Methods of preparing t cells for t cell therapy

Abstract

To provide methods for delaying or inhibiting T cell maturation or differentiation in vitro for a T cell therapy.SOLUTION: The present invention provides a method comprising a step of 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. In some embodiments, the method further comprises a step of administering the one or more T cells to a subject in need of a T cell therapy.SELECTED DRAWING: Figure 1-1

Description

[Technical Field]

[0001] Statement of Government Interest This invention was made in the fulfillment of a Cooperative Research and Development Agreement with the National Cancer Institute (NCI), an agency of the U.S. Department of Health and Human Services. The U.S. Government has certain rights in this invention.

[0002] FIELD OF THE INVENTION The present invention relates to methods for preparing one or more T cells for T cell therapy. In particular, the present invention relates to methods for improving the efficacy of T cell therapy by contacting one or more T cells with an AKT inhibitor ("AKTi") and at least one of exogenous interleukin-7 (IL-7) and exogenous interleukin-15 (IL-15). [Background technology]

[0003] background Human cancers are essentially composed of normal cells that have undergone genetic or epigenetic transformation to become abnormal cancer cells. In doing so, the cancer cells begin to express proteins and other antigens that differ from those expressed by normal cells. These abnormal tumor antigens can be used by the body's innate immune system to specifically target and kill cancer cells. However, cancer cells use various mechanisms to prevent immune cells such as T and B lymphocytes from successfully targeting cancer cells.

[0004] Human T cell therapy relies on the human T cells enriched or modified ex vivo to target and kill cancer cells in subjects (e.g., patients).Various techniques have been developed to enrich the concentration of natural T cells that can target tumor antigens, or genetically modify T cells to specifically target known cancer antigens.These therapies have been found to have promising effects on tumor size and patient survival.However, it has been found that it is difficult to predict whether a given T cell therapy will be effective in each patient.

[0005] Transplantation of mixed populations of T cells is among the factors that prevent T cell therapy from reaching its full potential. In conventional T cell therapy, donor T cells are harvested, optionally modified to target specific antigens (e.g., tumor cells) or selected for anti-tumor properties (e.g., tumor-infiltrating lymphocytes), expanded in vitro, and administered to a subject in need thereof. Typically, the resulting T cells contain a mixed population of large, mature cells, many of which are terminally differentiated. As a result, the expected in vivo persistence of these cells may be limited, and the initially observed positive effects may be reversed over time as the tumor rebounds in the absence of transplanted T cells. Thus, there remains a need to increase the in vivo persistence of T cells for use in T cell therapy. Summary of the Invention

[0006] The present disclosure provides a method for delaying or inhibiting T cell maturation or differentiation in vitro for T cell therapy, comprising contacting one or more T cells from a subject in need of T cell therapy with an AKTi and at least one of exogenous IL-7 and exogenous IL-15, wherein the resulting T cells exhibit delayed maturation or differentiation.

[0007] The present disclosure further provides a method for delaying or inhibiting T cell maturation or differentiation in vitro, comprising culturing one or more T cells in a medium comprising an AKTi and at least one of exogenous IL-7 and exogenous IL-15.

[0008] The present disclosure further provides a method for producing a stem cell-like CD8 T cell population comprising culturing one or more T cells in a medium. + A method for generating T cells is provided, comprising contacting one or more T cells with an AKTi and at least one of exogenous IL-7 and exogenous IL-15.

[0009] The present disclosure also provides a method for extending the in vivo persistence of one or more T cells in adoptive cell therapy, comprising contacting the one or more T cells with an AKTi and at least one of exogenous IL-7 and exogenous IL-15 prior to administration to a subject.

[0010] In certain embodiments, the methods disclosed herein further comprise administering one or more T cells to a subject in need thereof. In some embodiments, the subject is in need of T cell therapy.

[0011] The present disclosure further provides a method of treating a tumor in a subject in need of T cell therapy, comprising administering to the subject one or more T cells contacted with (i) an AKTi and (ii) exogenous IL-7 and / or exogenous IL-15.

[0012] The present disclosure also provides a method of reducing or diminishing tumor size or inhibiting tumor growth in a subject in need of T cell therapy, comprising administering to the subject one or more T cells that have been contacted with (i) an AKTi and (ii) exogenous IL-7 and / or exogenous IL-15.

[0013] In some embodiments, the T cell therapy comprises an engineered CAR cell therapy or an engineered TCR cell therapy. In one embodiment, the engineered CAR cell or engineered TCR cell therapy treats a tumor in a subject. [Brief explanation of the drawings]

[0014] [Figure 1]Figures 1A-1F show the phenotypes of CD4+ and CD8+ T cells following culture in the presence of IL-2 or IL-7 and IL-15 at 7 and 14 days. Figures 1A and 1C show the percentage of the total population of cultured CD4+ and CD8+ T cells, respectively, that were characterized as naive or central memory T cells (Tcm) at day 7 for IL-2 and IL-7 / IL-15-treated cells. Figures 1B and 1D show the percentage of the total population of cultured CD4+ and CD8+ T cells, respectively, that were characterized as effector memory T cells (Tem) or effector T cells (Teff) at day 7 for IL-2 and IL-7 / IL-15-treated cells. Figure 1E shows the percentage of the total population of cultured CD8+ T cells that were characterized as naive or central memory T cells (Tcm) at day 14 for IL-2 and IL-7 / IL-15-treated cells. Figure 1F shows the percent of the total population of cultured CD8+ T cells characterized as effector memory T cells (Tem) or effector T cells (Teff) at day 14 for IL-2 and IL-7 / IL-15 treated cells. Individual data points reflect individual samples. Horizontal lines represent the mean, and error bars indicate standard deviation. Statistical significance is indicated by p-value ("ns" stands for "not significant"). [Figure 2]Figures 2A-2D show the phenotypes of CD4+ and CD8+ T cells following culture in the presence of IL-7 and IL-15 or in the presence of IL-7, IL-15, and AKTi at 7 and 14 days. Figures 2A and 2C show the percentage of the total population of cultured CD4+ and CD8+ T cells, respectively, that were characterized as naive or Tcm cells at day 7 for IL-7 / IL-15-treated and IL-7 / IL-15 / AKTi-treated cells. Figures 2B and 2D show the percentage of the total population of cultured CD4+ and CD8+ T cells, respectively, that were characterized as Tem or Teff cells at day 7 for IL-7 / IL-15-treated and IL-7 / IL-15 / AKTi-treated cells. Individual data points reflect individual samples. Horizontal lines represent the mean values, and error bars indicate standard deviations. Statistical significance is indicated by p-values ​​("ns" stands for "not significant"). [Figure 3] Figure 3A shows the transduction efficiency of donor T cells contacted with IL-2 alone; IL-2 and AKTi; IL-7 and IL-15; and IL-7, IL-15, and AKTi. Transduction efficiency is indicated by the percentage of total T cells that are CD3+ and have positive soluble MHC-tetramer staining (Tet+). Dark gray bars indicate the percentage of CD3+ Tet+ cells in T cell samples contacted with IL-2. Downward-striped bars indicate the percentage of CD3+ Tet+ cells in T cell samples contacted with IL-2 and AKTi. Light gray bars indicate the percentage of CD3+ Tet+ cells in T cell samples contacted with IL-7 and IL-15. Upward-striped bars indicate the percentage of CD3+ Tet+ cells in T cell samples contacted with IL-7, IL-15, and AKTi. Error bars indicate standard deviation. Figure 3B shows the tetramer mean fluorescence intensity (MFI) for cells from donor 1 (circles), donor 2 (squares), and donor 3 (triangles) following culture in the presence of IL-2 alone; IL-2 and AKTi; IL-7 and IL-15; and IL-7, IL-15, and AKTi. Statistical analysis showed that none of the differences in tetramer MFI were significant (p = ns). [Figure 4] Figures 4A-4D show cell proliferation over the course of 7 days for cells from four donors cultured in the presence of IL-2 (circles); IL-2 and AKTi (squares); IL-7 and IL-15 (triangles); or IL-7, IL-15, and AKTi (inverted triangles). Each of Figures 4A-4D shows cell proliferation for a single donor cell line. The source material for the expansion protocol was peripheral blood mononuclear cells. [Figure 5] Figures 5A-5C show cell proliferation over the course of 9 days for cells from three donors transduced with a class I TCR (HPV-E6) and cultured in the presence of IL-2 (circles); IL-2 and AKTi (squares); IL-7 and IL-15 (triangles); or IL-7, IL-15, and AKTi (inverted triangles). Each of Figures 5A-5C shows cell proliferation for a single donor's cell line. The source material for the expansion protocol was peripheral blood mononuclear cells. [Figure 6] Figures 6A-6C show cell proliferation over the course of 10 days for isolated CD4+ and CD8+ cells from three donors transduced with a class II TCR (MAGE-A3) and cultured in the presence of IL-2 (circles); IL-2 and AKTi (squares); IL-7 and IL-15 (triangles); or IL-7, IL-15, and AKTi (inverted triangles). Each of Figures 6A-6C shows cell proliferation for a single donor's cell line. [Figure 7] Figures 7A-7C show cell proliferation over the course of 10 days for isolated CD4+ cells from three donors transduced with a class II TCR (MAGE-A3) and cultured in the presence of IL-2 (circles); IL-2 and AKTi (squares); IL-7 and IL-15 (triangles); or IL-7, IL-15, and AKTi (inverted triangles). Each of Figures 7A-7C shows cell proliferation for a single donor's cell line. [Figure 8]Figures 8A-8C show cell proliferation over the course of 10 days for isolated CD8+ cells from three donors transduced with a class II TCR (MAGE-A3) and cultured in the presence of IL-2 (circles); IL-2 and AKTi (squares); IL-7 and IL-15 (triangles); or IL-7, IL-15, and AKTi (inverted triangles). Each of Figures 8A-8C shows cells from a single donor's cell line. [Figure 9] Figures 9A-9D show cell expansion over the course of 8 days for CD4+ and CD8+ cells from three donors transduced with class II TCR (MAGE-A3). Cells were cultured in the presence of IL-7 and IL-15 (Figure 9A: circles; Figures 9B-9C: squares) or IL-7, IL-15, and AKTi (Figure 9A: squares; Figures 9B-9C: circles). Cells were expanded at a large-scale manufacturing scale in the XURI™ Cell Expansion System. Each of Figures 9A-9D shows cell expansion for a single donor cell line. The source material for the expansion protocol was isolated CD4+ and CD8+ cells. [Figure 10] Figure 1 shows transduction efficiency for T cells transduced with class I TCR (HPV-E6). Cells were cultured in the presence of IL-2 alone; IL-2 and AKTi; IL-7 and IL-15; or IL-7, IL-15, and AKTi. Cells were transduced on day 2, and transduction efficiency was measured on day 10 by staining the cells with an anti-mTCRb antibody that specifically recognizes the transduced TCR. The percentage of cells (y-axis) showing positive anti-mTCRb staining for each culture condition (x-axis) is shown. [Figure 11]Figures 11A-11F show the transduction efficiency of class II TCR (MAGE-A3)-transduced CD4+ / CD8+ T cells from two donors. Cells were cultured in the presence of IL-2 alone; IL-2 and AKTi; IL-7 and IL-15; or IL-7, IL-15, and AKTi. Cells were transduced on day 2, and transduction efficiency was measured on day 10 by staining the cells with an anti-mTCRb antibody (mC TCR PE) (Figures 11A and 11D). The MFI of anti-mTCRb staining for each culture condition is shown in Figures 11B and 11E. Figures 11C and 11F show FACS analysis of the distribution of cells expressing the CDR and transduced TCR for both donors. [Figure 12] Figure 1 shows the transduction efficiency for class II TCR (MAGE-A3) transduced T cells from four manufacturing-scale runs (16, 21, 22, and 23). For each run, cells were divided into two culture conditions: addition of IL-7 and IL-15, and addition of IL-7, IL-15, and AKTi, as indicated. Transduction efficiency was measured by staining cells with an anti-mTCRb antibody (mC TCR PE). The percentage of cells showing positive anti-mTCRb staining (y-axis) for each run (x-axis) is shown. [Figure 13] Figures 13A-13F show the differentiation status of CD4+ / CD8+ T cells transduced with class II TCR (MAGE-A3) and cultured under various conditions with or without AKTi. T cells from donor 1 (Figures 13A and 13D), donor 2 (Figures 13B and 13E), and donor 3 (Figures 13C and 13E) were cultured in the presence of IL-2 alone; IL-2 and AKTi; IL-7 and IL-15; or IL-7, IL-15, and AKTi, and then stained for CD62L expression, a marker of cells in the early stages of differentiation. The percentage of CD3+ and CD62L+ cells (y-axis) for each culture condition (x-axis) for each donor is presented in Figures 13A-13C. The MFI of CD62L staining (y-axis) for each culture condition (x-axis) for each donor cell line is shown in Figures 13D-13E. [Figure 14]Figures 14A-14B show the effect of culture conditions on T cell function, as evidenced by cytokine production by T cells from three manufacturing-scale runs (21, 22, and 23). Class II TCR (MAGE-A3)-transduced donor T cells were cultured in the XURI™ Bioreactor Cell Expansion System in the presence of IL-7 and IL-15, or IL-7, IL-15, and AKTi. The percent of cells staining positive for CD3 and IFNg (Figure 14A) and CD3 and TNFa (Figure 14B) are shown for cells cultured in the presence or absence of AKTi for each of runs 21, 22, and 23. [Figure 15] Figure 1 shows T cell activity, as evidenced by IFNg production, for donor T cells transduced with a class II TCR (MAGE-A3) and cocultured with positive and negative target tumor cell lines. T cells from two manufacturing-scale runs (21 and 22) were transduced with a class II TCR and cultured in the XURI™ Bioreactor Cell Expansion System in the presence of IL-7 and IL-15, or IL-7, IL-15, and AKTi. Cells were then cocultured overnight with tumor cell lines expressing the TCR target antigen (H1299, HT1197, or HT1367) or not expressing the TCR target antigen (DU145, SK MEL 28, or SK MEL 5). T cell activity is shown by IFNg production (y-axis), expressed as pg / mL, for each cell line (x-axis) for each culture condition. Error bars indicate standard deviation. [Figure 16]Figures 16A-16D show T cell activity, as evidenced by IFNg production, for three donor T cell lines transduced with a class I TCR (HPV-E6) and cultured with or without AKTi. Figure 16A shows the amount of IFNg (pg / mL; y-axis) produced by T cells from three donors (x-axis) following coculture with a tumor cell line (Caski; a cervical cancer cell line) expressing a TCR antigen in the presence of IL-2 alone; IL-2 and AKTi; IL-7 and IL-15; or IL-7, IL-15, and AKTi. Figures 17B-17D show IFNg production (pg / mL; y-axis) for donor 1 (Figure 16B), donor 2 (Figure 16C), and donor 3 (Figure 16D) T cells following coculture with T2 cells loaded with titrated amounts of SCR-specific peptide (target peptide; x-axis) in the presence of IL-2 alone (circles); IL-2 and AKTi (squares); IL-7 and IL-15 (triangles); or IL-7, IL-15, and AKTi (inverted triangles). Error bars indicate standard deviation. [Figure 17] Figures 17A-17D are FACS histograms showing T cell proliferation following culture in the presence (Figures 17B and 17D) or absence (Figures 17A and 17C) of AKTi. T cells from donor 3 were expanded in IL-2 (Figure 17A), IL-2 and AKTi (Figure 17B), IL-7 and IL-15 (Figure 17C), and IL-7, IL-15, and AKTi (Figure 17D) and cocultured with a tumor cell line transduced with a class II TCR (MAGE-A3) and expressing a TCR antigen for 4 days. T cell proliferation was measured by carboxyfluorescein succinimidyl ester (CFSE) staining (Figures 17A-17D). L = late proliferation; M = intermediate proliferation; and E = early proliferation. [Figure 18]Figures 18A and 18B are FACS histograms showing cell proliferation of T cells from two large-scale production culture runs: 21 (Figure 18A) and 22 (Figure 18B). T cells were grown in IL-2 and in IL-7, IL-15, and AKTi and transduced with a class II TCR (MAGE-A3). T cells were co-cultured for 4 days with either a tumor cell line expressing the TCR target antigen ("positive target") or a cell line not expressing the TCR target antigen ("negative target") in the presence of IL-7 and IL-15, or IL-7, IL-15, and AKTi. T cell proliferation was measured by CFSE staining, normalized to mode, and compared to comp-FITC-A staining, as shown for each of runs 21 (Figure 18A) and 22 (Figure 18B). DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description The present invention relates to methods for preparing T cells for use in T cell therapy. In particular, the present invention relates to a method for modulating, e.g., delaying or inhibiting, T cell maturation or differentiation in vitro by contacting one or more T cells with an AKTi and at least one of exogenous IL-7 and exogenous IL-15. By delaying or inhibiting T cell maturation or differentiation, a population of donor T cells can be enriched for less differentiated, immature T cells (e.g., naive T cells or central memory Tcm cells), increasing the persistence of one or more T cells after administration to a subject, e.g., a patient. As a result, a population of enriched immature T cells is more likely to produce a sustained anti-tumor effect than a population of T cells at mixed differentiation stages.

[0016] definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout this application.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains.For example, Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0018] Units, prefixes, and symbols are expressed in the format recognized by the International System of Units (SI). Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not intended to limit the various aspects of the present disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0019] As used herein, the indefinite article "a" or "an" should be understood to refer to "one or more" of any described or listed components.

[0020] The terms "about" or "essentially comprising" refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined (i.e., limitations of the measurement system). For example, "about" or "essentially comprising" can mean within one or more standard deviations according to practice in the art. Alternatively, "about" or "essentially comprising" can mean within a range of up to 10% (i.e., ±10%). For example, about 3 mg can include any value between 2.7 mg and 3.3 mg (about 10%). Furthermore, particularly with respect to biological systems or processes, these terms can mean up to an order of magnitude or up to five times the value. When a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of "about" or "essentially comprising" should be considered to be within an acceptable error range for that particular value or composition.

[0021] As described herein, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the range described, and, where appropriate, fractions thereof (e.g., tenths and hundredths of an integer), unless otherwise specified.

