Compositions and methods for improving cancer treatment

Abstract

The present disclosure relates generally to compositions and methods for treating cancer. More specifically, the present disclosure relates to compositions and their use in one of altering the transformation of epithelial cells to mesenchymal cells or the transformation of mesenchymal cells to epithelial cells of tumor cells. The method comprises administering to the subject a composition comprising a PI3K inhibitor, such as paxalisib (GDC-0084), and an immunotherapy that is not targeted to cancer stem cells (CSC), wherein the immunotherapy is an immune checkpoint antagonist or a PARP inhibitor.

Description

Technical Field

[0001] The present invention generally relates to compositions and their use in treating cancer. More specifically, the present invention relates to compositions and their use in altering one of epithelial to mesenchymal transition or mesenchymal to epithelial transition of tumor cells. Background Art

[0002] Any prior publication (or information derived therefrom) or any known matter mentioned in this specification is not, and should not be taken as, a confirmation, acknowledgement or any form of implication that the prior publication (or information derived therefrom) or known matter constitutes part of the common general knowledge in the field to which this specification relates.

[0003] Phosphatidylinositol is one of many phospholipids found in cell membrane, plays an important role in intracellular signal transduction.Cell signal transduction by 3'-phosphorylated phosphatidylinositol is relevant with multiple cellular processes, such as malignant transformation, growth factor signal transduction, inflammation and immunity (Rameh et al. (1999) J.Biol Chem, 274:8347-8350).The enzyme responsible for producing these phosphorylation signal products, phosphatidylinositol 3-kinase (also referred to as PI 3-kinase or PI3K), was initially identified to have the activity relevant to viral oncoprotein and growth factor receptor tyrosine kinase, these kinases are by phosphatidylinositol (PI) and its phosphorylation derivative phosphorylation (Panayotou et al. (1992) Trends Cell Biol 2:358-60) at the 3'-hydroxyl of inositol ring.

[0004] Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase that phosphorylates lipids at the 3-hydroxyl residues of the inositol ring (Whitman et al. (1988) Nature, 332: 664). The 3-phosphorylated phospholipids (PIP3) produced by PI3 kinase act as second messengers with lipid binding domains (including plekstrin homology (PH) regions) to recruit kinases such as Akt and phosphoinositide-dependent kinase-1 (PDK1). The binding of Akt to membrane PIP3 causes Akt to shift to the plasma membrane, bringing Akt into contact with PDK1, which is responsible for activating 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 important 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 major PI3K isoform in cancer is the class I PI3-kinase p110α (alpha) (as described in U.S. Patents 5,824,492; 5,846,824; 6,274,327). Other isoforms are associated with cardiovascular and immune-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 in cancer cells, reverse the inhibition of apoptosis in cancer cells, and overcome cancer cell resistance to cytotoxic agents (Folkes et al. (2008) J. Med. Chem. 51:5522-5532; Yaguchi et al. (2006) Jour, of the Nat. Cancer Inst. 98(8):545-556).

[0006] However, despite numerous clinical studies investigating the use of PI3K inhibitors for the treatment of many solid tumors, no candidate drugs have yet entered the clinic. Therefore, further work is needed to successfully utilize these promising drug candidates to improve efficacy and reduce toxicity for these challenging diseases. Summary of the Invention

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

[0008] In one aspect of the present invention, a method for altering epithelial-to-mesenchymal transition or mesenchymal-to-epithelial transition in PI3K-overexpressing cells is provided, 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 for 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 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 CSV, EGRF, and ABCB5, SoX9, SNAIL, and AKT1.

[0012] Epithelial cells are characterized by expressing one or both of EpCAM and MDA5. In addition, in some embodiments, the subject includes a CD8+ T cell population that expresses one or more of TIM3, TIGIT, PD1, TOX1, and EOMES. Typically, epithelial cells express epithelial cell markers including E-cadherin.

[0013] In some embodiments, the immunotherapy targeting CSC 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] For example, the PARP inhibitor may be suitably selected from 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 paxalisib (GDC-0084), idelalisib, LY294002, 3-methyladenine, apellisib, quercetin, wortmannin, GNE-490, PI3K-IN-36, 740Y-P, AZD-7648, bupanisib, inavolisib, dactolisib, copanlisib, eganelisib, pitilisib, SAR405, duvilisib, taselisib, recilisib, YM-201636, omipalisib, PI-103, α-linolenic acid, feminostat, and isorhamnetin.

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

[0018] In another aspect of the present invention, a PI3K inhibitor and an immunotherapy that does not target cancer stem cells (CSCs) are provided for treating T cell dysfunction disorders, or for enhancing the immune function (e.g., immune effector function, T cell function, etc.) of a cancer individual, for treating or delaying the progression of cancer, or for treating the recurrence of cancer.

[0019] In another aspect, the present invention provides uses of PI3K inhibitors and immunotherapies that do not target cancer stem cells (CSCs) in the preparation of a medicament for treating T cell dysfunction in an individual with cancer, or for enhancing their immune function (e.g., immune effector function, T cell function, etc.), for treating or delaying the progression of cancer, or for treating the recurrence of cancer.

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

[0021] In another aspect, the present invention provides a PI3K inhibitor, an immunotherapy that does not target cancer stem cells (CSCs), and an adjuvant (e.g., a chemotherapeutic agent) for treating or adjuvanting a T cell dysfunction disorder in an individual with cancer, or for enhancing immune function (e.g., immune effector function, T cell function, etc.), for treating or delaying the progression of cancer, or for treating the recurrence of cancer.

[0022] In another aspect, the present invention provides uses of PI3K inhibitors, immunotherapies that do not target cancer stem cells (CSCs), and adjuvants (e.g., chemotherapeutic agents) in the preparation of medicaments for treating or adjuvanting T cell dysfunction in individuals with cancer, or for enhancing immune function (e.g., immune effector function, T cell function, etc.), for treating or delaying cancer progression, or for treating cancer recurrence.

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

[0024] Suitably, 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 in a sample obtained from the subject prior to simultaneous administration (e.g., relative to the levels of TIM3, TIGIT, PD1, TOX2 and EOMES in activated T cells).

[0025] In another embodiment, the present invention provides a kit comprising a drug comprising a PI3K inhibitor and optionally a pharmaceutically acceptable carrier, and instructions for use comprising instructional materials for administering the drug concurrently with another drug comprising an immunotherapy that does not target cancer stem cells (CSCs) and optionally a medically acceptable carrier for treating a T cell dysfunction disorder in an individual with cancer, or for enhancing their immune function (e.g., immune effector function, T cell function, etc.), for treating or delaying the progression of cancer, or for treating cancer recurrence in an individual.

[0026] In another embodiment, the present invention provides a kit comprising a drug, an optional pharmaceutically acceptable carrier, and instructions for use, wherein the drug comprises an immunotherapy that does not target cancer stem cells (CSCs), the instructions for use comprising instructional materials for administering the drug concurrently with another drug, wherein the other drug comprises a PI3K inhibitor and an optional medically acceptable carrier, for treating a T cell dysfunction disorder in an individual with cancer, or for enhancing their immune function (e.g., immune effector function, T cell function, etc.), for treating or delaying the progression of cancer, or for treating cancer recurrence in an individual.

[0027] In another aspect of the present invention, a kit is provided, comprising a first drug and a second drug, wherein the first drug comprises a PI3K inhibitor and an optional pharmaceutically acceptable carrier, and the second drug comprises an immunotherapy that does not target cancer stem cells (CSCs) and an optional medically acceptable carrier for treating T cell dysfunction in individuals with cancer, or for enhancing their immune function (e.g., immune effector function, T cell function, etc.), for treating or delaying the progression of cancer, or for treating cancer recurrence in individuals. Suitably, the kit also includes instructions for use, which include instructional materials for administering the first drug and the second drug simultaneously, for treating T cell dysfunction in individuals with cancer, or for enhancing their immune function (e.g., immune effector function, T cell function, etc.), for treating or delaying the progression of cancer, or for treating cancer recurrence in individuals.

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

[0029] In some embodiments, the PI3K inhibitor is GDC-0084, and the unit dose is equivalent to administering to a subject a dose of about 11.25 mg / kg or less. Suitably, the PI3K inhibitor can be GDC-0084, and the unit dose is equivalent to administering to a subject a dose of about 7.5 mg / kg or less. Also contemplated is a PI3K inhibitor that is GDC-0084, and the unit dose is equivalent to administering to a subject a dose of about 4 mg / kg or less.

[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. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The following drawings constitute part of this 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 drawings in combination with the detailed description of specific embodiments presented herein.

[0033] Figure 1 : Provides KM survival plots of PI3K protein expression during the survival period of stage IV metastatic cancer.

[0034] Figure 2Liquid biopsies from cancer patients in either the immunotherapy-resistant or immunotherapy-responsive cohorts were processed using our optimized laboratory protocol to isolate PBMCs from the liquid biopsy samples. The samples were then stained for CSV, PI3KCA, and ABCB5.

[0035] Figure 3 Fluorescence intensity (FI) and population dynamics (percentage of cell population) of CSV, ABCB5, EpCAM, PI3KCA, and AKT1 (AKT1 plays a role in promoting invasion and metastasis) were measured using ASI Digital Pathology in CTCs from liquid biopsies of patients with stage IV metastatic solid tumors. CTCs were treated with GDC-0084 or vehicle control. (A) CSV, ABCB5, two mesenchymal markers, and PI3KCA / AKT1 are shown. (B) Percentage of CSV+ABCB5+ or EpCAM+ cells or PI3KC / AKT1+ cells are shown.

[0036] Figure 4 Liquid biopsies from patients with stage IV metastatic solid tumors were processed using our optimized laboratory protocol to isolate PBMCs from the liquid biopsy samples. The samples were then stimulated (ST: 4 hours with PMA / CaI in the presence of Brefeldin A) or left unstimulated (NS) and / or treated with the GDC-0084 inhibitor. The samples were then stained for three separate panels of markers. Depletion signature panel: EOMES, PD1, and CD8. Checkpoint panel: TIGIT, TIM3, and CD8. Effector signature panel: Perforin, GZNb, and CD8.

[0037] Figure 5 : (A) MCF7 (epithelial breast cancer), MDAMB231 (mesenchymal / CSC breast cancer), MDAM B231-Br (brain cancer version of MDA-MB-231), and 4T1 (highly invasive, immunotherapy-resistant mouse breast cancer) cell lines; and (B) H1299 (epithelial lung cancer), CT26 (epithelial, highly immunogenic mouse colon tumor), and (LLC) highly treatment-resistant Lewis lung cancer cell lines were stained for PI3KCA markers. Samples were permeabilized with Triton X-100 and stained with mouse primary antibodies to CSV and rabbit primary antibodies to PI3KCA, and detected with donkey AF anti-mouse 568 and anti-rabbit 647.

[0038] Figure 6 The effect of PI3K inhibitor GDC-0084 on the migration of MDA-MB-231 and CT26 cells is shown. The effect of GDC-0084 on cell migration was analyzed by wound healing (scratch assay), where the images represent the effect of DMSO control, IC 25GDC-0084 at a concentration of 1.25 μM and IC 50 Different cell lines treated with GDC-0084 at a concentration of 2.5 μM. (C) Graphical representation of the relative wound density of MDA-MB-231 cells treated with PI3K inhibitors. Black bars: DMSO control; light gray bars: IC 25 Concentration 1.25 μM; dark gray bar: IC 50 The concentration was 2.5 μM. Error bars correspond to the mean ± standard deviation of eight replicates. P values ​​were calculated using two-way ANOVA with Tukey's multiple comparison test, where ns indicates not significant, ns = p > 0.05; * indicates p < 0.05; ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001.

[0039] Figure 7 Shown are the IC values ​​of GDC-0084 for different cancer cell lines. 50 In a dose-response design, WST-1 cell proliferation reagent was added to CT26, 4T1, and MCF7 cells pretreated with PI3K inhibitors for 72 h. Cell proliferation was measured indirectly by formazan formation and absorbance at 450 nm was recorded. EC values ​​were calculated using Graphpad Prism software as described in the Methods. 50 Beforehand, the percentage (%) of proliferation was determined.

[0040] Figure 8 Olaparib treatment showed upregulation of several resistance and mesenchymal markers. MDA-MB-231 cells were treated with either vehicle ("CTRL") or olaparib ("Ola."). Expression of cancer stem cell-like resistance markers (ABCB5, ALDH1A) was quantified by immunofluorescence staining. Images show IF staining of (A) ABCB5 and (B) ALDH1A1, and demonstrate upregulation of ABCB5 and ALDH1A1 expression. ***P ≤ 0.001. This figure demonstrates that PARP inhibition can upregulate markers characteristic of cancer stem cell resistance.

[0041] Figure 9 Olaparib treatment failed to inhibit CSCs or cell proliferation: MDA-MB-231 cells were treated with olaparib for 24 hours. Cells were harvested and stained with APC / CD44 and PE / CD24 for flow cytometry. (A) Shown are the percentage inhibition of CSCs compared to the control group, followed by representative flow cytometry images. (B) MDA-MB-231 cells were treated with three different concentrations of olaparib or talazoparib, and cell proliferation was assessed using the WST-1 assay.

[0042] Figure 10Graphical and photographic representations of PARP knockdown are provided. PARP1 knockdown was performed using two different concentrations of PARP1 siRNA, 5 nM and 10 nM, with 5 nM being the optimal concentration previously determined by our laboratory in other cell lines. After incubation with siRNA for 48 hours, cells were snap-frozen at −80°C. Following RNA extraction and cDNA synthesis, TaqMan-qRT-PCR was performed to quantify mRNA expression. Both concentrations significantly reduced PARP1 mRNA expression (A). Panels (B) and (C) show the nuclear fluorescence intensity of PARP. The graphs represent the mean ± standard error of three independent experiments. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P < 0.0001, two-way analysis of variance.

[0043] Figure 11 PARP1 knockout was shown to increase the expression of resistance markers. MDA-MB-231 cells were incubated with 5nM PARP1 siRNA for 48 hours, and then RNA was extracted or cDNA was synthesized and amplified by qPCR, or the cells were fixed for immunofluorescence staining. (A, B) mRNA expression, (C, D) Immunofluorescence intensity of mesenchymal markers after PARP1 knockout, (E) PD-L1. The relative fold change of mRNA expression or the change of total nuclear fluorescence intensity (TNFI) or total cytoplasmic fluorescence intensity (TCFI) is shown. *P≤0.05, **P≤0.01, ***P≤0.001, ****P<0.0001, two-way analysis of variance. Wound healing (scratch assay) analysis of the effect of GDC-0084 combined with olaparib on cell migration. Images represent (F) DMSO control, (G) IC 25 IC values ​​of GDC-0084 (1.25 μM) and olaparib (5 nM) were 50 MDA-MB-231 cell lines treated with 2.5 μM GDC-0084 and 250 nM olaparib. Error bars correspond to the mean ± standard deviation of eight replicates (I). P values ​​were calculated using a two-way ANOVA with Tukey's multiple comparison test, where ns indicates not significant, ns = P>0.05; * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001.

[0044] Figure 12 αPD1 treatment in the CT26 colon cancer model and the 4T1 breast cancer model is shown. Treatment protocols have been demonstrated using the Balb / c CT26 (A) colon cancer model and the 4T1 (B) breast cancer model. Tumor volume of mice treated with vehicle (IgG) or αPD1 (10 mg / kg) (n=5 / group). *p<0.05, **p<0.01, unpaired t-test.

[0045] Figure 13 PI3K inhibition targets and suppresses the expression of mesenchymal resistance markers. 4T1 (A) or CT26 (B) cancer cell lines were treated with control or GDC-0084 (at two low concentrations, 0.2 or 0.3 μM). Samples were then permeabilized with Triton-X 100 and stained with mouse primary antibodies to CSV or EpCAM, rabbit primary antibodies to EGFR or FOXN2, or goat primary antibodies to 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 fields with scale bars (orange) are depicted above. Comparative analysis was performed for fluorescence intensity of EGFR, cytoplasmic fluorescence intensity (CFI) of CSV, or global fluorescence intensity of ABCB5. Data were plotted using PRISM, and one-way ANOVA was performed using Kruskal-Wallis. Significant differences are plotted.

[0046] Figure 14 : PI3K inhibition induces immunovisibility 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-X 100 and stained with mouse primary antibodies to CSV or EpCAM, rabbit primary antibodies to 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. Fluorescence intensities were compared and analyzed. Data were plotted using PRISM, and one-way ANOVA was performed using Kruskal-Wallis. Significant differences are plotted.

[0047] Figure 15PI3K inhibition targets and suppresses stem-like cancer stem cell-like features of cancer recurrence and induces epithelial features. The MDA-MB-231TNBC cancer cell line was treated with a control or two different PI3K inhibitors, idelalisib (two low concentrations of 12.5 μM or 25 μM) or LY294002 (two low concentrations of 2.5 μM or 5 μM). Samples were then permeabilized with Triton-X 100 and stained with mouse primary antibodies to CSV or EpCAM, rabbit primary antibodies to SoX9 (SoX9 is a marker of mesenchymal cancer stem cells and progressive, therapy-resistant cancer cells), or PI3K or goat primary antibodies to ABCB5 or SNAIL (11A and 11B), 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 40x objective. Example image fields with scale bars (orange) are depicted above. Comparative analysis was performed on the nuclear fluorescence intensity of SoX9, the cytoplasmic fluorescence intensity (CFI) of CSV and EpCAM, and the global fluorescence intensity of ABCB5 or SNAIL. Data were plotted using PRISM, and one-way analysis of variance was performed using the Kruskal-Wallis method. Significant differences were plotted.

[0048] Figure 16 GDC-0084 administration at 7.5 mg / kg reduces clinical abnormalities in the 4T1 syngeneic tumor model. GDC-0084 therapy in the 4T1 breast cancer model. Treatment regimen using the 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 with clinical abnormalities (reduced activity, hunched posture, piloerection, weight loss, and metastases) at the end of GDC-0084 monotherapy.

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

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

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

[0052] Figure 20 GDC-0084 at a dose of 7.5 mg / kg reduced the expression of SOX9 and TOX1. Images captured using Qupath analysis are depicted in bar graphs for:

[0053] A) CSC signature (SOX9, PDL1, panCK), n > 500,000 cells analyzed.

[0054] B) CD8 dysfunction signature (CD8, TOX1, PD1), n ​​> 10,000 cells analyzed.

[0055] C) CSC signatures (SOX9, PDL1, panCK), n > 10,000 cells analyzed.

[0056] D) CD8 dysfunction signature (CD8, TOX1, PD1), n>10,000 cells analyzed. Qupath analysis was used to select DAPI-positive cells, and then PDL1- and panCK-positive CSC signature cells were scored based on SOX9 fluorescence intensity. For the CD8 dysfunction signature, DAPI-positive cells were selected, followed by CD8 and PD1+, and scored based on TOX1 fluorescence intensity. Images were taken from an Aperio FL fluorescence slide scanner, which captured stained FFPE tumor sections of GDC mouse models treated with control, anti-PD1 immunotherapy, GDC-0084 (high dose C / D and low dose A / B), or the combination. FFPE sections of lung tissue were processed as described above and stained for SOX9, PDL1, panCK, or CD8, TOX1, PD1; DAPI was used for nuclear staining. Data are plotted as mean ± SD and represent the fluorescence intensity of SOX9 or TOX1. Tukey post hoc test, ns = not significant,

[0057] ***p<0.001, ****p<0.0001.

[0058] Figure 21 PI3K inhibition, either alone or in combination, targets mesenchymal characteristics and induces an epithelial phenotype in immunotherapy-resistant mouse models. FFPE primary tumor samples were processed on BONDRX using the Opal staining kit targeting PI3KCA, CSV, E-cadherin, or EGFR. (A)-(D) Quantification of fluorescence intensity (FI) for PI3KCA, CSV, E-cadherin, or EGFR was determined using ASI Digital Pathology.

[0059] Figure 22 PI3K inhibitors, either alone or in combination, induce an epithelial phenotype in an immunotherapy-resistant mouse model. FFPE primary tumor samples were processed on BONDRX using Opal staining kits targeting PI3KCA, CSV, E-cadherin, or EGFR. The percentage of cells positive for E-cadherin expression was determined using the ASI digital pathology platform.

[0060] Figure 23 PI3K monotherapy or combination therapy induces effector and TRM characteristics. FFPE tumor samples were stained with the Opal staining kit and images were analyzed using ASI Digital Pathology. CD8+IFNγ+ T cell-positive cells (A) or CD8+ T cell expression markers of tissue-resident memory cells (CD103, CD69, CD44, and CD8) (B) were quantified. Kruskal-Wallis nonparametric tests indicated significant differences.

[0061] Figure 24PI3K checkpoint depletion signature in CD8+ T cells, either monotherapy or in combination. FFPE primary tumor specimens were treated with tyramide staining kits targeting CD8, LAG3, and TIM3 checkpoint markers. The percentage of cells positive for each marker was determined using the ASI Digital Pathology Platform. The percentage of TIM3- and LAG3-positive CD8+ T cells was calculated among total CD8+ T cells. The figure represents n = 3 mice per group, and significant differences were calculated using the Kruskal-Wallis test. ns = not significant, *p = < 0.02.

[0062] Figure 25 Combining PI3K inhibition and immunotherapy eliminates metastatic spread in the 4T1 immunotherapy-resistant model. GDC-0084 + / - αPD1 treatment in the 4T1 model of metastatic breast cancer. (A) Treatment regimen using the Balb / c 4T1 breast cancer model. (B) Presence of metastatic tumor nodules in the lungs of individual mice at day 20 post-inoculation. **p < 0.01, Tukey post hoc test.

[0063] Figure 26 : Optimal therapeutic dose of GDC-0084 established in the 4T1 TNBC model. (A) Treatment regimen using the Balb / c 4T1 TNBC breast cancer model. (B) Flowchart of the dose-escalation experiment. In the first phase, GDC-0084 was administered daily at a dose of 15 mg / kg + / - αPD1 (10 mg / kg) on ​​days 0 and 4. In the second phase, GDC-0084 (up to 15 mg / kg) was co-administered with αPD1 on days 0 and 4, separated by 4 hours. In the final phase, GDC-0084 was co-administered with αPD1 at a single daily dose (up to 7.5 mg / kg) on ​​days 0 and 4. (C) Tumor volume (percentage of vehicle) of individual mice at each experimental phase before harvest, *p<0.05, **p<0.01, ***p<0.001, ns not significant, unpaired t-test or Dunnett post hoc test, n=4-5 / group.

[0064] Figure 27 Lowering the dose of GDC-0084 improves safety in mice. (A) Mouse body weights were monitored during the initial dose, dose-escalation, and single-dose-escalation experiments. # indicates mouse death. (B) Liver weights were measured at harvest. *p < 0.05, unpaired t-test, n = 4-5 per group.

[0065] Figure 28No hepatotoxicity was observed at the optimal therapeutic dose of GDC-0084. Pathologists scored H&E-stained FFPE livers from both the fractionated and single-dose de-escalation studies. (A) Changes in liver inflammation, (B) extramedullary hematopoiesis (EMH), and (C) hepatocyte changes (metabolic and / or degenerative). Each parameter was scored as 1 = mild, 2 = moderate, or 3 = severe. *p < 0.05, ***p < 0.001, ****p < 0.0001 compared to vehicle, Dunnett post hoc test, n = 4-5 / group.

[0066] Figure 29 PI3K-mTOR inhibition reduces splenomegaly and extramedullary hematopoiesis in the spleen. (A) Spleen weights were measured at harvest in both the fractionated and single-dose studies. *p<0.05, **p<0.01, ****p<0.0001, compared with vehicle, Dunnett post hoc test, n=4-5 / group. (B) Pathologists scored H&E-stained FFPE spleens. Changes in extramedullary hematopoiesis (EMH) were scored as 1 = mild, 2 = moderate, or 3 = severe compared with vehicle. *p<0.01, ***p<0.001, Dunnett post hoc test, n=4-5 / group.

[0067] Figure 30 PI3K-mTOR inhibition reduces leukocyte infiltration in the lungs. Expert pathologists scored H&E-stained FFPE lungs from both the fractionated and single-dose escalation studies. Changes in leukocytosis compared with vehicle were scored as 1 = mild, 2 = moderate, or 3 = severe. *p < 0.01, ***p < 0.001, Dunnett post hoc test. N = 4-5 per group.

[0068] Figure 31 Combination therapy with GDC-0084 and αPD1 prevents lymph node metastasis. (A) Images of H&E-stained FFPE lymph nodes from 4T1 mice treated with αPD1, GDC-0084 (7.5 mg / kg), and PD1. (B) Lymph node sections were examined by an independent expert pathologist and scored as 1 = 1-3 minor sites (width / length << 0.5 mm), 2 = 1-3, with at least one site measuring 0.5-1 mm in diameter, or 3 = 2-3 or more sites, all with macroscopic leukocytic infiltration and / or hemorrhage.

