sample examined using the diagnostic tests described in the Examples herein) comprises tumor-infiltrating lymphocytes (TILs), which are within or otherwise associated with the cancerous tissue. In such 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 may be used as biomarkers of T cell exhaustion, which is characterized, for example, by high levels of inhibitory co-receptors and a lack of ability to produce effector cytokines (Wherry, EJ 2011 Nature Immunology 12: 492-499; Rabinovich et al., 2007 Annual Review of Immunology 25: 267-296).

[0208] In some embodiments of the methods of the present disclosure, the individual has a T cell dysfunction manifesting as a T cell dysfunction disorder. The T cell dysfunction disorder may be characterized by T cell anergy or a reduced ability to achieve cytokine secretion, proliferation, or cytolytic activity. In some embodiments of the methods of the present disclosure, the T cell dysfunction disorder is characterized by suppressed T cell immune function. In some embodiments of the methods of the present disclosure, the T cell dysfunction disorder is characterized by mesenchymal phenotype T cells. In some embodiments of the methods of the present disclosure, the T cell dysfunction 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. According to the present invention, PI3K inhibitor treatment may increase the expression of biomarkers of T cell activation and effector capacity (e.g., IL-2, IFN-γ, and TNF), decrease the expression of biomarkers of T cell effector inhibition and cancer progression (e.g., ZEB1), decrease the expression of biomarkers of T cell exhaustion (e.g., PD-1 and EOMES), and / or increase the expression of the transcription factor TBET, which increases IFN-γ production in cells of the adaptive and innate immune systems. Notably, PI3K inhibitor treatment may confer enhanced sensitivity of exhausted T cells to reactivation by an ICM-binding antagonist. Thus, a PI3K inhibitor and an ICM-binding antagonist in a combined treatment may increase the priming, activation, and / or proliferation of T cells (e.g., CD4+ T cells, CD8+ T cells, memory T cells) compared to before administration of the combination. In some embodiments, the T cells are CD4+ and / or CD8+ T cells.

[0209] In some embodiments of the methods of the present disclosure, activated CD4+ and / or CD8+ T cells in the individual are characterized by enhanced cytolytic activity compared to before administration of the IFN-γ-producing CD4+ and / or CD8+ T cells and / or combination, and gamma IFN-γ can be measured by any means known in the art, including, for example, intracellular cytokine staining (ICS), which involves cell fixation, permeabilization, and staining with an antibody to IFN-γ. Cytolytic activity can be measured by any means known in the art, for example, using a cell killing assay with mixed effector and target cells.

[0210] In some embodiments, the CD8+ T cells are characterized, for example, by the presence of CD8b expression (e.g., by RT-PCR using Fluidigm or the like) (Cd8b is also known as T cell surface glycoprotein CD8 beta chain, CD8 antigen alpha polypeptide p3'7, accession number NM_172213). In some embodiments, the CD8+ T cells are from peripheral blood. In some embodiments, the CD8+ T cells are from a tumor.

[0211] In some embodiments, Treg cells are characterized, for example, by the presence of Fox3p expression (e.g., by RT-PCR using Fluidigm or the like) (Foxp3 is also known as forkhead box protein P3, scurfin, FOXP3delta7, immunodeficiency, polyendocrinopathy, enteropathy, X-linked, accession number NM_014009). In some embodiments, Tregs are from peripheral blood. In some embodiments, Treg cells are from tumors.

[0212] In some embodiments, the inflammatory or activated T cells are characterized, for example, by the presence of TBET and / or CXCR3 expression or a TBET:EOMES ratio that correlates with inflammatory or activated T cells (e.g., by RT-PCR using Fluidigm, etc.). In some embodiments, the inflammatory or activated T cells are from peripheral blood. In some embodiments, the inflammatory or activated T cells are from a tumor.

[0213] In some embodiments of the disclosed methods, the CD4+ and / or CD8+ T cells exhibit increased release of a cytokine selected from the group consisting of IFN-γ, TNF, and an interleukin, e.g., IL-2. Cytokine release can be measured by any means known in the art, for example, using Western blot, ELISA, or immunohistochemical assays to detect the presence of released cytokines in a sample containing CD4+ and / or CD8+ T cells.

[0214] 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 expression of CD44highCD62Llow. CD44highCD62Llow expression can be detected by any means known in the art, such as by preparing a single-cell suspension of tissue (e.g., cancer tissue) and performing surface staining and flow cytometry with commercially available 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 expression of CXCR3 (also known as CXC chemokine receptor type 3, Mig receptor, IPIO receptor, G protein-coupled receptor 9, interferon-inducible protein 10 receptor, accession number 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 a tumor.

[0215] In some embodiments of the disclosed methods, administration of an effective amount of a PI3K inhibitor and an ICM-binding antagonist, and optionally an adjunct, to an individual is characterized by an increased level of an inflammatory marker (e.g., CXCR3) on CD8+ T cells compared to before administration of the combination therapy. CXCR3 / CD8+ T cells can be measured by any means known in the art. In some embodiments, the CXCR3 / CD8+ T cells are from peripheral blood. In some embodiments, the CXCR3 / CD8+ T cells are from a tumor.

[0216] In some embodiments of the methods of the invention, Treg function is suppressed compared to before administration of the combination, hi some embodiments, T cell exhaustion is reduced compared to before administration of the combination.

[0217] In some embodiments, the number of Tregs is decreased compared to before administration of the combination. In some embodiments, the level of IFN-γ in plasma is increased compared to before administration of the combination. The number of Tregs can be assessed, for example, by determining the percentage of CD4+Fox3p+CD45+ cells (e.g., by FACS analysis). In some embodiments, for example, the absolute number of Tregs in a sample is determined. In some embodiments, the Tregs are from peripheral blood. In some embodiments, the Tregs are from a tumor.

[0218] In some embodiments, T cell priming, activation and / or proliferation is increased 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 the percentage of Ki67+CD8+ T cells (e.g., by FACS analysis). In some embodiments, T cell proliferation is detected by determining the 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 a tumor.

[0219] 5. Methods of detection and diagnosis According to the present invention, PD-1, TBET, and EOMES may be used to assess T cell exhaustion as known in the art. T cells may be obtained from T cell-containing patient samples, which are suitably selected tissue samples, such as tumors, 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, archived, fresh, or frozen. In some embodiments, the sample is whole blood. In some embodiments, the whole blood contains immune cells, circulating tumor cells, and any combination thereof.

[0220] The presence and / or expression level / amount of a biomarker (e.g., any one or more of TBET and EOMES, also collectively referred to herein as "T cell function biomarkers") may be determined qualitatively and / or quantitatively based on any suitable criteria known in the art, including, but not limited to, DNA, mRNA, cDNA, protein, protein fragment, and / or gene copy number. In certain embodiments, the presence and / or expression level / amount of the biomarker in a first sample is increased or elevated compared to its presence / absence and / or expression level / amount in a second sample (e.g., before treatment with the therapeutic combination). In certain embodiments, the presence / absence and / or expression level / amount of the biomarker in a first sample is decreased or decreased compared to its presence and / or expression level / amount in a second sample. In certain embodiments, the second sample is a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. Additional disclosure for determining the presence / absence and / or expression level / amount of a gene is described herein.

[0221] In some embodiments of any of the methods, increased expression refers to any overall increase of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more in the level of a biomarker (e.g., protein or nucleic acid (e.g., gene or mRNA)) as detected by standard art-known methods described herein compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, increased expression refers to an increase in the expression level / amount of the biomarker in the sample, wherein the increase is at least about 1.5×, 1.75×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 25×, 50×, 75×, or 100× the expression level / amount of the respective biomarker in the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, increased expression refers to an overall increase of 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 compared to a reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene). In some embodiments of any of the methods, decreased expression refers to an overall decrease of any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more in the level of a biomarker (e.g., protein or nucleic acid (e.g., gene or mRNA)) as detected by standard art-known methods described herein, compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.In certain embodiments, reduced expression refers to a decrease in the expression level / amount of a biomarker in a sample, wherein the decrease is at least about 0.9x, 0.8x, 0.7x, 0.6x, 0.5x, 0.4x, 0.3x, 0.2x, 0.1x, 0.05x, or 0.01x of the expression level / amount of the respective biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

[0222]

[0184] The presence and / or expression levels / amounts of various biomarkers in a sample may be analyzed by several methods, many of which are known in the art and understood by those of skill in the art, 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 (e.g., serum ELISA), biochemical enzyme 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-based detection methods such as branched DNA, SISBA, TMA, RNA-Seq, FISH, microarray analysis, gene expression profiling and / or serial analysis of gene expression ("SAGE"), and any one of a variety of assays that may be performed by protein, gene and / or tissue array analysis. Exemplary protocols for assessing the status of genes and gene products can be found, for example, in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting), and 18 (PCR Analysis). Multiplex immunoassays, such as those available from Rules-Based Medicine or Mesoscale Discovery ("MSD"), can also be used.

[0223] In some embodiments, the presence and / or expression level / amount of the biomarkers is determined using a method comprising: (a) performing gene expression profiling, PCR (e.g., rtPCR or qRT-PCR), RNA-seq, microarray analysis, SAGE, MassARRAY technology, or FISH on a sample (e.g., a subject cancer sample); and b) determining the presence and / or expression level / amount of the biomarkers in the sample. In some embodiments, the microarray method comprises the use of a microarray chip having one or more nucleic acid molecules hybridizable under stringent conditions to nucleic acid molecules encoding the above-mentioned genes, or having one or more polypeptides (e.g., peptides or antibodies) capable of binding to one or more of the proteins encoded by the above-mentioned genes. 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.