[0022] The term "and / or," as used herein, shall be considered a specific disclosure of each of the two specified features or components, with or without the other. Thus, when the term "and / or" is used herein in a phrase such as "A and / or B," it is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, when the term "and / or" is used in a phrase such as "A, B, and / or C," it is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0023] Whenever an aspect is described herein with the word "comprising," it is understood that otherwise similar aspects described with "consisting of" and / or "consisting essentially of" are also provided. The term "activation" or "activated" refers to a state of immune cells, such as T cells, that have been sufficiently stimulated to induce detectable cell proliferation. Activation may also be associated with induced cytokine production and detectable effector function. The term "activated T cells" particularly refers to T cells undergoing cell division. T cell activation may be characterized by increased T cell expression of one or more biomarkers, including, but not limited to, CD57, PD1, CD107a, CD25, CD137, CD69, and / or CD71.

[0024] "Administering" refers to the physical introduction of a drug into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary administration routes for T cells prepared by the methods disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral administration routes, for example, by injection or infusion. The phrase "parenteral administration," as used herein, refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, T cells prepared by the present methods are administered via a non-parenteral route, for example, orally. Other non-parenteral routes include topical, epidermal or mucosal administration routes, for example, intranasal, vaginal, rectal, sublingual or topical. Administration can also be, for example, once, multiple times, and / or over one or more extended periods of time.

[0025] The terms "AKT inhibitor," "AKTI," or "AKTi" can be used interchangeably and refer to any molecule (e.g., an AKT antagonist), including, but not limited to, a small molecule, a polynucleotide (e.g., DNA or RNA), or a polypeptide (e.g., an antibody or antigen-binding portion thereof) that can block, reduce, or inhibit the activity of AKT. AKT is a serine / threonine kinase, also known as protein kinase B or PKB. An AKT inhibitor can act directly on AKT, for example, by binding to AKT, or it can act indirectly, for example, by interfering with the interaction of AKT with a binding partner or by inhibiting the activity of another member of the PI3K-AKT-mTOR pathway. Non-limiting examples of AKTi are provided in other sections of this application.

[0026] The term "antibody" (Ab) includes, but is not limited to, immunoglobulins that specifically bind to an antigen. Generally, antibodies may comprise at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region may comprise three or four constant domains, CH1, CH2 CH3, and / or CH4. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region may comprise one constant domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), flanked by more conserved regions, called framework regions (FRs). Each VH and VL contains three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen, such as AKT.

[0027] Immunoglobulins can be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the Ab class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene. The term "antibody" includes, by way of example, both natural and non-natural Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or non-human Abs; fully synthetic Abs; and single-chain Abs. Non-human Abs can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless explicitly stated and unless the context dictates otherwise, the term "antibody" also includes antigen-binding fragments or portions of any of the aforementioned immunoglobulins, including monovalent and bivalent fragments or portions, as well as single-chain Abs.

[0028] An "antigen-binding molecule" or "antibody fragment" refers to any portion of an antibody that is less than the entire antibody. An antigen-binding molecule may include antigenic complementarity-determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules.

[0029] The term "autologous" refers to any material derived from the same individual to whom it is to be later reintroduced. For example, the modified autologous cell therapy (eACT™) described herein involves the collection of lymphocytes from a donor, e.g., a patient, which are then modified, e.g., to express a CAR construct, and then administered back to the same donor, e.g., patient.

[0030] The term "allogeneic" refers to any material derived from one individual that is then introduced into another individual of the same species; for example, allogeneic T cell transplantation.

[0031] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and proliferation leads to the formation of malignant tumors that can invade neighboring tissues and further metastasize to distant sites in the body via the lymphatic system or bloodstream. "Cancer" or "cancerous tissue" can include tumors of various stages. In some embodiments, a cancer or tumor is at Stage 0, i.e., the cancer or tumor is very early in its development and has not metastasized. In some embodiments, a cancer or tumor is at Stage I, i.e., the cancer or tumor is relatively small in size, has not spread to nearby tissues, and has not metastasized. In other embodiments, a cancer or tumor is at Stage II or Stage III, i.e., the cancer or tumor is larger than in Stage 0 or Stage I and has grown into neighboring tissues but has not metastasized, except possibly to lymph nodes. In other embodiments, a cancer or tumor is at Stage IV, i.e., the cancer or tumor has metastasized. Stage IV may also be referred to as advanced cancer or metastatic cancer.

[0032] " Antitumor effect " as used herein refers to the biological effect that can be manifested as a reduction in tumor volume, inhibition of tumor growth, reduction in tumor cell number, reduction in tumor cell proliferation, reduction in metastasis number, increase in overall survival or progression-free survival, increase in life expectancy, or improvement in various physiological symptoms associated with tumors.Antitumor effect can also be used to prevent the occurrence of tumors, for example, vaccines.

[0033] The term "progression-free survival" (which may be abbreviated as PFS), as used herein, refers to the time from the date of treatment to the date of disease progression according to the revised IWG Response Criteria for Malignant Lymphoma or death from any cause.

[0034] "Disease progression" is assessed by measuring malignant lesions on radiographs; other methods should not be reported as adverse events. Deaths due to disease progression in the absence of signs and symptoms should be reported under the primary tumor type (e.g., DLBCL).

[0035] "Duration of response" (which may be abbreviated as DOR), as used herein, refers to the time from a subject's first objective response to the date of confirmed disease progression by the revised "IWG Response Criteria for Malignant Lymphoma" or death.

[0036] The term "overall survival" (which may be abbreviated as OS) is defined as the time from the date of treatment to the date of death.

[0037] "Cytokine" as used herein refers to a non-antibody protein that can be released by immune cells, including macrophages, B cells, T cells, and mast cells, to communicate an immune response. In some embodiments, one or more cytokines are released in response to T cell therapy. In some embodiments, the cytokines secreted in response to T cell therapy can be an indication of effective T cell therapy.

[0038] As used herein, a "therapeutically effective amount" or "therapeutically effective dosage" refers to the amount of T cells or DC cells produced by the method and that, when used alone or in combination with another therapeutic agent, protects a subject from developing disease or promotes disease regression (as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free symptom-free periods, or prevention of functional impairment or disability resulting from disease exposure). The ability of T cells or DC cells to promote disease regression can be assessed using a variety of methods known to those skilled in the art, for example, by assaying the activity of an agent in human subjects during clinical trials, in an animal model system predictive of efficacy in humans, or in an in vitro assay.

[0039] The term "effective amount" or "effective dose" as used herein refers to the amount of one or more T cell maturation inhibitors (e.g., AKTi, IL-7, and IL-15) that together induce a desired response. Thus, the effective amount of AKTi, the effective amount of IL-7, and the effective amount of IL-15 for delaying or inhibiting T cell differentiation or maturation may be lower than the effective amount of AKTi alone, the effective amount of IL-7 alone, or the effective amount of IL-15 alone. In other embodiments, the effective dose of AKTi may refer to the amount, e.g., concentration, of AKTi that reduces AKT activity by a desired amount, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%.

[0040] The term "lymphocyte," as used herein, may include natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cell-toxic) lymphocyte that represents a major component of the innate immune system. NK cells reject tumor and virus-infected cells. They work through the process of apoptosis, or programmed cell death. They are named "natural killers" because they do not require activation to kill cells. T cells play a major role in cell-mediated immunity (without antibody involvement). Their T cell receptors (TCRs) distinguish between themselves and other lymphocyte types. The thymus, a specialized organ of the immune system, is primarily responsible for the maturation of T cells.

[0041] Several types of T cells exist: helper T cells (e.g., CD4+ cells), effector T cells, EFF cells), cytotoxic T cells (TCs, also known as cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8+ T cells or killer T cells), memory T cells ((i) stem memory T SCMThe cells are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, and IL-7Rα+, similar to naive cells, but they also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, exhibiting many functional attributes characteristic of memory cells; (ii) central memory T CM The cells express L-selectin and CCR7 + and CD45RO + , which secrete IL-2 but not IFNγ or IL-4; and (iii) effector memory T EM However, there are various types of T cells within tumors: T cells (which do not express L-selectin or CCR7 but do express CD45RO and produce effector cytokines such as IFNγ and IL-4), regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKT), and gamma delta T cells. T cells found within tumors are called "tumor-infiltrating lymphocytes" or "TILs." B cells, on the other hand, play a major role in humoral immunity (antibody-mediated). They produce antibodies and antigens, act as antigen-presenting cells (APCs), and become memory B cells after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow, hence their name.

[0042] "Naive" T cells refer to mature T cells that remain immunologically undifferentiated. Following positive and negative selection in the thymus, T cells become CD4 + or CD8 + In their naive state, T cells express L-selectin (CD62L + ), IL-7 receptor-α (IL-7R-α), and CD132, but they do not express CD25, CD44, CD69, or CD45RO. As used herein, "immature" also refers to T cells that exhibit a phenotype characteristic of either naive or immature T cells, e.g., T SCM Cells or T CMFor example, immature T cells express L-selectin (CD62L + ), IL-7Rα, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, and LFA-1. Naive or immature T cells can be differentiated into terminally differentiated effector T cells, e.g., T EM Cells and T EFF It can be contrasted with cells.

[0043] "T cell function," as referred to herein, refers to a normal characteristic of healthy T cells. In some embodiments, T cell function includes T cell proliferation. In some embodiments, T cell function includes T cell activity. In some embodiments, T cell function includes cytolytic activity. In some embodiments, the methods of the present invention, e.g., culturing T cells in the presence of an AKT inhibitor (and optionally IL-7 and / or IL-15), increase one or more T cell functions, thereby making the T cells more suitable and / or potent for T cell therapy. In some embodiments, T cells cultured according to the present methods have increased T cell function compared to T cells cultured under conditions lacking an AKT inhibitor (or AKTi, IL-7, and IL-15). In certain embodiments, T cells cultured according to the present methods have increased T cell proliferation compared to T cells cultured under conditions lacking an AKT inhibitor (or AKTi, IL-7, and IL-15). In certain embodiments, T cells cultured according to the present methods have increased T cell activity compared to T cells cultured under conditions lacking an AKT inhibitor (or AKTi, IL-7, and IL-15). In certain embodiments, T cells cultured according to the present methods have increased cytolytic activity compared to T cells cultured under conditions lacking an AKT inhibitor (or AKTi, IL-7, and IL-15).

[0044] As used herein, cell "proliferation" refers to the ability of T cells to increase in number through cell division. Proliferation can be measured by staining cells with carboxyfluorescein succinimidyl ester (CFSE). Cell proliferation can occur in vitro, for example, during T cell culture, or in vivo, for example, following administration of T cell therapy.

[0045] "T cell activity," as used herein, refers to any activity common to healthy T cells. In some embodiments, T cell activity includes cytokine production. In some embodiments, T cell activity includes the production of one or more cytokines selected from interferon gamma (IFNg), tissue necrosis factor alpha (TNFa), and both.

[0046] "Cytolytic activity" or "cytotoxicity," as used herein, refers to the ability of T cells to destroy target cells. In some embodiments, the target cells are cancer cells, e.g., tumor cells. In some embodiments, the T cells express a chimeric antigen receptor (CAR) or a T cell receptor (TCR), and the target cells express a target antigen.

[0047] The terms "genetically engineered," "gene editing," or "modified" refer to methods of modifying a cell's genome, including, but not limited to, deleting a coding or non-coding region or portion thereof, or inserting a coding region or portion thereof. In some embodiments, the modified cells are lymphocytes, e.g., T cells, which can be obtained from either a patient or a donor. These cells can be modified to express exogenous constructs, such as chimeric antigen receptors (CARs) or T cell receptors (TCRs), that are integrated into the cell's genome.

[0048] "Immune response" refers to the actions of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils), and soluble macromolecules (including Abs, cytokines, and complement) produced by either these cells or the liver, that result in the selective targeting, binding to, damaging, destroying, and / or eliminating from the vertebrate body invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.

[0049] The term "immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of suffering from a disease or recurrence of a disease by methods involving inducing, enhancing, suppressing, or otherwise modifying an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy can include adoptive T cell therapy, tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, modified autologous cell therapy (eACT™), and allogeneic T cell transplantation. However, those skilled in the art will recognize that the methods of preparing T cells disclosed herein will also enhance the effectiveness of any transplanted T cell therapy. Examples of T cell therapy are described in U.S. Patent Application Publication Nos. 2014 / 0154228 and 2002 / 0006409, U.S. Patent No. 5,728,388, and International Publication No. WO 2008 / 081035.

[0050] T cells for immunotherapy can be derived from any source known in the art. For example, T cells can be differentiated in vitro from hematopoietic stem cell populations, or T cells can be obtained from donors. The donor can be a subject, for example, a subject in need of anti-cancer treatment. T cells can be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumor. In addition, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. T cells can also be obtained from an artificial thymic organoid (ATO) cell culture system, which reproduces the human thymic environment and supports the efficient ex vivo differentiation of T cells from primary and reprogrammed pluripotent stem cells. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is incorporated by reference herein in its entirety.

[0051] The term "modified autologous cell therapy" (which may be abbreviated as "eACT™"), also known as adoptive cell transfer, is a process in which a patient's own T cells are harvested and then genetically modified to recognize and target one or more antigens expressed on the cell surface of one or more specific tumor cells or malignancies. T cells can be engineered to express, for example, a chimeric antigen receptor (CAR) or a T cell receptor (TCR). CAR-positive (+) T cells are engineered to express an extracellular single-chain variable fragment (scFv) with specificity for a particular tumor antigen linked to an intracellular signaling moiety containing a costimulatory domain and an activation domain.Examples of costimulatory domains include CD28, CTLA4, CD16, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), programmed death-ligand-1 (PD-L1), inducible T cell costimulatory molecule (ICOS), ICOS-L, lymphocyte function-associated antigen-1 (LFA-1) (CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, and CD276. (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.The activation domain can be derived from, for example, CD3: e.g., CD3 zeta, epsilon, delta, gamma, etc. In some embodiments, CARs are designed to have two, three, four, or more costimulatory domains. CAR scFvs can be designed, for example, to target CD19, a transmembrane protein expressed by cells of the B-cell lineage, including all normal B cells and B-cell malignancies (including, but not limited to, NHL, CLL, and non-T-cell ALL). Exemplary CAR+ T-cell therapies and constructs are described in U.S. Patent Application Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, which are incorporated by reference in their entireties.

[0052] As used herein, a "patient" includes any human suffering from cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein. The term "donor subject" refers herein to a subject from whom cells are obtained for further in vitro manipulation. The donor subject may be a cancer patient who will be treated with a population of cells produced by the methods described herein (i.e., an autologous donor), or may be an individual who provides a lymphocyte sample that will be used to treat a different individual or cancer patient after the population of cells produced by the methods described herein is produced (i.e., an allogeneic donor). The subject who receives the cells prepared by the present method may be referred to as a "recipient subject."

[0053] "Stimulation," as used herein, refers to a primary response elicited by the binding of a stimulatory molecule to its cognate ligand, where the binding mediates a signal transduction event. A "stimulatory molecule" is a molecule on a T cell, such as a T cell receptor (TCR) / CD3 complex, that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell. A "stimulatory ligand" is a ligand that, when present on an antigen-presenting cell (e.g., an artificial antigen-presenting cell (aAPC), a dendritic cell, a B cell, etc.), specifically binds to a stimulatory molecule on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, etc. Stimulatory ligands include, but are not limited to, peptide-loaded MHC class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies. "Activated" or "active," as used herein, refers to a T cell after stimulation. Activated T cells may be characterized by the expression of one or more markers selected from CD137, CD25, CD71, CD26, CD27, CD28, CD30, CD154, CD40L, and CD134.

[0054] The term "exogenous" refers to any substance derived from an external source. For example, exogenous IL-7 or exogenous IL-15 can be commercially obtained or recombinantly produced. When added to or contacted with one or more T cells, "exogenous IL-7" or "exogenous IL-15" indicates that the IL-7 and / or IL-15 is not produced by the T cells. In some embodiments, the T cells prior to being mixed with exogenous IL-7 or IL-15 may contain trace amounts of IL-7 and / or IL-15 (i.e., endogenous IL-7 or IL-15) produced by the T cells or isolated from a subject along with the T cells. One or more T cells described herein can be contacted with exogenous IL-7 and / or IL-15 through any means known in the art, including the addition of isolated IL-7 and / or IL-15 to the culture; the inclusion of IL-7 and / or IL-15 in the culture medium; or the expression of IL-7 and / or IL-15 by one or more cells in the culture other than the one or more T cells, e.g., by a feeder layer.

[0055] The term "persistence," as used herein, refers to the ability of one or more transplanted T cells or their progeny (e.g., differentiated or mature T cells) administered to a subject to remain at detectable levels in a subject for a period of time. As used herein, increasing the persistence of one or more transplanted T cells or their progeny (e.g., differentiated or mature T cells) refers to increasing the amount of time that the transplanted T cells are detectable in a subject after administration. For example, the in vivo persistence of one or more transplanted T cells can be increased by at least about at least 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 3 weeks, at least about 4 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months. Additionally, the persistence of one or more transplanted T cells in vivo may be increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold, compared to one or more transplanted T cells not prepared by the methods disclosed herein.

[0056] The terms "reduce" and "reduce" are used interchangeably herein and refer to any change less than the original. "Reduce" and "reduce" are relative terms that require a comparison between before and after measurement. "Reduce" and "reduce" include complete depletion. In some embodiments, the terms "reduce" and "reduce" include a comparison of T cell effects between T cells prepared by the methods disclosed herein (e.g., contacting with AKTi and at least one of IL-7 and IL-15) and T cells without this preparation.

[0057] The term "modulating" T cell maturation, as used herein, refers to the use of any of the interventions described herein to control the maturation, e.g., differentiation, of one or more T cells. In some embodiments, "modulating" refers to delaying or inhibiting T cell maturation. In other embodiments, "modulating" refers to accelerating or promoting T cell maturation. In particular, "delaying or inhibiting T cell maturation," as used herein, refers to maintaining one or more T cells in an immature or undifferentiated state. For example, "delaying or inhibiting T cell maturation" refers to the maintenance of one or more T cells in an immature or undifferentiated state. EM or T EFF T cells into a naive or T state, as opposed to progressing to a CM "Delaying or inhibiting T cell maturation" may also refer to maintaining immature or undifferentiated T cells (e.g., naive T cells and / or T CMThe term "immature" may refer to increasing or enriching the overall percentage of T cells (e.g., mature or immature). The status of T cells (e.g., as mature or immature) can be determined, for example, by screening for the expression of various genes and the presence of various proteins expressed on the surface of T cells. For example, the presence of one or more markers selected from the group consisting of L-selectin (CD62L+), IL-7R-α, CD132, CR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, LFA-1, and any combination thereof, may indicate less mature, undifferentiated T cells.

