Method for 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: A method comprises the step for 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. In some embodiments, the method further comprises administering the one or more T cells to a subject in need of T cell therapy.SELECTED DRAWING: None

Description

Technical Field

[0001] Statement of Government Interest This invention was made in the performance of a joint research and development contract with the National Cancer Institute (NCI), an agency of the United States Department of Health and Human Services. The United States Government has certain rights in this invention.

[0002] Field of the Invention The present invention relates to a method for preparing one or more T cells for T cell therapy. In particular, the present invention relates to a method 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 Art

[0003] Background Human cancer is essentially composed of normal cells that have undergone genetic or epigenetic transformation into abnormal cancer cells. In doing so, cancer cells begin to express proteins and other antigens that are different from those expressed by normal cells. These abnormal tumor antigens can be used by the body's natural 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] These therapies have been found to have promising effects on tumor size and patient survival. However, it has been found difficult to predict whether a given T cell therapy will be effective in each patient. to do.

[0005] The transplantation of a mixed population of T cells is among the factors that prevent T cell therapies from reaching their full potential. In conventional T cell therapies, donor T cells are harvested and optionally modified to target specific antigens (e.g., tumor cells) or selected for antitumor properties (e.g., tumor infiltrating lymphocytes), expanded in vitro, and administered to the subject in need thereof. Typically, the resulting T cells comprise a heterogeneous population of largely mature cells, many of which are terminally differentiated. As a result, the expected in vivo persistence of these cells can be limited, and the initial positive effects observed can be reversed over time as the tumor rebounds in the absence of the transplanted T cells. Accordingly, there remains a need to increase the in vivo persistence of T cells for use in T cell therapies. cells, many of which are terminally differentiated. As a result, the expected in vivo persistence of these cells can be limited, and the initial positive effects observed can be reversed over time as the tumor rebounds in the absence of the transplanted T cells. Accordingly, there remains a need to increase the in vivo persistence of T cells for use in T cell therapies. . SUMMARY OF THE INVENTION

[0006] The present disclosure provides a method for delaying or inhibiting the maturation or differentiation of T cells in vitro for use in T cell therapies, the method comprising contacting one or more T cells derived from a subject in need of T cell therapy with 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 the maturation or differentiation of T cells in vitro for ​​A method comprising culturing one or more T cells in a medium containing AKTi and at least one of exogenous IL-7 and exogenous IL-15 is provided.

[0008] The present disclosure further provides a method for generating stem cell-like CD8 + T cells, the method comprising contacting one or more T cells with AKTi and at least one of exogenous IL-7 and exogenous IL-15.

[0009] The present disclosure also provides a method for prolonging the in vivo persistence of one or more T cells in adoptive cell therapy, the method comprising contacting one or more T cells with 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. is needed.

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

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

[0013] In certain embodiments, the T cell therapy includes modified CAR cell therapy or modified TCR cell therapy. One In an aspect, the modified CAR cell or modified TCR cell therapy treats tumors in a subject.

Brief Description of the Drawings

[0014]

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[0015] Detailed Description The present invention relates to a method for preparing T cells for use in T cell therapy. In particular, the present invention relates to a method for preparing one or more T cells by inducing an AKTi and exogenous IL-7 and exogenous IL-15. The present invention relates to a method of modulating, e.g., delaying or inhibiting, maturation or differentiation of T cells in vitro by contacting the T cells with at least one of By promoting or inhibiting T cell proliferation, the donor T cell population becomes less differentiated and immature. The present invention can be used to enrich for T cells (e.g., naive T cells or central memory Tcm cells) that are highly conserved in the human T cell population. In some embodiments, the T cells may be enriched for immature T cells, which may increase 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 stages of differentiation.

[0016] definition To make the present disclosure more readily understandable, 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 shall have the same meaning as commonly understood by one of ordinary skill 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 many common dictionaries of terms used by one of ordinary skill in the art in this disclosure.

[0018] Units, prefixes, and symbols are represented in the form approved by the International System of Units (SI). Numerical ranges include the numbers defining the range. The headings provided herein do not limit the various aspects of the disclosure that can be obtained by referring to the entire specification. Accordingly, the terms defined immediately below are more fully defined by reference to the entire specification.

[0019] As used herein, the indefinite article "a" or "an" shall be understood to refer to "one or more" of any recited or enumerated component as appropriate.

[0020] The terms "about" or "consisting essentially of" will depend in part on the way in which a value or composition is measured or determined (i.e., the limitations of the measurement system), as determined by one of ordinary skill in the art and refers to a value or composition that is within an acceptable error range for a particular value or composition. For example, "about" or "consisting essentially of" may mean within one or more standard deviations in accordance with practices in the art. Alternatively, "about" or "consisting essentially of" may mean within a range of up to 10% (i.e., ±10%). For example, about 3 mg can include any numerical value from 2.7 mg to 3.3 mg (for 10%). Furthermore, especially with respect to biological systems or processes, these terms may mean up to one order of magnitude or up to five-fold of a value. In the present application and claims where a particular value or composition is provided, unless otherwise specified, the meaning of "about" or "consisting essentially of" should be considered to be within an acceptable error range for that particular value or composition.

[0021] As used herein, any concentration range, percentage range, ratio range or integer range is understood to include any integer within the recited range and, where appropriate, values of fractions thereof (e.g., one tenth and one hundredth of an integer), unless otherwise specified so as to.

[0022] The term "and / or" as used herein is considered to be a specific disclosure of each of two specified features or components, with or without the other thereof. Thus, the term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following situations : 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] When an aspect is described herein with the word "comprising", it is understood that other aspects similar in other respects described by "consisting of" and / or "consisting essentially of" are always provided. The terms "activated" or "activated" refer to the state of immune cells, such as T cells, that have been sufficiently stimulated to induce detectable cell proliferation. Activation can also be associated with induced cytokine production and detectable effector functions. The term "activated T cell" specifically refers to a T cell that is undergoing cell division. T cell activation can 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 an agent to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration for T cells prepared by the methods disclosed herein include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration by injection or infusion. The phrase "parenteral administration" as used herein, when used herein, usually means a mode of administration other than enteral and topical administration by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. In some embodiments, the T cells prepared by the method are not parenterally It is administered via a route, for example, orally. Other non - parenteral routes include topical, epidermal or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual or topical. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended periods.

[0025] The terms "AKT inhibitor", "AKTI", or "AKTi" can be used interchangeably and refer to any molecule (e.g., an AKT antagonist) that can block, reduce, or inhibit the activity of AKT and that includes, but is not limited to, small molecules, polynucleotides (e.g., DNA or RNA), or polypeptides (e.g., an antibody or its antigen - binding portion). 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 between AKT and its binding partner or by inhibiting the activity of another member of the PI3K - AKT - mTOR pathway. Non - limiting examples of AKTi are shown in other sections of this application. and can block, reduce, or inhibit the activity of AKT, and includes small molecules, polynucleotides (e.g., DNA or RNA), or polypeptides (e.g., an antibody or its antigen - binding portion). 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 between AKT and its binding partner or by inhibiting the activity of another member of the PI3K - AKT - mTOR pathway. Non - limiting examples of AKTi are shown in other sections of this application. molecules, polynucleotides (e.g., DNA or RNA), or polypeptides (e.g., an antibody or its antigen - binding portion), but is not limited thereto. 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 between AKT and its binding partner or by inhibiting the activity of another member of the PI3K - AKT - mTOR pathway. Non - limiting examples of AKTi are shown in other sections of this application. molecules, polynucleotides (e.g., DNA or RNA), or polypeptides (e.g., an antibody or its antigen - binding portion), but is not limited thereto. 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 between AKT and its binding partner or by inhibiting the activity of another member of the PI3K - AKT - mTOR pathway. Non - limiting examples of AKTi are shown in other sections of this application. and can block, reduce, or inhibit the activity of AKT, and includes small molecules, polynucleotides (e.g., DNA or RNA), or polypeptides (e.g., an antibody or its antigen - binding portion). 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 between AKT and its binding partner or by inhibiting the activity of another member of the PI3K - AKT - mTOR pathway. Non - limiting examples of AKTi are shown in other sections of this application. by interfering with the interaction between AKT and its binding partner or by inhibiting the activity of another member of the PI3K - AKT - mTOR pathway. Non - limiting examples of AKTi are shown in other sections of this application. Non - limiting examples of AKTi are shown in other sections of this application.