[0069] Figure 32:GDC-0084 showed efficacy in combination with a PARP inhibitor. (A) PARP inhibitor experimental process. In the first phase, GDC-0084 was administered daily at a dose of 7.5 mg / kg before (before) or 30 minutes after (after) olaparib (50 mg / kg). In the second phase, GDC-0084 was administered daily at a dose of 7.5 mg / kg 30 minutes after olaparib (50 mg / kg). (B) Treatment regimen using the Balb / c 4T1 TNBC breast cancer model. (C) Tumor volume (percentage of vehicle or olaparib) of individual mice at each experimental phase before harvest. **p<0.01, ns not significant, Tukey post hoc test, n=5 / group.

[0070] Figure 33 Combination therapy with GDC-0084 and olaparib did not induce toxicity. Body and liver weights were assessed in mice treated with GDC-0084 (7.5 mg / kg) following olaparib treatment. ns, not significant, Tukey post hoc test, n = 5 / group.

[0071] Figure 34 Combination treatment with GDC-0084 and olaparib reduces liver and lung inflammation. Independent expert pathologists scored H&E-stained FFPE livers and lungs following the GDC-0084 and olaparib experiments. (A) Changes in liver inflammation and extramedullary hematopoiesis (EMH), and (B) pulmonary leukocytosis, with each parameter scored as 1 = mild, 2 = moderate, or 3 = severe. *p < 0.05, **p < 0.001, ****p < 0.0001 compared to vehicle, Dunnett post hoc test, n = 5 / group.

[0072] Figure 35 Combination therapy with GDC-0084 and olaparib reduces splenomegaly and extramedullary hematopoiesis in the spleen. (A) Expert pathologists scored H&E-stained FFPE spleens from a GDC-0084 and olaparib experiment. Changes in extramedullary hematopoiesis (EMH) were scored as 1 = mild, 2 = moderate, or 3 = severe. (B) Spleen weights were measured at harvest. *p < 0.05, ***p < 0.001, ****p < 0.0001 compared to vehicle, Dunnett post hoc test, n = 5 / group. *p < 0.01, ***p < 0.001 compared to vehicle, Dunnett post hoc test, n = 5 / group.

[0073] Figure 36Comparison of PI3K-mTOR inhibitor combination therapy for cancer cell proliferation. In a dose-response design, WST proliferation reagent was added to MDA-MB-231 cells pretreated with PI3K inhibitors for 72 hours. Cell proliferation (%) was measured indirectly by formazan formation and absorbance at 450 nm.

[0074] Figure 37 Figure 3: GDC-0084 wound healing dose-response curve (scratch assay). Pre-stimulated (PMA / TGFβ) MCF-7 cells were scratched prior to treatment with GDC-0084 (0.078-2.5 μM) for 24 hours. Changes in wound density were monitored over 24 hours. (A) Representative images from the scratch assay comparing MCF-7 cells treated with vehicle and GDC-0084 (1.25 μM and 2.5 μM). (B) Dose-response curve comparing wound density (%) in GDC-0084-treated cells (0.078-2.5 μM) over a 24-hour treatment period.

[0075] Figure 38 Comparison of cancer cell migration treated with PI3K-mTOR inhibitors. Pre-stimulated (PMA / TGFβ) MCF-7 cells were scratch wounded prior to 24-hour treatment with the indicated doses of PI3K-mTOR inhibitors. (A) Dose-response curves comparing wound density (%) in cells treated with PI3K-mTOR inhibitors (0.078-2.5 μM) over the 24-hour treatment period. (B) Comparison of the relative wound density observed at 24 hours for each PI3K-mTOR inhibitor. ****p<0.0001 compared to vehicle, Dunnett post hoc test, n=5 / group. *p<0.01, ***p<0.001 compared to vehicle, Dunnett post hoc test, n=6 / group. DETAILED DESCRIPTION

[0076] 1. Definition

[0077] 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 the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

[0078] As used herein, the articles "a" and "an" refer to one or more (ie, at least one) of the grammatical object of the article. For example, "an element" refers to one element or a plurality of elements.

[0079] The term "about" as used herein refers to the typical error range of the corresponding value that is readily known to those skilled in the art. "About" values ​​or parameters mentioned herein include (and describe) embodiments for the value or parameter itself.

[0080] 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 skilled in the art and disclosed herein. The expression level or amount of the assessed biomarker can be used to determine the response to treatment.

[0081] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted as an alternative (or).

[0082] In this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" will be understood to mean including the stated step or element or group of steps or elements but not excluding any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present. "Consisting of" means including and limited to whatever follows "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory and no other elements may be present. "Consisting essentially of" means including any elements listed after the phrase and is limited to other elements that do not interfere with or contribute to the activity or action specified for the listed elements in this disclosure. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present, depending on whether they affect the activity or action of the listed elements.

[0083] "Chemotherapeutic agents" include compounds that are useful in treating cancer. Examples of chemotherapeutic agents include erlotinib ( Genentech / OSIPharm.), bortezomib ( Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant ( AstraZeneca), sunitib ( Pfizer / Sugen), letrozole ( Novartis), imatinib mesylate ( Novartis、finasunate( Novartis), oxaliplatin ( Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, Wyeth), lapatinib ( GSK572016, GlaxoSmithKline), lonafarnib (SCH66336), sorafenib ( Bayer Laboratories), gefitinib ( AstraZeneca), AG1478; alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines (such as benzodepa, carboquinone, metodepa, and uredepa); ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan) bryostatin; callystatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); adrenocortical hormones (including prednisone and prednisone acetate); cyproterone acetate; 5α-reductase inhibitors finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mosetinol, dolastatin, aldesleukin, talc-duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard;Nitroureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1I and calicheamicin omega 1I (Angewandte); 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, aclacinomycins, actinomycins, authramycins, azaserine, bleomycins, cactinomycins, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diaza-5-oxo-L-norleucine; (doxorubicin), linodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolodoxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins (e.g., mitomycin C), mycophenolic acid, acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, or zorubicin; antimetabolites, such as methotrexate, 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs, such as ancitabine (a incitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; antiadrenal agents, such as aminoglutethimide, mitotane, and trilostane; folic acid supplements, such as folinic acid; aceglatone; and aldophosphamide glycosides. glycoside); aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene;Edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate acetate; epothilones; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids (e.g., maytansine and ansamitocin); mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); polyurethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ), (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.) and (docetaxel, doxetaxel; Sanofi-Aventis); chlorambucil; (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; Vinorelbine; novantrone; teniposide; edatrexate; daunorubicin; aminopterin; capecitabine Ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

[0084] The chemotherapeutic agents also include (i) antihormonal drugs used to modulate or inhibit the effects of tumor hormones, such as antiestrogens and selective estrogen receptor modulators (SERMs), including tamoxifen (including tamoxifencitrate), raloxifene, droloxifene, iodoxifene, 4-hydroxytamoxifen, droloxifene, raloxifene hydrochloride (keoxifene), LY117018, onastone and (toremifine citrate); (ii) aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazole, aminoglutethimide, (Megestrol acetate), (exemestane, Pfizer), formestane, fadrozole, (Vorozole), (letrozole, Novartis) and (anastrozole; AstraZeneca); (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; buserelin, triptorelin, 17-hydroxyprogesterone acetate, ethynol, premarin, fluoxymesterone, all-trans retinoic acid, fenretinide, and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-α, Ralf, and H-Ras; (vii) ribozymes, such as VEGF expression inhibitors (e.g. ) and HER2 expression inhibitors; (viii) vaccines, such as gene therapy vaccines, e.g. and rIL-2; topoisomerase 1 inhibitors, such as rmRH; and (ix) pharmaceutically acceptable salts, acids and derivatives of any of the foregoing.

[0085] The chemotherapeutic agents also include antibodies such as alemtuzumab (Campath), bevacizumab ( Genentech); Cetuximab ( Imclone); Panitumumab ( Amgen), rituximab ( Genentech / / Biogen Idec), Pertuzumab ( 2C4, Genentech), trastuzumab ( Genentech), tositumomab (Bexxar, Corixia), and the antibody-drug conjugate gemtuzumab-ozogamicin ( Wyeth).

[0086] Other humanized monoclonal antibodies with therapeutic potential as drugs in combination with the compounds of the present invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol (certolizumab pegol), cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, p alivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumabTetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, and visilizumab, as well as the anti-interleukin-12 antibody (ABT-874 / J695, Wyeth Research and Abbott Laboratories), which is a recombinant, fully human, full-length IgG1λ antibody genetically modified to recognize the interleukin-12p40 protein.

[0087] Chemotherapeutic agents also include "EGFR inhibitors," which refer to compounds that bind to or otherwise directly interact with EGFR and prevent or reduce its signaling activity, and may also be referred to as "EGFR antagonists." Examples of such drugs include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see U.S. Patent No. 4,943,533, Mendelsohn et al.), and variants thereof, such as chimeric 225 (C225 or cetuximab; ) and reshaped human 225 (H225) (see WO 96 / 40210, Imclone Systems, Inc.); IMC-11F8, a fully human EGFR targeting antibody (Imclone); antibodies that bind to type II mutant EGFR (U.S. Patent 5212290); humanized and chimeric antibodies that bind to EGFR, such as U.S. Patent 5891996; and human antibodies that bind to EGFR, such as ABX-EGF or panitumumab (see WO 98 / 50433, Abgenix / Amgen); EMD 55900 (Stragliotto et al., Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab) is a humanized EGFR antibody against EGFR that competes with EGF and TGF-α for EGFR binding (EMD / Merck); human EGFR antibody HuMax-EGFR (GenMab); fully human antibodies designated E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E1.6.3, as described in U.S. Patent 623,583; MDX-447 (Medarex); and mAb 806 or humanized mAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). Anti-EGFR antibodies can be conjugated to cytotoxic agents to produce immunoconjugates (see, for example, EP659439A2, Merck Patent GmbH). EGFR antagonists include small molecules such as compounds described in U.S. Patents 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, 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, Genetech / OSI Pharmaceuticals); PD 183805 (Cl 1033, 2-acrylamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-dihydrochloride, Pfizer Inc.); ZD1839, gefitinib 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenylamino)quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluorophenyl)-N2-(1-methylpiperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol); (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino] -7H-pyrrolo[2,3-d]pyrimidine); CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(-dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU5271; Pfizer); dual EGFR / HER2 tyrosine kinase inhibitors, such as lapatinib ( GSK572016 or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[2-methylsulfonyl)ethyl]amino]methyl]-2-furyl]-4-quinazolinamine).

[0088] Chemotherapeutic agents also include "tyrosine kinase inhibitors," including the EGFR-targeted drugs described in the preceding paragraph, small molecule HER2 tyrosine kinase inhibitors, such as TAK165 available from Takeda; CP-724714, an oral selective inhibitor of ErbB2 receptor tyrosine kinase (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; lapatinib (GSK572016; available from GlaxoSmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; 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 provided by ISIS.

[0089] Drugs that inhibit Raf-1 signaling; non-HER-targeted TK inhibitors, such as imatinib mesylate ( purchased from GlaxoSmithKline); multi-targeted tyrosine kinase inhibitors, such as sunitinib ( available from Pfizer); VEGF receptor tyrosine kinase inhibitors, such as vatalanib (PTK787 / ZK222584, available from Novartis / Schering AG); MAPK extracellular-regulated kinase I inhibitor Cl-1040 (available from Pharmacia); quinazolines, such as PD 153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP60261, and CGP 62706; pyrazolopyrimidine, 4-(anilino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrosine containing a nitrothiophene moiety; PD-0183805 (Warner-Lambert); antisense molecules (e.g., molecules that bind to a nucleic acid encoding a HER); quinoxaline (U.S. Patent No. 5,804,396); tryphostins (U.S. Patent No. 5,804,396); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); pan-HER inhibitors such as Cl-1033 (Pfizer); Affinitac (ISIS 3521; ISIS / Lilly); imatinib mesylate PKI 166 (Novartis); GW2016 (GlaxoSmithKline); Cl-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone), rapamycin (sirolimus, ); or as described in any of the following patent publications: U.S. Patent No. 5,804,396; WO 1999 / 09016 (U.S. Cyanamid); WO 1998 / 43960 (U.S. Cyanamid); WO 1997 / 38983 (Warner-Lambert); WO 1999 / 06378 (Warner-Lambert); WO 1999 / 06396 (Warner-Lambert); WO 1996 / 30347 (Pfizer); WO 1996 / 33978 (Zeneca); WO 1996 / 3397 (Zeneca) and WO 1996 / 33980 (Zeneca).

[0090] Chemotherapeutic agents also include dexamethasone, interferon, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacizumab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab tiuxetan, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelleukin, Palifermin, pamidronate disodium, pegademase, pegaspargase, long-acting filgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronic acid and zoledronic acid, and pharmaceutically acceptable salts thereof.

[0091] Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortolone pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinolone acetonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, alclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone butyrate, clobetasol propionate, fluocortolone caproate, fluocortolone epivalate, and fluprednidine acetate; immunoselective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and its D-isomer form (feG) (IMULAN BioTherapeutics, LLC); antirheumatic drugs such as azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide, sulfasalazine; tumor necrosis factor (TNF) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), cetrizumab (Cimzia), golimumab (Simponi); interleukin-1 (IL-1) blockers such as anakinra (Kineret); T-cell costimulation blockers such as abatacept (Orencia); interleukin-6 (IL-6) blockers such as tocilizumab Interleukin-13 (IL-13) blockers, such as neratumumab; interferon-α (IFN-α) blockers, such as longitumumab; β7 integrin blockers, such as rhuMAb Beta7; IgE pathway blockers, such as anti-M1 prime; blockers of secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa1 / β2, 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 212 and radioisotopes of Lu); other investigational drugs, such as thioplatin, PS-341, phenyl butyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols, such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavins, flavanols, proanthocyanidins, betulinic acid, and their derivatives; autophagy inhibitors, such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, ); β-lapachone; lapachol; colchicine; betulinic acid; acetylcamptothecin, scopolamine, and 9-aminocamptothecin); podophyllotoxin; tegafur Bexarotene Bisphosphonates, such as clodronate (e.g., or ), etidronate NE-58095, Zoledronic Acid / Zoledronate Alendronate sodium Pamidronate sodium Tiludronate sodium or risdronate sodium Epidermal growth factor receptor (EGF-R); vaccines, such as Vaccines; perifosine, COX-2 inhibitors (such as celecoxib or etoricoxib), proteasome inhibitors (such as PS341); CCl-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors such as olimusen sodium Pixantrone maleate; farnesyltransferase inhibitors, such as lonafarnib (SCH 6636, SARASAR TM); and any pharmaceutically acceptable salts, acids or derivatives of the foregoing; and combinations of two or more of the foregoing, such as CHOP, which is an abbreviation for combined therapy with cyclophosphamide, doxorubicin, vincristine and prednisolone; and FOLFOX, which is an abbreviation for oxaliplatin (ELOXATIN TM ) is the abbreviation for the combined 5-FU and leucovorin treatment regimen.

[0092] Chemotherapeutic agents also include nonsteroidal anti-inflammatory drugs (NSAIDs) with analgesia, antipyretic and anti-inflammatory effects.NSAIDs include non-selective inhibitors of cyclooxygenase.The specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, rofecoxib and valdecoxib. NSAIDs are used to relieve symptoms of rheumatoid arthritis, osteoarthritis, inflammatory joint diseases, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headaches and migraines, postoperative pain, mild to moderate pain caused by inflammation and tissue damage, fever, intestinal obstruction, and renal colic.

[0093] The terms "correlation" and "association" are used interchangeably herein to refer to an association between two measurements (or measured entities). The present disclosure provides genetic and / or epigenetic variations whose levels are associated with disease diagnosis and / or prognosis and / or response to treatment.

[0094] As used herein, the terms "reduce", "lower", "decrease", "inhibit", "suppress", "attenuate" and the like all refer to statistically significant reductions. In some embodiments, these terms generally mean a reduction of at least 10% (e.g., in the absence of a given treatment or drug) compared to a reference level, 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 "inhibit" do not require complete inhibition or reduction compared to a reference level. "Complete inhibition" etc. is 100% inhibition compared to a reference level. Reduction can preferably be reduced to an acceptable level within the normal range (e.g., for an individual without a given disease).

[0095] As used herein, the term "epithelial-mesenchymal transition" (EMT) refers to the conversion of epithelial cells to mesenchymal phenotypes, which is a normal process of embryonic development. EMT is also the process in which damaged epithelial cells as ion and fluid transporters become matrix-remodeling mesenchymal cells. In cancer, this conversion typically results in changes in cell morphology, mesenchymal protein expression, and increased invasiveness. The criteria for defining EMT in vitro include the loss of epithelial cell polarity, separation into single cells, and subsequent dispersion after obtaining cell motility (see Vincent-Salomon and Thiery, Breast Cancer Res. 2003; 5 (2): 101-6). During EMT, changes in the expression, distribution and / or function of molecular classes, and the causes associated therewith, include growth factors (e.g., transforming growth factor (TGF)-β, wnts), transcription factors (e.g., SNAI, SMAD, LEF and nuclear β-catenin), molecules of the cell-cell adhesion axis (cadherins, catenins), cytoskeletal regulators (Rho family) and extracellular proteases (matrix metalloproteinases, plasminogen activators) (see Thompson and Newgreen, Cancer Res. 2005; 65(14):5991-5).

[0096] The terms "increased," "increase," "enhance," or "activate" as used herein refer to a statistically significant increase. In some embodiments, the terms "increased," "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and can include, for example, an increase of at least about 10% compared to a reference level, such as an increase of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or up to 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 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase from 2-fold to 10-fold or greater compared to a reference level. In the context of a marker or symptom, "increased" refers to a statistically significant increase in level.

[0097] "Measuring" or "measurement" refers to assessing the presence, absence, quantity, or amount (which may be an effective amount) of a given substance in a sample, including deriving the qualitative or quantitative concentration level of such a substance, or otherwise assessing the value or classification of a clinical parameter of a subject. Alternatively, the terms "testing," "detecting," or "detection" may be used to refer to all measuring or measurement described in this specification.

[0098] As used herein, the term "mesenchymal to epithelial transition" (MET) is a reversible biological process involving the conversion of motile, multipolar, or spindle-shaped mesenchymal cells to a planar polarized cell array called epithelial cells. MET is the reverse process of EMT. MET occurs in normal development, cancer metastasis, and induced pluripotent stem cell reprogramming.

[0099] As used herein, the terms "overexpress," "overexpression," "overexpressing," or "overexpressed" interchangeably refer to a higher level of detectable gene transcription or translation, typically in cancer cells, compared to normal cells (e.g., PI3KCA gene). Thus, overexpression refers to overexpression of protein and RNA (due to increased transcription, post-transcriptional processing, translation, post-translational processing, altered stability, and altered protein degradation), as well as local overexpression due to altered protein trafficking patterns and enhanced functional activity, such as increased enzymatic hydrolysis of substrates. Overexpression can also be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to normal cells or control cells (e.g., breast cells).

[0100] As used herein, the term "PI3K inhibitor" and its grammatical variants refer to molecules that reduce or inhibit at least one function or biological activity of PI3K. For example, a PI3K inhibitor can inhibit or reduce the enzymatic activity of PI3K and / or can inhibit or reduce the expression of PI3K.

[0101] As used herein, the term "PI3K overexpressing cell" refers to a vertebrate cell, particularly a mammalian cell, that expresses PI3K at a detectable level higher than that of a normal cell. The cell can be a vertebrate cell, such as a primate cell; an avian cell; a livestock cell (such as a sheep cell, a cow cell, a horse cell, a deer cell, a donkey cell, and a pig cell); a laboratory test animal cell (such as a rabbit cell, a mouse cell, a rat cell, a guinea pig cell, and a hamster cell); a companion animal cell (such as a cat cell and a dog cell); and a captive wild animal cell (such as a fox cell, a deer cell, and a wild dog cell). In a specific embodiment, the PI3K overexpressing cell is a human cell. In a specific embodiment, the PI3K overexpressing cell is a cancer stem cell or a non-cancer stem cell tumor cell; preferably a cancer stem cell tumor cell. Compared to a normal cell or a control cell (e.g., a breast cell), the overexpression can also be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

[0102] The term "selective" and its grammatical variants are used herein to refer to the inhibition of PI3K without substantially inhibiting one or more other PI3K enzymes or isoforms. Typically, a molecule that is selective for PI3K exhibits a PI3K selectivity greater than about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or greater than about 100-fold for the inhibition of one or more other PI3K enzymes. In other embodiments, the selective molecule has an inhibitory effect on PI3K that is at least 50-fold greater than its inhibitory effect on one or more other PI3K enzymes. In further embodiments, the selective molecule has an inhibitory effect on PI3K that is at least 100-fold greater than its inhibitory effect on one or more other PI3K enzymes. In further embodiments, the selective molecule has an inhibitory effect on PI3K that is at least 500-fold greater than its inhibitory effect on one or more other PI3K enzymes. In further embodiments, the selective molecule has an inhibitory effect on PI3K that is at least 100-fold greater than its inhibitory effect on one or more other PI3K enzymes.

[0103] As used herein, "subject" refers to a person or animal. Typically, the animal is a vertebrate, such as a primate, rodent, livestock, or game animal. Primates include chimpanzees, crab-eating macaques, spider monkeys, and macaques (such as rhesus monkeys). Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffaloes, felines (such as house cats), canines (such as dogs, foxes, wolves), birds (such as chickens, emus, ostriches), and fish (such as trout, catfish, and salmon). In some embodiments, the subject is a mammal (e.g., a primate (e.g., human)). The terms "individual," "patient," and "subject" are used interchangeably herein.

[0104] As used herein, the terms "treat," "treatment," and "treating," etc., refer to therapeutic treatments wherein the purpose is to reverse, alleviate, ameliorate, inhibit, slow down, or stop the development or severity of a condition associated with a disease or disorder (e.g., cancer or tumor). The term "treatment" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder associated with cancer or tumor. A treatment is typically "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of the disease is slowed or stopped. That is, "treatment" includes not only improvement of symptoms or markers, but also stopping or at least slowing the progression or worsening of symptoms compared to the absence of treatment. Beneficial or desired clinical results include, but are not limited to, relief of one or more symptoms, reduction in the scope of the disease, stabilization (i.e., non-worsening) of the disease state, delaying or slowing progression of the disease, improving or alleviating the disease state, relief (whether partial or complete), and / or reduction in mortality, whether detectable or undetectable. The term "treatment" of a disease also includes alleviating the symptoms or side effects of the disease (including palliative care). A treatment does not need to cure a disease (i.e., completely reverse or eliminate the disease) to be considered effective.

[0105] As used herein, the term "tumor" refers to any tumor cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized, in part, by unregulated cell growth. As used herein, the term "cancer" refers to non-metastatic and metastatic cancers, including early-stage and late-stage cancers. The term "precancerous lesion" refers to a disease or growth that typically precedes or develops into cancer. The term "non-metastatic" refers to a benign cancer, or one that remains at the primary site and has not infiltrated the lymphatic or vascular system or tissues outside the primary site. Generally speaking, a non-metastatic cancer is any stage 0, I, or II cancer. "Early stage cancer" refers to a cancer that is not invasive or metastatic, or is classified as stage 0, I, or II cancer. The term "late stage cancer" typically refers to stage III or IV cancer, but may also refer to stage II cancer or a substage of stage II cancer. Those skilled in the art will understand that the classification of stage II cancer as early stage cancer or late stage cancer depends on the specific 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, stomach cancer, liver cancer, bladder cancer, urethral cancer, thyroid cancer, kidney cancer, malignant tumors, 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.

[0106] Unless specifically stated otherwise, each embodiment described herein applies mutatis mutandis to every embodiment.

[0107] 2. Composition

[0108] The present invention is based in part on such determination that functionally suppressed T cells of mesenchymal phenotype are exposed to PI3K inhibitors, resulting in epigenetic reprogramming of T cells, with their immune effector functions de-suppressed, including elevated expression of biomarkers of T cell activation and effector capacity (e.g., IL-2, IFN-γ, and TNF), reduced expression of biomarkers of T cell effector inhibition and cancer progression (e.g., ZEB1), and reduced expression of biomarkers of T cell exhaustion (e.g., PD-1 and EOMES) and elevated expression of the transcription factor TBET (which increases the production of IFN-γ in adaptive and innate immune system cells). The inventors also found that PI3K inhibitor-mediated phosphorylation reprogramming enhances the sensitivity of exhausted T cells to reactivation by ICM-bound antagonists.

[0109] Therefore, according to the present invention, compositions and methods are provided for enhancing immune effector function and / or T cell (e.g., CD8+ T cell) function using PI3K inhibitors (e.g., PI3K activity inhibitors) and ICM binding antagonists, including increasing T cell activation and enhancing the sensitivity of exhausted T cells to reactivation by ICM binding antagonists. Therefore, the methods and compositions of the present invention are particularly suitable for treating T cell dysfunction diseases, including cancer and infection.

[0110] The present invention is based, at least in part, on the identification of compositions that inhibit EMT and induce MET in CSC tumor cells. Therefore, the inventors believe that the compositions of the present invention may be used to treat or prevent cancer, and / or improve the responsiveness of cancer or tumors to immunotherapies that do not target CSCs.