[0058] "Treatment" of a subject or "treating" a subject refers to any type of intervention or procedure performed on a subject, or the administration of one or more T cells prepared according to the present invention to a subject, for the purpose of reversing, mitigating, ameliorating, suppressing, delaying, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, or condition, or biochemical marker associated with a disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission.

[0059] Various aspects of the invention are described in further detail in the following subsections.

[0060] Method for preparing immune cells The present disclosure relates to a method for preparing immune cells (e.g., lymphocytes or dendritic cells) for use in cell therapy. Some in vitro manipulated cells (e.g., CAR T cells, TCR cells, or dendritic cells) are found to be less effective when administered to patients after in vitro manipulation. Without being bound by any theory, it is noted that one reason may be that lymphocytes may be prematurely differentiated in vitro before being administered to patients. In some embodiments, the present disclosure describes a method for delaying, preventing, or inhibiting premature differentiation of cells in vitro by adding an AKTi and at least one of exogenous IL-7 and exogenous IL-15.

[0061] In one embodiment, the present disclosure relates to a method for modulating, e.g., delaying or inhibiting, the maturation or differentiation of T cells or DC cells in vitro by contacting one or more cells obtained from a donor subject with an AKT inhibitor and at least one (or both) of exogenous IL-7 and exogenous IL-15. Delaying or inhibiting the maturation or differentiation of T cells or DC cells can increase the percentage of immature, less differentiated cells (e.g., naive T cells or central memory Tcm cells) in a population of harvested T cells or DC cells. Thus, the methods described herein can be used to increase the in vivo persistence of transplanted T cells or DC cells or their progeny in cell therapy (e.g., T cell therapy or DC cell therapy). Additionally, the present disclosure provides that the resulting T cells or DC cells exhibit increased proliferation and superior antitumor activity in vitro and in vivo.

[0062] In another aspect, the present invention includes a method for modulating, e.g., delaying or inhibiting, cell (e.g., T cell) maturation or differentiation in vitro for cell therapy (e.g., T cell therapy), comprising: (i) contacting one or more cells (e.g., T cells or DC cells) from a subject in need of cell therapy (e.g., 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. The contacting step can include adding (i) an AKT inhibitor and (ii) exogenous IL-7 and / or exogenous IL-15 directly to the one or more T cells or to a buffer or medium containing the T cells, mixing (i) an AKT inhibitor and (ii) exogenous IL-7 and / or exogenous IL-15 with other components, and / or adding the one or more cells to a medium containing (i) an AKT inhibitor and (ii) exogenous IL-7 and / or exogenous IL-15. In certain embodiments, the one or more T cells are not contacted with exogenous interleukin-2 (IL-2). Further preparation of T cells is described elsewhere herein.

[0063] The present disclosure provides a method for treating a subject with an AKT inhibitor, comprising contacting one or more T cells or DC cells in vitro with an AKT inhibitor and at least one of IL-7 and IL-15 to increase the concentration of naive T cells and T cells in a sample relative to the concentration of more terminally differentiated T cells. CM Thus, in another aspect, the present invention provides a method for increasing the concentration of stem cell-like CD4 + T cells or CD8 + In another embodiment, the present invention provides a method for generating T cells, the method comprising culturing one or more T cells in a medium comprising (i) an AKT inhibitor and (ii) exogenous IL-7, exogenous IL-15, or both. + / CD45RA + / CCR7 +Methods for enriching a population of T cells include: (a) obtaining one or more T cells from a subject; (b) contacting one or more T cells with (i) an AKT inhibitor and (ii) exogenous IL-7, exogenous IL-15, or both; and (c) expanding the one or more T cells in the presence of the AKT inhibitor and exogenous IL-7, exogenous IL-15, or both. Generating increased concentrations of immature and undifferentiated T cells or DC cells can increase the in vivo persistence of the cells upon transplantation into a subject in need of cell therapy (e.g., T cell therapy or DC cell therapy). Accordingly, in another aspect, the invention includes a method for prolonging the in vivo persistence of one or more T cells or DC cells in adoptive cell therapy, comprising contacting the one or more T cells or DC cells with (i) an AKT inhibitor and (ii) exogenous IL-7, exogenous IL-15, or both, prior to administration to a subject; wherein the in vivo persistence is prolonged compared to one or more transferred T cells that were not contacted with the AKT inhibitor and exogenous IL-7, exogenous IL-15, or both.

[0064] The methods disclosed herein include modulating, e.g., delaying or inhibiting, the maturation or differentiation of one or more T cells or DC cells in vitro. The delay or inhibition of maturation or differentiation of one or more T cells or DC cells can be measured by any method known in the art. For example, the delay or inhibition of maturation or differentiation of one or more T cells or DC cells can be measured by detecting the presence of one or more biomarkers. The presence of one or more biomarkers can be detected by any method known in the art, including, but not limited to, immunohistochemistry and / or fluorescence-activated cell sorting (FACS). In some embodiments, the one or more biomarkers are L-selectin (CD62L), +), IL-7Rα, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, LFA-1, or any combination thereof. In some embodiments, the delayed or inhibited maturation or differentiation of one or more T cells or DC cells is selected from the group consisting of L-selectin (CD62L +), IL-7Rα, and CD132. Those skilled in the art will recognize that the present methods can increase the relative proportion of immature and undifferentiated T cells or DC cells in a population of harvested cells, but that some mature and differentiated cells may still be present. As a result, delay or inhibition of maturation or differentiation of one or more T cells or DC cells can be measured by calculating the total percentage of immature and undifferentiated cells in the cell population before and after contacting one or more cells with an AKT inhibitor and at least one of exogenous IL-7 and exogenous IL-15. In some embodiments, the methods disclosed herein increase the percentage of immature and undifferentiated T cells in a T cell population. In certain embodiments, the one or more T cells contacted with an AKT inhibitor and at least one of exogenous IL-7 and exogenous IL-15 comprise at least about 10%, at least about 15%, 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%, or about 100%, immature or undifferentiated T cells. In other embodiments, the one or more T cells or DC cells contacted with an AKT inhibitor and at least one of exogenous IL-7 and exogenous IL-15 comprise at least about 10% to at least about 90%, at least about 20% to at least about 80%, at least about 30% to at least about 70%, at least about 40% to at least about 60%, at least about 10% to at least about 50%, at least about 20%, at least about 40%, at least about 35% to at least about 45%, at least about 20% to at least about 60%, or at least about 50% to at least about 90% immature or undifferentiated T cells or DC cells. In certain embodiments, the immature or undifferentiated T cells are naive T cells and / or central memory Tcm cells.

[0065] The methods disclosed herein include contacting one or more T cells or DC cells with an AKT inhibitor and one or more of exogenous IL-7 and exogenous IL-15. In some embodiments, the method includes contacting one or more T cells or DC cells with an AKT inhibitor, exogenous IL-7, and exogenous IL-15. In another embodiment, the method includes contacting one or more T cells or DC cells with an AKT inhibitor and exogenous IL-7. In another embodiment, the method includes contacting one or more T cells or DC cells with an AKT inhibitor and exogenous IL-15. In one particular embodiment, the one or more T cells or DC cells are also contacted with exogenous IL-2. In another embodiment, the one or more T cells or DC cells are not contacted with exogenous IL-2.

[0066] One or more T cells or DC cells can be contacted with an AKT inhibitor and exogenous IL-7 and / or IL-15 through any means known in the art. For example, the AKT inhibitor and IL-7 / IL-15 can be added to the culture medium used to culture one or more T cells or DC cells. Alternatively, the AKT inhibitor and IL-7 / IL-15 can be produced by one or more cells, e.g., a feeder cell layer, co-cultured with one or more T cells or DC cells. The AKT inhibitor, IL-7, and IL-15 can be added together or individually. For example, the AKT inhibitor can be added to the culture medium, and IL-7 and / or IL-15 can be produced by cells co-cultured with one or more T cells.

[0067] In addition, one or more T cells or DC cells can be contacted with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15 at the same time, at different times, overlapping times, or sequentially. For example, one or more T cells or DC cells can be contacted with exogenous IL-7 and / or exogenous IL-15 before being contacted with an AKT inhibitor. Alternatively, one or more T cells or DC cells can be contacted with an AKT inhibitor before being contacted with exogenous IL-7 and / or exogenous IL-15. In one specific embodiment, one or more T cells or DC cells are first contacted with exogenous IL-7 and / or exogenous IL-15 alone, and then simultaneously contacted with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15. In another embodiment, one or more T cells or DC cells are first contacted with an AKT inhibitor alone, and then simultaneously contacted with the AKT inhibitor and exogenous IL-7 and / or exogenous IL-15. In some embodiments, the one or more T cells or DC cells are washed to remove the AKT inhibitor, exogenous IL-7, and / or exogenous IL-15.

[0068] One or more T cells or DC cells of the present disclosure can be administered to a subject for use in T cell or DC cell therapy. Accordingly, one or more T cells or DC cells can be harvested from a subject in need of T cell therapy or from a donor. Once harvested, the one or more T cells can be treated for any suitable period of time before administration to a subject. During this period, the one or more T cells can be contacted with, e.g., cultured in the presence of, an AKT inhibitor, exogenous IL-7, and / or exogenous IL-15 for any period between harvesting the T cells from the donor and administering them to the subject. For example, the one or more T cells can be contacted with, e.g., cultured in the presence of, an AKT inhibitor, exogenous IL-7, and / or exogenous IL-15 for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days. In some embodiments, one or more T cells are contacted with, e.g., cultured in the presence of, an AKT inhibitor, exogenous IL-7, and / or exogenous IL-15 for about 1 day to about 14 days, about 1 day to about 10 days, about 1 day to about 7 days, about 1 day to about 6 days, about 1 day to about 5 days, about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, or about 2 days to about 6 days. In one particular embodiment, from the day the T cells are harvested (e.g., day 0) until the day the T cells are administered to the subject, one or more T cells are contacted with, e.g., cultured in the presence of, an AKT inhibitor, exogenous IL-7, and / or exogenous IL-15. In another embodiment, the T cells are contacted with, e.g., cultured in the presence of, an AKT inhibitor, exogenous IL-7, and / or exogenous IL-15 from day 0 until administration, from day 1 until administration, from day 2 until administration, from day 3 until administration, from day 4 until administration, from day 5 until administration, or from day 6 until administration.In some embodiments, the one or more T cells are washed prior to administration to remove the AKT inhibitor, exogenous IL-7, and / or exogenous IL-15.

[0069] In one embodiment, the present disclosure relates to a method for regulating, e.g., delaying or inhibiting, the maturation or differentiation of T cells or DC cells in vitro by contacting one or more T cells or DC cells obtained from a donor subject with an AKT inhibitor and at least one (or both) of exogenous IL-7 and exogenous IL-15, wherein the one or more cells are not contacted with exogenous IL-2. In one embodiment, one or more cells treated with an AKTi and at least one (or both) of IL-7 and IL-15, but not IL-2, exhibit more delayed or inhibited maturation or differentiation than one or more cells treated with IL-2 alone or IL-2 and AKTi. The one or more T cells or DC cells may exhibit an increased percentage of immature, less differentiated cells (e.g., naive T cells or central memory Tcm cells) compared to one or more T cells or DC cells treated with IL-2 alone or IL-2 and AKTi. Therefore, the methods described herein can be used to increase the in vivo persistence of transplanted T cells or DC cells or their progeny in cell therapy (e.g., T cell therapy or DC cell therapy). In addition, the present disclosure provides that the resulting T cells or DC cells exhibit increased proliferation in vitro and in vivo and excellent anti-tumor activity. In some embodiments, one or more T cells are CD4 cells. In other embodiments, one or more T cells are CD8 cells. In certain embodiments, the AKTi is contacted with at least one of IL-7 and IL-15 for at least 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, or about 13 days. In other embodiments, the AKTi is contacted with at least one of IL-7 and IL-15 for more than 1 day but less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, or less than 8 days.

[0070] The method described herein can further comprise enriching the lymphocyte population obtained from donor.Enrichment of lymphocyte population, for example, one or more T cells, can be achieved by any suitable separation method, including but not limited to, using separation medium (for example, FICOLL-PAQUE™, ROSETTESEP™ HLA Total Lymphocyte enrichment cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical Cat. No. 0850494X) etc.), cell size, shape or density separation by filtration or elution, immunomagnetic separation (for example, magnetic activated cell sorting system, MACS), fluorescent separation (for example, fluorescence activated cell sorting system, FACS) or bead-based column separation.

[0071] The methods described herein may further include stimulating the population of lymphocytes with one or more T cell stimulatory agents under suitable conditions to generate a population of activated T cells. Any combination of one or more suitable T cell stimulatory agents may be used to generate the population of activated T cells, including, but not limited to, antibodies or functional fragments thereof that target T cell stimulatory or costimulatory molecules (e.g., anti-CD2 antibodies, anti-CD3 antibodies, anti-CD28 antibodies, or functional fragments thereof), or any other suitable mitogen (e.g., tetradecanoylphorbol acetate (TPA), phytohemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)), or natural ligands for T cell stimulatory or costimulatory molecules.

[0072] Suitable conditions for stimulating a population of lymphocytes as described herein may include a temperature, a length of time, and / or the presence of a CO2 level. In some embodiments, the temperature for stimulation is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C. In some embodiments, the temperature for stimulation is about 34-38°C. In some embodiments, the temperature for stimulation is about 35-37°C. In some embodiments, the temperature for stimulation is about 36-38°C. In some embodiments, the temperature for stimulation is about 36-37°C or about 37°C.

[0073] Another condition for stimulating a lymphocyte population as described herein may include the time for stimulation. In some embodiments, the time for stimulation is about 24 to 72 hours. In some embodiments, the time for stimulation is about 24 to 36 hours, about 30 to 42 hours, about 36 to 48 hours, about 40 to 52 hours, about 42 to 54 hours, about 44 to 56 hours, about 46 to 58 hours, about 48 to 60 hours, about 54 to 66 hours, or about 60 to 72 hours. In one particular embodiment, the time for stimulation is about 48 hours or at least about 48 hours. In other embodiments, the time for stimulation is about 44 to 52 hours. In certain embodiments, the time for stimulation is about 40 to 44 hours, about 40 to 48 hours, about 40 to 52 hours, or about 40 to 56 hours.

[0074] Other conditions for stimulating lymphocyte populations as described herein may include CO2 levels. In some embodiments, the CO2 level for stimulation is about 1.0-10% CO2. In some embodiments, the CO2 level for stimulation is about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In one embodiment, the CO2 level for stimulation is about 3-7% CO2. In other embodiments, the CO2 level for stimulation is about 4-6% CO2. In yet other embodiments, the CO2 level for stimulation is about 4.5-5.5% CO2. In one particular embodiment, the CO2 level for stimulation is about 5% CO2.

[0075] The conditions for stimulating the population of lymphocytes can include any combination of a temperature, a length of time for stimulation, and / or the presence of a level of CO. For example, stimulating the population of lymphocytes can include stimulating the population of lymphocytes with one or more T cell stimulatory agents at a temperature of about 36-38° C. for a length of time of about 44-52 hours in the presence of a CO level of about 4.5-5.5% CO.

[0076] The concentration of lymphocytes useful in the methods herein is about 1.0-10.0 x 10 6 In one embodiment, the lymphocyte concentration is about 1.0 to 2.0 x 10 cells / mL. 6 cells / mL, approximately 1.0~3.0 x 10 6 cells / mL, approximately 1.0~4.0 x 10 6 cells / mL, approximately 1.0~5.0 x 10 6 cells / mL, approximately 1.0~6.0 x 10 6 cells / mL, approximately 1.0~7.0 x 10 6 cells / mL, approximately 1.0~8.0 x 10 6 cells / mL, 1.0~9.0 x 10 6 cells / mL, or approximately 1.0–10.0 x 10 6 In one embodiment, the lymphocyte concentration is about 1.0-2.0 x 10 cells / mL. 6 In one embodiment, the lymphocyte concentration is about 1.0 to 1.2 x 10 cells / mL. 6 cells / mL, approximately 1.0~1.4 x 10 6 cells / mL, approximately 1.0~1.6 x 10 6 cells / mL, approximately 1.0~1.8 x 10 6 cells / mL, or approximately 1.0–2.0 x 10 6 In some embodiments, the concentration of lymphocytes is at least about 1.0 x 10 cells / mL. 6 At least approximately 1.1 x 10 cells / mL 6 At least approximately 1.2 x 10 cells / mL 6 At least approximately 1.3 x 10 cells / mL6 At least approximately 1.4 x 10 cells / mL 6 At least approximately 1.5 x 10 cells / mL 6 At least approximately 1.6 x 10 cells / mL 6 At least approximately 1.7 x 10 cells / mL 6 At least approximately 1.8 x 10 cells / mL 6 At least approximately 1.9 x 10 cells / mL 6 At least approximately 2.0 x 10 cells / mL 6 At least approximately 4.0 x 10 cells / mL 6 At least approximately 6.0 x 10 cells / mL 6 At least approximately 8.0 x 10 cells / mL 6 cells / mL, or at least approximately 10.0 x 10 6 cells / mL.