[0026] The term "antibody" (Ab) includes, without limitation, immunoglobulins that specifically bind to an antigen. Generally, an antibody can comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each H chain comprises a heavy - chain variable region (abbreviated as VH herein) and a heavy - chain constant region. The heavy - chain constant region comprises three or four constant domains, CH1, CH2, CH3, and a heavy - chain constant region. The heavy - chain constant region comprises three or four constant domains, CH1, CH2, CH3, and / or may contain CH4. Each light chain includes a variable light chain region (abbreviated as VL herein) and a constant light chain region. The constant light chain region may include one constant domain, CL. The VH and VL regions can 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 in the following order from the amino terminus to the carboxy terminus as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen, such as AKT.

[0027] The immunoglobulin can be derived from any of the generally known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also known to those skilled in the art and include but are not limited to human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to an Ab class or subclass encoded by a heavy chain constant region gene (e.g., , IgM or IgG1). 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 otherwise and unless otherwise specified in the context, the term "antibody" also includes any antigen-binding fragment or antigen-binding portion of any of the foregoing immunoglobulins, including monovalent and bivalent fragments or portions, as well as single-chain Abs.

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

[0029] The term "autologous" refers to any material derived from the same individual that is intended to be re-introduced later. For example, the modified autologous cell therapy (eACT™) described herein involves 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., the patient.

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

[0031] "Cancer" refers to a broad group of various diseases characterized by uncontrolled growth of abnormal cells in the body. The disordered cell division and growth form malignant tumors that can invade adjacent tissues and spread to distant sites in the body through the lymphatic system or bloodstream. "Cancer" or "cancer tissue" can include tumors at various stages. In one aspect, the cancer or tumor is stage 0 and thus, for example, the cancer or tumor is in a relatively early stage of development and has not metastasized. In some aspects, the cancer or tumor is stage I and thus, for example, the cancer or tumor is relatively small in size, has not spread to nearby tissues, and has not metastasized. In other aspects, the cancer or tumor is stage II or stage III and thus, for example, the cancer or tumor is larger than in stage 0 or stage I, has grown into adjacent tissues, but has not metastasized except possibly to lymph nodes. In other aspects, the cancer or tumor is stage IV and thus, for example, the cancer or tumor has metastasized. Stage IV can also be referred to as advanced cancer or metastatic cancer.

[0032] The "anti-tumor effect", as used herein, refers to a biological effect that can manifest as a reduction in tumor volume, inhibition of tumor growth, reduction in the number of tumor cells, reduction in tumor cell proliferation, reduction in the number of metastases, increase in overall survival or progression-free survival, increase in mean lifespan, or improvement of various physiological symptoms related to the tumor. The anti-tumor effect can also be used for the prevention of tumor development, for example, for 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 or death from any cause according to the Revised IWG Response Criteria for Malignant Lymphoma. refers to.

[0034] "Disease progression" is evaluated by the measurement of malignant lesions on radiographs, i.e., other methods should be reported as adverse events. Deaths resulting from disease progression in the absence of signs and symptoms should be reported as the primary tumor type (e.g., DLBCL).

[0035] The "duration of response" (which may be abbreviated as DOR), as used herein, refers to the period from the first objective response of the subject to the date of confirmed disease progression or death according to the Revised IWG Response Criteria for Malignant Lymphoma.

[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 convey an immune response. In some embodiments, one or more cytokines are released in response to T cell therapy. In one embodiment, those The cytokines may be signs of effective T cell therapy.

[0038] As used herein, "therapeutically effective amount" or "therapeutically effective dosage" refers to an amount of T cells or DC cells produced by the methods herein and used alone or in combination with another therapeutic agent that protects a subject from the onset of a 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 periods, or prevention of functional or physical impairment resulting from disease affliction). The ability of T cells or DC cells to promote disease regression can be evaluated using various methods known to those of skill in the art, such as in human subjects during clinical trials, in animal model systems for predicting efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0039] As used herein, the term "effective amount" or "effective dosage" refers to the amount of one or more T cell maturation inhibitors (e.g., AKTi, IL-7, and IL-15) that together elicit 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 dosage of AKTi may refer to the amount, e.g., concentration, of AKTi that reduces AKT activity by a desired amount, such as at least about 10%, at least 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] As used herein, the term "lymphocyte" may include natural killer (NK) cells, T cells, or B cells. NK cells are a type of cytotoxic (cytotoxic) lymphocyte that represents a major component of the innate immune system. NK cells reject tumors and virus-infected cells. It acts through the process of apoptosis or programmed cell death. They are named "natural killer" 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 receptor (TCR) distinguishes themselves from other lymphocyte types. The thymus is mainly responsible for the maturation of T cells.

[0041] Some types of T cells, namely, helper T cells (e.g., CD4+ cells, effector T EFF cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTL, T killer cells, cytolytic T cells, CD8+ T cells or killer T cells), memory T cells ((i) Stem memory T SCM cells, like naive cells, are CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Rα+, but they also express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1 and exhibit a number of functional characteristics specific to memory cells); (ii) Central memory T CM cells express L-selectin, CCR7 + and CD45RO + and they secrete IL-2 but do not secrete IFNγ or IL-4; and (iii) Effector memory T EM cells, however do not express L-selectin or CCR7 but express CD45RO and secrete effs such as IFNγ and IL-4 There are effector cytokines-producing regulatory T cells (Tregs, suppressor T cells, or CD4+CD25+ regulatory T cells), natural killer T cells (NKTs), and γδ T cells. T cells found within tumors are referred to as "tumor infiltrating lymphocytes" or "TILs". On the other hand, B cells play a major role in humoral immunity (with antibody involvement). It makes antibodies and antigens, acts as an antigen presenting cell (APC), and becomes a memory B cell after activation by antigen interaction. In mammals, immature B cells are formed in the bone marrow and are named after their origin .

[0042] "Naive" T cells refer to mature T cells that remain immunologically undifferentiated. Following positive and negative selection in the thymus, T cells emerge as either CD4 + or CD8 + naive T cells. 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 specific to either naive T cells or immature T cells, e.g., T SCM cells or T CM cells. For example, immature T cells may express one or more of 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 contrasted with terminally differentiated effector T cells, e.g., T EM cells and T EFF cells.

[0043] "T cell function", when referred to in this specification, refers to the normal properties of healthy T cells. In some embodiments, T cell function includes T cell proliferation. In some embodiments, T cell function includes T cell activation. In some embodiments, T cell function includes cytolytic activity. In some embodiments, the methods of the invention, for example, 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 more potent for T cell therapy. In some embodiments, T cells cultured according to the method have increased T cell function compared to T cells cultured under conditions lacking an AKT inhibitor ( or AKTi, IL-7, and IL-15). In one embodiment, T cells cultured according to the method have increased T cell proliferation compared to T cells cultured under conditions lacking an AKT inhibitor (or AKTi, IL-7, and IL-15). In one embodiment, T cells cultured according to the method have increased T cell activation compared to T cells cultured under conditions lacking an AKT inhibitor (or AKTi, IL-7, and IL-15). In one embodiment, T cells cultured according to the method have increased cytolytic activity compared to T cells cultured under conditions lacking an AKT inhibitor (or AKTi, IL-7, and IL-15).

[0044] Cell "proliferation", as used herein, 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 certain embodiments, T cell activity includes the production of one or more cytokines selected from interferon gamma (IFNg), tumor 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, such as 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 a portion thereof, or inserting a coding region or a portion thereof. In some embodiments, the cells to be modified are lymphocytes, such as T cells, that can be obtained from either a patient or a donor. These cells can be modified to express an exogenous construct, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR), that is integrated into the cell's genome.

