CN120569200A - Compositions and methods for improving cancer treatment - Google Patents

Abstract

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

Description

Compositions and methods for improving cancer treatment

Technical Field

The present invention relates generally to compositions and their use in the treatment of cancer. More particularly, the present invention relates to compositions and their use in altering one of epithelial to mesenchymal cell transformation of tumor cells or mesenchymal to epithelial cell transformation.

Background

Any reference in this specification to a prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as, an acknowledgement or any form of suggestion that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Phosphatidylinositol is one of many phospholipids found in cell membranes and plays an important role in intracellular signal transduction. Cell signaling through 3' -phosphoinositides is involved in a variety of cellular processes, such as malignant transformation, growth factor signaling, inflammation and immunization (Rameh et al (1999) J.biol Chem, 274:8347-8350). The enzymes responsible for the production of these phosphorylated signal products, phosphatidylinositol 3-kinases (also known as PI 3-kinases or PI 3K), were originally identified as having activity associated with viral oncoproteins and growth factor receptor tyrosine kinases that phosphorylate Phosphatidylinositol (PI) and its phosphorylated derivatives at the 3' -hydroxy group of the inositol ring (Panayotou et al (1992) TRENDS CELL Biol 2:358-60).

Phosphatidylinositol 3-kinase (PI 3K) is a lipid kinase that phosphorylates lipids at the 3-hydroxy residue of the inositol ring (Whitman et al (1988) Nature, 332:664). The 3-phosphorylated phospholipids (PIP 3) produced by PI3 kinases act as second messenger recruiting kinases with lipid binding domains, including Plekstrin Homology (PH) regions, such as Akt and phosphoinositide-dependent kinase-1 (PDK 1). Binding of Akt to membrane PIP3 results in translocation of Akt to the plasma membrane, contacting Akt with PDK1, PDK1 responsible for activating Akt. Tumor-inhibiting phosphatase PTEN dephosphorylates PIP3 and thus acts as a negative regulator of Akt activation. PI 3-kinase 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).

The major PI3K subtype in cancer is the class I PI 3-kinase p110α (alpha) (as described in U.S. patent nos. 5824492;5846824; 62774327). Other subtypes have been implicated in cardiovascular and immunoinflammatory disorders (Workman P (2004) Biochem Soc Trans 32:393-396; patel et al (2004)Proceedings ofthe American Association ofCancer Research(Abstract LB-247)95thAnnual Meeting,March 27-31,Orlando,Florida,USA;Ahmadi K and Waterfield MD(2004)Encyclopedia ofBiological Chemistry(Lennarz W J,Lane M D eds)ElsevierAcademic Press).PI3K/Akt/PTEN pathways are attractive targets for cancer drug development because such modulators or inhibitors are expected to inhibit proliferation in cancer cells, reverse inhibition of apoptosis in cancer cells, and overcome resistance of cancer cells 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).

However, despite many clinical studies investigating the treatment of many solid tumors with PI3K inhibitors, no drug candidate has entered the clinic. Thus, further work is needed to successfully utilize these promising candidate drugs to improve efficacy and reduce toxicity for these challenging diseases.

Disclosure of Invention

The present invention is based on the discovery that PI3K inhibitors have significant activity in inhibiting EMT, inhibiting the formation and maintenance of Cancer Stem Cells (CSCs), and inducing mesenchymal transition to epithelium (MET), which makes them useful in the treatment of a range of cancers (e.g., solid tumors), including recurrent cancers.

In one aspect of the invention, there is provided a method of altering epithelial to mesenchymal cell transformation or mesenchymal to epithelial cell transformation of PI 3K-overexpressing cells comprising contacting PI 3K-overexpressing cells with a composition comprising a PI3K inhibitor and an immunotherapy that does not target Cancer Stem Cells (CSCs).

In another aspect, the invention provides a method of treating or preventing cancer in a subject, wherein the cancer comprises at least one PI3K overexpressing cell, the method comprising administering to the subject a composition comprising a PI3K inhibitor and an immunotherapy that does not target Cancer Stem Cells (CSCs). The expression of one or more biomarkers CSV, EGRF, ABCB, soX9, SNAIL, AKT1, epCAM, MDA5, TIM3, TIGIT, PD1, TOX1, EOMES, and E-cadherein in a subject can be screened prior to administration of the composition to the subject.

In some embodiments, the PI3K overexpressing cell is a CSC. In some embodiments, the PI3K overexpressing cell is a CSC tumor cell.

In some embodiments, the PI3K overexpressing cells express one or more mesenchymal/cancer stem cell markers selected from CSV, EGRF, and ABCB5, soX, SNAIL, and AKT1.

Epithelial cells are characterized by expression of one or both of EpCAM and MDA 5. Furthermore, in some embodiments, the subject comprises a cd8+ T cell population that expresses one or more of TIM3, TIGIT, PD1, TOX1, and EOMES. Typically, the epithelial cells express an epithelial cell marker comprising E-cadherin.

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

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

For example, the PARP inhibitor may be suitably selected from the group consisting of olaparib (Olaparib), tazopanib (talazoparib), veliparib (veliparib), nilaparib (niraparib), and Lu Kapa ni (rucaparib).

Typically, the PI3K inhibitor is a catalytic PI3K inhibitor. As illustrative examples, PI3K inhibitors may be selected from paxalisib (GDC-0084), idarubicin, LY294002, 3-methyladenine, apicalis, quercetin (quercetin), wortmannin, GNE-490, PI3K-IN-36, 740Y-P, AZD-7648, bupirinotecan, irairiser (inavolisib), daclizumab (dactolisib), coupannix (Copanlisib), eganelisib, pitelist, SAR405, dulcitol, taselisib, recilisib, YM-201636, ox Mi Pali sibutran (omipalisib), PI-103, alpha-linolenic acid, feminostat, and isorhamnetin.

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

In another aspect of the invention there is provided the use of PI3K inhibitors and immunotherapy that does not target Cancer Stem Cells (CSCs) for treating a T cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) in a cancer individual, for treating or delaying progression of cancer, or for treating recurrence of cancer.

In another aspect, the invention provides the use of PI3K inhibitors and immunotherapy that does not target Cancer Stem Cells (CSCs) in the manufacture of a medicament for treating a T cell dysfunctional disorder in an individual suffering from cancer, or for enhancing their immune function (e.g., immune effector function, T cell function, etc.), for treating or delaying progression of cancer, or for treating recurrence of cancer.

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

In another aspect, the invention provides the use of PI3K inhibitors, immunotherapy and adjuvants that do not target Cancer Stem Cells (CSCs) (e.g., chemotherapeutic agents) for treating or aiding in the treatment of a T cell dysfunction disorder in an individual suffering from 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.

In another aspect, the invention provides the use of PI3K inhibitors, immunotherapy and adjuvants (e.g., chemotherapeutic agents) that do not target Cancer Stem Cells (CSCs) in the manufacture of a medicament for treating or adjunctively treating a T cell dysfunction disorder in an individual having cancer, or for enhancing immune function (e.g., immune effector function, T cell function, etc.), for treating or delaying progression of cancer, or for treating cancer recurrence.

In some preferred embodiments, the PI3K inhibitor, immunotherapy, and adjuvant (e.g., chemotherapeutic agent) are formulated for simultaneous administration.

Suitably, the use may further comprise the step of detecting an increase in the level of one or more of TIM3, TIGIT, PD1, TOX1 and EOMES in T cells in a sample obtained from the subject (e.g., relative to the level of TIM3, TIGIT, PD1, TOX2 and EOMES in activated T cells) prior to simultaneous administration.

In another embodiment, the invention provides a kit comprising a medicament comprising a PI3K inhibitor and optionally a pharmaceutically acceptable carrier, and instructions for use of the material for simultaneous administration of the medicament with another medicament comprising an immunotherapy that does not target Cancer Stem Cells (CSCs) and optionally a pharmaceutically acceptable carrier, for treating a T cell dysfunction disorder in a subject suffering from cancer, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) thereof, for treating or delaying progression of cancer, or for treating cancer recurrence in a subject.

In yet another embodiment, the invention provides a kit comprising a medicament comprising an immunotherapy that does not target Cancer Stem Cells (CSC), optionally a pharmaceutically acceptable carrier, and instructions for use comprising instructional material for administering the medicament concurrently with another medicament comprising a PI3K inhibitor and optionally a pharmaceutically acceptable carrier, for treating a T cell dysfunctional disorder in an individual having cancer, or for enhancing immune function (e.g., immune effector function, T cell function, etc.), for treating or delaying progression of cancer, or for treating cancer recurrence in an individual.

In another aspect of the invention, a kit is provided comprising a first medicament comprising a PI3K inhibitor and optionally a pharmaceutically acceptable carrier and a second medicament comprising an immunotherapy that does not target Cancer Stem Cells (CSCs) and optionally a medically acceptable carrier for treating a T cell dysfunctional disorder in an individual suffering from cancer, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) thereof, for treating or delaying progression of cancer, or for treating cancer recurrence in an individual. Suitably, the kit further comprises instructions for use comprising instructional material for simultaneously administering the first and second medicaments, for treating a T cell dysfunctional disorder in an individual having cancer, or for enhancing immune function (e.g., immune effector function, T cell function, etc.) thereof, for treating or delaying progression of cancer, or for treating cancer recurrence in an individual.

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

In some embodiments, the PI3K inhibitor is GDC-0084 in a unit dose equivalent to a dose of about 11.25mg/kg or less administered to a subject. Suitably, the PI3K inhibitor may be GDC-0084 in a unit dose equivalent to a dose of about 7.5mg/kg or less administered to a subject. Also contemplated as PI3K inhibitors is GDC-0084, the unit dose corresponding to a dose of about 4mg/kg or less administered to a subject.

In some embodiments, the composition further comprises immunotherapy that does not target Cancer Stem Cells (CSCs).

In some embodiments, the immunotherapy is selected from ICM antagonists (e.g., PD1 antagonists, PDL1 antagonists, CTLA4 antagonists, etc.) or PARP inhibitors.

Drawings

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

FIG. 1 is a KM survival diagram providing phase IV metastatic cancer survival PI3K protein expression.

Figure 2 liquid biopsies from cancer patients with immunotherapy tolerance or immunotherapy response cohorts were treated to isolate PBMCs from liquid biopsy samples using our optimized laboratory protocol. Samples were then stained for CSV, PI3KCA, ABCB 5.

FIG. 3 determination of Fluorescence Intensity (FI) and population dynamics (percent cell population) in liquid biopsied CTCs from stage IV metastatic solid tumor patients using ASI digital pathology for CSV, ABCB5, epCAM, PI3KCA, AKT1 (AKT 1 plays a role in promoting invasion and metastasis), CTCs were treated with GDC-0084 or vector controls. (A) The percentage population of CSV, ABCB5 two mesenchymal markers and PI3KCA/AKT1 (B) CSV+ABCB5+ or EpCAM+ cells or PI3KC/AKT1 are shown.

FIG. 4 liquid biopsies of patients with stage IV metastatic solid tumors were treated to isolate PBMC from liquid biopsy samples using our optimized laboratory protocol. The samples were then stimulated (ST: stimulated with PMA/CaI for 4 hours in the presence of Bu Lei Feier dine A) or Not Stimulated (NS) and/or treated with GDC-0084 inhibitor. The samples were then stained for three separate sets of markers. Depletion profile index group EOMES, PD1 and CD8. Checkpoint group TIGIT, TIM3 and CD8. Perforin, GZNb and CD8.

FIG. 5 (A) PI3KCA marker staining of MCF7 (epithelial breast cancer), MDAMB231 (mesenchymal/CSC breast cancer), MDAM B231-Br (brain cancer version of MDA-MB-231) and 4T1 (highly invasive, immune treatment resistant mouse breast cancer) cell lines, and (B) H1299 (epithelial-like lung cancer), CT26 (epithelial-like, highly immunogenic mouse colon tumor) and (LLC) highly resistant Lewis lung cancer cell lines. Samples were infiltrated with Triton X-100, stained with mouse primary antibodies to CSV, rabbit primary antibodies to PI3KCA, and detected with donkey AF anti-mouse 568, anti-rabbit 647.

FIG. 6 shows the effect of the PI3K inhibitor GDC-0084 on MDA-MB-231 and CT26 cell migration. Wound healing (scratch test) the effect of GDC-0084 on cell migration was analyzed, where the images represent different cell lines treated with DMSO control, GDC-0084 at IC ₂₅ concentration of 1.25. Mu.M, and GDC-0084 at IC ₅₀ concentration of 2.5. Mu.M. (C) Graphical representation of relative wound density of MDA-MB-231 treated with PI3K inhibitor. Black bars, DMSO control, light gray bars, IC ₂₅ concentration 1.25 μm, dark gray bars, IC ₅₀ concentration 2.5 μm. Error bars correspond to mean ± standard deviation of eight replicates. P values were calculated using a two-way anova with Tukey multiple comparison test, where ns represents no significance, ns=p >0.05, P <0.01, P <0.001, and P <0.0001.

FIG. 7 shows the IC ₅₀ of GDC-0084 against different cancer cell lines. In the dose-response design, WST-1 cell proliferation reagents were added to CT26, 4T1 and MCF7 cells pretreated with PI3K inhibitor for 72 hours. Cell proliferation was measured indirectly by formazan formation and absorbance at 450nm was recorded. The percent proliferation (%) was determined as described in the methods before EC ₅₀ was calculated using GRAPHPAD PRISM software.

Figure 8 shows that olaparib treatment upregulated some drug resistance and mesenchymal markers. MDA-MB-231 cells are treated with a solvent ("CTRL") or Olaparib ("olap"). The expression of cancer stem cell-like drug resistance markers (ABCB 5, ALDH 1A) was quantitatively analyzed by immunofluorescent staining. Images show IF staining of (a) ABCB5 and (B) ALDH1A1 and show up-regulation of ABCB5, ALDH1A1 expression. * P is less than or equal to 0.001. The figure shows that PARP inhibition can up-regulate cancer stem cell resistance signature markers.

FIG. 9 shows that Olaparib treatment failed to inhibit CSCs or cell proliferation by treating MDA-MB-231 cells with Olaparib for 24 hours. Cells were collected and stained with APC/CD44 and PE/CD24 followed by flow cytometry. (A) The percent inhibition of CSCs compared to the control group is shown, followed by representative flow cytometry images. (B) MDA-MB-231 cells were treated with three different concentrations of either Olaparib or Taraxazopali and cell proliferation was assessed using the WST-1 assay.

Figure 10 provides a graphical and photographic representation of PARP knockout. PARP1 knockdown was performed with two different concentrations of PARP1 siRNA, 5nM and 10nM, where 5nM is the optimal concentration that we laboratory predetermine in other cell lines. After 48 hours of incubation with siRNA, cells were flash frozen at-80 ℃. After 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 graph shows the mean ± standard error of three independent experiments. * P≤0.05, p≤0.01, p≤0.001, P <0.0001, bi-directional analysis of variance.

Figure 11 shows that PARP1 knockout increased expression of resistance markers. MDA-MB-231 cells were incubated with 5nM PARP1 siRNA for 48 hours, then RNA was extracted or cDNA was synthesized, and then amplified by qPCR, or cells were fixed for immunofluorescent staining. (a, B) mRNA expression, (C, D) immunofluorescence intensity of mesenchymal markers after PARP1 knockout, (E) PD-L1. Shows the relative fold change in mRNA expression or the change in Total Nuclear Fluorescence Intensity (TNFI) or Total Cytoplasmic Fluorescence Intensity (TCFI). * P≤0.05, p≤0.01, p≤0.001, P <0.0001, bi-directional analysis of variance. Wound healing (scratch test) the effect of GDC-0084 in combination with olaparib on cell migration was analyzed. The images represent MDA-MB-231 cell lines treated with (F) a DMSO control, (G) GDC-0084 at an IC ₂₅ concentration of 1.25. Mu.M and 5nM Olaparib (H) IC ₅₀ at a concentration of 2.5. Mu.M GDC-0084 and 250nM Olaparib. Error bars correspond to mean ± standard deviation (I) of eight replicates. P values were calculated using a two-way anova with Tukey multiple comparison test, where ns represents no significance, ns=p >0.05, P <0.01, P <0.001, and P <0.0001.

FIG. 12 shows αPD1 treatment in colon cancer CT26 model and 4T1 breast cancer model. Treatment regimens using the Balb/c CT26 (A) colon cancer model and the 4T1 (B) breast cancer model have been demonstrated. Tumor volume of mice treated with solvent (IgG) or αpd1 (10 mg/kg) (n=5/group). * p <0.05, < p <0.01, unpaired t-test.

FIG. 13 PI3K inhibits target and inhibits mesenchymal drug resistance marker expression. The 4T1 (A) or CT26 (B) cancer cell lines were treated with control or GDC-0084 (at two low concentrations of 0.2 or 0.3. Mu.M). Samples were then infiltrated with Triton-X100, stained with primary mouse antibodies to CSV or EpCAM, primary rabbit antibodies to EGFR or FOXN2, or primary goat antibodies to ABCB5, and detected with secondary donkey AF antibodies to mouse 488, anti-rabbit 568, or anti-goat 647. The ASI digital pathology system using a 100-fold objective lens images a protein target. An example of an image field of view with a scale (orange) is depicted above. The fluorescence intensity of EGFR, the Cytoplasmic Fluorescence Intensity (CFI) of CSV or the overall fluorescence intensity of ABCB5 were analyzed for comparison. Data were plotted using PRISM and single-factor analysis of variance was performed using Kruskal-Wallis. Significant differences are plotted.

FIG. 14 PI3K inhibition induces immune visibility markers. The 4T1 (A) or CT26 (B) cancer cell lines were treated with control or GDC-0084 (0.2 or 0.3mM concentration). Samples were then permeabilized with Triton-X100, 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. The ASI digital pathology system using a 100-fold objective lens images a protein target. An example of an image field of view with a scale (orange) is depicted above. The fluorescence intensity was analyzed by comparison. Data were plotted using PRISM and single-factor analysis of variance was performed using Kruskal-Wallis. Significant differences are plotted.

Fig. 15 pi3k inhibition targets and inhibits stem cell-like cancer stem cell cancer recurrence characteristics and induces epithelial characteristics. MDA-MB-231TNBC cancer cell lines were treated with a control or two different PI3K inhibitors, idalrisin (two low concentrations 12.5. Mu.M or 25. Mu.M) or LY294002 (two low concentrations 2.5. Mu.M or 5. Mu.M). Samples were then infiltrated with Triton-X100 and stained with mouse primary antibodies to CSV or EpCAM, rabbit primary antibodies to SoX (SoX is a marker for mesenchymal stem cells and progressive, treatment resistant cancer cells) or PI3K or goat primary antibodies to ABCB5 or SNAIL (11A and 11B), and detected with donkey AF anti-mouse 488, anti-rabbit 568 or anti-goat 647 secondary antibodies. The ASI digital pathology system using a 40-fold objective was used to image protein targets. An example of an image field of view with a scale (orange) is depicted above. Comparative analyses were performed on the nuclear fluorescence intensity of SoX, the Cytoplasmic Fluorescence Intensity (CFI) of CSV and EpCAM, or the overall fluorescence intensity of ABCB5 or SNAIL. Data were plotted using PRISM and single-factor analysis of variance was performed using Kruskal-Wallis. Significant differences are plotted.

FIG. 16 shows that 7.5mg/kg of GDC-0084 administration reduced clinical abnormalities in 4T 1-homologous tumor models. GDC-0084 therapy in a 4T1 breast cancer model. Treatment regimen using Balb/c 4T1 breast cancer model. GDC-0084 (B) was administered daily at 7.5mg/kg (A) or 15 mg/kg. (C) At the end of GDC-0084 monotherapy, the percentage of mice with clinical abnormalities (reduced activity, humpback posture, erectile hair, weight loss and metastasis) occurred.

FIG. 17 tumor burden was eliminated by administration of GDC-0084 at a dose of 7.5 mg/kg. Representative images of tumors harvested from mice administered (A) 7.5mg/kg GDC-0084 (C) 15mg/kg +/- αPD1 in Balb/C4T 1 breast cancer model. (B, D) primary tumor volume and final tumor weight of mice treated with (B) 7.5mg/kg GDC-0084 (D) 15mg/kg once daily +/- αPD1 (10 mg/kg). Data are expressed as mean ± SEM. One-way anova with Tukey post-hoc analysis, p <0.001, p <0.0001 (n=5/group).

FIG. 18 shows that GDC-0084 administration at a dose of 7.5mg/kg reduced tumor inflammation, including mononuclear and neutrophil infiltration. In the Balb/c 4T1 breast cancer model, mice administered either (A) 7.5mg/kg GDC-0084 or (B) 15mg/kg once daily +/- αPD1 were given representative images of H & E stained tumors and pathology scores of tumor inflammation. Data are expressed as mean ± SEM. One-way anova with Tukey post-hoc analysis, p <0.01, p <0.001, p <0.0001, ns=insignificant (n=5/group).

FIG. 19 shows that administration of GDC-0084 at a dose of 7.5mg/kg reduced splenomegaly in a 4T1 syngeneic tumor model. Representative images of spleens harvested from mice administered (A) 7.5mg/kg GDC-0084 or (B) 15mg/kg once daily +/- αPD1 in a Balb/c 4T1 breast cancer model. Spleen weights are expressed as mean ± SEM. One-way analysis of variance for Tukey post-hoc analysis, p <0.05, p <0.01, p <0.001, p <0.0001, ns=insignificant (n=5/group).

FIG. 20 shows that GDC-0084 was administered at a dose of 7.5mg/kg to reduce SOX9 and TOX1 expression. The captured image is analyzed using Qupath to depict a bar graph for:

A) CSC characteristics (SOX 9, PDL1, panCK), n >500000 cells were analyzed.

B) CD8 dysfunction features (CD 8, TOX1, PD 1), n >10000 cells were analyzed.

C) CSC characteristics (SOX 9, PDL1, panCK), n >10000 cells were analyzed.

D) CD8 dysfunction features (CD 8, TOX1, PD 1), n >10000 cells were analyzed. Qupath analysis was used to select DAPI positive cells and then CSC-characterized cells positive for PDL1 and panCK were scored according to SOX9 fluorescence intensity. For CD8 dysfunction features DAPI positive cells were selected, then CD8 and pd1+ were selected and scored according to TOX1 fluorescence intensity. Images were taken from an Aperio FL fluorescence slide scanner, which photographs stained FFPE tumor sections of GDC mouse models treated with controls, anti-PD 1 immunotherapy, GDC-0084 (high dose C/D and low dose A/B), or a combination. FFPE sections of lung tissue were treated as described before and stained for SOX9, PDL1, panCK or CD8, TOX1, PD1, DAPI for nuclear staining. Data are plotted as mean ± standard deviation, representing fluorescence intensity of SOX9 or TOX 1. Tukey post-hoc, ns=no significance,

***p<0.001,****p<0.0001。

Fig. 21 pi3k alone or in combination inhibits targeted mesenchymal characteristics and induces an epithelial phenotype in an immunotherapeutic-resistant mouse model. FFPE primary tumor samples treated with Opal staining kit targeting PI3KCA, CSV, E-cadherin or EGFR at BONDRX. (A) - (D) quantification of PI3KCA, CSV, E-cadherin or EGFR Fluorescence Intensity (FI) using ASI digital pathology.

FIG. 22 PI3K alone or in combination induces an epithelial phenotype in an immunotherapeutic resistant mouse model. FFPE primary tumor samples treated with Opal staining kit targeting PI3KCA, CSV, E-cadherin or EGFR at BONDRX. The percentage of E-cadherin expressing positive cells was determined using the ASI digital pathology platform.

FIG. 23 PI3K monotherapy or combination therapy induces effector and TRM characteristics. Cd8+ ifnγ+ T cell positive cells (a) or cd8+ T cell expression markers (B) of tissue resident memory cells (CD 103, CD69, CD44 and CD 8) were also quantified by staining FFPE tumor samples with Opal staining kit and analyzing images with ASI digital pathology. The Kruskal-Wallis nonparametric test showed significant differences.

FIG. 24 shows that PI3K alone or in combination inhibits checkpoint depletion characteristics in CD8+ T cells. FFPE primary tumor samples treated with tyramine staining kit targeting CD8, LAG3 and TIM3 checkpoint markers. The percentage of positive cells for each marker was determined using the ASI digital pathology platform. The percent expression of TIM3 and LAG3 positive cd8+ T cells in total cd8+ T cells was calculated. The graph shows n=3 mice per group, with significant differences calculated using the Kruskal-Wallis test. ns=no significance, p= <0.02.

FIG. 25 shows that combination of PI3K inhibition and immunotherapy can eliminate metastatic spread in 4T1 immunotherapy-resistant models. GDC-0084+/- αPD1 treatment in a 4T1 model of metastatic breast cancer. (A) treatment regimen using Balb/c 4T1 breast cancer model. (B) On day 20 post-inoculation, individual mice had metastatic tumor nodules in the lungs, p <0.01, tukey post-hoc trial.

FIG. 26 optimal therapeutic dose of GDC-0084 established in the 4T1 TNBC model. (A) Treatment regimen using Balb/c 4T1 TNBC breast cancer model. (B) procedure of dose-decreasing experiment. In the first phase, GDC-0084 was administered at a dose of 15mg/kg +/- αPD1 (10 mg/kg) daily on days 0 and 4. In the second phase, GDC-0084 (up to 15 mg/kg) was administered in combination with alpha PD 1at 4 hours intervals on days 0 and 4. In the final stage, GDC-0084 was administered in combination with alpha PD 1at single daily doses (up to 7.5 mg/kg) on days 0 and 4. (C) Tumor volume (percent solvent) of individual mice at each experimental stage prior to harvest, < p <0.05, < p <0.01, < p <0.001, ns insignificant, unpaired t-test or Dunnett post-hoc test, n=4-5/group.