[0077] Anti-CD3 antibodies (or functional fragments thereof), anti-CD28 antibodies (or functional fragments thereof), or a combination of anti-CD3 and anti-CD28 antibodies can be used in the step of stimulating the lymphocyte population. Any soluble or immobilized anti-CD2, anti-CD3, and / or anti-CD28 antibodies or functional fragments thereof can be used (e.g., clone OKT3 (anti-CD3), clone 145-2C11 (anti-CD3), clone UCHT1 (anti-CD3), clone L293 (anti-CD28), clone 15E8 (anti-CD28)). In some aspects, antibodies can be commercially purchased from suppliers known in the art, including, but not limited to, Miltenyi Biotec, BD Biosciences (e.g., MACS GMP CD3 pure 1 mg / mL, Part No. 170-076-116), and eBioscience, Inc. Furthermore, those skilled in the art will understand how to generate anti-CD3 and / or anti-CD28 antibodies by standard methods. In some embodiments, the one or more T cell stimulatory agents that can be used in the step of stimulating a lymphocyte population include an antibody or functional fragment thereof that targets a T cell stimulatory molecule or costimulatory molecule in the presence of a T cell cytokine. In one aspect, the one or more T cell stimulatory agents include an anti-CD3 antibody and IL-2. In certain embodiments, the T cell stimulatory agent comprises an anti-CD3 antibody at a concentration of about 20 ng / mL to 100 ng / mL. In certain embodiments, the concentration of the anti-CD3 antibody is about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL. In one particular embodiment, the concentration of the anti-CD3 antibody is about 50 ng / mL. In alternative embodiments, T cell activation is not required. In such embodiments, the step of stimulating the population of lymphocytes to generate a population of activated T cells is omitted from the method, and the population of lymphocytes (which may be enriched for T lymphocytes) is transduced according to the steps described below.

[0078] The methods described herein may include transducing a population of activated T cells with a viral vector comprising a nucleic acid molecule encoding a cell surface receptor using single-cycle transduction to generate transduced T cells. Several recombinant viruses have been used as viral vectors for delivering genetic material to cells. The viral vector that may be used following the transduction step may be any ecotropic or amphotropic viral vector, including, but not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, and recombinant adeno-associated viral (AAV) vectors. In some embodiments, the method further includes transducing one or more T cells with a retrovirus. In one embodiment, In some embodiments, the viral vector used to transduce the population of activated T cells is the MSGV1 gammaretroviral vector. In some embodiments, the viral vector used to transduce the population of activated T cells is the PG13-CD19-H3 vector described by Kochenderfer, J. Immunother. 32(7): 689-702 (2009). According to one aspect of this embodiment, the viral vector is propagated in suspension culture in a viral vector production medium, referred to herein as the "viral vector inoculum." Any suitable growth medium and / or supplements for propagating viral vectors can be used in the viral vector inoculum according to the methods described herein. According to some aspects, the viral vector inoculum is then added to a serum-free culture medium, described below, during the transduction step.

[0079] In some embodiments, one or more T cells can be transduced with a retrovirus. In one embodiment, the retrovirus contains a heterologous gene encoding a cell surface receptor. In one particular embodiment, the cell surface receptor can bind to an antigen on the surface of a target cell, for example, on the surface of a tumor cell.

[0080] Conditions for transducing a population of activated T cells as described herein can include a specific time, at a specific temperature, and / or in the presence of a specific CO2 level. In certain embodiments, the temperature for transduction is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C. In one embodiment, the temperature for transduction is about 34-38°C. In another embodiment, the temperature for transduction is about 35-37°C. In another embodiment, the temperature for transduction is about 36-38°C. In yet another embodiment, the temperature for transduction is about 36-37°C. In one particular embodiment, the temperature for transduction is about 37°C.

[0081] In certain embodiments, the time for transduction is about 12 to 36 hours. In some embodiments, the time for transduction is about 12 to 16 hours, about 12 to 20 hours, about 12 to 24 hours, about 12 to 28 hours, or about 12 to 32 hours. In other embodiments, the time for transduction is about 20 hours or at least about 20 hours. In one embodiment, the time for transduction is about 16 to 24 hours. In other embodiments, the time for transduction is at least about 14 hours, at least about 16 hours, at least about 18 hours, at least about 20 hours, at least about 22 hours, at least about 24 hours, or at least about 26 hours.

[0082] In certain embodiments, the CO2 level for transduction is about 1.0-10% CO2. In other embodiments, the CO2 level for transduction is about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In one embodiment, the CO2 level for transduction is about 3-7% CO2. In another embodiment, the CO2 level for transduction can be about 4-6% CO2. In another embodiment, the CO2 level for transduction is about 4.5-5.5% CO2. In one specific embodiment, the CO2 level for transduction is about 5% CO2.

[0083] In some embodiments, transduction of a population of activated T cells as described herein can be carried out at any combination of a particular length of time, a particular temperature, and / or in the presence of a particular CO2 level: at a temperature of about 36-38°C for about 16-24 hours, and in the presence of a CO2 level of about 4.5-5.5% CO2.

[0084] The methods described herein can include expanding one or more populations of transduced T cells for a specified period of time to generate a population of modified T cells. The predetermined time for expansion can be any suitable time that allows for the generation of: (i) a sufficient number of cells in the modified T cell population for at least one dose administered to a patient, (ii) a population of modified T cells containing a favorable proportion of immature cells compared to typical longer processes, or (iii) both (i) and (ii). This time will depend on the cell surface receptors expressed by the T cells, the vector used, the dose required to have a therapeutic effect, and other variables. Thus, in some embodiments, the predetermined time for expansion can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or more than 21 days. In some aspects, the time for expansion is shorter than expansion methods known in the art. For example, the predetermined time for expansion can be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or more than 75% shorter. In one aspect, the time for expansion is about 3 days, and the time from enrichment of the population of lymphocytes to generation of modified T cells is about 6 days.

[0085] Conditions for expanding a population of transduced T cells may include a temperature and / or the presence of a CO2 level. In certain embodiments, the temperature is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C. In one embodiment, the temperature is about 34-38°C. In another embodiment, the temperature is about 35-37°C. In another embodiment, the temperature is about 36-38°C. In yet another embodiment, the temperature is about 36-37°C. In one particular embodiment, the temperature is about 37°C. In certain embodiments, the CO2 level is 1.0-10% CO2. In other embodiments, the CO2 level is about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO2. In one embodiment, the CO2 level is about 4.5-5.5% CO2. In another embodiment, the level of CO2 is about 5% CO2. In other embodiments, the level of CO2 is about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, or about 6.5% CO2. In some embodiments, the conditions for expanding a population of transduced T cells include the presence of a temperature and / or a CO2 level, in any combination. For example, the conditions for expanding a population of transduced T cells include a temperature of about 36-38°C and a CO2 level of about 4.5-5.5% CO2.

[0086] Each step of the methods described herein can be performed in a closed system. In some embodiments, the closed system is a closed-bag culture system using any suitable cell culture bag (e.g., Miltenyi Biotec MACS® GMP Cell Differentiation Bag, Origen Biomedical PermaLife Cell Culture bag). In some embodiments, the cell culture bag used in the closed-bag culture system is coated with a recombinant human fibronectin fragment during the transduction step. The recombinant human fibronectin fragment may contain three functional domains: a central cell-binding domain, a heparin-binding domain II, and a CS1 sequence. The recombinant human fibronectin fragment can be used to enhance the gene efficiency of retroviral transduction of immune cells by aiding in colocalization of target cells and viral vectors. In some embodiments, the recombinant human fibronectin fragment is RETRONECTIN® (Takara Bio, Japan). In some embodiments, the cell culture bag is coated with the recombinant human fibronectin fragment at a concentration of about 1 to 60 μg / mL or about 1 to 40 μg / mL. In other embodiments, the cell culture bag is coated with a recombinant human fibronectin fragment at a concentration of about 1-20 μg / mL, 20-40 μg / mL, or 40-60 μg / mL. In some embodiments, the cell culture bag is coated with a recombinant human fibronectin fragment at a concentration of about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, about 10 μg / mL, about 11 μg / mL, about 12 μg / mL, about 13 μg / mL, about 14 μg / mL, about 15 μg / mL, about 16 μg / mL, about 17 μg / mL, about 18 μg / mL, about 19 μg / mL, or about 20 μg / mL. In other embodiments, the cell culture bag is coated with about 2-5 μg / mL, about 2-10 μg / mL, about 2-20 μg / mL, about 2-25 μg / mL, about 2-30 μg / mL, about 2-35 μg / mL, about 2-40 μg / mL, about 2-50 μg / mL, or about 2-60 μg / mL of recombinant human fibronectin fragment.In some embodiments, the cell culture bag is coated with at least about 2 μg / mL, at least about 5 μg / mL, at least about 10 μg / mL, at least about 15 μg / mL, at least about 20 μg / mL, at least about 25 μg / mL, at least about 30 μg / mL, at least about 40 μg / mL, at least about 50 μg / mL, or at least about 60 μg / mL of recombinant human fibronectin fragment. In one particular embodiment, the cell culture bag is coated with at least about 10 μg / mL of recombinant human fibronectin fragment. Cell culture bags used in closed-bag culture systems can optionally be blocked with human albumin serum (HSA) during the transduction step. In an alternative embodiment, the cell culture bag is not blocked with HSA during the transduction step.

[0087] In other aspects, at least one of (a) contacting a population of lymphocytes with an AKT inhibitor and at least one of exogenous IL-7 and exogenous IL-15, (b) stimulating the population of lymphocytes, (c) transducing a population of activated T cells, and (d) expanding the population of transduced T cells is performed using a serum-free culture medium to which serum is not added. In some aspects, each of (a) through (d) is performed using a serum-free culture medium to which serum is not added. In another aspect, at least one of (a) contacting a population of lymphocytes with an AKT inhibitor and at least one of exogenous IL-7 and exogenous IL-15, (b) stimulating the population of lymphocytes, (c) transducing a population of activated T cells, and (d) expanding the population of transduced T cells is performed using a serum-free culture medium. In some aspects, each of (a) to (d) is performed using a serum-free culture medium to which no serum has been added. As referred to herein, the term "serum-free medium" or "serum-free culture medium" means that the growth medium used is not supplemented with serum (e.g., human serum or bovine serum). In other words, in some embodiments, serum is not added to the culture medium as an individually separated and distinct component for the purpose of supporting the viability, activation, and growth of the cultured cells. Any suitable culture medium, such as a T cell growth medium, can be used to culture cells in suspension according to the methods described herein. For example, the T cell growth medium may include, but is not limited to, a sterile, low-glucose solution containing appropriate amounts of buffer, magnesium, calcium, sodium pyruvate, and sodium bicarbonate. In one embodiment, the T cell growth medium is OPTMIZER™ (Life Technologies). In contrast to typical methods for generating modified T cells, the methods described herein can use a culture medium that is not supplemented with serum (e.g., human or bovine).

[0088] AKT inhibitors The AKT kinase family has three highly homologous isoforms: AKT1 (PKBα), AKT2 (PKBβ), and AKT3 (PKBγ), each with unique and overlapping functions. As part of the PI3K-AKT-mTOR signaling pathway, AKT acts downstream of PI3K to activate mTOR, inducing various responses in cells, including survival, growth, proliferation, migration, and metabolism.

[0089] Any AKT inhibitor known in the art can be used in the present invention, including any inhibitor of AKT1, AKT2, AKT3, or any combination thereof. AKT inhibitors include 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, and GSK 690693 (CAS # 937174-76-0), FPA 124 (CAS # 902779-59-3), Miltefosine, PHT-427 (CAS # 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-111A, 3-[1-[[4-(7-phenyl-3H-imidazo[4,5-g]quinoxalin-6-yl)phenyl]methyl]piperidin-4-yl]-1H-benzimidazol-2-one, and any combination thereof.AKT inhibitors also include 1-{1-[4-(7-phenyl-1H-imidazo[4,5-g]quinoxalin-6-yl)benzyl]piperidin-4-yl}-1,3-dihydro-2H-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-6-yl)benzyl]piperidin-4-yl}-1,3-dihydro-2H-benzimidazol-2-one; 1-{1-[4-(7-phenyl-1H-imidazo[4,5-g]quinoxalin-6-yl)benzyl]piperidin-4-yl}-1,3-dihydro-2H-benzimidazol-2-one;1-{1-[4-(7-phenyl-1H-imidazo[4,5-g]quinoxalin-6-yl)benzyl]piperidin-4-yl}-1,3-dihydro-2H-benzimidazol-2-one;N,N-Dimethyl-1-[4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl] Meth-namine; 1-{1-[4-(3-phenylbenzo[g]quinoxalin-2-yl)benzyl]piperidin-4-yl}-1,-3-dihydro-2H-benzimidazol-2-one (also known as 3-[1-[[4-(7-phenyl-3H-imidazo[4,5-g]quinoxalin-6-yl)phenyl]methyl]piperidin-4-yl]-1H-benzimidazol-2-one); U.S. Patent No. 7, issued September 25, 2007 No. 7,273,869, issued Sep. 25, 2007, which is incorporated herein by reference in its entirety; and any combination thereof. In one example, the AKTi comprises Formula I.

[0090] In one particular embodiment, 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. In another embodiment, the AKT inhibitor is Akt inhibitor VIII.

[0091] In some embodiments, the AKT comprises the formula shown by Formula I, or a pharmaceutically acceptable salt or stereoisomer thereof: t is 2, 3, 4, 5, or 6; u, v, and x are independently selected from CH and N; w is a bond, CH, and N; y and z are independently selected from CH and N, with the proviso that at least one of y and z is N; R 1 is:1) (C=O) a O b C1-C 10 Alkyl, 2) (C=O) a O b aryl, 3) C2-C 10 Alkenyl, 4) C2-C 10 Alkynyl, 5) (C=O) a O b Heterocyclyl, 6) (C=O) a O b C3-C8 cycloalkyl, 7) CO2H, 8) halo, 9) CN, 10) OH, 11) O b C1-C6 perfluoroalkyl, 12) O a (C=O) b NR 7 R8, 13) NR c (C=O)NR 7 R 8 , 14) S(O) m R a , 15) S(O)2NR 7 R 8 , 16) NR c S(O) m R a , 17) Oxo, 18) CHO, 19) NO2, 20) NR c (C=O)O b R a , 21) O(C=O)O b C1-C 10Alkyl, 22) O(C=O)O b C3-C8 cycloalkyl, 23) O(C=O)O b Aryl, and 24) O(C=O)O b -heterocycle; said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl are independently selected from R z may be substituted with one or more substituents selected from the group consisting of: 2 is:1) (C=O) a O b C1-C 10 Alkyl, 2) (C=O) a O b aryl, 3) C2-C 10 Alkenyl, 4) C2-C 10 Alkynyl, 5) (C=O) a O b Heterocyclyl, 6) (C=O) a O b C3-C8 cycloalkyl, 7) CO2H, 8) halo, 9) CN, 10) OH, 11) O b C1-C6 perfluoroalkyl, 12) O a (C=O) b NR 7 R 8 , 13) NR c (C=O)NR7R 8 , 14) S(O) m R a , 15) S(O)2NR 7 R 8 , 16) NR c S(O) m R a , 17) CHO, 18) NO2, 19) NR c (C=O)O b R a , 20) O(C=O)O b C1-C 10 Alkyl, 21) O(C=O)O b C3-C8 cycloalkyl, 22) O(C=O)O b Aryl, and 23) O(C=O)O b-heterocycle; said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl are independently selected from R z optionally substituted with 1, 2 or 3 substituents selected from: R 3 and R 4 are independently selected from H, C-C-alkyl and C-C-perfluoroalkyl, or R 3 and R 4 are combined to form -(CH2) t -, where one of the carbon atoms is O, S(O) m , -N(R b )C(O)-, and -N(COR a )-; R 5 and R 6 are 1) H, 2) (C=O)O b R a , 3) C1-C 10 Alkyl, 4) Aryl, 5) C2-C 10 Alkenyl, 6) C2-C 10 alkynyl, 7) heterocyclyl, 8) C3-C8 cycloalkyl, 9) SO2R a , and 10) (C=O)NR b 2, wherein said alkyl, cycloalkyl, aryl, heterocyclyl, alkenyl, and alkynyl are independently selected from R z or R 5 and R 6 can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle having 5 to 7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, which monocyclic or bicyclic heterocycle can be substituted with Q and further substituted with R z Q is optionally substituted with one or more substituents selected from -NR 7 R 8 , aryl and heterocyclyl, wherein said aryl and heterocyclyl are selected from R zmay be substituted with 1 to 3 substituents selected from the following; R 7 and R 8 are 1) H, 2) (C=O)O b C1-C 10 Alkyl, 3) (C=O)O b C3-C8 cycloalkyl, 4) (C=O)O b Aryl, 5) (C=O)O b Heterocyclyl, 6) C1-C 10 Alkyl, 7) Aryl, 8) C2-C 10 Alkenyl, 9) C2-C 10 10) Alkynyl, 11) Heterocyclyl, 12) C3-C8 Cycloalkyl, 13) SO2R a , and 13) (C=O)NR b 2; said alkyl, cycloalkyl, aryl, heterocyclyl, alkenyl, and alkynyl are independently selected from R z or R 7 and R 8 can be taken together with the nitrogen to which they are attached to form a monocyclic or bicyclic heterocycle having 5 to 7 members in each ring and optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, which monocyclic or bicyclic heterocycle is z may be substituted with one or more substituents selected from the group consisting of: z 1) (C=O) r O s (C1-C 10 ) alkyl, 2) O r (C1-C3) perfluoroalkyl, 3) (C0-C6) alkylene-S(O) m R a , 4) Oxo, 5) OH, 6) Halo, 7) CN, 8) (C=O) r O s (C2-C 10 ) alkenyl, 9) (C=O) r O s (C2-C 10 ) alkynyl, 10) (C=O) r O s(C3-C6)cycloalkyl, 11) (C=O) r O s (C0-C6) alkylene-aryl, 12) (C=O) r O s (C0-C6) alkylene-heterocyclyl, 13) (C=O) r O s (C0-C6) alkylene-N(R b )2, 14) C(O)R a , 15) (C0-C6) alkylene-CO2R a , 16) C(O)H, 17) (C0-C6) alkylene-CO2H, 18) C(O)N(R b )2, 19) S(O) m R a , 20) S(O)2N(R b )2, 21) NR c (C=O)O b R a , 22) O(C=O)O b C1-C 10 Alkyl, 23) O(C=O)O b C3-C8 cycloalkyl, 24) O(C=O)O b Aryl, and 25) O(C=O)O b -heterocycle; said alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heterocyclyl are selected from R b , OH, (C1-C6)alkoxy, halogen, CO2H, CN, O(C=O)C1-C6 alkyl, oxo, and N(R b ) optionally substituted with up to three substituents selected from R a is substituted or unsubstituted (C-C) alkyl, substituted or unsubstituted (C-C) alkenyl, substituted or unsubstituted (C-C) alkynyl, substituted or unsubstituted (C-C) cycloalkyl, substituted or unsubstituted aryl, (C-C) perfluoroalkyl, 2,2,2-trifluoroethyl, or substituted or unsubstituted heterocyclyl; and R bis H, (C1-C6)alkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted heterocyclyl, (C3-C6)cycloalkyl, (C=O)O-C1-C6 alkyl, (C=O)C1-C6 alkyl or S(O)2R a and;R c are 1) H, 2) C1-C 10 Alkyl, 3) Aryl, 4) C2-C 10 Alkenyl, 5) C2-C 10 6) alkynyl, 7) C3-C8 cycloalkyl, 8) C1-C6 perfluoroalkyl, wherein said alkyl, cycloalkyl, aryl, heterocyclyl, alkenyl, and alkynyl are selected from R z It may be substituted with one or more substituents selected from the following:

[0092] In some embodiments, AKT signaling can be inhibited directly, for example, by a molecule that binds to AKT, or indirectly, for example, by interfering with another member of the PI3K-AKT-mTOR signaling pathway. Thus, an AKT inhibitor can be a molecule that inhibits the activity of one or more members of the PI3K-AKT-mTOR signaling pathway. For example, one or more T cells can be contacted with an AKT inhibitor, a PI3K inhibitor, an mTOR inhibitor, or any combination thereof.