[0048] An "immune response" is the selective targeting, binding to, damaging, destroying, and / or elimination of 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, by cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils), and the production by either these cells or the liver​ Refers to the action of soluble polymers (including Abs, cytokines, and complement) that are involved.

[0049] The term "immunotherapy" refers to the treatment of a subject who has a disease, is at risk of developing a disease, or is suffering from a recurrence of a disease, by a method that includes 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 lymphocytes (TIL) immunotherapy, autologous cell therapy, modified autologous cell therapy (eACT™), and allogeneic T cell transplantation. However, one of ordinary skill in the art will recognize that the methods for preparing T cells disclosed herein will enhance the effectiveness of any adoptive T cell therapy. Examples of T cell therapy are described in US Patent Application Publication Nos. 2014 / 0154228 and 2002 / 0006409, US Patent No. 5,728,388, and International Publication No. WO 2008 / 081035.

[0050] The T cells of immunotherapy can be derived from any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a donor. 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 from, ascites, pleural effusion, spleen tissue, and tumors. 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 of skill 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 is a human ​​​Reproduce the thymic environment and support 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 US Patent Application Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety. The term "engineered 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) specific for a particular tumor antigen linked to an intracellular signaling moiety that includes a co-stimulatory domain and an activation domain. Co-stimulatory domains include, for example, 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 co-stimulatory molecule (ICOS), ICOS-L, lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18)), CD3 gamma

[0051] , CD3 delta, CD3 epsilon, CD247, 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 Body, integrin, signal transduction lymphocyte activation molecule (SLAM protein), activated 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, CD83 and ligands that specifically bind thereto or may be derived from any combination thereof. The activation domain may be derived from, for example, CD3 : for example, CD3 zeta, epsilon, delta, gamma, etc. In one aspect , the CAR is designed to have 2, 3, 4, or more co-stimulatory domains. The CAR scFv is a transmembrane protein expressed, for example, by cells of the B cell lineage including all normal B cells as well as B cell malignancies (including but not limited to NHL, CLL, and non-T cell ALL) It can be designed to target CD19, a protein. Exemplary CAR + T cell therapies and constructs are described in US Patent Application Publication Nos. 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, and these references are incorporated by reference in their entirety.

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

[0053] "Stimulation", as used herein, refers to the primary response induced by the binding of a stimulatory molecule to its cognate ligand, where the binding mediates a signaling event. A "stimulatory molecule" is a molecule on a T cell that specifically binds to a cognate stimulatory ligand present on an antigen-presenting cell, such as the T cell receptor (TCR) / CD3 complex. There is. A "stimulatory ligand" specifically binds to a stimulatory molecule on a T cell when present on an antigen-presenting cell (e.g., an artificial antigen-presenting cell (aAPC), dendritic cell , B cell, etc.), thereby A ligand that can mediate a primary response by T cells, including but not limited to activation, initiation of an immune response, proliferation, etc. Examples of stimulatory ligands include MHC loaded with peptides. ​Examples include, but are not limited to, class I molecules, anti-CD3 antibodies, superagonist anti-CD28 antibodies, and superagonist anti-CD2 antibodies. "Activated" or "active", as used herein, refers to T cells after being stimulated. Activated T cells can 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 obtained commercially or produced recombinantly. When added to or contacted with one or more T cells, "exogenous IL-7" or "exogenous IL-15" indicates that this IL-7 and / or IL-15 is not produced by the T cells. In some embodiments, the T cells before 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 the subject together with the T cells. One or more T cells described herein can be contacted with exogenous IL-7 and / or exogenous IL-15 through any means known in the art, including addition of isolated IL-7 and / or IL-15 to the culture; including IL-7 and / or IL-15 in the culture medium; or expression of IL-7 and / or IL-15 by one or more cells in a culture other than the one or more T cells, for example by a feeder layer.

[0055] As used herein, the term "persistence" refers to the ability to continue at a detectable level in a subject over a period of time, e.g., the ability of one or more transplanted T cells or their progeny (e.g., differentiated or mature T cells) administered to the subject. 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 the subject after administration. For example, the in vivo persistence of one or more transplanted T cells can be increased to 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, 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. In addition, the in vivo persistence of one or more transplanted T cells can 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. For example, 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 the subject after administration. For example, the in vivo persistence of one or more transplanted T cells can be increased to 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, 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. For example, the in vivo persistence of one or more transplanted T cells can be increased to 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, 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. For example, the in vivo persistence of one or more transplanted T cells can be increased to 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, 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. For example, the in vivo persistence of one or more transplanted T cells can be increased to 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, 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. In addition, the in vivo persistence of one or more transplanted T cells can 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. In addition, the in vivo persistence of one or more transplanted T cells can 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. In addition, the in vivo persistence of one or more transplanted T cells can 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 "decrease" are used interchangeably herein and refer to any change that is less than the original. "Reduce" and "decrease" are relative terms that require a comparison between before and after measurement. "Reduce" and "decrease" include complete depletion. In some embodiments, the terms "reduce" and "decrease" refer to 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 such preparation. include a comparison of T cell effects.

[0057] The term "modulate" when used herein with respect to T cell maturation refers to the use of any intervention described herein to control the maturation, e.g., differentiation, of one or more T cells. In some embodiments, "modulate" refers to delaying or inhibiting T cell maturation. In other embodiments, "modulate" refers to accelerating or promoting T cell maturation. In particular, "delay or inhibit T cell maturation" when used herein refers to maintaining one or more T cells in an immature or undifferentiated state. For example, "delay or inhibit T cell maturation" may refer to maintaining T cells in a naive or T state as opposed to progressing to a T or T state. "Delay or inhibit T cell maturation" may also refer to increasing or enriching the overall percentage of immature or undifferentiated T cells (e.g., naive T cells and / or T cells) in a mixed population of T cells. The state of a T cell (e.g., as mature or immature) can be screened, for example, by screening for the expression of various genes and the presence of various proteins expressed on the surface of the T cell. For example, "delay or inhibit T cell maturation" may refer to maintaining T cells in a naive or T state as opposed to progressing to a T or T state. "Delay or inhibit T cell maturation" may also refer to increasing or enriching the overall percentage of immature or undifferentiated T cells (e.g., naive T cells and / or T cells) in a mixed population of T cells. The state of a T cell (e.g., as mature or immature) can be screened, for example, by screening for the expression of various genes and the presence of various proteins expressed on the surface of the T cell. EM or T EFF state, "delay or inhibit T cell maturation" may refer to maintaining T cells in a naive or T state as opposed to progressing to a T or T state. "Delay or inhibit T cell maturation" may also refer to increasing or enriching the overall percentage of immature or undifferentiated T cells (e.g., naive T cells and / or T cells) in a mixed population of T cells. The state of a T cell (e.g., as mature or immature) can be screened, for example, by screening for the expression of various genes and the presence of various proteins expressed on the surface of the T cell. CM state, "delay or inhibit T cell maturation" may refer to maintaining T cells in a naive or T state as opposed to progressing to a T or T state. "Delay or inhibit T cell maturation" may also refer to increasing or enriching the overall percentage of immature or undifferentiated T cells (e.g., naive T cells and / or T cells) in a mixed population of T cells. The state of a T cell (e.g., as mature or immature) can be screened, for example, by screening for the expression of various genes and the presence of various proteins expressed on the surface of the T cell. In a mixed population of T cells, "delay or inhibit T cell maturation" may also refer to increasing or enriching the overall percentage of immature or undifferentiated T cells (e.g., naive T cells and / or T cells). The state of a T cell (e.g., as mature or immature) can be screened, for example, by screening for the expression of various genes and the presence of various proteins expressed on the surface of the T cell. cells) in a mixed population of T cells. CM cells) in a mixed population of T cells. In a mixed population of T cells, "delay or inhibit T cell maturation" may also refer to increasing or enriching the overall percentage of immature or undifferentiated T cells (e.g., naive T cells and / or T cells). The state of a T cell (e.g., as mature or immature) can be screened, for example, by screening for the expression of various genes and the presence of various proteins expressed on the surface of the T cell. by screening for the expression of various genes and the presence of various proteins expressed on the surface of the T cell. For example, L-selectin (CD62L+), IL-7R-α, CD132, CR7, CD45RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, LFA-1, and their related proteins can be determined. The presence of one or more markers selected from the group consisting of any combination is less May represent immature, 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 administration to a subject of one or more T cells prepared according to the present invention, 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 indicator 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 methods for producing immune cells (e.g., lymphocytes or dendritic cells) for use in cell therapy. The present invention relates to a method for preparing certain in vitro engineered cells (e.g., CAR T cells). It has been found that lymphocytes (e.g., TCR cells, or dendritic cells) are not as effective when administered to a patient 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 a patient. The present disclosure relates to a combination of, in one embodiment, AKTi with exogenous IL-7 and exogenous IL-15. and Methods for slowing, preventing or inhibiting the