FIG. 27A-D reduction of GDC-0084 dose improves safety in mice. (A) Mice body weight was monitored during the initial dose, split dose decrementing, and single dose decrementing experiments. # indicates death of the mice. (B) liver weight was measured at harvest. * p <0.05, unpaired t test, n=4-5/group.

FIG. 28 No hepatotoxicity was observed with the optimal therapeutic dose of GDC-0084. Pathologists scored FFPE livers for H & E staining in split dose escalation and single dose escalation experiments. The changes in (a) liver inflammation, (B) extramedullary hematopoiesis (EMH) and (C) hepatocyte changes (metabolism and/or degeneration) score 1 = mild, 2 = moderate or 3 = severe for each parameter. P <0.05, p <0.001, p <0.0001, dunnett post hoc test, n=4-5/group compared to solvent.

FIG. 29 shows that PI3K-mTOR inhibition reduces splenomegaly and extramedullary hematopoiesis in the spleen. (A) At harvest, spleen weight was measured by split dose decrementing and single dose decrementing experiments. * p <0.05, < p <0.01, < p <0.0001, < n=4-5/group as compared to solvent, dunnett post hoc test. (B) pathologists scored the H & E stained FFPE spleens. The change score for extramedullary hematopoiesis (EMH) was 1=mild, 2=moderate, or 3=severe compared to solvent. * p <0.01, p <0.001, dunnett post hoc test, n=4-5/group.

FIG. 30 shows that PI3K-mTOR inhibition reduces leukocyte infiltration into the lung. Expert pathologists scored FFPE lungs for H & E staining in split dose escalation and single dose escalation experiments. The change score for leukocytosis was 1=mild, 2=moderate or 3=severe, p <0.01, p <0.001, dunnett post hoc test, n=4-5/group compared to solvent.

FIG. 31 GDC-0084 and alpha PD1 combination therapy prevents lymph node metastasis. (A) Images of H & E stained FFPE lymph nodes from αpd1 and GDC-0084 (7.5 mg/kg) and PD1 treated 4T1 mice. (B) Lymph node sections were examined by independent expert pathologists and given a score of 1=1-3 tiny sites (width/length < <0.5 mm), 2=1-3, with at least one diameter of 0.5-1mm, or 3=2-3 or more sites, all visible with white blood cell infiltration and/or bleeding.

FIG. 32 shows that GDC-0084 is useful in combination with PARP inhibitors. (A) PARP inhibitor protocol. In the first phase, GDC-0084 was administered at a dose of 7.5mg/kg daily either before (before) or 30 minutes after (after) Olaparib (50 mg/kg). In the second phase, GDC-0084 was administered at a dose of 7.5mg/kg daily 30 minutes after Olaparib (50 mg/kg). (B) Treatment regimen using Balb/c 4T1 TNBC breast cancer model. (C) Tumor volume (percent of solvent or olapanib) of individual mice at each experimental stage prior to harvest. * P <0.01, ns is not significant, tukey post hoc test, n=5/group.

FIG. 33 GDC-0084 and Olaparib combination therapy did not cause toxicity. Mice body weight and liver weight were assessed following treatment with GDC-0084 (7.5 mg/kg) following Olaparib treatment. ns, not significant, tukey post hoc, n=5/group.

FIG. 34 reduction of liver and lung inflammation following GDC-0084 and Olaparib combination therapy. Independent expert pathologists scored the H & E stained FFPE livers and lungs after GDC-0084 olaparib experiments. (A) Changes in liver inflammation and extramedullary hematopoiesis (EMH), and (B) pulmonary leukocytosis, each parameter scored as 1 = mild, 2 = moderate, or 3 = severe. P <0.05, p <0.001, p <0.0001, dunnett post hoc detection, n=5/group compared to solvent.

FIG. 35 shows that GDC-0084 in combination with Olaparib reduces splenomegaly and extramedullary hematopoiesis. (A) The expert pathologist scored FFPE spleens for H & E staining from GDC-0084 olaparib after the experiment. The change score for extramedullary hematopoiesis (EMH) was 1=mild, 2=moderate or 3=severe. (B) Spleen weight was measured at harvest and p <0.05, p <0.001, p <0.0001, dunnett post hoc, n=5/group compared to solvent. P <0.01, p <0.001, dunnett post hoc detection, n=5/group compared to solvent.

FIG. 36 comparison of proliferation of PI3K-mTOR inhibitor combination therapy cancer cells. In the dose-response design, WST proliferation reagents were added to MDA-MB-231 cells pretreated with PI3K inhibitor for 72 hours. Cell proliferation (%) was measured indirectly by formazan formation and absorbance at 450nm was recorded.

FIG. 37 dose response curve for GDC-0084 wound healing (scratch test). Pre-stimulated (PMA/TGF beta) MCF-7 cells were scraped prior to 24 hours of treatment with GDC-0084 (0.078-2.5. Mu.M). The change in wound density was monitored over 24 hours. (A) Representative images from scratch assays compare solvent and GDC-0084 (1.25. Mu.M and 2.5. Mu.M) treated MCF-7 cells. (B) Dose response curves for GDC-0084 treated cells (0.078-2.5 μm) were compared for wound density (%) over a 24 hour treatment period.

FIG. 38 comparison of PI3K-mTOR inhibitors to treat cancer cell migration. Pre-stimulated (PMA/tgfβ) MCF-7 cells were scratched before 24 hours of treatment with the indicated doses of PI3K-mTOR inhibitor. (A) Dose response curves comparing wound density (%) of PI3K-mTOR inhibitor treated cells (0.078-2.5 μm) over a 24 hour treatment period. (B) The relative wound densities observed at 24 hours for each PI3K-mTOR inhibitor were compared. P <0.0001 compared to solvent, dunnett post hoc assay, n=5/group. P <0.01, p <0.001, dunnett post hoc detection, n=6/group compared to solvent.

Detailed Description

1. Definition of the definition

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

The articles "a" and "an" as used herein refer to one or more (i.e., to at least one) of the grammatical object of the article. For example, "an element" refers to an element or elements.

The term "about" as used herein refers to the usual error range of the corresponding value as readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to that value or parameter itself.

The "amount" or "level" of a biomarker is the level detectable 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 biomarker assessed can be used to determine the response to the treatment.

As used herein, "and/or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted as alternatives (or).

In this specification, unless the context requires otherwise, the words "comprise", "comprises", "comprising" and "includes" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" or the like means that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. "consisting of" is meant to include and be limited to anything after "consisting of (consisting of)". Thus, the term "consisting of" means that the listed elements are necessary or mandatory and that no other elements are present. "consisting essentially of" is meant to include any element listed after a phrase and is limited to other elements not interfering with or contributing to the activity or action specified for the listed element in this disclosure. Thus, the phrase "consisting essentially of" means that the listed elements are necessary or mandatory, but other elements are optional, and may or may not be present, depending on whether they affect the activity or effect of the listed elements.

"Chemotherapeutic agents" include compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib @Genentech/oscham.) bortezomib @Millennium pharm), disulfiram, epigallocatechin gallate, salinosporamideA, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase a (LDH-a), fulvestrant @AstraZeneca)、sunitib(The composition contains the components of the compositionNovartis), imatinib mesylateNovartis)、finasunate(Novartis, oxaliplatin @Cynophenanthrene), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus),Huisha, lapatinib @GSK572016, ghatti Smith), lonafanil (SCH 66336), sorafenib @Bayer laboratory), gefitinibAselerant), AG1478, alkylating agents such as thiotepa andCyclophosphamide, alkyl sulfonates such as busulfan, imperoshu and piposhu, aziridines such as benzotepa, carboquinone, metirizine and ureirizine, ethyleneimines (ETHYLENIMINES) and methyl melamines (METHYLAMELAMINES) including altretamine (altretamine), triethylenemelamine (TRIETHYLENEMELAMINE), triethylenephosphoramide (triethylenephosphoramide), Triethylenethiophosphamide (triethylenethiophosphoramide) and trimethylol melamine (trimethylolomelamine), annonaceous acetogenins (acetogenin), especially bullatacin and bullatacin (bullatacinone), camptothecins, including topotecan and irinotecan, bryostatin, card Li Yisu (callystatin), CC-1065, including adoxine (adozelesin), and combinations thereof, The synthetic analogues of carzelesin and bizelesin, cryptophycins (cryptophycins) (especially cryptophycin 1 and cryptophycin 8), adrenocorticoids (including prednisone and prednisone acetate), cyproterone acetate, 5α -reductase (including finasteride and dutasteride), vorinostat, romidepsin, panobinostat, valproic acid, mofetil, dolastatin, aldesinterleukin, talc-fold carcinomycin (including synthetic analogues KW-2189 and CB1-TM 1), eiutherobin (eleutherobin), water-ghost-abane (panorastatin), sarcandol (sarcodictyin), spongostatin (spongistatin), nitrogen mustards (nitrogen mustards), such as chlorambucil (chlorambucil), dolac, Naphthol (chlornaphazine), cholesteryl phosphoramide (cholophosphamide), estramustine (estramustine), isophosphamide (ifosfamide), mechlorethamine (mechlorethamine), mechlorethamine hydrochloride (mechlorethamine oxidehydrochloride), melphalan, mechlorethamine (novembichin), mechlorethamine cholesterol (PHENESTERINE), prednisoline (prednimustine), and pharmaceutical compositions, Trefosfamide (trofosfamide), uracil mustard (uracil mustard), nitroureas (nitrosureas) such as carmustine (carmustine), chlorourea (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), and the like, Nimustine (nimustine) and ramustine (ranimustine), antibiotics, such as enediyne (enediyne) antibiotics (e.g. calicheamicin (calicheamicin), especially calicheamicin gamma 1I and calicheamicin omega 1I (Angew chem. Intl. Ed. Engl.199433: 183-186)), dactinomycin (dynemicin) including dactinomycin A, bisphosphonates (biphosate), such as chlorophosphonate (clodronate), epothilone (esperamicin), and new oncostatin (neocarzinostatin) chromophores and related chromoprotein enediyne antibiotics, Azithromycin (aclacinomysins), actinomycin, anthramycin (authramycin), azaserine (azaserine), bleomycin, actinomycin C (cactinomycin), cartonecin (carabicin), carminomycin (carminomycin), carcinophilic mycin (carzinophilin), chromomycin (chromomycinis), actinomycin D (dactinomycin), daunorubicin (daunorubicin), dithiubicin (detorubicin), 6-diaza-5-oxo-L-norleucine,(Doxorubicin), pindolorubin, cyanomorpholino-doxorubicin, 2-pyrrolo-doxorubicin and deoxydoxorubicin), epirubicin, eldroubicin (esorubicin), idarubicin (idarubicin), doxycycline (marcellomycin), mitomycin (e.g., mitomycin C), mycophenolic acid (mycophenolic acid), norgamycin (nogalamycin), olivomycin (olivomycin), perlecithromycin (peplomycin), pofeomycin (potfiromycin), doxycycline (52), Puromycin, trifolicin (quelamycin), rodobicin (rodorubicin), streptozotocin (streptonigrin), streptozotocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), jingstadine (zinostatin) or zorubicin (zorubicin), antimetabolites such as methotrexate, 5-fluorouracil (5-FU), folic acid analogs such as dimethyl folic acid (denopterin), methotrexate, Pterin (pteropterin), trimetric (trimerexate), purine analogs such as fludarabine (fludarabine), 6-mercaptopurine, thioxanthine, thioguanine, pyrimidine analogs such as ambcitabine (ancitabine), azacytidine (azacitidine), 6-azauridine, carmofur (carmofur), cytarabine, dideoxyuridine (dideoxyuridine), doxifluridine (doxifluridine), Enocitabine (enocitabine), fluorouridine (floxuridine), androgens such as carbo Lu Gaotong (calusterone), drotasone propionate (dromostanolone propionate), epithioandrosterol (epitiostanol), emastrane (mepitiostane), testosterone (testolactone), anti-adrenal agents such as aminoglutethimide (aminoglutethimide), mitotane (mitotane), mitotane, and the like, Trolesteine (trilostane), folic acid supplements such as folinic acid, acetoglucurolactone (aceglatone), aldehyde phosphoramide glycoside (aldophosphamide glycoside), aminoketovaleric acid (aminolevulinic acid), eniluracil (eniluracil), amfenadine (amsacrine), amostatin (bestrabucil), bisacodyl (bisantrene), idatroxacin (edatraxate), cyclophosphamide (defofamine), dimethacin (demecolcine), deazoquinone (diaziquone), epothilone (elformithine), oxypicolazole acetate (elliptinium acetate), epothilones (epothilone), etoxagline (etoglucid), gallium nitrate, hydroxyurea, lentinan (lentinan), lonidamine (lonidamine), maytansinoids such as maytansine (maytansine) and ansamitocin (ansamitocin)), mitoguazone (mitoguazone), mitoxantrone (mitoxantrone), mo Pai danol (mopidamol), diamine nitroacridine (nitracrine), penstadine (diaziquone), fluxazine (nitracrine), and piroxicam (nitracrine-3) are provided.Polysaccharide complexes (JHS Natural Products, eugene, oreg.); radzosin (razoxane), rhizomycin (rhizoxin), sisofilan (sizofiran), gemfibrospiramine (spirogermanium), tenascosporanic acid (tenuazonic acid), trisaminoquinone (triaziquone), 2' -trichlorotriethylamine, trichothecenes (trichothecenes) (especially T-2 toxin, wart-sporine A (verracurinA), baculosporine A (roridinA) and serpentine (anguidine)), polyurethanes, vindesine (vindesine), dacarbazine (dacarbazine), mannitol nitrogen mustard (mannomustine), dibromomannitol (mitobronitol), dibromodulcitol (mitolactol), pipobroman (pipobroman), ganciclovir (gacytosine), cytarabine ("Ara-C"); cyclophosphamide, thiotepa; taxanes (taxanes), such as TAXOL (paclitaxel), bristol (Myers Squibb Oncology, pricoxel, N.))Albumin engineered nanoparticulate formulations (albumin-engineered nanoparticle formulations of paclitaxel)(American Pharmaceutical Partners、Schaumberg、Ill.) and of paclitaxel (Cremophor-free)(Docetaxel, doxetaxel; sanofi-Aventis); chlorambucil; (Gemcitabine), 6-thioguanine, mercaptopurine, methotrexate, platinum coordination complexes such as cisplatin, oxaliplatin and carboplatin, vinblastine (vinblastine), platinum, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine; (vinorelbine), nor An Tuo (novantrone), teniposide, idazoxifloxacin, 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.

The chemotherapeutic agents also include (i) anti-hormonal agents such as antiestrogens and Selective Estrogen Receptor Modulators (SERMs) for modulating or inhibiting tumor hormonal effects, including tamoxifen (includingTamoxifen citrate), raloxifene, droloxifene, ioxyfene, 4-hydroxy tamoxifen, trawoxifene, raloxifene hydrochloride (keoxifene), LY117018, onapristone and(Toremifine citrate), and (ii) aromatase inhibitors which inhibit aromatase which regulates estrogen production in the adrenal gland, e.g. 4 (5) -imidazole, aminoglutethimide,(Megestrol acetate),(Exemestane, pyroxene), formestane, fazodone,(Wo Luo),(Letrozole, nohua) and(Anastrozole; assiconazole), (iii) antiandrogens such as flutamide, nilutamide, bicalutamide, leuprorelin and goserelin, (buserelin, triptorelin (tripterelin), 17-hydroxyprogesterone acetate, ethylestrol, betamethadone, fluoxytestosterone, all-trans retinoic acid, feitinib (fenretinide) and troxacitabine (a 1, 3-dioxolane nucleoside cytosine analogue), (iv) protein kinase inhibitors, (v) lipid kinase inhibitors, (vi) antisense oligonucleotides, particularly those oligonucleotides which inhibit gene expression in signal pathways associated with abnormal cell proliferation, such as PKC-alpha, ralf and H-Ras, (vii) ribozymes such as VEGF expression inhibitors (such as VEGF expression inhibitors) And HER2 expression inhibitor (viii) vaccines, such as gene therapy vaccines, e.gAnd RIL-2, topoisomerase 1 inhibitors, e.g. RmRH, and (ix) pharmaceutically acceptable salts, acids and derivatives of any of the above.

The chemotherapeutic agent also comprises antibodies such as alemtuzumab (Campath) and bevacizumab @, andGenentech); cetuximab @Imclone @ panitumumab @Anin company, rituximab @Genentech// Biogen Idec), pertuzumab @2C4, genntech, trastuzumab @Genentech), tositumomab (Bexxar, corixia) and antibody-drug conjugate gemtuzumab-ozumeab @Wheatstone).

Other humanized monoclonal antibodies having therapeutic potential as drugs in combination with the compounds of the present invention include apolizumab (apremizumab), aselizumab (alemtuzumab), atlizumab (attitumomab), bapineuzumab (bapiduzumab), bivatuzumab mertansine (bivalizumab maytansine), cantuzumab mertansine (katuzumab maytansine), cedelizumab (cerizumab), certolizumab pegol (polyethylene glycol cetuximab), and, cidfusituzumab (cetuximab), cidtuzumab (cetuximab), daclizumab (daclizumab), ecalizumab (eculizumab), efalizumab (efalizumab), epratuzumab (epalizumab), erlizumab (erlizumab), felvizumab (non-valizumab), fontolizumab (aryltuzumab), gemtuzumab ozogamicin (gemtuzumab ozuzumab), ozuzumab, inotuzumab ozogamicin (oibituzumab), ipilimumab (ipilimumab), labetuzumab (la Bei Tuozhu mab), lintuzumab (lintuzumab), matuzumab (matuzumab), mepolizumab (mepolizumab), motavizumab (mot Wei Zhushan antibody), motovizumab (Mo Tuozhu mab), natalizumab (natalizumab), nimotuzumab (nituzumab), nolovizumab (noluzumab), numavizumab (Nomamizumab), ocrelizumab (Orivizumab), omalizumab (omalizumab), palivizumab (palivizumab), pascolizumab (pasmodizumab), pecfusituzumab (petatuzumab), pectuzumab (petatuzumab), pexelizumab (pekzumab), ralivizumab (Lalizumab), ranibizumab (ranibizumab), reslivizumab (Ralizumab), reslizumab (Rayleigh), resyvizumab (Rayleigh), rovelizumab (Luo Weizhu), ruplizumab (Lu Lizhu), sibrotuzumab (cetrimide), siplizumab (cetrimide), sontuzumab (pinocembrin), tacatuzumab tetraxetan (Takatuzumab Titrastuzumab), tadocizumab (Tadolizumab), talizumab (Talizumab), taraxacum, tefibazumab (tefeizumab), tocilizumab (tolizumab), toralizumab (tolizumab), tucotuzumab celmoleukin (toxilizumab celer interleukin), tucusituzumab (toxilizumab), umavizumab (Wu Mazhu mab), urtoxazumab (Wu Tuozhu mab), ustekinumab (Wu Sinu mab), visilizumab (wixilizumab), and anti-interleukin-12 antibodies (ABT-874/J695, wheatstone research and yaban laboratory), which are full length IgG1 lambda type antibodies of a recombinant fully human sequence genetically modified to recognize interleukin-12 p40 proteins.

Chemotherapeutic agents also include "EGFR inhibitors," which refer to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity, also referred to as "EGFR antagonists. Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB 8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see U.S. patent 4943533, mendelsohn et al) and variants thereof, such as chimeric 225 (C225 or cetuximab; ) And engineered human 225 (reshaped human, H25) (see WO 96/40210,Imclone Systems, inc.), IMC-11F8, fully human EGFR-targeting antibody (Imclone), antibodies that bind to type II mutant EGFR (U.S. Pat. No. 5212290), humanized and chimeric antibodies that bind to EGFR, such as U.S. Pat. No. 5891996, and human antibodies that bind to EGFR, such as ABX-EGF or panitumumab (see WO98/50433, abgenix/Amgen), EMD 55900 (Stragliotto et al), eur.J. Cancer 32A:636-640 (1996)), EMD7200 (matuzumab) is a humanized EGFR antibody that binds to EGFR and EGFR-. Alpha.competing with EGFR (EMD/Merck), human EGFR antibodies HuMax-EGFR (GenMab), fully human antibodies called E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 and E1.6.3, such as MDX.J. Cancer 32A:636-640 (1996), and Max, see U.S. Pat. No. 4, and Max.K 2.K-6.K. 6) are human antibodies that compete with EGFR and EGFR (EMD/Merck), and Max-alpha.K) are human EGFR antibodies (Mab). The anti-EGFR antibody may be conjugated with a cytotoxic agent, thereby producing an immunoconjugate (see e.g. EP659439A2, MERCK PATENT GmbH). EGFR antagonists include small molecules such as the compounds described in U.S. Pat. Nos. 5616582、5457105、5475001、5654307、5679683、6084095、6265410、6455534、6521620、6596726、6713484、5770599、6140332、5866572、6399602、6344459、6602863、6391874、6344455、5760041、6002008 and 5747498, and PCT publications WO98/14451, WO98/50038, WO99/09016 and WO99/24037. Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, Genetech/OSI pharmaceutical Co.), PD 183805 (Cl 1033,2-acrylamide, N- [4- [ (3-chloro-4-fluorophenyl) amino ] -7- [3- (4-morpholinyl) propoxy ] -6-quinazolinyl ] -dihydrochloride, part of the company, PYRIGHT, inc.), ZD1839, gefitinib4- (3 ' -Chloro-4 ' -fluoroanilino) -7-methoxy-6- (3-morpholinopropoxy) quinazoline, aslican; 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, boringer's lattice-Han); PKI-166 ((R) -4- [4- [ (1-phenethyl) amino ] -1H-pyrrolo [2,3-d ] pyrimidin-6-yl ] -phenol); (R) -6- (4-hydroxyphenyl) -4- [ (1-phenethyl) amino ] -7H-pyrrolo [2,3-d ] pyrimidine); CL-387785 (N- [4- [ (3-bromophenyl) amino ] -6-butynamide); EKB-9 (N- [4- [ (3-chloro-4-fluorophenyl) amino ] -3-cyano-7-ethoxy-2-butenyl ] -2- (-quinolinyl) (Hui-4- (-c-phenyl) amino) AG1478 (gabion), AG1571 (SU 5271; gabion), dual EGFR/HER2 tyrosine kinase inhibitors, e.g. Lapattinib @GSK572016 or N- [ 3-chloro-4- [ (3-fluorophenyl) methoxy ] phenyl ] -6[5[ [ [ 2-methylsulfonyl) ethyl ] amino ] methyl ] -2-furyl ] -4-quinazolinamine.

Chemotherapeutic agents also include "tyrosine kinase inhibitors", including EGFR-targeting 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 (both pyroxene and OSI)), dual HER inhibitors such as EKB-569 (available from Wheatstone) which preferentially bind EGFR but inhibit HER2 and EGFR overexpressing cells, lapatinib (GSK 572016; available from Gelanin Smith), an oral HER2 and EGFR tyrosine kinase inhibitor, PKI-166 (available from Norhua corporation), pan HER inhibitors such as Canatinib (Cl-1033; pharmacia), raf-1 inhibitors such as the antisense agent ISIS-5132 provided by ISIS.

Agents that inhibit Raf-1 signaling, non-HER targeted TK inhibitors, such as imatinib mesylatePurchased from ghanin smic), multi-targeted tyrosine kinase inhibitors such as sunitinib @Purchased from a pyro); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK 787/ZK222584, available from North/Hill Co., ltd.); MAPK extracellular regulated kinase I inhibitor Cl-1040 (available from Pharmacia); quinazolines such as PD 153035,4- (3-chloroanilino) quinazoline, pyridopyrimidines, pyrimidopyrimidines such as CGP 59326, CGP 60261 and CGP 62706, pyrazolopyrimidines, 4- (anilino) -7H-pyrrolo [2,3-d ] pyrimidine; curcumin (diferuloyl methane,4, 5-bis (4-fluoroanilino) phthalimide); tyrosine containing a nitrothiophene moiety; PD-0183805 (Wana-lanbo); antisense molecules such as molecules that bind to nucleic acids encoding HER); quinoxaline (U.S. Pat. No. 5804396); tryphostins (U.S. No. 5804396); ZD6474 (Asp-787 (Nowa/Hill Co., ltd.); pan HER inhibitor such as SiS. No. 3, IS35; islands 35/Islands sulfonic acid) and the like 1, 35 S. IslandsPKI 166 (Nohua), GW2016 (Gellan Seck), cl-1033 (J.P.), EKB-569 (Wheatstone), semaxinib (J.P.), ZD6474 (Azimut), PTK-787 (Nohua/Hill Co.), INC-1C11 (Imclone), rapamycin (sirolimus, sichuan-N.C.),) Or as described in any of the following patent publications, U.S. Pat. No. 5804396, WO 1999/09016 (cyanamide), WO 1998/43960 (cyanamide), WO 1997/38983 (Wana-Lanbert), WO 1999/06678 (Wana-Lanbert), WO 1999/06396 (Wana-Lanbert), WO 1996/30347 (pyrotechnical company), WO 1996/33978 (Zeneca), WO 1996/3397 (Zeneca) and WO 1996/33980 (Zeneca).