[0224]

[0186] Methods for assessing mRNA in cells are well known and include, for example, hybridization assays using complementary DNA probes (e.g., in situ hybridization, Northern blots and related techniques using labeled riboprobes specific for one or more genes) and various nucleic acid amplification assays (e.g., RT-PCR and other amplification-based detection methods using complementary primers specific for one or more genes, e.g., branched DNA, SISBA, TMA, etc.).

[0225]

[0187] Samples from mammals can be conveniently assayed for mRNA using Northern, dot blot, or PCR analysis. In addition, such methods can include one or more steps that allow the level of target mRNA in a biological sample to be determined (e.g., by simultaneously examining the level of 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.

[0226]

[0188] Optional methods include protocols for examining or detecting mRNA, such as target mRNA, in tissue or cell samples using microarray technology. 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 so that the sequence and location of each member of the array is known. For example, a selection of genes whose expression correlates with increased or decreased clinical benefit of antiangiogenic therapy can 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.

[0227] According to some embodiments, the presence and / or expression level / amount is measured by monitoring the protein expression levels of the aforementioned genes. In certain embodiments, the method comprises contacting a biological sample with an antibody against a biomarker described herein (e.g., an anti-PD-1 antibody, an anti-PI3K antibody, an anti-TBET antibody, an anti-EOMES antibody) under conditions that allow binding of the biomarker, and detecting whether a complex is formed between the antibody and the biomarker. Such methods can be in vitro or in vivo methods. 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.

[0228] In certain embodiments, the presence and / or expression level / amount of biomarker protein in a sample is determined using IHC and staining protocols. IHC staining of tissue sections has been shown to be a reliable method of determining or detecting the presence of a protein in a sample. In some embodiments, the 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, the expression level of the biomarker is determined using a method comprising: (a) performing IHC analysis of a sample (e.g., a subject cancer sample) using an antibody; and (b) determining the expression level of the biomarker in the sample. In some embodiments, the 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., a control cell line stained sample or a tissue sample from a non-cancerous patient).

[0229] In some embodiments, T cell function biomarker expression is assessed in tumors or tumor samples. As used herein, a tumor or tumor sample may encompass some or all of the tumor area occupied by tumor cells. In some embodiments, a tumor or tumor sample may further encompass tumor areas occupied by tumor-associated intratumoral cells and / or tumor-associated stroma (e.g., continuous peritumoral desmoplastic stroma). Tumor-associated intratumoral cells and / or tumor-associated stroma may include areas of immune infiltrate (e.g., tumor-infiltrating immune cells described herein) directly adjacent to and / or contiguous with the main tumor mass. In some embodiments, T cell function biomarker expression is assessed in tumor cells. In some embodiments, T cell function biomarker expression is assessed in immune cells within the tumor area, such as tumor-infiltrating immune cells.

[0230] In an alternative method, a sample can be contacted with an antibody specific for the biomarker under conditions sufficient to form an antibody-biomarker complex, and then the complex can be detected. The presence of a biomarker can be detected in several ways, such as Western blotting and ELISA procedures, for assaying a variety of tissues and samples, including plasma or serum. A wide range of immunoassay techniques using such assay formats are available. See, for example, U.S. Patent Nos. 4,016,043, 4,424,279, and 4,018,653. These include both single-site and two-site assays or non-competitive "sandwich" assays, as well as traditional competitive binding assays. These assays also include direct binding of labeled antibodies to target biomarkers.

[0231] The presence and / or expression level / amount of a selected T cell function biomarker in a tissue or cell sample can also be examined via a functional or activity-based assay. For example, if the biomarker is an enzyme (e.g., PI3K), an assay known in the art (e.g., a kinase assay) can be performed to determine or detect the presence of a given enzyme activity in a tissue or cell sample.

[0232] In certain embodiments, samples may be normalized for both differences in the amount of biomarkers assayed and variations in the quality of the samples used and between assay runs. Such normalization may be achieved by detecting and incorporating the expression of specific normalizing biomarkers, including well-known housekeeping genes.

[0233] Alternatively, normalization can be based on the average or median signal of all of the assay genes or a large subset thereof (global normalization approach). On a gene-by-gene basis, the measured normalized amount of a subject tumor mRNA or protein is compared to the amount found in a reference set. The normalized expression level of each mRNA or protein per test tumor 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 being analyzed will fall at some percentile within this range. This can be determined by methods well known in the art.

[0234] 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 tumor. Tissue biopsy is often used to obtain a representative piece of tumor tissue. Alternatively, tumor cells can be obtained indirectly in the form of tissue or fluid known or suspected to contain the target tumor cells. For example, samples of lung cancer lesions can be obtained by resection, bronchoscopy, fine needle aspiration, bronchial scraping, or from sputum, pleural fluid, or blood. Genes or gene products can be detected from cancer or tumor tissue or other biological samples, such as urine, sputum, serum, or plasma. The same techniques discussed above for detecting target genes or gene products in cancerous samples can be applied to other biological samples. Cancer cells can be excised from cancer lesions and present in such biological samples. By screening such biological samples, simple and early diagnosis of these cancers can be achieved. In addition, the progress of therapy can be more easily monitored by testing such biological samples for target genes or gene products.

[0235] In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or a combined plurality of samples from the same subject or individual obtained at one or more time points different from the time point at which the test sample is obtained. For example, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from the same subject or individual at an earlier time point than the time point at which the test sample is obtained. Such reference samples, reference cells, reference tissues, control samples, control cells, or control tissues may be useful when the reference sample is obtained at the time of initial diagnosis of cancer and when the test sample is obtained later, when the cancer has become metastatic.

[0236] In certain embodiments, the 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 other than the subject or individual. In certain embodiments, the 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) other than the subject or individual. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from normal tissue or pooled plasma or serum samples from one or more individuals other than the subject or individual. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample from tumor tissue or pooled plasma or serum samples from one or more individuals with a disease or disorder (e.g., cancer) other than the subject or individual.

[0237] In some embodiments, the sample is a tissue sample from an individual. In some embodiments, the tissue sample is a tumor tissue sample (e.g., biopsy tissue). In some embodiments, the tissue sample is lung tissue. In some embodiments, the tissue sample is kidney 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 stomach 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 node 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, etc.).

[0238] In some embodiments, a tumor tissue sample (the term "tumor sample" is used interchangeably herein) may include some or all of the tumor area occupied by tumor cells. In some embodiments, a tumor or tumor sample may further include tumor areas occupied by tumor-associated intratumoral cells and / or tumor-associated stroma (e.g., continuous peritumoral desmoplastic stroma). Tumor-associated intratumoral cells and / or tumor-associated stroma may include areas of immune infiltrate (e.g., tumor-infiltrating immune cells as described herein) directly adjacent to and / or contiguous with the main tumor mass.

[0239] In some embodiments, tumor cell staining is expressed as a percentage of all tumor cells that exhibit membrane staining of any intensity. Infiltrating immune cell staining can be expressed as a percentage of the total tumor area occupied by immune cells that exhibit staining of any intensity. The total tumor area encompasses malignant cells as well as tumor-associated stroma, including areas of immune infiltrate directly adjacent to and contiguous with the main tumor mass. Additionally, infiltrating immune cell staining can be expressed as a percentage of all tumor-infiltrating immune cells.

[0240] In some embodiments of any of the methods, the disease or disorder is a tumor. In some embodiments, the tumor is a malignant cancerous tumor (i.e., cancer). In some embodiments, the tumor and / or cancer is a solid tumor.

[0241] Solid tumors include any cancer of body tissues other than blood, bone marrow, and the lymphatic system. Solid tumors can be further divided into those of epithelial cell origin and those of non-epithelial cell origin. Examples of epithelial cell solid tumors include tumors of the gastrointestinal tract, colon, colorectum (e.g., basal-like 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, gallbladder, lip, nasopharynx, skin, uterus, male genital tract, urinary tract (e.g., urothelial carcinoma, dysplastic urothelial carcinoma, transitional cell carcinoma), bladder, and skin. Solid tumors of non-epithelial origin include sarcoma, brain tumor, and bone tumor. 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 an adenocarcinoma. In some embodiments, the cancer is a 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 tumor. In some embodiments, the cancer is a metastatic tumor at a second site derived from any of the above types of cancer.

[0242] In some embodiments of any of the methods, the cancer exhibits human effector cells (e.g., is infiltrated by human effector cells). Methods of detecting human effector cells are well known in the art, including, for example, by IHC. In some embodiments, the cancer exhibits high levels of human effector cells. In some embodiments, the human effector cells are one or more of NK cells, macrophages, and 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.

[0243] In some embodiments of any of the methods, the cancer exhibits cells that express FcR (e.g., is infiltrated by cells that express FcR). Methods of detecting FcR are well known in the art, including, for example, by IHC. In some embodiments, the cancer exhibits high levels of cells that express FcR. In some embodiments, the FcR is an FcyR. In some embodiments, the FcR is an 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.

[0244] 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 technology, 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, PD-1 expression is detected in a blood sample. In some embodiments, PD-1 expression is detected on circulating immune cells in the blood sample. In some embodiments, the circulating immune cells are CD3+ / CD8+ T cells. In some embodiments, the immune cells are isolated from the blood sample prior to analysis. Any suitable method of isolating / enriching such cell populations may be used, including but not limited to cell sorting. In some embodiments, PD-1 expression is reduced in samples from individuals who respond to treatment with a PI3K inhibitor and / or an ICM-binding antagonist, e.g., an anti-PD-1 antibody. In some embodiments, PD-1 expression is elevated on circulating immune cells, such as CD3+ / CD8+ T cells, in blood samples.