[0061] In one aspect, the present disclosure relates to a method of regulating the maturation or differentiation of T cells or DC cells in vitro, for example, delaying or inhibiting, 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). In addition, the present disclosure provides that the resulting T cells or DC cells exhibit increased proliferation both in vitro and in vivo as well as excellent anti-tumor activity. For example, it relates to a method of delaying or inhibiting. 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). In addition, the present disclosure provides that the resulting T cells or DC cells exhibit increased proliferation both in vitro and in vivo as well as excellent anti-tumor activity.

[0062] In another aspect, the present invention provides a method for regulating, for example, delaying or inhibiting cell (e.g., T cell) maturation or differentiation in vitro for cell therapy (e.g., T cell therapy), the method comprising 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 (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. The contacting step comprises adding (i) an AKT inhibitor and (ii) exogenous IL-7 and / or exogenous IL-15 directly to one or more T cells or to a buffer or medium containing the T cells, or adding (i) an AKT inhibitor and (ii) exogenous IL-7 and / or exogenous IL-15 together with other components to the T cells. The contacting step comprises adding (i) an AKT inhibitor and (ii) exogenous IL-7 and / or exogenous IL-15 directly to one or more T cells or to a buffer or medium containing the T cells, or adding (i) an AKT inhibitor and (ii) exogenous IL-7 and / or exogenous IL-15 together with other components to the T cells.​ a step of mixing with the component of , and / or a step of adding one or more cells into a medium containing (i) an AKT inhibitor and (ii) exogenous IL-7 and / or exogenous IL-15 may be included. In certain embodiments, one or more T cells are not contacted with exogenous interleukin-2 (IL-2). Further preparation of T cells is described elsewhere in this specification.

[0063] The present disclosure shows that 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 can increase the concentration of naive T cells and T cells in the sample compared to the concentration of more terminally differentiated T cells. Accordingly, in another aspect, the present invention is a method for generating stem cell-like CD4 CM T cells or CD8 + T cells, comprising the step of culturing one or more T cells in a medium containing (i) an AKT inhibitor and (ii) exogenous IL-7, exogenous IL-15, or both. + In other aspects, the present invention is a method for enriching a population of CD8 / CD45RA + / CCR7 + T cells, comprising: (a) obtaining one or more T cells from a subject; (b) contacting the 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 an AKT inhibitor and exogenous IL-7, exogenous IL-15, or both. Generating increased concentrations of immature and undifferentiated T cells or DC cells increases 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). DC cell therapy). This 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 This is possible. Thus, in another aspect, the present invention provides a method for prolonging the in vivo persistence of one or more T cells or DC cells in adoptive cell therapy, the method comprising contacting 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 transplanted T cells that have not been contacted with the AKT inhibitor and exogenous IL-7, exogenous IL-15, or both. 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. Delaying or inhibiting the maturation or differentiation of one or more T cells or DC cells can be measured by any method known in the art. For example, delaying or inhibiting the 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 aspects, the one or more biomarkers are selected from the group consisting of L-selectin (CD62L

[0064] + ), IL-7Rα, CD132, CCR7, CD4 5RA, CD45RO, CD27, CD28, CD95, IL-2Rβ, CXCR3, LFA-1, or any combination thereof. In one aspect, delaying or inhibiting the maturation or differentiation of one or more T cells or DC cells can be measured by detecting the presence of one or more of L-selectin (CD62L + ), IL-7Rα, and CD132. Those skilled in the art will appreciate that the present + + ), IL-7Rα, and CD132. Those skilled in the art will appreciate that the present The method can increase the relative proportion of immature and undifferentiated T cells or DC cells in a population of cells taken, but it will be recognized that some mature and differentiated cells may still be present. As a result, the delay or inhibition of the 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 one embodiment, one or more T cells contacted with an AKT inhibitor and at least one of exogenous IL-7 and exogenous IL-15 contain 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, 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 contain 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 one embodiment, the immature or undifferentiated T cells are naive T cells and / or central memory Tcm cells. ​

[0065] The methods disclosed herein include administering to the patient one or more T cells or DC cells an AKT inhibitor. In some embodiments, the method comprises contacting one or more T cells or DC cells with exogenous IL-7 and exogenous IL-15. In another embodiment, the method comprises contacting the cells with exogenous IL-7 and exogenous IL-15. The method comprises 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] The one or more T cells or DC cells can be contacted with the 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 administered to one or more cultured T cells or DC cells. Alternatively, the AKT inhibitor and IL-7 / IL-15 can be produced by one or more cells that are co-cultured with one or more T cells or DC cells, for example, by a feeder cell layer. IL-7 and IL-15 can be added together or individually. For example, an AKT inhibitor can be added to the culture medium, and IL-7 and / or IL-15 can be produced by cells that are 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 - and can be contacted with exogenous IL-7 and / or exogenous IL-15 . 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 particular 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 an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15. In some embodiments, 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 . Thus, one or more T cells or DC cells can be obtained from a subject in need of T cell therapy or from a donor. After collection, one or more T cells can be processed for any suitable period of time before being administered to the subject. During this time, one or more T cells can be contacted with an AKT inhibitor, exogenous IL-7, and / or exogenous IL-15 for any period between collection of the T cells from the donor and administration to the subject. For example, one or more T cells can be contacted for at least about 1 day, at least about 2 days, at least about 3 days ​for 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, with an AKT inhibitor , exogenous IL-7, and / or exogenous IL-15, for example, can be cultured in the presence thereof . In some embodiments, one or more T cells are contacted with 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, for example, cultured in the presence thereof. 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 an AKT inhibitor, exogenous IL-7, and / or exogenous IL-15, for example, cultured in the presence thereof. In another embodiment, the T cells are, from day 0 until administration, from day 1 until administration, from day 2 until administration, from day 3 until administration, 4 until administration, from day 5 until administration, or from day 6 until administration, contacted with an AKT inhibitor, exogenous IL-7, and / or exogenous IL-15, for example, cultured in the presence thereof . In some embodiments, 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 certain aspects, the present disclosure provides a method of modulating, 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 aspect, one or more cells treated with an AKTi and at least one (or both) of IL-7 and IL-15 but not with IL-2 exhibit a more highly delayed or inhibited maturation or differentiation than one or more cells treated with IL-2 alone or with IL-2 and an AKTi. 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 with IL-2 and an AKTi. 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). In addition, the present disclosure provides that the resulting T cells or DC cells exhibit increased proliferation and superior anti-tumor activity in vitro and in vivo. In some aspects, one or more T cells are CD4 cells. In other aspects, one or more T cells are CD8 cells. In certain aspects, the contacting of the AKTi with at least one of IL-7 and IL-15 is carried out 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 aspects, the contacting of the AKTi with at least one of IL-7 and IL-15 is carried out for more than 1 day and up to 14 days. days. ​​ It is carried out until 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 may further include a step of enriching a population of lymphocytes obtained from a donor. Enrichment of a population of lymphocytes, for example, one or more T cells, can be achieved by using a separation medium (e.g., 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 (e.g ., magnetic-activated cell sorting system, MACS), fluorescence-activated cell sorting (e.g., fluorescence-activated cell sorting system, FACS), or bead-based column separation, including but not limited to these, and can be achieved by any suitable separation method.