Chemotherapeutic agents also include dexamethasone, interferon, colchicine, chlorphenidine (metoprine), cyclosporin, amphotericin, metronidazole, alemtuzumab, aliskiric acid, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacizumab, bexarotene, cladribine, clofazocine, dapoxetine alpha, dimesleukin (denileukin,), dexrazoxane, alfazoxetine, elotinib, feaglutin, histamine acetate, temozolomide, interferon alpha-2 a, interferon alpha-2-b, lenalidomide, levamisole, mesna, methoxaline, nandrolone, nelarabine, nofetumomab, opril, palivimin, pamidronate disodium, pegademase, columbrotene, meldonium, melitemide, plicin, phenoxacin, flupirtine, fludrozole, flugline, fluzoxazole, ATRADIX (ATRACTone, ATRAZone, ATRAXone, ATRADIX, zone, ATRAXone, and pharmaceutical compositions thereof).

Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tibetasone pivalate, triamcinolone acetonide, triamcinolone (triamcinolone alcohol), mometasone, ambetanide, budesonide, deanenide, fluocinolone acetonide, betamethasone sodium phosphate, dexamethasone sodium phosphate, flucortisone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, beclomethasone dipropionate, betamethasone dipropionate, prednisolide butyrate, clobetasol propionate, fluclobetane caproate, fluclocortlone chlorate (fluocortolone pivalate), and fluprednisodine acetate; immunoselective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and D-isomer forms (feG) thereof (IMULAN BioTherapeutics, LLC), antirheumatic drugs, such as azathioprine, cyclosporin (cyclosporin A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide Mi Tehuan, sulfasalazine, tumor Necrosis Factor (TNF) blockers, such as etanercept (Enbrel), infliximab (Remicode), adalimumab (Humira), pezilimumab (Cimzia), golimumab (Simmoni), interleukin 1 (IL-1) blockers, such as anakinra (Kineret), T-cell co-stimulatory blockers, such as Abauzepine (Orencia), interleukin 6 (IL-6) blockers, such as tolizumabInterleukin 13 (IL-13) blockers, such as Leuconostoc, interferon-alpha (IFN-alpha) blockers, such as Long Li group mAb, beta7 integrin blockers, such as rhuMAb Beta7, igE pathway blockers, such as anti-M1 prime, secretion homotrimer LTa3 and membrane-bound heterotrimer LTa 1/Beta 2 blockers, such as anti-lymphotoxin alpha (LTa), radioisotopes (e.g., ,At²¹¹、I¹³¹、I¹²⁵、Y⁹⁰、Re¹⁸⁶、Re¹⁸⁸、Sm¹⁵³、Bi²¹²、P³²、Pb²¹² and the radioisotope of Lu), other investigative drugs, such as carbosulfan (thioplatin), PS-341, phenylbutyrate, ET-18-OCH ₃ or farnesyl transferase inhibitors (L-739749, L-744832), polyphenols, such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavins, flavanols, procyanidins, betulinic acid and derivatives thereof, autophagy inhibitors, such as chloroquine, delta-9-tetrahydrocannabinol (cannabinol,) Beta-lapachone, lapachol, colchicine, betulinic acid, acetylcamptothecin, scopolamine and 9-aminocamptothecin), podophyllotoxin and tegafurBexaroteneBisphosphonates, such as chlorophosphonates (e.g.,Or (b)) Etidronate saltsNE-58095, zoledronic acid/zoledronic acid esterAlendronate sodiumPamidronate sodiumLu Linsuan sodium saltOr risedronate sodiumEpidermal growth factor receptor (EGF-R), vaccines, e.gVaccine, pirifadine, COX-2 inhibitors (e.g. celecoxib or etoricoxib), proteasome inhibitors (e.g. PS 341), CCl-779, tipifanib (R11577), orafenib, ABT510, bcl-2 inhibitors, e.g. sodium orelmesonPixeron maleate, farnesyl transferase inhibitors such as lenafani (SCH 6636, SARASAR ^TM), and pharmaceutically acceptable salts, acids or derivatives of any of the foregoing, and combinations of two or more of the foregoing, such as CHOP, which is an abbreviation for cyclophosphamide, doxorubicin, vincristine, and prednisolone in combination therapy, and FOLFOX, which is an abbreviation for oxaliplatin (ELOXATIN ^TM) in combination with 5-FU and leucovorin therapy.

Chemotherapeutic agents also include non-steroidal anti-inflammatory drugs (NSAIDs) with analgesic, antipyretic and anti-inflammatory effects. NSAIDs include non-selective inhibitors of cyclooxygenase enzymes. 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, droxic, lornoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid and COX-2 inhibitors such as celecoxib, etoricibuzole, lumiracoxib, rofecoxib and valdecoxib. The nonsteroidal anti-inflammatory drugs can be used for relieving symptoms such as mild to moderate pain, fever, intestinal obstruction, renal colic and the like caused by rheumatoid arthritis, osteoarthritis, inflammatory joint diseases, ankylosing spondylitis, psoriatic arthritis, reiter syndrome, acute gout, dysmenorrhea, metastatic bone pain, headache and migraine, postoperative pain, inflammation and tissue injury.

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

The terms "reduce", "decrease", "inhibit", "suppress", "attenuate" and the like as used herein all refer to a statistically significant reduction. In some embodiments, these terms generally mean at least a 10% reduction (e.g., in the absence of a given treatment or drug) from a reference level, and may include, for example, 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, "reducing," "suppressing," and "suppressing" do not require complete suppression or reduction compared to a reference level. "complete inhibition" and the like are 100% inhibition compared to reference levels. The reduction may preferably be reduced to an acceptable level within the normal range (e.g., for individuals without a given disease).

As used herein, the term "epithelial-mesenchymal transition" (EMT) refers to the transition from epithelial cells to a mesenchymal phenotype, which is the normal process of embryo development. EMT is also the process by which damaged epithelial cells, which are ion and fluid transport proteins, become matrix remodelling mesenchymal cells, and in cancer this transformation generally results in altered cell morphology, increased mesenchymal protein expression and invasiveness. Criteria for defining EMT in vitro include loss of epithelial cell polarity, isolation into individual cells, and subsequent dispersion after cell motility is achieved (see Vincent-Salomon and Thiery, breast cancer rRs.2003; 5 (2): 101-6). The changes in expression, distribution and/or function of molecular classes during EMT, and reasons related thereto, include growth factors (e.g., transforming Growth Factors (TGF)) -P, wnts), transcription factors (e.g., SNAI, SMAD, LEF and nuclear beta-catenin), molecules of the cell-cell adhesion axis (cadherin, catenin), cytoskeletal modulators (Rho family) and extracellular proteases (matrix metalloproteinases, plasminogen activators) (see Thompson and Newgreen, cancer rres.2005;65 (14): 5991-5).

The terms "increase (increase)", "increase" or "activation" herein refer to a statistically significant increase. In some embodiments, the term "increase (increase)", "increase" or "increase" may mean an increase of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and may 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 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 of 2-fold to 10-fold or more compared to a reference level. In the context of markers or symptoms, "increase" refers to a statistically significant level of increase.

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

As used herein, the term "mesenchymal-to-epithelial transformation" (MET) is a reversible biological process involving the transformation of a moving, multipolar or spindle-shaped mesenchymal cell into a planar polarized cell array called an epithelial cell. MET is the inverse of EMT. MET occurs in normal development, cancer metastasis, and induction of pluripotent stem cell reprogramming.

As used herein, the terms "over-expression", "over-expression" or "over-expressed" interchangeably refer to a level of transcription or translation of a gene (e.g., PI3KCA gene) that is generally detectable in a cancer cell as compared to a normal cell. Thus, overexpression refers to overexpression of proteins and RNAs (due to increased transcription, post-transcriptional processing, translation, post-translational processing, altered stability and altered protein degradation), as well as localized overexpression due to altered protein transport patterns and enhanced functional activity, e.g., increased enzymatic hydrolysis of substrates. Overexpression may also be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to normal cells or control cells (e.g., breast cells).

The term "PI3K inhibitor" and grammatical variations thereof as used herein refers to molecules that reduce or inhibit at least one function or biological activity of PI 3K. For example, a PI3K inhibitor may inhibit or reduce the enzymatic activity of PI3K and/or may inhibit or reduce the expression of PI 3K.

As used herein, the term "PI3K overexpressing cell" refers to a vertebrate cell, particularly a mammalian cell, that expresses PI3K at a detectable level that is higher than normal cells. The cells may be vertebrate cells such as primate cells, avian cells, livestock cells (e.g., sheep cells, cow cells, horse cells, deer cells, donkey cells and pig cells), laboratory test animal cells (e.g., rabbit cells, mouse cells, rat cells, guinea pig cells and hamster cells), companion animal cells (e.g., cat cells and dog cells), and wild animal cells (e.g., fox cells, deer cells and wild dog cells) in stock. In a specific embodiment, the PI3K overexpressing cell is a human cell. In specific embodiments, the PI 3K-overexpressing cells are cancer stem cells or non-cancer stem cell tumor cells, preferably cancer stem cell tumor cells. Overexpression may also be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to normal cells or control cells (e.g., breast cells).

The term "selective" and grammatical variations thereof is used herein to refer to inhibition of PI3K without substantial inhibition of one or more other PI3K enzymes or isoforms. Typically, a molecule selective for PI3K exhibits PI3K selectivity of greater than about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or greater than about 100-fold inhibition of one or more other PI3K enzymes. In other embodiments, the inhibitory effect of the selective molecule on PI3K is at least 50-fold greater than the inhibitory effect on one or more other PI3K enzymes. In further embodiments, the inhibitory effect of the selective molecule on PI3K is at least 100-fold greater than the inhibitory effect on one or more other PI3K enzymes. In further embodiments, the inhibitory effect of the selective molecule on PI3K is at least 500-fold greater than the inhibitory effect on one or more other PI3K enzymes. In further embodiments, the inhibitory effect of the selective molecule on PI3K is at least 100-fold greater than the inhibitory effect on one or more other PI3K enzymes.

As used herein, "subject" refers to a human or animal. Typically the animal is a vertebrate, such as a primate, rodent, livestock or hunting animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (e.g., rhesus monkeys). Rodents include mice, rats, woodchuck, ferrets, rabbits, and hamsters. Domestic animals and hunting animals include cattle, horses, pigs, deer, wild cattle, buffalo, felines (e.g., domestic cats), canines (e.g., dogs, foxes, wolves), birds (e.g., chickens, emus, ostriches), and fish (e.g., trout, catfish, and salmon). In some embodiments, the subject is a mammal (e.g., a primate (e.g., a human)). The terms "individual," "patient," and "subject" are used interchangeably herein.

As used herein, the terms "treatment", "treatment" and the like refer to therapeutic treatments in which the purpose is to reverse, reduce, ameliorate, inhibit, slow or stop the progression or severity of a disorder associated with a disease or disorder (e.g., cancer or tumor). The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder associated with cancer or tumor. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Or if the progression of the disease slows or stops, the treatment is "effective". That is, "treatment" includes not only improvement of symptoms or markers, but also stopping or at least slowing the progression or worsening of symptoms as compared to the case without treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the state of disease, remission (whether partial or total), and/or reduction of mortality, whether detectable or undetectable. The term "treatment" of a disease also includes alleviation of symptoms or side effects of the disease (including palliative treatment). Treatment is not required to cure a disease (i.e., completely reverse or no disease) to be considered effective.

As used herein, the term "tumor" refers to any tumor cell growth and proliferation, whether malignant or benign, as well as all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe the physiological condition of a mammal, often characterized in part by unregulated cell growth. As used herein, the term "cancer" refers to non-metastatic and metastatic cancers, including early and late stage cancers. The term "precancerous lesion" refers to a disease or growth that is usually preceded by, or progresses to, cancer. The term "non-metastatic" refers to benign cancers, or tissues that remain at the primary site, do not penetrate the lymphatic or vascular system or beyond the primary site. Generally, a non-metastatic cancer is any stage 0, I or II cancer. "early stage cancer" refers to cancer that is not invasive or metastatic, or is classified as stage 0, 1 or II cancer. The term "advanced cancer" generally refers to stage III or IV cancer, but may also refer to stage II cancer or sub-stage of stage II cancer. Those of skill in the art will appreciate that the classification of stage II cancer as early stage cancer or late stage cancer depends on the particular type of cancer. Illustrative examples of cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, pancreatic cancer, colorectal cancer, lung cancer, hepatocellular cancer, gastric cancer, liver cancer, bladder cancer, urinary tract cancer, thyroid cancer, renal cancer, malignant tumor, melanoma, brain cancer, non-small cell lung cancer, head and neck squamous cell cancer, endometrial cancer, multiple myeloma, mesothelioma, rectal cancer, and esophageal cancer. In an exemplary embodiment, the cancer is breast cancer.

Unless specifically stated otherwise, each embodiment described herein applies mutatis mutandis to each embodiment.

2. Composition and method for producing the same

The present invention is based in part on the determination that exposure of functionally inhibited T cells to PI3K inhibitors of the mesenchymal phenotype results in epigenetic reprogramming of T cells, with derepression of their immune effector functions, including increased expression of biomarkers of T cell activation and effector capacity (e.g., IL-2, IFN- γ, and TNF), decreased expression of biomarkers of T cell effector inhibition and cancer progression (e.g., ZEB 1), as well as decreased expression of biomarkers of T cell depletion (e.g., PD-1 and EOMES) and increased expression of the transcription factor TBET (which increases adaptability and production of IFN- γ in cells of the innate immune system). The inventors have also found that PI3K inhibitor mediated reprogramming of phosphorylation enhances sensitivity of depleted T cells to ICM binding antagonist reactivation.

Thus, in accordance with the present invention, compositions and methods are provided for enhancing immune effector function and/or enhancing T cell (e.g., cd8+ T cell) function utilizing PI3K inhibitors (e.g., inhibitors of PI3K activity) and ICM binding antagonists, including increasing T cell activation and enhancing the sensitivity of depleted T cells to ICM binding antagonist reactivation. Thus, the methods and compositions of the invention are particularly useful for treating T cell dysfunctional disorders, including cancer and infections.

The present invention is based, at least in part, on the identification of compositions that inhibit CSC tumor cell EMT and induce CSC tumor cell MET. Thus, the inventors believe that the compositions of the invention may be useful in the treatment or prevention of cancer, and/or to improve the responsiveness of cancer or tumors to non-CSC-targeting immunotherapy.

Thus, in one aspect of the invention, a composition is provided comprising a PI3K inhibitor and an immunotherapy that does not target CSCs.

2.1PI3K inhibitors

The compositions of the invention comprise inhibitors, including and comprising any agent that reduces the accumulation, function (e.g., enzymatic activity, localization, etc.) or stability of PI3K, or reduces the expression of PI3KCA genes, such inhibitors include, but are not limited to, small and large molecules, such as nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, polysaccharides, lipopolysaccharides, lipids, or other organic (carbon-containing) or inorganic molecules.

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

Representative transcripts of this type include nucleotide sequences corresponding to any one of (1) the human PI3K nucleotide sequences set forth in GenBank accession Nos. NM-006219.3, NM-001256045, NM-005026, NM-001350234 and NM-0013500235, (2) nucleotide 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 one of the sequences set forth in (1), (3) nucleotide sequences hybridizing to any one of the sequences set forth in (1) under at least low, medium or high stringency conditions, (4) nucleotide sequences encoding any one of the amino acid sequences, e.g., the human PI3K amino acid sequences set forth in UniProt accession Nos. P42336, Q8NEB9, O00710, O75747, P48736 and P42338, (5) 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 one of the sequences set forth in (4), and nucleotide sequences encoding 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 one of the sequences set forth in (4).

Exemplary antagonistic nucleic acid molecules include antisense molecules, aptamers, ribozymes, and triplex forming molecules, RNAi, and external targeting sequences. The nucleic acid molecule may act as an effector, inhibitor, modulator, and stimulator of the specific activity possessed by the target molecule, or the functional nucleic acid molecule may possess de novo activity independent of any other molecule.

Antagonistic nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, antagonistic nucleic acid molecules can interact with genomic DNA of PI3KmRNA or PIK3 genes (e.g., PIK3 CA), or they can interact with PI3K polypeptides. In general, antagonistic nucleic acid molecules are designed to interact with other nucleic acids based on sequence homology between the target molecule and the antagonistic nucleic acid molecule. In other cases, the specific recognition between the antagonistic nucleic acid molecule and the target molecule is not based on the homology of the sequence between the antagonistic nucleic acid molecule and the target molecule, but on the formation of tertiary structure allowing specific recognition to occur.

In some embodiments, antisense RNA or DNA molecules are used to directly block PI3K translation by binding to target mRNA and preventing protein translation. Antisense molecules are designed to interact with a target nucleic acid molecule through canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule can be designed to promote destruction of the target molecule, e.g., by RNAseH-mediated RNA-DNA hybridization degradation. Alternatively, the antisense molecule can be designed to interrupt a processing function, such as transcription or replication, that normally occurs on the target molecule. Antisense molecules can be designed based on the sequence of the target molecule. There are many ways to optimize antisense efficiency by finding the most accessible region of the target molecule. Non-limiting methods include in vitro selection experiments and DNA modification studies using DMS and DEPC. In a specific example, the antisense molecule binds to the target molecule with a dissociation constant (K _d) of less than or equal to 10 ^-6、10^-8、10^-10 or 10 ^-12. In particular embodiments, antisense oligonucleotides derived from the translation initiation site are used, for example, the-10 to +10 region.

An aptamer is a molecule that interacts with a target molecule in a specific manner. The aptamer is typically a small nucleic acid between 15 and 50 bases in length that folds into defined secondary and tertiary structures, such as a stem-loop or G-quadruplex. The aptamer may bind small molecules such as ATP and theophylline, as well as large molecules such as reverse transcriptase and thrombin. The aptamer can bind very tightly to K _d in target molecules smaller than 10 ^-12 M. Suitably, the aptamer binds the target molecule with a K _d of less than 10 ^-6、10^-8、10^-10 or 10 ^-12. The aptamer can bind to the target molecule with very high specificity. For example, the aptamer that has been isolated has a binding affinity that differs more than 10000 times between the target molecule and another molecule that differs only at a single location on the molecule. Desirably, K _d of the aptamer to the target molecule is at least 10, 100, 1000, 10000, or 100000 times lower than K _d of the background binding molecule. A suitable method for generating an aptamer of interest, such as PI3K, is "systematic evolution of ligand enrichment" described in the SELEX ^TM).SELEX^TM method, U.S. Pat. No. 5475096 and U.S. Pat. No. 5270163 (see also International publication No. WO 91/19813). Briefly, a mixture of nucleic acids is contacted with a target molecule under conditions conducive to binding.

In other embodiments, the anti-PI 3K ribozyme is used to catalyze the specific cleavage of PI3K RNA. The mechanism of ribozyme action involves sequence-specific hybridization of a ribozyme molecule to a complementary target RNA, followed by endo-cleavage. There are several different types of ribozymes that catalyze nuclease or nucleic acid polymerase type reactions that are based on ribozymes found in natural systems, such as hammerhead ribozymes, hairpin ribozymes, and tetrahymena ribozymes. Still other ribozymes are not found in natural systems, but they are engineered to catalyze specific reactions de novo. Representative ribozymes cleave RNA or DNA substrates. In some embodiments, a ribozyme that cleaves RNA substrates is used. Specific ribozyme cleavage sites within a potential RNA target were initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences, GUA, GUU, and GUC. Once confirmed, short RNA sequences of 15 to 20 ribonucleotides corresponding to the target gene region 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 a candidate target can also be assessed by testing its accessibility to hybridization with a complementary oligonucleotide using a ribonuclease protection assay.

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, a structure called a triplex is formed in which three DNA strands form a complex that depends on Watson Crick and Hoogsteen base pairing. Three chain molecules are preferred because they can bind to the target with high affinity and specificity. It is generally desirable that triplex forming molecules bind to target molecules with a K _d of less than 10 ^-6、10^-8、10^-10 or 10 ^-12.

An 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 that cleaves the target molecule. EGS can be designed to specifically target selected RNA molecules. RNAse P aids in processing of the transfer RNA (tRNA) within the cell. Bacterial RNAse P can be recruited to cleave almost any RNA sequence by using an EGS that mimics the target RNA, EGS complex, to a natural tRNA substrate. Likewise, eukaryotic EGS/RNAse P-directed RNA cleavage can be used to cleave a desired target within eukaryotic cells.

In other embodiments, RNA molecules that mediate RNA interference (RNAi) of PI3K genes or PI3K transcripts may be used to reduce or eliminate gene expression. RNAi refers to a product that interferes with or disrupts a target gene by introducing single-stranded or, typically, double-stranded RNA (dsRNA) homologous to the target gene transcript. RNAi methods, including double-stranded RNA interference (dsRNAi) or small interfering RNA (siRNA), have been widely documented in many organisms, including mammalian cells and caenorhabditis elegans (Fire et al, 1998.Nature 391,806-811). RNAi can be triggered in mammalian cells by a 21-23 nucleotide (nt) duplex of small interfering RNA (siRNA) (Chiu et al, 2002mol. Cell.10:549-561; elbashir et al, 2001.Nature 411:494-498), or by microRNAs (miRNAs), functional small hairpin RNAs (shRNAs) or other dsRNAs expressed in vivo using DNA templates with RNA polymerase III promoters (Zeng et al, 2002.Mol.Cell 9:1327-1333; paddison et al, 2002.Genes Dev.16:948-958; lee et al, 2002.Nature Biotechnol.20:500-505; paul et al, 2002.Nature Biotechnol.20:505-508; tuschl, T.,2002.Nature Biotechnol.20:440-448; yu et al, 2002.Proc.Natl.Acad.Sci.USA 99 (6047-6052; 2002-2002, 2002-842; mc42-556; 556, 556).

In particular embodiments, dsRNA itself, particularly dsRNA production constructs corresponding to at least a portion of the PIK3 gene, are used to reduce or eliminate its expression. RNAi-mediated inhibition of gene expression can be achieved using any of the techniques reported in the art, for example, by transfection of a nucleic acid construct encoding a stem loop or hairpin RNA structure into the genome of the target cell, or by expression of a transfected nucleic acid construct having homology to the PIK3 gene from between 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 is capable of self-folding and producing dsRNA, or as long as it produces two separate RNA transcripts that are then annealed to form dsRNA that has homology to the target gene.

RNAi does not require absolute homology, for dsRNA of about 200 base pairs, the lower threshold is described as about 85% homology (plasmid and Ketting,2000,Current Opinion in Genetics and Dev.10:562-67). Thus, depending on the length of the dsRNA, the level of homology of the nucleic acid encoding RNAi to the target gene transcript may vary, i.e., a dsRNA of 100 to 200 base pairs has at least about 85% homology to the target gene, while a longer dsRNA (i.e., 300 to 100 base pairs) has at least about 75% homology to the target gene. The RNA encoding construct expresses a single RNA transcript designed to anneal to the separately expressed RNA, or the RNA encoding construct expresses a separate transcript from the Xiang Qi promoter (convergent promoter), suitably at least about 100 nucleotides in length. RNA encoding constructs that express a single RNA designed to form a dsRNA by internal folding are typically at least about 200 nucleotides in length.

The promoter used to express the dsRNA-forming construct may be any type of promoter if the dsRNA produced is specific for targeting gene products in the disrupted cell lineage. Or 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 induced by external control factors or by intracellular environmental factors.

In some embodiments, RNA molecules of about 21 to about 23 nucleotides that direct cleavage of their corresponding specific mRNA, such as described in U.S. patent publication No. US2002/0086356, can be used to mediate RNAi. Such 21-23nt RNA molecules may contain a 3' hydroxyl group, may be single-stranded or double-stranded (as two 21-23nt RNAs), wherein the dsRNA molecule may be blunt ended or contain a overhanging end (e.g., 5', 3 ').

In some embodiments, the antagonist nucleic acid molecule is an siRNA. The siRNA may be prepared by any suitable method. For example, reference may be made to International publication WO 02/44321, which discloses siRNAs capable of sequence-specific degradation of target 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. The siRNA may be synthesized chemically or in vitro, or may be the result of intracellular processing of short double-stranded hairpin RNAs (shrnas) into siRNA. Synthetic siRNAs are typically designed using algorithms and conventional DNA/RNA synthesizers. Suppliers include Ambion (Austin, texas), chemGENs (Alsh, massachusetts), dharmacon (Lafeit, colorado), GLEN RESEARCH (Stirling, virginia), MWB Biotech (Esburgh, germany), proligo (Boerdec, colorado), and Qiagen (Netherlands Wen Tuo). The siRNA can also be synthesized in vitro by using a kit such as a SILENCER ^TM siRNA construction kit of Ambion.

Production of siRNA from vectors is typically accomplished by transcription of short hairpin RNAs (shrnas). Kits for producing vectors comprising shRNA are available, such as the GENESUPPRESSOR ^TM construction kit of Imgenex and BLOCK-IT ^TM inducible RNAi plasmids and lentiviral vectors of Invitrogen. In addition, methods of preparing and delivering siRNA to a subject are also well known in the art. See, for example, U.S. patent 2005/0282188, U.S. patent 2005/0239031, U.S. patent 2005/0234132, U.S. patent 2005/0176018, U.S. patent 2005/0059817, U.S. patent 2005/002025, U.S. patent 2004/0192626, U.S. patent 2003/0073340, U.S. patent 2002/0150936, U.S. patent 2002/0142980, and U.S. patent 2002/0101010129, each of which is incorporated herein by reference.

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. (St. Joule, california), oriGene Technologies, inc. (Rockwell, md.) and Sigma-ALDRICH PTY LTD (Kaschin, new Navigil, australia).