[0111] Therefore, in one aspect of the present invention, a composition is provided, comprising a PI3K inhibitor and an immunotherapy that does not target CSCs.

[0112] 2.1 PI3K inhibitors

[0113] The compositions of the present invention include inhibitors, which include and comprise any active agent that reduces the accumulation, function (e.g., enzymatic activity, localization, etc.) or stability of PI3K; or reduces the expression of the PI3KCA gene, 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.

[0114] In some embodiments, the PI3K inhibitor is an antagonistic nucleic acid molecule that functions to inhibit the transcription or translation of a PI3K transcript.

[0115] Representative transcripts of this type include nucleotide sequences corresponding to any of the following sequences: (1) human PI3K nucleotide sequences such as those set forth in GenBank Accession Nos. NM_006219.3, NM_001256045, NM_005026, NM_001350234, and NM_0013500235, (2) nucleotide sequences that have 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%, 200%, 201%, 202%, 203%, 204%, 205%, 206%, 207%, 208%, 209%, 210%, 211%, 212%, 213%, 214%, 215 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity; (3) nucleotide sequences that hybridize to the sequence described in (1) under at least low, medium or high stringency conditions; (4) nucleotide sequences encoding any of the following amino acid sequences: for example, UniProt accession numbers P42336, Q8NEB9, O007 50, the human PI3K amino acid sequence described in O75747, P48736 and P42338; (5) a nucleotide sequence encoding an amino acid sequence that has 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% or more of the same amino acid sequence as any one of the sequences mentioned in (4). %, 96%, 97%, 98%, 99% sequence similarity; and nucleotide sequences encoding amino acid sequences having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of the sequences mentioned in (4).

[0116] Exemplary antagonistic 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, regulators and stimulators of a specific activity possessed by a target molecule, or functional nucleic acid molecules can have de novo activity independent of any other molecules.

[0117] Antagonist nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptide or carbohydrate chain.Therefore, antagonist nucleic acid molecules can interact with the genomic DNA (such as PIK3CA) of PI3KmRNA or PIK3 gene, or they can interact with PI3K polypeptide.Usually, antagonist nucleic acid molecules are designed to interact with other nucleic acids based on the sequence homology between target molecule and antagonist nucleic acid molecules.In other cases, the specific recognition between antagonist nucleic acid molecules and target molecule is not based on the homology of sequence between antagonist nucleic acid molecules and target molecule, but is based on the formation of the tertiary structure that allows specific recognition to occur.

[0118] In some embodiments, antisense RNA or DNA molecules are used to directly block the translation of PI3K by binding to the target mRNA and preventing protein translation. Antisense molecules are designed to interact with target nucleic acid molecules through canonical or non-canonical base pairing. The interaction between antisense molecules and target molecules can be designed to promote the destruction of target molecules, such as RNAseH-mediated RNA-DNA hybrid degradation. Alternatively, antisense molecules can be designed to interrupt processing functions that normally occur on target molecules, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. There are many methods for optimizing antisense efficiency by finding the most accessible region of the target molecule. Non-limiting methods include in vitro selection experiments and DNA modification studies using DMS and DEPC. In a specific example, antisense molecules are used with a concentration of less than or equal to 10 -6 , 10 -8 , 10 -10 or 10 -12 The dissociation constant (K d In a specific embodiment, antisense oligonucleotides are used that are derived from the translation start site, for example the -10 to +10 region.

[0119] Aptamers are molecules that interact with target molecules in a specific manner. Aptamers are typically small nucleic acids between 15 and 50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quadruplexes. Aptamers can bind to small molecules, such as ATP and theophylline, as well as macromolecules, such as reverse transcriptase and thrombin. Aptamers can bind to molecules smaller than 10 -12 K in the target molecule of M d Suitably, the aptamer binds very tightly to a -6 , 10 -8 , 10 -10 or 10 -12 K dBinding to target molecules. Aptamers can bind to target molecules with very high specificity. For example, aptamers have been isolated that have binding affinities exceeding 10,000-fold between a target molecule and another molecule that differs at only a single position on the molecule. Ideally, the K of the aptamer to the target molecule should be d Ratio to the K of background binding molecules d A suitable method for generating aptamers of interest (e.g., PI3K) is "Systematic Evolution of Ligands by Exponential Enrichment" (SELEX). TM ). SELEX TM Method is described in U.S. Patent No. 5,475,096 and U.S. Patent No. 5,270,163 (see also International Publication No. WO 91 / 19813). In short, the nucleic acid mixture is contacted with the target molecule under conditions conducive to binding. Unbound nucleic acid is separated from the bound nucleic acid, and the nucleic acid-target complex dissociates. The dissociated nucleic acid is then amplified to produce a ligand-rich nucleic acid mixture, which is subjected to repeated cycles of binding, distribution, dissociation, and amplification as needed to produce a high-affinity nucleic acid ligand highly specific to the target molecule.

[0120] In other embodiments, anti-PI3K ribozymes are used to catalyze specific cleavage of PI3K RNA. The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to a complementary target RNA, followed by endonucleolytic cleavage. Several different types of ribozymes catalyze nuclease- or nucleic acid polymerase-type reactions based on ribozymes found in natural systems, such as hammerhead ribozymes, hairpin ribozymes, and Tetrahymena ribozymes. Other ribozymes are not found in natural systems but have been engineered to catalyze specific reactions de novo. Representative ribozymes cleave RNA or DNA substrates. In some embodiments, ribozymes that cleave RNA substrates are used. Specific ribozyme cleavage sites within potential RNA targets are initially identified by scanning the target molecule for ribozyme cleavage sites, which include the sequences GUA, GUU, and GUC. Once identified, short RNA sequences of 15 to 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated to predict structural features, such as secondary structures that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be assessed by testing their accessibility for hybridization with complementary oligonucleotides using ribonuclease protection assays.

[0121] Triplex-forming functional nucleic acid molecules are molecules that can interact with double-stranded or single-stranded nucleic acids. When a triplex molecule interacts with a target region, it forms a structure called a triplex, in which three DNA strands form a complex that relies on Watson Crick and Hoogsteen base pairing. Triplex molecules are preferred because they can bind to the target region with high affinity and specificity. It is generally desirable that the number of triplex-forming molecules is less than 10-6 , 10 -8 , 10 -10 or 10 -12 K d Bind to target molecule.

[0122] External guide sequence (EGS) is a molecule that binds to a target nucleic acid molecule to form a complex that is recognized by RNAse P, which cuts the target molecule. EGS can be designed to specifically target a selected RNA molecule. RNAse P helps process transfer RNA (tRNA) within the cell. By using EGS, bacterial RNAse P can be recruited to cut almost any RNA sequence, and EGS enables the target RNA:EGS complex to mimic the natural tRNA substrate. Similarly, eukaryotic EGS / RNAse P-guided RNA cutting can be used to cut desired targets within eukaryotic cells.

[0123] In other embodiments, RNA molecules that mediate RNA interference (RNAi) of the PI3K gene or PI3K transcript can be used to reduce or eliminate gene expression. RNAi refers to the interference or destruction of the product of a target gene by introducing single-stranded or usually double-stranded RNA (dsRNA) homologous to the target gene transcript. RNAi approaches, including double-stranded RNA interference (dsRNAi) or small interfering RNA (siRNA), have been extensively documented in many organisms, including mammalian cells and the nematode Caenorhabditis 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 by microRNA (miRNA), functional small hairpin RNA (shRNA), or other dsRNAs expressed in vivo using DNA templates 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).

[0124] In a specific embodiment, dsRNA itself is used, in particular, a dsRNA production construct corresponding to at least a portion of the PIK3 gene is used to reduce or eliminate its expression. RNAi-mediated gene expression inhibition can be achieved using any technology reported in the art, such as by transfecting the nucleic acid construct encoding stem loop or hairpin RNA structure into the genome of the target cell, or by expressing a transfection nucleic acid construct with homology to the PIK3 gene between the convergent promoters, or as a head-to-head or tail-to-tail replication from behind a single promoter. Any similar construct can be used, as long as it produces an RNA that can self-fold and produce dsRNA, or as long as it produces two separate RNA transcripts, which are then annealed to form a dsRNA with homology to the target gene.

[0125] RNAi does not require absolute homology, and for dsRNAs of about 200 base pairs, a lower threshold is described as about 85% homology (Plasterk and Ketting, 2000, Current Opinion in Genetics and Dev. 10:562-67). Thus, depending on the length of the dsRNA, the level of homology between the nucleic acid encoding RNAi and the target gene transcript can vary, i.e., dsRNAs of 100 to 200 base pairs have at least about 85% homology to the target gene, while 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 a separately expressed RNA, or RNA encoding constructs expressing a single transcript from a convergent promoter, are suitably at least about 100 nucleotides in length. RNA encoding constructs expressing a single RNA designed to form a dsRNA by internal folding are typically at least about 200 nucleotides in length.

[0126] The promoter used to express the construct forming the dsRNA can be any type of promoter, provided that the dsRNA produced is specific for the gene product in the cell lineage being targeted for disruption. Alternatively, the promoter may be lineage-specific, in that it is expressed only in cells of a particular developmental lineage. This may be advantageous when there is some overlap in homology with genes expressed in non-targeted cell lineages. Promoters may also be inducible by external control factors or intracellular environmental factors.

[0127] In some embodiments, RNA molecules of about 21 to about 23 nucleotides that direct the cleavage of their corresponding specific mRNAs, such as those described in U.S. Patent Publication No. US2002 / 0086356, can be used to mediate RNAi. Such 21-23 nt RNA molecules can include a 3' hydroxyl group and can be single-stranded or double-stranded (as two 21-23 nt RNAs), wherein the dsRNA molecules can be blunt-ended or include overhanging ends (e.g., 5', 3').

[0128] In some embodiments, the antagonist nucleic acid molecule is siRNA. siRNA can be prepared by any suitable method. For example, reference can be made to International Publication WO 02 / 44321, which discloses siRNAs that can sequence-specifically degrade target mRNA when bases are paired with 3' overhanging ends, which is incorporated herein by reference. Sequence-specific gene silencing can be achieved in mammalian cells using synthetic short double-stranded RNAs that mimic siRNAs produced by the enzyme dicer. siRNAs can be synthesized chemically or in vitro, or they can be the result of intracellular processing of short double-stranded hairpin RNAs (shRNAs) into siRNAs. Synthetic siRNAs are typically designed using algorithms and traditional DNA / RNA synthesizers. Suppliers include Ambion (Austin, Texas), ChemGenes (Ashland, Massachusetts), Dharmacon (Lafayette, Colorado), Glen Research (Sterling, Virginia), MWB Biotech (Espersberg, Germany), Proligo (Boulder, Colorado), and Qiagen (Ventor, The Netherlands). siRNAs can also be synthesized using Ambion's SILENCER TM siRNA construction kits and other kits are synthesized in vitro.

[0129] Production of siRNA from vectors is usually accomplished by transcribing short hairpin RNA (shRNA). Kits for producing vectors containing shRNA are available, such as Imgenex's GENESUPPRESSOR TM Construction kit and Invitrogen's BLOCK-IT TMInducible RNAi plasmids and lentiviral vectors. In addition, methods for preparing siRNA and delivering it to a subject are also well known in the art. See, for example, U.S. Patent No. 2005 / 0282188, U.S. Patent No. 2005 / 0239731, U.S. Patent No. 2005 / 0234232, U.S. Patent No. 2005 / 0176018, U.S. Patent No. 2005 / 0059817, U.S. Patent No. 2005 / 002025, U.S. Patent No. 2004 / 0192626, U.S. Patent No. 2003 / 0073640, U.S. Patent No. 2002 / 0150936, U.S. Patent No. 2002 / 0142980, and U.S. Patent No. 2002 / 0120129, each of which is incorporated herein by reference.

[0130] Exemplary RNAi molecules (e.g., PI3K siRNA and shRNA) are described in the art (e.g., Ma et al., 2013. BMC Biochem. 14:20; 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 (Castlehill, NSW, Australia).

[0131] The present invention further contemplates inhibitor compounds based on peptides or polypeptides. The PI3K inhibitory peptides described above can be modified as part of a fusion protein. The fusion protein can include a transporter or peptide whose function is to increase the cellular uptake of the peptide inhibitor, having another desired biological effect, such as a therapeutic effect, or having both functions. The fusion protein can be prepared by methods known to those skilled in the art. The inhibitor peptide can be combined with another peptide in a variety of ways known in the art or conjugated in other ways. For example, the inhibitor peptide can be combined with a carrier peptide or other peptides described herein by cross-linking, wherein both peptides in the fusion protein maintain their activity. As another example, the peptide can be connected from the C-terminus of one peptide or otherwise coupled to the N-terminus of another peptide by an amide bond. The connection between the inhibitor peptide and the other members of the fusion protein can be non-cleavable, can be with a peptide bond, or can be cut with, for example, an ester or other cleavable bond known in the art.

[0132] In some embodiments, the transporter or peptide can be, for example, a Drosophila Antennapedia homeodomain-derived sequence comprising the amino acid sequence CRQIKIWFQNRRMKWKK [SEQ ID NO: 1] and can be cross-linked to the inhibitor via an N-terminal Cys-Cys bond (e.g., as described in Theodore et al., 1995. J. Neurosci. 15:7158-7167; Johnson et al., 1996. Circ. Res 79:1086). Alternatively, the inhibitor can be modified with a transporter polypeptide derived from the transactivating regulatory protein (Tat) of human immunodeficiency virus type 1 (e.g., amino acids 47-57 of Tat as set forth 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 inhibitor can be modified by other methods known to those skilled in the art to increase cellular uptake of the inhibitor.

[0133] The PI3K inhibitory peptide can also be introduced into cells by introducing a nucleic acid comprising a nucleotide sequence encoding the PI3K inhibitory peptide into the cells. 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 exists in a free state in the cell.

[0134] Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia virus-based viral vectors, 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., Hum Gene Ther 9:8186, 1998; Flannery et al., PNAS 94:69166921,1997; Bennett et al., Invest Opthalmol Vis Sci 38:28572863,1997; Jomary et al., Gene Ther 4:683690,1997, Rolling et al., Hum Gene Ther 10:641648,1999; Ali et al., Hum Mol Genet. 5:591594,1996; Srivastava's WO 93 / 09239, Samulski et al., J. Vir. 63:3822-3828, 1989; Mendelson et al., Virol. 166:154-165, 1988; and Flotte et al., PNAS (1993) 90:10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:1031923, 1997; Takahashi et al., J Virol 73:78127816, 1999); retroviral vectors (e.g., mouse 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), etc.

[0135] The present invention also contemplates small molecule agents that reduce the functional activity of PI3K (e.g., reducing PI3K-mediated phosphorylation, inhibiting the binding of PI3K to the CD44 or uPAR promoter, reducing the binding of PI3K (e.g., active PI3K) to chromatin; reducing PI3K-mediated inhibition of the guanine exchange factor GIV / Girdin, reducing PI3K-mediated inhibition of regulatory T cell function, reducing PI3K-mediated EMT, etc.).

[0136] Small molecule agents that reduce PI3K functional activity suitable for use in the present invention include, for example, compounds selected from Formula I:

[0137]

[0138] and stereoisomers, geometric isomers, tautomers and pharmaceutically acceptable salts thereof, wherein:

[0139] Dashed lines represent optional double bonds, and at least one dashed line is a double bond;

[0140] X 1 It is S, O, N, NR a , CR 1 、C(R 1 )2 or -C(R 1) 2O-;

[0141] X 2 It is C, CR 2 or N;

[0142] X 3 It is C, CR 3 or N;

[0143] A is with X 2 and X 3 A fused 5-, 6- or 7-membered carbocyclic or heterocyclic ring, optionally substituted by one or more R 5 group substitution;

[0144] R a is s H, C1-C 12 Alkyl, C2-C8 alkenyl, C2-C8 alkynyl, -(C1-C 12 Alkylene)-(C3-C 12 Carbocyclic), -(C1-C 12 Alkylene)-(heterocyclic group having 3-20 ring atoms), -(C1-C 12 Alkylene)-C(=O)-(heterocyclyl having 3-20 ring atoms), -(C1-C 12 Alkylene)-(C6-C 20 Aryl) and -(C1-C 12-alkylene)-(heteroaryl having 5-20 ring atoms), wherein alkyl, alkenyl, alkynyl, alkylene, carbocyclyl, heterocyclyl, aryl and heteroaryl are optionally substituted with one or more groups independently selected from the group consisting of 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;

[0145] R 1 、R 2 and R 3 independently selected from H, F, Cl, Br, I, -CH3, -CH2CH3, -C(CH3)3, -CH2OH, -CH2CH2OH, -C(CH3)2OH, -CH2OCH3, -CN, -CF3, -CO2H, -COCH3, -COC(CH3)3, -CO2CH3, -CONH2, -CONHCH3, -CON(CH3)2, -C(CH3)2CONH2, -NO 2, -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;

[0146] R 4 Selected from C6-C 20 Aryl, heterocyclyl having 3 to 20 ring atoms and heteroaryl having 5 to 20 ring atoms, each of which is optionally substituted by one or more R 6 Group substituted, the R 6Groups are independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -CH(CH3)2, -CH2CH(CH3)2, -CH2CH3, -CH2CN, -CN, -CF3, -CH2OH, -CO2H, -CONH2, -CONH(CH3), -CON(CH3)2, -NO2, -NH2, -NHCH3, -NHCOCH3, -OH, -OCH3, -OCH2CH3, -OCH(CH3)2, -SH, -NHC(=O)NHCH3, -NHC(=O)NHCH2CH3, -NHC(=O)NHCH(CH3)2, -NHS(O)2CH3, -N(CH3)C(=O)OC(CH3)3, -S(O)2CH3, benzyl, benzyloxy, morpholinyl, morpholinomethyl, and 4-methylpiperazin-1-yl; and

[0147] R 5 Independently selected from C1-C 12 Alkyl, C2-C8 alkenyl, C2-C8 alkynyl, (C1-C 12 Alkylene)-(C3-C 12 Carbocyclic), -(C1-C 12 Alkylene)-(heterocyclic group having 3-20 ring atoms), -(C1-C 12 Alkylene)-C(=O)-(heterocyclyl having 3-20 ring atoms), -(C1-C 12 Alkylene)-(C6-C 20 Aryl), and -(C1-C 12 alkylene)-(heteroaryl having 5-20 ring atoms); or two pairs of R 5The group forms a 3-, 4-, 5- or 6-membered carbocyclic or heterocyclic ring, wherein the alkyl, alkenyl, alkynyl, alkylene, carbocyclic, heterocyclic, aryl and heteroaryl groups are optionally substituted with one or more groups independently selected from the group consisting of 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;

[0148] mor is selected from:

[0149]

[0150] Optionally, one or more R 7 Group substituted, the R 7 The groups are independently selected from F, Cl, Br, I, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, -C(CH3)3, -CH2OCH3, -CHF2, -CN, -CF3, -CH2OH, -CH2OCH3, -CH2CH2OH, -CH2C(CH3)2OH, -CH(CH3)OH, -CH(CH2CH3)OH, -CH2CH(OH)CH3, -C(CH3)2OH, -C(CH3)2OCH3, -CH(CH3)F, -C(CH3)F2, -CH(CH2CH3)F, -C(CH2CH3)2F, -CO2H, -CONH2, -CON(CH2CH3)2,

[0151] -COCH3, -CON(CH3)2, -NO2, -NH2, -NHCH3, -N(CH3)2, -NHCH2CH3,

[0152] -NHCH(CH3)2、-NHCH2CH2OH、-NHCH2CH2OCH3、-NHCOCH3、-NHCOCH2CH3、

[0153] -NHCOCH2OH, -NHS(O)2CH3, -N(CH3)S(O)2CH3, =O, -OH, -OCH3, -OCH2CH3,

[0154] -OCH(CH3)2, -SH, -NHC(=O)NHCH3, -NHC(=O)NHCH2CH3, -S(O)CH3,

[0155] -S(O)CH2CH3, -S(O)2CH3, -S(O)2NH2, -S(O)2NHCH3, -S(O)2N(CH3)2 and

[0156] -CH2S(O)2CH3.

[0157] In some other embodiments, the small molecule PI3K inhibitor is selected from the group consisting of:

[0158]

[0159] The present invention allows for the administration of lower doses of PI3K inhibitor therapy to a subject. The ability to utilize lower doses of PI3K therapy 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).

[0160] In some embodiments, the lower amount / dose of the PI3K inhibitor reduces or minimizes any undesirable side effects associated with PI3K therapy.

[0161] 2.2 Immunotherapy

[0162] The compositions of the present invention also include anti-cancer therapies, which are typically immunotherapies. Any immunotherapy that does not target cancer stem cells (CSCs) is generally considered suitable for use in the compositions and methods of the present invention. Checkpoint inhibitor molecule antagonists and PARP inhibitors are generally considered to be particularly suitable.

[0163] 2.2.1 Checkpoint inhibitor molecule (ICM) antagonists

[0164] Any suitable ICM antagonist that can be used for treatment can be used 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.

[0165] ICMs antagonized by the therapeutic compositions 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.

[0166] In some preferred embodiments, the ICM antagonist included in the therapeutic composition is a PD-1 antagonist. In this regard, "PD-1 antagonists" include any compound or biological molecule that blocks the binding of PD-L1 (e.g., PD-L1 expressed on the surface of cancer cells) to PD-1 expressed on immune cells (e.g., T cells, B cells, or NKT cells). 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 number Q15116) is as follows:

[0167] PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHP SPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL[SEQ ID NO:3].

[0168] 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 Publication Nos. US2003 / 0039653, US2004 / 0213795, US2006 / 0110383, US2007 / 0065427, US2007 / 0122378, US2012 / 237522 and International PCT Publication Nos. WO2004 / 072286, WO2006 / 121168, WO2006 / 133396, WO2007 / 005874, WO2008 / 083174, WO2008 / 156 712, WO2009 / 024531, WO2009 / 014708, WO2009 / 114335, WO2010 / 027828, WO2010 / 027423, WO2010 / 036959, WO2010 / 029435, WO2010 / 029434, WO2010 / 063011, WO2010 / 089411, WO2011 / 066342, WO2011 / 110604, WO2011 / 110621 and WO2012 / 145493 (the entire contents of which are incorporated herein by reference). Specific monoclonal antibodies that can be used for the purposes of the present invention include the anti-PD-1 monoclonal antibodies nivolumab, pembrolizumab, and pidilizumab, as well as the humanized anti-PD-1 antibodies h409A11, h409A16, and h409A17 described in International Patent Publication No. WO2008 / 156712.

[0169] The anti-PD-1 antigen binding molecules of the present invention preferably bind to a region of the PD-1 extracellular domain. For example, the anti-PD-1 antigen binding molecules can specifically bind to a region of the human PD-1 extracellular domain comprising the amino acid sequence SFVLNWYRMSPSNQTDKLAAFPEDR [SEQ ID NO: 4] (i.e., residues 62 to 86 of the native PD-1 sequence set forth in SEQ ID NO: 3) and SGTYLCGAISLAPKAQIKE [SEQ ID NO: 5] (i.e., residues 118 to 136 of the native PD-1 sequence set forth in SEQ ID NO: 3) or both. In another example, the anti-PD-1 antigen binding molecules bind to a region of the human PD-1 extracellular domain comprising the amino acid sequence NWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRV [SEQ ID NO: 6] (i.e., corresponding to residues 66 to 97 of the native human PD-1 sequence set forth in SEQ ID NO: 3).

[0170] In certain embodiments, the anti-PD-1 antigen-binding molecule comprises the fully humanized IgG4 monoclonal antibody nivolumab (as described in detail in U.S. Patent No. 8,008,449 (referred to as "5C4"), the entire contents of which are incorporated herein by reference) or an antigen-binding fragment thereof. In a representative example of this type, the anti-PD-1 antigen-binding molecule comprises the CDR sequences listed in Table 5.

[0171] Table 5

[0172]

[0173] In a more specific embodiment, the anti-PD-1 antigen binding molecule comprises the heavy chain amino acid sequence of nivolumab, for example as shown below:

[0174] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEW

[0175] VAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCAT

[0176] NDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPv

[0177] TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP

[0178] SNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV

[0179] VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD

[0180] WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS

[0181] LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK[SEQ ID NO:13];

[0182] or an antigen-binding fragment thereof, comprising, consisting of, or consisting essentially of the following amino acid sequence:

[0183] QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEW

[0184] VAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS[SEQ ID NO:14].

[0185] In some of the same and other embodiments, the anti-PD-1 antigen binding molecule can comprise the light chain amino acid sequence of nivolumab, such as shown below:

[0186] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ IDNO:15];

[0187] or an antigen-binding fragment thereof, comprising, consisting of, or consisting essentially of the following amino acid sequence:

[0188] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK[SEQ ID NO:16].

[0189] In an alternative embodiment, the anti-PD-1 antigen binding molecule comprises the humanized IgG4 mAb pembrolizumab or an antigen-binding fragment thereof. In a non-limiting example of this type, the anti-PD-1 antigen binding molecule comprises the CDR sequences listed in Table 6.

[0190] Table 6

[0191]

[0192]

[0193] In some embodiments, the anti-PD-1 antigen binding molecule competes with the mAb pembrolizumab for binding to PD-1.