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

[0072] Suitable conditions for stimulating a population of lymphocytes as described herein may include a certain temperature, a certain length of time, and / or the presence of a certain CO 2 level. In certain embodiments , the temperature for stimulation is about 34°C, about 35°C, about 36°C, about 37°C, or about 38°C. In certain embodiments, the temperature for stimulation is about 34 - 38°C. In certain embodiments, the temperature for stimulation is about 35 - 37°C. In certain embodiments, the temperature for stimulation is about 36 - 38°C. In certain embodiments, the temperature for stimulation is about 36 - 37°C or about 37°C.

[0073] Another condition for stimulating a population of lymphocytes as described herein may include the time for stimulation. In some embodiments, the time for stimulation is about 24 - 72 hours. In some embodiments, the time for stimulation is about 24 - 36 hours, about 30 - 42 hours, about 36 - 48 hours, about 40 - 52 hours, about 42 - 54 hours, about 44 - 56 hours, about 46 - 58 hours, about 48 - 60 hours, about 54 - 66 hours, or about 60 - 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 - 52 hours. In certain embodiments, the time for stimulation is about 40 - 44 hours, about 40 - 48 hours, about 40 - 52 hours, or about 40 - 56 hours.

[0074] Another condition for stimulating a population of lymphocytes as described herein is the CO 2 level that may be included. In some embodiments, the level of CO 2 for stimulation is about 1.0 - 10% CO 2 . In some embodiments, the level of CO for stimulation is about 1.0%, about 2.0%, about 3.0%, 2 about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO . In one embodiment 2 , the level of CO for stimulation is2 The level is about 3 - 7% CO 2 In other embodiments, the CO for stimulation 2 The level is about 4 - 6% CO 2 In yet other embodiments, the CO for stimulation 2 The le The level is about 4.5 - 5.5% CO 2 In one particular embodiment, the CO for stimulation 2 The level is about 5% CO 2 is.

[0075] The conditions for stimulating a population of lymphocytes can include, in any combination, a certain temperature, a certain length of time for stimulation, and / or the presence of a certain CO 2 level. For example, the step of stimulating a population of lymphocytes can include stimulating the population of lymphocytes with one or more T cell stimulants at a temperature of about 36 - 38°C, for a length of time of about 44 - 52 hours, in the presence of a CO 2 CO 2 level of about 4.5 - 5.5%.

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

[0077] An anti-CD3 antibody (or a functional fragment thereof), an anti-CD28 antibody (or a functional fragment thereof), or a combination of an anti-CD3 antibody and an anti-CD28 antibody is used according to the step of stimulating a population of lymphocytes It can be used. Any soluble or immobilized anti-CD2, anti-CD3, and / or anti-CD28 antibody or its functional fragment 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, the antibody 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 1mg / mL, Part No. 170-076-116), and eBioscience, Inc. Further, those skilled in the art will understand how to produce anti-CD3 antibodies and / or anti-CD28 antibodies by standard methods. In some embodiments, one or more T cell stimulants that can be used according to the step of stimulating a population of lymphocytes include antibodies or their functional fragments that target T cell stimulatory molecules or costimulatory molecules in the presence of T cell cytokines. In one aspect, one or more T cell stimulants include an anti-CD3 antibody and IL-2. In certain embodiments, the T cell stimulant includes 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 can be enriched for T lymphocytes) is transduced according to the steps below.

[0078] The methods described herein use a single cycle of transduction to transduce a population of activated T cells with a viral vector containing a nucleic acid molecule encoding a cell surface receptor to generate transduced T cells. A number of recombinant viruses have been used as viral vectors for delivering genetic material to cells. Viral vectors that can be used according to the transduction step can 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 virus (AAV) vectors. In some embodiments, the method further comprises transducing one or more T cells with a retrovirus. In one embodiment, the viral vector used to transduce a population of activated T cells is the MSGV1 gamma-retroviral vector. In certain embodiments, the viral vector used to transduce a 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 grown in suspension culture in a medium for producing viral vectors, referred to herein as a "viral vector inoculum." Any suitable growth medium and / or adjuvant for growing the viral vector 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 the serum-free culture medium described below during the transduction step. using transduction to transduce a population of activated T cells with a viral vector containing a nucleic acid molecule encoding a cell surface receptor A number of recombinant viruses have been used as viral vectors for delivering genetic material to cells. Viral vectors that can be used according to the transduction step can 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 virus (AAV) vectors. In some embodiments, the method further comprises transducing one or more T cells with a retrovirus. In one embodiment, the viral vector used to transduce a population of activated T cells is the MSGV1 gamma-retroviral vector. In certain embodiments, the viral vector used to transduce a 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 grown in suspension culture in a medium for producing viral vectors, referred to herein as a "viral vector inoculum." Any suitable growth medium and / or adjuvant for growing the viral vector 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 the serum-free culture medium described below during the transduction step. viral vector, including but not limited to recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, and recombinant adeno-associated virus (AAV) vectors. In some embodiments, the method further comprises transducing one or more T cells with a retrovirus. In one embodiment, the viral vector used to transduce a population of activated T cells is the MSGV1 gamma-retroviral vector. In certain embodiments, the viral vector used to transduce a 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 grown in suspension culture in a medium for producing viral vectors, referred to herein as a "viral vector inoculum." Any suitable growth medium and / or adjuvant for growing the viral vector 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 the serum-free culture medium described below during the transduction step. In some embodiments, the method further comprises transducing one or more T cells with a retrovirus. In one embodiment, the viral vector used to transduce a population of activated T cells is the MSGV1 gamma-retroviral vector. In certain embodiments, the viral vector used to transduce a 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 grown in suspension culture in a medium for producing viral vectors, referred to herein as a "viral vector inoculum." Any suitable growth medium and / or adjuvant for growing the viral vector 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 the serum-free culture medium described below during the transduction step. In some embodiments, the method further comprises transducing one or more T cells with a retrovirus. In one embodiment, the viral vector used to transduce a population of activated T cells is the MSGV1 gamma-retroviral vector. In certain embodiments, the viral vector used to transduce a 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 grown in suspension culture in a medium for producing viral vectors, referred to herein as a "viral vector inoculum." Any suitable growth medium and / or adjuvant for growing the viral vector 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 the serum-free culture medium described below during the transduction step. In one embodiment, the viral vector used to transduce a population of activated T cells is the MSGV1 gamma-retroviral vector. In certain embodiments, the viral vector used to transduce a 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 grown in suspension culture in a medium for producing viral vectors, referred to herein as a "viral vector inoculum." Any suitable growth medium and / or adjuvant for growing the viral vector 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 the serum-free culture medium described below during the transduction step. In certain embodiments, the viral vector used to transduce a 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 grown in suspension culture in a medium for producing viral vectors, referred to herein as a "viral vector inoculum." Any suitable growth medium and / or adjuvant for growing the viral vector 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 the 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 comprises a heterologous gene that encodes 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 include: At a specific time, at a specific temperature and / or with a specific CO 2 may include being in the presence of a 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 some embodiments, the time for transduction is about 12-36 hours. In some embodiments, the time for transduction is about 12-16 hours, about 12-20 hours, about 12-24 hours, about 12-28 hours, or about 12-32 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 16-24 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 one embodiment, the CO for transduction 2 The level is about 1.0-10% CO 2 Other aspects 2. CO for Transduction in 2The levels are 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% CO 2 . In one embodiment, the CO for transfection 2 level is about 3 - 7% CO 2 . In another embodiment, the CO for transfection 2 level is about 4 - 6% CO 2 . In another embodiment, the CO for transfection 2 level is about 4.5 - 5.5% CO 2 . In one particular embodiment, the CO for transfection 2 level is about 5% CO 2 .

[0083] In some embodiments, the transfection of a population of activated T cells as described herein can be performed in any combination of a specific length of time, a specific temperature, and / or the presence of a specific CO 2 level: at a temperature of about 36 - 38°C, for about 16 - 24 hours, and in the presence of a CO level of about 4.5 - 5.5% CO 2 in the presence of a CO 2 level.