[0245]

[0207] Also provided herein is a method for monitoring the pharmacodynamic activity of an ICM-binding antagonist treatment by measuring the expression level of one or more T cell functional biomarkers described herein in a sample comprising leukocytes obtained from a subject, wherein the subject is treated with an ICM-binding antagonist and a PI3K inhibitor, and one or more of the T cell functional biomarkers are selected from TBET, PD-1, and EOMES, as well as a method for determining a treatment as demonstrating pharmacodynamic activity based on the expression level of one or more T cell functional biomarkers in a sample obtained from the subject compared to a reference, wherein an increased expression level of the one or more T cell functional biomarkers compared to the reference is indicative of pharmacodynamic activity for the PD-1 antagonist treatment. These methods may further include measuring the expression level of one or more additional biomarkers of T cell function and / or T cell composition (e.g., the percentage of Tregs and / or the absolute number of Tregs, e.g., the number of CD8+ effector T cells), where the additional biomarkers of T cell function include cytokines such as IFN-γ or T cell markers or memory T cell markers (e.g., markers of T effector memory cells), and determining a treatment as demonstrating pharmacodynamic activity based on the expression level of the one or more biomarkers of T cell function, the one or more additional biomarkers of T cell function, and / or the T cell composition in a sample obtained from the subject compared to a reference, where an increase in the expression level of the one or more biomarkers of T cell function, the one or more additional biomarkers of T cell function, and / or the T cell composition compared to the reference is indicative of pharmacodynamic activity for the PD-1 antagonist treatment. The expression level of the biomarkers and / or the cell composition may be measured by one or more methods described herein.

[0246] As used herein, "pharmacodynamic (PD) activity" can refer to the effect of treatment (e.g., combination treatment with a PI3K inhibitor and an ICM-binding antagonist) on a subject. Examples of PD activity can include modulation of the expression levels of one or more genes. Without wishing to be bound by theory, it is believed that monitoring PD activity, such as by measuring the expression of one or more T cell function biomarkers, can be advantageous during clinical trials testing PI3K inhibitors and ICM-binding antagonists. Monitoring PD activity can be used, for example, to monitor response to treatment, toxicity, etc.

[0247] In some embodiments, the expression levels and / or cellular composition 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 treatment (e.g., combination treatment with a PI3K inhibitor and an ICM binding antagonist). In some embodiments, the reference may include a sample from the same subject prior to receiving treatment (e.g., combination treatment with a PI3K inhibitor and an ICM binding antagonist). In some embodiments, the reference may include reference values ​​from one or more samples from other subjects receiving treatment (e.g., combination treatment with a PI3K inhibitor and an ICM binding antagonist). For example, a patient population may be treated, and a mean, representative, or median value for the expression level of one or more genes may be generated from the population as a whole. A set of samples obtained from cancers with shared characteristics (e.g., the same cancer type and / or stage or administration of a common treatment, such as combination treatment with a PI3K inhibitor and an ICM binding antagonist) may be tested from a population, for example, in a clinical outcome study. This set may be used to derive a reference, such as a reference number, to which a subject's sample can be compared. Any of the references described herein can be used as a reference for monitoring PD activity.

[0248] Certain aspects of the present disclosure relate to measuring the expression levels of one or more biomarkers (e.g., gene expression products including mRNA and protein) in a sample. In some embodiments, the sample may include leukocytes. In some embodiments, the sample may be a peripheral blood sample (e.g., from a patient with a tumor). In some embodiments, the sample is a tumor sample. A tumor sample may include cancer cells, lymphocytes, leukocytes, stroma, blood vessels, connective tissue, basement membrane, and any other cell types associated with the tumor. In some embodiments, the sample is a tumor tissue sample containing tumor-infiltrating leukocytes. In some embodiments, the 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.

[0249] Tumor samples can be obtained from a subject by any method known in the art, including, but not limited to, biopsy, endoscopy, or surgical procedures. In some embodiments, tumor samples can be prepared by methods such as freezing, fixing (e.g., by using formalin or a similar fixative), and / or embedding in paraffin wax. In some embodiments, tumor samples can be sectioned. In some embodiments, fresh tumor samples (i.e., not prepared by the above methods) can be used. In some embodiments, tumor samples can be prepared by incubation in a solution to preserve the integrity of mRNA and / or protein.

[0250] In some embodiments, the sample may be a peripheral blood sample. The peripheral blood sample may include white blood cells, PBMCs, etc. Any technique known in the art for isolating white blood cells from a peripheral blood sample may be used. For example, a blood sample may be aspirated, red blood cells may be lysed, and a white blood cell pellet may be isolated and used in the sample. In another example, density gradient separation may be used to separate white blood cells (e.g., PBMCs) from red blood cells. In some embodiments, a fresh peripheral blood sample (i.e., one not prepared by the methods described above) may be used. In some embodiments, the peripheral blood sample may be prepared by incubation in a solution to preserve the integrity of the mRNA and / or protein.

[0251] In some embodiments, responsiveness to treatment can mean any one or more of: prolonging survival (including overall survival and progression-free survival), producing an objective response (including a complete response or a partial response), or improving signs or symptoms of cancer. In some embodiments, responsiveness can mean improvement in one or more factors according to the published set of RECIST guidelines for determining tumor status in cancer patients, i.e., response, stabilization, or progression. 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 shows improvement, for example, by one or more factors based on the RECIST criteria. A non-responsive subject may refer to a subject whose cancer does not show improvement, for example, by one or more factors based on the RECIST criteria.

[0252] Traditional response criteria may be inadequate for characterizing the antitumor activity of the therapeutic agents of the present invention and may result in delayed responses that may be preceded by early apparent radiological progression, including the appearance of new lesions. Therefore, modified response criteria have been developed that take into account the possibility of new lesion appearance and that allow for radiological progression to be confirmed at subsequent evaluations. Thus, in some embodiments, responsiveness may refer to improvement in one or more factors according to immune-related response criteria (irRC). See, e.g., Wolchok et al. (2009, supra). In some embodiments, new lesions are added to a defined tumor burden and are tracked for radiological progression, e.g., at subsequent evaluations. In some embodiments, the presence of non-target lesions is included in the assessment of complete response, but not in the assessment of radiological progression. In some embodiments, radiological progression may be determined solely based on measurable disease and / or confirmed by serial evaluations for ≥4 weeks from the first confirmed date.

[0253] In some embodiments, responsiveness can include immune activation. In some embodiments, responsiveness can include therapeutic efficacy. In some embodiments, responsiveness can include immune activation and therapeutic efficacy.

[0254] 6. Kit

[0216] In another aspect of the invention, therapeutic kits are provided that include a PI3K inhibitor and an ICM-binding antagonist. In some embodiments, the therapeutic kits further include a package insert containing instructions for administering the PI3K inhibitor and the ICM-binding antagonist in combination to treat a T cell dysfunction disorder or enhance immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, or to treat or slow cancer progression or treat an infection in an individual. Any PI3K inhibitor and ICM-binding antagonist described herein or known in the art can be included in the kit.

[0255] In some embodiments, the PI3K inhibitor and ICM-binding antagonist are in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers can be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), metal alloys (e.g., stainless steel or Hastelloy), etc. In some embodiments, the container holds the formulation, and a label on or associated with the container can indicate usage. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the kit further includes one or more other agents (e.g., chemotherapeutic agents and anti-neoplastic agents). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes.

[0256] In other embodiments of the present invention, diagnostic kits for determining expression of biomarkers, including T cell function biomarkers disclosed herein, are provided, comprising reagents that allow for detection and / or quantification of the biomarkers. Such reagents include, for example, a compound or material, or a set of compounds or materials, that allow for quantification of the biomarkers. In specific embodiments, the compound, material, or set of compounds or materials allows for the determination of the expression level of a gene (e.g., a T cell function biomarker gene), including, but not limited to, extraction of RNA material, determination of the level of the corresponding RNA, primers for synthesis of the corresponding cDNA, primers for amplification of the DNA, and / or probes capable of specifically hybridizing to the RNA encoded by the gene (or the corresponding cDNA), TaqMan probes, proximity assay probes, ligases, antibodies, etc.

[0257] The kit may also optionally include appropriate reagents for detection of the label, positive and negative controls, wash 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 the T cell function biomarker polynucleotide. Enzymes suitable for amplifying nucleic acids, including various polymerases (e.g., reverse transcriptase, Tag, Sequenase™, DNA ligase, etc., depending on the nucleic acid amplification technique used), deoxynucleotides, and buffers may also be included to provide the reaction mixture necessary for amplification. Such kits will generally also include, in a suitable manner, identifiable containers for each individual reagent and enzyme and 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); and (ii) an antibody that specifically binds to the T cell function biomarker polypeptide. The kits may 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 instructions for using the kit to quantitate expression of T cell function biomarker genes. The reagents described herein, which may optionally be associated with a detectable label, may be presented in the form of a microfluidics card, chip or chamber, microarray, or kit adapted for use in the assays described in the Examples or below, e.g., the RT-PCR or QPCR techniques described herein.