[0093] An amount of an AKT inhibitor useful in the methods described herein can be an amount (i.e., an effective amount) that can reduce or inhibit the activity of AKT in one or more T cells. In another embodiment, an amount of an AKT inhibitor useful in the present invention can be an amount that can delay or inhibit the maturation or differentiation of T cells or DC cells in vitro in combination with exogenous IL-7 and / or exogenous IL-15. Thus, in one embodiment, one or more T cells are treated with an AKT inhibitor, e.g., 3-[1-[[4-(7-phenyl-3H-imidazo[4,5-g]quinoxalin-6-yl)phenyl]methyl]piperidin-4-yl]-1H-benzimidazol-2-one, at least about 1 nM, at least about 10 nM, at least about 50 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 600 nM, at least about 700 nM, at least about 800 nM, at least about 900 nM, at least about 1000 nM, at least about 1500 nM, at least about 2000 nM, at least about 3000 nM, at least about 4000 nM, at least about 5000 nM, at least about 1000 nM, at least about 2500 nM, at least about 3500 nM, at least about 4500 nM, at least about 5000 nM, at least about 1000 nM, at least about 15000 nM, at least about 10000 nM, at least about 10000 nM, at least about 25000 nM, at least about 15000 nM, at least about 10 nM, at least about 1 μM, at least about 2 μM, at least about 3 μM, at least about 4 μM, at least about 5 μM, at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM, at least about 10 μM, at least about 11 μM, at least about 12 μM, at least about 13 μM, at least about 14 μM, at least about 15 μM, at least about 16 μM, at least about 17 μM, at least about 18 μM, at least about The contact may be at a concentration of about 19 μM, at least about 20 μM, at least about 25 μM, at least about 30 μM, at least about 35 μM, at least about 40 μM, at least about 45 μM, at least about 50 μM, at least about 60 μM, at least about 70 μM, at least about 80 μM, at least about 90 μM, at least about 100 μM, at least about 200 μM, at least about 300 μM, at least about 400 μM, at least about 500 μM, or at least about 1 mM.In another embodiment, the one or more T cells are treated with an AKT inhibitor, e.g., 3-[1-[[4-(7-phenyl-3H-imidazo[4,5-g]quinoxalin-6-yl)phenyl]methyl]piperidin-4-yl]-1H-benzimidazol-2-one, at a concentration of about 1 nM to about 1 mM, about 10 nM to about 1 mM, about 100 nM to about 1 mM, about 1 μM to about 1 mM, about 10 μM to about 1 mM, about 100 μM to about 1 mM, about 1 nM to about 100 μM, about 1 nM to about 10 μM, about 1 nM to about 1 μM, about 1 nM to about 100 nM, about 1 nM to about 50 nM, about 100 nM to about 100 μM, about 500 The contact can be carried out at a concentration of about nM to about 50 μM, about 1 μM to about 50 μM, about 1 μM to about 10 μM, or about 5 μM to about 10 μM.

[0094] Any reduction in AKT activity can be achieved according to the present method. For example, AKT activity can be reduced or inhibited by an AKT inhibitor by at least about 5%, at least about 10%, at least about 15%, 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 99%, or about 100%.

[0095] Exogenous IL-7 and exogenous IL-15 Interleukin-7 (IL-7) is a cytokine that promotes lymphocyte homeostasis and is required for T cell development. Endogenous IL-7 is produced by epithelial cells in the thymus and bone marrow, and its receptor, IL-7 receptor-α (IL-7R-α), is expressed by naive T cells and T CM IL-7 signaling is expressed by a subset of T cells, including IL-7-positive T cells. IL-7 signaling results in the activation of various tyrosine kinases, including the Janus kinase / signal transducer and activator of transcription (Jak / STAT) pathway, PI3K, and Src family tyrosine kinases.

[0096] Any exogenous IL-7 can be used in the methods described herein. In some embodiments, the exogenous IL-7 is human IL-7. In some embodiments, the exogenous IL-7 is wild-type IL-7. In other embodiments, the exogenous IL-7 is recombinant IL-7. IL-7 can be produced and obtained by any method known in the art, including, but not limited to, isolating IL-7 from one or more IL-7-producing cells or obtaining commercially available IL-7.

[0097] Any concentration of IL-7 can be used in the methods described herein. For example, the method can include injecting one or more T cells with at least about 0.001 ng / ml IL-7, at least about 0.005 ng / ml IL-7, at least about 0.01 ng / ml IL-7, at least about 0.05 ng / ml IL-7, at least about 0.1 ng / ml IL-7, at least about 0.5 ng / ml IL-7, at least about 1.0 ng / ml IL-7, at least about 1 ng / ml IL-7, at least about 2 ng / ml IL-7, at least about 3 ng / ml IL-7, at least about 4 ng / ml IL-7, at least about 5 ng / ml IL-7, at least about 6 ng / ml IL-7, at least about 7 ng / ml IL-7, at least about 8 ng / ml IL-7, at least about 9 ng / ml IL-7, at least about 10 ng / ml IL-7, at least about 11 ng / ml The method may include contacting the IL-7 with at least about 12 ng / ml IL-7, at least about 13 ng / ml IL-7, at least about 14 ng / ml IL-7, at least about 15 ng / ml IL-7, at least about 20 ng / ml IL-7, at least about 25 ng / ml IL-7, at least about 30 ng / ml IL-7, at least about 35 ng / ml IL-7, at least about 40 ng / ml IL-7, at least about 45 ng / ml IL-7, at least about 50 ng / ml IL-7, at least about 100 ng / ml IL-7, at least about 200 ng / ml IL-7, at least about 300 ng / ml IL-7, at least about 400 ng / ml IL-7, at least about 500 ng / ml IL-7, or at least about 1000 ng / ml IL-7. In one embodiment, the one or more T cells are contacted with about 0.001 to about 500 ng / ml IL-7, about 0.01 to about 100 ng / ml IL-7, about 0.1 to about 50 ng / ml IL-7, about 1 to about 10 ng / ml IL-7, about 1 to about 5 ng / ml IL-7, about 5 to about 10 ng / ml IL-7, about 3 to about 7 ng / ml IL-7, or about 4 to about 6 ng / ml IL-7.In one particular embodiment, the one or more T cells are contacted with about 5 ng / ml IL-7.

[0098] Interleukin-15 (IL-15) is a cytokine that promotes T cell proliferation. It is expressed by members of the monocyte / macrophage lineage, blood-derived dendritic cells, bone marrow stromal cells, and thymic epithelial cells. IL-15 signals through its receptor, the IL-15 receptor, to activate, for example, the Jak / STAT pathway, stimulate the Ras / Raf / MAPK pathway, and activate NF-κB.

[0099] Any exogenous IL-15 can be used in the methods described herein. In some embodiments, the exogenous IL-15 is human IL-15. In some embodiments, the exogenous IL-15 is wild-type IL-15. In other embodiments, the exogenous IL-15 is recombinant IL-15. IL-15 can be produced and obtained by any method known in the art, including, but not limited to, isolating IL-15 from one or more IL-15-producing cells or obtaining commercially available IL-15.

[0100] Any concentration of IL-15 can be used in the methods described herein. For example, the method can include infecting one or more T cells with at least about 0.001 ng / ml IL-15, at least about 0.005 ng / ml IL-15, at least about 0.01 ng / ml IL-15, at least about 0.05 ng / ml IL-15, at least about 0.1 ng / ml IL-15, at least about 0.5 ng / ml IL-15, at least about 1.0 ng / ml IL-15, at least about 1 ng / ml IL-15, at least about 2 ng / ml IL-15, at least about 3 ng / ml IL-15, at least about 4 ng / ml IL-15, at least about 5 ng / ml IL-15, at least about 6 ng / ml IL-15, at least about 7 ng / ml IL-15, at least about 8 ng / ml IL-15, at least about 9 ng / ml IL-15, at least about 10 ng / ml IL-15, at least about 11 ng / ml IL-15, at least about 12 ng / ml IL-15, at least about 13 ng / ml IL-15, at least about 14 ng / ml IL-15, at least about 15 ng / ml IL-15, at least about 20 ng / ml IL-15, at least about 25 ng / ml IL-15, at least about 30 ng / ml IL-15, at least about 35 ng / ml IL-15, at least about 40 ng / ml IL-15, at least about 45 ng / ml IL-15, at least about 50 ng / ml IL-15, at least about 100 ng / ml IL-15, at least about 200 ng / ml IL-15, at least about 300 ng / ml IL-15, at least about 400 ng / ml IL-15, at least about 500 ng / ml The method may include contacting the cells with IL-15, or at least about 1000 ng / ml IL-15.In one embodiment, the one or more T cells are contacted with about 0.001 to about 500 ng / ml IL-15, about 0.01 to about 100 ng / ml IL-15, about 0.1 to about 50 ng / ml IL-15, about 1 to about 10 ng / ml IL-15, about 1 to about 5 ng / ml IL-15, about 5 to about 10 ng / ml IL-15, about 3 to about 7 ng / ml IL-15, or about 4 to about 6 ng / ml IL-15. In one particular embodiment, the one or more T cells are contacted with about 5 ng / ml IL-15.

[0101] In some embodiments, one or more T cells are contacted with exogenous IL-7 but not with exogenous IL-15. In other embodiments, one or more T cells are contacted with exogenous IL-15 but not with exogenous IL-7. In still other embodiments, one or more T cells are contacted with both exogenous IL-7 and exogenous IL-15. When one or more T cells are contacted with both exogenous IL-7 and exogenous IL-15, the one or more T cells can be contacted with equal or different concentrations of exogenous IL-7 and exogenous IL-15. In certain embodiments, one or more T cells are contacted with equal concentrations of exogenous IL-7 and exogenous IL-15. In other embodiments, one or more T cells are contacted with different concentrations of exogenous IL-7 and exogenous IL-15. In one embodiment, one or more T cells are contacted with a higher concentration of exogenous IL-7 than exogenous IL-15. In another embodiment, the one or more T cells are contacted with a lower concentration of exogenous IL-7 than exogenous IL-15. In one particular embodiment, the one or more T cells are contacted with about 5 ng / ml of exogenous IL-7 and about 5 ng / ml of exogenous IL-15.

[0102] Furthermore, one or more T cells can be contacted with exogenous IL-7 and exogenous IL-15 at the same time, e.g., simultaneously, or at different times, e.g., sequentially. In some embodiments, one or more T cells are contacted with exogenous IL-7 before exogenous IL-15. In other embodiments, one or more T cells are contacted with exogenous IL-15 before exogenous IL-7. In some embodiments, one or more T cells are contacted with exogenous IL-7 and exogenous IL-15 at the same time.

[0103] T cells The one or more T cells described herein can be obtained from any source, including, for example, a human donor. The donor can be a subject in need of anti-cancer treatment (e.g., treatment with one or more T cells produced by the methods described herein) (i.e., an autologous donor), or can be an individual who provides a lymphocyte sample that will be used to treat a different individual or cancer patient after the population of cells produced by the methods described herein is produced (i.e., an allogeneic donor). The population of lymphocytes can be obtained from the donor by any suitable method used in the art. For example, the population of lymphocytes can be obtained by any suitable extracorporeal method, venipuncture, or other blood collection method that obtains a blood and / or lymphocyte sample. In one embodiment, the population of lymphocytes is obtained by apheresis. The one or more T cells can be collected from any tissue that contains one or more T cells, including, but not limited to, a tumor. In some embodiments, a tumor, or a portion thereof, is removed from a subject, and one or more T cells are isolated from the tumor tissue. Any T cell can be used in the method disclosed herein, including any T cell suitable for T cell therapy.For example, one or more cells useful in the present invention can be selected from the group consisting of tumor-infiltrating lymphocytes (TIL), cytotoxic T cells, CAR T cells, modified TCR T cells, natural killer T cells, dendritic cells, and peripheral blood lymphocytes.In one particular embodiment, the T cell is a tumor-infiltrating leukocyte.In some embodiments, one or more T cells express CD8, for example, CD8 + In other embodiments, one or more T cells express CD4, e.g., CD4 + T cells.

[0104] The methods described herein can be used to delay or inhibit T cell maturation or differentiation in vitro by contacting one or more T cells from a donor with an AKT inhibitor and at least one of exogenous IL-7 and exogenous IL-15. The inventors have found that treating one or more T cells with an AKT inhibitor and IL-7 and / or IL-15 increases the concentration of naive and immature T cells in vitro. In particular, following treatment, one or more T cells can express one or more genes indicative of undifferentiated or immature T cells. The one or more genes indicative of undifferentiated or immature T cells can be selected from the group consisting of CD8, CD45RA, CCR7, CD45RO, CD62L, CD28, CD95, IL-7Rα, CXCR4, TCF7, FOXO1, ID3, BCL6, and any combination thereof. For example, contacting one or more T cells with an AKT inhibitor and IL-7 and / or IL-15 can be performed by contacting one or more T cells with an AKT inhibitor selected from CD8, CD45RA, CCR7, and any combination thereof. The percentage of cells expressing one or more genes indicative of undifferentiated or immature T cells may be increased.

[0105] In other embodiments, one or more T cells express CCR7 and CD45RO following contact with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15. In one particular embodiment, a greater percentage of one or more T cells express CCR7 and CD45RO after contact with an AKT inhibitor and at least one of exogenous IL-7 and exogenous IL-15 compared to before contact. In another embodiment, one or more T cells express CCR7 and CD45RA after contact with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15. In one particular embodiment, a greater percentage of one or more T cells express CCR7 and CD45RA after contact with an AKT inhibitor and at least one of exogenous IL-7 and exogenous IL-15 compared to before contact. In another embodiment, following contact with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15, T cells exhibit increased expression of CCR7, CD45RO, CD45RA, or any combination thereof, compared to expression of CCR7, CD45RO, and CD45RA by T cells that were not contacted with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15.

[0106] In other embodiments, one or more T cells express CD62L, CD28, or both following contact with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15. In one particular embodiment, a greater percentage of one or more T cells express CD62L, CD28, or both following contact with an AKT inhibitor and at least one of exogenous IL-7 and exogenous IL-15 compared to before contact. In another embodiment, one or more T cells exhibit increased expression of CD62L, CD28, or both following contact with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15 compared to expression of CD62L and CD28 by T cells that were not contacted with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15.

[0107] In one particular embodiment, T cells exhibit increased expression of CD95, IL-7 receptor alpha (IL-7Rα), CXCR4, TCF7, FOXO1, ID3, BCL6, CD62L, CD45RA, or any combination thereof, following contact with an AKT inhibitor and exogenous IL-7, exogenous IL-15, or both, compared to expression of CD95, IL-7 receptor alpha (IL-7Rα), CXCR4, TCF7, FOXO1, ID3, BCL6, CD62L, and CD45RA by T cells that were not contacted with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15.

[0108] T cell therapy The present invention provides methods for modulating, e.g., delaying or inhibiting, T cell maturation or differentiation in vitro for T cell therapy, comprising contacting one or more T cells from a subject in need of T cell therapy with (i) 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. In some embodiments, the method further comprises administering the one or more T cells to a subject in need thereof. Those skilled in the art will understand that one or more T cells generated by the methods described herein can be used in any method of treating a patient, including administering one or more T cells to the patient.

[0109] For example, without limitation, the methods described herein can enhance the efficacy of T cell therapy, which can be an adoptive T cell therapy selected from the group consisting of tumor-infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, modified autologous cell therapy (eACT™), allogeneic T cell transplantation, non-T cell transplantation, and any combination thereof. Adoptive T cell therapy broadly includes any method of selecting, enriching in vitro, and administering to a patient autologous or allogeneic T cells capable of recognizing and binding to tumor cells. TIL immunotherapy is a type of adoptive T cell therapy in which lymphocytes capable of infiltrating tumor tissue are isolated, enriched in vitro, and administered to a patient. TIL cells can be either autologous or allogeneic. Autologous cell therapy is an adoptive T cell therapy that involves isolating T cells capable of targeting tumor cells from a patient, enriching the T cells in vitro, and administering the T cells back to the same patient. Allogeneic T cell transplantation can include the transplantation of ex vivo expanded natural T cells or genetically engineered T cells. As described in more detail above, modified autologous cell therapy is adoptive T cell therapy in which a patient's own lymphocytes are isolated, genetically modified to express tumor-targeting molecules, expanded in vitro, and administered back to the patient. Non-T cell transplantation can include autologous or allogeneic therapy with non-T cells, such as, but not limited to, natural killer (NK) cells.