[0084] The methods described herein may include the step of expanding a population of one or more transfected T cells for a specific period of time to generate a population of modified T cells. The predetermined time for expansion can be any suitable time that enables the following generation: (i) a minimum amount for administration to a patient that A sufficient number of cells in the modified T cell population for at least 1 dose, (ii) a population of modified T cells containing a preferred proportion of naive cells compared to a typical longer process, 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 is 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 the 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% shorter, or more than 75% shorter. In one aspect the time for expansion is about 3 days and the time from enrichment of the lymphocyte population to generation of the modified T cells is about 6 days.

[0085] The conditions for expanding the population of transduced T cells can include a certain temperature and / or the presence of a certain CO 2 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 CO 2 level is 1.0 - 10% CO 2 is. In other embodiments, the CO 2The levels are 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% CO 2 is. In one aspect, CO 2 The level is about 4.5 - 5.5% CO 2 is. Another In the aspect, CO 2 The level is about 5% CO 2 is. In other aspects, CO 2 The level is about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, or about 6.5% CO 2 is. In some aspects, the conditions for growing the population of transduced T cells are at a certain temperature and / or a certain CO 2 level in any combination. For example, the conditions for growing the population of transduced T cells are at a temperature of about 36 - 38°C and in the presence of about 4.5 - 5.5% CO 2 of CO 2 level.

[0086] Each step of the methods described herein can be performed in a closed system. In one aspect, the closed system is a closed bag culture system using any suitable cell culture bag (e.g., Miltenyi Biotec MACS (registered trademark) GMP Cell Differentiation Bag, Origen Biomedical PermaLife Cell Culture bag). In some aspects, 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 can include 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 assisting in the co-localization of target cells and viral vectors. In certain embodiments, the recombinant human fibronectin fragment is RETRONECTIN® (Takara Bio, Japan). In certain embodiments, the cell culture bag is coated with the recombinant human fibronectin fragment at a concentration of about 1-60 μg / mL or about 1-40 μg / mL ). In other embodiments, the cell culture bag is coated with the 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 the recombinant human fibronectin fragment at 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 the recombinant human fibronectin fragment at 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. In certain 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 a recombinant human fibronectin fragment. In one particular embodiment, the cell culture bag is coated with at least about 10 μg / mL of a recombinant human fibronectin fragment. The cell culture bag used in a closed bag culture system 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 HAS 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 no serum has been added. In some aspects, each of (a)-(d) is performed using a serum-free culture medium to which no serum has been 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 lymph ocytes, (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)-(d) is performed using a serum-free culture medium to which no serum has been added. As used herein, the term "serum-free medium" or "serum-free culture medium" refers to a growth medium in which serum (e.g., human serum or bovine This means that the serum is not supplemented. In other words, in some embodiments, serum is not added to the culture medium as separate, isolated components individually to support the viability, activation, and growth of the cultured cells. Any suitable culture medium, T cell growth medium, can be used to culture the cells in suspension according to the methods described herein. For example, the T cell growth medium can 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 (trademark) (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). For the purpose of supporting the viability, activation, and growth of the cultured cells, serum is not added to the culture medium as separate, isolated components individually. Any suitable culture medium, T cell growth medium, can be used to culture the cells in suspension according to the methods described herein. For example, as the T cell growth medium, a sterile low glucose solution containing appropriate amounts of buffer, magnesium, calcium, sodium pyruvate, and sodium bicarbonate can be mentioned, but it is not limited thereto. In one embodiment, the T cell growth medium is OPTMIZER (trademark) (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). The methods described herein can use a culture medium that is not supplemented with serum (e.g., human or bovine).

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

[0089] Any AKT inhibitor known in the art that includes an inhibitor of AKT1, AKT2, AKT3, or any combination thereof can be used in the present invention. 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, elsyl phosphocholine, 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 # 1191951-57-1), 10-DEBC hydrochloride, Akt in hibitor 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-041164 (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-benzimidazo -ol-2-one, and those which may be selected from any combination thereof. The AKT inhibitor may also be 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-2-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-benzimid -ol-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 (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); compounds having a structure comprising Formula I, Formula II, Formula III, Formula IV, Formula V, Formula VI, Formula VII, and Formula VIII disclosed in U.S. Patent No. 7,273,869, published September 25, 2007 (which is hereby incorporated by reference in its entirety); their stereoisomers; any AKTi disclosed in U.S. Patent No. 7,273,869, published September 25, 2007 (which is hereby incorporated by reference in its entirety); and those which may be selected from 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, AKT comprises a formula represented by Formula I, or a pharmaceutically acceptable salt or stereoisomer thereof: TIFF0007684370000001.tif48128wherein: a is 0 or 1; b is 0 or 1; m is 0, 1 or 2; n is 0, 1 or 2; p is 0, 1, 2 or 3; r is 0 or 1; s is 0 or 1; t is 2, 3, 4, 5 or 6; u, v and x are independently selected from CH and N; w is selected from a bond, CH and N; y and z are independently selected from CH and N, provided that at least one of y and z is N; R 1 is: 1) (C=O) a O b C 1 -C 10 alkyl, 2) (C=O) a O b aryl, 3) C 2 -C 10 alkenyl, 4) C 2 -C 10 alkynyl, 5) (C=O) a O b heterocyclyl, 6) (C=O) a O b C 3 -C 8 cycloalkyl, 7) CO 2 H, 8) halo, 9) CN, 10) OH, 11) O b C 1 -C 6 perfluoroalkyl, 12) O a (C=O) b NR 7 R 8 , 13) NR c (C=O)NR 7 R8 , 14) S(O) m R a , 15) S(O) 2 NR 7 R 8 , 16) NR c S(O) m R a , 17) Oxo, 18) CHO, 19) NO 2 , 20) NR c (C=O)O b R a , 21) O(C=O)O b C 1 -C 10 Alkyl, 22) O(C=O)O b C 3 -C 8 Cycloalkyl, 23) O(C=O)O b Aryl, and 24) O(C=O)O b -Heterocycle are independently selected; said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl are substituted with one or more substituents selected from z R ; R 2 is: 1) (C=O) a O b C 1 -C 10 Alkyl, 2) (C=O) a O b Aryl, 3) C 2 -C 10 Alken yl, 4) C 2 -C 10 Alkynyl, 5) (C=O) a O b Heterocyclyl, 6) (C=O) a O b C 3 -C 8 Cycloalkyl, 7) CO 2 H, 8) Halo, 9) CN, 10) OH, 11) O b C 1 -C 6 Perfluoroalkyl, 12) O a (C=O) bNR 7 R 8 、13) NR c (C=O)NR7R 8 、14) S(O) m R a 、15) S(O) 2 NR 7 R 8 、16) NR c S(O) m R a 、17) CHO 、18) NO 2 、19) NR c (C=O)O b R a 、20) O(C=O)O b C 1 -C 10 alkyl, 21) O(C=O)O b C 3 -C 8 cycloalkyl, 22) O(C=O)O b aryl, and 23) O(C=O)O b -independently selected from heterocycles; said alkyl, aryl, alkenyl, alkynyl, heterocyclyl, and cycloalkyl may be substituted with 1, 2 or 3 substituents selected from R z ; R 3 and R 4 are independently selected from H, C 1 -C 6 -alkyl and C 1 -C 6 -perfluoroalkyl, or R 3 and R 4 combine to form -(CH 2 ) t -, where one of the carbon atoms may be replaced by a moiety selected from 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) C 2 -C 10 alkenyl, 6) C 2 -C 10 alkynyl, 7) heterocyclyl, 8) C 3 -C 8 cycloalkyl, 9) SO 2 R a and 10) (C=O)NR b 2 independently selected from, said alkyl, cycloalkyl, aryl, heterocyclyl, alkenyl, and alkynyl may be substituted with one or z more substituents selected from R or R 5 and R 6 together with the nitrogen to which they are attached may form a monocyclic or bicyclic heterocycle having 5 to 7 members in each ring, and optionally, in addition to nitrogen, containing one or two additional heteroatoms selected from N, O and S, which may be substituted with Q and further may be substituted with one or more substituents selected from R z ; Q is selected from -NR R 7 R 8 aryl and heterocyclyl, said aryl and heterocyclyl may be substituted with 1 z ~3 substituents selected from R ; R 7 and R 8 are 1) H, 2) (C=O)O b C 1 -C 10 alkyl, 3) (C=O)O b C 3 -C 8 cycloalkyl, 4) (C=O)O b aryl, 5) (C=O)O b heterocyclyl, 6) C 1 -C 10 alkyl, 7) aryl, 8) C 2 -C10 Alkenyl, 9) C 2 -C 10 Alkynyl, 10) Heterocyclyl, 11) C 3 -C 8 Cycloalkyl, 12) SO 2 R a and 13) (C=O)NR b 2 Independently selected from; said alkyl, cycloalkyl, aryl, heterocyclyl, alkenyl, and alkynyl may be substituted with one or more substituents selected from R z or R 7 and R 8 together with the nitrogen to which they are attached may form a monocyclic or bicyclic heterocycle having 5 to 7 members in each ring and further optionally, in addition to nitrogen, one or two additional heteroatoms selected from N, O, and S and may be substituted with one or more substituents selected from R z ; R z is 1) (C=O) r O s (C 1 -C 10 )alkyl, 2) O r (C 1 -C 3 )perfluoroalkyl, 3) (C 0 -C 6 )alkylene - S(O) m R a , 4) oxo, 5) OH, 6) halo, 7) CN, 8) (C=O) r O s (C 2 -C 10 )alkenyl, 9) (C=O) r O s (C 2 -C 10 )alkynyl, 10) (C=O) r O s (C 3 -C 6 )cycloalkyl, 11) (C=O) r Os (C 0 -C 6 ) alkylene-aryl, 12) (C=O) r O s (C 0 -C 6 ) alkylene - heterocyclyl, 13) (C=O) r O s (C 0 -C 6 ) alkylene - N(R b ) 2 , 14) C(O)R a , 15) (C 0 -C 6 ) alkylene - CO 2 R a , 16) C(O)H, 17) (C 0 -C 6 ) alkylene - CO 2 H, 18) C(O)N(R b ) 2 , 19) S(O) m R a , 20) S(O) 2 N(R b ) 2 , 21) NR c (C=O)O b R a , 22) O(C=O)O b C 1 -C 10 alkyl, 23) O(C=O)O b C 3 -C 8 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 R b , OH, (C 1 -C 6 ) alkoxy, ha logen, CO 2 H, CN, O(C=O)C 1 -C 6 alkyl, oxo, and N(R b ) 2may be substituted with up to 3 substituents selected from; R a is substituted or unsubstituted (C 1 -C 6 ) alkyl, substituted or un substituted (C 2 -C 6 ) alkenyl, substituted or unsubstituted (C 2 -C 6 ) alkynyl, substituted or unsubstituted (C 3 -C 6 ) cycloalkyl, substituted or unsubstituted aryl, (C 1 -C 6 ) perfluoroalkyl, 2,2,2-trifluoroethyl, or substituted or unsubstituted heterocyclyl; and, R b is H, (C 1 -C 6 ) alkyl, substituted or unsubstituted aryl, substituted or unsubstituted benzyl, substituted or unsubstituted heterocyclyl, (C 3 -C 6 ) cycloalkyl, (C=O)OC 1 -C 6 alkyl, (C=O)C 1 -C 6 alkyl or S(O) 2 R a ; R c is 1) H, 2) C 1 -C 10 alkyl, 3) aryl, 4) C 2 -C 10 alk enyl, 5) C 2 -C 10 alkynyl, 6) heterocyclyl, 7) C 3 -C 8 cycloalkyl, 8) C 1 -C 6 pe rfluoroalkyl, selected from, and the alkyl, cycloalkyl, aryl, heterocyclyl, alkenyl, and alkynyl are optionally substituted with one or more substituents selected from R z ; and may be substituted with one or more substituents selected from .