[0258] Suitable materials for packaging the components of the diagnostic kit may include crystal, plastic (polyethylene, polypropylene, polycarbonate, etc.), bottles, vials, paper, envelopes, etc. In addition, the kit of the present invention may include instructions for simultaneous, sequential, or separate use of the various components included in the kit. The instructions may be in the form of printed matter or electronic support, such as an electronic storage medium (magnetic disk, tape, etc.), optical medium (CD-ROM, DVD), etc., capable of storing the instructions so that they are readable by the subject.

[0259]

[0221] Alternatively or additionally, the medium may include an internet address providing the instructions.

[0260]

[0222] In order that the present invention may be readily understood and put into practical effect, certain preferred embodiments will now be described by the following non-limiting experimental examples. [Example]

[0261] Example Example 1 PI3K expression is associated with reduced cancer survival PI3KCA is part of the cytoplasmic / plasma membrane PI3KCA / AKT / MTOR signaling pathway (Hassan et al., 2013). PI3KCA mutational burden has been linked to 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 signaling pathway is associated with cancer stem cell (CSC) and EMT (Chen et al., 2020; Xia et al., 2015). PI3KCA contributes to immune evasion by phosphorylating AKT(P), inducing PDL1 expression, and mediating immune evasion (Yang et al., 2017). 35% of breast cancer patients have mutation-positive tumors (Fusco et al., 2021), and PI3KCA mutations were present in approximately 6% of TNBC patients (Mosele et al., 2020). This does not account for the majority of patients who relapse or have resistance to immunotherapy.

[0262] Using the web tool KM Plotter (Lanczky and Gyorffy, 2021), we plotted overall survival probability against low and high protein expression of PI3K (Figure 1). This analysis revealed a significant reduction in survival in the high PI3K expression cohort in both metastatic breast cancer and non-small cell lung cancer patient cohorts. This analysis was performed to demonstrate that PI3K enrichment is associated with a significant reduction in survival probability across multiple cancer types.

[0263] Liquid biopsy-based stratification detecting enrichment of PI3K CSC and CD8 T cell signatures and inhibition of PI3K targets CSC relapse signatures and exhausted T cell signatures in patient liquid biopsies We identified dual CSC and T cell signatures in patient liquid biopsies from stage IV solid tumor cohorts (i.e., melanoma, NSCLC, RCC, HCC, breast cancer, and GBM). Using our novel liquid biopsy platform, we identified a novel cancer stem cell-like mesenchymal signature enriched for PI3K in stage IV cancer patients (Figure 2). This CSC is a key cell type involved in cancer recurrence and is not targeted by chemotherapy, immunotherapy, or radiotherapy, i.e., traditional cancer therapies. However, we hypothesized that these patients would respond to our monotherapy and / or combination therapy approach. These CTCs are positive for the mesenchymal CSC markers CSV and ABCB5.

[0264]

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

[0265] Analysis revealed that the overall expression intensity of CSV and ABCB5, as measured by immunofluorescence analysis using a digital pathology system, was significantly reduced in GDC-0084-treated samples (Figure 3A). In contrast, EpCAM, a non-mesenchymal epithelial cell marker, showed little change in expression (Figure 3A). Population dynamic analysis using a digital pathology system revealed that the CSV- and ABCB5-positive cell populations decreased by approximately 56%, while EpCAM increased by 38% (Figure 3B). PI3KCA inhibition by GDC-0084 also inhibited the % AKT1-positive cells (AKT1 contributes to the mesenchymal metastatic signature) and global expression of AKT1 (AKT1 is a key tumorigenic marker regulated by PI3KCA). This suggests that targeting PI3KCA targets key regulators of metastasis, mesenchymal transition, and cancer stem cell signatures (CSCs).

[0266] Liquid biopsy-based assay from stage IV solid tumor cancer patients demonstrates that PI3K inhibition suppresses T cell dysfunction and induces effector signatures CD8+ T cells were analyzed for overall expression intensity measured by immunofluorescence analysis using our laboratory's digital pathology system. The 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 demonstrated significant inhibition of TIM3 in both NS and ST CD8+ T cells by GDC-0084 treatment, while GDC-0084 significantly inhibited TIGIT only in NS CD8+ T cells (Figure 4B). Figure 4C shows the effector signature panel, which, in contrast to Figures 4A and 4B, demonstrates significant increases in perforin and GZNb expression in GDC-0084-treated CD8+ T cells in both NS and ST groups.

[0267] PI3K enriched in cancer cell lines enriched for mesenchymal CSC signatures Whole-cell intensity analysis was performed using the ASI Digital Pathology System according to normalization settings. Analysis of PI3KCA expression in breast cancer cell lines (Figure 5A) revealed a pattern of increasing expression as the cell line transitioned from an epithelial signature to a more mesenchymal, metastatic, immunotherapy-resistant signature (MCF7 is an epithelial breast cancer cell line with a low CSC population; MDA-MB-231 is a TNBC cell line with >90% mesenchymal SCs; MDA-MB-231-BR is a brain metastatic cancer clone of MDA-MB-231 with >90% mesenchymal CSCs; and finally, 4T1 is an immunotherapy-resistant, metastatic, aggressive breast cancer model with >90% CSCs).

[0268] Interestingly, a similar pattern was observed in 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, while LLC is highly immunotherapy-resistant and aggressive. Overall, these data suggest that PI3KCA is enriched in metastatic treatment-resistant cancer cell lines in terms of whole-cell expression. This suggests the importance of targeting PI3KCA to treat metastatic treatment-resistant cancers, which represent a significant unmet medical need. ·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, H1299: epithelial-like lung cancer cell line, CT26: an epithelial-like highly immunogenic murine colon tumor cell line; and ·LLC: Highly treatment-resistant immunotherapy-resistant Lewis lung cancer cell line (Li et al., 2021).

[0269] Effects of PI3K inhibitor GDC-0084 on cell migration and invasion of MDA-MB-231 and CT26 Interestingly, the PI3K / Akt / mTOR pathway is activated in up to 70% of patients with breast cancer brain metastases. Upregulation of this pathway is associated with a worse prognosis, yet no approved drugs are available for these patients. The PI3K inhibitor GDC-0084 is a brain-penetrating agent that has shown promising activity in preclinical models of glioblastoma. GDC-0084 exhibits high activity with its IC25 (1.25 μM) and IC 50Both GDC-0084 and GDC-0084-treated mice (2.5 μM) inhibited breast cancer cell migration, with greater inhibition of MDA-MB231 cell migration at IC25 concentrations (Figure 6). Wound healing analysis demonstrated that treatment with GDC-0084 (a PI3K inhibitor) inhibited CT26 cell migration. DMSO treatment was used as a control. These data show that 0.3 μM GDC-0084 had a more pronounced effect on wound healing compared to lower concentrations (i.e., 0.2 μM) (Figure 6C). This suggests that GDC-0084 is capable of inhibiting cancer cell migration at higher concentrations. GDC-0084 exhibited the greatest inhibitory effect on the responsive colon cancer cell line, CT26. We used DMSO treatment as a control.

[0270] IC of GDC-0084 against different cancer cell lines 50 Decision The data clearly demonstrate EC50 data for GDC-0084 in different cell lines. Individual dose-response curves for CT26, 4T1, MDA-MB-231, MDA-MB-231-Brm, and MCF7 are shown in Figure 7. These data were obtained to demonstrate the efficacy of targeting PI3KCA with GDC-0084 in immunotherapy-responsive or epithelial cancer cell lines (CT26, MCF7) compared with immunotherapy-resistant or mesenchymal cancer cell lines (4T1, MDA-MB-231). These data demonstrate that targeting PI3K with GDC-0084 can significantly suppress the proliferation of both epithelial and mesenchymal cancer cell lines.

[0271] The effect of PARP inhibition on mesenchymal CSC signatures Olaparib and veliparib are PARP inhibitors used to treat several types of breast and ovarian cancer. However, olaparib does not target cancer stem cells (CSCs) or mesenchymal signatures, and does not affect ABCB5 or ALDH1A (Figure 8). We next examined the effects 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). To further explore the importance of monotherapy targeting of PARP, we examined the effect of siRNA knockdown of PARP on the mesenchymal CSC signature (Figure 10). We confirmed that PARP knockdown did not inhibit these markers of the mesenchymal CSC signature (Figure 11). Combined treatment with the PI3K pathway inhibitor GDC-0084 and the PARP inhibitor olaparib restricted MDA-MB-231 cell migration (Figure 17). The inventors demonstrated that the combination of GDC-0084 and olaparib inhibited breast cancer cell migration, with combination "2" (i.e., 1.25 μM GDC-0084 + 250 nM olaparib) having the greatest inhibition of MDA-MB-231 migration. This demonstrates that PARP inhibitors are not effective monotherapeutic agents but may be beneficial in combination with PI3K inhibitors.

[0272] αPD1 therapy in CT26 colon cancer model and 4T1 breast cancer model We sought to demonstrate that αPD1 monotherapy causes a significant reduction in tumor volume in the immunotherapy-responsive CT26 colon cancer model compared to the 4T1 breast cancer model ( FIG. 12 ). This data establishes that CT26 is an immunotherapy-responsive line, while the 4T1 model clearly demonstrates that 4T1 is an immunotherapy-resistant model, as demonstrated by the efficacy of primary tumor burden in response to αPD1 immunotherapy.

[0273] PI3K inhibition targets and blocks the expression of mesenchymal resistance markers Next, we demonstrated that treatment with GDC-0084 significantly inhibited CSC / mesenchymal markers, induced expression of the epithelial marker EpCAM, and also increased expression of the viral mimicry / immunovisible marker MDA5 (Figures 13 and 14). This demonstrates that targeting PI3KCA also inhibits metastatic and therapy-resistant signatures, further rendering tumors more immunovisible and therefore more responsive to combined immunotherapy.