[0194] In another embodiment, the anti-PD-1 antigen binding molecule comprises the heavy chain amino acid sequence of pembrolizumab, for example, as shown below:

[0195] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQ GTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPvTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK[SEQ ID NO:23];

[0196] or an antigen-binding fragment thereof, comprising, consisting of, or consisting essentially of the following amino acid sequence:

[0197] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS[SEQ ID NO:24].

[0198] Similarly, the anti-PD-1 antigen binding molecule may comprise the light chain amino acid sequence of pembrolizumab as follows:

[0199] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ ID NO:25];

[0200] or an antigen-binding fragment thereof, comprising, consisting of, or consisting essentially of the following amino acid sequence:

[0201] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK[SEQ ID NO:26].

[0202] In 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 shown in Table 7.

[0203] Table 7

[0204]

[0205] In a more specific embodiment, the anti-PD-1 antigen binding molecule comprises the heavy chain amino acid sequence of pidilizumab as described below:

[0206] QVQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVNTAYLQITSLTAEDTGMYFCVRVGYDALDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO:27];

[0207] or an antigen-binding fragment thereof, comprising, consisting of, or consisting essentially of the following amino acid sequence:

[0208] QVQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVNTAYLQITSLTAEDTGMYFCVRVGYDALDYWGQGTLVTVSS[SEQ ID NO:28].

[0209] In some of the same and other embodiments, the anti-PD-1 antigen binding molecule comprises the light chain amino acid sequence of pidilizumab as shown below:

[0210] EIVLTQSPSSLSSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTSYCLTINSLQPEDFATYYCQQRSSFPLTFGGGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ IDNO:29],

[0211] or an antigen-binding fragment thereof, comprising, consisting of, or consisting essentially of the following amino acid sequence:

[0212] EIVLTQSPSSLSSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTSYCLTINSLQPEDFATYYCQQRSSFPLTFGGGTKLEIK[SEQ ID NO:30].

[0213] Other suitable monoclonal antibodies are described in International Patent Publication No. WO 2015 / 026634, the entire contents of which are incorporated herein by reference. These include mAbs or antigen-binding fragments thereof comprising: (a) light chain CDRs (CDR1, CDR2, and CDR3, respectively) having the amino acid sequences of RASKSVSTSGFSYLH [SEQ ID NO:31], LASNLES [SEQ ID NO:32], and QHSWELPLT [SEQ ID NO:33] and heavy chain CDRs (CDR1, CDR2, and CDR3, respectively) having the amino acid sequences of SYYLY [SEQ ID NO:34], GVNPSNGGTNFSEKFKS [SEQ ID NO:35], and RDSNYDGGFDY [SEQ ID NO:36]; or (b) light chain CDRs (CDR1, CDR2, and CDR3, respectively) having the amino acid sequences of RASKGVSTSGYSYLH [SEQ ID NO:37], LASYLES [SEQ ID NO:38], and QHSRDLPLT [SEQ ID NO:39] and heavy chain CDRs (CDR1, CDR2, and CDR3, respectively) having the amino acid sequences of NYYMY [SEQ ID NO:31], GVNPSNGGTNFSEKFKS [SEQ ID NO:35], and RDSNYDGGFDY [SEQ ID NO:36]; The heavy chain CDRs (CDR1, CDR2 and CDR3, respectively) of GINPSNGGTNFN EKFKN [SEQ ID NO:40], GINPSNGGTNFN EKFKN [SEQ ID NO:41] and RDYRFDMGFDY [SEQ ID NO:42] are shown.

[0214] For example, such a monoclonal antibody may comprise (a) a heavy chain variable region comprising:

[0215] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS[SEQ ID NO:43],

[0216] or a variant or antigen-binding fragment thereof; and

[0217] A light chain variable region comprising an amino acid sequence selected from the group consisting of:

[0218] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK[SEQ ID NO:44],

[0219] IVLTQSPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIYLASYLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSRDLPLTFGQGTKLEIK[SEQ ID NO:45], or

[0220] DIVMTQTPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIYLASYLESGVPDRFSGSGSGTAFTLKISRVEAEDVGLYYCQHSRDLPLTFGQGTKLEIK [SEQ ID NO: 46], or a variant or antigen-binding fragment thereof.

[0221] In a further exemplary embodiment, the anti-PD-1 monoclonal antibody may comprise an IgG1 heavy chain comprising:

[0222] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK [SEQ ID NO:47], or its variants or antigen-binding fragments; and a light chain, comprising any of the following:

[0223] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC [SEQ ID NO:48],

[0224] EIVLTQSPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIYLASYLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSRDLPLTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC[SEQ ID NO:49]

[0225] DIVMTQTPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIYLASYLESGVPDRFSGSGSGTAFTLKISRVEAEDVGLYYCQHSRDLPLTFGQGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC[SEQ ID NO:50],

[0226] or a variant or antigen-binding fragment thereof.

[0227] In other embodiments, the ICM antagonist is a PD-L1 antagonist. Alternative names or synonyms for PD-L1 include PDCD1L1, PDL1, B7H1, B7-4, CD274, and B7-H. Typically, a PD-L1 antagonist specifically binds to the natural amino acid sequence of human PD-L1 (UniProt accession number Q9NZQ7), as shown below:

[0228] MRIFAVFFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPV TSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET[SEQ IDNO:51].

[0229] Suitably, the PD-L1 antagonist is an anti-PD-L1 antigen binding molecule. For example, the anti-PD-L1 antigen binding molecules suitable for the present invention include 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 International Publication Nos. WO2007 / 005874 and WO2010 / 077634 and U.S. Patent Nos. 8217149 and 8779108, the entire contents of each antibody are incorporated herein by reference. Other anti-PD-L1 monoclonal antibodies are described in International PCT Patent Publication No. WO2016 / 007235, the entire contents of which are also incorporated herein by reference.

[0230] The anti-PD-L1 antigen binding molecule is suitably bound to a region of the extracellular domain of PD-L1. For example, the anti-PD-L1 antigen binding molecule can specifically bind to a region of the extracellular domain of human PD-L1, the region comprising the amino acid sequence SKKQSDTHLEET [SEQ ID NO: 13] (i.e., residues 279 to 290 of the native PD-L1 sequence set forth in SEQ ID NO: 14). In certain embodiments, the anti-PD-L1 antigen binding molecule comprises a fully humanized IgG1 mAb durvalumab (as described in International PCT Publication No. WO2011 / 066389 and U.S. Patent Publication No. 2013 / 034559, "MEDI4736," the entire contents of which are incorporated herein by reference) or an antigen-binding fragment thereof. In a representative embodiment of this type, the anti-PD-L1 antigen binding molecule comprises the CDR sequences listed in Table 8.

[0231] Table 8

[0232]

[0233]

[0234] In a more specific embodiment, the anti-PD-L1 antigen binding molecule comprises the heavy chain amino acid sequence of durvalumab, for example as shown below:

[0235] VQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQG TLVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG[SEQ ID NO:52],

[0236] or an antigen-binding fragment thereof, which comprises an amino acid sequence consisting of or consisting essentially of the following amino acid sequence:

[0237] VQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS[SEQ ID NO:53].

[0238] In some of the same and other embodiments, the anti-PD-L1 antigen binding molecule can comprise a light chain amino acid sequence:

[0239] EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQID NO:54],

[0240] or an antigen-binding fragment thereof, comprising, consisting of, or consisting essentially of the following amino acid sequence:

[0241] EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIK[SEQ ID NO:55].

[0242] Alternatively, the anti-PD-L1 antigen binding molecule competes with the mAb durvalumab for binding to PD-L1.

[0243] 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 representative embodiments of this type, the anti-PD-L1 antigen binding molecule comprises the CDR sequences listed in Table 9.

[0244] Table 9

[0245]

[0246] In a more specific embodiment, the anti-PD-L1 antigen binding molecule comprises the heavy chain amino acid sequence of atezolizumab, for example as shown below:

[0247] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTL VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 56], or an antigen-binding fragment thereof comprising, consisting of, or consisting essentially of the following amino acid sequence:

[0248] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS[SEQ ID NO:57].

[0249] In some of the same and other embodiments, the anti-PD-L1 antigen binding molecule comprises the light chain amino acid sequence of atezolizumab, e.g., as shown below:

[0250] DIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ IDNO:58],

[0251] or an antigen-binding fragment thereof, comprising, consisting of, or consisting essentially of the following amino acid sequence:

[0252] DIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK[SEQ ID NO:59].

[0253] Alternatively, the anti-PD-L1 antigen binding molecule competes with the monoclonal antibody atezolizumab for binding to PD-L1.

[0254] 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 representative embodiments of this type, the anti-PD-L1 antigen binding molecule comprises the CDR sequences listed in Table 10.

[0255] Table 10

[0256]

[0257] In a specific embodiment, the anti-PD-L1 antigen binding molecule comprises the heavy chain amino acid sequence of avelumab, for example as shown below:

[0258] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQG TLVTVSSASTKGPSVFPLAPSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK [SEQ ID NO:60], or an antigen-binding fragment thereof comprising, consisting of, or consisting essentially of the following amino acid sequence:

[0259] EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS [SEQ ID NO: 61].

[0260] In some of the same and other embodiments, the anti-PD-L1 antigen binding molecule comprises the light chain amino acid sequence of avelumab, e.g., as shown below:

[0261] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLG QPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS[SEQID NO:62],

[0262] or an antigen-binding fragment thereof, comprising, consisting of, or consisting essentially of the following amino acid sequence:

[0263] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL [SEQ ID NO: 63].

[0264] Alternatively, the anti-PD-L1 antigen binding molecule competes with avelumab for binding to PD-L1.

[0265] In some embodiments, the ICM antagonist is an antagonist of CTLA4. Alternative names or synonyms for CTLA4 include ALPS5, CD, CD152, CELIAC3, CTLA-4, GRD4, GSE, IDDM 12. Typically, a CTLA4 antagonist specifically binds to the mature amino acid sequence of human CTLA4 (UniProt accession number P16410), for example as shown below:

[0266] KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN[SEQ ID NO:64].

[0267] Suitably, the CTLA4 antagonist is an anti-CTLA4 antigen binding molecule. For example, anti-CTLA4 antigen binding molecules suitable for use in the present invention include the anti-CTLA4 monoclonal antibody ipilimumab (BMS-734016, MDX-010, MDX-101) and tremelimumab (ticilimumab, CP-675206).

[0268] The anti-CTLA4 antigen binding molecules suitably bind to a region of the extracellular domain of CTLA4. For example, the anti-CTLA4 antigen binding molecules can specifically bind to a region of the extracellular domain of human CTLA4 comprising any one or more of the following amino acid sequences: YASPGKATEVRVTVLRQA [SEQ ID NO: 65] (i.e., residues 26 to 42 of the native CTLA4 sequence shown in SEQ ID NO: 64), DSQVTEVCAATYMMGNELTFLDD [SEQ ID NO: 66] (i.e., residues 43 to 65 of the native CTLA4 sequence shown in SEQ ID NO: 64), and VELMYPPPYYLGIG [SEQ ID NO: 67] (i.e., residues 96 to 109 of the native CTLA4 sequence shown in SEQ ID NO: 64). Alternatively or additionally, the anti-CTLA4 antigen binding molecule can specifically bind to a region of the extracellular domain of human CTLA4 comprising any one or more, and preferably all, of the following residues in the mature form: K1, A2, M3, E33, R35, Q41, S44, Q45, V46, E48, L91, 193, K95, E97, M99, P102, P103, Y104, Y105, L106, 1108, N110.

[0269] In certain embodiments, the anti-CTLA4 antigen binding molecule comprises the human IgG1 mAb ipilimumab (e.g., as described in International Publication No. WO 2014 / 209804 and U.S. Patent Publication No. 2015 / 0283234, the entire contents of which are incorporated herein by reference) or an antigen-binding fragment thereof. In representative embodiments of this type, the anti-CTLA4 antigen binding molecule comprises the CDR sequences listed in Table 11.

[0270] Table 11

[0271]

[0272] In a more specific embodiment, the anti-CTLA4 antigen binding molecule comprises the heavy chain amino acid sequence of ipilimumab, e.g., as described below:

[0273] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFD YWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK [SEQ ID NO: 68], or an antigen-binding fragment thereof, non-limiting examples of which include, consist of, or consist essentially of the following amino acid sequence:

[0274] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS[SEQ ID NO:69].

[0275] In some of the same and other embodiments, the anti-CTLA4 antigen binding molecule comprises the light chain amino acid sequence of ipilimumab, e.g., as shown below:

[0276] EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQID NO:70],

[0277] or an antigen-binding fragment thereof, representative examples of which include, consist of, or consist essentially of the following amino acid sequence:

[0278] EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK[SEQ ID NO:71].

[0279] In some embodiments, the anti-CTLA4 antigen binding molecule comprises the human IgG2 mAb tremelimumab (e.g., as described in U.S. Patent Publication No. 2009 / 0074787, the entire contents of which are incorporated herein by reference) or an antigen-binding fragment thereof. In representative embodiments of this type, the anti-CTLA4 antigen binding molecule comprises the CDR sequences listed in Table 12.

[0280] Table 12

[0281]

[0282] In a more specific embodiment, the anti-CTLA4 antigen binding molecule comprises the heavy chain amino acid sequence of tremelimumab, e.g., as described below:

[0283] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYIQMNSLRAEDTAVYYCARDPRGATLYYYYYGMD VWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTIS KTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO:172],

[0284] or an antigen-binding fragment thereof, non-limiting examples of which include, consist of, or consist essentially of the following amino acid sequence:

[0285] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS [SEQ ID NO:73].

[0286] In some of the same and other embodiments, the anti-CTLA4 antigen binding molecule comprises the light chain amino acid sequence of tremelimumab, e.g., as shown below:

[0287] DIQMTQSPSSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ IDNO:74],

[0288] or an antigen-binding fragment thereof, representative examples of which include, consist of, or consist essentially of the following amino acid sequence:

[0289] DIQMTQSPSSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIK[SEQ ID NO:75].

[0290] In other embodiments, the ICM antagonist is a B7-H3 antagonist. Typically, the B7-H3 antagonist of the present invention specifically binds to the natural amino acid sequence of human B7-H3 (UniProt accession number Q5ZPR3), as described below:

[0291] [SEQ ID NO:76].

[0292] Suitably, the B7-H3 antagonist is an anti-B7-H3 antigen binding molecule. For example, an anti-B7-H3 antigen binding molecule suitable for use in the present invention is the monoclonal antibody 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.

[0293] Table 13

[0294]

[0295] In a more specific embodiment, the anti-B7-H3 antigen binding molecule comprises the heavy chain amino acid sequence of enoxaparin, for example as shown below:

[0296] VQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISSRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQ GTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO:77],

[0297] or an antigen-binding fragment thereof, representative examples of which include, consist of, or consist essentially of the following amino acid sequence:

[0298] VQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISSRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSS[SEQ ID NO:78].

[0299] In some of the same and other embodiments, the anti-B7-H3 antigen binding molecule comprises the light chain amino acid sequence of enoxaparin, e.g., as shown below.

[0300] DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ IDNO:79],

[0301] or an antigen-binding fragment thereof, representative examples of which include, consist of, or consist essentially of the following amino acid sequence:

[0302] DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIK[SEQ ID NO:80].

[0303] In some alternative embodiments, the anti-B7-H3 antigen binding molecule competes with the monoclonal antibody enoxaparin for binding to B7-H3.

[0304] In other embodiments, the ICM antagonist is an indoleamine 2,3-dioxygenase (IDO) antagonist. The mature amino acid sequence of human IDO (UniProt accession number P14902) is shown below:

[0305] MAHAMENSWTISKEYHIDEEVGFALPNPQENLPDFYNDWMFIAKHLPDLIESGQLRERVEKLNMLSIDHLTDHKSQRLARLVLGCITMAYVWGKGHGDVRKVLPRNIAVPYCQLSKKLELPPILVYADCVLANWKKKDPNKPLTYENMDVLFSFRDGDCSKGFFLVSLLVEIAAASAIK VIPTVFKAMQMQERDTLLKALLEIASCLEKALQVFHQIHDHVNPKAFFSVLRIYLSGWKGNPQLSDGLVYEGFWEDPKEFAGGSAGQSSVFQCFDVLLGIQQTAGGGHAAQFLQDMRRYMPPAHRNFLCSLESNPSVREFVLSKGDAGLREAYDACVKALVSLRSYHLQIVTKYILIPAS QQPKENKTSEDPSKLEAKGTGGTDLMNFLKTVRSTTEKSLLKEG[SEQID NO:81].

[0306] Any IDO antagonist is suitable for use as a therapeutic agent in the present invention. Currently, three small molecule IDO inhibitors are being developed for clinical use: GDC-0919 (1-cyclohexyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)ethanol), indole oxime (1-methyl-D-tryptophan), and epaccardostat (1,2,5-oxadiazole-3-carboxamidine, 4-(2-((aminosulfonyl)amino)ethyl)amino)-N-(3-bromo-4-fluorophenyl)-N'-hydroxy-, (C(Z))-). The molecular structure of each of these molecules is provided below.

[0307]

[0308] (Indoximod)

[0309] In some embodiments, the ICM antagonist is a killer cell immunoglobulin (KIR) antagonist. In a preferred embodiment of this type, the KIR antagonist blocks the interaction between KIR2-DL-1, -2 and -3 and their ligands. For example, the mature amino acid sequence of human KIR, i.e., KIR2-DL1 (UniProt accession number P43626), is provided below:

[0310] HEGVHRKPSLLAHPGPLVKSEETVILQCWSDVMFEHFLLHREGMFNDTLRLIGEHHDGVSKANFSISRMTQDLAGTYRCYGSVTHSPYQVSAPSDPLDIVIIGLYEKPSLSAQPGPTVLAGENVTLSCSSRSSYDMYHLSREGEAHERRLPAGPKVNGTFQADFP LGPATHGGTYRCFGSFHDSPYEWSKSSDPLLVSVTGNPSNSWPSPTEPSSKTGNPRHLHILIGTSVVIILFILLFFLLHRWCSNKKNAAVMDQESAGNRTANSEDSDEQDPQEVTYTQLNHCVFTQRKITRPSQRPKTPPTDIIVYTELPNAESRSKVVSCP[SEQ ID NO:82].

[0311] Anti-KIR antigen binding molecules suitable for use in the present invention can be produced using methods well known in the art. Alternatively, industry-recognized KIR antigen binding molecules can be used. For example, anti-KIR antigen binding molecules include, for example, the fully humanized mAb lirelumab or an antigen-binding fragment thereof described in International Publication No. WO2014 / 066532, the entire contents of which are incorporated herein in their entirety. Suitably, the anti-KIR antigen binding molecules comprise the CDR regions listed in Table 14.

[0312] Table 14

[0313]

[0314]

[0315] In a representative embodiment of this type, the anti-KIR antigen binding molecule can comprise the heavy chain variable domain amino acid sequence of lirelumab, for example as shown below:

[0316] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYAISWVRQAPGQGLEWMGGFIPIFGAANYAQKFQGRVTITADESTSTAYMELSSLRSDDTAVYYCARIPSGSYYYDYDMDVW GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPvTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGP PCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK [SEQ ID NO: 83], or an antigen-binding fragment thereof, representative examples of which include, consist of, or consist essentially of the following amino acid sequence:

[0317] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYAISWVRQAPGQGLEWMGGFIPIFGAANYAQKFQGRVTITADESTSTAYMELSSLRSDDTAVYYCARIPSGSYYYDYDMDVWGQGTTVTVSS [SEQ ID NO:84].

[0318] In some of the same and other embodiments, the anti-KIR antigen binding molecule can comprise the light chain variable domain amino acid sequence of riluumab, e.g., as shown below:

[0319] EIVLTQSPVTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWMYTFGQGTKLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ IDNO:85],

[0320] or an antigen-binding fragment thereof, representative examples of which include, consist of, or consist essentially of the following amino acid sequence:

[0321] EIVLTQSPVTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWMYTFGQGTKLEIKRT[SEQ ID NO:86].

[0322] In an alternative embodiment, the ICM antagonist is a LAG-3 antagonist. LAG3 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 dendritic cells. A representative mature amino acid sequence of human LAG-3 (UniProt accession number P18627) is as follows:

[0323] LQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEY RAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYA GAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPW LEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLvTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPE QL [SEQ ID NO:87].

[0324] In some embodiments, the LAG-3 antagonist is an anti-LAG-3 antigen binding molecule. For example, a suitable anti-LAG antigen binding molecule is the anti-LAG3 humanized mAb BMS-986016. Other anti-LAG-3 antibodies are described in U.S. Patent Publication No. 2011 / 0150892 and International PCT Publication Nos. WO2010 / 019570 and WO2014 / 008218, the entire contents of which are incorporated herein by reference.

[0325] In some embodiments, the anti-LAG-3 antigen binding molecule comprises the CDR sequences listed in Table 15.

[0326] Table 15

[0327]

[0328]

[0329] The anti-LAG-3 antigen binding molecule suitably comprises mAb BMS-986016 or an antigen-binding fragment thereof. More specifically, in some embodiments, the anti-LAG-3 antigen binding molecule has the heavy chain amino acid sequence of BMS-986016, for example, as shown below:

[0330] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPP CPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK [SEQ ID NO: 88], or an antigen-binding fragment thereof, representative examples of which include, consist of, or consist essentially of the following amino acid sequence:

[0331] QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSS[SEQ ID NO:89].

[0332] Similarly, the anti-LAG-3 antigen binding molecule can comprise the light chain amino acid sequence of BMS-986016, as set forth in SEQ ID NO:45, as shown below:

[0333] EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ IDNO:90],

[0334] or an antigen-binding fragment thereof, representative examples of which include, consist of, or consist essentially of the following amino acid sequence:

[0335] EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIK[SEQ ID NO:91].

[0336] 2.2.2 PARP inhibitors

[0337] Poly (ADP-ribose) polymerase (PARP) enzymes are a family of enzymes that cleave NAD+, release nicotinamide, and continuously add ADP-ribose units to form ADP-ribose polymers. Activation of PARP enzymes can lead to depletion of cellular NAD+ levels (e.g., PARP acts as a consumer of NAD+) 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 double-stranded or single-stranded DNA breaks. It is well known that antialkylating agents can deplete the NAD+ content of tumor cells, and the discovery of PARPs explains this phenomenon (PARP Inhibitors and Cancer Therapy. Curtin N. in Poly ADP Ribosylation. ed. Alexander Burke, Lands Bioscience and Springer Bioscience, 2006: 218-233). Antialkylating agents induce DNA chain breaks, thereby activating PARP-1, which is part of the DNA repair pathway. Poly(ADP-ribosylation) of nuclear proteins by PARP-1 converts DNA damage into intracellular signals that can activate DNA repair (e.g., via the base excision repair (BER) pathway) or trigger cell death if DNA damage is too extensive to be effectively repaired.

[0338] PARP-2 contains a catalytic domain that catalyzes poly (ADP-ribosylation) reactions. PARP-2 displays auto-modification properties similar to those of PARP-1. The protein is localized to the nucleus in vivo, potentially explaining the residual poly (ADP-ribosylation) synthesis observed in PARP-1-deficient cells treated with alkylating agents or hydrogen peroxide. Some drugs that inhibit PARP (e.g., those primarily designed to inhibit PARP-1) may also inhibit PARP-2 (e.g., niraparib).

[0339] The role of PARP enzymes in the DNA damage response (e.g., DNA repair in response to genotoxic stress) provides compelling suggestions that PARP inhibitors may be useful anticancer agents. PARP inhibitors may be particularly effective in treating cancers caused by germline or sporadic defects in the homologous recombination DNA repair pathway, such as BRCA-1 and / or BRCA-2-deficient cancers.

[0340] Preclinical in vitro and in vivo studies have demonstrated that PARP inhibitors are selectively cytotoxic against tumors harboring homozygous inactivation of BRCA-1 and / or BRCA-2 genes, 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 BRCA-1 and / or BRCA-2-deficient cancers is that PARP-1 and PARP-2 are essential for base excision repair (BER) of damaged DNA. Following the formation of a single-strand DNA break, PARP-1 and PARP-2 bind to the lesion, become activated, and catalyze the addition of long polymers of ADP-ribose (PAR chains) to several chromatin-associated proteins, including histones. This leads to chromatin relaxation and the rapid recruitment of DNA repair factors, which can access and repair the DNA break. Normal cells repair up to 10,000 DNA defects daily, 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. When the replication machinery passes through the break site, the pre-existing single-strand break is converted into a double-strand break. Double-strand breaks that occur during S phase are preferentially repaired by the error-free HR pathway. Cells with inactivation of genes required for HR (such as BRCA-1 and / or BRCA-2) accumulate stalled replication forks during S phase and may use error-prone non-homologous end joining (NHEJ) to repair damaged DNA. Both the inability to complete S phase (due to stalled replication forks) and the error-prone repair by NHEJ are thought to lead to cell death.