[0092] In certain aspects, 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. The amount of 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 aspect

[0093] the amount of 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 aspect, one or more T cells are contacted with an AKT inhibitor, for example, 3 - [1 - [[4 - (7 - phenyl - 3H - imidazo[4,5 - g]quinoxalin - 6 - yl)phenyl]methyl]piperidin - 4 - yl] - 1H - benzimidazol - 2 - one, and 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 yl)phenyl]methyl]piperidin - 4 - yl] - 1H - benzimidazol - 2 - one, and 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 a concentration of at least 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 aspect, one or more T cells are contacted with an AKT inhibitor, such as 3-[1-[[4-(7-phenyl-3H- imidazo[4,5-g]quinoxalin-6-yl)phenyl]methyl]piperidin-4-yl]-1H-benzo imidazol-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 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 this method. For example, AKT activity can be reduced or inhibited 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% by 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%. can be obtained.

[0095] Exogenous IL-7 and exogenous IL-15 Interleukin-7 (IL-7) is a cytokine that promotes lymphocyte homeostasis and is necessary 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 subsets of T cells including naive T cells and T cells. IL-7 signaling gives rise to various tyrosine kinases including the Janus kinase / signal transducer and activator of transcription (Jak / STAT) pathway, PI3K, and Src family tyrosine kinases. CM subsets of T cells including naive T cells and T cells. IL-7 signaling gives rise to various tyrosine kinases including the Janus kinase / signal transducer and activator of transcription (Jak / STAT) pathway, PI3K, and Src family tyrosine kinases. to occur.

[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 comprise contacting 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 IL-7, 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 aspect, the method can comprise contacting one or more T cells with from about 0.001 to about 500 ng / ml IL-7, from about 0.01 to about 100 ng / ml IL-7 , contact with 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, contact one or more T cells 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 involve contacting 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 n g / 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 IL-15, or at least about 1000 ng / ml IL-15 and contacting may include the step of contacting. In one aspect, 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 aspect, 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 and not with exogenous IL-15. In other embodiments, one or more T cells are contacted with exogenous IL-15 and 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 one embodiment , 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, one or more T cells are contacted with a lower concentration of exogenous IL-7 than exogenous IL-15. In one particular embodiment, one or more T cells are contacted with approximately 5 ng / ml of exogenous IL-7 and approximately 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 cell ​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 an individual who donates a lymphocyte sample that will be used to treat a different individual or cancer patient after a population of cells produced by the methods described herein has been generated (i.e., an allogeneic donor). A population of lymphocytes can be obtained from a donor by any suitable method used in the art. For example, a population of lymphocytes can be obtained by any suitable ex vivo method, venipuncture, or other blood collection method from which a sample of blood and / or lymphocytes can be obtained. In one aspect, a population of lymphocytes is obtained by apheresis. One or more T cells can be harvested from any tissue containing one or more T cells, including but not limited to a tumor. In some aspects, a tumor or a portion thereof is harvested from a subject and one or more T cells are isolated from the tumor tissue. Any T cell containing T cells suitable for T cell therapy can be used in the methods disclosed herein. For example, one or more cells useful in the present invention can be selected from the group consisting of tumor infiltrating lymphocytes (TILs), cytotoxic T cells, CAR T cells, modified TCR T cells, natural killer T cells, dendritic cells, and peripheral blood lymphocytes. In one particular aspect, the T cell is a tumor infiltrating leukocyte. In one aspect, one or more T cells express CD8 and are, for example, CD8 T cells. In other aspects, one or more T cells express CD4 and are, for example, CD4 T cells. After a population of cells produced by the methods described herein has been generated, it can be used to treat a different individual or cancer patient. A population of lymphocytes can be obtained from a donor by any suitable method used in the art. For example, a population of lymphocytes can be obtained by any suitable ex vivo method, venipuncture, or other blood collection method from which a sample of blood and / or lymphocytes can be obtained. In one aspect, a population of lymphocytes is obtained by apheresis. One or more T cells can be harvested from any tissue containing one or more T cells, including but not limited to a tumor. In some aspects, a tumor or a portion thereof is harvested from a subject and one or more T cells are isolated from the tumor tissue. Any T cell containing T cells suitable for T cell therapy can be used in the methods disclosed herein. For example, one or more cells useful in the present invention can be selected from the group consisting of tumor infiltrating lymphocytes (TILs), cytotoxic T cells, CAR T cells, modified TCR T cells, natural killer T cells, dendritic cells, and peripheral blood lymphocytes. + In one particular aspect, the T cell is a tumor infiltrating leukocyte. In one aspect, one or more T cells express CD8 and are, for example, CD8 T cells. In other aspects, one or more T cells express CD4 and are, for example, CD4 T cells. +

[0104] The methods described herein involve treating one or more donor-derived T cells with an AKT inhibitor. ​It can be used to delay or inhibit the maturation or differentiation of T cells in vitro by contacting with at least one of exogenous IL-7 and exogenous IL-15. The present The inventors have found that the treatment of 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 may express one or more genes that are markers of undifferentiated or immature T cells. One or more genes that are markers of undifferentiated or immature T cells may be selected from the group of CD8, CD45RA, CCR7, CD45RO, CD62L, CD28, CD95, IL-7Rα, CXCR4, TCF7, FOXO1, ID3, BCL6, and any combination thereof. For example, 1 contacting one or more T cells with an AKT inhibitor and IL-7 and / or IL-15 can increase the percentage of cells expressing one or more genes that are markers of undifferentiated or immature T cells selected from CD8, CD45RA, CCR7, and any combination thereof .