[0274] Next, we performed further inhibition analysis using different inhibitors targeting PI3K to confirm the efficacy of targeting PI3K to inhibit the mesenchymal CSC signature. Two different PI3K inhibitors were used: idelalisib, which is used to treat certain hematological cancers such as chronic lymphocytic leukemia after relapse, and LY294002, a potent PI3K inhibitor currently undergoing clinical trials in patients with recurrent or progressive HNSCC and patients with mutations in PI3KCA and / or PI3K pathway genes (Figure 15). Treatment with both PI3K inhibitors (Figure 15) significantly inhibited the CSC / stem-like cancer recurrence markers CSV, SoX9, ABCB5, and SNAIL at two different low doses. The 12.5 μM idelalisib dose had the least significant effect. Additionally, 25 μM idelalisib and both concentrations of LY294002 all induced expression of the epithelial marker EpCAM. These data demonstrate that targeting PI3K with an alternative inhibitor to GDC-0084 is effective in inhibiting the mesenchymal cancer stem cell signature and inducing the epithelial signature.

[0275] GDC-0084 administration at 7.5 mg / kg reduces clinical abnormalities in a 4T1 syngeneic tumor model Next, we demonstrated that GDC-0084 monotherapy at 7.5 mg / kg reduced the clinical abnormalities observed in the 4T1 breast cancer model. The group of mice receiving the higher dose (i.e., 15 mg / kg) suffered from reduced activity and hunched posture, as well as multiple cases of significant piloerection (Figure 16). Compared to 15 mg / kg, GDC-0084 administration at 7.5 mg / kg reduced the clinical abnormalities previously observed at the higher dose. Forty percent of mice treated with 15 mg / kg GDC-0084 demonstrated reduced activity, hunched posture, piloerection, and weight loss, whereas no clinical abnormalities were present in the 7.5 mg / kg treatment group. These data highlight reduced toxicity at lower doses.

[0276] GDC-0084 administration at 7.5 mg / kg inhibits tumor burden Next, we demonstrated that "low-dose" GDC-0084 therapy (7.5 mg / kg) abrogates primary tumor burden in a 4T1 breast cancer model (see Figure 17). GDC-0084 administration at 7.5 mg / kg, both as monotherapy and in combination with αPD1, substantially reduces tumor volume and final tumor weight, achieving a reduction of more than 50% in the 4T1 breast cancer model. Of note, no significant reduction in tumor burden was observed in mice administered 15 mg / kg GDC-0084 with or without αPD1.

[0277] GDC-0084 at 7.5 mg / kg reduces tumor inflammation, including monocyte and neutrophil infiltration Administration of "low dose" GDC-0084 (i.e., 7.5 mg / kg) reduces tumor inflammation, including monocyte and neutrophil infiltration (Figure 18). Administration of 7.5 mg / kg GDC-0084, both as monotherapy and in combination with αPD1, dramatically reduces tumor inflammation associated with the 4T1 breast cancer model. While tumor inflammation was scored as "moderate" and "severe" in the control and αPD1-treated groups, only mild inflammation was observed when administered in combination with 7.5 mg / kg GDC-0084.

[0278] Additionally, pathologists noted a substantial reduction in myeloid populations, including monocytes and neutrophils, in the GDC-0084 7.5 mg / kg treatment group. Notably, no significant reduction in inflammation was observed in mice receiving a daily dose of 15 mg / kg GDC-0084.

[0279] GDC-0084 at 7.5 mg / kg reduces splenomegaly in a 4T1 syngeneic tumor model GDC-0084 administration at 7.5 mg / kg reduces splenomegaly in the 4T1 breast cancer model. At lower doses of GDC, the spleen appears normal in size compared to higher doses (Figure 19). GDC administration at 7.5 mg / kg reduces the splenomegaly typically associated with the 4T1 breast cancer model.

[0280]

[0185] No significant difference in spleen weight was observed between the 15 mg / kg treatment group and the corresponding controls, but a dramatic reduction in splenomegaly was observed at the lower dose of GDC-0084 (7.5 mg / kg), both as monotherapy and in combination with αPD1.

[0281] GDC-0084 administration at 7.5 mg / kg reduces SOX9 and TOX1 expression In both experiments, combination treatment significantly reduced SOX9 expression (SOX9 is a marker for mesenchymal therapy-resistant cancer stem cells), with 7.5 mg / kg GDC-0084 having a more pronounced effect and showing exceptional reduction as monotherapy. Interestingly, 15 mg / kg GDC-0084 showed induction of TOX1 (TOX1 is a marker of T cell exhaustion and dysfunction) in CD8+ T cells, while at 7.5 mg / kg, a significant reduction of TOX1 was observed in CD8+ T cells (Figure 20). These data demonstrate that "high-dose" GDC-0084 induces dysregulated inflammation, contributing to increased T cell dysfunction (higher TOX1 expression), and does not regulate SOX9, a marker for cancer stem cell-like mesenchymal signatures, metastasis, and cancer recurrence. In contrast, "low-dose" GDC-0084, either as monotherapy or in combination, significantly inhibited SOX9+ expression and significantly reduced TOX1 expression in CD8+ T cells. Notably, the combination of anti-PD1 and "low-dose" GDC-0084 (7.5 mg) had the strongest effect in suppressing TOX1 expression (Figure 20).

[0282]

[0187] PI3K alone or in combination inhibition targets the mesenchymal signature to induce an epithelial phenotype in an IO resistance mouse model.

[0283]

[0188] Combination treatment with GDC-0084 and anti-PD1 significantly inhibited PI3KCA, mesenchymal markers CSV and EGFR, and furthermore, this combination also resulted in a significant induction of the epithelial marker E-cadherin (Figure 21).

[0284] Both GDC-0084 monotherapy and combination treatment with anti-PD1 significantly induced a cell population positive for the epithelial marker E-cadherin (see Figure 22). GDC-0084 with or without αPD1 therapy did not affect mouse body weight in a 4T1 allogeneic tumor model (Figure 17). GDC-0084 combination therapy with αPD1 significantly suppressed tumor burden, but not as a high-dose monotherapy, whereas a low-dose monotherapy could suppress tumor burden (Figure 17). Notably, this combination also worked in the 4T1 immunotherapy-resistant tumor model. Combination treatment with GDC-0084 and an anti-PD1 inhibitor significantly inhibited PI3KCA and the cancer stem cell-like mesenchymal markers CSV and EGFR. Expression of the epithelial marker E-cadherin was induced (Figure 21). Notably, GDC-0084 monotherapy or combined treatment with an anti-PD1 inhibitor significantly induced a cell population positive for the epithelial marker E-cadherin (Figure 22). These data demonstrate that PI3KCA inhibitors in combination with immunotherapy can significantly affect tumor burden compared with immunotherapy alone and further reprogram cancer cell or tumor microenvironment signatures toward a more epithelial signature and therapeutic responsiveness. This suggests a viable therapeutic approach to enhance durable responses to immunotherapy.

[0285] PI3K monotherapy or combination therapy induces effector and TRM signatures Combination treatment significantly increased the infiltration of CD8+IFNγ+ T cells into tumors compared with monotherapy (Figure 23A). In addition, the percentage of CD8+ T cells expressing TRM markers (i.e., CD44, CD103, and CD69) among the total CD8+ T cell population was significantly increased in the combination group, with superior induction compared with PI3K inhibitor monotherapy (Figure 23B). Combination treatment also significantly increased tumor-infiltrating CD8+IFNγ+ T cell-positive cells (Figure 23A). In addition, the percentage of CD8+ T cells expressing TRM markers (i.e., CD44, CD103, and CD69) among the total CD8+ T cell population was significantly increased in the combination group (Figure 23B). This assay was performed to understand whether PI3K inhibition monotherapy or combination with immunotherapy reprograms immune cell signatures in addition to cancer cell signatures. The combination treatment was able to significantly influence and induce immune effector signatures and improve antitumor immunity.

[0286] PI3K monotherapy or combination therapy inhibits checkpoint exhaustion signatures in CD8+ T cells Combined treatment with GDC-0084 and an anti-PD1 inhibitor significantly reduced the TIM3- and LAG-positive CD8+ T cell population compared to either PD1 or GDC-0084 monotherapy alone (Figure 24). This assay was performed to examine whether the signature for dysfunctional immune CD8+ T cells could be reprogrammed. Analysis revealed that the combined therapy could significantly reduce the immune dysfunction signature.

[0287] Combining PI3K inhibition with immunotherapy abrogates metastatic spread in the 4T1 IO-resistant model GDC-0084 combination therapy with αPD1 significantly inhibits metastatic tumor burden, but not as a single agent. This combination was also efficacious in the 4T1 immunotherapy-resistant tumor model (Figure 25). These data demonstrate that PI3K inhibitors in combination with immunotherapy can significantly affect tumor burden compared to immunotherapy alone and can further reprogram cancer cell or tumor microenvironment signatures in an immunotherapy-resistant mouse breast cancer model (4T1) to become more responsive to treatment and prevent metastatic spread of cancer to the lung. This suggests therapeutic approaches that can be implemented to enhance durable responses to immunotherapy.