[0110] In one particular embodiment, the T cell therapy of the present invention is an engineered autologous cell therapy (eACT™). According to this embodiment, the method may include a step of collecting blood cells from a donor. The isolated blood cells (e.g., T cells) can then be contacted with an AKT inhibitor and one or more of exogenous IL-7 and exogenous IL-15. The T cells can then be engineered to express a chimeric antigen receptor ("engineered CAR T cells") or a T cell receptor ("engineered TCR T cells"). In one particular embodiment, the engineered CAR T cells or engineered TCR T cells contacted with an AKT inhibitor and one or more of exogenous IL-7 and exogenous IL-15 are administered to a subject. In some embodiments, the engineered T cells treat a tumor in the subject.

[0111] In some embodiments, one or more T cells are transduced with a retrovirus containing a heterologous gene encoding a cell surface receptor. In one particular embodiment, the cell surface receptor can bind to an antigen on the surface of a target cell, for example, on the surface of a tumor cell. In some embodiments, the cell surface receptor is a chimeric antigen receptor or a T cell receptor.

[0112] In one embodiment, one or more T cells can be engineered to express a chimeric antigen receptor. The chimeric antigen receptor can include a binding molecule for a tumor antigen. The binding molecule can be an antibody or its antigen-binding molecule. For example, the antigen-binding molecule can be selected from scFv, Fab, Fab', Fv, F(ab')2, and dAb, as well as any fragment or combination thereof.

[0113] The chimeric antigen receptor may further comprise a hinge region. The hinge region may be derived from an IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8 alpha hinge region. In one specific embodiment, the hinge region is derived from an IgG4 hinge region.

[0114] The chimeric antigen receptor can also contain a transmembrane domain. The transmembrane domain can be that of any transmembrane molecule that is a coreceptor on immune cells, or that of a member of the immunoglobulin superfamily. In some embodiments, the transmembrane domain is derived from the transmembrane domain of CD28, CD28T, CD8 alpha, CD4, or CD19. In one specific embodiment, the transmembrane domain comprises a domain derived from the CD28 transmembrane domain. In another embodiment, the transmembrane domain comprises a domain derived from the CD28T transmembrane domain.

[0115] The chimeric antigen receptor may further comprise one or more costimulatory signaling regions. For example, the costimulatory signaling region may be the signaling region of CD28, CD28T, OX-40, 41BB, CD27, inducible T cell costimulatory molecule (ICOS), CD3 gamma, CD3 delta, CD3 epsilon, CD247, Ig alpha (CD79a), or Fc gamma receptor. In one specific embodiment, the costimulatory signaling region is the CD28 signaling region. In another embodiment, the costimulatory signaling region is the CD28T signaling region.

[0116] In one embodiment, the chimeric antigen receptor further comprises a CD3 zeta signaling domain.

[0117] Chimeric antigen receptors can be engineered to target specific tumor antigens. In some embodiments, the tumor antigen is 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 / mutant), 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, exon 7 / 8), CAMEL (CTL-recognized antigen on melanoma), CAP-1 (carcinoembryonic antigen peptide-1), CASP-8 (caspase-8), CDC27m (cell division cycle 27 mutant), CDK4 / m (cyclin-dependent kinase 4 mutant), 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 mutant), 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 mutant), HST-2 (human signet ring tumor-2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxylesterase), 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:bD-galactosidase 2-aL fucosyltran spherase), LeY (Lewis Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), MAGE-A3, MAGE-A6, mesothelin, mouse CMV-infected cells, MART-1 / Melan-A (melanoma antigen-1 / melanoma antigen A recognized by T cells), MC1R (melanocortin 1 receptor), Myosin / m (myosin variant), MUC1 (mucin 1), MUM-1, -2, -3 (melanoma ubiquitous variants 1, 2, 3), NA88-A (NA cDNA clone from 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 (190KD bcr-abl protein), Pml / RARa (promyelocytic leukemia / retinoic acid receptor a), PRAME (preferentially expressed antigen in melanoma), PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), RAGE (kidney antigen), RU1 or RU2 (kidney ubiquitous 1 or 2), SAGE (sarcoma antigen), SART-1 or SART-3 (tumor rejection squamous cell carcinoma antigen 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 mutant), 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. In one particular embodiment, the tumor antigen is CD19.

[0118] In another embodiment, T cell therapy involves administering to a patient engineered T cells that express a T cell receptor ("engineered TCR T cells"). The T cell receptor (TCR) can comprise a binding molecule for a tumor antigen. In some embodiments, the tumor antigen is 707-AP, AFP, ART-4, BAGE, BCMA, Bcr-abl, CAIX, CD19, CD20, CD22, CD30, CD33, CD44v7 / 8, CAMEL, CAP-1, CASP-8, CDC27m, CDK4 / m, CEA, CT, Cyp-B, DAM, EGFR, EGFRvIII, EGP-2, EGP-40, Erbb2, 3, 4, ELF2M, ETV6-AML1, FBP, fAchR, G250, GAGE, GD2, GD3, GnT-V, Gp100, HAGE, HER-2 / neu, HLA-A, HPV, HSP70-2M, HST-2, hTERT or hTRT, iCE, IL- 13R-a2, KIAA0205, KDR, κ-light chain, LAGE, LDLR / FUT, LeY, L1CAM, MAGE, MAGE-A1, mesothelin, mouse CMV infected cells, MART-1 / Melan-A, MC1R, Myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NKG2D ligand, NY-BR-1, NY-ESO-1, oncofetal antigen, P15, p190 minor Selected from the group consisting of bcr-abl, Pml / RARa, PRAME, PSA, PSCA, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SSX1, -2, -3, 4, TAA, TAG-72, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, VEGF-R2, WT1, and any combination thereof.

[0119] In one embodiment, the TCR comprises a binding molecule for a viral oncogene. In one particular embodiment, the viral oncogene is selected from human papillomavirus (HPV), Epstein-Barr virus (EBV), and human T-lymphotropic virus (HTLV).

[0120] In yet another embodiment, the TCR comprises a binding molecule for a testicular, placental, or fetal tumor antigen. In one particular embodiment, the testicular, placental, or fetal tumor antigen is selected from the group consisting of NY-ESO-1, synovial sarcoma X breakpoint 2 (SSX2), melanoma antigen (MAGE), and any combination thereof.

[0121] In another embodiment, the TCR comprises a binding molecule for a lineage-specific antigen. In one particular embodiment, the lineage-specific antigen is selected from the group consisting of melanoma antigen-1 recognized by T cells (MART-1), gp100, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), and any combination thereof.

[0122] In one embodiment, the T cell therapy comprises administering to the patient modified CAR T cells that express a chimeric antigen receptor that binds to CD19 and further comprises a CD28 costimulatory domain and a CD3 zeta signaling region. In a particular embodiment, the T cell therapy comprises administering to the patient KTE-C19.

[0123] In one embodiment, the antigenic moiety also includes an Epstein-Barr virus (EBV) antigen (e.g., EBNA-1, EBNA-2, EBNA-3, LMP-1, LMP-2), a hepatitis A viral antigen (e.g., VP1, VP2, VP3), a hepatitis B viral antigen (e.g., HBsAg, HBcAg, HBeAg), a hepatitis C viral antigen (e.g., envelope glycoproteins E1 and E2), a herpes simplex virus type 1, 2, or 8 (HSV1, HSV2, or HSV8) viral antigen (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM, UL20, UL32, US43, UL45, UL49A), a cytomegalovirus (CMV) antigen (e.g., CMV ... V) viral antigens (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM, or other envelope proteins), human immunodeficiency virus (HIV) viral antigens (glycoproteins gp120, gp41, or p24), influenza viral antigens (e.g., hemagglutinin (HA) or neuraminidase (NA)), measles or mumps viral antigens, human papillomavirus (HPV) viral antigens (e.g., L1, L2), parainfluenza virus antigens, rubella virus antigens, respiratory syncytial virus (RSV) viral antigens, or varicella-zoster virus antigens, but are not limited to these. In such embodiments, the cell surface receptor can be any TCR or any CAR that recognizes any of the aforementioned viral antigens on virus-infected target cells.

[0124] In other embodiments, the antigenic moiety is associated with cells having immune or inflammatory dysfunction. Such antigenic moieties may include, but are not limited to, myelin basic protein (MBP), myelin proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), carcinoembryonic antigen (CEA), proinsulin, glutamine decarboxylase (GAD65, GAD67), heat shock proteins (HSP), or any other tissue-specific antigen involved in or associated with pathogenic autoimmune processes.

[0125] The methods disclosed herein can involve T cell therapy, which involves the transfer of one or more T cells to a patient. The T cells can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells, e.g., modified CAR+ T cells or modified TCR+ T cells, can be at least about 10 4 cells, at least about 10 5 cells, at least about 10 6 cells, at least about 10 7 cells, at least about 10 8 cells, at least about 10 9 , or at least about 10 10 In another embodiment, the therapeutically effective amount of T cells, e.g., modified CAR+ T cells or modified TCR+ T cells, can be about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells, or approximately 10 8 In one particular embodiment, a therapeutically effective amount of T cells, e.g., modified CAR+ T cells or modified TCR+ T cells, is about 2×10 6 cells / kg, approximately 3 X 10 6 cells / kg, approximately 4 X 10 6 cells / kg, approximately 5 X 10 6 cells / kg, approximately 6 X 10 6 cells / kg, approximately 7 X ​​10 6 cells / kg, approximately 8 X 10 6 cells / kg, approximately 9 X 10 6 cells / kg, approximately 1 X 10 7 cells / kg, approximately 2 X 10 7 cells / kg, approximately 3 X 10 7 cells / kg, approximately 4 X 10 7 cells / kg, approximately 5 X 10 7 cells / kg, approximately 6 X 10 7 cells / kg, approximately 7 X ​​10 7 cells / kg, approximately 8 X 10 7 cells / kg, or approximately 9 X 10 7 cells / kg.

[0126] In some embodiments, patients are preconditioned before administering T cell therapy.Patients can be preconditioned according to any method known in the art, including but not limited to, treatment with one or more chemotherapeutic agents and / or radiation therapy.In some embodiments, preconditioning can include any treatment before T cell therapy that reduces the number of endogenous lymphocytes, removes cytokine sinks, increases the serum level of one or more homeostatic cytokines or inflammatory factors, enhances the effector function of T cells administered after conditioning, enhances antigen-presenting cell activation and / or availability, or any combination thereof.In one embodiment, preconditioning includes increasing the serum level of one or more cytokines in the subject.

[0127] Cancer treatment The methods of the present invention can be used to treat cancer in a subject, reduce tumor size, kill tumor cells, prevent tumor cell proliferation, prevent tumor growth, eliminate tumors from a patient, prevent tumor recurrence, prevent tumor metastasis, induce remission in a patient, or any combination thereof. In some embodiments, the methods induce a complete response. In other embodiments, the methods induce a partial response.

[0128] In one embodiment, the invention relates to a method of treating a tumor in a subject in need of T cell therapy, comprising administering to the subject one or more T cells that have been contacted with (i) an AKT inhibitor and (ii) exogenous IL-7 and / or exogenous IL-15. In another embodiment, the invention relates to a method of reducing or decreasing tumor size or inhibiting tumor growth in a subject in need of T cell therapy, comprising administering to the subject one or more T cells that have been contacted with (i) an AKT inhibitor and (ii) exogenous IL-7 and / or exogenous IL-15. In certain embodiments, the one or more T cells have not been contacted with exogenous IL-2.

[0129] The cancer that can be treated includes tumor that does not form blood vessels, does not yet form blood vessels substantially, or forms blood vessels.Cancer can also include solid or non-solid tumor.In some embodiments, cancer can be selected from tumors originating from: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenocortical carcinoma, AIDS-related cancer, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, central nervous system, B-cell leukemia, lymphoma or other B-cell malignant tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytosis. tumors, brainstem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, germinoma, central nervous system, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma family tumors, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer Fibrous histiocytoma of bone, malignant and osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), soft tissue sarcoma, germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, cardiac cancer, hepatocellular (liver) carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (endocrine pancreas), Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, lymphoma, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, medulloepithelioma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary Midline tract carcinoma involving the NUT gene, mouth cancercancer), multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myeloma, multiple myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer cancer), oral cavity cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediately differentiated pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasm / multiple myeloma, pleuropulmonary blastoma, gestational breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvic urinary Transitional cell carcinoma of the duct, retinoblastoma, rhabdomyosarcoma, salivary gland carcinoma, sarcoma, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, cervical squamous cell carcinoma, gastric cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, skin, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor.

[0130] In one embodiment, the method can be used to treat tumors, where the tumor is lymphoma or leukemia. Lymphoma and leukemia are blood cancers that specifically affect lymphocytes. All white blood cells in the blood originate from a single type of pluripotent hematopoietic stem cell found in the bone marrow. This stem cell produces both myeloid progenitor cells and lymphoid progenitor cells, which then give rise to the various types of white blood cells found in the body. White blood cells derived from myeloid progenitor cells include T lymphocytes (T cells), B lymphocytes (B cells), natural killer cells, and plasma cells. White blood cells derived from lymphoid progenitor cells include megakaryocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, and macrophages. Lymphoma and leukemia can affect one or more of these cell types in patients.

[0131] In general, lymphomas can be divided into at least two subgroups: Hodgkin lymphoma and non-Hodgkin lymphoma. Non-Hodgkin lymphoma (NHL) is a heterogeneous group of cancers originating from B lymphocytes, T lymphocytes, or natural killer cells. In the United States, B-cell lymphomas account for 80–85% of reported cases. In 2013, it was estimated that approximately 69,740 new cases of NHL and more than 19,000 deaths related to the disease occurred. Non-Hodgkin lymphoma is the most common hematologic malignancy and the seventh leading new cancer site among men and women, accounting for 4% of all new cancer cases and 3% of cancer-related deaths.

[0132] Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL, accounting for approximately 30% of NHL cases. There are approximately 22,000 new diagnoses of DLBCL in the United States each year. It is classified as an aggressive lymphoma, and the majority of patients are treated with conventional chemotherapy (NCCN Guidelines NHL 2014).

[0133] First-line therapy for DLBCL typically involves an anthracycline-containing regimen, such as R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), which has an objective response rate of approximately 80% and a complete response rate of approximately 50% (Coiffier 2002), with approximately one-third of patients having refractory disease to first-line therapy or relapsing after R-CHOP (Sehn 2005). For patients who relapse after a response to first-line therapy, approximately 40-60% of patients can achieve a secondary response with additional chemotherapy. Standard treatment for second-line therapy for autologous stem cell transplant (ASCT)-eligible patients includes rituximab and combination chemotherapy, such as R-ICE (rituximab, ifosfamide, carboplatin, and etoposide) and R-DHAP (rituximab, dexamethasone, cytarabine, and cisplatin), which have an objective response rate of approximately 63% and a complete response rate of approximately 26%, respectively (Gisselbrecht 2010). Patients who respond to second-line therapy and are considered sufficiently suitable for transplantation undergo consolidation therapy with high-dose chemotherapy and ASCT, which is curative in approximately half of transplanted patients (Gisselbrecht 2010). Patients who fail ASCT have a very poor prognosis and no curative options.

[0134] Primary mediastinal large B-cell lymphoma (PMBCL) has distinct clinical, pathological, and molecular features compared with DLBCL. PMBCL is thought to arise from thymic (medullary) B cells and accounts for approximately 3% of patients diagnosed with DLBCL. PMBCL is typically identified in young adults in their 30s, with a slight female predominance. Gene expression profiling suggests that deregulated pathways in PMBCL overlap with those in Hodgkin lymphoma. First-line therapy for PMBCL generally includes anthracycline-containing regimens, including rituximab, such as infusion-adjusted etoposide, doxorubicin, and cyclophosphamide, as well as vincristine, prednisone, and rituximab (DA-EPOCH-R), with or without regional radiation therapy.

[0135] Follicular lymphoma (FL), a B-cell lymphoma, is the most common indolent (slow-growing) form of NHL, accounting for approximately 20% to 30% of all NHL cases. Some patients with FL undergo histological transformation to DLBCL (TFL), which is more aggressive and associated with a poorer outcome. Histological transformation to DLBCL occurs at an annual rate of approximately 3% over a 15-year period, with the risk of transformation continuing to decline in subsequent years. The biological mechanism of histological transformation is unknown. First-line treatment for TFL is influenced by previous therapy for follicular lymphoma but generally involves an anthracycline-containing regimen, including rituximab, to eliminate the aggressive component of the disease.

[0136] Treatment options for relapsed / refractory PMBCL and TFL are similar to those for DLBCL. Given the low prevalence of these diseases, no large-scale prospective randomized studies have been conducted in these patient populations. Patients with chemotherapy-refractory disease have a similar or worse prognosis than those with refractory DLBCL.

[0137] In summary, subjects with refractory aggressive NHL (e.g., DLBCL, PMBCL, and TFL) have a large unmet medical need, and further research into novel treatments is needed in these populations.

[0138] Thus, in some embodiments, the method can be used to treat lymphoma or leukemia, wherein the lymphoma or leukemia is a B-cell malignancy. Examples of B-cell malignancies include, but are not limited to, non-Hodgkin's lymphoma (NHL), small lymphocytic lymphoma (SLL / CLL), mantle cell lymphoma (MCL), FL, marginal zone lymphoma (MZL), extranodal (MALT lymphoma), nodal (monocytic B-cell lymphoma), splenic, diffuse large cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, Burkitt's lymphoma, and lymphoblastic lymphoma. In some embodiments, the lymphoma or leukemia is selected from the group consisting of B-cell chronic lymphocytic leukemia / small cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (e.g., Waldenstrom's macroglobulinemia), splenic marginal zone lymphoma, hairy cell leukemia, plasma cell neoplasms (e.g., plasma cell myeloma (i.e., multiple myeloma), or plasmacytoma), extranodal marginal zone B-cell lymphoma (e.g., For example, MALT lymphoma, nodal marginal zone B-cell lymphoma, follicular lymphoma (FL), transformed follicular lymphoma (TFL), primary cutaneous follicle center lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma (DLBCL), Epstein-Barr virus positive DLBCL, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma (PMBCL), intravascular large B-cell Lymphoma, ALK+ large B-cell lymphoma, plasmablastic lymphoma, primary effusion lymphoma, large B-cell lymphoma arising in HHV8-associated multicentric Castleman disease, Burkitt lymphoma / leukemia, T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, enteropathy-associated T-cell lymphoma, liver Selected from splenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides / Sezary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large cell lymphoma, B-lymphoblastic leukemia / lymphoma, B-lymphoblastic leukemia / lymphoma with recurrent genetic abnormalities, T-lymphoblastic leukemia / lymphoma, and Hodgkin's lymphoma.In some embodiments, the cancer is refractory to one or more prior treatments and / or has relapsed after one or more prior treatments.