The invention further contemplates peptide or polypeptide based inhibitor compounds. The PI3K inhibitory peptides as described above may be modified as part of a fusion protein. The fusion protein may comprise a transporter or peptide, which functions to increase cellular uptake of the peptide inhibitor, has another desired biological effect, such as a therapeutic effect, or may have both functions. Fusion proteins can be prepared by methods known to those skilled in the art. The inhibitor peptide may be conjugated or otherwise conjugated to another peptide in a variety of ways known in the art. For example, the inhibitor peptide may be conjugated to a carrier peptide or other peptide described herein by cross-linking, wherein both peptides in the fusion protein retain their activity. As another example, a peptide may be linked or otherwise coupled from the C-terminus of one peptide to the N-terminus of another peptide through an amide bond. The linkage between the inhibitor peptide and the other member of the fusion protein may be non-cleavable, may be by a peptide bond, or may be cleaved by, for example, an ester or other cleavable bond known in the art.

In some embodiments, the transporter or peptide may be, for example, a drosophila antennapedia homeodomain derived sequence comprising amino acid sequence CRQIKIWFQNRRMKWKK [ SEQ ID NO:1], and may be linked to the inhibitor by N-terminal Cys-Cys linkage (e.g., as described in Theodore et al, 1995J. Neurosci.15:7158-7167; johnson et al, 1996.Circ.Res 79:1086). Alternatively, the inhibitor may be modified by a trans-activating regulator protein (Tat) -derived transport polypeptide from human immunodeficiency virus type 1 (amino acid positions 47-57 of Tat as shown in SEQ ID NO: 2; YGRKRRQRRR), as described in Vives et al, 1997J. Biol. Chem,272:16010-16017, U.S. Pat. No. 5,804,604 and GenBank accession number AAT48070; or by polyarginine, as described in MITCHELL ET al, 2000.J.Peptide Res.56:318-325 and Rolhbard et al, 2000.Nature Med.6:1253-1257). The inhibitors may be modified by other methods known to those skilled in the art to increase cellular uptake of the inhibitors.

The PI3K inhibitory peptide may also be introduced into a cell by introducing into the cell a nucleic acid comprising a nucleotide sequence encoding the PI3K inhibitory peptide. The nucleic acid may be in the form of a recombinant expression vector. The PI3K inhibitory peptide coding sequence may be operably linked to a transcriptional control element, such as a promoter, in an 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 is present in the cell in a free state.

Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia virus-based viral vectors, polioviruses, adenoviruses (see, e.g., li et al, invest Opthalmol Vis Sci:25432549, 1994; borras et al, gene Ther 6:515524,1999; li and Davidson, PNAS 92:770077704, 1995; sakamoto et al, H GENE THER 5:10881097,1999;WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated viruses (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:28572863, 1997; jomark 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; WO 93/09239 by Srivastava, 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, JVirol: 78127816,1999), retroviral vectors (e.g., mouse leukemia virus, spleen necrosis virus and vectors derived from retroviruses, such as Rous sarcoma virus, hav sarcoma virus, avian leukemia virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus and mammary tumor virus), and the like.

The invention also contemplates small molecule agents that reduce PI3K functional activity (e.g., reduce PI 3K-mediated phosphorylation, inhibit PI3K binding to CD44 or uPAR promoters, reduce PI3K (e.g., active PI 3K) binding to chromatin; reduce PI 3K-mediated inhibition of guanine exchange factor GIV/Girdin, reduce PI 3K-mediated inhibition of regulatory T cell function, reduce PI 3K-mediated EMT, etc.).

Suitable small molecule agents for use in the present invention that reduce PI3K functional activity include, for example, compounds selected from formula I:

And stereoisomers, geometric isomers, tautomers and pharmaceutically acceptable salts thereof, wherein:

the dashed lines represent optional double bonds, and at least one dashed line is a double bond;

X ¹ is S, O, N, NR ^a、CR¹、C(R¹)₂ or-C (R ¹⁾ ₂ O-;

x ² is C, CR ² or N;

X ³ is C, CR ³ or N;

A is a 5, 6 or 7 membered carbocycle or heterocycle fused to X ² and X ³, optionally substituted with one or more R ⁵ groups;

R ^a is s H, C ₁-C₁₂ alkyl, C ₂-C₈ alkenyl, C ₂-C₈ alkynyl, - (C ₁-C₁₂ alkylene) - (C ₃-C₁₂ carbocyclyl), - (C ₁-C₁₂ alkylene) - (heterocyclyl having 3-20 ring atoms), - (C ₁-C₁₂ alkylene) -C (=O) - (heterocyclyl having 3-20 ring atoms), - (C ₁-C₁₂ alkylene) - (C ₆-C₂₀ aryl) and- (C ₁-C₁₂ 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 ：F、Cl、Br、I、-CH₃、-CH₂CH₃、-C(CH₃)₃、-CH₂OH、-CH₂CH₂OH、-C(CH₃)₂OH、-CH₂OCH₃、-CN、-CH₂F、-CHF₂、-CF₃、-CO₂H、-COCH₃、-COC(CH₃)₃、-CO₂CH₃、-CONH₂、-CONHCH₃、-CON(CH₃)₂、-C(CH₃)₂CONH₂、-NO₂、-NH₂、-NHCH₃、-N(CH₃)₂、-NHCOCH₃、-NHS(O)₂CH₃、-N(CH₃)C(CH₃)₂CONH₂、-N(CH₃)CH₂CH₂S(O)₂CH₃、＝O、-OH、-OCH₃、-S(O)₂N(CH₃)₂、-SCH₃、-S(O)₂CH₃、 cyclopropyl, cyclobutyl, oxetanyl, morpholino and 1, 1-dioxo-thiopyran-4-yl;

r ¹、R² and R ³ are independently selected from H、F、Cl、Br、I、-CH₃、-CH₂CH₃、-C(CH₃)₃、-CH₂OH、-CH₂CH₂OH、-C(CH₃)₂OH、-CH₂OCH₃、-CN、-CF₃、-CO₂H、-COCH₃、-COC(CH₃)₃、-CO₂CH₃、-CONH₂、-CONHCH₃、-CON(CH₃)₂、-C(CH₃)₂CONH₂、-NO₂、-NH₂、-NHCH₃、-N(CH₃)₂、-NHCOCH₃、-NHS(O)₂CH₃、-N(CH₃)C(CH₃)₂CONH₂、-N(CH₃)CH₂CH₂S(O)₂CH₃、＝O、-OH、-OCH₃、-S(O)₂N(CH₃)₂、-SCH₃、-S(O)₂CH₃、 cyclopropyl, cyclobutyl, oxetanyl, morpholino and 1, 1-dioxo-thiopyran-4-yl;

R ⁴ is selected from the group consisting of C ₆-C₂₀ aryl, heterocyclyl having 3 to 20 ring atoms, and heteroaryl having 5 to 20 ring atoms, each of which is optionally substituted with one or more R ⁶ groups, said R ⁶ groups being independently selected from the group consisting of 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

R ⁵ is independently selected from C ₁-C₁₂ alkyl, C ₂-C₈ alkenyl, C ₂-C₈ alkynyl, (C ₁-C₁₂ alkylene) - (C ₃-C₁₂ carbocyclyl), - (C ₁-C₁₂ alkylene) - (heterocyclyl having 3-20 ring atoms), - (C ₁-C₁₂ alkylene) -C (=O) - (heterocyclyl having 3-20 ring atoms), - (C ₁-C₁₂ alkylene) - (C ₆-C₂₀ aryl), and- (C ₁-C₁₂ alkylene) - (heteroaryl having 5-20 ring atoms), or two paired R ⁵ groups form a 3,4,5, or 6 membered carbocycle or heterocyclyl ring wherein alkyl, alkenyl, alkynyl, alkylene, carbocyclyl, heterocyclyl, aryl, and heteroaryl are optionally substituted ：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 groups with one or more groups independently selected from the group consisting of;

mor is selected from:

Optionally substituted with one or more R ⁷ groups, said R ⁷ groups being independently selected from F、Cl、Br、I、-CH₃、-CH₂CH₃、-CH₂CH₂CH₃、-CH(CH₃)₂、-C(CH₃)₃、-CH₂OCH₃、-CHF₂、-CN、-CF₃、-CH₂OH、-CH₂OCH₃、-CH₂CH₂OH、-CH₂C(CH₃)₂OH、-CH(CH₃)OH、-CH(CH₂CH₃)OH、-CH₂CH(OH)CH₃、-C(CH₃)₂OH、-C(CH₃)₂OCH₃、-CH(CH₃)F、-C(CH₃)F₂、-CH(CH₂CH₃)F、-C(CH₂CH₃)₂F、-CO₂H、-CONH₂、-CON(CH₂CH₃)₂、

-COCH₃、-CON(CH₃)₂、-NO₂、-NH₂、-NHCH₃、-N(CH₃)₂、-NHCH₂CH₃、

-NHCH(CH₃)₂、-NHCH₂CH₂OH、-NHCH₂CH₂OCH₃、-NHCOCH₃、-NHCOCH₂CH₃、

-NHCOCH₂OH、-NHS(O)₂CH₃、-N(CH₃)S(O)₂CH₃、＝O、-OH、-OCH₃、-OCH₂CH₃、

-OCH(CH₃)₂、-SH、-NHC(＝O)NHCH₃、-NHC(＝O)NHCH₂CH₃、-S(O)CH₃、

-S(O)CH₂CH₃、-S(O)₂CH₃、-S(O)₂NH₂、-S(O)₂NHCH₃、-S(O)₂N(CH₃)₂ And

-CH₂S(O)₂CH₃。

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

the invention allows for administration of lower doses of PI3K inhibitor therapy to a subject. The ability to utilize lower doses of PI3K therapy reduces 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., a cancer solid tumor).

In some embodiments, lower amounts/doses of PI3K inhibitor reduce or minimize any undesirable side effects associated with PI3K therapy.

2.2 Immunotherapy

The compositions of the present invention also include anti-cancer therapies, which are typically immunotherapy. Any immunotherapy that does not target Cancer Stem Cells (CSCs) is generally considered suitable for use in the compositions and methods of the invention. Checkpoint inhibitor molecular antagonists and PARP inhibitors are generally considered particularly suitable.

2.2.1 Point inhibitor molecule (ICM) antagonists

Any suitable ICM antagonist useful in therapy may 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.

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.

In some preferred embodiments, the ICM antagonist included in the therapeutic composition is a PD-1 antagonist. In this regard, a "PD-1 antagonist" includes any compound or biological molecule that blocks the binding of PD-L1 (e.g., PD-L1 expressed on the surface of a cancer cell) to PD-1 expressed on an immune cell (e.g., T cell, B cell, or NKT cell). Alternative names or synonyms for PD-1 include PDCD1, PD1, CD279 and SLEB2. A representative mature amino acid sequence of human PD-1 (UniProt accession number Q15116) is as follows:

PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL[SEQ ID NO:3].

Examples of monoclonal antibodies (mabs) that bind to human PD-1 and are therefore useful in the present invention are in U.S. patent publication nos. US2003/0039653, US2004/0213795, US2006/0110383, US2007/0065427, US 2007/012378, US2012/237522, and international PCT publications nos. WO2004/072286、WO2006/121168、WO2006/133396、WO2007/005874、WO2008/083174、WO2008/156712、WO2009/024531、WO2009/014708、WO2009/114335、WO2010/027828、WO2010/027423、WO2010/036959、WO2010/029435、WO2010/029434、WO2010/063011、WO2010/089411、WO2011/066342、WO2011/110604、WO2011/110621 and WO2012/145493 (the entire contents of which are incorporated herein by reference). Specific monoclonal antibodies useful for the purposes of the present invention include the anti-PD-1 monoclonal antibodies nivolumab (nivolumab), palivizumab (palivizumab) and pilizumab (pilizumab), as well as the humanized anti-PD-1 antibodies h409Al, h409a16 and h409a17 described in international patent publication No. WO 2008/156712.

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

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. 8008449 (referred to as "5C 4"), 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.

TABLE 5

In more specific embodiments, the anti-PD-1 antigen-binding molecule comprises the heavy chain amino acid sequence of nivolumab, e.g., as shown below:

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEW

VAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCAT

NDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPv

TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP

SNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV

VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQD

WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVS

LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK[SEQ ID NO:13];

or an antigen binding fragment thereof, comprising, consisting of, or consisting essentially of the amino acid sequence:

QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEW

VAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSS[SEQ ID NO:14]。

In some of the same and other embodiments, the anti-PD-1 antigen-binding molecule may comprise a light chain amino acid sequence of nivolumab, e.g., as shown below:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ ID NO:15];

or an antigen binding fragment thereof, comprising, consisting of, or consisting essentially of the amino acid sequence:

EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIK[SEQ ID NO:16].

In alternative embodiments, the anti-PD-1 antigen-binding molecule comprises a humanized IgG4 mAb palbociclib mAb or 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.

TABLE 6

In some embodiments, the anti-PD-1 antigen-binding molecule competes with mAb palbociclib to bind PD-1.

In further embodiments, the anti-PD-1 antigen-binding molecule comprises the heavy chain amino acid sequence of palbociclib, e.g., as set forth below:

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPvTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK[SEQ ID NO:23];

or an antigen binding fragment thereof, comprising, consisting of, or consisting essentially of the amino acid sequence:

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS[SEQ ID NO:24].

similarly, the anti-PD-1 antigen-binding molecule may comprise the light chain amino acid sequence of palbociclizumab, as follows:

EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ ID NO:25];

or an antigen binding fragment thereof, comprising, consisting of, or consisting essentially of the amino acid sequence:

EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK[SEQ ID NO:26].

In other embodiments of this type, the anti-PD-1 antigen-binding molecule comprises mAb pioglitazone or an antigen-binding fragment thereof. In some related embodiments, the anti-PD-1 antigen-binding molecule comprises CDR sequences as set forth in table 7.

TABLE 7

In a more specific embodiment, the anti-PD-1 antigen-binding molecule comprises the heavy chain amino acid sequence of pilzumab, as follows:

QVQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVNTAYLQITSLTAEDTGMYFCVRVGYDALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO:27];

or an antigen binding fragment thereof, comprising, consisting of, or consisting essentially of the amino acid sequence:

QVQLVQSGSELKKPGASVKISCKASGYTFTNYGMNWVRQAPGQGLQWMGWINTDSGESTYAEEFKGRFVFSLDTSVNTAYLQITSLTAEDTGMYFCVRVGYDALDYWGQGTLVTVSS[SEQ ID NO:28].

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

EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTSYCLTINSLQPEDFATYYCQQRSSFPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ ID NO:29],

or an antigen binding fragment thereof, comprising, consisting of, or consisting essentially of the amino acid sequence:

EIVLTQSPSSLSASVGDRVTITCSARSSVSYMHWFQQKPGKAPKLWIYRTSNLASGVPSRFSGSGSGTSYCLTINSLQPEDFATYYCQQRSSFPLTFGGGTKLEIK[SEQ ID NO:30].

Other suitable monoclonal antibodies are described in International patent publication No. WO2015/026634, the entire contents of which are incorporated herein by reference. These include mAbs or antigen binding fragments thereof comprising (a) light chain CDRs (CDR 1, CDR2 and CDR3, respectively) having amino acid sequences RASKSVSTSGFSYLH [ SEQ ID NO:31], LASNLES [ SEQ ID NO:32] and QHSWELPLT [ SEQ ID NO:33] and heavy chain CDRs (CDR 1, CDR2 and CDR3, respectively) having amino acid sequences SYYLY [ SEQ ID NO:34], GVNPSNGGTNFSEKFKS [ SEQ ID NO:35] and RDSNYDGGFDY [ SEQ ID NO:36], or (b) light chain CDRs (CDR 1, CDR2 and CDR3, respectively) having amino acid sequences RASKGVSTSGYSYLH [ SEQ ID NO:37], LASYLES [ SEQ ID NO:38] and QHSRDLPLT [ SEQ ID NO:39], and heavy chain CDRs (CDR 1, CDR2 and CDR3, respectively) having amino acid sequences NYYMY [ SEQ ID NO:40], GINPSNGGTNFN EKFKN [ SEQ ID NO:41] and RDYRFDMGFDY [ SEQ ID NO:42 ].

For example, such monoclonal antibodies may comprise (a) a heavy chain variable region comprising:

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSS[SEQ ID NO:43],

Or a variant or antigen binding fragment thereof, and

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

EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIK[SEQ ID NO:44],

IVLTQSPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIYLASYLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSRDLPLTFGQGTKLEIK[SEQ ID NO:45], Or (b)

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

In further exemplary embodiments, an anti-PD-1 monoclonal antibody may comprise an IgG1 heavy chain comprising:

QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK[SEQ ID NO:47], Or a variant or antigen binding fragment thereof, and a light chain comprising any one of the following:

EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ ID NO:48],

EIVLTQSPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIYLASYLESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQHSRDLPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC[SEQ ID NO:49]

DIVMTQTPLSLPVTPGEPASISCRASKGVSTSGYSYLHWYLQKPGQSPQLLIYLASYLESGVPDRFSGSGSGTAFTLKISRVEAEDVGLYYCQHSRDLPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC[SEQ ID NO:50],

or a variant or antigen-binding fragment thereof.

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 native amino acid sequence of human PD-L1 (UniProt accession number Q9 NZQ) as follows:

MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET[SEQ ID NO:51].

Suitably, the PD-L1 antagonist is an anti-PD-L1 antigen-binding molecule. For example, anti-PD-L1 antigen binding molecules suitable for use in the present invention include anti-PD-L1 mAbs validly You Shan antibody (MEDI 4736), atilizumab (TECENTRIQ), BMS-936559/MDX-1105, MSB0010718C, LY3300054, CA-170, GNS-1480, MPDL3280A, and Avstuzumab. These and other anti-PD-Ll 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 of which are incorporated herein by reference. Other anti-PD-L1 monoclonal antibodies are described in International PCT patent publication No. WO 2016/007435, the entire contents of which are also incorporated herein by reference.

The anti-PD-L1 antigen-binding molecule suitably binds to a region of the PD-L1 extracellular domain. For example, the anti-PD-L1 antigen-binding molecule may specifically bind to a region of the extracellular domain of human PD-L1 that comprises 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 rivarotid You Shan antibody (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.

TABLE 8

In a more specific embodiment, the anti-PD-L1 antigen-binding molecule comprises the heavy chain amino acid sequence of the dulcis You Shan antibody, for example as shown below:

VQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG[SEQ ID NO:52],

Or an antigen binding fragment thereof, comprising, consisting of, or consisting essentially of the amino acid sequence:

VQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREGGWFGELAFDYWGQGTLVTVSS[SEQ ID NO:53].

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

EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ ID NO:54],

or an antigen binding fragment thereof, comprising, consisting of, or consisting essentially of the amino acid sequence:

EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLLIYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLPWTFGQGTKVEIK[SEQ ID NO:55].

or the anti-PD-L1 antigen binding molecule competes with the mAb rivarotid You Shan for binding to PD-L1.

In other embodiments, the anti-PD-L1 antigen-binding molecule comprises the fully humanized IgG1 mAb atilizumab (as described in U.S. patent No. 8217148, incorporated herein by reference in its entirety) 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 9.

TABLE 9

In a more specific embodiment, the anti-PD-L1 antigen-binding molecule comprises the heavy chain amino acid sequence of atilizumab, e.g., as shown below:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO:56], Or an antigen binding fragment thereof, comprising, consisting of, or consisting essentially of the amino acid sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS[SEQ ID NO:57].

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

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ ID NO:58],

or an antigen binding fragment thereof, comprising, consisting of, or consisting essentially of the amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK[SEQ ID NO:59].

Or the anti-PD-L1 antigen binding molecule competes with the monoclonal antibody, atilizumab, for binding to PD-L1.

In other embodiments, the anti-PD-L1 antigen-binding molecule comprises the fully humanized IgG1 mAb avermectin (as described in U.S. patent No. 8217148, incorporated herein by reference in its entirety) 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 10.

Table 10

In specific embodiments, the anti-PD-L1 antigen-binding molecule comprises the heavy chain amino acid sequence of avermectin, e.g., as shown below:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK[SEQ ID NO:60], Or an antigen binding fragment thereof, comprising, consisting of, or consisting essentially of the amino acid sequence:

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS[SEQ ID NO:61].

in some of the same and other embodiments, the anti-PD-Ll antigen-binding molecule comprises a light chain amino acid sequence of avermectin, e.g., as shown below:

QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS[SEQ ID NO:62],

or an antigen binding fragment thereof, comprising, consisting of, or consisting essentially of the amino acid sequence:

QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL[SEQ ID NO:63].

Or the anti-PD-L1 antigen binding molecule competes with avirulent for binding to PD-L1.

In some embodiments, the ICM antagonist is an antagonist of CTLA 4. Alternative names or synonyms for CTLA4 include ALPS5, CD152, CELIAC, CTLA-4, GRD4, GSE, IDDM 12. Typically, CTLA4 antagonists specifically bind to the mature amino acid sequence of human CTLA4 (UniProt accession number P16410), e.g., as follows:

KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN[SEQ ID NO:64].

Suitably, the CTLA4 antagonist is an anti-CTLA 4 antigen-binding molecule. For example, anti-CTLA 4 antigen binding molecules suitable for use in the present invention include the anti-CTLA 4 monoclonal antibodies ipilimumab (ipilimumab) (BMS-734016, MDX-010, MDX-101) and tremelimumab (tremelilimumab, CP-675206).

The anti-CTLA 4 antigen-binding molecule suitably binds to a region of the CTLA4 extracellular domain. For example, the anti-CTLA 4 antigen-binding molecule can specifically bind to a region of the human CTLA4 extracellular domain comprising any one or more of the amino acid sequences YASPGKATEVRVTVLRQA [ SEQ ID NO:65] (i.e., residues 26 to 42 of the native CTLA4 sequence 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-CTLA 4 antigen-binding molecule can specifically bind to a region of the extracellular domain of human CTLA4 comprising any one or more, preferably all, of the mature form of residues Kl, A2, M3, E33, R35, Q41, S44, Q45, V46, E48, L91, 193, K95, E97, M99, P102, P103, Y104, Y105, L106, 1108, N110.

In certain embodiments, the anti-CTLA 4 antigen-binding molecule comprises human IgG1 mAb ipilimumab (e.g., as described in international publication WO2014/209804 and U.S. patent publication No. 2015/0283234, the entire contents of which are incorporated herein by reference) or an antigen-binding fragment thereof. In a representative embodiment of this type, the anti-CTA 4 antigen binding molecule comprises the CDR sequences listed in table 11.

TABLE 11

In more specific embodiments, the anti-CTLA 4 antigen-binding molecule comprises the heavy chain amino acid sequence of ipilimumab, e.g., as follows:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK[SEQ ID NO:68], Or antigen binding fragments thereof, non-limiting examples of which include, consist of, or consist essentially of the amino acid sequences:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS[SEQ ID NO:69].

in some of the same and other embodiments, the anti-CTLA 4 antigen-binding molecule comprises a light chain amino acid sequence of ipilimumab, e.g., as shown below:

EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ ID NO:70],

Or an antigen binding fragment thereof, representative examples of which include, consist of, or consist essentially of the amino acid sequence:

EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK[SEQ ID NO:71].

In some embodiments, the anti-CTAL 4 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-CTLA 4 antigen-binding molecule comprises the CDR sequences listed in table 12.

Table 12

In more specific embodiments, the anti-CTLA 4 antigen-binding molecule comprises the heavy chain amino acid sequence of tremelimumab, e.g., as follows:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVKGRFTISRDNSKNTLYIQMNSLRAEDTAVYYCARDPRGATLYYYYYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO:172],

or antigen binding fragments thereof, non-limiting examples of which include, consist of, or consist essentially of the amino acid sequences:

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS[SEQ ID NO:73].

In some of the same and other embodiments, the anti-CTLA 4 antigen-binding molecule comprises a light chain amino acid sequence of tremelimumab, e.g., as shown below:

DIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ ID NO:74],

Or an antigen binding fragment thereof, representative examples of which include, consist of, or consist essentially of the amino acid sequence:

DIQMTQSPSSLSASVGDRVTITCRASQSINSYLDWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYSTPFTFGPGTKVEIK[SEQ ID NO:75].

in other embodiments, the ICM antagonist is a B7-H3 antagonist. In general, the B7-H3 antagonists of the invention bind specifically to the natural amino acid sequence of human B7-H3 (UniProt accession number Q5ZPR 3) as follows:

MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEENAGAEDQDGEGEGSKTALQPLKHSDSKEDDGQEIA[SEQ ID NO:76].

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 enotuzumab (enoblituzumab) or an antigen-binding fragment thereof. In some embodiments, the anti-B7-H3 antigen binding molecule comprises CDR sequences as set forth in table 13.

TABLE 13

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

VQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK[SEQ ID NO:77],

Or an antigen binding fragment thereof, representative examples of which include, consist of, or consist essentially of the amino acid sequence:

VQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAYISSDSSAIYYADTVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCGRGRENIYYGSRLDYWGQGTTVTVSS[SEQ ID NO:78].

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

DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ ID NO:79],

Or an antigen binding fragment thereof, representative examples of which include, consist of, or consist essentially of the amino acid sequence:

DIQLTQSPSFLSASVGDRVTITCKASQNVDTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPFTFGQGTKLEIK[SEQ ID NO:80].

In some alternative embodiments, the anti-B7-H3 antigen binding molecule competes with the monoclonal antibody enotuzumab to bind to B7-H3.