[0288] Consideration The present inventors have discovered a novel method for reducing the overall toxicity of PI3K inhibitors (e.g., GDC-0084) while simultaneously increasing the overall efficacy of treatment. This is achieved by administering a reduced dose of 7.5 mg of GDC-0084 (the usual dose is a single 15 mg dose), further reducing overall adverse side effects and increasing the overall efficacy of GDC-0084 therapy. In addition, the present inventors have confirmed that the efficacy of combining PI3K inhibitors with immunotherapy is similarly enhanced. Therapeutic administration of low concentrations of GDC-0084 in combination with immunotherapy can be further reduced with a similar reduction in the dose of immunotherapy (e.g., anti-PD1), resulting in fewer adverse events and enhanced overall efficacy at significantly lower doses.

[0289] Summary of efficacy of monotherapy versus combination therapy Monotherapy at low doses inhibits checkpoint proteins (Figure 4: TIGIT, Tim3, PD1) and induces effector signatures (Figure 4). Reprograms the exhaustion signature (EOMES) Figure 4.

[0290]

[0195] Monotherapy at the lower dose has significantly fewer clinical abnormalities (Figure 12).

[0291] Monotherapy GDC-0084 at lower doses was able to significantly inhibit tumor weight (FIG. 13).

[0292] However, the low-dose combination exhibited characteristics of sustained tumor control. Furthermore, the low-dose combination was more effective in inhibiting inflammation (Figures 14 and 15).

[0293]

[0198] Lower doses in combination are more effective in inhibiting cancer stem cells (which are important for recurrence and metastasis) and reprogramming and inhibiting dysfunctional T cell signatures and inducing effector T cell signatures (Figures 16, 19-20).

[0294]

[0199] In combination with immunotherapy, the "low dose" is more effective at inducing epithelial signatures compared to the "high dose" or "effective dose" (Figures 17-18). The combination was also superior in inhibiting metastasis (Figure 21).

[0295] Dosing at 15 mg / kg represents a high dose of GDC-0084 (i.e., in clinical practice, humans are generally dosed at 20-60 mg), and our low dose represents only 50% of this dose. 25% of the original dose is also expected to be effective as a single agent and in combination.

[0296] Materials and Methods

[0201] All materials and reagents used in the synthesis and testing of the described compositions are commercially available, for example, from Sigma-Aldrich Co., Novabiochem, Abeam, and American Type Culture Collection (ATCC), unless otherwise specified.

[0297] In vivo animal studies Groups of 5 female BALB / c mice per treatment group were used in the xenograft study. 5 ) cells were injected into the right fourth inguinal mammary fat pad of 6-week-old BALB / c nude mice. For 4T1 tumor cell injection, 1 × 10 5 Cells / mouse were prepared in PBS.

[0298] In the CT26 colon carcinoma mouse model, 5 × 10 5 Cells were injected into the abdomen of 6-week-old BALB / c nude mice. Tumor growth was measured three times a week by caliper measurement. On days 0 and 5, mice were treated with 10 mg / kg anti-PD-1 via intraperitoneal injection. Tumor volume (½ (length × width)) was measured using a digital caliper. 2 )) were measured and presented as mean ± SEM.

[0299] Six-week-old female Balb / c mice were purchased from the Animal Resources Centre (ARC) and allowed to acclimate for one week before use. All experimental procedures were performed in accordance with the guidelines and regulations approved by the QIMR Berghofer Animal Ethics committee. After shaving the inoculation site of the mice, 1 × 10 mice were inoculated with 100 μl PBS. 5 4T1 cells were injected subcutaneously into the right mammary gland. The tumor grew to approximately 100 mm 3 Treatment was initiated when tumors reached a mean of 1 / 2 (a / b). Tumors were measured using external calipers and calculated using the modified ellipsoid formula 1 / 2 (a / b 2 ) (where a = longest side and b = shortest side) was used to calculate volume. For combination therapy experiments, mice were treated twice weekly by intraperitoneal injection with the indicated doses of vehicle (saline) or GDC-0084 (via oral gavage) in combination with anti-PD1 or isotype control (10 mg / kg). Mice were monitored daily for clinical abnormalities (reduced activity, hunched posture, piloerection, weight loss, and metastases), and tumor volume and body weight were measured three times weekly. Tumors were measured at the maximum limit (1000 mm) in the vehicle group. 3 ), all tumors and associated metastatic organs (lungs, liver, spleen) were harvested, weighed, imaged, fixed in 4% paraformaldehyde, and subjected to relevant analyses.

[0300] WST-1 cell proliferation assay The adherent cell lines CT26, 4T1, and MCF7 were seeded in triplicate at optimized cell densities (100, 100, 3000, and 300 cells / well, respectively) in 96-well flat-bottom tissue culture plates in a total volume of 100 μL / well. Cells were allowed to adhere overnight at 37°C and 5% CO2. The cells were then treated with a panel of inhibitors at different concentrations and placed in an incubator at 37°C and 5% CO2 for 72 hours, after which the medium was removed and replaced with 100 μl / well of WST-1 cell proliferation reagent (Sigma-Aldrich, 11644807001) at a final dilution of 1:10 in complete cell culture medium. The WST-1 reagent contains a water-soluble tetrazolium salt that is cleaved by the cellular mitochondrial dehydrogenase enzyme to produce formazan. The amount of formazan produced per reaction was then quantified using a microplate spectrophotometer to measure metabolic activity. The directly measured absorbance correlates with the number of metabolically active cells in culture. Absorbance at 450 nm was recorded using a microplate spectrophotometer at 0.5, 1, and 2 hours of incubation (after a 30-second mixing period). Percent proliferation was calculated by subtracting the mean absorbance of the background control (blank) from the sample absorbance. GraphPad Prism 8 software was used to calculate the EC of each inhibitor using a log(inhibitor) vs. response variable slope (4 parameters). 50 value was determined.

[0301] Scratch wound healing assay The adherent breast cancer cell line MDA-MB-231 was seeded in triplicate at an optimized cell density of 2500 cells / well in 96-well Incucyte® Image Lock plates in a total volume of 100 μL / well in low-serum medium (2%). Cells were left to adhere overnight at 37°C and 5% CO2. Cells were checked to ensure consistent wounds were created and were at 100% confluency. After 24 hours, wounds were created using a 96-pin InCucyte® WoundMaker tool. After wounding, cells were washed with low-serum medium to remove non-adherent cells and then treated with a panel of different concentrations of inhibitors (IC) prepared in low-serum medium.25 and IC 50 The plates were then placed in an Incucyte® Live Cell Analysis System, and wound healing was followed using the InCucyte® Zoom Live Cell Analysis System with scanning intervals every 6 hours.

[0302] In vitro metastatic cancer PI3K inhibitory signature 4T1 or CT26 cancer cell lines were treated with control or GDC-0084 (0.2 μM or 0.3 μM), idelalisib (two lower concentrations: 12.5 or 25 μM), or LY294002 (two lower concentrations: 2.5 or 5 μM). Samples were then permeabilized with Triton-X100 and stained with primary mouse antibodies against CSV or EpCAM, primary rabbit antibodies against EGFR, FOXN2, or MDA5, or primary goat antibodies against ABCB5, and detected with donkey AF anti-mouse 488, anti-rabbit 568, or anti-goat 647 secondary antibodies. Protein targets were imaged using an ASI digital pathology system with a 100x objective. Example image regions with scale bars (orange) are depicted above. Analysis was performed by comparing the fluorescence intensity for EGFR, the cytoplasmic fluorescence intensity (CFI) for CSV, or the total fluorescence intensity for ABCB5. Data were plotted in PRISM and analyzed using a Kruskal-Wallis one-way ANOVA nonparametric test. Significant differences are plotted.

[0303] OPAL tissue microscopy The OPAL staining kit for automated staining on the automated BONDRX platform was used according to the manufacturer's instructions. Proteins were then deployed and localized. Protein targets were localized by digital pathology laser scanning microscopy. Single 0.5 μm sections were acquired using an ASI digital pathology microscope with a 100× 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), and automated thresholding and background correction of the mean fluorescence intensity were performed to automatically determine the distribution and intensity, allowing for specific targeting of the expression of the protein of interest.

[0304] Isolation of CTCs from liquid biopsies of stage IV metastatic cancer patients Using our optimized laboratory protocol, liquid biopsies from cancer patients were processed to enrich for circulating tumor cells (CTCs) with CD45+ cell depletion or to isolate PBMCs from the liquid biopsy without depletion. Briefly, our optimized laboratory protocol is as follows: Whole blood was stored in EDTA tubes for circulating tumor cell identification. Tumor cells (CTCs) were enriched from whole blood by depleting CD45+ cells using RosetteSep™ human CD45 depletion cocktail. Unwanted cells were targeted for depletion using a tetrameric antibody conjugate that recognizes CD45, CD66b, and glycophorin A on red blood cells (RBCs). Unwanted cells were then removed via centrifugation using buoyant density medium Lymphoprep™ (catalog #07801). Purified tumor cells were then extracted as a highly enriched population from the interface between the plasma and buoyant density medium and collected in 20% FBS in PBS.

[0305] Samples were then stained for CSV (mesenchymal metastatic marker for CTCs), ABCB5 (cancer stem cell and chemo-resistance marker), and EpCAM (epithelial cell marker). Coverslips were mounted on glass microscope slides with ProLong nucblue transparent anti-fade reagent (Life Technologies). Protein targets were localized by confocal laser scanning microscopy. Single 0.5 μm sections were acquired using an ASI digital pathology system with a 100× oil immersion lens and running ASI software. Final images were obtained by averaging four sequential images of the same section. Digital images were analyzed using both ASI software for population dynamics and ImageJ software (ImageJ, NIH, Bethesda, MD, USA) for total cell number and fluorescence intensity (protein expression).