[0139] In some embodiments, the cancer is selected from follicular lymphoma, transformed follicular lymphoma, diffuse large B-cell lymphoma, and primary mediastinal (thymic) large B-cell lymphoma. In one particular embodiment, the cancer is diffuse large B-cell lymphoma.

[0140] In some embodiments, the cancer is refractory to or has relapsed following one or more of chemotherapy, radiation therapy, immunotherapy (including T cell therapy and / or treatment with an antibody or antibody-drug conjugate), autologous stem cell transplant, or any combination thereof. In one particular embodiment, the cancer is refractory diffuse large B-cell lymphoma.

[0141] In some embodiments, cancer is treated by administering to a subject one or more T cells that have been contacted with (i) an AKT inhibitor and (ii) exogenous IL-7 and / or exogenous IL-15. In certain embodiments, the one or more T cells are washed to remove the AKT inhibitor, exogenous IL-7, and / or exogenous IL-15 before administering the one or more T cells to the subject. In some embodiments, the one or more T cells comprise modified CAR cells or modified TCR cells. In one embodiment, the modified CAR cells or modified T cells treat a tumor in a subject.

[0142] kit Further included within the scope of the present invention is a kit, e.g., a pharmaceutical kit, comprising an AKT inhibitor and one or more of exogenous IL-7 and exogenous IL-15 for contacting one or more T cells in vitro. The kit typically includes a label and instructions indicating the intended use of the contents of the kit. The term "label" includes any writing or recorded material provided on or with the kit, or which otherwise accompanies the kit.

[0143] In some embodiments, the present invention provides a kit for preparing one or more T cells for T cell therapy for a subject in need thereof, the kit comprising: (i) AKT inhibitors, (ii) exogenous IL-7, and (iii) Instructions for contacting one or more T cells intended for use in T cell therapy with an AKT inhibitor and exogenous IL-7.

[0144] In other embodiments, the present invention provides a kit for preparing one or more T cells for T cell therapy for a subject in need thereof, the kit comprising: (i) AKT inhibitors, (ii) exogenous IL-15, and (iii) Instructions for contacting one or more T cells intended for use in T cell therapy with an AKT inhibitor and exogenous IL-15.

[0145] In other embodiments, the present invention provides a kit for preparing one or more T cells for T cell therapy for a subject in need thereof, the kit comprising: (i) AKT inhibitors, (ii) exogenous IL-7; (iii) exogenous IL-15, and (iii) Instructions for contacting one or more T cells intended for use in T cell therapy with an AKT inhibitor, exogenous IL-7, and / or exogenous IL-15.

[0146] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references cited throughout this application are expressly incorporated herein by reference.

[0147] The following examples are intended to illustrate various aspects of the present invention. Accordingly, the specific aspects discussed should not be construed as limitations on the scope of the present invention. For example, although the examples below relate to T cells transduced with an anti-CD19 chimeric antigen receptor (CAR), those skilled in the art will understand that the methods described herein can be applied to T cells transduced with any CAR. It will be apparent to those skilled in the art that various equivalents, modifications, and variations can be made without departing from the scope of the invention, and it is understood that such equivalent aspects are encompassed herein. Furthermore, all references cited in this disclosure are incorporated by reference in their entirety as if fully set forth herein.

[0148] Aspects E1. A method for delaying or inhibiting T cell maturation or differentiation in vitro for T cell therapy, comprising contacting one or more T cells from a subject in need of 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 compared to the T cell function of T cells cultured in the absence of the AKT inhibitor.

[0149] E2. A method for improving T cell function in vitro for T cell therapy, comprising contacting one or more T cells from a subject in need of 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 improved T cell function compared to the T cell function of T cells cultured in the absence of the AKT inhibitor.

[0150] E3. Improved T cell function (i) increased T-cell proliferation; (ii) increased cytokine production; (iii) increased cytolytic activity; and (iv) Any combination of (i) to (iii) The method of E1 or E2, selected from the group consisting of:

[0151] E4. A method for increasing T cell proliferation in vitro prior to T cell therapy, comprising contacting one or more T cells from a subject in need of 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 increased T cell proliferation compared to T cell proliferation of T cells cultured in the absence of the AKT inhibitor.

[0152] E5. A method for increasing T cell cytokine production in vitro prior to T cell therapy, comprising contacting one or more T cells from a subject in need of 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 increased cytokine production compared to the cytokine production of T cells cultured in the absence of the AKT inhibitor.

[0153] E6. The method of E3 or E5, 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) increased both IFNg and TNFa production.

[0154] E7. A method for increasing T cell cytolytic activity in vitro for T cell therapy, comprising contacting one or more T cells from a subject in need of 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 increased cytolytic activity compared to the T cell cytolytic activity of T cells cultured in the absence of the AKT inhibitor.

[0155] E8. A method for delaying or inhibiting T cell maturation or differentiation in vitro for T cell therapy, comprising contacting one or more T cells from a subject in need of 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.

[0156] E9. Any of the methods of E1-E8, wherein the contacting step comprises culturing the one or more T cells in medium comprising (i) an AKT inhibitor and (ii) exogenous IL-7 and / or exogenous IL-15.

[0157] E10. Any of the methods E1-E9, wherein the one or more T cells are not contacted with exogenous interleukin-2 (IL-2).

[0158] E11. Any of the methods E1-E10, wherein the T cells are washed to remove the AKT inhibitor, exogenous IL-7, and / or exogenous IL-15.

[0159] E12.AKT inhibitors include 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, and 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 # 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 A, 3-[1-[[4-(7-phenyl-3H-imidazo[4,5-g]quinoxalin-6-yl)phenyl]methyl]piperidin-4-yl]-1H-benzimidazol-2-one, and any combination thereof.

[0160] E13. Any of the methods E1-E12, wherein the AKT inhibitor comprises a compound selected from the group consisting of: (i) 3-[1-[[4-(7-phenyl-3H-imidazo[4,5-g]quinoxalin-6-yl)phenyl]methyl]piperidin-4-yl]-1H-benzimidazol-2-one; (ii) N,N-dimethyl-1-[4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl]meth-namine; or (iii) 1-{1-[4-(3-phenylbenzo[g]quinoxalin-2-yl)benzyl]piperidin-4-yl}-1,-3-dihydro-2H-benzimidazol-2-one.

[0161] E14. Any of the methods E1-E13, wherein the AKT inhibitor is a compound selected from the group consisting of: (i) 3-[1-[[4-(7-phenyl-3H-imidazo[4,5-g]quinoxalin-6-yl)phenyl]methyl]piperidin-4-yl]-1H-benzimidazol-2-one; (ii) N,N-dimethyl-1-[4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl]meth-namine; or (iii) 1-{1-[4-(3-phenylbenzo[g]quinoxalin-2-yl)benzyl]piperidin-4-yl}-1,-3-dihydro-2H-benzimidazol-2-one.

[0162] E15. Any of the methods E1-E14, 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.

[0163] E16. The method of E15, wherein the AKT inhibitor is in an amount of about 1 nM to about 1 mM.

[0164] E17. The AKT inhibitor is at least about 1 nM, at least about 10 nM, at least about 50 nM, at least about 100 nM, at least about 200 nM, at least about 300 nM, at least about 400 nM, at least about 500 nM, at least about 1 μM, at least about 2 μM, at least about 3 μM, at least about 4 μM, at least about 5 μM, at least about 6 μM, at least about 7 μM, at least about 8 μM, at least about 9 μM, at least about 10 μM, at least about 11 μM, at least about 12 μM, at least about 13 μM, at least about 14 μM, at least about 15 μM, at least about 16 μM, at least about 17 μM, at least about 18 μM, at least about 20 μM, at least about 21 μM, at least about 22 μM, at least about 23 μM, at least about 24 μM, at least about 25 μM, at least about 26 μM, at least about 27 μM, at least about 28 μM, at least about 29 μM, at least about 30 μM, at least about 31 μM, at least about 32 μM, at least about 33 μM, at least about 34 μM, at least about 35 μM, at least about 36 μM, at least about 37 μM, at least about 38 μM, at least about 39 μM, at least about 40 μM, at least about 41 μM, at least about 42 μM, at least about 43 μM, at least about 44 μM, at least about 45 μM, at least about 46 μM, at least about 47 μM, at least about 48 μM, at least about 49 μM, at least The method of E15, wherein the amount is an amount selected from the group consisting of about 19 μM, at least about 20 μM, at least about 25 μM, at least about 30 μM, at least about 35 μM, at least about 40 μM, at least about 45 μM, at least about 50 μM, at least about 60 μM, at least about 70 μM, at least about 80 μM, at least about 90 μM, at least about 100 μM, at least about 200 μM, at least about 300 μM, at least about 400 μM, at least about 500 μM, or at least about 1 mM.

[0165] E18. The method of E15, wherein the AKT inhibitor is in an amount of about 8 μM.

[0166] E19. Any of the methods E1-E18, wherein the exogenous IL-7 is in an amount of about 0.001 to about 500 ng / ml IL-7.

[0167] E20. Any of the methods E1-E18, wherein the exogenous IL-7 is in an amount of about 1 to about 10 ng / ml IL-7.

[0168] E21. The method of any of E1-E18, wherein the exogenous IL-7 is in an amount of at least about 5 ng / ml IL-7.

[0169] E22. Any of the methods E1-E21, wherein the exogenous IL-15 is in an amount of about 0.001 to about 500 ng / ml IL-15.

[0170] E23. Any of the methods E1-E21, wherein the exogenous IL-15 is in an amount of about 1 to about 10 ng / ml IL-15.

[0171] E24. The method of any of E1-E21, wherein the exogenous IL-15 is in an amount of at least about 5 ng / ml IL-15.

[0172] E25. Any method from E1 to E24, wherein one or more T cells express CD8.

[0173] E26. The method of E25, wherein the one or more T cells are selected from the group consisting of tumor-infiltrating lymphocytes, cytotoxic T cells, CAR T cells, modified TCR T cells, natural killer T cells, and peripheral blood lymphocytes.

[0174] E27. Any of the methods E1-E26, wherein one or more T cells are obtained from a subject in need of anti-cancer treatment.

[0175] E28. The method of E27, wherein one or more T cells are obtained from a tumor in a subject in need of anti-cancer treatment.

[0176] E29. The method of E20 or E28, wherein the one or more T cells comprise one or more tumor infiltrating leukocytes (TILs).

[0177] E30: Activating T cells using any of the methods from E1 to E29.

[0178] E31. The method of E30, wherein T cell activation is carried out in a closed system.

[0179] E32. The method of E31, wherein the closed system comprises a closed bag system.

[0180] E33. Proliferating T cells, using any of the methods E1 to E32.

[0181] E34. The method of E32 for expanding T cells in vitro.

[0182] E35. The method of E32 for expanding T cells in vivo.

[0183] E36. Any of the methods E1-E35, wherein contacting one or more T cells with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15 extends the persistence of the T cells in vivo.

[0184] E37. Any of the methods of E1-E36, wherein following contacting the one or more T cells with an AKT inhibitor and at least one of exogenous IL-7 and exogenous IL-15, the resulting T cells express one or more genes indicative of undifferentiated or immature T cells.

[0185] E38. The method of E37, wherein the one or more genes indicative of undifferentiated or immature T cells are selected from the group consisting of CD8, CD45RA, CCR7, and any combination thereof.

[0186] E39. The method of any of E1-E38, further comprising retroviral transduction of the T cells.

[0187] E40. The method of E39, wherein the retrovirus comprises a heterologous gene encoding a cell surface receptor.

[0188] E41. The method of E40, wherein the cell surface receptor is capable of binding to an antigen on the surface of a target cell.

[0189] E42. The method of E41, wherein the target cell is a tumor cell.

[0190] E43. The method of E41 or E42, wherein the cell surface receptor is a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0191] E44. Cell surface receptors include 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 / mutant), 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, exon 7 / 8), CAMEL (CTL-recognized antigen on melanoma), CAP-1 (carcinoembryonic antigen peptide-1), CASP-8 (caspase-8), CDC27m (cell division cycle 27 mutant), CDK4 / m (cyclin-dependent kinase 4 mutant), 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 mutant), 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 (helicase 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 mutant), HST-2 (human signet ring tumor-2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxylesterase), 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:bD-galactosidase 2-a L-fucosyltransferase), LeY (Lewis Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), mesothelin, mouse CMV-infected cells, MART-1 / Melan-A (melanoma antigen-1 / melanoma antigen A recognized by T cells), MC1R (melanocortin 1 receptor), Myosin / m (myosin variant), MUC1 (mucin 1), MUM-1, -2, -3 (melanoma ubiquitous variants 1, 2, 3), NA88-A (NA cDNA clone from 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 (190KD bcr-abl protein), Pml / RARa (promyelocytic leukemia / retinoic acid receptor a), PRAME (preferentially expressed antigen in melanoma), PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), RAGE (kidney antigen), RU1 or RU2 (kidney ubiquitous 1 or 2), SAGE (sarcoma antigen), SART-1 or SART-3 (tumor rejection squamous cell carcinoma antigen 1 or 3), SSX1, -2, -3, 4 (synovial sarcoma X1, -2, -3, Any of the methods E41 to E43, wherein the antibody is capable of binding to an antigen selected from the group consisting of IL-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 mutant), 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.

[0192] E45. The method of any of E1-E44, further comprising administering the resulting T cells to a subject in need thereof.

[0193] E46. A method of treating a tumor in a subject in need of T cell therapy, comprising administering to the subject one or more T cells contacted with (i) an AKT inhibitor and (ii) exogenous IL-7 and / or exogenous IL-15.

[0194] E47. A method of reducing or diminishing tumor size or inhibiting tumor growth in a subject in need of T cell therapy comprising administering to the subject one or more T cells contacted with (i) an AKT inhibitor and (ii) exogenous IL-7 and / or exogenous IL-15.

[0195] E48. The method of E46 or E47, wherein the one or more T cells have not been contacted with exogenous IL-2.

[0196] E49. Any of the methods E46-E48, wherein the T cells express CCR7 and CD45RO following contact with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15.

[0197] E50. Any of the methods E46-E48, wherein the T cells express CCR7 and CD45RA following contact with an AKT inhibitor and exogenous IL-7, exogenous IL-15, or both.

[0198] E51. Any of the methods E46-E48, wherein the T cells exhibit increased expression of CCR7, CD45RO, CD45RA, or any combination thereof, following contact with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15, compared to expression of CCR7, CD45RO, and CD45RA by T cells that have not been contacted with the AKT inhibitor and exogenous IL-7 and / or exogenous IL-15.

[0199] E52. Any of the methods E46-E51, wherein the T cells express CD62L, CD28, or both following contact with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15.

[0200] E53. Any of the methods E46-E52, wherein the T cells exhibit increased expression of CD62L, CD28, or both following contact with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15, compared to expression of CD62L and CD28 by T cells that have not been contacted with the AKT inhibitor and exogenous IL-7 and / or exogenous IL-15.

[0201] E54. Any of the methods E46-E53, wherein the T cells exhibit increased expression of CD95, IL-7 receptor alpha (IL-7Rα), CXCR4, TCF7, FOXO1, ID3, BCL6, CD62L, CD45RA, or any combination thereof, following contact with an AKT inhibitor and exogenous IL-7, exogenous IL-15, or both, compared to expression of CD95, IL-7 receptor alpha (IL-7Rα), CXCR4, TCF7, FOXO1, ID3, BCL6, CD62L, and CD45RA by T cells that have not been contacted with the AKT inhibitor and exogenous IL-7 and / or exogenous IL-15.

[0202] E55. Any of the methods E46-E54, wherein one or more T cells are isolated from the donor.

[0203] E56. The method of E55, in which the donor is the subject.

[0204] E57. Any of methods E46 to E56, wherein the tumor is cancer.

[0205] E58. Cancer is bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, 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), esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and any combination thereof.

[0206] E59. Any of the methods of E1-E58, wherein the T cell therapy comprises an engineered CAR cell therapy or an engineered TCR cell therapy.

[0207] E60. The method of E59, wherein the engineered CAR cell or engineered TCR cell therapy treats a tumor in a subject. [Example]

[0208] Example 1 Donor T cells were cultured at equal plating densities for 10 days in the presence of IL-2, IL-7, IL-15, and / or AKT inhibitors. The T cell phenotype of cultured T cells was assessed for (i) CD4 T cells cultured for 7 and 14 days in IL-2 alone compared with IL-7 and IL-15, and (ii) CD4 T cells cultured in IL-7 and IL-15 compared with IL-7, IL-15, and AKT inhibitors. + T cells and CD8 +T cells were determined (Figure 1A-F). A trend toward more immature T cells was observed when cells were grown in the presence of IL-7 and IL-15. In particular, a significantly higher (p = 0.03, n = 6) percentage of naive and Tcm cells was observed (Figure 1B). + Although a significant increase in the percentage of more mature effector T cells was observed in IL-7 / IL-15-treated cells compared to IL-2-treated cells (Fig. 1A), no difference was observed in the percentage of more mature effector T cells (Fig. 1B). + The effect was not maintained after long-term culture in the CD8 compartment (data now shown). + A significant increase in CD4+ / CD4+ T cells was observed in the CD4+ / CD4+ T cells compartment, with a significantly higher (p = 0.03, n = 6) percentage of naive and Tcm cells (Fig. 1C) and a significantly lower (p = 0.03, n = 6) percentage of effector T cells (Fig. 1D) in IL-7 / IL-15-treated cell cultures compared with IL-2-treated cell cultures. + Unlike in the CD8 compartment, this effect + compartment after long-term culture (Fig. 1E, p = 0.03, n = 6; and Fig. 1F, p = 0.03, n = 6).