In other embodiments, the ICM antagonist is an indoleamine 2, 3-dioxygenase (IDO) antagonist. The mature amino acid sequence of human IDO (UniProt accession P14202) is shown below:

MAHAMENSWTISKEYHIDEEVGFALPNPQENLPDFYNDWMFIAKHLPDLIESGQLRERVEKLNMLSIDHLTDHKSQRLARLVLGCITMAYVWGKGHGDVRKVLPRNIAVPYCQLSKKLELPPILVYADCVLANWKKKDPNKPLTYENMDVLFSFRDGDCSKGFFLVSLLVEIAAASAIKVIPTVFKAMQMQERDTLLKALLEIASCLEKALQVFHQIHDHVNPKAFFSVLRIYLSGWKGNPQLSDGLVYEGFWEDPKEFAGGSAGQSSVFQCFDVLLGIQQTAGGGHAAQFLQDMRRYMPPAHRNFLCSLESNPSVREFVLSKGDAGLREAYDACVKALVSLRSYHLQIVTKYILIPAS QQPKENKTSEDPSKLEAKGTGGTDLMNFLKTVRSTTEKSLLKEG[SEQID NO:81].

Any IDO antagonist is suitable for use in the therapeutic agents of the 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), indoxime (1-methyl-D-tryptophan) and Epakadoxostat (1, 2, 5-oxadiazole-3-carboxamidine, 4- (2- ((sulfamoyl) amino) ethyl) amino) -N- (3-bromo-4-fluorophenyl) -N' -hydroxy-, (C (Z)) (-). The molecular structure of each of these molecules is provided below.

(Indoximod)

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 its ligands. For example, the mature amino acid sequence of human KIR, namely KIR2-DL1 (UniProt accession number P43626), is provided below:

HEGVHRKPSLLAHPGPLVKSEETVILQCWSDVMFEHFLLHREGMFNDTLRLIGEHHDGVSKANFSISRMTQDLAGTYRCYGSVTHSPYQVSAPSDPLDIVIIGLYEKPSLSAQPGPTVLAGENVTLSCSSRSSYDMYHLSREGEAHERRLPAGPKVNGTFQADFPLGPATHGGTYRCFGSFHDSPYEWSKSSDPLLVSVTGNPSNSWPSPTEPSSKTGNPRHLHILIGTSVVIILFILLFFLLHRWCSNKKNAAVMDQESAGNRTANSEDSDEQDPQEVTYTQLNHCVFTQRKITRPSQRPKTPPTDIIVYTELPNAESRSKVVSCP[SEQ ID NO:82].

anti-KIR antigen-binding molecules suitable for use in the present invention may be produced using methods well known in the art. Alternatively, KIR antigen binding molecules recognized in the industry may be used. For example, the anti-KIR antigen-binding molecules include fully humanized mAb Li Ruilu mAb or antigen-binding fragment thereof, e.g., as described in international publication No. WO2014/066532, the entire contents of which are incorporated herein in their entirety. Suitably, the anti-KIR antigen-binding molecule comprises the CDR regions listed in table 14.

TABLE 14

In representative embodiments of this type, the anti-KIR antigen-binding molecule may comprise the heavy chain variable domain amino acid sequence of Li Ruilu mab, e.g., as shown below:

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYAISWVRQAPGQGLEWMGGFIPIFGAANYAQKFQGRVTITADESTSTAYMELSSLRSDDTAVYYCARIPSGSYYYDYDMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPvTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK[SEQ ID NO:83], Or an antigen binding fragment thereof, representative examples of which include, consist of, or consist essentially of the amino acid sequence:

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSFYAISWVRQAPGQGLEWMGGFIPIFGAANYAQKFQGRVTITADESTSTAYMELSSLRSDDTAVYYCARIPSGSYYYDYDMDVWGQGTTVTVSS[SEQ ID NO:84].

in some of the same and other embodiments, the anti-KIR antigen-binding molecule may comprise a Li Lushan antibody light chain variable domain amino acid sequence, e.g., as set forth below:

EIVLTQSPVTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWMYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ ID NO:85],

Or an antigen binding fragment thereof, representative examples of which include, consist of, or consist essentially of the amino acid sequence:

EIVLTQSPVTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWMYTFGQGTKLEIKRT[SEQ ID NO:86].

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:

LQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLvTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPE QL[SEQ ID NO:87].

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 anti-LAG 3 humanized mAb BMS-986016. Other anti-LAG-3 antibodies are described in U.S. patent publication No. 2011/0150892 and international PCT publications nos. WO2010/019570 and WO2014/008218, the entire contents of which are incorporated herein by reference.

In some embodiments, the anti-LAG-3 antigen-binding molecule comprises a CDR sequence set forth in table 15.

TABLE 15

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:

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK[SEQ ID NO:88], Or an antigen binding fragment thereof, representative examples of which include, consist of, or consist essentially of the amino acid sequence:

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSS[SEQ ID NO:89].

similarly, the anti-LAG-3 antigen binding molecule may comprise the light chain amino acid sequence of BMS-986016, as shown in SEQ ID NO:45, as follows:

EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC[SEQ ID NO:90],

Or an antigen binding fragment thereof, representative examples of which include, consist of, or consist essentially of the amino acid sequence:

EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIK[SEQ ID NO:91].

2.2.2PARP inhibitors

Poly ADP-ribose polymerase (PARP) enzymes are a family of enzymes that cleave nad+, liberate nicotinamide and add ADP-ribose units in succession to form ADP-ribose polymers. Activation of PARP enzymes can lead to depletion of cellular nad+ levels (e.g., PARP as a consumer of nad+) and mediate cellular signaling through ADP ribosylation of downstream targets. PARP-1 is a zinc finger DNA binding enzyme that is activated by binding to DNA double or single strand breaks. It is well known that anti-alkylating agents can deplete the nad+ content of tumor cells and the discovery of PARPs explains this phenomenon (PARP Inhibitors and CancerTherapy.CurtinN.in PolyADP Ribosylation.ed.Alexander Burke,Lands Bioscience and Springer Bioscience,2006:218-233). that anti-alkylating agents induce DNA strand 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 to intracellular signals that can activate DNA repair (e.g., via the Base Excision Repair (BER) pathway) or initiate cell death in the event that DNA damage is too extensive and not able to be repaired effectively.

PARP-2 contains a catalytic domain capable of catalyzing poly ADP ribosylation reactions. PARP-2 shows similar auto-modification properties as PARP-1. The localization of this protein in the nucleus of cells in vivo may explain the residual poly-ADP-ribose synthesis observed in PARP-1 deficient cells treated with alkylating agents or hydrogen peroxide. Some drugs that inhibit PARP (e.g., drugs that are primarily intended to inhibit PARP-1) may also inhibit PARP-2 (e.g., nilaparil).

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

Preclinical ex vivo and in vivo experiments have shown that PARP inhibitors are selectively cytotoxic to tumors with homozygous inactivation of BRCA-1 and/or BRCA-2 genes, which are known to be important in the Homologous Recombination (HR) DNA repair pathway. The biological basis for the use of PARP inhibitors as single agents in cancers deficient in BRCA-1 and/or BRCA-2 is that PARP-1 and PARP-2 are essential for Base Excision Repair (BER) of damaged DNA. After single-stranded DNA breaks are formed, PARP-1 and PARP-2 bind at the lesion site, are activated, and catalyze the addition of long polymers of ADP-ribose (PAR chain) to several proteins associated with chromatin, including histones. This results in relaxation of the chromatin and rapid recruitment of DNA repair factors that can contact and repair DNA breaks. Normal cells repair up to 10000 DNA defects per day, single strand breaks being the most common form of DNA damage. Cells with BER pathway defects have an unrepaired single strand break when they enter S phase. When the replication machinery passes the breakpoint, the pre-existing single strand break is converted to a double strand break. Double strand breaks occurring in S phase are preferentially repaired by the error-free HR pathway. Cells with an inactivation of genes required for HR (e.g., BRCA-1 and/or BRCA-2) accumulate arrested replication forks in S phase and may use error-prone non-homologous end joining (NHEJ) to repair damaged DNA. Failure to complete S phase (due to replication fork arrest) and error-prone repair by NHEJ, both are considered to be responsible for cell death.

Without wishing to be bound by theory, it is hypothesized that treatment with PARP inhibitors can selectively kill subpopulations of cancer cells lacking DNA repair pathways (e.g., BRCA-1 and/or BRCA-2 inactivation). For example, tumors produced in patients with germline BRCA mutations have defective homologous recombination DNA repair pathways, and will increasingly rely on BER, a pathway blocked by PARP inhibitors, to maintain genome integrity. This concept of inducing death by blocking one DNA repair pathway in tumors with a pre-existing defect in the complementary DNA repair pathway using PARP inhibitors is known as synthetic lethality.

The therapeutic potential of PARP inhibitors is further expanded because PARP inhibitors have been observed to have monotherapy activity not only in HR deficient tumors, but also in preclinical models in combination with cisplatin, carboplatin, alkylating and methylating agents, radiation therapy and topoisomerase I inhibitors among other agents. In contrast to the rationale underlying monotherapy, where PARP alone inhibits sufficient cell death to cause HR deficient cancers (due to endogenous DNA damage), PARP is required to repair DNA damage induced by standard cytotoxic chemotherapy. In some cases, the specific role of PARP is not clear, but PARP is known to be necessary for release of captured topoisomerase I/irinotecan from DNA. Temozolomide-induced DNA damage is repaired by the BER pathway, which requires PARP recruitment repair proteins. Combination therapies that potentiate or coordinate cancer treatment without significantly increasing toxicity would provide substantial benefit to cancer patients, including ovarian cancer patients.

In some embodiments, treatment with PARP inhibitors (e.g., PARP-1/2 inhibitors) as provided herein for 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 potential defects in DNA repair. These defects make cancer cells more dependent on the remaining DNA repair pathways, and targeting these pathways is expected to have an effect on tumor cell survival rather than on normal cells.

In some embodiments, the PARP inhibitor is selected from ABT-767, AZD 2461, BGB-290, BGP 15, CEP 8983, CEP 9722, DR 2313, E7016, E7449, fluxazopanib (SHR 3162), IMP4297, INO1001, JPI 289, JPI 547, monoclonal antibody B3-LysPE conjugate, MP 124, nilaparil (ZEJULA), NU 1025, NU 1064, NU 1076, NU1085, olapanib, ONO2231, PD 128763, R503, R554, lu Kapa ni (RUBRACA), SBP 101, WW 101914, simmerrill, talazapani (BMN-673), vilippani (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.

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.

In some embodiments, the PARP inhibitor is selected from the group consisting of olaparib, nilaparib, lu Kapa n, talazapanib, veliparib, or any combination thereof. In some embodiments, the PARP inhibitor is prepared as a pharmaceutically acceptable salt. In some embodiments, the salt form may exist as a solvated or hydrated polymorph.

Thus, the PARP inhibitor may be selected from the group consisting of olaparib, nilaparib, lu Kapa n and talazapanib, or a pharmaceutically acceptable salt thereof.

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

Olaparib (AZD 2281, KU-0059436) is a potent PARP inhibitor (PARP 1,2 and 3) and is currently being developed as a monotherapy, also in combination with chemotherapy, ionizing radiation and other anti-cancer drugs, including new drugs and immunotherapy.

PARP inhibition is a novel approach to tumors that are defective in DNA repair mechanisms. PARP enzymes are critical for repair of DNA Single Strand Breaks (SSBs).

Inhibition of PARP results in the persistence of SSB, which is 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 a lack of Homologous Recombination (HRD), such as ovarian cancer in patients with a mutation in breast cancer susceptibility gene 1/2 (BRCA 1/2), cannot accurately repair DNA damage, and can be fatal to cells as DNA abnormalities accumulate. Of such tumor types, olaparib may provide a potentially effective and less toxic cancer treatment than the currently available chemotherapy regimens. Olaparib captures the inactive form of PARP at the SSB site of DNA, thereby preventing its repair.

2.3 Adjuvants

In some embodiments, the PI3K inhibitor and the ICM binding antagonist are administered concurrently with the adjuvant for the treatment or co-treatment of a T cell dysfunctional disorder. Non-limiting examples of adjuvants include cytotoxic agents, gene therapy agents, DNA therapy agents, viral therapy agents, RNA therapy agents, immunotherapeutic agents, bone marrow transplant agents, nanotherapeutic agents, or combinations of the foregoing. The adjuvant may 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 limiting agent (e.g., an agent intended to reduce the occurrence and/or severity of a therapeutic side-effect, such as an anti-emetic agent, etc.). In some embodiments, the adjuvant is a radiation therapeutic agent. In some embodiments, the adjuvant is an agent that targets the PI3K/AKT/mTOR pathway, an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and/or a chemopreventive agent. In some embodiments, the adjuvant is an immunotherapeutic agent, e.g., blocking antibody, ipilimumab (also known as MDX-010, MDX-101, or) Antagonists such as blocking antibodies, MGA271, antagonists against TGF-beta (e.g., metimab (metelimumab, also known as CAT-192), fumerino (fresolimumab, also known as GC 1008) or LY 2157299), T cells expressing Chimeric Antigen Receptor (CAR) (e.g., cytotoxic T cells or CTL), T cells containing dominant negative TGF-beta receptor (e.g., dominant negative TGF-beta type II receptor), agonists against CD137 (also known as TNFRSF9, 4-1BB or ILA), such as activating antibodies, uririnotecan (urelumab, also known as BMS-663513), agonists against CD40, such as activated antibodies, CP-870893, agonists against OX40 (also known as CD 134), such as activated antibodies, are administered in combination with anti-OX 40 antibodies (e.g., agonOX), agonists against CD27, such as activated antibodies, CDX-1127, indoleamine-2, 3-dioxygenase (IDO), 1-methyl-D-tryptophan (also known as 1-D-MT), antibody-drug conjugates (in some embodiments, containing mertansine or monomethyl auristatin E (MMAE)), anti-NaPi 2B antibody-MMAE conjugates (also known as DNIB0600A or RG 7599), trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansines or RG 7599)Genentech), DMUC5754A, an endothelin B receptor-targeting antibody-drug conjugate (EDNBR), e.g., an antibody to EDNBR that binds MMAE, an angiogenesis inhibitor, an antibody to VEGF, such as VEGF-A, bevacizumab (also known asGenentech), antibodies to angiopoietin 2 (also known as Ang 2), MEDI3617, antineoplastic agents, agents to CSF-IR (also known as M-CSFR or CD 115), anti-CSF-lR (also known as IMC-CS 4), 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, sargrastim or CD 115)) IL-2 (also known as aldesleukin or) IL-12, CD 20-targeting antibody (in some embodiments, CD 2-targeting antibody is atozumab (also known as GA101 or) Or rituximab), GITR-targeting antibody (in some embodiments, the GIRT-targeting antibody is TRX 518), in combination with an adjuvant, a TLR agonist such as Poly-ICLC (also known as "Poly-ICLC"), in some embodiments, a peptide cancer vaccine (in some embodiments, a personalized peptide vaccine; in some embodiments, a peptide cancer vaccine is a multivalent long peptide, polypeptide, peptide mixture, hybrid peptide, or peptide-driven dendritic cell vaccine (peptide-pulsed DENDRITIC CELL VACCINE, see, e.g., yamada et al, CANCER SCI,104:14-21,2013)) LPS, MPL or CpG ODN, TNF, IL-1, HMGB1, IL-10 antagonist, IL-4 antagonist, IL-13 antagonist, HVEM antagonist, ICOS agonist, e.g., by administration of ICOS-L, or an agonistic antibody to ICOS, a CX3CL 1-targeting agent, a CXCL 10-targeting agent, a CCL 5-targeting agent, LFA-1 or ICAM1 agonist, a selectin agonist, a targeted therapeutic agent, a B-Raf inhibitor, vitamin Mo Feini (also known asDarafenib (also known as) Erlotinib (also known as) MEK inhibitors, such as MEK1 (also known as MAP2K 1) or MEK2 (also known as MAP2K 2), cobratinib (also known as GDC-0973 or XL-518), trametinib (also known as) K-Ras inhibitor, c-Met inhibitor, orantuximab (also known as MetMAb), alk inhibitor, AF802 (also known as CH5424802 or Alatinib), BKM120, aidalrisi (also known as GS-1101 or CAL-101), pirifunew (also known as KRX-0401), akt, MK2206, GSK690693, GDC-0941, mTOR inhibitor, sirolimus (also known as rapamycin), temsirolimus (also known as CCl-779 or CAL-101)) Everolimus (also known as RAD 001), diphenfos (also known as AP-23573, MK-8669 or deforolimus), OSI-027, AZD8055, INK128, dual PI3K/mTOR inhibitor, XL765, GDC-0980, BEZ235 (also known as NVP-BEZ 235), 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.

In some embodiments, the adjuvant is an anti-infective drug. The anti-infective agent is suitably selected from antibacterial agents including, but not limited to, compounds that kill or inhibit the growth of microorganisms such as viruses, bacteria, yeasts, fungi, protozoa, and the like, thus including antibiotics, amoebacides, antifungals, antiprotozoals, antimalarials, antituberculosis agents, and antiviral agents. Anti-infective agents also include insect repellents and nematicides. Exemplary antibiotics include quinolone drugs (e.g., amifloxacin, cinnoxacin, ciprofloxacin, enoxacin, fluorofloxacin, flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, lomefloxacin, oxaquinic acid, pefloxacin, roxacin, temafloxacin, tosfloxacin, sparfloxacin, clinafloxacin, gatifloxacin, moxifloxacin, gemefloxacin, and ganaxoxacin mesylate), tetracycline tiredness, glycylcycline, and oxazolidinones (e.g., aureomycin, noraureomycin, doxycycline, lai Jia tetracycline, methacycline, minocycline, oxytetracycline, tetracycline, tigecycline, Linezolid, epezilamine), glycopeptides, aminoglycosides (such as amikacin, arbekacin, ding Ganjun, daptomycin, fotirmycin, gentamicin, kanamycin, neomycin, netilmicin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin), beta-lactams (e.g., imipenem, meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefathiamidine, cefazepine, ceftizoxime, cefmenoxime, cefdezine, cefonicid, cefoperazone, cefradine, cefotaxime, cefotiam, ceftizane, cefimidazole, cefpiramide, cefpodoxime ester, Cefsulodin, ceftazidime, cefteam, ceftezole ceftibuten, ceftizoxime, ceftriaxone, cefuroxime ceftibuten, ceftizoxime ceftriaxone, cefuroxime cefradine, cefmetazole (cefinetazole), cefoxitin, cefotetan aztreonam, carmomon, flomoxef, moxef, laxef aztreonam, carbomer flomoxef, moxef, laxef, Methicillin, mezlocillin, nafcillin, oxacillin, penicillin G, piperacillin, sulbenicillin, temoxicillin, 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, LY 206763), rifamycin, macrolides (such as azithromycin, clarithromycin, azithromycin, erythromycin, marcomycin, rotamycin, roseamycin, roxithromycin, and vinegared marcomycin), ketolides (e.g., telithromycin, quinicin), coumarins, lincomamides (e.g., clindamycin, lincomycin), and chloramphenicol. Exemplary antiviral agents include abacavir sulfate, acyclovir sodium, amantadine hydrochloride, amprenavir, cidofovir, dela Wei Dingjia sulfonate, didanosine, efavirenz, famciclovir, fosamil sodium, foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine/zidovudine, nelfinavir mesylate, nevirapine phosphate, oseltamivir, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir, methanesulfonate saquinavir, stavudine, valacyclovir hydrochloride, zalcitabine, zanami Wei Heji dovudine. Non-limiting examples of antimuscarinic or antimotogenic agents include atovaquone, chloroquine hydrochloride, chloroquine phosphate, metronidazole hydrochloride and pentamidine isethionate. The insect repellent may be at least one selected from the group consisting of toldazole, thiapyrimidine pamoate, albendazole, ivermectin and thiabendazole. Exemplary antifungal agents may be selected from amphotericin B, amphotericin B-cholesterol sulfate complex, amphotericin B-lipid complex, amphotericin B liposome, fluconazole, flucytosine, micronized 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. antitubercular drugs include, but are not limited to, clofazimine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine, and streptomycin sulfate.

3. Pharmaceutical composition and formulation

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 adjuvants such as those described herein.

The pharmaceutical compositions and formulations described herein may 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 th edition, osol, a.ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed, including but not limited to buffers such as phosphates, citrates and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzyl ammonium chloride, hexamethyl diammonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol or benzyl alcohol, alkyl p-hydroxybenzoates such as p-hydroxybenzoates or propyl esters, 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 non-polyethylene glycol (e.g., PEG) surfactants. Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, e.g., rHuPH20 #BaxterInternational, incorporated). Certain exemplary shasegps and methods of use, including rHuPH20, are described in U.S. patent publication nos. 2005/026086 and 2006/0104968. In one aspect, sHASEGP is conjugated to one or more additional glycosaminoglycanases, such as a chondroitinase.

In some embodiments, particularly where peptide and polypeptide active agents (e.g., antibodies, inhibitory peptides, and immunoadhesins) are involved, the active agent and optionally the 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. 6267958. Aqueous antibody formulations include those described in U.S. patent No. 6171586 and WO2006/044908, the latter formulations comprising histidine acetate buffer.

The compositions and formulations herein may also contain other active ingredients, preferably those having complementary activities without adversely affecting each other, as desired for the particular indication being treated. Such active ingredients are present in an appropriate combination in an amount effective to achieve the intended purpose.

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

Can be prepared into sustained release preparation. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Formulations for in vivo administration are typically sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

The formulations may be administered systemically or locally, depending on the particular situation being treated. For example, suitable routes may include, for example, oral, rectal, transmucosal, or intestinal administration, parenteral administration, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections. Formulations and application techniques can be found in "Remington's Pharmaceutical Sciences," Mack Publishing co., easton, pa., last edition.

4. Therapeutic use

PI3K inhibitors and ICM-binding antagonists (also referred to herein as "therapeutic combinations" or "combination therapies") are disclosed as being useful for treating T cell dysfunctional disorders in individuals having cancer, or for enhancing immune function (e.g., immune effector function, T cell function, etc.), for treating or delaying progression of cancer, or for treating infections in individuals. In particular embodiments, therapeutic combinations for treating or delaying progression of cancer (including metastatic cancer) and for preventing recurrence of cancer are disclosed. Any PI3K inhibitor and ICM binding antagonist known in the art or described herein may be used in this regard.

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

Suitably, the individual treated with the combination therapy comprises T cells (e.g. cd8+ T cells) having a mesenchymal phenotype, e.g. T cells expressing CSV, EGRF and ABCB5, soX, SNAIL and AKT1 in the same T cell, and/or having a higher level than activated T cells. The T cells may be tumor infiltrating lymphocytes or circulating lymphocytes. T cells suitably exhibit T cell depletion or failure, in representative examples of this type, the T cells express elevated levels of EOMES above TBET and/or PD-1 expression. In some embodiments, T cells have impaired or suppressed immune function and appropriately express biomarkers of reduced T cell activation (e.g., reduced production and/or secretion of cytokines such as IL-2, IFN-gamma, and TNF). Thus, TBET, PD1, and EOMES (also referred to herein as "T cell function biomarkers") can be used to determine immune function of T cells of a patient to assess T cell immune status of the patient, including sensitivity to ICM binding antagonist treatment.

In some embodiments, the subject is a human.

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

In some embodiments, the individual has a cancer that is resistant (has been demonstrated to be resistant) to one or more ICM-binding antagonists. In some embodiments, resistance to ICM-binding antagonists includes recurrence of cancer or refractory cancer. Recurrence may refer to the recurrence of cancer at the primary or new site after treatment. In some embodiments, resistance to ICM-binding antagonists includes progression of cancer during treatment with ICM-binding blockers. In some embodiments, resistance to ICM-binding antagonists includes the failure of the cancer to respond to treatment. Cancers may become resistant at the beginning of treatment or may become resistant during treatment. In some embodiments, the cancer is in an early or late stage.

In some embodiments of any of the methods, assays and/or kits, any one or more T cell functional biomarkers are detected in the sample using a method selected from FACS, western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunodetection method, HPLC, surface plasmon resonance, spectrometry, 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 of any of the methods, assays and/or kits, any one or more T cell functional biomarkers are detected in the sample by protein expression. In some embodiments, protein expression is determined by Immunohistochemistry (IHC). In some embodiments, any one or more T cell functional biomarkers are detected using antibodies that specifically bind the respective biomarker.

In some embodiments, the combination therapy of the invention comprises administration of a PI3K inhibitor and an ICM binding antagonist. The PI3K inhibitor and ICM binding antagonist may be administered in any suitable manner known in the art. For example, the PI3K inhibitor and ICM binding blocker may be administered sequentially (at different times) or simultaneously (simultaneously). 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. Thus, combination therapy may involve the administration of PI3K inhibitors alone, simultaneously or sequentially with ICM binding antagonists. In some embodiments, this may be achieved by administering a single composition or pharmacological formulation comprising both types of agents, or by simultaneously administering two separate compositions or formulations. One of the compositions comprises a PI3K inhibitor and the other composition comprises an ICM binding antagonist. In other embodiments, treatment with PI3K inhibitor may be at intervals ranging from minutes to days before or after treatment with ICM binding antagonist. In embodiments where the PI3K inhibitor is administered separately from the ICM binding antagonist, it is generally ensured that there is no expiration of a long period of time between each delivery, so that the PI3K inhibitor is still able to have a beneficial effect on the inhibited function of T cells (e.g. mesenchymal T cells) as described above, particularly to confer enhanced immune function to T cells, including sensitivity of T cells to reactivation of ICM binding blockers. In this case, it is contemplated that the two modes will be applied within about 1-12 hours of each other, more suitably about 2-6 hours. However, in some cases, it may be desirable to significantly extend the treatment time, with hours (2, 3,4, 5,6, or 7) to days (1, 2,3, 4, 5,6, 7, or 8) between each administration.