[0306] Isolation of CTCs from liquid biopsies of stage IV metastatic cancer patients Method II Liquid biopsies from cancer patients were processed using our optimized laboratory protocol (as described above in Liquid Biopsy Method I) to enrich for circulating tumor cells (CTCs) in the liquid biopsy by CD45+ cell depletion. Samples were then stained for CSV (a mesenchymal metastatic marker for CTCs), ABCB5 (a cancer stem cell and chemoresistant marker), and PI3K (a kinase). Coverslips were mounted on glass microscope slides with ProLong nucblue transparent anti-fade reagent (Life Technologies). Protein targets were localized by confocal laser scanning microscopy. Single 0.5 μm sections were acquired using an ASI digital pathology system with a 40× oil immersion lens, running ASI software. Final images were obtained by averaging four sequential images of the same section. Digital images were analyzed using both ASI software for population dynamics and ImageJ software (ImageJ, NIH, Bethesda, MD, USA) for total cell number and fluorescence intensity (protein expression).

[0307] Example 2 Establishing the optimal therapeutic dose for PI3K-mTOR inhibitor efficacy To establish the optimal therapeutic dose for the GDC-0084 PI3K-mTOR inhibitor, we used the highly aggressive immunotherapy-resistant 4T1 breast cancer model. Once tumors were established (approximately 100 mm 3 ), GDC-0084 was administered daily by oral gavage in combination with αPD1 immunotherapy (Figure 26A). Sequential experiments were performed daily, with an initial study using a high starting dose of GDC of 15 mg / kg + / - αPD1. This was followed by a split-dose de-escalation study, in which daily GDC doses of 1.875 mg / kg to 15 mg / kg in combination with αPD1 were administered as split doses 4 hours apart, and finally, a single-dose de-escalation study, in which daily GDC doses of 3.75 and 7.5 mg / kg in combination with αPD1 were administered as single doses (Figure 26B).

[0308] Administration of a high daily dose of 15 mg / kg GDC-0084 significantly reduced tumor volume by 69% when delivered in combination with immunotherapy (Figure 26C, panel 1). Split-dose experiments revealed comparable tumor volume reductions 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 a lower dose of 7.5 mg / kg GDC in combination with αPD1 reduced primary tumor volume to the same extent as the higher dose (Figure 26C, panel 3), confirming 7.5 mg / kg as the optimal therapeutic dose.

[0309] Reducing the dose of GDC-0084 prevents toxic complications Next, we 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 loss after the fourth day of treatment, ultimately resulting in mouse lethality in both the starting dose and split-dose de-escalation experiments (Figure 27A, panels 1 and 2). However, at the optimal therapeutic dose of 7.5 mg / kg, mice maintained their body weight throughout the duration of the experiment, and no mouse lethality occurred (Figure 27A, panels 2 and 3).

[0310] Liver enlargement, or hepatomegaly, is associated with organ damage. To determine GDC-0084 treatment-induced hepatotoxicity, we measured liver weight at the time of harvest (Figure 2B). Administration of a high daily dose of 15 mg / kg of GDC-0084 increased liver weight by approximately 20% by the end of treatment (Figure 27B, panel 1). In contrast, no difference in liver weight was observed when the lower dose of 7.5 mg / kg was administered (Figure 27B, panels 2 and 3). Collectively, our data confirm that administration of GDC-0084 at the optimal therapeutic dose of 7.5 mg / kg was free of toxicity-related complications.

[0311] Reducing the dose of a PI3K-mTOR inhibitor reduces liver inflammation To assess changes in liver pathology after GDC treatment, H&E-stained liver sections were examined (Figure 28). Using established scoring criteria, inflammatory changes, extramedullary hematopoiesis, and hepatocellular injury were scored as 1 = mild, 2 = moderate, or 3 = severe for each parameter. Importantly, reducing the GDC-0084 dose 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 the extramedullary hematopoiesis typically associated with the 4T1 model (Figure 28B, panel 2). Finally, supporting our liver weight data, GDC-0084 treatment at a high daily dose of 15 mg / kg induced hepatocellular injury, thus confirming hepatotoxicity (Figure 28C, panel 1). Notably, significant hepatocellular changes were not observed at the optimal therapeutic dose of GDC-0084 (Figure 28C, panel 2).

[0312] GDC-0084 αPD1 treatment reduces 4T1-associated splenomegaly and extramedullary hematopoiesis In addition to extramedullary hematopoiesis, an enlarged spleen or splenomegaly is commonly associated with the 4T1 model. To examine splenic changes after GDC-0084 treatment, we measured spleen weight in both split-dose and single-dose deescalation experiments. Administration of a lower dose of GDC-0084, 7.5 mg / kg, reduced splenomegaly in both dosing regimens (Figure 29A). Furthermore, assessment of splenic pathology revealed a significant reduction in extramedullary hematopoiesis after treatment with GDC-0084 at the optimal therapeutic dose of 7.5 mg / kg (Figure 29B, panel 2).

[0313] GDC-0084 inhibits pulmonary leukocyte infiltration after αPD1 treatment In light of the above data demonstrating that GDC-0084 αPD1 treatment reduces lung metastasis, we next examined lung immune cell populations after GDC-0084 treatment. Examination of lung pathology revealed a significant reduction in leukocyte infiltration into the lung at all concentrations, including the optimal therapeutic dose of 7.5 mg / kg (Figure 30). This highlights that GDC-0084 treatment not only has no effect on primary tumor and metastasis, but also on inflammation.

[0314] GDC-0084 αPD1 treatment reduces lymph node metastasis In addition to lung metastasis, the 4T1 model is also associated with lymph node metastasis. To assess lymph node metastasis after GDC-0084 treatment, H&E-stained lymph node sections were examined by an independent, expert pathologist and scored for leukocyte infiltration and / or hemorrhage as follows: 1 = 1 to 3 very small areas (width / length << 0.5 mm), 2 = 1 to 3 areas (at least one of which was 0.5 to 1 mm in diameter), or 3 = 2 to 3 or more areas (all visible to the naked eye). In the absence of GDC-0084 treatment, large areas of metastatic tumor growth were observed; however, these areas were not visible when immunotherapy was administered in combination with GDC-0084 (7.5 mg / kg) (Figure 31A). Therefore, lymph node metastasis scores were substantially lower in GDC-0084 αPD1-treated mice, confirming that GDC-0084 not only inhibits lung metastasis but also lymph node metastasis (Figure 31B).

[0315] PI3K-mTOR treatment reduces primary tumor volume Next, to determine whether GDC-0084 demonstrates efficacy when administered in combination with the PARP inhibitor olaparib, we utilized the 4T1 model of TNBC. In the first experiment, GDC-0084 was administered pre- or post-olaparib, and the second experiment repeated this optimal dosing regimen (Figure 32A). GDC-0084 was administered once daily by oral gavage at an optimal therapeutic dose of 7.5 mg / kg in combination with a once-daily intraperitoneal (ip) injection of olaparib (Figure 32B). GDC-0084 treatment substantially reduced tumor volume by 53% and 60% both pre- and post-PARP inhibitor, respectively (Figure 32C, panel 1). We 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 GDC-0084 also demonstrates efficacy when administered in combination with a PARP inhibitor, strengthening its application in addition to immunotherapy.

[0316] Combined treatment with GDC-0084 and a PARP inhibitor does not induce toxicity To examine toxicity-related complications, we monitored the body weight and liver weight of mice in the PARP inhibitor study (Figure 33). At the optimal therapeutic dose of 7.5 mg / kg, GDC-0084 did not alter the body weight of mice when administered post-olaparib (Figure 33A). Similarly, no changes in liver weight were observed, indicating no toxicity issues (Figure 33B).

[0317] Inhibition of inflammation in the lung and liver after combined treatment with GDC-0084 and olaparib Given the effects of GDC-0084 and immunotherapy on inflammation, we next sought to examine liver and lung pathology in PARP inhibitor studies. When administered at 7.5 mg / kg post-olaparib, GDC-0084 substantially reduced liver inflammation and extramedullary hematopoiesis (Figure 34A). Examination of lung pathology also revealed a significant reduction in lung leukocytosis in GDC-0084- and olaparib-treated mice (Figure 34B), confirming the impact of GDC-0084 not only on inflammation in primary tumors but also at metastatic sites.

[0318]

[0223] Finally, assessment of splenic pathology demonstrated a reduction in splenic weight as well as extramedullary hematopoiesis after GDC-0084 and olaparib treatment (Figures 35A and 35B).

[0319] PI3K-mTOR inhibitors reduce cancer cell proliferation We next sought to compare the efficacy of PI3K-mTOR inhibitors such as GDC-0084, and to this end performed proliferation assays using a broader range of inhibitors, including the more general PI3K inhibitors: wortmannin (general PI3K inhibitor), idelalisib (p110δ,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 were omipalisib, apitolisib, and dactolisib (all with IC<1.0 μM). 50 GDC-0084 has the most dramatic effect on MDA-MB-231 cell proliferation with an IC value of 2.5 μM (Table 16). 50 More common PI3K inhibitors exhibited substantially higher values, confirming that inhibition of PI3K and mTOR are both important in preventing cancer cell proliferation.

[0320] [Table 12]

[0321] Inhibition of PI3K and mTOR affects cancer cell migration To establish the effect of PI3K-mTOR inhibition on cancer cell migration, we performed scratch assays using the human breast cancer cell line MCF-7 pre-stimulated with PMA-TGFβ, which induces a mesenchymal phenotype (Figure 37A). In a GDC-0084 dose-response experiment, approximately 50% inhibition was observed at concentrations of 1.25 μM and 2.5 μM 24 hours after treatment (Figure 37B).

