Methods of Preparing T Cells for T Cell Therapy

1 . A method for delaying or inhibiting T cell maturation or differentiation in vitro for a T cell therapy, comprising contacting one or more T cells from a subject in need of a T cell therapy with an AKT inhibitor and at least one of exogenous Interleukin-7 (IL-7) and exogenous Interleukin-15 (IL-15), wherein the resulting T cells exhibit delayed maturation or differentiation, and/or wherein the resulting T cells exhibit improved T cell function relative to a T cell function of a T cell cultured in the absence of an AKT inhibitor.

2 . The method of claim 1 , wherein the improved T cell function is selected from the group consisting of:

(i) increased T cell proliferation;

(ii) increased cytokine production;

(iii) increased cytolytic activity; and

(iv) any combination of (i)-(iii).

3 . The method of claim 2 , wherein the increased cytokine production is selected from the group consisting of (i) increased interferon gamma (IFNg) production, (ii) increased tissue necrosis factor alpha (TNFa) production, and (iii) both increased IFNg and TNFa production.

4 . The method of claim 1 , further comprising administering the resulting T cells to a subject in need thereof.

5 . A method of treating a tumor in a subject in need of a T cell therapy comprising administering to the subject one or more T cells, wherein the one or more T cells have been contacted with (i) an AKT inhibitor and (ii) exogenous IL-7 and/or exogenous IL-15.

6 . A method of reducing or decreasing the size of a tumor or inhibiting growth of a tumor in a subject in need of a T cell therapy comprising administering to the subject one or more T cells, wherein the one or more T cells have been contacted with (i) an AKT inhibitor and (ii) exogenous IL-7 and/or exogenous IL-15.

7 . The method of claim 1 , wherein the one or more T cells are not contacted with exogenous Interleukin-2 (IL-2).

8 . The method of claim 1 , wherein the AKT inhibitor is selected from the group consisting of A6730, B2311, 124018, GSK2110183 (afuresertib), Perifosine (KRX-0401), GDC-0068 (ipatasertib), RX-0201, VQD-002, LY294002, A-443654, A-674563, Akti-1, Akti-2, Akti-1/2, AR-42, API-59CJ-OMe, ATI-13148, AZD-5363, erucylphosphocholine, GSK-2141795 (GSK795), KP372-1, L-418, NL-71-101, PBI-05204, PIA5, PX-316, SR13668, triciribine, GSK 690693 (CAS #937174-76-0), FPA 124 (CAS #902779-59-3), Miltefosine, PHT-427 (CAS #1 191951-57-1), 10-DEBC hydrochloride, Akt inhibitor III, Akt inhibitor VIII, MK-2206 dihydrochloride (CAS #1032350-13-2), SC79, AT7867 (CAS #857531-00-1), CCT128930 (CAS #885499-61-6), A-674563 (CAS #552325-73-2), AGL 2263, AS-041 164 (5-benzo[1,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione), BML-257 (CAS #32387-96-5), XL-418, CAS #612847-09-3, CAS #98510-80-6, H-89 (CAS #127243-85-0), OXY-1 1 1 A, 3-[1-[[4-(7-phenyl-3H-imidazo [4,5-g]quinoxalin-6-yl)phenyl]methyl]piperidin-4-yl]-1H-benzimidazol-2-one, N,N-dimethyl-1-[4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl]metha-namine, 1-{1-[4-(3-phenylbenzo[g]quinoxalin-2-yl)benzyl]piperidin-4-yl}-1,-3-dihydro-2H-benzimidazol-2-one and any combination thereof.

9 . The method of claim 1 , wherein the AKT inhibitor is 3-[1-[[4-(7-phenyl-3H-imidazo[4,5-g]quinoxalin-6-yl)phenyl]methyl]piperidin-4-yl]-1H-benzimidazol-2-one.

10 . The method of claim 1 , wherein

the AKT inhibitor is at an amount of from about 1 nM to about 1 mM.

11 . The method of claim 1 , wherein

the AKT inhibitor is at an amount of about 8 μM.

12 . The method of claim 1 , wherein the one or more T cells are selected from the group consisting of tumor infiltrating lymphocytes, cytotoxic T cells, CAR T cells, engineered TCR T cells, natural killer T cells, peripheral blood lymphocytes, and tumor infiltrating leukocytes.

13 . The method of claim 1 , further comprising transducing the T cells with a retrovirus.

14 . The method of claim 13 , wherein the retrovirus comprises a heterologous gene encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