[0209] CD4 + and CD8 + In both compartments, the addition of an AKT inhibitor further increased the trend towards more immature T cells. At day 7, there was a slightly higher percentage of naive and Tcm CD4 + A significantly higher percentage of effector T cells was observed in IL-7 / IL-15 / AKTi-treated cells compared to IL-7 / IL-15-treated cells (Fig. 2A), and a slightly lower percentage of effector T cells was observed in IL-7 / IL-15 / AKTi-treated cells compared to IL-7 / IL-15-treated cells (Fig. 2B). No significant difference was observed in cells cultured for 14 days (data not shown). However, CD8 +Significant differences were observed in the compartments at day 7. In particular, at day 7, the naive and Tcm CD4 + The percentage of effector T cells was significantly higher in IL-7 / IL-15 / AKTi-treated cells compared to IL-7 / IL-15-treated cells (p = 0.03, n = 6) (Fig. 2C), and the percentage of effector T cells was significantly lower in IL-7 / IL-15 / AKTi-treated cells compared to IL-7 / IL-15-treated cells (p = 0.03, n = 6) (Fig. 2D). However, this effect was not observed on day 14 (data not shown).

[0210] To determine whether contacting T cells with one or more of IL-2, IL-7, and / or IL-15 and an AKT inhibitor has an effect on transduction efficiency, T cells collected from three donors were transduced with a retrovirus carrying a class I TCR in OriGen PERMALIFE™ PL30 bags two days after stimulation. The transduced cells were then cultured for 10 days in the presence of (i) IL-2; (ii) IL-2 and an AKT inhibitor; (iii) IL-7 and IL-15; and (iv) IL-7, IL-15, and an AKT inhibitor. T cells were then assayed for CD3 expression and positive soluble MHC-tetramer staining (Tet), indicators of successful transduction. + ) were analyzed. No significant differences in transduction efficiency were observed depending on the culture condition (Figure 3A), and no significant differences in tetramer mean fluorescence intensity (MFI) were observed across culture conditions (Figure 3B).

[0211] Example 2 The effects of AKTi inhibitors on cell proliferation were investigated under various conditions. First, the effects of AKTi culture conditions on various donor cell sources were evaluated as follows. Apheresis products from four healthy donors were processed using high-density centrifugation to obtain peripheral blood mononuclear cells (PBMCs) (Figures 4A–4D). Cells from the four donors were counted, stimulated with OKT3 (a monoclonal antibody against CD3), and cultured for 7–10 days in the presence of IL-2 (circles); IL-2 and AKTi (squares); IL-7 and IL-15 (triangles); or IL-7, IL-15, and AKTi (inverted triangles) (Figures 4A–4D). Cell proliferation was monitored for each donor cell line under each culture condition, and AKTi did not have a negative effect on cell proliferation (Figures 4A–4D).

[0212] Next, we evaluated PBMCs transduced with class I TCR (HPV-E6). Apheresis products from three healthy donors were again processed using high-density centrifugation to obtain PBMCs, which were counted and stimulated using OKT3. Cells from the three donors were then cultured in the presence of IL-2 (circles); IL-2 and AKTi (squares); IL-7 and IL-15 (triangles); or IL-7, IL-15, and AKTi (inverted triangles) (Figures 5A-5C). On day 2, cells were transduced with class I TCR (HPV-E6). Cell proliferation was monitored for each donor cell line under each culture condition, and AKTi did not have a negative effect on cell proliferation (Figures 5A-5C).

[0213] Next, CD4 + / CD8 + The effects of AKTi culture conditions on T cells were evaluated. Apheresis products from three healthy donors were again processed using high-density centrifugation to obtain PBMCs. PBMCs were then cultured with anti-CD4 and anti-CD8 Ab beads to obtain CD4 T cells. + and CD8 + Cells were selected using the CLINIMACS® system (Miltenyi Biotec). CD4+ and CD8 + Cells were counted and stimulated using OKT3 and anti-CD28 Ab. Cells were then cultured in the presence of IL-2 (circles); IL-2 and AKTi (squares); IL-7 and IL-15 (triangles); or IL-7, IL-15, and AKTi (inverted triangles) (Figures 6A-6C). On day 2, cells were transduced with a class II TCR (MAGE-A3). Cell proliferation was monitored for each donor cell line under each culture condition, and AKTi had no negative effect on cell proliferation (Figures 6A-6C).

[0214] CD4+ and CD8+ T cells were then evaluated separately. Apheresis products from three healthy donors were again processed using high-density centrifugation to obtain PBMCs. PBMCs were then cultured with either anti-CD4 beads (Figures 7A-7C) or anti-CD8 beads (Figures 8A-8C), and target cells were selected using the CLINIMACS® system (Miltenyi Biotec). Cells from the three donors were then counted and stimulated using OKT3 and anti-CD28 Ab. Cells were then cultured in the presence of IL-2 (circles); IL-2 and AKTi (squares); IL-7 and IL-15 (triangles); or IL-7, IL-15, and AKTi (inverted triangles). On day 2, cells were transduced with a class II TCR (MAGE-A3). Cell proliferation was assessed by CD4+ T cells from each donor cell line under each culture condition. + (Figures 7A-7C) and CD8 + (Figures 8A-8C) When cells were observed, AKTi did not have a negative effect on cell proliferation.

[0215] Next, we evaluated the effects of culture conditions during large-scale manufacturing. Apheresis products from four healthy donors were again processed using high-density centrifugation to obtain peripheral blood mononuclear cells (PBMCs) (Figures 9A-9D). PBMCs were then cultured with anti-CD4 and anti-CD8 beads to obtain CD4. + / CD8 +Cells were selected using the CLINIMACS® system. + / CD8 + Cells were counted and stimulated using OKT3 and anti-CD28. Cells were then cultured at a large-scale in the XURI™ Cell Expansion System (GE Healthcare Life Sciences) for 8 days in the presence of IL-7 and IL-15 (Figure 9A: circles; Figures 9B-9C: squares) or IL-7, IL-15, and AKTi (Figure 9A: squares; Figures 9B-9C: circles). On the second day of culture, cells were transduced with a class II TCR (MAGE-A3). Cell proliferation was monitored for each donor cell line under each culture condition, and AKTi had no negative effect on cell proliferation (Figures 9A-9D).

[0216] Example 3 The effect on T cell transduction efficiency following culture in the presence of AKTi was examined. Previously frozen donor T cells were stimulated and then cultured for 10 days in the presence of IL-2, IL-2 and AKTi; IL-7 and IL-15; or IL-7, IL-15, and AKTi. Cells were transduced with a class I TCR (HPV-E6) in T-75 tissue culture flasks (FIG. 10) or a class II TCR (MAGE-A3) in OriGen PERMALIFE™ bags (FIGS. 11A-11F) on day 2 after stimulation. T cell transduction efficiency was measured by anti-mTCRb antibody staining on day 10 (FIGS. 10 and 11A-11F). Although anti-mTCRb staining MFI shows slightly greater overall intensity for cells cultured in the presence of IL-7, IL-15, and AKTi compared with IL-7 and IL-15 alone (Figures 11C and 11F), AKTi had no negative effect on transduction efficiency (Figures 10, 11A, 11B, 11D, and 11E).

[0217] Similar results were observed for T cells cultured at manufacturing scale (Figure 12). Previously frozen donor T cells from four manufacturing-scale runs (21, 22, and 23) were cultured in OriGen PERMALIFE™ bags in the presence of IL-7 and IL-15 or IL-7, IL-15, and AKTi. Two days after stimulation, cells were transduced with a class II TCR (MAGE-A3). Cells were then expanded in a XURI™ Bioreactor Cell Expansion System. T cell transduction efficiency was determined on day 8 by anti-mTCRb (mC TCR PE) antibody staining. CD3 T cells expressing the transduced TCR for each culture condition for each run were transduced with a class II TCR (MAGE-A3). + The percentage of cells that transduced under large-scale manufacturing conditions was shown (Figure 12). Cells grown in the presence of IL-7, IL-15, and AKTi have greater transduction efficiency than cells cultured in IL-7 and IL-15 alone (Figure 12).

[0218] Example 4 To determine the effect of various culture conditions on the differentiation state, CD4 + / CD8 + T cells were transduced with a class II TCR (MAGE-A3) and cultured in the presence of IL-2; IL-2 and AKTi; IL-7 and IL-15; and IL-7, IL-15, and AKTi. Cells were then stained with an antibody directed against CD62L, a marker of early differentiation. The percentage of cells staining positive for CD62L expression was determined for cells from each culture condition for donors 1, 2, and 3 (Figures 13A, 13B, and 13C, respectively). Mean fluorescence intensity (MFI) indicated that cells cultured in the presence of AKTi had greater levels of CD62L on the surface of positive cells compared to cells cultured in the absence of AKTi (Figures 13D-13E).

[0219] Example 5 To determine the effect of AKTi on T cell function, cytokine production and T cell proliferation were assessed following culture under various conditions. T cells from four manufacturing-scale runs (21, 22, and 23) were cultured in OriGen PERMALIFE™ bags in the presence of IL-2; IL-2 and AKTi; IL-7 and IL-15; and IL-7, IL-15, and AKTi. On day 2, T cells were transduced with a class II TCR (MAGE-A3) and then expanded in a XURI™ Bioreactor Cell Expansion System in the presence of IL-7 and IL-15, or IL-7, IL-15, and AKTi. T cells were stimulated with PMA, ionomycin, brephadin A, and monensin for 5.5 hours. Intracellular flow cytometry demonstrated increased T cell activity for cells cultured in the presence of AKTi, as evidenced by increased production of the cytokines IFNg (Figure 14A) and TNFa (Figure 14B).

[0220] To further confirm that AKTi increases T cell activity, T cells from two manufacturing-scale runs (21 and 22) were cultured as described above and cocultured overnight with positive (H1299, HT1197, and HT1367) and negative (DU145, SK MEL 28, and SK MEL 5) target tumor cell lines. Cells cultured in the presence of AKTi showed greater IFNg production under each culture condition tested (Figure 15), indicating that AKTi-cultured cells were more potent in responding to stimulation than cells cultured in the absence of AKTi.

[0221] Similar results were observed for small-scale cultures of donor T cells. Cells from donors 1, 2, and 3 were stimulated and cultured in the presence of IL-2; IL-2 and AKTi; IL-7 and IL-15; or IL-7, IL-15, and AKTi, and transduced with class I TCRs on day 2 post-stimulation, as described above. T cells were cocultured overnight with tumor cell lines (Caski cells; Figure 16A) or with T2 cells loaded with titrated amounts of TCR-specific peptides (Figures 16B–16D). As observed in the large-scale production experiments described above, cells cultured in the presence of AKTi produced higher levels of IFNg than cells cultured in the absence of AKTi (Figures 16A–16D). Titration of TCR-specific peptides showed that AKTi culture conditions induced greater IFNg production at almost all levels.

[0222] T cell proliferation was also found to increase following culture in the presence of AKTi. T cells from donors 1, 2, and 3 were transduced with class II TCRs 2 days after stimulation, as described above. T cells were stained with CFSE and cocultured with tumor cell lines (positive control) for 4 days. Increased T cell proliferation was observed in cells grown in the presence of AKTi (Figures 17A and 17C) compared with cells grown without AKTi (Figures 17B and 17D). Figures 17A–17D show representative data from donor 3, in which a greater percentage of cells were characterized as being in late (L) or intermediate (M) phases of proliferation for cells cultured in IL-2 and AKTi (Figure 17B) and IL-7, IL-15, and AKTi (Figure 17D) than for cells cultured in the absence of AKTi (Figures 17A and 17C).

[0223] Increased T cell proliferation was also observed under large-scale manufacturing culture conditions. T cells from two large-scale manufacturing runs (21 and 22) were transduced with class II TCRs on day 2 post-stimulation as described above. T cells were stained with CFSE and co-cultured with positive or negative tumor cell lines for 4 days. Increased T cell proliferation was observed in cells grown in the presence of AKTi for runs 21A / 21B (Figure 18A) and 22A / 22B (Figure 18B), respectively.

[0224] Example 6 To determine the effect of AKTi on T cell cytolytic activity, luciferase-expressing target cells are cocultured with T cells grown under various culture conditions (IL-2 alone; IL-2 and AKTi; IL-7 and IL-15; and IL-7, IL-15, and AKTi) as described above for periods ranging from 16 to 96 hours. T cells are then cocultured with luciferase-expressing target cells. Target cell viability is measured by luciferase intensity; a decrease in luciferase intensity indicates T cell recognition and target-specific killing. Therefore, a reduction in luciferase levels is a direct indicator of T cell cytotoxicity. Cells cultured in the presence of an AKT inhibitor are expected to have greater cytotoxicity than cells cultured in the absence of an AKT inhibitor.

Claims

1. 1. A method for delaying or inhibiting T cell maturation or differentiation in vitro for T cell therapy, comprising contacting one or more T cells from a subject in need of 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 compared to the T cell function of T cells cultured in the absence of the AKT inhibitor.

2. Improved T cell function (i) increased T-cell proliferation; (ii) increased cytokine production; (iii) increased cytolytic activity, and (iv) Any combination of (i) to (iii) 2. The method of claim 1, selected from the group consisting of:

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

4. 4. The method of any one of claims 1 to 3, further comprising administering the resulting T cells to a subject in need thereof.

5. (i) AKT inhibitors and (ii) exogenous IL-7 and / or exogenous IL-15 10. A method of treating a tumor in a subject in need of T cell therapy, comprising administering to the subject one or more T cells that have been contacted with

6. (i) AKT inhibitors and (ii) exogenous IL-7 and / or exogenous IL-15 10. A method of reducing or diminishing the size of a tumor or inhibiting the growth of a tumor in a subject in need of T cell therapy, comprising administering to the subject one or more T cells that have been contacted with

7. 7. The method of any one of claims 1 to 6, wherein the one or more T cells are not contacted with exogenous interleukin-2 (IL-2).

8. AKT inhibitors include 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, and GSK 690693 (CAS # 937174-76-0), FPA 124 (CAS # 902779-59-3), Miltefosine, PHT-427 (CAS # 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 8. The method of any one of claims 1 to 7, wherein A is selected from the group consisting of 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. 9. The method of any one of claims 1 to 8, 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. (i) the AKT inhibitor is in an amount of about 1 nM to about 1 mM; (ii) the exogenous IL-7 is in an amount of about 0.001 to about 500 ng / ml IL-7; (iii) the exogenous IL-15 is in an amount of about 0.001 to about 500 ng / ml IL-15; or (iv) any combination of (i)-(iii). The method according to any one of claims 1 to 9.

11. (i) the AKT inhibitor is in an amount of about 8 μM; (ii) the exogenous IL-7 is in an amount of at least about 5 ng / ml IL-7; (iii) the exogenous IL-15 is in an amount of at least about 5 ng / ml IL-15; or (iv) any combination of (i)-(iii). The method according to any one of claims 1 to 10.

12. 12. The method of any one of claims 1-11, wherein the one or more T cells are selected from the group consisting of tumor-infiltrating lymphocytes, cytotoxic T cells, CAR T cells, modified TCR T cells, natural killer T cells, peripheral blood lymphocytes, and tumor-infiltrating leukocytes.

13. 13. The method of any one of claims 1 to 12, further comprising the step of retrovirally transducing the T cells.

14. 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. TCR or CAR may be 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 / mutant), 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, exon 7 / 8), CAMEL (CTL-recognized antigen on melanoma), CAP-1 (carcinoembryonic antigen peptide-1), CASP-8 (caspase-8), CDC27m (cell division cycle 27 mutant), CDK4 / m (cyclin-dependent kinase 4 mutant), 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 mutant), 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 100kD), 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 mutant), HST-2 (human signet ring tumor-2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxylesterase), 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:bD-galactosidase 2-aL fucosyltran spherase), LeY (Lewis Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma-associated antigen 1), MAGE-A3, MAGE-A6, mesothelin, mouse CMV-infected cells, MART-1 / Melan-A (melanoma antigen-1 / melanoma antigen A recognized by T cells), MC1R (melanocortin 1 receptor), Myosin / m (myosin variant), MUC1 (mucin 1), MUM-1, -2, -3 (melanoma ubiquitous variants 1, 2, 3), NA88-A (NA cDNA clone from 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 (190KD bcr-abl protein), Pml / RARa (promyelocytic leukemia / retinoic acid receptor a), PRAME (preferentially expressed antigen in melanoma), PSA (prostate-specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate-specific membrane antigen), RAGE (kidney antigen), RU1 or RU2 (kidney ubiquitous 1 or 2), SAGE (sarcoma antigen), SART-1 or SART-3 (tumor rejection squamous cell carcinoma antigen 1 or 3), SSX1, -2, -3, 4 (synovial sarcoma X1, -2, -3, 15. The method of claim 14, wherein the antibody can bind to an antigen selected from the group consisting of tyrosinase-related protein 1 (TRP-1), tyrosinase-related protein 2 (TRP-2), VEGF-R2 (vascular endothelial growth factor receptor 2), WT1 (Wilms tumor gene), TAA (tumor-associated antigen), TAG-72 (tumor-associated glycoprotein 72), TEL / AML1 (translocation Ets family leukemia / acute myeloid leukemia 1), TPI / m (triosephosphate isomerase mutant), 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. Following contact with an AKT inhibitor and 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, compared to expression of CCR7, CD45RO, and CD45RA by T cells that have not been contacted with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15; (iv) expressing CD62L, CD28, or both; (v) exhibits increased expression of CD62L, CD28, or both, compared to expression of CD62L and CD28 by T cells that have not been contacted with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15; (vi) following contact with an AKT inhibitor and exogenous IL-7, exogenous IL-15, or both, exhibits increased expression of CD95, IL-7 receptor alpha (IL-7Rα), CXCR4, TCF7, FOXO1, ID3, BCL6, CD62L, CD45RA, or any combination thereof, compared to expression of CD95, IL-7 receptor alpha (IL-7Rα), CXCR4, TCF7, FOXO1, ID3, BCL6, CD62L, and CD45RA by T cells that have not been contacted with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15; or (vii) any combination of (i)-(vi).

16. The method according to any one of claims 1 to 15.

17. Tumors include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, 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), esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute bone marrow cancer, 17. The method of any one of claims 5 to 16, wherein the cancer is selected from myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), chronic lymphocytic leukemia (CLL), childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and any combination thereof.

Images