[0341] 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 deficient DNA repair pathways (e.g., BRCA-1 and / or BRCA-2 inactivation). For example, tumors arising in patients with germline BRCA mutations have defective homologous recombination DNA repair pathways and will become increasingly dependent on BER (a pathway blocked by PARP inhibitors) to maintain genomic integrity. This concept of inducing death by blocking one DNA repair pathway in tumors with pre-existing defects in the complementary DNA repair pathway using PARP inhibitors is known as synthetic lethality.

[0342] The therapeutic potential of PARP inhibitors has been further expanded by the observation that PARP inhibitors are not only active as monotherapies in HR-deficient tumors but also effective in combination with other agents such as cisplatin, carboplatin, alkylating and methylating drugs, radiation therapy, and topoisomerase I inhibitors in preclinical models. In contrast to the rationale for monotherapy 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. In some cases, the specific role of PARP is unclear, but it is known that PARP is required for the release of trapped topoisomerase I / irinotecan complexes from DNA. Temozolomide-induced DNA damage is repaired via the BER pathway, which requires PARP to recruit repair proteins. Combination therapies that enhance or synergize cancer treatment without significantly increasing toxicity would provide substantial benefit to cancer patients, including those with ovarian cancer.

[0343] In some embodiments, treatment with a PARP inhibitor (e.g., a PARP-1 / 2 inhibitor) as provided herein for use in the various methods and kits disclosed herein can selectively kill a subset of cancer cell types by exploiting their defects in DNA repair. Human cancers exhibit genomic instability and increased mutation rates due to underlying defects in DNA repair. These defects render cancer cells more dependent on remaining DNA repair pathways, and targeting these pathways is expected to have an impact on the survival of tumor cells rather than normal cells.

[0344] In some embodiments, the PARP inhibitor is selected from ABT-767, AZD 2461, BGB-290, BGP 15, CEP8983, CEP 9722, DR 2313, E7016, E7449, fluzoparib (SHR 3162), IMP4297, INO1001, JPI 289, JPI 547, monoclonal antibody B3-LysPE40 conjugate, MP 124, niraparib (ZEJULA), NU 1025, NU 1064, NU1076, NU1085, olaparib, ONO2231, PD 128763, R 503, R554, rucaparib (RUBRACA), SBP 101, SC 101914, ximinperide, talazoparib (BMN-673), veliparib (ABT-888) and WW46, 2-(4-(trifluoromethyl)phenyl)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-4-ol, or a pharmaceutically acceptable salt thereof.

[0345] 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.

[0346] In some embodiments, the PARP inhibitor is selected from olaparib, niraparib, rucaparib, talazoparib, veliparib, or any combination thereof. In some embodiments, the PARP inhibitor is prepared as a pharmaceutically acceptable salt. In some embodiments, the salt form can exist as a solvated or hydrated polymorph.

[0347] Thus, the PARP inhibitor may be selected from olaparib, niraparib, rucaparib and talazoparib, or a pharmaceutically acceptable salt thereof.

[0348] In some embodiments, the PARP inhibitor is olaparib or a pharmaceutically acceptable salt thereof. In some embodiments, the PARP inhibitor is olaparib.

[0349] Olaparib (AZD2281, KU-0059436) is a potent PARP inhibitor (PARP 1, 2, and 3) currently being developed as a monotherapy and in combination with chemotherapy, ionizing radiation, and other anticancer agents, including novel agents and immunotherapies.

[0350] PARP inhibition is a new approach to target tumors with defects in DNA repair mechanisms. The PARP enzyme is essential for repairing single-strand breaks (SSBs) in DNA.

[0351] Inhibition of PARP leads to the persistence of SSBs, which are then converted into more severe DNA double-strand breaks (DSBs) during DNA replication. During 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 can be lethal to cells as DNA abnormalities accumulate. In these tumor types, olaparib may provide a potentially effective and less toxic cancer treatment compared to currently available chemotherapy regimens. Olaparib traps the inactive form of PARP at SSB sites on DNA, preventing their repair.

[0352] 2.3 adjuvants

[0353] In some embodiments, the PI3K inhibitor and the ICM binding antagonist are administered simultaneously with an adjuvant for the treatment or adjuvant treatment of T cell dysfunction diseases. Non-limiting examples of adjuvants include cytotoxic agents, gene therapy agents, DNA therapy agents, viral therapy agents, RNA therapy agents, immunotherapy agents, bone marrow transplant agents, nanotherapy agents, or a combination of the foregoing. The adjuvant can be in the form of an adjuvant therapy or a neoadjuvant therapy. In some embodiments, the adjuvant is a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the adjuvant is a side effect limiter (e.g., an agent intended to reduce the occurrence and / or severity of treatment side effects, such as an antiemetic, etc.). In some embodiments, the adjuvant is a radiotherapeutic agent. In some embodiments, the adjuvant is an agent targeting the PI3K / AKT / mTOR pathway, an HSP90 inhibitor, a microtubule inhibitor, an apoptosis inhibitor, and / or a chemopreventive agent. In some embodiments, the adjuvant is an immunotherapeutic agent, such as a blocking antibody, ipilimumab (also known as MDX-010, MDX-101, or ), tremelimumab (also known as ticilimumab or CP-675206), antagonists to B7-H3 (also known as CD276), such as the blocking antibody, MGA271, antagonists to TGF-β (e.g., metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299), T cells (e.g., cytotoxic T cells or CTLs) expressing a chimeric antigen receptor (CAR), T cells comprising a dominant-negative TGF-β receptor (e.g., a dominant-negative TGF-β type II receptor), agonists to CD137 (also known as TNFRSF9, 4-1BB, or ILA), such as the activating antibody, urelumab (also known as BMS-663513), agonists for CD40, such as an activating antibody, CP-870893, agonists for OX40 (also known as CD134), such as an activating antibody, administered in combination with an anti-OX40 antibody (e.g., AgonOX), agonists for CD27, such as an activating antibody, CDX-1127, indoleamine-2,3-dioxygenase (IDO), 1-methyl-D-tryptophan (also known as 1-D-MT), antibody-drug conjugates (in some embodiments, comprising mertansine or monomethyl auristatin E (MMAE)), anti-NaPi2b antibody-MMAE conjugates (also known as DNIB0600A or RG7599), trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine or Genentech), DMUC5754A, antibody-drug conjugates targeting endothelin B receptor (EDNBR), e.g., anti-EDNBR antibodies conjugated to MMAE, angiogenesis inhibitors, anti-VEGF antibodies, such as VEGF-A, bevacizumab (also known as Genentech), antibodies against angiopoietin 2 (also known as Ang2), MEDI3617, anti-tumor agents, agents against CSF-IR (also known as M-CSFR or CD115), anti-CSF-1R (also known as IMC-CS4), interferons such as IFN-α or IFN-γ, Roferon-A, GM-CSF (also known as recombinant human granulocyte macrophage colony-stimulating factor, rhu GM-CSF, sargramostim or ), IL-2 (also known as aldesleukin or ), IL-12, antibodies targeting CD20 (in some embodiments, antibodies targeting CD2 are atezolizumab (also known as GA101 or ) or rituximab), antibodies targeting GITR (in some embodiments, the antibody targeting GIRT is TRX518), combined with cancer vaccines (in some embodiments, the cancer vaccine is a peptide cancer vaccine, which in certain embodiments is a personalized peptide vaccine; in some embodiments, the peptide cancer vaccine is a multivalent long peptide, a polypeptide, a peptide mixture, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (peptide-pulsed dendritic cell vaccine, see, e.g., Yamada et al., Cancer Sci, 104: 14-21, 2013)), in combination with adjuvants, TLR agonists, such as Poly-ICLC (also known as ), LPS, MPL or CpGODN, TNF, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, HVEM antagonists, ICOS agonists, such as by administering ICOS-L, or agonistic antibodies to ICOS, agents targeting CX3CL1, agents targeting CXCL10, agents targeting CCL5, LFA-1 or ICAM1 agonists, selectin agonists, targeted therapeutics, B-Raf inhibitors, vemurafenib (also known as Dabrafenib (also known as ), erlotinib (also known as ), MEK inhibitors 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 ), K-Ras inhibitors, c-Met inhibitors, onatumumab (also known as MetMAb), Alk inhibitors, AF802 (also known as CH5424802 or alectinib), BKM120, idelalisib (also known as GS-1101 or CAL-101), perifosine (also known as KRX-0401), Akt, MK2206, GSK690693, GDC-0941, mTOR inhibitors, sirolimus (also known as rapamycin), temsirolimus (also known as CCl-779 or ), everolimus (also known as RAD001), deforolimus (also known as AP-23573, MK-8669 or deforolimus), OSI-027, AZD8055, INK128, dual PI3K / mTOR inhibitors, XL765, GDC-0980, BEZ235 (also known as NVP-BEZ235), BGT226, GSK2126458, PF-04691502, PF-05212384 (also known as PKI-587). The adjuvant may be one or more of the cytotoxic or chemotherapeutic agents described herein.

[0354] In some embodiments, the adjuvant is an anti-infective drug. The anti-infective drug is suitably selected from antimicrobials, which include, but are not limited to, compounds that kill or inhibit the growth of microorganisms such as viruses, bacteria, yeasts, fungi, and protozoa, and thus include antibiotics, amebicides, antifungals, antiprotozoal drugs, antimalarials, antituberculosis drugs, and antivirals. Anti-infective drugs also include 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, rofloxacin, temafloxacin, tosulfloxacin, sparfloxacin, clinafloxacin, gatifloxacin, moxifloxacin, gemefloxacin, and ganoxacin mesylate), tetracyclines, glycylcyclines, and oxazolidinones (e.g., chlortetracycline, demeclocycline, doxycycline, lymecycline, methetracycline, minocycline, telocycline, chlortetracycline, chloramphenicol ... amikacin, tetracycline, tigecycline, linezolid, eprazolid), glycopeptides, aminoglycosides (e.g., amikacin, arbekacin, daptomycin, fortinomycin, gentamicin, kanamycin, neomycin, netilmicin, ribosomycin, sisomicin, spectinomycin, streptomycin, tobramycin), beta-lactams (e.g., imipenem, meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefathiamidine, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicidin, cefoperazone, cefoperazone, ceforamate, cefadroxil, cefamandole, Fotaxime, cefotiam, cefuroxime, cefpiramide, cefpodoxime proxetil, cefsulodin, ceftazidime, cefditoren, ceftizoxime, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefazolin, cephalacetrile, cephalexin, cephaloglycin, cephalothin, cephapirin, cefradine, cefinetazole, cefoxitin, cefotetan, aztreonam, carumonam, fluoxetine, latamoxef, and amazocet Lin, amoxicillin, ampicillin, azlocillin, benzylpenicillin, carfilzocillin, 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, cefazolin, ME1228, KP-736, CP-6232, Ro 09-1227, OPC-20000, LY206763), rifamycins, macrolides (such as azithromycin, clarithromycin, erythromycin, oleandomycin, rotamycin, roseomycin, roxithromycin, and troleandomycin), ketolides (such as telithromycin and quinomicin), coumarins, lincosamides (such as clindamycin and lincomycin), and chloramphenicol.Exemplary antiviral drugs 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 anti-amoebic drugs or anti-protozoal drugs include atovaquone, chloroquine hydrochloride, chloroquine phosphate, metronidazole, metronidazole hydrochloride and pentamidine isethionate. The anthelmintic can be at least one selected from mebendazole, pyrantel pamoate, albendazole, ivermectin and thiabendazole. Exemplary antifungal drugs can be selected from amphotericin B, amphotericin B-cholesterol sulfate complex, amphotericin B-lipid complex, amphotericin B liposomes, fluconazole, flucytosine, micronized griseofulvin, ultramicronized griseofulvin, itraconazole, ketoconazole, nystatin and terbinafine hydrochloride. Non-limiting examples of antimalarial drugs include chloroquine hydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine and pyrimethamine with sulfadoxine. Anti-tuberculosis drugs include but are not limited to clofazimine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampicin, rifapentine and streptomycin sulfate.

[0355] 3. Pharmaceutical compositions and preparations

[0356] 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, such as described herein.

[0357] The pharmaceutical compositions and formulations described herein can be prepared by mixing an active ingredient (e.g., a small molecule, nucleic acid, or polypeptide) of 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 include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol); low molecular weight (less than about 10 residues) polypeptides. ; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., Zn protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 ( Baxter International Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

[0358] In some embodiments, particularly when 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 WO2006 / 044908, the latter's formulations including histidine acetate buffer.

[0359] The compositions and preparations herein may also contain other active ingredients as required for the specific indication being treated, preferably those having complementary activities and not adversely affecting each other. Such active ingredients are present in appropriate combinations in amounts effective to achieve the intended purpose.

[0360] The active ingredient can be embedded in microcapsules, for example, prepared by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, embedded in colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions. These techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0361] Sustained-release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which are in the form of shaped articles, such as films or microcapsules. Formulations for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0362] The formulations can be administered systemically or topically, depending on the specific condition being treated. For example, suitable routes may include oral, rectal, transmucosal, or intestinal administration; parenteral administration includes intramuscular, subcutaneous, and intramedullary injections, as well as intrathecal, direct intracerebroventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Formulations and administration techniques can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition.

[0363] 4. Therapeutic Uses

[0364] The present invention discloses that PI3K inhibitors and ICM-binding antagonists (also referred to herein as "therapeutic combinations" or "combination therapies") can be used to treat T cell dysfunction disorders in individuals with cancer, or to enhance immune function (e.g., immune effector function, T cell function, etc.), to treat or delay the progression of cancer, or to treat infection in individuals. In specific embodiments, therapeutic combinations for treating or delaying the progression of cancer (including metastatic cancer) and for preventing cancer recurrence are disclosed. Any PI3K inhibitor and ICM-binding antagonist known in the art or described herein can be used in this regard.

[0365] In some embodiments, combination therapy further comprises the use or administration of an adjuvant agent (eg, a chemotherapeutic agent), as described herein.

[0366] Suitably, individuals treated with combination therapy include T cells with a mesenchymal phenotype (e.g., CD8+ T cells), for example, T cells expressing CSV, EGRF, and ABCB5, SoX9, SNAIL, and AKT1 in the same T cell, and / or their levels are higher than those of activated T cells. The T cells may be tumor-infiltrating lymphocytes or circulating lymphocytes. The T cells appropriately exhibit T cell exhaustion or anergy, and in representative examples of this type, the levels of EOMES expressed by the T cells are higher than TBET and / or PD-1 expression is elevated. In some embodiments, the immune function of the T cells is impaired or suppressed, and biomarkers of reduced T cell activation (e.g., reduced production and / or secretion of cytokines such as IL-2, IFN-γ, and TNF) are appropriately expressed. Therefore, TBET, PD1, and EOMES (also referred to herein as "T cell function biomarkers") can be used to determine the immune function of the patient's T cells to assess the patient's T cell immune status, including sensitivity to ICM combined with antagonist treatment.

[0367] In some embodiments, the subject is a human.

[0368] In some embodiments, the individual has been treated with an ICM binding antagonist prior to treatment with the combination of the ICM binding antagonist and the PI3K inhibitor.

[0369] In some embodiments, the individual has a cancer that is resistant (has been shown to be resistant) to one or more ICM-binding antagonists. In some embodiments, resistance to an ICM-binding antagonist comprises recurrence of the cancer or a refractory cancer. Recurrence may mean that the cancer reappears at the original site or a new site after treatment. In some embodiments, resistance to an ICM-binding antagonist comprises progression of the cancer during treatment with an ICM-binding blocker. In some embodiments, resistance to an ICM-binding antagonist comprises the cancer not responding to treatment. The cancer may be resistant at the beginning of treatment or may become resistant during treatment. In some embodiments, the cancer is in the early or late stages.

[0370] In some embodiments of any of the methods, assays, and / or kits, any one or more T cell function biomarkers are detected in a sample using a method selected from FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunodetection methods, HPLC, surface plasmon resonance, spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technology, and FISH, and combinations thereof.

[0371] In some embodiments of any of the methods, assays, and / or kits, any one or more 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 T cell function biomarkers are detected using antibodies that specifically bind to the corresponding biomarkers.

[0372] In some embodiments, the combination therapy of the present invention includes administering a PI3K inhibitor and an ICM binding antagonist. The PI3K inhibitor and the ICM binding antagonist can be administered in any suitable manner known in the art. For example, the PI3K inhibitor and the ICM binding blocker can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the PI3K inhibitor is present in a separate composition as an ICM binding antagonist. In some embodiments, the PI3K inhibitor and the ICM binding antagonist are in the same composition. Therefore, the combination therapy may involve administering the PI3K inhibitor separately, simultaneously or sequentially together with the ICM binding antagonist. In some embodiments, this can be achieved by administering a single composition or pharmacological formulation comprising two types of agents, or by administering two separate compositions or formulations simultaneously. One composition comprises a PI3K inhibitor and the other composition comprises an ICM binding antagonist. In other embodiments, treatment with the PI3K inhibitor can be before or after treatment with the ICM binding antagonist, with intervals ranging from a few minutes to several days. In embodiments where the PI3K inhibitor and the ICM binding antagonist are administered separately, it is generally ensured that there is no expiration for a long period of time between each delivery, so that the PI3K inhibitor can still have a beneficial effect on functionally suppressed T cells (e.g., mesenchymal T cells) as described above, particularly to enhance T cell immune function, including sensitivity of T cells to reactivation by the ICM binding blocker. In this case, it is expected that the two modes will be administered within about 1-12 hours of each other, more preferably, about 2-6 hours apart. However, in some cases, it may be necessary to significantly extend the treatment time, wherein the administrations are separated by several hours (2, 3, 4, 5, 6, or 7) to several days (1, 2, 3, 4, 5, 6, 7, or 8).

[0373] It is conceivable that multiple administrations of the PI3K inhibitor or ICM binding antagonist may be required. Various combinations may be employed, wherein the PI3K inhibitor is "A" and the ICM binding antagonist is "B," as shown below:

[0374] 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.

[0375] The PI3K inhibitor and the ICM binding antagonist can be administered by the same route of administration or different routes of administration. In some embodiments, the ICM binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intraventricularly, or intranasally. In some embodiments, the PI3K inhibitor is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implanted, inhaled, intrathecally, intraventricularly, or intranasally. An effective amount of the PI3K inhibitor and the ICM binding antagonist can be administered to prevent or treat a disease. The appropriate dosage of the PI3K inhibitor and the ICM binding antagonist can be determined based on the type of disease to be treated, the type of PI3K inhibitor and ICM binding blocker, the severity and course of the disease, the individual's clinical condition, the individual's medical history and response to treatment, and the judgment of the attending physician. In some embodiments, combination therapy with a PI3K inhibitor (e.g., GDC-0084) and an ICM binding antagonist (e.g., an anti-PD-1 antibody) is synergistic, such that the effective dose of the ICM binding antagonist (e.g., an anti-PD-1 antibody) in the combination therapy is reduced relative to the effective dose of the ICM binding blocker (e.g., an anti-PD-1 antibody) as a single agent.

[0376] As a general suggestion, no matter by one or more administration, the therapeutically effective amount of the peptide or polypeptide active agent (such as antibody, peptide inhibitor, immunoadhesin, etc.) applied to the mankind is in the range of about 0.01 to about 50mg / kg patient weight. In some embodiments, the antibody used is for example about 0.01 to about 45mg / kg, about 0.01 to about 40mg / kg, about 0.01mg / kg to about 35mg / kg, about 0.01mg / kg to about 30mg / kg, about 0.01% to about 25mg / kg, about 0.01mg / kg to about 20mg / kg, about 0.01mg / kg to about 15mg / kg, about 0.01mg / kg to about 10mg / kg, about 0.01g / kg to about 5mg / kg or about 0.01mg / kg to about 1mg / kg daily. In some embodiments, the peptide or polypeptide active agent (such as antibody, peptide inhibitor, immunoadhesin, etc.) is administered with a dosage of 15mg / kg. However, other dosage regimens may also be useful. In one embodiment, on day 1 of a 21-day cycle, an anti-PDL1 antibody as 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. The dose can be administered as a single dose or multiple doses (e.g., 2 or 3 doses), such as by infusion. Compared to monotherapy, the dose of the peptide or polypeptide active agent (e.g., antibody, peptide inhibitor, immunoadhesin, etc.) administered in the combination therapy may be reduced. The progress of this therapy is easily monitored by conventional techniques.

[0377] Small molecule compounds are typically administered at an initial dose of about 0.0001 mg / kg to about 1000 mg / kg per day. A daily dose range of about 0.01 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 200 mg / kg, or about 1 mg / kg to about 100 mg / kg, or about 10 mg / kg to about 50 mg / kg may be used. However, the dosage may vary depending on the patient's requirements, the severity of the disease being treated, and the compound being used.

[0378] In any case, the dosage can be determined empirically, taking into account the type and stage of disease diagnosed in a particular patient. In the context of the present invention, the dosage administered to the patient should be sufficient to produce a beneficial therapeutic response in the patient over time. The size of the dosage will also be determined by the presence, nature, and extent of any adverse side effects that occur when a particular compound is administered to a particular patient. Determining the appropriate dosage for a particular situation is within the skill of the practitioner. Typically, treatment begins with a smaller dose that is less than the optimal dose of the compound. Thereafter, the dosage is increased in small increments until the optimal effect is achieved in the particular situation. For convenience, if necessary, the total daily dose can be divided and administered in portions during the day. The dosage can be administered daily or every other day at the discretion of the attending physician. The dosage can also be administered regularly or continuously over a longer period of time (weeks, months, or years), for example, by using subcutaneous capsules, pouches, or reservoirs, or by patch or pump. In some embodiments, the PI3K inhibitor, ICM binding antagonist, and optionally an adjuvant (e.g., chemotherapeutic agent) are administered on a regular schedule. Alternatively, combination therapy can be administered when symptoms occur.

[0379] As used herein, a "routine schedule" refers to a predetermined specified time period. A routine schedule can include time periods of the same or different lengths, as long as the schedule is predetermined. For example, a routine plan may involve administering a PI3K inhibitor, an ICM binding antagonist, and an optional adjuvant every day, every two days, every three days, every four days, every five days, every six days, every week, every month, or any set number of days or weeks therebetween, every two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, etc. Alternatively, a predetermined routine schedule may involve administering a PI3K inhibitor, an ICM binding antagonist, and an optional adjuvant simultaneously every day for the first week, then monthly for a few months, and then every three months. Any particular combination will be included in the routine schedule as long as it is determined in advance that the appropriate schedule involves administration on a particular day.

[0380] In some embodiments, the methods of treatment and uses may also include additional treatments. Additional treatments may be radiation therapy, surgery (e.g., lumpectomy and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination thereof. In some embodiments, the additional treatment is radiation therapy. In some embodiments, the additional treatment is surgery. In some embodiments, the additional treatment is a combination of radiation therapy and surgery. In some embodiments, the additional treatment is gamma radiation.

[0381] The efficacy of any of the methods described herein (e.g., combination therapy, comprising administering an effective amount of a PI3K inhibitor, an ICM-binding antagonist, and optionally a combination of adjuvants) can be tested in various models known in the art, such as clinical or preclinical models. Suitable preclinical models are exemplified herein, which 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 Claudemann melanoma cancer models.

[0382] The efficacy of any of the methods described herein (e.g., a combination therapy comprising administering an effective amount of a PI3K inhibitor, an ICM-binding antagonist, and optionally an adjuvant) can be tested in a GEM model of developing tumors, including but not limited to GEM models of non-small cell lung cancer, pancreatic ductal adenocarcinoma, or melanoma. For example, as described in Jackson et al., 2001 Genes Dev. 15(24):3243-8 (describing KrasG12D) and Lee et al. (2012 Dis. Model Mech. 5(3):397-402) (FRT-mediated p53null alleles), mice expressing KrasG12D in a p53null background after adenoviral recombinase treatment can be used as a preclinical model of non-small cell lung cancer. As another example, mice expressing KrasG12D in a p16 / p19null background can be used as a preclinical model for pancreatic ductal adenocarcinoma (PDAC), as described by Jackson et al. (2001, supra) (describing KrasG12D) and Aguirre et al. (2003 Genes Dev. 17(24):3112-26) (p16 / p19null allele). As another example, mice with melanocytes expressing BrafV600E in a melanocyte-specific PTENnull background following inducible (e.g., 4-OHT-treated) recombinase treatment can be used as a preclinical model for melanoma, as described by Dankort et al. (2007 Genes Dev. 21(4):379-84) (describing Braf.sup.V600E) and Trotman et al. (2003 PLoS Biol. 1(3):E59) (PTENnull allele). For any of these exemplary models, after tumor development, mice are randomly enrolled in treatment groups that receive a PI3K inhibitor, an ICM binding antagonist, and optionally an adjuvant or control treatment. Tumor size (e.g., tumor volume) is measured during treatment, and overall survival is monitored.

[0383] In some embodiments of the methods of the present disclosure, cancer (in some embodiments, a sample of a patient's cancer examined using a diagnostic test, such as described in the Examples herein) includes tumor infiltrating lymphocytes (TILs), wherein TILs are within or otherwise associated with cancer tissue. In these embodiments, it is assessed whether TILs express any one or more T cell function biomarkers disclosed herein. For example, TBET, PD-1, and EOMES can be used as biomarkers of T cell exhaustion, characterized by, for example, high levels of inhibitory co-receptors and the ability to produce effector cytokines (Wherry, EJ 2011 Nature Immunology 12: 492-499; Rabinovich et al., 2007 Annual Review of Immunology 25: 267-296).