15 . The method of claim 14 , wherein the TCR or the CAR is capable of binding an antigen selected from the group consisting of 707-AP (707 alanine proline), AFP (alpha (a)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen; b-catenin/m, b-catenin/mutated), BCMA (B cell maturation antigen), Bcr-abl (breakpoint cluster region-Abelson), CAIX (carbonic anhydrase IX), CD19 (cluster of differentiation 19), CD20 (cluster of differentiation 20), CD22 (cluster of differentiation 22), CD30 (cluster of differentiation 30), CD33 (cluster of differentiation 33), CD44v7/8 (cluster of differentiation 44, exons 7/8), CAMEL (CTL-recognized antigen on melanoma), CAP-1 (carcinoembryonic antigen peptide-1), CASP-8 (caspase-8), CDC27m (cell-division cycle 27 mutated), CDK4/m (cycline-dependent kinase 4 mutated), CEA (carcinoembryonic antigen), CT (cancer/testis (antigen)), Cyp-B (cyclophilin B), DAM (differentiation antigen melanoma), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor, variant III), EGP-2 (epithelial glycoprotein 2), EGP-40 (epithelial glycoprotein 40), Erbb2, 3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3, 4), ELF2M (elongation factor 2 mutated), ETV6-AML1 (Ets variant gene 6/acute myeloid leukemia 1 gene ETS), FBP (folate binding protein), fAchR (Fetal acetylcholine receptor), G250 (glycoprotein 250), GAGE (G antigen), GD2 (disialoganglioside 2), GD3 (disialoganglioside 3), GnT-V (N-acetylglucosaminyltransferase V), Gp100 (glycoprotein 100 kD), HAGE (helicose antigen), HER-2/neu (human epidermal receptor-2/neurological; also known as EGFR2), HLA-A (human leukocyte antigen-A) HPV (human papilloma virus), HSP70-2M (heat shock protein 70-2 mutated), HST-2 (human signet ring tumor-2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxyl esterase), IL-13R-a2 (Interleukin-13 receptor subunit alpha-2), KIAA0205, KDR (kinase insert domain receptor), κ-light chain, LAGE (L antigen), LDLR/FUT (low density lipid receptor/GDP-L-fucose: b-D-galactosidase 2-a-Lfucosyltransferase), LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (Melanoma-associated antigen 1), MAGE-A3, MAGE-A6, mesothelin, Murine CMV infected cells, MART-1/Melan-A (melanoma antigen recognized by T cells-1/Melanoma antigen A), MC1R (melanocortin 1 receptor), Myosin/m (myosin mutated), MUC1 (mucin 1), MUM-1, -2, -3 (melanoma ubiquitous mutated 1, 2, 3), NA88-A (NA cDNA clone of patient M88), NKG2D (Natural killer group 2, member D) ligands, NY-BR-1 (New York breast differentiation antigen 1), NY-ESO-1 (New York esophageal squamous cell carcinoma-1), oncofetal antigen (h5T4), P15 (protein 15), p190 minor bcr-abl (protein of 190 KD bcr-abl), Pml/RARa (promyelocytic leukaemia/retinoic acid receptor a), PRAME (preferentially expressed antigen of melanoma), PSA (prostate-specific antigen), PSCA (Prostate stem cell antigen), PSMA (prostate-specific membrane antigen), RAGE (renal antigen), RU1 or RU2 (renal ubiquitous 1 or 2), SAGE (sarcoma antigen), SART-1 or SART-3 (squamous antigen rejecting tumor 1 or 3), SSX1, -2, -3, 4 (synovial sarcoma X1, -2, -3, -4), TAA (tumor-associated antigen), TAG-72 (Tumor-associated glycoprotein 72), TEL/AML1 (translocation Ets-family leukemia/acute myeloid leukemia 1), TPI/m (triosephosphate isomerase mutated), TRP-1 (tyrosinase related protein 1, or gp75), TRP-2 (tyrosinase related protein 2), TRP-2/INT2 (TRP-2/intron 2), VEGF-R2 (vascular endothelial growth factor receptor 2), WT1 (Wilms' tumor gene), and any combination thereof.

16 . The method of claim 1 , wherein, following the contact with the AKT inhibitor and the exogenous IL-7 and/or exogenous IL-15, the T cells:

(i) express CCR7 and CD45RO;

(ii) express CCR7 and CD45RA;

(iii) exhibit increased expression of CCR7, CD45RO, CD45RA, or any combination thereof, as compared to the expression of CCR7, CD45RO, and CD45RA by T cells not contacted with the AKT inhibitor and the exogenous IL-7 and/or exogenous IL-15;

(iv) express CD62L, CD28, or both;

(v) exhibit increased expression of CD62L, CD28, or both, as compared to the expression of CD62L and CD28 by T cells not contacted with the AKT inhibitor and the exogenous IL-7 and/or exogenous IL-15;

(vi) exhibit increased expression of CD95, IL-7 receptor alpha (IL-7Rα), CXCR4, TCF7, FOXO1, ID3, BCL6, CD62L, CD45RA, or any combination thereof following the contact with the AKT inhibitor and the exogenous IL-7, exogenous IL-15, or both, as compared to the expression of CD95, IL-7Rα, CXCR4, TCF7, FOXO1, ID3, BCL6, CD62L, and CD45RA by T cells not contacted with the AKT inhibitor and the exogenous IL-7 and/or exogenous IL-15; or

(vii) any combination of (i) to (vi).

17 . The method of claim 5 , wherein the tumor is a cancer selected from bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B cell lymphoma (PMBC), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B cell malignancies, and any combination thereof.

18 . The method of claim 1 , wherein:

(i) the exogenous IL-7 is at an amount of about 0.001 to about 500 ng/ml IL-7;

(ii) the exogenous IL-15 is at an amount of about 0.001 to about 500 ng/ml IL-15; or

(iii) both (i) and (ii).

19 . The method of claim 1 , wherein:

(i) the exogenous IL-7 is at an amount of at least about 5 ng/ml IL-7;

(ii) the exogenous IL-15 is at an amount of at least about 5 ng/ml IL-15; or

(iii) both (i) and (ii).

20 . The method of claim 1 , wherein:

(i) the AKT inhibitor is at an amount of about 8 μM;

(ii) the exogenous IL-7 is at an amount of at least about 5 ng/ml IL-7; and

(iii) the exogenous IL-15 is at an amount of at least about 5 ng/ml IL-15.