It is envisioned that multiple administrations of PI3K inhibitor or ICM binding antagonist are required. Various combinations may be employed, wherein the PI3K inhibitor is "a" and the ICM binding antagonist is "B", as follows:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/B.

The PI3K inhibitor and the ICM binding antagonist may be administered by the same route of administration or by different routes of administration. In some embodiments, the ICM binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the PI3K inhibitor is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, implantable, inhaled, intrathecally, intraventricularly, or intranasally. Effective amounts of PI3K inhibitors and ICM binding antagonists may be administered to prevent or treat a disease. The appropriate dosage of PI3K inhibitor and ICM binding antagonist may be determined according to the type of disease to be treated, the type of PI3K inhibitor and ICM binding blocker, the severity and course of the disease, the clinical condition of the individual, the medical history of the individual and the response to treatment, and the discretion of the attending physician. In some embodiments, the combination treatment with a PI3K inhibitor (e.g., GDC-0084) and an ICM binding antagonist (e.g., an anti-PD-1 antibody) is synergistic, whereby the effective dose of the ICM binding antagonist (e.g., an anti-PD-1 antibody) in the combination treatment is reduced relative to the effective dose of the ICM binding blocker (e.g., an anti-PD-1 antibody) as a single agent.

As a general proposition, whether by one or more administrations, a therapeutically effective amount of a peptide or polypeptide active agent (e.g., antibody, peptide inhibitor, immunoadhesin, etc.) administered to a human is in the range of about 0.01 to about 50mg/kg of patient body weight. In some embodiments, the antibody used is administered daily, e.g., at about 0.01 to about 45mg/kg, about 0.01 to about 40mg/kg, about 0.01 to about 35mg/kg, about 0.01 to about 30mg/kg, about 0.01% to about 25mg/kg, about 0.01 to about 20mg/kg, about 0.01 to about 15mg/kg, about 0.01 to about 10mg/kg, about 0.01g to about 5mg/kg, or about 0.01 to about 1 mg/kg. In some embodiments, the peptide or polypeptide active agent (e.g., antibody, peptide inhibitor, immunoadhesin, etc.) is administered at a dose of 15 mg/kg. However, other dosage regimens may also be useful. In one embodiment, the anti-PDL 1 antibodies described herein are administered to a human at a dose of about 100mg, about 200mg, about 300mg, about 400mg, about 500mg, about 600mg, about 700mg, about 800mg, about 900mg, about 1000mg, about 1100mg, about 1200mg, about 1300mg, or about 1400mg on day 1 of a 21-day cycle. The dose may be administered in a single dose or in multiple doses (e.g., 2 or 3 doses), such as by infusion. The dosage of peptide or polypeptide active agents (e.g., antibodies, peptide inhibitors, immunoadhesins, etc.) administered in combination therapy may be reduced as compared to monotherapy. The progress of this therapy is readily monitored by conventional techniques.

The small molecule compound is typically administered at an initial dose of about 0.0001mg/kg to about 1000mg/kg per day. Daily dosage ranges of about 0.01mg/kg to about 500mg/kg, or about 0.1mg/kg to about 200mg/kg, or about 1mg/kg to about 100mg/kg, or about 10mg/kg to about 50mg/kg may be used. However, the dosage may vary depending on the requirements of the patient, the severity of the disease being treated and the compound being used.

In any event, the dosage may be determined empirically, taking into account the type and stage of disease that a particular patient is diagnosing. In the context of the present invention, the dose administered to a patient should be sufficient to produce a beneficial therapeutic response in the patient over time. The size of the dose 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 is initiated with a smaller dose than the optimal dose of the compound. Thereafter, the dosage is increased in small increments until the optimal effect is reached under the particular circumstances. For convenience, the total daily dose may be administered in portions and divided throughout the day, if desired. The dosage may be administered daily or at intervals as determined by the attending physician. The dose may also be administered periodically or continuously over a longer period of time (weeks, months or years), for example by using subcutaneous capsules, sachets or reservoirs, or by patches or pumps. In some embodiments, the PI3K inhibitor, ICM binding antagonist, and optionally an adjuvant (e.g., a chemotherapeutic agent) are administered on a regular schedule. Or when symptoms occur, a combination therapy may be administered.

As used herein, "routine schedule" refers to a predetermined specified period of time. The routine schedule may include time periods of the same or different lengths, as long as the schedule is predetermined. For example, a routine schedule may involve daily, 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. administration of the PI3K inhibitor, ICM binding antagonist, and optional adjuvants. Or the predetermined routine schedule may involve daily administration of PI3K inhibitor, ICM binding antagonist and optional adjuvants simultaneously for the first week, followed by monthly administration for several months, and then once every three months. Any particular combination will be incorporated into the routine schedule as long as the appropriate schedule is determined in advance to relate to the administration of a certain day.

In some embodiments, the methods of treatment and uses may also include additional treatments. The additional treatment 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 of the above. 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.

Efficacy of any of the methods described herein (e.g., combination therapy, including administration of an effective amount of a combination of PI3K inhibitor, ICM-binding antagonist, and optionally an adjuvant) can be tested in various models known in the art, such as clinical or preclinical models. Suitable preclinical models are illustrated herein, which further may include, but are not limited to, ID8 ovarian cancer, GEM models, B16 melanoma, RENCA renal cell carcinoma, CT26 colorectal cancer, MC38 colorectal cancer, and claudeman melanoma cancer models.

Efficacy of any of the methods described herein (e.g., a combination therapy comprising administration of an effective amount of a combination of a PI3K inhibitor, an ICM-binding antagonist, and optionally an adjuvant) can be tested in a GEM model of developing a tumor, including but not limited to a GEM model of non-small cell lung cancer, pancreatic ductal adenocarcinoma, or melanoma. For example, mice expressing KrasG12D in the p53null background after adenovirus recombinase treatment can be used as preclinical models of non-small cell lung cancer as described in Jackson et al, 2001genes Dev.15 (24): 3243-8) (describing KrasG 12D) and Lee et al (2012Dis.Model Mech.5 (3): 397-402) (FRT-mediated p53null allele). As another example, mice expressing KrasG12D in the p16/p19null background can be used as preclinical models of pancreatic adenocarcinoma (PDAC) as described by Jackson et al (2001, supra) (describing KrasG 12D) and Aguirre et al (2003 genes Dev.17 (24): 3112-26) (p 16/p19null allele). As another example, mice with melanocytes expressing BrafV E in the background of melanocyte-specific PTENnull can be used as preclinical models of melanoma after treatment with an inducible (e.g., 4-OHT treatment) recombinase 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 were randomly recruited to treatment groups receiving PI3K inhibitors, ICM binding antagonists, and optionally adjuvant or control treatments. Tumor size (e.g., tumor volume) was measured during treatment and overall survival was monitored.

In some embodiments of the methods of the present disclosure, the cancer (in some embodiments, a sample of a patient's cancer examined using a diagnostic test, e.g., as described in the examples herein) comprises tumor-infiltrating lymphocytes (TILs), wherein the TILs are within or otherwise associated with the cancer tissue. In these embodiments, it is assessed whether TIL expresses any one or more of the T cell functional biomarkers disclosed herein. For example, TBET, PD-1 and EOMES can be used as biomarkers for T cell depletion, characterized by, for example, high levels of inhibitory co-receptors and a lack of capacity to produce effector cytokines (Wherry, E.J.2011Nature Immunology 12:492-499; rabinovich et al, 2007Annual Review ofImmunology 25:267-296).

In some embodiments of the methods of the present disclosure, the subject has T cell dysfunction, manifesting as a T cell dysfunctional disease. T cell dysfunction may be characterized by a failure of T cells or a reduced ability to secrete cytokines, proliferate or perform cytolytic activity. In some embodiments of the methods of the present disclosure, the T cell dysfunctional disorder is characterized by an inhibition of T cell immune function. In some embodiments of the methods of the present disclosure, the T cell dysfunctional disorder is characterized by T cells of the mesenchymal phenotype. In some embodiments of the methods of the present disclosure, the T cell dysfunctional disorder is characterized by T cell depletion. In some embodiments of the methods of the present disclosure, the T cells are cd4+ and/or cd8+ T cells. According to the invention, PI3K inhibitor treatment may increase the expression of biomarkers of T cell activation and effector capacity (e.g., IL-2, IFN- γ, and TNF), decrease the expression of biomarkers of T cell effector inhibition and cancer progression (e.g., ZEBl), decrease the expression of biomarkers of T cell failure (e.g., PD-1 and EOMES), and/or increase the expression of the transcription factor TBET, which increases the adaptability and production of IFN- γ in cells of the innate immune system. Notably, PI3K inhibitor treatment may enhance sensitivity of depleted T cells to ICM binding antagonist reactivation. Thus, combination therapy of PI3K inhibitors and ICM binding antagonists may increase the initiation, activation, and/or proliferation of T cells (e.g., cd4+ T cells, cd8+ T cells, memory T cells) compared to prior to administration of the combination. In some embodiments, the T cells are cd4+ and/or cd8+ T cells.

In some embodiments of the methods of the present disclosure, the activated cd4+ and/or cd8+ T cells in the subject are characterized as IFN- γ -producing cd4+ and/or cd8+ T cells and/or enhance cytolytic activity as compared to prior to administration of the combination γ. IFN-gamma can be measured by any method known in the art, including, for example, intracellular Cytokine Staining (ICS), involving cell fixation, permeation, and staining with anti-IFN-gamma antibodies. Cytolytic activity may be measured by any method known in the art, for example, cell killing assays using mixed effector cells and target cells.

In some embodiments, cd8+ T cells are characterized, for example, by the presence of CD8b expression (e.g., by RT-PCR, e.g., using Fluidigm) (CD 8b is also known as the T cell surface glycoprotein CD8 beta chain; CD8 antigen, alpha polypeptide p3'7; accession No. nm_ 172213). In some embodiments, the cd8+ T cells are from peripheral blood. In some embodiments, the cd8+ T cells are from a tumor.

In some embodiments, treg cells are characterized, for example, by the presence of Fox3P expression (e.g., by RT-PCR, e.g., using Fluidigm) (Foxp 3 is also known as fork protein P3; scurfin; foxp3delta7; immunodeficiency, multicrinopathy, enteropathy, X linkage; accession No. nm_ 014009). In some embodiments, tregs are from peripheral blood. In some embodiments, the Treg cells are from a tumor.

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

In some embodiments of the methods of the present disclosure, CD4+ and/or CD8+ T cells exhibit increased cytokine release selected from IFN-gamma, TNF, and an interleukin such as IL-2. Cytokine release may be measured by any method known in the art, for example using Westblot, ELISA or immunohistochemical assays to detect the presence of released cytokines in samples containing cd4+ and/or cd8+ T cells.

In some embodiments of the methods of the present disclosure, the cd4+ and/or cd8+ T cells are effector memory T cells. In some embodiments of the methods of the present disclosure, the cd4+ and/or cd8+ effector memory T cells are characterized as having low expression of CD44 high CD 62L. The high CD44 and low CD62L expression can be detected by any means known in the art, for example, by preparing a single cell suspension of tissue (e.g., cancer tissue) and surface staining and flow cytometry using commercial antibodies to CD44 and CD 62L. In some embodiments of the methods of the present disclosure, the cd4+ and/or cd8+ effector memory T cells are characterized by having expression of CXCR3 (also known as C-X-C chemokine receptor type 3, mig receptor, IPIO receptor, G protein-coupled receptor 9, interferon inducible protein 10 receptor, accession No. nm— 001504). In some embodiments, the cd4+ and/or cd8+ effector memory T cells are from peripheral blood. In some embodiments, the cd4+ and/or cd8+ effector memory T cells are from a tumor.

In some embodiments of the methods of the present disclosure, administering to the individual an effective amount of a PI3K inhibitor and an ICM binding antagonist, and optionally an adjuvant, is characterized by an elevated level of an inflammatory marker (e.g., CXCR 3) on cd8+ T cells, as compared to prior to administration of the combination therapy. CXCR3/CD8+ T cells may be measured by any method known in the art. In some embodiments, the CXCR3/CD 8- + T cells are from peripheral blood. In some embodiments, the CXCR3/cd8+ T cells are from a tumor.

In some embodiments of the methods of the invention, treg function is inhibited compared to prior to administration of the combination. In some embodiments, T cell depletion is reduced compared to prior to administration of the combination.

In some embodiments, the number of tregs is reduced compared to prior to administration of the combination. In some embodiments, the plasma IFN- γ levels are elevated compared to prior to administration of the combination. Treg numbers can be assessed, for example, by determining the percentage of cd4+fox3p+cd45+ cells (e.g., by FACS analysis). In some embodiments, for example, the absolute number of tregs in a sample is determined. In some embodiments, tregs are from peripheral blood. In some embodiments, tregs are from tumors.

In some embodiments, T cells are activated, and/or proliferated to increase as compared to prior to administration of the combination. In some embodiments, the T cells are cd4+ and/or cd8+ T cells. In some embodiments, T cell proliferation is detected by determining the percentage of ki67+cd8+ T cells (e.g., by FACS analysis). In some embodiments, T cell proliferation is detected by determining the percentage of ki67+cd4+ T cells (e.g., by FACS analysis). In some embodiments, the T cells are from peripheral blood. In some embodiments, the T cells are from a tumor.

5. Detection and diagnostic methods

According to the invention, PD-1, TBET and EOMES can be used to assess T cell depletion as known in the art. T cells can be obtained from patient samples containing T cells, which are suitably selected tissue samples, such as tumor and fluid samples, such as peripheral blood. In some embodiments, the sample is obtained prior to treatment with the therapeutic combination. In some embodiments, the tissue sample is formalin fixed and paraffin embedded, archived, fresh or frozen. In some embodiments, the sample is whole blood. In some embodiments, whole blood includes immune cells, circulating tumor cells, and any combination thereof.

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 functional biomarkers") can be determined qualitatively and/or quantitatively according to any suitable criteria known in the art, including, but not limited to DNA, mRNA, cDNA, proteins, protein fragments, and/or gene copy numbers. In certain embodiments, the presence and/or expression level/amount of the biomarker in the first sample is increased or elevated compared to the presence/absence and/or expression level/amount of the biomarker in the second sample (e.g., prior to treatment with the therapeutic combination). In certain embodiments, the presence/absence and/or expression level/amount of the biomarker in the first sample is reduced or decreased as compared to the presence and/or expression level/amount of the biomarker in the 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 genes are described herein.

In some embodiments of any of the methods, increased expression as compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue refers to an overall increase in the level of a biomarker (e.g., protein or nucleic acid (e.g., gene or mRNA)) detected by a standard technique known method described herein of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more. In certain embodiments, increased expression refers to an increase in the level/amount of expression 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 greater than the level/amount of expression of the corresponding biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, increased expression as compared to a reference sample, a reference cell, a reference tissue, a control sample, a control cell, a control tissue, or an internal control (e.g., housekeeping gene) 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. In some embodiments of any method, reduced expression refers to a total reduction in the level of a biomarker (e.g., protein or nucleic acid (e.g., gene or mRNA)) detected by standard techniques known methods described herein of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more as compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, reduced expression refers to a reduction in the expression level/amount of a biomarker in a sample, wherein the reduction 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 of the expression level/amount of the corresponding biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

The presence and/or expression levels/amounts of various biomarkers in a sample may be analyzed by a variety of methods, many of which are known in the art, and those of skill in the art will understand, including but not limited to immunohistochemistry ("IHC"), westernblot analysis, immunoprecipitation, molecular binding analysis, ELISA, ELIFA, fluorescence activated cell sorting ("FACS"), massaray, proteomics, quantitative blood detection (e.g., serum ELISA), biochemical enzyme activity detection, in situ hybridization, southern analysis, northern analysis, whole genome sequencing, polymerase chain reaction ("PCR"), including quantitative real-time PCR ("qRT-PCR") and other amplification type detection methods, e.g., branch DNA, SISBA, TMA, etc.), RNA-Seq, FISH, microarray analysis, gene expression profiling and/or gene expression series analysis ("SAGE"), as well as any of a wide variety of assays that may 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,CurrentProtocols In MolecularBiology,Units2 (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.

In some embodiments, the presence and/or expression level/amount of a biomarker is determined using a method comprising (a) performing a gene expression profiling analysis, PCR (e.g., rtPCR or qRT-PCR), RNA-seq, microarray analysis, SAGE, massARRAY technology, or FISH on a sample (e.g., a cancer sample of a subject), and b) determining the presence and/or expression level/amount of a biomarker in the sample. In some embodiments, microarray methods include the use of microarray chips having one or more nucleic acid molecules that can hybridize under stringent conditions to nucleic acid molecules encoding the above genes, or having one or more polypeptides (e.g., peptides or antibodies) that can bind to one or more proteins encoded by the above 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.

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 ribose probes 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 branch DNA, SISBA, TMA, etc.).

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

The optional method includes a protocol for examining or detecting mRNA (e.g., target mRNA) in a tissue or cell sample by microarray techniques. Test and control mRNA samples from the test and control tissue samples were reverse transcribed and labeled using a nucleic acid microarray to generate cDNA probes. The probes are then hybridized to a nucleic acid array immobilized on a solid support. The array is configured such that the order and location of each member of the array is known. For example, the selection of genes whose expression correlates with increased or decreased clinical benefit of anti-angiogenic therapy may be arranged on a solid support. Hybridization of the labeled probe to a particular array member indicates that the sample from which the probe was derived expresses the gene.

According to some embodiments, the presence and/or expression level/amount is measured by observing the protein expression level of the above genes. In certain embodiments, the methods comprise contacting a biological sample with a biomarker antibody described herein (e.g., an anti-PD-1 antibody, an anti-PI 3K antibody, an anti-TBET antibody, an anti-EOMES antibody) under conditions that allow for biomarker binding, and detecting whether a complex is formed between the antibody and the biomarker. Such methods may be in vitro or in vivo. In some embodiments, one or more anti-biomarker antibodies are used to select subjects eligible for combination therapy with PI3K inhibitors and ICM binding antagonists.

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

In some embodiments, expression of a T cell functional biomarker is assessed on a tumor or tumor sample. As used herein, a tumor or tumor sample may include a portion or all of the tumor area occupied by tumor cells. In some embodiments, the tumor or tumor sample may also include a tumor region occupied by tumor-associated intratumoral cells and/or tumor-associated stroma (e.g., adjacent peri-tumor connective tissue proliferation stroma). Tumor-associated intratumoral cells and/or tumor-associated stroma may include an immunoinfiltrate region immediately adjacent and/or adjacent to the primary tumor mass (e.g., tumor-infiltrating immune cells as described herein). In some embodiments, expression of a T cell functional biomarker is assessed on tumor cells. In some embodiments, expression of the T cell functional biomarker is assessed on immune cells within the tumor region (e.g., tumor infiltrating immune cells), as described above.

In an alternative method, the sample may be contacted with an antibody specific for the biomarker under conditions sufficient to form an antibody-biomarker complex, and then the complex detected. The presence of biomarkers can be detected in a variety of ways, for example by Westernblotting and ELISA procedures to analyze various tissues and samples, including plasma or serum. A variety of immunoassay techniques are available using this assay format, see for example U.S. patent nos. 4016043, 4424279, and 4018653. These include both single-site and double-site or "sandwich" assays of non-competitive types, as well as traditional competitive binding assays. These assays also include direct binding of the labeled antibodies to the target biomarker.

The presence and/or expression level/amount of a selected T cell functional biomarker in a tissue or cell sample can also be checked by functional or activity-based assays. For example, if the biomarker is an enzyme (e.g., PI 3K), an assay known in the art (e.g., a kinase assay) can be performed to determine or detect the presence of a given enzyme activity in a tissue or cell sample.

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

Alternatively, normalization may be based on the mean or median signal of all or a majority of the genes tested (global normalization method). The measured normalized amount of tumor mRNA or protein of the subject is compared to the amount of the reference set on a gene-by-gene basis. Normalized expression levels of each mRNA or protein for each tumor tested for each subject can be expressed as a percentage of the expression levels measured for the reference set. The presence and/or expression level/amount measured in a particular subject sample to be analyzed will fall within a certain percentile of that range, which can be determined by methods well known in the art.

In some embodiments, the sample is a clinical sample. In other embodiments, the sample is used in a diagnostic assay. In some embodiments, the sample is obtained from a primary or metastatic tumor. Tissue biopsies are commonly used to obtain representative tumor tissue. Or the tumor cells may be obtained indirectly in the form of a tissue or fluid known or believed to contain the tumor cells of interest. For example, a sample of a lung cancer lesion may be obtained by excision, bronchoscopy, fine needle puncture, bronchobrushing, or from sputum, pleural fluid, or blood. The gene or gene product may be detected from cancer or tumor tissue or other body samples (e.g., urine, sputum, serum or plasma). The same techniques described above for detecting a target gene or gene product in a cancer sample 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, a simple early diagnosis of these cancers can be made. Furthermore, by detecting a target gene or gene product in these body samples, the progress of the treatment can be more easily monitored.

In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or a combined plurality of samples from the same subject or individual, which are obtained at one or more different time points than when the test sample was obtained. For example, a reference sample, a reference cell, a reference tissue, a control sample, a control cell, or a control tissue is obtained from the same subject or individual at an earlier point in time 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 initial diagnosis of cancer and the test sample is subsequently obtained at the time of cancer metastasis.

In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more healthy individuals other than the subject or individual. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more individuals who are not subjects or individuals who have a disease or disorder (e.g., cancer). In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample or pooled plasma or serum sample from normal tissue of one or more non-subjects or individuals. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled RNA sample or pooled plasma or serum sample from tumor tissue of one or more individuals who are not subjects or individuals who have a disease or disorder (e.g., cancer).

In some embodiments, the sample is a tissue sample from an individual. In some embodiments, the tissue sample is a tumor tissue sample (e.g., biopsy tissue). In some embodiments, the tissue sample is lung tissue. In some embodiments, the tissue sample is kidney tissue. In some embodiments, the tissue sample is skin tissue. In some embodiments, the tissue sample is pancreatic tissue. In some embodiments, the tissue sample is stomach tissue. In some embodiments, the tissue sample is bladder tissue. In some embodiments, the tissue sample is esophageal tissue. In some embodiments, the tissue sample is mesothelial tissue. In some embodiments, the tissue sample is breast tissue. In some embodiments, the tissue sample is thyroid tissue. In some embodiments, the tissue sample is colorectal tissue. In some embodiments, the tissue sample is head and neck tissue. In some embodiments, the tissue sample is osteosarcoma tissue. In some embodiments, the tissue sample is prostate tissue. In some embodiments, the tissue sample is ovarian tissue, HCC (liver), blood cells, lymph nodes, and/or bone/bone marrow tissue. In some embodiments, the tissue sample is colon tissue. In some embodiments, the tissue sample is endometrial tissue. In some embodiments, the tissue sample is brain tissue (e.g., glioblastoma, neuroblastoma, etc.).

In some embodiments, a tumor tissue sample (the term "tumor sample" is used interchangeably herein) may include part or all of the tumor area occupied by tumor cells. In some embodiments, the tumor or tumor sample may also include a tumor region occupied by tumor-associated intratumoral cells and/or tumor-associated stroma (e.g., adjacent peri-tumor connective tissue proliferation stroma). Tumor-associated intratumoral cells and/or tumor-associated stroma may include an immunoinfiltrate region immediately adjacent and/or adjacent to the primary tumor mass (e.g., tumor-infiltrating immune cells as described herein).

In some embodiments, tumor cell staining is expressed as a percentage of total tumor cells showing any intensity of membrane staining. Invasive immune cell staining can be expressed as a percentage of total tumor area of immune cells showing any intensity staining. The total tumor area includes malignant cells and tumor-associated stroma, including the area of immune infiltration immediately adjacent and contiguous to the major tumor mass. Furthermore, the staining of infiltrating immune cells can be expressed as a percentage of all tumor infiltrating immune cells.

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.

Solid tumors include any cancer of body tissue other than the blood, bone marrow, or lymphatic system. Solid tumors can be further divided into solid tumors of epithelial origin and solid tumors of non-epithelial origin. Examples of epithelial solid tumors include tumors of the gastrointestinal tract, colon, colorectal (e.g., basal-like colorectal cancer), breast, prostate, lung, kidney, liver, pancreas, ovary (e.g., 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 (e.g., urothelial cancer, dysplastic urothelial cancer (DYSPLASTIC UROTHELIUM CARCINOMA) and transitional cell carcinoma), bladder and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors, and bone tumors. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is second-line or third-line locally advanced or metastatic non-small cell lung cancer. In some embodiments, the cancer is an adenocarcinoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., cancer 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 cancers at a second site.

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

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

In some embodiments, the T cell functional biomarker is detected in the sample using a method selected from FACS, westernblot, 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, FACS analysis is used to detect T cell functional biomarkers. In some embodiments, the T cell functional 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, the 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 of isolating/enriching such cell populations may be used, including but not limited to cell sorting. In some embodiments, PD-1 expression is reduced in a sample from an individual who is responsive 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 a blood sample.