[0384] In some embodiments of the disclosed methods, the individual has T cell dysfunction, manifested as T cell dysfunction disease. The characteristic of T cell dysfunction may be that T cells are incompetent or have reduced ability to secrete cytokines, proliferate or perform cytolytic activity. In some embodiments of the disclosed methods, the characteristic of T cell dysfunction disease is that T cell immune function is suppressed. In some embodiments of the disclosed methods, the characteristic of T cell dysfunction disease is T cells of mesenchymal phenotype. In some embodiments of the disclosed methods, the characteristic of T cell dysfunction disease is T cell exhaustion. In some embodiments of the disclosed methods, T cells are CD4+ and / or CD8+ T cells. According to the present invention, PI3K inhibitor treatment can increase the expression of biomarkers of T cell activation and effector capacity (e.g., IL-2, IFN-γ and TNF), reduce the expression of biomarkers of T cell effector inhibition and cancer progression (e.g., ZEB1), reduce 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 the production of IFN-γ in cells of the adaptive and innate immune systems. Notably, treatment with a PI3K inhibitor may enhance the sensitivity of exhausted T cells to reactivation with an ICM binding antagonist. Thus, combined treatment with a PI3K inhibitor and an ICM binding antagonist 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.

[0385] In some embodiments of the methods of the present disclosure, compared to before the administration of combination γ, the CD4+ and / or CD8+ T cells activated in the individual are characterized by producing IFN-γ CD4+ and / or CD8+ T cells and / or enhancing cytolytic activity. IFN-γ can be measured by any method known in the art, including, for example, intracellular cytokine staining (ICS), involving cell fixation, infiltration and staining with anti-IFN-γ antibodies. Cytolytic activity can be measured by any method known in the art, such as a cell killing assay using mixed effector cells and target cells.

[0386] In some embodiments, the CD8+ T cells are characterized by, for example, the presence of CD8b expression (e.g., by RT-PCR, e.g., using Fluidigm) (CD8b is also known as the 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.

[0387] In some embodiments, Treg cells are characterized by, for example, the presence of expression of Fox3p (e.g., by RT-PCR, e.g., using Fluidigm) (Foxp3 is also known as forkhead protein P3; scurfin; FOXP3delta7; immunodeficiency, polyendocrine disease, enteropathy, X-linked; accession number NM_014009). In some embodiments, Tregs are from peripheral blood. In some embodiments, Treg cells are from tumors.

[0388] In some embodiments, inflammatory or activated T cells are characterized by, for example, the presence of TBET and / or CXCR3 expression, or a TBET:EOMES ratio associated with inflammatory or activated T cells (e.g., by RT-PCR, e.g., using Fluidigm). In some embodiments, inflammatory or activated T cells are from peripheral blood. In some embodiments, inflammatory or activated T cells are from tumors.

[0389] In some embodiments of the disclosed methods, CD4+ and / or CD8+ T cells exhibit an increase in the release of cytokines selected from IFN-γ, TNF, and interleukins such as IL-2. Cytokine release can be measured by any method known in the art, such as using a West blot, ELISA, or immunohistochemical assay to detect the presence of cytokines released in a sample containing CD4+ and / or CD8+ T cells.

[0390] In some embodiments of the methods of the present disclosure, CD4+ and / or CD8+ T cells are effector memory T cells. In some embodiments of the methods of the present disclosure, CD4+ and / or CD8+ effector memory T cells are characterized by having CD44 high CD62L low expression. The expression of CD44 high CD62L low can be detected by any means known in the art, for example, by preparing a single cell suspension of tissue (such as cancer tissue), and using commercial antibodies for CD44 and CD62L for surface staining and flow cytometry. In some embodiments of the methods of the present disclosure, CD4+ and / or CD8+ effector memory T cells are characterized by having CXCR3 (also known as CXC chemokine receptor type 3, Mig receptor, IPIO receptor, G protein coupled receptor 9, interferon-induced protein 10 receptor, accession number NM_001504) expression. In some embodiments, CD4+ and / or CD8+ effector memory T cells are from peripheral blood. In some embodiments, CD4+ and / or CD8+ effector memory T cells are from tumors.

[0391] In some embodiments of the disclosed methods, compared to before the combination therapy is administered, an effective amount of a PI3K inhibitor and an ICM binding antagonist and optionally an adjuvant are administered to an individual characterized by elevated levels of inflammatory markers (e.g., CXCR3) on CD8+ T cells. CXCR3 / CD8+ T cells can be measured by any method known in the art. In some embodiments, CXCR3 / CD8~+ T cells are from peripheral blood. In some embodiments, CXCR3 / CD8+ T cells are from tumors.

[0392] In some embodiments of the methods of the invention, Treg function is suppressed compared to before administration of the combination. In some embodiments, T cell exhaustion is reduced compared to before administration of the combination.

[0393] In some embodiments, compared with before the administration of the combination, the number of Treg is reduced. In some embodiments, compared with before the administration of the combination, plasma IFN-γ levels are increased. The Treg number can be assessed by, for example, determining the percentage of CD4+Fox3p+CD45+ cells (e.g., by FACS analysis). In some embodiments, the absolute number of Treg in, for example, a sample is determined. In some embodiments, Treg is from peripheral blood. In some embodiments, Treg is from a tumor.

[0394] In some embodiments, compared with before the administration of the combination, T cell initiation, activation and / or proliferation increase. In some embodiments, T cells are CD4+ and / or CD8+ T cells. In some embodiments, T cell proliferation is detected by measuring the percentage of Ki67+CD8+ T cells (for example, by FACS analysis). In some embodiments, T cell proliferation is detected by measuring the percentage of Ki67+CD4+ T cells (for example, by FACS analysis). In some embodiments, T cells are from peripheral blood. In some embodiments, T cells are from tumors.

[0395] 5. Detection and Diagnosis Methods

[0396] According to the present invention, PD-1, TBET and EOMES can be used to assess T cell exhaustion as known in the art. T cells can be obtained from patient samples containing T cells, which are appropriately selected tissue samples, such as tumors and fluid samples, such as peripheral blood. In some embodiments, the sample is obtained before 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, whole blood includes immune cells, circulating tumor cells, and any combination thereof.

[0397] The presence and / or expression level / amount of a biomarker (e.g., any one or more of TBET and EOMES, collectively referred to herein as "T cell function biomarkers") can be qualitatively and / or quantitatively determined according to any suitable standard 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 / quantity of a biomarker in a first sample increases or increases compared to the presence / absence and / or expression level / amount of a biomarker in a second sample (e.g., before treatment with a therapeutic combination). In certain embodiments, the presence / absence and / or expression level / quantity of a biomarker in a first sample decreases or decreases compared to the presence and / or expression level / amount of a biomarker in a second sample. In certain embodiments, the second sample is a reference sample, a reference cell, a reference tissue, a control sample, a control cell, or a control tissue. Other disclosures for determining the presence / absence and / or expression level / amount of a gene are described herein.

[0398] In some embodiments of any of the methods, elevated expression refers to an 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 methods known in the art as described herein, compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, elevated expression refers to an increase in the expression level / amount of a biomarker in a sample, wherein the increase is at least about 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold the expression level / amount of the corresponding biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, increased expression refers to an overall increase of greater than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold compared to a reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., a housekeeping gene). In some embodiments of any method, decreased expression refers to an overall decrease 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)) detected by standard methods known in the art as 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 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, 0.1-fold, 0.05-fold, or 0.01-fold the expression level / amount of the corresponding biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

[0399] The presence and / or expression level / amount of various biomarkers in a sample can be analyzed by a variety of methods, many of which are known in the art and will be understood by those skilled 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 tests (such as 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, e.g., branched DNA, SISBA, TMA, etc.), RNA-Seq, FISH, microarray analysis, gene expression profiling and / or serial analysis of gene expression ("SAGE"), and any of a wide range of analyses that can be performed by protein, gene and / or tissue array analysis. Typical protocols for assessing the status of genes and gene products can be found 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 provided in Rules Based Medicine or Meso Scale Discovery ("MSD"), can also be used.

[0400] In some embodiments, a method is used to determine the presence and / or expression level / amount of a biomarker, the method comprising: (a) performing gene expression profiling, PCR (such as rtPCR or qRT-PCR), RNA-seq, microarray analysis, SAGE, MassARRAY technology, or FISH on a sample (such as a subject's cancer sample); and b) determining the presence and / or expression level / amount of the biomarker in the sample. In some embodiments, the microarray method comprises using a microarray chip having one or more nucleic acid molecules that can hybridize under stringent conditions with nucleic acid molecules encoding the above-mentioned genes, or having one or more polypeptides (such as peptides or antibodies) that can bind to one or more 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.

[0401] Methods for assessing mRNAs in cells are well known and include, for example, hybridization assays using complementary DNA probes (e.g., in situ hybridization using labeled riboprobes specific for one or more genes, Northern blotting, and related techniques) and various nucleic acid amplification assays (e.g., RT-PCR using complementary primers specific for one or more genes, as well as other amplification-type detection methods, such as branched DNA, SISBA, TMA, etc.).

[0402] Mammalian samples can be conveniently assayed for mRNA using Northern, dot blot, or PCR analysis. In addition, these methods can include one or more steps that allow determination of the level of target mRNA in a biological sample (e.g., by simultaneously examining the levels of a comparative control mRNA sequence for a "housekeeping" gene, such as an actin family member). Optionally, the sequence of the amplified target cDNA can be determined.

[0403] Optional methods include schemes for examining or detecting mRNA (such as target mRNA) in tissue or cell samples by microarray technology. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to produce cDNA probes. The probes are then hybridized with a nucleic acid array fixed on a solid support. The array is configured so that the order and position of each member of the array are known. For example, the selection of genes whose expression is associated with the clinical benefit of an increase or decrease in anti-angiogenic therapy can be arranged on a solid support. Hybridization of the labeled probe with a specific array member shows that the sample from which the probe comes expresses the gene.

[0404] According to some embodiments, the presence and / or expression level / amount is measured by observing the protein expression level of the above-mentioned genes. In certain embodiments, the method includes contacting the biological sample with a biomarker antibody as described herein (e.g., anti-PD-1 antibody, anti-PI3K antibody, anti-TBET antibody, antibody, anti-EOMES antibody) under conditions that allow biomarker binding, and detecting whether a complex is formed between the antibody and the biomarker. This method can be an in vitro or in vivo method. In some embodiments, one or more anti-biomarker antibodies are used to select subjects who are eligible for combined treatment with a PI3K inhibitor and an ICM binding antagonist.

[0405] In certain embodiments, IHC and staining protocols are used to examine the presence and / or expression level / amount of biomarker proteins in a sample. IHC staining of tissue sections has been shown to be a reliable method for determining or detecting the presence of proteins in a sample. In some embodiments, the expression of T cell function biomarkers in a sample from an individual is elevated protein expression, which is determined using IHC in a further embodiment. In one embodiment, a method is used to determine the expression level of a biomarker, the method comprising: (a) performing IHC analysis on a sample (e.g., a subject cancer sample) with 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 staining sample or a tissue sample from a non-cancerous patient).

[0406] In some embodiments, the expression of T cell function biomarkers is assessed on a tumor or tumor sample. As used herein, a tumor or tumor sample can include part or all of the tumor area occupied by tumor cells. In some embodiments, a tumor or tumor sample can also include a tumor area occupied by tumor-related intratumor cells and / or tumor-related matrix (e.g., adjacent peritumoral desmoplastic matrix). Tumor-related intratumor cells and / or tumor-related matrix may include an immune infiltration area (e.g., tumor-infiltrating immune cells as described herein) adjacent to and / or adjacent to the main tumor mass. In some embodiments, the expression of T cell function biomarkers is assessed on a tumor cell. In some embodiments, as described above, the expression of T cell function biomarkers is assessed on immune cells (e.g., tumor-infiltrating immune cells) in the tumor area.

[0407] In an alternative approach, the sample can be contacted with an antibody specific for the biomarker under conditions sufficient to form an antibody-biomarker complex, and the complex is then detected. The presence of the biomarker can be detected in a variety of ways, such as by Western blotting and ELISA procedures to analyze various tissues and samples, including plasma or serum. A variety of immunoassay techniques using this assay format are available, for example, see U.S. Patents Nos. 4,016,043, 4,424,279, and 4,018,653. These include single-site and two-site or "sandwich" assays of the non-competitive type, as well as traditional competitive binding assays. These assays also include direct binding of labeled antibodies to target biomarkers.

[0408] The presence and / or expression level / amount of selected T cell function biomarkers in a tissue or cell sample can also be examined by functional or activity-based assays. For example, if the biomarker is an enzyme (e.g., PI3K), assays known in the art (e.g., kinase assays) can be performed to determine or detect the presence of a given enzyme activity in a tissue or cell sample.

[0409] In certain embodiments, samples are normalized for differences in the amount of biomarkers assayed and for variability in the quality of the samples used and variability between assay runs. Such normalization can be achieved by detecting and combining the expression of certain normalizing biomarkers, including well-known housekeeping genes.

[0410] Alternatively, standardization can be based on the mean or median signal of all tested genes or most of its subsets (global normalization method). On a gene-by-gene basis, the measured standardized amount of subject tumor mRNA or protein is compared with the amount of the reference set. The standardized expression level of each mRNA or protein of each tested tumor of each subject can be expressed as a percentage of the expression level measured by the reference set. The presence and / or expression level / amount measured in the specific subject sample to be analyzed will fall within a certain percentile within this range, which can be determined by methods well known in the art.

[0411] In some embodiments, the sample is a clinical sample. In other embodiments, the sample is used for diagnostic determination. In some embodiments, the sample is obtained from a primary or metastatic tumor. Tissue biopsy is generally used to obtain representative tumor tissue. Alternatively, tumor cells can be obtained indirectly in the form of tissue or liquid that is known or believed to contain target tumor cells. For example, samples of lung cancer lesions can be obtained by resection, bronchoscopy, fine needle aspiration, bronchial brushing or from sputum, pleural fluid or blood. Genes or gene products can be detected from cancer or tumor tissue or other body samples (such as urine, sputum, serum or plasma). The same technology used to detect target genes or gene products in cancer samples can be applied to other body samples. Cancer cells may fall off from cancer lesions and appear in such body samples. By screening these body samples, simple early diagnosis of these cancers can be performed. In addition, by detecting target genes or gene products in these body samples, the progress of treatment can be more easily monitored.

[0412] In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or a combination of multiple samples from the same subject or individual that are obtained at one or more time points different from when the test sample was 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 when the test sample was obtained. Such a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be useful if the reference sample is obtained during the initial diagnosis of the cancer and the test sample is subsequently obtained when the cancer metastasizes.

[0413] In certain embodiments, reference sample, reference cell, reference tissue, control sample, control cell or control tissue is a combination of multiple samples from one or more non-subjects or individual healthy individuals. In certain embodiments, 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 condition (e.g., cancer) that are not a subject or individual. In certain embodiments, reference sample, reference cell, reference tissue, control sample, control cell or control tissue is a mixed RNA sample or mixed plasma or serum sample from one or more non-subjects or individual normal tissues. In certain embodiments, reference sample, reference cell, reference tissue, control sample, control cell or control tissue is a combined RNA sample or mixed plasma or serum sample from one or more individual tumor tissues that are not a subject or individual with a disease or condition (e.g., cancer).

[0414] 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., a biopsy). 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 nodes, and / or bone / bone marrow tissue. In some embodiments, the tissue sample is colon tissue. In some embodiments, the tissue sample is endometrial tissue. In some embodiments, the tissue sample is brain tissue (eg, glioblastoma, neuroblastoma, etc.).

[0415] In some embodiments, a tumor tissue sample (the term "tumor sample" is used interchangeably herein) may include part or all of a tumor region occupied by tumor cells. In some embodiments, a tumor or tumor sample may also include a tumor region occupied by tumor-associated intratumoral cells and / or tumor-associated stroma (e.g., adjacent peritumoral desmoplastic stroma). Tumor-associated intratumoral cells and / or tumor-associated stroma may include immune-infiltrating regions (e.g., tumor-infiltrating immune cells as described herein) immediately adjacent to and / or adjacent to the main tumor mass.

[0416] In some embodiments, tumor cell staining is expressed as a percentage of all tumor cells showing membrane staining of any intensity. Infiltrating immune cell staining can be expressed as a percentage of the total tumor area showing immune cells staining of any intensity. The total tumor area includes malignant cells and tumor-associated stroma, including areas of immune infiltration 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.

[0417] 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.

[0418] Solid tumors include any body tissue cancer except blood, bone marrow or lymphatic system.Solid tumors can be further divided into solid tumors of epithelial cell origin and solid tumors of non-epithelial cell origin.The example of epithelial cell solid tumors includes gastrointestinal tract, colon, colorectal (such as basal sample colorectal cancer), breast, prostate, lung, kidney, liver, pancreas, ovary (such as endometrioid ovarian cancer), head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gall bladder, labia, nasopharynx, skin, uterus, male reproductive organs, urinary organs (such as urothelial carcinoma, dysplastic urothelial carcinoma (dysplasticurothelium carcinoma) and transitional cell carcinoma), bladder and skin tumors.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 adenocarcinoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple negative breast cancer), gastric cancer, colorectal cancer, or hepatocellular carcinoma. In some embodiments, the cancer is a primary tumor. In some embodiments, the cancer is a metastatic tumor derived from any of the above types of cancer at a second site.

[0419] In some embodiments of any of the methods, the cancer displays human effector cells (e.g., is infiltrated by human effector cells). Methods for detecting human effector cells are well known in the art, including, for example, by IHC. In some embodiments, the cancer displays high levels of human effector cells. In some embodiments, the human effector cells are one or more of NK cells, macrophages, monocytes. In some embodiments, the cancer is any cancer described herein. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple-negative breast cancer), gastric cancer, colorectal cancer, or hepatocellular carcinoma.

[0420] In some embodiments of any of the methods, the cancer exhibits cells expressing FcRs (e.g., infiltrated by cells expressing FcR'). Methods for detecting FcRs are well known in the art, including, for example, by IHC. In some embodiments, the cancer exhibits high levels of cells expressing FcRs. In some embodiments, the FcR is an FcγR. In some embodiments, the FcR is activating an FcγR. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple-negative breast cancer), gastric cancer, colorectal cancer, or hepatocellular carcinoma.

[0421] In some embodiments, T cell function biomarkers are detected in a sample using a method selected from FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunodetection method, HPLC, surface plasmon resonance, 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, T cell function biomarkers are 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 a blood sample. In some embodiments, circulating immune cells are CD3+ / CD8+ T cells. In some embodiments, immune cells are isolated from a blood sample prior to analysis. Any suitable method for isolating / enriching such a cell population can 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 (e.g., CD3+ / CD8+ T cells) in blood samples.

[0422] Also provided herein are methods for monitoring the pharmacodynamic activity of an ICM binding antagonist therapy by measuring the expression level of one or more T cell function biomarkers as described herein in a sample comprising leukocytes obtained from a subject, wherein the subject has been treated with an ICM binding antagonist and a PI3K inhibitor, and wherein the one or more T cell function biomarkers are selected from TBET, PD-1, and EOMES, and determining that the therapy exhibits pharmacodynamic activity based on the expression level of the one or more T cell function biochemical markers in the sample obtained from the subject as compared to a reference, wherein an increase in the expression level of the one or more T cell function biochemical markers as compared to the reference indicates pharmacodynamic activity of the PD-1 antagonist therapy. These methods can also include measuring the expression level of one or more additional biomarkers of T cell function and / or cellular composition (e.g., the percentage of Tregs and / or the absolute number of Tregs; e.g., the number of CD8+ effector T cells), wherein the additional biomarkers of T cell function include cytokines, such as IFN-γ, T cell markers, or memory T cell markers (e.g., markers of T effector memory cells); and based on the expression level of one or more T cell function biomarkers, one or more additional T cell function biomarkers, and / or cellular composition in a sample obtained from the subject compared to a reference, determining that the treatment shows pharmacodynamic activity, wherein an increase in the expression level of one or more T cell function biomarkers, one or more additional T cell function biomarkers, and / or cellular composition compared to a reference indicates pharmacodynamic activity of the PD-1 antagonist treatment. The expression level of the biomarker and / or cellular composition can be measured by one or more methods described herein.

[0423] As used herein, "pharmacodynamic (PD) activity" can refer to the effect of a treatment (e.g., a combination of a PI3K inhibitor and an ICM binding antagonist treatment) on a subject. An example of PD activity can include modulating the expression level of one or more genes. Without wishing to be bound by theory, it is believed that in clinical trials examining PI3K inhibitors and ICM binding antagonists, it may be advantageous to monitor PD activity, for example, by measuring the expression of one or more T cell function biomarkers. For example, monitoring PD activity can be used to monitor response to treatment, toxicity, etc.

[0424] In some embodiments, the expression levels of one or more marker genes, proteins and / or cell compositions can be compared with a reference, which can include samples from subjects who have not received treatment (e.g., a combination of PI3K inhibitor treatment and ICM binding antagonists). In some embodiments, the reference can include samples from the same subject before receiving treatment (e.g., a combination of PI3K inhibitor treatment and ICM binding antagonists). In some embodiments, the reference can include reference values ​​for one or more samples from other subjects who received treatment (e.g., a combination of PI3K inhibitor treatment and ICM binding antagonists). For example, a group of patients can be treated and the average value, mean or median of one or more gene expression levels can be generated from the entire patient population. A group of samples obtained from cancers with common characteristics (e.g., the same cancer type and / or stage, or exposed to a common treatment, such as a combination of a PI3K inhibitor and an ICM-binding antagonist) can be studied from a population, such as a clinical outcome study. The set can be used to derive a reference value, such as a reference numerical value, with which the subject's sample can be compared. Any reference value described herein can be used as a reference for monitoring PD activity.

[0425] Certain aspects of the present disclosure relate to the measurement of the expression level 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, basal layer, and any other cell type 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.

[0426] Tumor samples can be obtained from a subject by any method known in the art, including but not limited to biopsy, endoscopy or surgery. In some embodiments, tumor samples can be prepared by freezing, fixing (e.g., by using formalin or similar fixatives) and / or embedding in paraffin. In some embodiments, tumor samples can be sliced. In some embodiments, fresh tumor samples (i.e., samples that have not yet been prepared by the above methods) can be used. In some embodiments, tumor samples can be prepared by incubating in a solution to maintain the integrity of mRNA and / or protein.

[0427] In some embodiments, the sample can be a peripheral blood sample. The peripheral blood sample can include white blood cells, PBMC, etc. Any technique known in the art for separating white blood cells from a peripheral blood sample can be used. For example, a blood sample can be drawn, red blood cells can be dissolved, white blood cell particles can be separated and used as a sample. In another example, density gradient separation can be used to separate white blood cells (such as PBMC) from red blood cells. In some embodiments, a fresh peripheral blood sample (i.e., a sample not yet prepared by the above method) can be used. In some embodiments, a peripheral blood sample can be prepared by incubating in a solution to maintain the integrity of mRNA and / or protein.

[0428] In some embodiments, responsiveness to treatment may refer to any one or more of the following: prolonging survival (including overall survival and progression-free survival); resulting in an objective response (including complete response or partial response); or improving signs or symptoms of cancer. In some embodiments, responsiveness may refer to an improvement in one or more factors used to determine the status of a tumor in a cancer patient according to the published RECIST guidelines, i.e., responding, stabilizing, or progressing. For a more detailed discussion of these guidelines, see Eisenhauer et al. (2009 Eur J Cancer 45: 228-47), Topalian et al. (2012 N Engl J Med 366: 2443-54), Wolchok et al. (2009 Clin Can Res 15: 7412-20), and Therasse et al. (2000 J. Natl. Cancer Inst. 92: 205-16). A responsive subject may refer to a subject whose cancer shows improvement (e.g., according to one or more factors based on RECIST criteria). A non-responsive subject can refer to a subject whose cancer does not show improvement (eg, according to one or more factors of RECIST criteria).

[0429] Conventional response criteria may not be sufficient to characterize the anti-tumor activity of the therapeutic agents of the present invention, which may produce a delayed response that may be preceded by initial significant radiographic progression, including the appearance of new lesions. Therefore, modified response criteria have been developed that take into account the possible appearance of new lesions and allow for confirmation of radiographic progression in subsequent assessments. Therefore, in some embodiments, responsiveness can refer to an improvement in one or more factors according to immune-related response criteria (irRC). For example, see Wolchok et al. (2009, supra). In some embodiments, new lesions are counted in the defined tumor burden and followed up, for example, to confirm radiographic progression in subsequent assessments. In some embodiments, the presence of non-target lesions is included in the assessment of complete remission and is not included in the assessment of radiographic progression. In some embodiments, radiographic progression can be determined based solely on measurable disease and / or can be confirmed by continuous assessment for ≥4 weeks from the date of first recording.