[0322] Again, to compare the efficacy of GDC-0084 with other PI3K-mTOR inhibitors, we performed a second experiment using IC50 concentrations from the proliferation assay with apitolisib, dactolisib, and omipalisib (Figure 38A). Similar to GDC-0084, the PI3K-mTOR inhibitors all significantly reduced wound healing density, confirming that inhibition of both PI3K and mTOR prevents cell migration (Figure 38B).

[0323] Materials and Methods Animal testing Six- to eight-week-old female BALB / c mice were procured from the Animal Resources Centre (ARC). After acquisition, the mice were allowed a one-week acclimatization period before being housed in a holding room at the Berghofer Medical Research Institute (QIMRB). All experimental procedures were performed in accordance with the guidelines and regulations approved by the QIMRB Ethics Committee. 1 × 10 mice suspended in phosphate-buffered saline (PBS) were used. 5 4T1 cells were injected into the mammary fat pad of BALB / c mice. Tumors were approximately 50-100 mm 3Treatment was initiated when tumor size reached 100 μg / cm. For the 4T1 model, mice were subjected to oral administration of the indicated doses of GDC once daily in combination with either anti-PD-1 treatment (10 mg / kg) administered intraperitoneally on days 0 and 4 or olaparib (50 mg / kg) administered once daily. Tumors were measured using external calipers and calculated using the modified ellipsoid formula 1 / 2 (a / b). 2 ) (where a = longest side and b = shortest side) was used to calculate the volume. Mice were monitored daily for clinical abnormalities (reduced activity, hunched posture, piloerection, weight loss, and metastasis), and tumor volumes were measured three times a week. Tumors reached their maximum limit (1000 mm) in the vehicle group. 3 ), all tumors and associated metastatic organs (lungs, liver, spleen) were harvested, weighed, imaged, fixed in 4% paraformaldehyde, and subjected to relevant analyses.

[0324] Pathological analysis H&E-stained FFPE liver, lung, lymph node, and spleen were subjected to pathological analysis by an independent, expert pathologist. Changes in inflammation, extramedullary hematopoiesis (EMH), hepatocellular, and pulmonary leukocytosis were scored as 1 = mild, 2 = moderate, or 3 = severe. Lymph node sections were scored for leukocyte infiltration and / or hemorrhage as 1 = 1 to 3 very small areas (width / length << 0.5 mm), 2 = 1 to 3 areas (at least one of which was 0.5 to 1 mm in diameter), or 3 = 2 to 3 or more areas (all visible to the naked eye).

[0325] WST-1 cell proliferation assay MDA-MB-231 cells were seeded at an optimized density in a 96-well flat-bottom tissue culture plate in a total volume of 100 μL per well. Cells were allowed to adhere overnight at 37°C and 5% CO2. The cells were then treated with the indicated dose of a PI3K inhibitor and placed in an incubator at 37°C and 5% CO2 for 72 hours. The medium was then removed and replaced with 100 μL / well of WST-1 cell proliferation reagent (Sigma-Aldrich, 11644807001) at a final dilution of 1:10 in complete cell culture medium. The WST-1 reagent contains a water-soluble tetrazolium salt that is cleaved by the cellular mitochondrial dehydrogenase enzyme to produce formazan. The amount of formazan produced per reaction was then quantified using a microplate spectrophotometer to measure metabolic activity. The directly measured absorbance correlates with the number of metabolically active cells in culture. Absorbance at 450 nm was recorded using a microplate spectrophotometer at 0.5, 1, and 2 hours of incubation (after a 30-second mixing period). Percent growth was calculated by subtracting the mean absorbance of the background control (blank) from the sample absorbance. IC50 values ​​for each inhibitor were determined using GraphPad Prism 8 software by log(inhibitor) vs. response variable slope (4 parameters).

[0326] Scratch wound healing assay MCF7 cells were stimulated with PMA / TGFβ for 24 hours and then seeded at optimized cell density in 96-well Incucyte® Image Lock plates in a total volume of 100 μL / well in low-serum medium (2%). Cells were left to adhere overnight at 37°C and 5% CO2. Cells were checked to ensure consistent wounds were created and were 100% confluent. After 24 hours, wounds were created using a 96-pin Incucyte® WoundMaker tool. After injury, cells were washed with low-serum medium to remove non-adherent cells and then treated with the indicated doses of PI3K inhibitors prepared in low-serum medium. Plates were then placed in an Incucyte® Live-Cell Analysis System, and wound healing was tracked using an InCucyte® Zoom Live-Cell Analysis System with scanning intervals of every 4 hours.

[0327] 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). 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, Marchio 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

1. A method for modifying epithelial-mesenchymal or mesenchymal-epithelial transition of PI3K-overexpressing cells, comprising contacting the PI3K-overexpressing cells with a composition comprising a PI3K inhibitor and an immunotherapy that does not target cancer stem cells (CSCs).

2. A method for treating or preventing cancer in a subject, wherein the cancer contains 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 (CSCs).

3. The method of claim 1 or 2, wherein the PI3K-overexpressing cells are CSCs.

4. The method of claim 3 , wherein the PI3K-overexpressing cells are CSC tumor cells.

5. 5. The method of any one of claims 1 to 4, wherein the PI3K-overexpressing cells express 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 cells are characterized by 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 population of CD8+ T cells that express one or more of TIM3, TIGIT, PD1, TOX1, and EOMES.

8. The method of any one of claims 1 to 7, wherein the epithelial cells express an epithelial cell signature comprising E-cadherin.

9. The method of any one of claims 1 to 8, wherein the immunotherapy targeting CSCs 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. 11. The method of claim 10, wherein the PARP inhibitor is selected from the group comprising olaparib, talazoparib, veliparib, niraparib, and rucaparib.

15. 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, 740Y-P, AZD-7648, buparlisib, inavolisib, dactolisib, copanlisib, eganelisib, pictilisib, SAR405, duvelisib, taselisib, resilisib, YM-201636, omipalisib, PI-103, alpha-linolenic acid, fimepinostat, and isorhamnetin.

16. The method of any one of claims 1 to 15, wherein the PI3K inhibitor is paxalisib (GDC-0084).

17. 17. The method of any one of claims 2 to 16, wherein 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 (CSCs) to treat a T cell dysfunction disorder, or to enhance immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, to treat or delay the progression of cancer, or to treat the recurrence of cancer.

19. Use of a PI3K inhibitor and an immunotherapy that does not target cancer stem cells (CSCs) in the manufacture of a medicament for treating a T cell dysfunction disorder, or enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, treating or delaying the progression of cancer, or treating the recurrence of cancer.

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

21. Use of a PI3K inhibitor, an immunotherapy that does not target cancer stem cells (CSCs), and an adjuvant (e.g., a chemotherapeutic agent) to treat or assist in the treatment of a T cell dysfunction disorder, or to enhance immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, to treat or delay the progression of cancer, or to treat the recurrence of cancer.

22. Use of a PI3K inhibitor, an immunotherapy that does not target cancer stem cells (CSCs), and an adjuvant (e.g., a chemotherapeutic agent) in the manufacture of a medicament for treating or assisting in the treatment of a T cell dysfunction disorder, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, for treating or delaying the progression of cancer, or for treating the recurrence of cancer.

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

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

25. 25. The method of any one of claims 18 to 24, further comprising detecting elevated levels of one or more of TIM3, TIGIT, PD1, TOX1, and EOMES in T cells (e.g., relative to the levels of TIM3, TIGIT, PD1, TOX1, and EOMES in activated T cells) in a sample obtained from the subject prior to the combined administration.

26. A kit comprising a drug comprising a PI3K inhibitor and an optional pharmaceutically acceptable carrier for treating a T cell dysfunction disorder, or enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, or treating or delaying the progression of cancer, or treating recurrent cancer in an individual, and a package insert containing instructions for administering the drug in combination with another drug comprising an immunotherapy that does not target cancer stem cells (CSCs) and an optional pharmaceutically acceptable carrier.

27. A kit comprising a drug comprising an immunotherapy that does not target cancer stem cells (CSCs) and an optional pharmaceutically acceptable carrier for treating a T cell dysfunction disorder, or enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, treating or delaying the progression of cancer, or treating the recurrence of cancer in an individual, and a package insert containing instructions for administering the drug in combination with another drug comprising a PI3K inhibitor and an optional pharmaceutically acceptable carrier.

28. A kit comprising a first agent comprising a PI3K inhibitor and optionally a pharmaceutically acceptable carrier, and a second agent comprising an immunotherapy that does not target cancer stem cells (CSCs) and optionally a pharmaceutically acceptable carrier, for treating a T cell dysfunction disorder, or enhancing immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, or treating or delaying the progression of cancer, or treating the recurrence of cancer in an individual.

29. 30. The kit of claim 28, further comprising a package insert containing instructions for administering the first agent and the second agent in combination to treat a T cell dysfunction disorder, or to enhance immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, to treat or slow the progression of cancer, or to treat the recurrence of cancer in an individual.

30. A pharmaceutical composition for treating cancer in a subject, 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. 31. The composition of claim 30, wherein the PI3K inhibitor is GDC-0084 and the unit dose corresponds to administration of about 11.25 mg / kg or less to the subject.

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

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

34. 32. The composition of claim 30 or 31, further comprising an immunotherapy that does not target cancer stem cells (CSCs).

35. 25. 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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