Also provided herein are methods of monitoring the pharmacodynamic activity of an ICM binding antagonist treatment by measuring the level of expression 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 treatment exhibits pharmacodynamic activity based on the level of expression of the one or more T cell function biomedical markers in the sample obtained from the subject as compared to a reference, wherein an increase in the level of expression of the one or more T cell function biochemical markers as compared to the reference is indicative of pharmacodynamic activity for the PD-1 antagonist treatment. The methods may further comprise measuring the expression level (e.g., percentage of Treg and/or absolute number of Treg; e.g., number of cd8+ effector T cells) of one or more additional biomarkers of T cell function and/or cellular composition, wherein the additional biomarkers of T cell function comprise cytokines, e.g., IFN- γ, T cell markers, or memory T cell markers (e.g., markers of T effector memory cells), and determining that the treatment exhibits pharmacodynamic activity based on the expression level of the 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 as compared to the reference, wherein an increase in the expression level of the one or more T cell function biomarkers, one or more additional T cell function biomarkers, and/or cellular composition as compared to the reference is indicative of pharmacodynamic activity for the PD-1 antagonist treatment. The expression level of the biomarker and/or cellular composition may be measured by one or more of the methods described herein.

As used herein, "Pharmacodynamic (PD) activity" may refer to the effect of a treatment (e.g., a combination of a PI3K inhibitor and ICM binding antagonist treatment) on a subject. One example of PD activity may include modulating the expression level of one or more genes. Without wishing to be bound by theory, it is believed that in a clinical trial 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 functional biomarkers. For example, monitoring PD activity can be used to monitor response to therapy, toxicity, and the like.

In some embodiments, the expression levels of one or more marker genes, proteins, and/or cellular constituents may be compared to a reference, which may include a sample from a subject that is not receiving treatment (e.g., a combination of PI3K inhibitor treatment and ICM binding antagonist). In some embodiments, the reference may include a sample from the same subject prior to receiving the treatment (e.g., a combination of PI3K inhibitor treatment and ICM binding antagonist). In some embodiments, the reference may include reference values from one or more samples of other subjects receiving treatment (e.g., a combination of PI3K inhibitor treatment and ICM binding antagonist). For example, a group of patients may be treated and an average, mean, or median of one or more gene expression levels generated from the entire patient population. A set of samples obtained from cancers having common characteristics (e.g., the same type and/or stage of cancer, or exposure to a common treatment, such as a combination therapy of a PI3K inhibitor with an ICM-binding antagonist) may be studied from a population of people, for example, a clinical outcome study. The set may be used to derive a reference value, for example a reference value, against which a sample of the subject may be compared. Any of the reference values described herein may be used as a reference for monitoring PD activity.

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 comprise white blood cells. 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. Tumor samples may include cancer cells, lymphocytes, leukocytes, interstitials, blood vessels, connective tissue, basal lamina, and any other cell type associated with a tumor. In some embodiments, the sample is a tumor tissue sample containing tumor-infiltrating leukocytes. In some embodiments, the sample may be processed to isolate (separate) or to isolate (isolate) one or more cell types (e.g., white blood cells). In some embodiments, the sample may be used without isolating or separating the cell type.

Tumor samples may be obtained from a subject by any method known in the art, including but not limited to biopsy, endoscope, or surgery. In some embodiments, the tumor sample may be prepared by methods such as freezing, fixing (e.g., by using formalin or similar fixative), and/or embedding in paraffin. In some embodiments, a tumor sample may be sectioned. In some embodiments, fresh tumor samples (i.e., samples that have not been prepared by the methods described above) may be used. In some embodiments, tumor samples may be prepared by incubation in solution to preserve mRNA and/or protein integrity.

In some embodiments, the sample may be a peripheral blood sample. The peripheral blood sample may include white blood cells, PBMCs, and the like. Any technique known in the art for separating leukocytes from a peripheral blood sample may be used. For example, a blood sample may be withdrawn, red blood cells lysed, and white blood cell particles separated and used as a sample. In another example, density gradient separation can be used to separate leukocytes (e.g., PBMCs) from erythrocytes. In some embodiments, a fresh peripheral blood sample (i.e., a sample that has not been prepared by the above-described method) may be used. In some embodiments, the peripheral blood sample may be prepared by incubation in solution to maintain mRNA and/or protein integrity.

In some embodiments, responsiveness to treatment may refer to any one or more of extending survival (including total survival and progression free survival), leading to an objective response (including complete or partial response), or ameliorating signs or symptoms of cancer. In some embodiments, reactivity may refer to an improvement, i.e., remission, stabilization, or progression, of one or more factors used to determine the status of a tumor in a cancer patient according to a published set of RECIST guidelines. For a more detailed discussion of these guidelines, see Eisenhauer et al, (2009Eur J Cancer45:228-47), topalian et al (2012N Engl J Med 366:2443-54), wolchok et al (2009Clin Can Res 15:7412-20) and Therasse et al (2000J.Natl.Cancer Inst.92:205-16). A reactive subject may refer to a subject in which the cancer exhibits improvement (e.g., according to one or more factors based on RECIST criteria). A non-reactive subject may refer to a subject in which the cancer does not exhibit improvement (e.g., according to one or more factors of RECIST criteria).

Conventional response criteria may not be sufficient to characterize the anti-tumor activity of the therapeutic agents of the invention, which may produce delayed responses, and initial apparent imaging progress, including the appearance of new lesions, may occur prior to the delayed responses. Thus, modified response criteria have been developed that take into account the possible occurrence of new lesions and allow for the confirmation of imaging progression in subsequent evaluations. Thus, in some embodiments, reactivity may refer to an improvement in one or more factors according to immune related response criteria (irRC). See, for example, wolchok et al (2009, supra). In some embodiments, new lesions are counted in a defined tumor burden and follow-up, e.g., confirmation of imaging progression in a subsequent assessment. In some embodiments, the presence of non-target lesions is included in the assessment of complete remission, and not in the assessment of imaging progression. In some embodiments, imaging progression may be determined based solely on measurable disease, and/or may be confirmed by continuous assessment for ≡4 weeks from the date of first recording.

In some embodiments, the reactivity may include immune activation. In some embodiments, the reactivity may include therapeutic efficacy. In some embodiments, reactivity may include immune activation and therapeutic efficacy.

6. Kit for detecting a substance in a sample

In other aspects of the invention, therapeutic kits comprising a PI3K inhibitor and an ICM binding antagonist are provided. In some embodiments, the therapeutic kit further comprises instructions comprising instructions for simultaneous administration of the PI3K inhibitor and the ICM-binding antagonist to treat a T cell dysfunctional disorder, or to enhance immune function (e.g., immune effector function, T cell function, etc.), or to treat or delay progression of cancer, or to treat an infection in an individual suffering from cancer. Any of the PI3K inhibitors and ICM binding antagonists described herein or known in the art may be included in a kit.

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 may be made of a variety of materials such as glass, plastic (e.g., polyvinyl chloride or polyolefin) or metal alloys (e.g., stainless steel or hastelloy). In some embodiments, the container contains the formulation and the label on or associated with the container may indicate instructions for use. The kit may also include other materials as desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructional materials for use. In some embodiments, the kit further comprises one or more additional agents (e.g., chemotherapeutic agents and antineoplastic agents). Suitable containers for one or more reagents include, for example, bottles, vials, bags, and syringes.

In other embodiments of the invention, diagnostic kits for determining biomarker expression are provided, including the T cell functional biomarkers disclosed herein, comprising reagents that allow detection and/or quantification of the biomarkers. Such agents include, for example, compounds or materials, or groups of compounds or materials, that allow for the quantification of the biomarker. In particular embodiments, the compound, material or group of compounds or materials allows for determining the expression level of a gene (e.g., a T cell functional biomarker gene), including but not limited to extracting RNA material, determining the level of the corresponding RNA, etc., primers for synthesizing the corresponding cDNA, primers for amplifying DNA and/or probes capable of specifically hybridizing to RNA (or corresponding cDNA) encoded by the gene, taqMan probes, proximity analysis probes (proximity assayprobe), ligases, antibodies, etc.

The kit may also optionally include suitable reagents for detecting labels, positive and negative controls, wash solutions, blotting membranes, microtiter plates, dilution buffers, and the like. For example, a nucleic acid-based detection kit may include (i) a T cell functional biomarker polynucleotide (which may be used as a positive control), (ii) a primer or probe that specifically hybridizes to the T cell functional biomarker polynucleotide. Enzymes suitable for amplifying nucleic acids are also included, including various polymerases (reverse transcriptases, tag, sequenase ^TM, DNA ligases, etc., depending on the nucleic acid amplification technique employed), deoxynucleotides, and buffers to provide the reaction mixture required for amplification. Such a kit will also typically include different containers for each individual reagent and enzyme, as well as each primer or probe, in an appropriate manner. Alternatively, the protein-based assay kit may comprise (i) a T cell functional biomarker polypeptide (which may be used as a positive control), (ii) an antibody that specifically binds to the T cell functional biomarker polypeptide. The kit may also have various means (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 functional 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 embodiments or assays described below, such as RT-PCR or Q-PCR techniques described herein.

Materials suitable for packaging the diagnostic kit components may include crystals, plastics (polyethylene, polypropylene, polycarbonate, etc.), bottles, vials, papers, envelopes, etc. Furthermore, the kit of the present invention may comprise instructional materials for simultaneously, sequentially or separately using the different components contained in the kit. The instructional material may be in the form of a printed material or in the form of an electronic support capable of storing instructions so that the subject can read, for example, an electronic storage medium (magnetic disk, tape, etc.), an optical medium (CD-ROM, DVD), etc.

Alternatively or additionally, the medium may contain an internet address that provides instructional material.

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

Examples

Example 1

PI3K expression is associated with reduced cancer survival.

PI3KCA is part of the cytoplasmic/plasma membrane PI3KCA/AKT/MTOR signaling pathway (Hassan et al, 2013). PI3KCA mutant burden is associated with cancer progression through the AKT pathway (playing a role in promoting invasion and metastasis) (Jiang et al 2020). Recent studies have shown that the PI3KCA signaling pathway is associated with Cancer Stem Cells (CSC) and EMT (Chen et al 2020; xia et al 2015). PI3KCA plays a role in immune evasion by phosphorylating AKT (P) to induce PDL1 expression and mediating immune evasion (Yang et al, 2017). 35% of breast cancer patients have mutation-positive tumors (Fusco et al, 2021), and about 6% of patients have PI3KCA mutations in TNBC (Mosele et al, 2020). This does not account for relapse or immunotherapeutic tolerance in most patients.

The inventors plotted the overall survival probability of PI3K low and high protein expression using a network tool KM plotter (Lanczky and Gyorffy, 2021) (fig. 1). This analysis shows that the survival rate of PI3K high expression cohorts is significantly reduced in metastatic breast cancer and non-small cell lung cancer patient cohorts. This analysis was intended to demonstrate that PI3K enrichment was associated with a significant decrease in survival probability for a variety of cancer types.

Stratification based on liquid biopsies detects enrichment of PI3K CSCs and CD 8T cell characteristics and PI3K target CSC relapse characteristics and depletion of T cell characteristics in liquid biopsies of patients.

The inventors identified dual CSC and T cell characteristics in a liquid biopsy of patients from a stage IV solid tumor cohort (i.e., melanoma, NSCLC, RCC, HCC, breast cancer, GBM). Using our novel liquid biopsy platform, a novel PI 3K-enriched cancer stem cell, stem cell-like mesenchymal signature was identified in stage IV cancer patients (fig. 2). These CSCs are critical cell types for cancer recurrence and are not targeted by chemotherapy, immunotherapy, or radiotherapy (i.e., conventional cancer treatment). However, the inventors hypothesize that these patients will respond to the monotherapy and/or the combination therapy methods of the invention. These CTCs were positive for the mesenchymal markers, CSC markers CSV and ABCB 5.

The inventors have also identified a novel T cell depletion/dysfunction profile enriched in checkpoint proteins (TIM 3, TIGIT, PD 1) and depletion markers (TOX 1, EOMES).

Analysis showed that the overall expression intensity measured using immunofluorescence analysis of CSV and ABCB5 using the digital pathology system was significantly reduced in GDC-0084 treated samples (fig. 3A). In contrast, epCAM is a marker of non-mesenchymal epithelial cells, whose expression changes little (fig. 3A). Population dynamics analysis using the digital pathology system showed a reduction of approximately 56% in CSV and ABCB5 positive cell populations, while EpCAM increased by 38% (fig. 3B). Inhibition of PI3KCA by GDC-0084 also inhibited the percentage of AKT1 positive cells (AKT 1 contributes to the mesenchymal transfer characteristics) and overall expression of AKT1 (AKT 1 is a key tumorigenic marker for PI3KCA modulation). This suggests that targeting PI3KCA also targets metastasis, mesenchymal transformation and key modulation of cancer stem cell characteristics (CSCs).

Liquid biopsy-based assays from stage IV solid tumor cancer patients indicate that PI3K inhibition inhibits T cell dysfunction and induces effector features

The overall expression intensity of cd8+ T cells was measured by immunofluorescence analysis using the digital pathology system of the inventors laboratory. The depletion group showed that GDC-0084 significantly inhibited expression of EOMES and PD1 in NS and ST samples (fig. 4A). The immune checkpoint inhibitor group showed that GDC-0084 treatment had significant inhibition of TIM3 in both NS and ST cd8+ T cells, whereas GDC-0084 significantly inhibited TIGIT only in NS cd8+ T cells (fig. 4B). FIG. 4C shows the effector feature set, in contrast to FIGS. 4A and 4B, showing a significant increase in perforin and GZNb expression in CD8+ T cells treated with GDC-0084 from both the NS and ST groups.

PI3K is enriched in cancer cell lines with enriched mesenchymal CSC characteristics.

Whole cell intensity analysis was performed according to standardized settings using ASI digital pathology system. Analysis of PI3KCA expression in breast cancer cell lines (fig. 5A) revealed a pattern of increased expression, i.e., the cell line was transformed from epithelial characteristics to more mesenchymal, metastatic and immunotherapeutic-resistant characteristics (MCF 7 is a breast cancer epithelial cell line with a low CSC population, MDA-MB-231 is a TNBC cell line with >90% mesenchymal CSC, MDRA-MB-231-BR is a brain metastatic cancer clone with MDA-MB-231 with >90% mesenchymal stem cells, and finally 4T1 is a breast cancer model with >90% CSC-resistant, metastatic high invasive).

Interestingly, similar patterns also appeared for H1299 (epithelial lung cancer), CT26 (IO-reactive, epithelial colon cancer) and LLC (highly invasive, metastatic lung cancer model) (fig. 5B). Both H1299 and CT26 are epithelial-like immunogenic cell lines, whereas LLC is highly resistant to immunotherapy and aggressive. Overall, these data indicate that PI3KCA is enriched in metastatic, drug-resistant cancer cell lines from the perspective of whole cell expression. This suggests the importance of targeting PI3KCA for the treatment of metastatic, drug-resistant cancers, a significant unsolved challenge.

MCF7 epithelial breast cancer cell line;

MDAMB231 mesenchymal/CSC breast cancer cell line;

MDAMB231-Br brain cancer version MDA-MB-231;

4T1 highly invasive, IO resistant mouse breast cancer model;

an epithelial-like lung cancer cell line;

CT26 epithelial-like, highly immunogenic mouse colon tumor cell line, and

LLC, a highly resistant Lewis lung cancer cell line, resistant to immunotherapy (Li et al 2021).

Effects of the PI3K inhibitor GDC-0084 on MDA-MB-231 and CT26 cell migration and invasion.

Interestingly, the PI3K/Akt/mTOR pathway is activated in up to 70% of breast cancer brain metastasis patients. Up-regulation of this pathway is associated with a poorer prognosis, however, no approved drug is currently available for these patients. GDC-0084 is a PI3K inhibitor, a brain penetrating agent, showing promising activity in preclinical models of glioblastoma. GDC-0084 inhibited breast cancer cell migration at IC ₂₅ (1.25. Mu.M) and IC ₅₀ (2.5. Mu.M), with IC ₂₅ concentrations having greater inhibition of MDA-MB231 cell migration (FIG. 6). Wound healing analysis showed that CT26 cell migration was inhibited after GDC-0084 (PI 3K inhibitor) treatment. DMSO treatment was used as control. These data show that 0.3 μm GDC-0084 has a more pronounced effect on wound healing than the lower concentration (i.e., 0.2 μm) (fig. 6C). This suggests that GDC-0084 is able to inhibit cancer cell migration at higher concentrations. GDC-0084 has the greatest inhibitory effect on the responsive colon cancer cell line CT 26. The inventors used DMSO treatment as a control.

The IC ₅₀ of GDC-0084 was assayed against different cancer cell lines.

The data clearly show the EC ₅₀ data for GDC-0084 in different cell lines. Individual dose response curves for CT26, 4T1, MDA-MB-231 and MDA-MB-231-Brm are shown in FIG. 7 as MCF7. These data were obtained to demonstrate the efficacy of GDC-0084 in targeting PI3KCA in an immunotherapy responder or an epithelial cancer cell line (CT 26, MCF 7) compared to an immunotherapy-resistant or mesenchymal cancer cell line (4 t1, mda-MB-231 cell line). These data indicate that GDC-0084 targeting PI3 ks is capable of significantly inhibiting proliferation of epithelial and mesenchymal cancer cell lines.

PARP inhibits the effects on mesenchymal and CSC characteristics.

Olaparib and Uliptinib are PARP inhibitors and have been used to treat certain types of breast and ovarian cancers. However, olaparib did not target Cancer Stem Cells (CSC) or mesenchymal characteristics, nor did it affect ABCB5 or ALDH1A (fig. 8). The inventors next studied the effect of olaparib and veliparib on CSC characteristics or proliferation of the mesenchymal TNBC cell line MDA-MB-231, neither of which inhibited CSC characteristics or proliferation (fig. 9). Next, to further investigate the importance of PARP single drug therapy targeting, the inventors studied the effect of siRNA knockout of PARP (fig. 10) on mesenchymal, CSC characteristics. PARP knockdown was identified to have no inhibitory effect on these markers of mesenchymal and CSC characteristics (fig. 11). Combination therapy of the PI3K pathway inhibitor GDC-0084 and the PARP inhibitor olaparib limited migration of MDA-MB-231 cells (fig. 17). The inventors demonstrated that the combination of GDC-0084 and Olaparib inhibited breast cancer cell migration, with combination "2" (i.e., 1.25. Mu.M GDC-0084+250nM Olaparib) having the greatest inhibitory effect on MDA-MB-231 migration. This suggests that PARP inhibitors are not effective single therapeutic agents, but may be beneficial in combination with PI3K inhibitors.

Αpd1 therapy in colon cancer CT26 model and breast cancer 4T1 model.

The inventors tried to demonstrate that αpd1 single drug treatment resulted in a significant reduction in tumor volume in the immunotherapy-responsive CT26 colon cancer model compared to the 4T1 breast cancer model (fig. 12). These data indicate that CT26 is an immunotherapeutic response line, while the 4T1 model clearly indicates that 4T1 is an immunotherapeutic tolerance model, as demonstrated by its efficacy on primary tumor burden of αpd1 immunotherapy.

Pi3K inhibitors target and inhibit expression of mesenchymal drug-resistant markers

Next, the inventors demonstrated that treatment with GDC-0084 significantly inhibited CSC/mesenchymal markers, induced expression of the epithelial marker EpCAM, and also increased expression of the viral mimicking/immune visibility marker MDA5 (fig. 13 and 14). This suggests that targeting PI3KCA can also inhibit metastasis and treatment tolerance characteristics and make tumors more immune-visible and thus more responsive to combination therapy with immunotherapy.

Next, the inventors performed further inhibition assays on different inhibitors targeting PI3K to confirm the effectiveness of targeting PI3K to inhibit mesenchymal CSC features. Two different PI3K inhibitors were used (fig. 15), idary has been used to treat certain hematological cancers, such as chronic lymphocytic leukemia after relapse, LY294002 is a potent inhibitor of PI3K, and is currently tested in patients with recurrent or progressive HNSCC and patients with mutations in PI3KCA and/or PI3K pathway genes. Treatment with either of the two PI3K inhibitors (fig. 15) at two different low doses significantly inhibited CSC/stem-like cancer recurrence markers CSV, soX9, ABCB5 and SNAIL. The effect of idarubicin at a dose of 12.5 μm was least pronounced. Furthermore, all 25 μm of idarubis and two concentrations of LY294002 induced expression of the epithelial marker EpCAM. This data demonstrates that targeting PI3 ks with alternative inhibitors of GDC-0084 is effective in inhibiting mesenchymal cancer stem cell characteristics and inducing epithelial characteristics.

The administration of 7.5mg/kg GDC-0084 reduces clinical abnormalities in 4T1 syngeneic tumor models.

Next, the inventors demonstrated that single drug treatment with GDC-0084 at 7.5mg/kg reduced the clinical abnormalities observed in the 4T1 breast cancer model. Groups of mice receiving high doses (i.e., 15 mg/kg) exhibited multiple cases of reduced activity, humpback posture and pronounced hair erections (fig. 16). Administration of GDC-0084 at 7.5mg/kg reduced the clinical abnormalities previously observed at higher doses compared to 15 mg/kg. Although 40% of mice treated with 15mg/kg GDC-0084 showed reduced activity, humpback posture, vertical hair and weight loss, no clinical abnormalities were seen in the 7.5mg/kg treated group. These data underscores the reduced toxicity of lower doses.

GDC-0084 was administered at a dose of 7.5mg/kg to eliminate tumor burden.

The inventors next demonstrated that "low dose" GDC-0084 treatment (7.5 mg/kg) eliminated primary tumor burden in the 4T1 breast cancer model (see FIG. 17). In the 4T1 breast cancer model, GDC-0084 single drug therapy at 7.5mg/kg and co-administration with αpd1 significantly reduced tumor volume, resulting in a reduction in tumor weight of more than 50%. Surprisingly, no significant reduction in tumor burden was observed in mice administered with 15mg/kg GDC-0084, with or without the administration of alpha PD 1.

GDC-0084 administration at a dose of 7.5mg/kg reduced tumor inflammation, including mononuclear cell and neutrophil infiltration.

Administration of "low dose" GDC-0084 (i.e., 7.5 mg/kg) reduced tumor inflammation, including mononuclear and neutrophil infiltration (FIG. 18). The single treatment with GDC-0084 at 7.5mg/kg and the combined administration with alpha PD1 significantly reduced tumor inflammation associated with the 4T1 breast cancer model. Although the control and αpd1 treated groups scored "moderate" and "severe" tumor inflammation, only mild inflammation was observed when administered in combination with GDC-0084 at a dose of 7.5 mg/kg.

Furthermore, pathologists noted a substantial decrease in myeloid cell populations (including monocytes and neutrophils) in the treatment group administered 7.5 mg/kgGDC-0084. Notably, no significant reduction in inflammation was observed in mice administered 15mg/kg GDC-0084 per day.

In the 4T1 syngeneic tumor model, administration of GDC-0084 at a dose of 7.5mg/kg reduced splenomegaly.

Administration of GDC-0084 at 7.5mg/kg reduced splenomegaly in the 4T1 breast cancer model. Spleen size and appearance were normal at low doses of GDC compared to high doses (fig. 19). GDC administration of 7.5mg/kg reduced splenomegaly normally associated with the 4T1 breast cancer model.

Although there was no significant difference in spleen weights between the 15mg/kg treated group and the corresponding control group, a significant reduction in splenomegaly was observed at the lower dose of GDC-0084 (7.5 mg/kg), either as monotherapy or in combination with alpha PD 1.

GDC-0084 was administered at a dose of 7.5mg/kg to reduce SOX9 and TOX1 expression.

The combination treatment in both experiments significantly reduced the expression of SOX9 (SOX 9 is a marker of mesenchymal, treatment-tolerant cancer stem cells), however, GDC-0084 at 7.5mg/kg had a more significant effect in the combination treatment and was especially reduced when treated as a single drug. Interestingly, 15mg/kg GDC-0084 showed induction of TOX1 in CD8+ T cells (TOX 1 is a marker of T cell depletion and dysfunction), whereas at 7.5mg/kg TOX1 was significantly reduced in CD8+ T cells (FIG. 20). These data indicate that "high dose" GDC-0084 induces deregulated inflammation and increases T cell dysfunction (higher expression of TOX 1), and is unable to control SOX9, SOX9 being a marker of cancer stem cell-like mesenchymal characteristics, metastasis and cancer recurrence. In contrast, "low dose" GDC-0084 as monotherapy or in combination significantly inhibited SOx9+ expression and significantly reduced TOX1 expression in CD8+ T cells. Notably, the combination of anti-PD 1 and "low dose" GDC-0084 (7.5 mg) was most effective in eliminating TOX1 expression (fig. 20).

PI3K single or combined inhibition targets mesenchymal characteristics and induces an epithelial phenotype in an IO-resistant mouse model.

Combination therapy with GDC-0084 and anti-PD 1 significantly inhibited PI3KCA, the mesenchymal markers CSV and EGFR, and this combination therapy also induced better expression of the epithelial marker E-cadherin (fig. 21).