[0430] In some embodiments, responsiveness may include immune activation. In some embodiments, responsiveness may include therapeutic efficacy. In some embodiments, responsiveness may include immune activation and therapeutic efficacy.

[0431] 6. Test kit

[0432] In other aspects of the present invention, a therapeutic kit comprising a PI3K inhibitor and an ICM binding antagonist is provided. In some embodiments, the therapeutic kit further comprises instructions for use, the instructions for use comprising instructional materials for simultaneously administering the PI3K inhibitor and the ICM-binding antagonist to treat T cell dysfunction in individuals with cancer, or to enhance their immune function (e.g., immune effector function, T cell function, etc.), or to treat or delay cancer progression, or to treat infection in individuals. Any of the PI3K inhibitors and ICM binding antagonists described herein or known in the art can be included in the kit.

[0433] In some embodiments, the PI3K inhibitor and the ICM binding antagonist are in the same container or separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The container can be made of a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin) or metal alloy (such as stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and a label on or associated with the container can indicate instructions for use. The kit can also include other materials required from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts with instructional materials. In some embodiments, the kit also includes one or more other reagents (such as chemotherapeutic agents and anti-tumor agents). Suitable containers for one or more reagents include, for example, bottles, vials, bags, and syringes.

[0434] In other embodiments of the present invention, there is provided a diagnostic kit for determining biomarker expression, including T cell function biomarkers disclosed herein, which includes reagents that allow detection and / or quantification of biomarkers. Such reagents include, for example, compounds or materials, or compound or material groups that allow biomarkers to be quantified. In a specific embodiment, compounds, materials or compound or material groups allow determination of the expression level of genes (such as T cell function biomarker genes), including but not limited to extracting RNA materials, determining the level of corresponding RNA, primers for synthesizing corresponding cDNA, primers for amplifying DNA and / or probes that can specifically hybridize with gene-encoded RNA (or corresponding cDNA), TaqMan probes, proximity assay probes (proximity assay probe), ligases, antibodies, etc.

[0435] The kit may also optionally include appropriate reagents for detection labels, positive and negative controls, washing solutions, blotting membranes, microtiter plates, dilution buffers, etc. For example, a nucleic acid-based detection kit may include (i) T cell function biomarker polynucleotides (which may be used as positive controls), (ii) primers or probes that specifically hybridize to T cell function biomarker polynucleotides. It may also include enzymes suitable for amplifying nucleic acids, including various polymerases (reverse transcriptase, Tag, Sequenase TM, DNA ligase, etc., depending on the nucleic acid amplification technology used), deoxynucleotides and buffers to provide the reaction mixture required for amplification. Such kits will also typically include different containers for each individual reagent and enzyme and each primer or probe in an appropriate manner. Alternatively, a protein-based detection kit may include (i) a T cell function biomarker polypeptide (which can be used as a positive control), (ii) an antibody that specifically binds to a T cell function biomarker polypeptide. The kit may also have various devices (e.g., one or more) and reagents (e.g., one or more) for performing an assay described herein; and / or printed instructional materials for quantifying T cell function biomarker gene expression using the kit. The reagents described herein, which may optionally be associated with a detectable label, may be presented in the form of a microfluidic card, chip or chamber, microarray or kit, suitable for use in the embodiments or assays described below, such as the RT-PCR or Q PCR techniques described herein.

[0436] Suitable materials for packaging the components of the diagnostic kit may include crystals, plastics (polyethylene, polypropylene, polycarbonate, etc.), bottles, vials, paper, envelopes, etc. In addition, the kit of the present invention may include instructional materials for the simultaneous, sequential, or separate use of the various components contained in the kit. The instructional materials may be in the form of printed materials or in the form of electronic support capable of storing instructions so that the subject can read them, such as electronic storage media (disks, tapes, etc.), optical media (CD-ROMs, DVDs), etc.

[0437] Alternatively or additionally, the media may include an Internet address where instructional material is provided.

[0438] In order that the present invention may be readily understood and put into practical effect, particularly preferred embodiments will now be described by way of the following non-limiting experimental examples.

[0439] Example

[0440] Example 1

[0441] PI3K expression is associated with decreased cancer survival.

[0442] PI3KCA is part of the cytoplasmic / plasma membrane PI3KCA / AKT / mTOR signaling pathway (Hassan et al., 2013). PI3KCA mutational burden is associated with cancer progression through the AKT pathway, which plays a role in promoting invasion and metastasis (Jiang et al., 2020). Recent studies have implicated the PI3KCA signaling pathway in cancer stem cells (CSCs) and EMT (Chen et al., 2020; Xia et al., 2015). PI3KCA plays a role in immune evasion by phosphorylating AKT (P), inducing PDL1 expression and mediating immune evasion (Yang et al., 2017). 35% of breast cancer patients have mutation-positive tumors (Fusco et al., 2021), and approximately 6% of TNBC patients harbor PI3KCA mutations (Mosele et al., 2020). This does not explain recurrence or immunotherapy resistance in most patients.

[0443] The inventors used the web tool KM plotter (Lanczky and Gyorffy, 2021) to plot the overall survival probability of low and high PI3K protein expression ( Figure 1 ). This analysis showed that in a cohort of patients with metastatic breast cancer and non-small cell lung cancer, the survival rate of the PI3K high expression cohort was significantly reduced. This analysis aimed to prove that PI3K enrichment was associated with a significantly reduced survival probability in multiple cancer types.

[0444] Enrichment of PI3K CSC and CD8 T cell signatures in liquid biopsies of patients based on liquid biopsy stratification and PI3K-targeted CSC recurrence characteristics and exhausted T cell characteristics .

[0445] The inventors identified a dual CSC and T cell signature in liquid biopsies from patients with stage IV solid tumors (i.e., melanoma, NSCLC, RCC, HCC, breast cancer, GBM). Using our novel liquid biopsy platform, we identified a novel, PI3K-enriched cancer stem cell, stem cell-like mesenchymal signature in stage IV cancer patients ( Figure 2 These CSCs are key cell types for cancer recurrence and are not targeted by chemotherapy, immunotherapy, or radiotherapy (i.e., conventional cancer treatments). However, the inventors hypothesized that these patients would respond to the monotherapy and / or combination therapy of the present invention. These CTCs are positive for mesenchymal markers, CSC markers CSV and ABCB5.

[0446] The inventors also identified a novel T cell exhaustion / dysfunction signature enriched in checkpoint proteins (TIM3, TIGIT, PD1) and exhaustion markers (TOX1, EOMES).

[0447] The analysis showed 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 3 A). In contrast, EpCAM is a marker for non-mesenchymal epithelial cells and its expression changes little ( Figure 3 A). Population dynamics analysis using the digital pathology system showed that the CSV and ABCB5-positive cell populations decreased by approximately 56%, while EpCAM increased by 38% ( Figure 3 B). Inhibition of PI3KCA by GDC-0084 also suppressed the percentage of AKT1-positive cells (AKT1 contributes to mesenchymal metastatic characteristics) as well as overall AKT1 expression (AKT1 is a key tumorigenic marker regulated by PI3KCA). This suggests that targeting PI3KCA also targets key regulators of metastasis, mesenchymal transition, and cancer stem cell (CSC) characteristics.

[0448] Liquid biopsy-based assays from stage IV solid tumor cancer patients suggest that PI3K inhibition suppresses T cell dysfunction Characteristics of the inhibitory and inductive effects

[0449] The overall expression intensity of CD8+ T cells was measured by immunofluorescence analysis using the digital pathology system of the inventor's laboratory. The depletion group showed that GDC-0084 significantly inhibited the expression of EOMES and PD1 in NS and ST samples ( Figure 4 A). The immune checkpoint inhibitor group showed that GDC-0084 treatment significantly inhibited TIM3 in both NS and ST CD8+ T cells, while GDC-0084 only significantly inhibited TIGIT in NS CD8+ T cells ( Figure 4 B). Figure 4 C shows the effector signature group, and Figure 4 In contrast, Figures A and 4B show that the expression of perforin and GZNb was significantly increased in GDC-0084-treated CD8+ T cells in the NS and ST groups.

[0450] PI3K is enriched in cancer cell lines with enriched mesenchymal CSC signatures .

[0451] Whole cell intensity analysis was performed using an ASI digital pathology system according to standardized settings. Analysis of PI3KCA expression in breast cancer cell lines ( Figure 5 A) Reveals a pattern of increased expression as cell lines transition from an epithelial identity to a more mesenchymal, metastatic, and immunotherapy-resistant profile (MCF7 is a breast cancer epithelial cell line with a low CSC population, MDA-MB-231 is a TNBC cell line with >90% mesenchymal CSCs, MDRA-MB-231-BR is a brain-metastatic cancer clone of MDA-MB-231 with >90% mesenchymal stem cells, and finally 4T1 is an immunotherapy-resistant, metastatic, highly aggressive breast cancer model with >90% CSCs).

[0452] Interestingly, similar patterns were observed in H1299 (epithelial lung cancer), CT26 (IO-responsive, epithelial colon cancer), and LLC (highly aggressive, metastatic lung cancer model) ( Figure 5 B) H1299 and CT26 are both epithelial-like, immunogenic cell lines, while LLC is highly resistant to immunotherapy and exhibits aggressive behavior. Overall, these data demonstrate that PI3KCA is enriched in metastatic, drug-resistant cancer cell lines based on whole-cell expression. This highlights the importance of targeting PI3KCA for the treatment of metastatic, drug-resistant cancers, a significant unresolved problem.

[0453] MCF7 epithelial breast cancer cell line;

[0454] MDAMB231 mesenchymal / CSC breast cancer cell line;

[0455] MDAMB231-Br, the brain cancer version of MDA-MB-231;

[0456] 4T1 highly invasive, IO-resistant mouse breast cancer model;

[0457] H1299: epithelial lung cancer cell line;

[0458] CT26: an epithelial-like, highly immunogenic mouse colon tumor cell line; and

[0459] LLC: A highly drug-resistant Lewis lung cancer cell line that is resistant to immunotherapy (Li et al., 2021).

[0460] Effects of PI3K inhibitor GDC-0084 on migration and invasion of MDA-MB-231 and CT26 cells .

[0461] 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; however, there are currently no approved drugs for these patients. GDC-0084 is a PI3K inhibitor that is a brain-penetrant agent that has shown promising activity in preclinical models of glioblastoma. GDC-0084 has a high IC of 1. 25 (1.25 μM) and IC 50 (2.5μM) inhibits breast cancer cell migration, IC 25 concentration has a greater inhibitory effect on the migration of MDA-MB231 cells ( Figure 6 ). Wound healing assays showed that CT26 cell migration was inhibited after treatment with GDC-0084 (PI3K inhibitor). DMSO treatment was used as a control. These data showed that 0.3 μM of GDC-0084 had a more significant effect on wound healing compared to a lower concentration (i.e., 0.2 μM). Figure 6 C). This indicates that GDC-0084 can inhibit cancer cell migration at higher concentrations. GDC-0084 has the greatest inhibitory effect on the responsive colon cancer cell line CT26. The inventors used DMSO treatment as a control.

[0462] Determination of the IC of GDC-0084 against different cancer cell lines 50 .

[0463] The data clearly show that GDC-0084 EC 50 The individual dose-response curves for CT26, 4T1, MDA-MB-231, and MDA-MB-231-Brm are shown in Figure 2. Figure 7 Shown is MCF7. These data were obtained to demonstrate the efficacy of GDC-0084 targeting PI3KCA in immunotherapy responders or epithelial cancer cell lines (CT26, MCF7) compared to immunotherapy resistant or mesenchymal cancer cell lines (4T1, MDA-MB-231 cell lines). These data demonstrate that GDC-0084 targeting PI3K significantly inhibits the proliferation of epithelial and mesenchymal cancer cell lines.

[0464] Effects of PARP inhibition on mesenchymal and CSC characteristics.

[0465] Olaparib and veliparib are PARP inhibitors that have been used to treat certain types of breast and ovarian cancer. However, olaparib does not target cancer stem cells (CSCs) or mesenchymal features, nor does it affect ABCB5 or ALDH1A ( Figure 8 The inventors next investigated the effects of olaparib and veliparib on CSC characteristics or proliferation of the mesenchymal TNBC cell line MDA-MB-231, and neither drug inhibited CSC characteristics or proliferation ( Figure 9 Next, to further investigate the importance of PARP monotherapy targeting, the inventors investigated siRNA knockdown of PARP ( Figure 10 ) on mesenchymal and CSC characteristics. It was identified that PARP knockout had no inhibitory effect on these markers of mesenchymal and CSC characteristics ( Figure 11 Combination treatment with the PI3K pathway inhibitor GDC-0084 and the PARP inhibitor olaparib limited the migration of MDA-MB-231 cells ( 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 inhibitory effect on MDA-MB-231 migration. This suggests that PARP inhibitors are not effective monotherapy drugs but may be beneficial in combination with PI3K inhibitors.

[0466] αPD1 therapy in the CT26 colon cancer model and the 4T1 breast cancer model.

[0467] The inventors sought to demonstrate that αPD1 monotherapy resulted in a significant reduction in tumor volume in the immunotherapy-responsive CT26 colon cancer model compared to the 4T1 breast cancer model ( Figure 12 These data demonstrate that CT26 is an immunotherapy-responsive line, while the 4T1 model clearly demonstrates that 4T1 is an immunotherapy-resistant model, as evidenced by its primary tumor burden response to αPD1 immunotherapy.

[0468] PI3K inhibitors target and suppress the expression of mesenchymal resistance markers

[0469] Next, the inventors demonstrated that treatment with GDC-0084 significantly suppressed CSC / mesenchymal markers, induced the expression of the epithelial marker EpCAM, and also increased the expression of the viral mimicking / immune visibility marker MDA5 ( Figure 13 and 14 This suggests that targeting PI3KCA could also suppress metastasis and therapy resistance characteristics and render tumors more immune-visible and therefore more responsive to combination treatment with immunotherapies.

[0470] Next, the inventors conducted further inhibition analysis on different inhibitors targeting PI3K to confirm the effectiveness of targeting PI3K in inhibiting mesenchymal CSC characteristics. Two different PI3K inhibitors ( Figure 15 ), idelalisib has been used to treat certain blood cancers, such as relapsed chronic lymphocytic leukemia, and LY294002, a strong PI3K inhibitor, is currently being tested in patients with relapsed or progressive HNSCC and patients with mutations in PI3KCA and / or PI3K pathway genes. Figure 15 Treatment with two different low doses of idelalisib significantly suppressed CSC / stem-like cancer recurrence markers CSV, SoX9, ABCB5, and SNAIL. The effect was least pronounced at the 12.5 μM dose of idelalisib. Furthermore, both 25 μM doses of idelalisib and both concentrations of LY294002 induced expression of the epithelial marker EpCAM. This data suggests that targeting PI3K with an alternative inhibitor to GDC-0084 is effective in suppressing mesenchymal cancer stem cell characteristics and inducing epithelial characteristics.

[0471] Administration of 7.5 mg / kg of GDC-0084 reduced clinical abnormalities in the 4T1 syngeneic tumor model .

[0472] Next, the inventors demonstrated that monotherapy with 7.5 mg / kg of GDC-0084 reduced the clinical abnormalities observed in the 4T1 breast cancer model. The group of mice receiving the high dose (i.e., 15 mg / kg) showed multiple cases of decreased activity, hunched posture, and pronounced piloerection ( Figure 16Administration of GDC-0084 at 7.5 mg / kg reduced the clinical abnormalities previously observed at higher doses compared to 15 mg / kg. While 40% of mice treated with 15 mg / kg GDC-0084 exhibited decreased activity, hunched posture, piloerection, and weight loss, no clinical abnormalities were observed in the 7.5 mg / kg treatment group. These data highlight the reduced toxicity observed with the lower dose.

[0473] GDC-0084 at a dose of 7.5 mg / kg eliminated tumor burden .

[0474] The inventors next demonstrated that "low-dose" GDC-0084 treatment (7.5 mg / kg) abolished primary tumor burden in the 4T1 breast cancer model (see Figure 17 In the 4T1 breast cancer model, GDC-0084 monotherapy at 7.5 mg / kg and in combination with αPD1 significantly reduced tumor volume, ultimately reducing tumor weight by more than 50%. Surprisingly, no significant reduction in tumor burden was observed in mice treated with 15 mg / kg GDC-0084, with or without αPD1.

[0475] GDC-0084 administered at a dose of 7.5 mg / kg reduced tumor inflammation, including monocyte and neutrophil infiltration. Run .

[0476] Administration of a “low dose” of GDC-0084 (i.e., 7.5 mg / kg) reduced tumor inflammation, including monocyte and neutrophil infiltration ( Figure 18 GDC-0084 monotherapy at 7.5 mg / kg and in combination with αPD1 significantly reduced tumor inflammation associated with the 4T1 breast cancer model. While tumor inflammation scores were "moderate" and "severe" in the control and αPD1-treated groups, only mild inflammation was observed when GDC-0084 was co-administered at a dose of 7.5 mg / kg.

[0477] In addition, pathologists noted a significant reduction in myeloid cell populations, including monocytes and neutrophils, in the group treated with 7.5 mg / kg GDC-0084. Notably, no significant reduction in inflammation was observed in mice given 15 mg / kg GDC-0084 daily.

[0478] GDC-0084 reduced splenomegaly at a dose of 7.5 mg / kg in the 4T1 syngeneic tumor model .

[0479] GDC-0084 administered at 7.5 mg / kg reduced splenomegaly in the 4T1 breast cancer model. Spleen size and appearance were normal at low doses of GDC compared to high doses ( Figure 19 Administration of 7.5 mg / kg of GDC reduced the splenomegaly commonly associated with the 4T1 breast cancer model.

[0480] While there were no significant differences in spleen weights between the 15 mg / kg treated groups and the corresponding control groups, a significant reduction in splenomegaly was observed at a lower dose of GDC-0084 (7.5 mg / kg), both as monotherapy and in combination with αPD1.

[0481] GDC-0084 administration at a dose of 7.5 mg / kg reduced the expression of SOX9 and TOX1 .

[0482] Combination therapy in both experiments significantly reduced the expression of SOX9 (a marker of mesenchymal, therapy-resistant cancer stem cells), however, GDC-0084 at 7.5 mg / kg had a more pronounced effect in combination therapy and was particularly effective as a monotherapy. Interestingly, 15 mg / kg GDC-0084 was shown to induce TOX1 (a marker of T cell exhaustion and dysfunction) in CD8+ T cells, while 7.5 mg / kg significantly reduced TOX1 in CD8+ T cells. Figure 20 ). These data suggest that "high-dose" GDC-0084 induced dysregulated inflammation and increased T cell dysfunction (higher expression of TOX1) and failed to control SOX9, a marker of cancer stem cell-like mesenchymal characteristics, metastasis, and cancer recurrence. In contrast, "low-dose" GDC-0084 significantly suppressed SOX9+ expression as monotherapy or in combination therapy and significantly reduced TOX1 expression in CD8+ T cells. Notably, the combination of anti-PD1 and "low-dose" GDC-0084 (7.5 mg) was the most effective in eliminating TOX1 expression ( Figure 20 ).

[0483] PI3K inhibition, either alone or in combination, targets mesenchymal characteristics and induces an epithelial phenotype in an IO-resistant mouse model.

[0484] The combination of GDC-0084 and anti-PD-1 significantly inhibited PI3KCA, the mesenchymal marker CSV, and EGFR, and this combination also better induced the expression of the epithelial marker E-cadherin ( Figure 21 ).

[0485] Both GDC-0084 monotherapy and combination therapy containing GDC-0084 and anti-PD-1 significantly induced the cell population positive for the epithelial marker E-cadherin (see Figure 22 GDC-0084 with or without concomitant αPD1 treatment had no effect on mouse body weight in the 4T1 syngeneic tumor model ( Figure 17 The combination of GDC-0084 and αPD1 significantly suppressed tumor burden, but high-dose monotherapy did not, while low-dose monotherapy was able to suppress tumor burden ( Figure 17). Notably, this combination also worked in the 4T1 immunotherapy-resistant tumor model. Combination treatment with GDC-0084 and anti-PD1 inhibitors significantly inhibited PI3KCA and cancer stem cell-like mesenchymal markers CSV and EGFR, and induced the expression of the epithelial marker E-cadherin ( Figure 21 Strikingly, GDC-0084 monotherapy or combination therapy with GDC-0084 and anti-PD1 inhibitors significantly induced a cell population positive for the epithelial marker E-cadherin ( Figure 22 These data suggest that PI3KCA inhibition combined with immunotherapy can significantly impact tumor burden and reprogram cancer cell or tumor microenvironment characteristics to be more epithelial and responsive to treatment compared to immunotherapy alone. This suggests that this is a viable therapeutic approach to enhance durable responses to immunotherapy.

[0486] PI3K monotherapy or combination therapy induces effector and TRM characteristics .

[0487] Combination therapy is superior to single-drug therapy and significantly increases the infiltration of CD8+IFNγ+ T cells into tumors ( Figure 23 A). In addition, among the total CD8 T cell population, the combination therapy group expressed T cells significantly higher than that of the PI3K inhibitor monotherapy group. RM The proportion of CD8+ T cells with markers (i.e., CD44, CD103, CD69) also increased significantly, and its induction effect was stronger ( Figure 23 B). Combination therapy also significantly increased the infiltration of CD8+IFNγ+ T cells into tumors ( Figure 23 A). In addition, in the total CD8 T cell population, the combined group expressed T RM The proportion of CD8+T cells with markers (i.e., CD44, CD103, CD69) also increased significantly ( Figure 23 B) This assay was performed to understand whether PI3K inhibition, either monotherapy or in combination with immunotherapy, reprograms immune cell signatures beyond those of cancer cells. Combination therapy significantly impacts and induces immune effector signatures, enhancing anti-tumor immunity.

[0488] PI3K inhibition alone or in combination with other therapies characterizes checkpoint exhaustion in CD8+ T cells .

[0489] Compared with PD1 or GDC-0084 monotherapy alone, the combination of GDC-0084 and anti-PD1 inhibitors significantly reduced the number of TIM3 and LAG3 positive CD8+ T cells ( Figure 24 This assay was performed to investigate whether the characteristics of dysfunctional immune CD8+ T cells were reprogrammed. The analysis showed that the combination therapy significantly reduced the dysregulated immune characteristics.

[0490] Combination of a PI3K inhibitor and immunotherapy abolishes metastatic spread in the 4T1-IO tolerant model .

[0491] Combination therapy of GDC-0084 and αPD1 significantly suppressed metastatic tumor burden, but neither agent alone could. This combination was also effective in the 4T1 immunotherapy-resistant tumor model ( Figure 25 These data suggest that PI3K inhibition combined with immunotherapy can significantly affect tumor burden compared to immunotherapy alone and is able to reprogram cancer cells or tumor microenvironmental features to become more responsive to treatment in an immunotherapy-resistant mouse breast cancer model (4T1) and eliminate metastatic spread of cancer to the lungs. This suggests that this is a viable therapeutic approach that can enhance durable responses to immunotherapy.

[0492] discuss

[0493] The inventors have obtained a new method that can reduce the overall toxicity of PI3K inhibitors (such as GDC-0084) and improve the overall efficacy of treatment. This is achieved by administering a reduced amount of GDC-0084, namely 7.5 mg (the normal dose is a single 15 mg), which reduces the overall adverse side effects and improves the overall efficacy of GDC-0084 therapy. In addition, the inventors found that the efficacy of PI3K inhibitors in combination with immunotherapy was also enhanced. The therapeutic administration of low concentrations of GDC-0084 in combination with immunotherapy can further reduce the dose of immunotherapy (such as anti-PD1), thereby reducing adverse events and improving the overall efficacy with a significantly reduced dose.

[0494] Summary of the efficacy of monotherapy and combination therapy

[0495] Lower doses of monotherapy inhibiting checkpoint proteins ( Figure 4 :TIGIT, Tim3, PD1) and induce effector characteristics ( Figure 4 ). Reprogramming Exhaustion Signature (EOMES) Figure 4 .

[0496] Lower doses of monotherapy resulted in significantly fewer clinical abnormalities ( Figure 12 ).

[0497] Lower doses of monotherapy GDC-0084 were able to significantly suppress tumor weight ( Figure 13 ).

[0498] However, combination therapy with lower doses showed sustained tumor control. In addition, the low-dose combination therapy was more effective in suppressing inflammation ( Figure 14 and Figure 15 ).

[0499] Lower doses of the combination therapy were more effective in suppressing cancer stem cells (important for relapse and metastasis), reprogramming and suppressing dysfunctional T cell characteristics, and inducing effector T cell characteristics ( Figure 16 , 19-20).

[0500] Compared with "high dose" or "effective dose", "low dose" combined with immunotherapy was more effective in inducing epithelial characteristics ( Figure 17-18 The combined therapy is also superior in inhibiting metastasis ( Figure 21 ).

[0501] Administration of 15 mg / kg represents a high dose of GDC-0084 (i.e., in the clinic, humans are typically administered 20-60 mg), and our low dose is only 50% of that dose. 25% of the original dose is also expected to be effective as a single agent and in combination.