Both GDC-0084 monotherapy and combination therapy comprising GDC-0084 and anti-PD 1 significantly induced the epithelial marker E-cadherin positive cell population (see FIG. 22). GDC-0084 with or without alpha PD1 treatment had no effect on mouse body weight in the 4T1 syngeneic tumor model (FIG. 17). Combination therapy of GDC-0084 with αpd1 significantly inhibited tumor burden, but high doses of single drug failed, while low doses of single drug inhibited tumor burden (fig. 17). Notably, this combination is also applicable to 4T1 immunotherapeutic resistant tumor models. Combination therapy with GDC-0084 and anti-PD 1 inhibitors significantly inhibited PI3KCA and cancer stem cell-like mesenchymal markers CSV and EGFR, inducing expression of the epithelial marker E-cadherin (fig. 21). Remarkably, GDC-0084 single drug treatment or combination treatment with GDC-0084 and an anti-PD 1 inhibitor significantly induced the epithelial marker E-cadherin positive cell population (fig. 22). These data indicate that PI3KCA inhibitors in combination with immunotherapy can significantly affect tumor burden and can reprogram cancer cells or tumor microenvironment features to more epithelial and treatment responsive features than immunotherapy alone. This suggests that this is a viable treatment that enhances the sustained response of immunotherapy.

PI3K single or combination therapy induces effector and TRM characteristics.

The combination therapy significantly increased infiltration of cd8+ ifnγ+ into the tumor over the monotherapy (fig. 23A). Furthermore, the proportion of cd8+ T cells expressing the T _RM marker (i.e. CD44, CD103, CD 69) was also significantly increased in the combination treatment group in the total CD 8T cell population, which induced more potent, compared to PI3K inhibitor monotherapy (fig. 23B). The combination treatment also significantly increased infiltration of cd8+ ifnγ+ T cell positive cells into the tumor (fig. 23A). Furthermore, the proportion of cd8+ T cells in the combined set expressing the T _RM marker (i.e. CD44, CD103, CD 69) was also significantly increased in the total CD 8T cell population (fig. 23B). The assay was performed to see if PI 3K-inhibiting monotherapy or combination immunotherapy reprogrammed immune cell characteristics other than cancer cell characteristics. The combined treatment can obviously influence and induce immune effector characteristics and improve the anti-tumor immunity.

PI3K alone or in combination therapy inhibits checkpoint depletion features in cd8+ T cells.

Combination treatment with GDC-0084 and anti-PD 1 inhibitors significantly reduced the number of TIM3 and LAG3 positive cd8+ T cells compared to PD1 or GDC-0084 monotherapy alone (fig. 24). This test was performed in order to investigate whether the characteristics of dysfunctional immune cd8+ T cells were reprogrammed. Analysis shows that combination therapy can significantly reduce the characteristics of immune dysfunction.

The combination of PI3K inhibitor and immunotherapy eliminates metastatic spread in the 4T1-IO tolerance model.

Combination therapy of GDC-0084 with αpd1 significantly inhibited metastatic tumor burden, but single drugs were not. This combination was also effective in 4T1 immunotherapy-resistant tumor models (fig. 25). These data demonstrate that PI3K inhibitors in combination with immunotherapy can significantly affect tumor burden and can reprogram cancer cells or tumor microenvironment features to become more therapeutically responsive in an immunotherapy-tolerant mouse breast cancer model (4T 1) and eliminate metastatic spread of cancer to the lung, as compared to immunotherapy alone. This suggests that this is a viable treatment that can enhance the sustained response of immunotherapy.

Discussion of the invention

The inventors have obtained a new approach that can reduce the overall toxicity of PI3K inhibitors (e.g., GDC-0084) and increase the overall efficacy of the treatment. This is achieved by administering reduced GDC-0084, i.e., 7.5mg (normal dose is 15mg at a single time), which reduces overall adverse side effects and increases the overall efficacy of GDC-0084 therapy. Furthermore, the inventors have found that the efficacy of PI3K inhibitors in combination with immunotherapy is also enhanced. Therapeutic administration of low concentrations of GDC-0084 in combination with immunotherapy may further reduce the dosage of immunotherapy (e.g., anti-PD 1), thereby reducing adverse events and improving overall efficacy with significantly reduced dosages.

Summary of efficacy of monotherapy and combination therapy

Lower doses of monotherapy inhibited checkpoint proteins (FIG. 4: TIGIT, tim3, PD 1) and induced effector features (FIG. 4). Reprogramming depletion feature (EOMES) fig. 4.

Lower doses of monotherapy were significantly less clinically abnormal (fig. 12).

Lower doses of monotherapy GDC-0084 were able to significantly inhibit tumor weight (fig. 13).

However, combination therapy with lower doses showed a feature of sustained tumor control. Furthermore, the low dose combination therapy was more effective in inhibiting inflammation (fig. 14 and 15).

The combination therapy at lower doses was more effective in inhibiting cancer stem cells (important for recurrence and metastasis), reprogramming and inhibiting dysfunctional T cell characteristics and inducing effector T cell characteristics (fig. 16, 19-20).

The "low dose" combined treatment with immunotherapy was more effective in inducing epithelial characteristics than the "high dose" or "effective dose" (fig. 17-18). The combination therapy was also superior in inhibiting metastasis (fig. 21).

Administration at 15mg/kg represents a high dose of GDC-0084 (i.e., clinically, humans typically administer 20-60 mg), with our low dose accounting for only 50% of this dose. 25% of the original dose is also expected to be effective as single and combination drug.

Materials and methods

Unless otherwise indicated, all materials and reagents used to synthesize and test the compositions are commercially available from Sigma-Aldrich corporation, novabiochem, abeam and American Type Culture Collection (ATCC).

In vivo animal studies.

5 Female BALB/c mice per treatment group were used for xenograft studies. 4T1 breast cancer cells (1X 10 ⁵) were injected into the fourth pair of breast fat pads in the right inguinal region of six-week-old BALB/c nude mice. For 4T1 tumor cell injection, 1×10 ⁵ cells prepared with PBS were used per mouse.

For the CT26 colon cancer mouse model, 5X 10 ⁵ cells were injected into the abdomen of 6-week-old BALB/c nude mice by intraperitoneal injection. Tumor growth was measured three times per week by calipers. On day 0 and day 5, mice were treated by intraperitoneal injection of 10mg/kg of anti-PD-1. Tumor volumes (1/2 (length x width ²)) were measured using digital calipers and expressed as mean ± SEM.

Six week old female Balb/c mice were purchased from the Automatic Resource Center (ARC) and acclimatized for one week prior to use. All experimental procedures were performed according to guidelines and regulations approved by the QIMR Berghofer animal ethics committee. Shave at the inoculation site and then subcutaneously inject 1×10 ⁵ T1 cells in 100 μl pbs into the right mammary gland of the mice. Treatment was started when the tumor reached approximately 100mm ³. The tumors were measured using external calipers and volumes were calculated using the modified elliptical formula 1/2 (a/b ²), where a = longest side, b = shortest side. For combination therapy experiments, mice were treated with indicated doses of vehicle (normal saline) or GDC-0084 (oral gavage) and combined with anti-PD 1 or isotype control (10 mg/kg), twice weekly by intraperitoneal injection. Mice were monitored daily for clinical abnormalities (reduced activity, humpback posture, erectile hair, weight loss and metastasis) and tumor volumes and body weights were measured three times a week. Once the tumors of the solvent 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 correlation analysis.

WST-1 cell proliferation assay.

Adherent cell lines CT26, 4T1 and MCF7 were seeded at optimized cell densities (100, 3000 and 300 per well, respectively) in 96-well flat bottom tissue culture plates in a total volume of 100 μl/well in triplicate. Cells were attached overnight at 37 ℃ and 5% co ₂. Cells were then treated with a series of different concentrations of inhibitor and placed in an incubator at 37 ℃ and 5% co ₂ for 72 hours, after which the 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 cellular mitochondrial dehydrogenases to produce formazan. The amount of formazan produced per 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. Absorbance at 450nm was recorded using a microplate spectrophotometer at incubation periods of 0.5, 1 and 2 hours (after 30 seconds of mixing time). The percent proliferation was calculated by subtracting the average absorbance of the background control (blank) from the sample absorbance. EC ₅₀ values for each inhibitor were determined by log (inhibitor) versus response-variable slope (four parameters) using GRAPHPAD PRISM software.

Scratch wound healing test

At 96 cellsIn Image Lock plates, the adherent breast cancer cell line MDA-MB-231 was plated in triplicate in low serum medium (2%) at an optimized cell density of 2500 cells per well, in a total volume of 100 μl/well. Cells were attached overnight at 37 ℃ and 5% co ₂. Cells were checked for 100% confluence to ensure that a consistent wound was created. After 24 hours, 96 needles were usedWoundMaker Tool to make a wound. Following injury, cells were washed with low serum medium to remove non-adherent cells, and then treated with a series of different concentrations of inhibitors (IC ₂₅ and IC ₅₀) prepared in low serum medium. The board is then placed inIn living cell analysis systems, use is made ofThe zoo living cell analysis device was scanned every 6 hours to track wound healing.

In vitro metastatic cancer PI3K inhibition characterization

The 4T1 or CT26 cancer cell lines were treated with control or GDC-0084 (at concentrations of 0.2. Mu.M or 0.3. Mu.M), idalrism (at two low concentrations of 12.5. Mu.M or 25. Mu.M) or LY294002 (at two low concentrations of 2.5. Mu.M or 5. Mu.M). Samples were then infiltrated with Triton X-100, stained with either a mouse primary antibody to CSV or EpCAM, a rabbit primary antibody to EGFR or FOXN2 or MDA5, or a goat primary antibody to ABCB5, and detected with donkey AF anti-mouse 488, anti-rabbit 568, or anti-goat 647 secondary antibodies. The ASI digital pathology system using a 100-fold objective lens images a protein target. The upper diagram depicts an example image field of view with a scale (orange). The fluorescence intensity of EGFR, the Cytoplasmic Fluorescence Intensity (CFI) of CSV or the overall fluorescence intensity of ABCB5 were analyzed for comparison. Data were plotted using PRISM and single-factor analysis of variance was performed using Kruskal-Wallis. Significant differences are plotted.

OPAL tissue microscopy.

Automated staining was performed using the OPAL staining kit of the automated BONDRX platform according to the manufacturer's instructions. Proteins are then mounted and localized. Protein targets were located by digital pathology laser scanning microscopy. Using ASI digital pathology microscopy, individual 0.5 μm sections were obtained using a 100-fold oil immersion objective running ASI software. The final image is obtained by averaging four consecutive images of the same slice. The digital images were analyzed using automated ASI software (APPLIED SPECTRAL IMAGING, carlsb, california) to automatically determine distribution and intensity by background correction of automatic threshold and average fluorescence intensity, so that target protein expression could be specifically targeted.

Method I for isolating CTCs from liquid biopsies of stage IV metastatic cancer patients.

Liquid biopsies of cancer patients were either subjected to cd45+ cell depletion treatment to enrich Circulating Tumor Cells (CTCs) or not to depletion treatment, PBMCs were isolated in liquid biopsies, using our optimized laboratory protocol. Briefly, we optimized laboratory protocol to store whole blood in EDTA tubes for circulating tumor cell identification. Rosetteep ^TM human CD45 depleted cocktail is used to enrich tumor cells (CTCs) from whole blood by depleting cd45+ cells. The use of tetrameric antibody complexes that recognize CD45, CD66b and glycophorin a on Red Blood Cells (RBCs) targets the depletion of unwanted cells. Unwanted cells were then removed by centrifugation on buoyant density medium Lymphoprep ^TM (catalog # 07801). Purified tumor cells were then extracted from the interface between plasma and buoyant density medium as a highly enriched population and harvested in PBS containing 20% fbs.

CSV (mesenchymal, metastatic markers of CTCs), ABCB5 (cancer stem cell markers and chemotherapeutical resistance markers) and EpCAM (epithelial cell markers) were then stained in the samples. The coverslip was mounted on a glass microscope slide using ProLong nucblue clearAntifade reagents (Life Technologies). And positioning the protein target point by a confocal laser scanning microscope. Using an ASI digital pathology system, individual 0.5 μm slices were obtained using a 100-fold oil immersion objective running ASI software. The final image is obtained by averaging four consecutive images of the same slice. Group dynamic analysis was performed using ASI software and total cell count and fluorescence intensity (protein expression) analysis was performed using ImageJ software (ImageJ, national institutes of health, bezieda, maryland, usa).

Method II for isolating CTC from liquid biopsies of patients with stage IV metastatic cancer

Liquid biopsies of cancer patients were subjected to cd45+ cell depletion treatment using our optimized laboratory protocol (as described above under liquid biopsy method I) to enrich Circulating Tumor Cells (CTCs) in the liquid biopsies. CSV (mesenchymal, metastatic markers of CTCs), ABCB5 (cancer stem cell markers and chemotherapeutical resistance markers) and PI3K (kinase) in the samples were then stained. The coverslip was mounted on a glass microscope slide using ProLong nucblue clearAntifade reagents (Life Technologies). And positioning the protein target point by a confocal laser scanning microscope. Using an ASI digital pathology system, individual 0.5 μm slices were obtained using a 40x oil immersion objective running ASI software. The final image is obtained by averaging four consecutive images of the same slice. Group dynamic analysis was performed using ASI software and total cell count and fluorescence intensity (protein expression) analysis was performed using ImageJ software (ImageJ, national institutes of health, bezieda, maryland, usa).

Example 2

The optimal therapeutic dose for the efficacy of the PI3K-mTOR inhibitor is determined.

To determine the optimal therapeutic dose of GDC-0084PI3K-mTOR inhibitor, the inventors used a 4T1 breast cancer model that was tolerated by highly invasive immunotherapy. Once the tumor had formed (about 100mm ³), GDC-0084 was administered daily by oral gavage in combination with αpd1 immunotherapy (fig. 26A). Experiments were performed sequentially, with preliminary studies using high initial doses of GDC of 15mg/kg +/- αPD1 per day. A split dose escalation study was then performed with daily GDC doses ranging from 1.875mg/kg to 15mg/kg, administered at 4 hour intervals and in combination with αpd1, and finally a single dose escalation study was performed with daily GDC doses of 3.75 and 7.5mg/kg, administered in single dose and in combination with αpd1 (fig. 26B).

When used in combination with immunotherapy, GDC-0084 administration at a high daily dose of 15mg/kg significantly reduced tumor volume by 69% (fig. 26C, fig. 1). Split-dose experiments showed a corresponding decrease in tumor volume when used in combination with anti-PD 1 at GDC doses of 15mg/kg and 7.5mg/kg (fig. 26C, fig. 2). In the last single dose escalation study, GDC at the lower dose of 7.5mg/kg in combination with αpd1 administration reduced primary tumor volume to the same extent as the high dose (fig. 26C, fig. 3), so 7.5mg/kg was confirmed as the optimal therapeutic dose.

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

The inventors next attempted to further investigate toxicity-related complications of GDC-0084 treatment by monitoring mouse body weight and liver weight in initial, divided and single dose reduction experiments (fig. 27). Mice treated with a high daily dose of 15mg/kg GDC-0084 alpha PD1 lost weight after treatment day 4, ultimately resulting in death of mice in the initial dose and in the split dose escalation experiments (FIGS. 27A, 1 and 2). However, at the optimal therapeutic dose of 7.5mg/kg, mice remained in weight throughout the experiment, with no mice dying (fig. 27A, fig. 2 and 3).

Liver enlargement or hepatomegaly is associated with organ damage. To determine whether GDC-0084 treatment induced hepatotoxicity, the inventors measured liver weight at harvest (fig. 2B). GDC-0084 was administered at a high daily dose of 15mg/kg, with an increase in liver weight of about 20% at the end of treatment (FIG. 27B, FIG. 1). In contrast, when administered at a lower dose of 7.5mg/kg, no difference in liver weight was observed (fig. 27B, groups 2 and 3). Taken together, our data confirm that the administration of GDC-0084 at an optimal therapeutic dose of 7.5mg/kg has no toxicity-related complications.

Reducing the dose of PI3K-mTOR inhibitor may reduce liver inflammation.

To assess changes in liver pathology following GDC treatment, H & E stained liver sections were examined (fig. 28). Changes in inflammation, extramedullary hematopoiesis and hepatocyte 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 7.5mg/kg significantly reduced liver inflammation (fig. 28A, fig. 2). Treatment of GDC-0084 at optimal therapeutic doses also reduced extramedullary hematopoiesis, which is typically associated with the 4T1 model (fig. 28B, fig. 2). Finally, supporting our liver weight data, GDC-0084 treatment at a high daily dose of 15mg/kg induced hepatocyte damage, confirming hepatotoxicity (fig. 28C, fig. 1). Notably, no significant changes were observed in hepatocytes at the optimal therapeutic dose of GDC-0084 (fig. 28C, fig. 2).

GDC-0084 alpha PD1 treatment reduced 4T 1-related splenomegaly and extramedullary hematopoiesis.

In addition to extramedullary hematopoiesis, enlargement of the spleen or splenomegaly is often associated with the 4T1 model. To examine spleen changes following GDC-0084 treatment, the inventors measured spleen weight in split-dose and single-dose decrementing experiments. In both administration regimens, administration of GDC-0084 at a lower dose of 7.5mg/kg reduced splenomegaly (FIG. 29A). In addition, spleen pathology assessment also showed significant reduction in extramedullary hematopoiesis following treatment with GDC-0084 at an optimal therapeutic dose of 7.5mg/kg (fig. 29B, fig. 2).

After GDC-0084 alpha PD1 treatment, lung leukocyte infiltration was inhibited.

In view of the above data, which indicate that GDC-0084 αPD1 treatment can reduce lung metastasis, the inventors next sought to assess changes in the lung immune cell population following GDC-0084 treatment. Pulmonary pathology examination showed a significant reduction in leukocyte infiltration into the lung at all concentrations, including the optimal therapeutic dose of 7.5mg/kg (fig. 30). This underscores that GDC-0084 treatment affects not only primary tumors and metastases, but also inflammation.

GDC-0084 alpha PD1 treatment reduced lymph node metastasis.

In addition to lung metastasis, the 4T1 model is also associated with lymph node metastasis. To assess lymph node metastasis following GDC-0084 treatment, independent expert pathologists examined H & E stained lymph node sections and gave scores of 1=1-3 tiny sites (width/length < <0.5 mm), 2=1-3, at least one of which was 0.5-1mm in diameter, or 3=2-3 or more sites, all of which were macroscopic, 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 used in combination with GDC-0084 (7.5 mg/kg) (FIG. 31A). Thus, GDC-0084 αpd1 treated mice showed significantly reduced lymph node metastasis scores, confirming that GDC-0084 inhibited not only lung metastasis but also lymph node metastasis (fig. 31B).

PI3K-mTOR treatment may reduce primary tumor volume.

To determine whether GDC-0084 shows efficacy when administered in combination with PARP inhibitors, olaparib, the inventors have subsequently used the 4T1 model of TNBC. In the initial experiments, GDC-0084 was administered before or after olaparib, and then repeated in the second experiment according to the optimal administration regimen (fig. 32A). GDC-0084 was administered daily by oral gavage, with an optimal therapeutic dose of 7.5mg/kg, and daily intraperitoneal injection of Olaparib (FIG. 32B). GDC-0084 treatment was performed before and after PARP inhibitor, and tumor volumes were significantly reduced by 53% and 60%, respectively (FIG. 32C, FIG. 1). The inventors then confirmed these results in a second experiment using the optimal dosing regimen of GDC-0084 after 30 minutes of olaparib administration (fig. 32C-fig. 2). Taken together, these data indicate that in addition to immunotherapy, GDC-0084 also shows therapeutic effects when used in combination with PARP inhibitors, thereby enhancing its use.

Combination therapy with GDC-0084 and PARP inhibitors does not cause toxicity.

To examine complications associated with toxicity, the inventors monitored the body weight and liver weight of mice in PARP inhibitor experiments (fig. 33). At the optimal therapeutic dose of 7.5mg/kg, GDC-0084 did not change mouse body weight when administered post-Olaparib (FIG. 33A). Also, no change in liver weight was observed, confirming no toxicity problem (fig. 33B).

GDC-0084 and Olaparib combined therapy inhibited lung and liver inflammation.

In view of the effects of GDC-0084 and immunotherapy on inflammation, the inventors next attempted to examine liver and lung pathology in PARP inhibitor studies. GDC-0084 significantly reduced liver inflammation and extramedullary hematopoiesis when administered post-Olaparib at a dose of 7.5mg/kg (FIG. 34A). Pulmonary pathology examination also emphasized that GDC-0084 and olaparib treated mice had significantly reduced pulmonary leukocytosis (fig. 34B), confirming that GDC-0084 has an effect not only on primary tumors but also on inflammation at the metastatic sites.

Finally, spleen pathology assessment showed that after GDC-0084 and olaparib treatment, extramedullary hematopoiesis was also reduced in addition to spleen weight (fig. 35A and 35B).

PI3K-mTOR inhibitors reduce cancer cell proliferation.

The inventors then tried to compare the efficacy of PI3K-mTOR inhibitors such as GDC-0084 and thus use a number of inhibitors for proliferation assays, including the more general PI3K inhibitors wortmannin (general PI3K inhibitor), idarrass (p110δ, γ inhibitor), apertural (Alpelisib) (p110α inhibitor) and LY294002 (p110α, β, δ inhibitor), as well as PI3K-mTOR inhibitors o Mi Lisai (omipalisib), apitolisib, dactorisib and GDC-0084 (p110α, β, δ, γ, mTOR inhibitor) (fig. 36). The effect of PI3K-mTOR inhibitors on MDA-MB-231 cell proliferation was most pronounced, with IC ₅₀ values for all of austenite Mi Lisai, apitolisib and dactolisib below 1.0 μm (table 16). In contrast, GDC-0084 has an IC ₅₀ value of 2.5. Mu.M. More general PI3K inhibitors show significantly higher values, confirming that inhibition of PI3K and mTOR is critical in preventing cancer cell proliferation.

Table 16

Not measured

Inhibition of PI3K and mTOR affects cancer cell migration.

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 (fig. 37A). In the GDC-0084 dose response experiment, about 50% inhibition was observed at concentrations of 1.25. Mu.M and 2.5. Mu.M after 24 hours of treatment (FIG. 37B).

Similarly, to compare the efficacy of GDC-0084 with other PI3K-mTOR inhibitors, the inventors performed a second experiment on apitolisib, dactolisib and omipalisib using IC ₅₀ concentrations from the proliferation assay (FIG. 38A). Similar to GDC-0084, all PI3K-mTOR inhibitors significantly reduced wound healing density (fig. 38B), confirming that inhibition of both PI3K and mTOR impeded cell migration.

Materials and methods

Animal study

Female BALB/c mice aged 6-8 weeks purchased from the Automation Resource Center (ARC). After harvest, mice were subjected to a one week adaptation period in the isolator of QIMR Berghofer medical institute (QIMRB). All experimental procedures were performed according to guidelines and regulations approved by the QIMRB ethics committee. 1X 10 ⁵ 4T1 cells suspended in Phosphate Buffered Saline (PBS) were applied to mammary fat pads of BALB/c mice. Treatment was started when the tumor reached approximately 50-100mm ³. In the 4T1 model, mice were given daily oral doses of GDC in combination with intraperitoneal injection of anti-PD-1 therapy (10 mg/kg) on day 0 and day 4 or daily administration of Olaparib (50 mg/kg). The tumors were measured using external calipers and volumes were calculated using the modified elliptical formula 1/2 (a/b ²), where a = longest side, b = shortest side. Mice were monitored daily for clinical abnormalities (reduced activity, humpback posture, erectile hair, weight loss and metastasis) and tumor volumes were measured three times per week. Once the tumors of the solvent 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 correlation analysis.

Pathological analysis

Independent expert pathologists performed pathological analyses on H & E stained FFPE livers, lungs, lymph nodes and spleens. Changes in inflammation, extramedullary hematopoiesis (EMH), hepatocytes and pulmonary leukocytosis were scored 1=mild, 2=moderate or 3=severe. Lymph node sections were scored 1=1-3 tiny sites (width/length < <0.5 mm), 2=1-3 where at least one diameter was 0.5-1mm, or 3=2-3 or more sites, all visible with the naked eye, without leukocyte infiltration and/or bleeding.

WST-1 cell proliferation assay.

MDA-MB-231 cells were seeded at an optimized density in 96-well flat bottom tissue culture plates in a total volume of 100. Mu.L/well. Cells were attached overnight at 37 ℃ and 5% co ₂. Cells were then treated with the indicated dose of PI3K inhibitor and placed in an incubator at 37 ℃ and 5% co ₂ for 72 hours, after which the medium was removed, replaced with 100 μl/well of WST-1 cell proliferation reagent (Sigma-Aldrich, 11644807001) and diluted 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 cellular mitochondrial dehydrogenases to produce formazan. The amount of formazan produced per 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. Absorbance was recorded at 450nm using a microplate spectrophotometer at incubation periods of 0.5, 1 and 2 hours (after 30 seconds of mixing time). The percent proliferation was calculated by subtracting the average absorbance of the background control (blank) from the sample absorbance. IC ₅₀ values for each inhibitor were determined by log (inhibitor) versus reaction-variable slope (four parameters) using GraphPadPrism 8 software.

Scratch wound healing test.

MCF7 cells were stimulated with PMA/TGF beta for 24 hours prior to seeding, and then seeded at an optimized cell density in 96 wellsIn Image Lock plates, a total volume per well of 100 μl, low serum medium (2%) was used. Cells were attached overnight at 37 ℃ and 5% co ₂. Cells were checked for 100% confluence to ensure that a consistent wound was created. After 24 hours, 96 needles were usedWoundMaker Tool to make a wound. Following injury, cells are washed with low serum medium to remove non-adherent cells, and then treated with prescribed doses of PI3K inhibitor prepared in low serum medium. The board is then placed inIn the living cell analysis system, the intucyte ^TM Zoom living cell analysis apparatus was used to scan every 4 hours to track wound healing.

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Claims (35)

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.