[0502] Materials and Methods

[0503] Unless otherwise stated, all materials and reagents used to synthesize and test the compositions were purchased from Sigma-Aldrich, Novabiochem, Abeam, and the American Type Culture Collection (ATCC).

[0504] In vivo animal studies.

[0505] Five female BALB / c mice were used for xenograft studies in each treatment group. 4T1 breast cancer cells (1×10 5 ) were injected into the fourth pair of mammary fat pads in the right inguinal region of six-week-old BALB / c nude mice. For 4T1 tumor cell injection, 1×10 5 cells.

[0506] For the CT26 colon cancer mouse model, 5 × 10 5 10 cells were injected into the abdomen of 6-week-old BALB / c nude mice. Tumor growth was measured three times a week by caliper. On days 0 and 5, mice were treated with 10 mg / kg anti-PD-1 intraperitoneal injection. Tumor volume (1 / 2 (length × width)) was measured using a digital caliper. 2 )) and expressed as mean ± SEM.

[0507] Six-week-old female Balb / c mice were purchased from the Animal Resource Center (ARC) and acclimatized 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. The inoculation site was shaved and 1 × 10 mice in 100 μl of PBS were placed in the inoculation site. 54T1 cells were injected subcutaneously into the right mammary gland of mice. When the tumor reached approximately 100 mm 3 Treatment was started at 4 hr. Tumors were measured using external calipers and the modified ellipse formula 1 / 2 (a / b 2 ) where a = longest side and b = shortest side. For the combination therapy experiments, mice were treated with the indicated doses of vehicle (normal saline) or GDC-0084 (oral gavage) and co-treated with anti-PD1 or isotype control (10 mg / kg) twice a week by intraperitoneal injection. Mice were monitored daily for clinical abnormalities (decreased activity, hunched posture, piloerection, weight loss, and metastasis), and tumor volume and body weight were measured three times a week. Once the tumors in the vehicle group reached their maximum limit (1000 mm 3 ), all tumors and related metastatic organs (lung, liver, spleen) were harvested, weighed, imaged, and fixed in 4% paraformaldehyde for relevant analysis.

[0508] WST-1 cell proliferation assay .

[0509] Adherent cell lines CT26, 4T1, and MCF7 were seeded in 96-well flat-bottom tissue culture plates at optimized cell densities (100, 100, 3000, and 300 cells per well, respectively) in a total volume of 100 μl / well in triplicate. The cells were allowed to adhere overnight at 37°C and 5% CO2. The cells were then treated with a series of inhibitors at different concentrations and placed in an incubator at 37°C and 5% CO2 for 72 hours, after which the culture medium was removed and replaced with 100 μl / well of WST-1 cell proliferation reagent (Sigma-Aldrich, 11644807001) in complete cell culture medium at a final dilution of 1:10. The WST-1 reagent contains a water-soluble tetrazolium salt that is cleaved by cell mitochondrial dehydrogenases to produce formazan. The amount of formazan produced in each reaction was then quantified using a microplate spectrophotometer to measure metabolic activity. The absorbance measured is directly related to the number of metabolically active cells in the culture. The absorbance at 450 nm was recorded at 0.5, 1, and 2 hours of incubation (after a 30-second mixing time) using a microplate spectrophotometer. The percentage of proliferation was calculated by subtracting the mean absorbance of the background control (blank) from the sample absorbance. The EC value for each inhibitor was determined using GraphPad Prism 8 software as log(inhibitor) versus response-variable slope (four parameters). 50 value.

[0510] Scratch wound healing assay

[0511] In 96 cells In Image Lock plates, adherent breast cancer cell line MDA-MB-231 was seeded in triplicate at an optimized cell density of 2500 cells per well in low serum medium (2%) in a total volume of 100 μl / well. The cells were allowed to adhere overnight at 37°C and 5% CO2. The cells were checked to be 100% confluent to ensure consistent wounding. After 24 hours, the cells were stained using a 96-well needle. Wounds were made with the WoundMaker Tool. After wounding, cells were washed with low serum medium to remove non-adherent cells, and then treated with a series of inhibitors (IC 25 and IC 50 ) treatment. Then place the plate in Live cell analysis system, using The Zoom live cell analysis device scans every six hours to track wound healing.

[0512] In vitro characterization of PI3K inhibition in metastatic cancer

[0513] 4T1 or CT26 cancer cell lines were treated with control or GDC-0084 (at concentrations of 0.2 μM or 0.3 μM), 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 X-100 and stained with mouse primary antibodies against CSV or EpCAM, rabbit primary antibodies against EGFR, FOXN2, or MDA5, or goat primary antibodies against ABCB5, 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. Comparative analysis was performed for fluorescence intensity of EGFR, cytoplasmic fluorescence intensity (CFI) of CSV, or global fluorescence intensity of ABCB5. Data were plotted using PRISM, and one-way analysis of variance was performed using Kruskal-Wallis. Significant differences are plotted.

[0514] OPAL tissue microscopy.

[0515] Automated staining was performed using the OPAL staining kit for the automated BONDRX platform according to the manufacturer's instructions. Proteins were then mounted and localized. Protein targets were localized by digital pathology laser scanning microscopy. Single 0.5 μm sections were acquired using an ASI digital pathology microscopy system with a 100x oil immersion objective running ASI software. The final image was obtained by averaging four consecutive images of the same section. Digital images were analyzed using automated ASI software (Applied Spectral Imaging, Carlsbad, CA), which automatically determined distribution and intensity using automatic thresholding and background correction of mean fluorescence intensity, allowing specific targeting of target protein expression.

[0516] Method I for isolating CTCs from liquid biopsies of patients with stage IV metastatic cancer.

[0517] Liquid biopsies from cancer patients were enriched for circulating tumor cells (CTCs) using our optimized laboratory protocol, either with CD45+ cell depletion or without depletion, to isolate PBMCs from liquid biopsies. In short, our optimized laboratory protocol involves storing whole blood in EDTA tubes for circulating tumor cell identification. RosetteSep TM The Human CD45 Depletion Cocktail is used to enrich tumor cells (CTCs) from whole blood by depleting CD45+ cells. Targeted depletion of unwanted cells is achieved using a tetrameric antibody complex that recognizes CD45, CD66b, and glycophorin A on red blood cells (RBCs). TM Unwanted cells were removed by centrifugation over PBS (Catalog #07801). Purified tumor cells were then extracted as a highly enriched population from the interface between the plasma and the buoyant density medium and harvested in PBS containing 20% ​​FBS.

[0518] The samples were then stained for CSV (a mesenchymal, metastatic marker of CTCs), ABCB5 (a cancer stem cell marker and chemotherapy resistance marker), and EpCAM (an epithelial cell marker). Coverslips were mounted on glass microscope slides using ProLong nucblueclearAntifade 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 100x oil-immersion objective running ASI software. The final image was obtained by averaging four consecutive images of the same section. Population dynamics analysis was performed using ASI software, and total cell counts and fluorescence intensity (protein expression) analysis were performed using ImageJ software (ImageJ, National Institutes of Health, Bethesda, MD, USA).

[0519] Method II for isolating CTCs from liquid biopsies of patients with stage IV metastatic cancer

[0520] Using our optimized laboratory protocol (as described above under Liquid Biopsy Method I), liquid biopsies from cancer patients were treated with CD45+ cell depletion to enrich circulating tumor cells (CTCs) in the liquid biopsy. The samples were then stained for CSV (a mesenchymal and metastatic marker of CTCs), ABCB5 (a cancer stem cell marker and a chemoresistance marker), and PI3K (a kinase). Coverslips were mounted on glass microscope slides using ProLong nucblue clearAntifade reagent (Life Technologies). Protein targets were localized by confocal laser scanning microscopy. Single 0.5-μm images were acquired using an ASI digital pathology system using a 40x oil immersion objective running ASI software. 5 μm sections. Final images were obtained by averaging four consecutive images of the same section. Population dynamics were analyzed using ASI software, and total cell counts and fluorescence intensity (protein expression) were analyzed using ImageJ software (ImageJ, National Institutes of Health, Bethesda, MD, USA).

[0521] Example 2

[0522] Determining the optimal therapeutic dose for efficacy of PI3K-mTOR inhibitors .

[0523] To determine the optimal therapeutic dose of the GDC-0084 PI3K-mTOR inhibitor, the inventors used the highly aggressive, immunotherapy-resistant 4T1 breast cancer model. 3 ), GDC-0084 combined with αPD1 immunotherapy ( Figure 26 A). The experiments were performed sequentially, with the initial study using a high starting dose of GDC at 15 mg / kg daily + / - αPD1. This was followed by a dose-decreasing study in which GDC doses were administered from 1.875 mg / kg to 15 mg / kg daily in divided doses 4 hours apart, in combination with αPD1; and finally a single dose-decreasing study in which GDC doses of 3.75 and 7.5 mg / kg daily were administered as a single dose, in combination with αPD1 ( Figure 26 B).

[0524] When used in combination with immunotherapy, GDC-0084 administered at a high daily dose of 15 mg / kg significantly reduced tumor volume by 69% ( Figure 26 C, Figure 1 ). Fractionated dose experiments showed that when GDC was used at doses of 15 mg / kg and 7.5 mg / kg, the tumor volume was reduced accordingly when used in combination with anti-PD1 ( Figure 26C, Figure 2 In the final single-dose-tapering study, GDC at a lower dose of 7.5 mg / kg combined with αPD1 reduced primary tumor volume to the same extent as the higher dose ( Figure 26 C, Figure 3 ), therefore 7.5 mg / kg was confirmed as the optimal therapeutic dose.

[0525] Reducing the dose of GDC-0084 may prevent toxic complications .

[0526] The inventors next sought to further investigate the toxicity-related complications of GDC-0084 treatment by monitoring the body and liver weights of mice in initial dose, fractionated dose, and single dose reduction experiments ( Figure 27 Mice treated with a high daily dose of 15 mg / kg GDC-0084αPD1 experienced weight loss after day 4 of treatment, ultimately leading to death in both the starting dose and the dose-escalating studies ( Figure 27 A, Figure 1 and 2 However, at the optimal therapeutic dose of 7.5 mg / kg, mice maintained their body weight throughout the experiment and no mice died ( Figure 27 A, Figure 2 and 3 ).

[0527] Liver enlargement or hepatomegaly is associated with organ damage. To determine whether GDC-0084 treatment induced hepatotoxicity, the inventors measured liver weight at harvest ( Figure 2 B). GDC-0084 administered at a high daily dose of 15 mg / kg increased liver weight by approximately 20% by the end of treatment ( Figure 27 B, Figure 1 In contrast, no difference in liver weight was observed when the lower dose of 7.5 mg / kg was administered ( Figure 27 B, Groups 2 and 3). Taken together, our data demonstrate that GDC-0084 administered at the optimal therapeutic dose of 7.5 mg / kg is free of toxicity-related complications.

[0528] Reducing the dose of PI3K-mTOR inhibitors can reduce liver inflammation .

[0529] To evaluate the changes in liver pathology after GDC treatment, H&E-stained liver sections were examined ( Figure 28 Changes in inflammation, extramedullary hematopoiesis, and hepatocellular injury were scored using established scoring criteria: 1 = mild, 2 = moderate, or 3 = severe for each parameter. Importantly, reducing the dose of GDC-0084 to 7.5 mg / kg significantly reduced liver inflammation ( Figure 28 A, Figure 2Treatment with GDC-0084 at the optimal therapeutic dose also reduced extramedullary hematopoiesis, which is typically associated with the 4T1 model ( Figure 28 B, Figure 2 Finally, supporting our liver weight data, GDC-0084 treatment at a high daily dose of 15 mg / kg induced hepatocellular injury, confirming hepatotoxicity ( Figure 28 C, Figure 1 Notably, no significant changes were observed in hepatocytes at the optimal therapeutic dose of GDC-0084 ( Figure 28 C, Figure 2 ).

[0530] GDC-0084αPD1 treatment reduces 4T1-associated splenomegaly and extramedullary hematopoiesis .

[0531] In addition to extramedullary hematopoiesis, enlarged spleen or splenomegaly is often associated with the 4T1 model. To examine changes in the spleen after GDC-0084 treatment, the inventors measured spleen weight in split-dose and single-dose taper experiments. In both administration regimens, administration of GDC-0084 at the lower dose of 7.5 mg / kg reduced splenomegaly ( Figure 29 A). In addition, spleen pathology evaluation also showed that extramedullary hematopoiesis was significantly reduced after treatment with the optimal therapeutic dose of 7.5 mg / kg GDC-0084 ( Figure 29 B, Figure 2 ).

[0532] GDC-0084 inhibits lung leukocyte infiltration after αPD1 treatment.

[0533] Given the above data indicating that GDC-0084αPD1 treatment can reduce lung metastasis, the inventors next sought to evaluate changes in lung immune cell populations after GDC-0084 treatment. Lung pathological examination showed that at all concentrations, including the optimal therapeutic dose of 7.5 mg / kg, leukocyte infiltration of the lungs was significantly reduced ( Figure 30 This emphasizes that GDC-0084 treatment affects not only primary tumors and metastases, but also inflammation.

[0534] GDC-0084αPD1 treatment reduces lymph node metastasis.

[0535] In addition to lung metastasis, the 4T1 model is also associated with lymph node metastasis. To assess lymph node metastasis after GDC-0084 treatment, independent expert pathologists examined H&E-stained lymph node sections and assigned a score: 1 = 1-3 tiny sites (width / length << 0.5 mm); 2 = 1-3 sites, at least one of which was 0.5-1 mm in diameter; or 3 = 2-3 or more sites, all of which were visible to the naked eye with leukocyte infiltration and / or hemorrhage. In the absence of GDC-0084 treatment, large areas of metastatic tumor growth were observed, but these areas were not visible when immunotherapy was combined with GDC-0084 (7.5 mg / kg) ( Figure 31 A). Therefore, the lymph node metastasis score of mice treated with GDC-0084αPD1 was significantly reduced, confirming that GDC-0084 inhibited not only lung metastasis but also lymph node metastasis ( Figure 31 B).

[0536] PI3K-mTOR treatment reduced primary tumor volume.

[0537] To determine whether GDC-0084 showed efficacy when combined with the PARP inhibitor, olaparib, the inventors then used the 4T1 model of TNBC. In the initial experiment, GDC-0084 was administered before or after olaparib, and then the administration was repeated in a second experiment based on the optimal administration schedule ( Figure 32 A). GDC-0084 was administered orally daily with an optimal therapeutic dose of 7.5 mg / kg, and olaparib was injected intraperitoneally daily ( Figure 32 B). GDC-0084 treatment before and after PARP inhibition significantly reduced tumor volume by 53% and 60%, respectively ( Figure 32 C, Figure 1 The inventors then confirmed these results in a second experiment using an optimal administration schedule of GDC-0084 30 minutes after olaparib ( Figure 32 C Figure 2 Taken together, these data suggest that GDC-0084 may be effective when combined with PARP inhibitors in addition to immunotherapy, thus enhancing its utility.

[0538] Combination therapy with GDC-0084 and a PARP inhibitor did not cause toxicity.

[0539] To examine complications related to toxicity, the inventors monitored body and liver weights of mice in PARP inhibitor experiments ( Figure 33 At the optimal therapeutic dose of 7.5 mg / kg, GDC-0084 did not change the body weight of mice when administered after olaparib ( Figure 33 A). Again, no changes in liver weight were observed, confirming no toxicity issues ( Figure 33 B).

[0540] Combination treatment with GDC-0084 and olaparib suppressed lung and liver inflammation.

[0541] Given the effects of GDC-0084 and immunotherapy on inflammation, the inventors next sought to examine liver and lung pathology in their PARP inhibitor studies. When administered after olaparib at a dose of 7.5 mg / kg, GDC-0084 significantly reduced liver inflammation and extramedullary hematopoiesis ( Figure 34 A). Lung pathology examination also highlighted a significant reduction in leukocytosis in the lungs of mice treated with GDC-0084 and olaparib ( Figure 34 B), demonstrating that GDC-0084 has an effect not only on primary tumors but also on inflammation at metastatic sites.

[0542] Finally, spleen pathology evaluation demonstrated that, in addition to spleen weight, extramedullary hematopoiesis was reduced after GDC-0084 and olaparib treatment ( Figure 35 A and 35B).

[0543] PI3K-mTOR inhibitors reduce cancer cell proliferation.

[0544] The inventors then sought to compare the efficacy of PI3K-mTOR inhibitors such as GDC-0084 and therefore performed proliferation assays using a large number 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), as well as the PI3K-mTOR inhibitors: omipalisib, apitolisib, dactorisib, and GDC-0084 (p110α, β, δ, γ, mTOR inhibitors) Figure 36 PI3K-mTOR inhibitors had the most significant effect on the proliferation of MDA-MB-231 cells, among which omiliximab, apitolisib and dactolisib had the highest IC values. 50 The values ​​were all below 1.0 μM (Table 16). In contrast, the IC 50 The value was 2.5 μM. The more general PI3K inhibitor showed significantly higher values, confirming that inhibition of PI3K and mTOR is crucial in preventing cancer cell proliferation.

[0545] Table 16

[0546]

[0547]

[0548] Not determined

[0549] Inhibition of PI3K and mTOR affects cancer cell migration.

[0550] To determine the effect of PI3K-mTOR inhibition on cancer cell migration, the inventors performed a scratch assay using the human breast cancer cell line MCF-7 pre-stimulated with PMA-TGFβ to induce a mesenchymal phenotype ( Figure 37 A). In the GDC-0084 dose response experiment, approximately 50% inhibition was observed at concentrations of 1.25 μM and 2.5 μM after 24 hours of treatment ( Figure 37 B).

[0551] Similarly, to compare the efficacy of GDC-0084 with other PI3K-mTOR inhibitors, the inventors used the IC 50 A second study was conducted on apitolisib, dactolisib, and omipalisib at different concentrations ( Figure 38 A). Similar to GDC-0084, all PI3K-mTOR inhibitors significantly reduced wound healing density ( Figure 38 B), demonstrated that inhibition of both PI3K and mTOR impedes cell migration.

[0552] Materials and Methods

[0553] Animal studies

[0554] Female BALB / c mice aged 6–8 weeks were purchased from the Animal Resource Center (ARC). After collection, the mice underwent a one-week acclimation period in an isolation room at the QIMRB Berghofer Institute for Medical Research (QIMRB). All experimental procedures were performed in accordance with the guidelines and regulations approved by the QIMRB Ethics Committee. 1 x 10 5 4T1 cells were administered into the mammary fat pad of BALB / c mice. When the tumors reached approximately 50-100 mm 3 In the 4T1 model, mice were treated with the indicated doses of GDC orally daily and combined with intraperitoneal injection of anti-PD-1 therapy (10 mg / kg) on ​​days 0 and 4 or daily olaparib (50 mg / kg). Tumors were measured using an external caliper and the modified ellipse formula of 1 / 2 (a / b 2 ) where a = longest side and b = shortest side. Mice were monitored daily for clinical abnormalities (reduced activity, hunched posture, piloerection, weight loss, and metastasis) and tumor volume was measured three times a week. Once the tumors in the vehicle group reached their maximum size (1000 mm 3), all tumors and related metastatic organs (lung, liver, spleen) were harvested, weighed, imaged, and fixed in 4% paraformaldehyde for relevant analysis.

[0555] Pathological analysis

[0556] Independent expert pathologists performed pathological analysis of H&E-stained FFPE livers, lungs, lymph nodes, and spleens. Changes in inflammation, extramedullary hematopoiesis (EMH), hepatocytes, and pulmonary leukocytosis were scored as 1 = mild, 2 = moderate, or 3 = severe. Lymph node sections were scored as 1 = 1-3 microscopic sites (<0.5 mm in width / length); 2 = 1-3 sites, at least one of which was 0.5-1 mm in diameter; or 3 = 2-3 or more sites, all macroscopically visible without leukocytic infiltration and / or hemorrhage.

[0557] WST-1 cell proliferation assay .

[0558] 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 / well. The cells were allowed to adhere overnight at 37°C and 5% CO2. The cells were then treated with the specified dose of PI3K inhibitor and placed in an incubator at 37°C and 5% CO2 for 72 hours, after which the culture medium was removed and replaced with 100 μL / well of WST-1 cell proliferation reagent (Sigma-Aldrich, 11644807001), diluted to 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 cellular mitochondrial dehydrogenases to produce formazan. The amount of formazan produced in each reaction was then quantified using a microplate spectrophotometer to measure metabolic activity. The measured absorbance is directly correlated to the number of metabolically active cells in the culture. The absorbance was recorded at 450 nm using a microplate spectrophotometer at 0.5, 1, and 2 hours of incubation (after a 30-second mixing time). The percentage of proliferation was calculated by subtracting the mean absorbance of the background control (blank) from the sample absorbance. The IC value of each inhibitor was determined using GraphPad Prism 8 software as log(inhibitor) versus response-variable slope (four parameters). 50 value.

[0559] Scratch wound healing assay.

[0560] Before plating, MCF7 cells were stimulated with PMA / TGFβ for 24 hours and then plated in 96-well plates at an optimized cell density. In Image Lock plates, the total volume per well was 100 μL, using low serum medium (2%). The cells were allowed to adhere overnight at 37°C and 5% CO2. The cells were checked to ensure that they were 100% confluent to ensure consistent wounding. After 24 hours, the cells were stained using a 96-well needle. Wounds were created using the WoundMaker Tool. After wounding, 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. The plate was then placed Live cell analysis system using Incucyte TM The Zoom live cell analysis device scans every four hours to track wound healing.

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Claims

1. A method for altering epithelial-to-mesenchymal transition or mesenchymal-to-epithelial transition in tumor cells that overexpress PI3K, 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 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). The method according to claim 1 or 2 , wherein the PI3K-overexpressing cells are CSCs. The method according to claim 3 , wherein the PI3K-overexpressing cells are CSC tumor cells. 5 . The method according to claim 1 , wherein the PI3K-overexpressing cells express one or more mesenchymal / cancer stem cell markers selected from 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 expressing one or more of TIM3, TIGIT, PD1, TOX1, and EOMES.

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

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

10. The method of claim 9, wherein the ICM antagonist is a PD1 antagonist, a PD-L1 antagonist, a CTLA4 antagonist, or a PD-L2 antagonist. The method of claim 10 , wherein the ICM antagonist is an antigen binding molecule (eg, an antibody).

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

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

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

15. The method according to any one of claims 1 to 14, wherein the PI3K inhibitor is selected from paxalisib (GDC-0084), idelalisib, LY294002, 3-methyladenine, apellisib, quercetin, wortmannin, GNE-490, PI3K-IN-36, 740Y-P, AZD-7648, bupanisib, inarlisib, daclisib, cupanisib, eganelisib, pitilisib, SAR405, duvilisib, taselisib, recilisib, YM-201636, omipalisib, PI-103, α-linolenic acid, ferminostat and isorhamnetin.

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

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

18. Use of PI3K inhibitors and immunotherapies that do not target cancer stem cells (CSCs) for treating T cell dysfunction in individuals with cancer, or for enhancing immune function (e.g., immune effector function, T cell function, etc.), for treating or delaying the progression of cancer, or for treating the recurrence of cancer.

19. Use of PI3K inhibitors and immunotherapies that do not target cancer stem cells (CSCs) in the preparation of a medicament for treating T cell dysfunction in an individual with cancer, or for enhancing their immune function (e.g., immune effector function, T cell function, etc.), for treating or delaying the progression of cancer, or for treating the recurrence of cancer.

20. The use according to claim 18 or 19, wherein the PI3K inhibitor and the immunotherapy are formulated for simultaneous administration.

21. Use of PI3K inhibitors, immunotherapies that do not target cancer stem cells (CSCs), and adjuvants (e.g., chemotherapeutic agents) for treating or adjuvanting the treatment of T cell dysfunction in individuals with cancer, or for enhancing immune function (e.g., immune effector function, T cell function, etc.), for treating or delaying the progression of cancer, or for treating the recurrence of cancer.

22. Use of PI3K inhibitors, immunotherapies that do not target cancer stem cells (CSCs), and adjuvants (e.g., chemotherapeutic agents) in the preparation of medicaments for treating or adjuvanting T cell dysfunction in individuals with cancer or for enhancing their immune function (e.g., immune effector function, T cell function, etc.), for treating or delaying the progression of cancer, or for treating cancer recurrence.

23. The use according to claim 21 or 22, wherein the PI3K inhibitor, immunotherapy and adjuvant (eg chemotherapeutic agent) are formulated for simultaneous administration.

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 in a sample obtained from the subject prior to simultaneous administration (e.g., relative to the levels of TIM3, TIGIT, PD1, TOX1, and EOMES in activated T cells).

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 in a sample obtained from the subject prior to simultaneous administration (e.g., relative to the levels of TIM3, TIGIT, PD1, TOX1, and EOMES in activated T cells).

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

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

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

29. The kit of claim 28, further comprising instructions for use comprising guiding material for administering the first drug and the second drug simultaneously to treat a T cell dysfunction disorder in an individual suffering from cancer, or to enhance their immune function (e.g., immune effector function, T cell function, etc.), to treat or delay the progression of cancer in the individual, or to treat cancer recurrence in the individual.

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

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

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

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

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