[0105] In another aspect, 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 aspect, a greater percentage of one or more T cells express CCR7 and CD45RO after contact compared to before contact with an AKT inhibitor and at least one of exogenous IL-7 and exogenous IL-15. In another aspect, one or more T cells are AKT-in hibitor and exogenous IL-7 and exogenous IL-15. contact with at least one of exogenous IL-7 and exogenous IL-15, after contact, a greater percentage of one or more T cells express CCR7 and CD45RO. In another aspect, one or more T cells are AKT Following contact with a splitter and exogenous IL-7 and / or exogenous IL-15, they express CCR7 and CD45RA. 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 the contact. In another embodiment, the T cells show 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 as compared to the 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 after contact with an AKT inhibitor and at least one of exogenous IL-7 and exogenous IL-15 compared to before the contact. In another embodiment, one or more T cells show increased expression of CD62L, CD28 or both following contact with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15 as compared to the 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, the T cells, following contact with an AKT inhibitor and exogenous IL-7, exogenous IL-15 or both, show increased expression of one or more of the following markers as compared to T cells that were not contacted with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15: CCR7, CD45RO, CD45RA, CD62L, CD28 or any combination thereof. ​​​Expression of CD95, interleukin-7 receptor alpha (IL-7Rα), CXCR4, TCF7, FOXO1, ID3, BCL6, CD62L, and CD45RA by T cells not contacted with native IL-15, compared to increased expression of IL-7 receptor alpha (IL-7Rα), CXCR4, TCF7, FOXO1, ID3, BCL6, CD62L, CD45RA, or any combination thereof is shown.

[0108] T cell therapy The present invention is a method for regulating, e.g., delaying or inhibiting, the maturation or differentiation of T cells in vitro for T cell therapy, comprising contacting one or more T cells derived 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 the 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 that includes administering the one or more T cells to the patient. For example, without limitation, the methods described herein can enhance the efficacy of adoptive T cell therapies that can be, for example, 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 therapies select autologous or allogeneic T cells that can recognize and bind to tumor cells, enrich them in vitro, and

[0109] For example, without limitation, the methods described herein can enhance the efficacy of adoptive T cell therapies that can be, for example, 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 therapies select autologous or allogeneic T cells that can recognize and bind to tumor cells, enrich them in vitro, and thereby. select autologous or allogeneic T cells that can recognize and bind to tumor cells, enrich them in vitro, and broadly includes any method of administration to a patient. TIL immunotherapy is a type of adoptive T cell therapy, where lymphocytes that can infiltrate tumor tissue are isolated, enriched in vitro, and administered to the patient. TIL cells can be either autologous or allogeneic. Autologous Cell therapy is an adoptive T cell therapy that involves isolating T cells from a patient that can target tumor cells, enriching the T cells in vitro, and administering the T cells back to the same patient. Allogeneic T cell transplantation can involve the transplantation of natural T cells expanded ex vivo or genetically engineered T cells. As described in more detail above, modified autologous cell therapy is an adoptive T cell therapy in which a patient's own lymphocytes are isolated, genetically modified to express a molecule that targets the tumor, expanded in vitro, and administered back to the patient. Non-T cell transplantation can involve 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 modified autologous cell therapy (eACT™). According to this embodiment, the method can include the 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 (“modified CAR T cells”) or a T cell receptor (“modified TCR T cells”). In one particular embodiment, modified CAR T cells or modified 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 modified 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, such as on the surface of a tumor cell. In some embodiments, the cell surface receptor is a chimeric antigen receptor or a T cell receptor is.

[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 an antigen-binding molecule thereof. 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 can further include a hinge region. The hinge region can be derived from the hinge region of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM, CD28, or CD8 alpha. One particular embodiment, the hinge region is derived from the hinge region of IgG4.

[0114] The chimeric antigen receptor can also include a transmembrane domain. The transmembrane domain can be the transmembrane domain of any transmembrane molecule that is a co-receptor on an immune cell, or the transmembrane domain of a member of the immunoglobulin superfamily. In one embodiment, the transmembrane domain is derived from the transmembrane domain of CD28, CD28T, CD8 alpha, CD4, or CD19. One particular embodiment, the transmembrane domain includes a domain derived from the CD28 transmembrane domain. In another embodiment, the transmembrane domain includes a domain derived from the CD28T transmembrane domain contains.

[0115] The chimeric antigen receptor can further include one or more co-stimulatory signaling regions. For example For example, the co-stimulatory signaling region can be the signaling region of CD28, CD28T, OX-40, 41BB, CD27, inducible T cell co-stimulator (ICOS), CD3 gamma, CD3 delta, CD3 epsilon, CD247, Ig alpha (CD79a), or the Fc gamma receptor. In one particular embodiment, the co-stimulatory signaling region is the CD28 signaling region. In another embodiment, the co-stimulatory signaling region is the CD28T signaling region. In one embodiment, the chimeric antigen receptor further comprises a CD3 zeta signaling domain. The chimeric antigen receptor can be engineered to target a specific tumor antigen. 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 / variant), 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)

[0116]

[0117] , CD44v7 / 8 (Cluster of Differentiation 44, Exon 7 / 8), CAMEL (CTL-recognized antigen on melanoma), CAP-1 (Cancer-embryonic 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 papillomavirus), HSP70-2M (Heat shock protein 70 - 2 mutant) ), HST-2 (Human ring finger 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 lipoprotein receptor / GDP-L-fucose:b-D-galactoside 2-a-L-fucosyltransferase), LeY (Lewis Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (Melanoma antigen) ​​Original), MAGE-A1 (Melanoma-associated antigen 1), MAGE-A3, MAGE-A6, Mesothelin, Mouse CMV-infected cells, MART-1 / Melan-A (Melanoma antigen recognized by T cells-1 / Melanoma antigen A), MC1R (Melanocortin 1 receptor), Myosin / m (Myosin variant), MUC1 (Mucin 1), MUM-1, -2, -3( Melanoma ubiquitous variant 1, 2, 3), NA88-A (NA cDNA clone of patient M88), NKG2D (Natural killer group 2, member D) ligand, NY-BR-1 (New York breast differentiation antigen 1), NY-ESO-1 (New York esophageal squamous cell carcinoma-1), Tumor fetal antigen (h5T4), P15 (Protein 15), p190 minor bcr-abl (190KD bcr-abl protein), Pml / RARa (Promyelocytic leukemia / Retinoic acid receptor a), PRAME (Antigen preferentially expressed in melanoma), PSA (Prostate-specific antigen), PS CA (Prostate stem cell antigen), PSMA (Prostate-specific membrane antigen), RAGE (Renal antigen), RU1 or RU2 (Renal ubiquitous 1 or 2), SAGE (Sarcoma antigen), SART-1 or SART-3 (Tumor rejection squamous 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 (Translocated Ets-family leukemia / Acute myeloid leukemia 1), TPI / m (Triose phosphate isomerase variant), 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 is selected. In one particular embodiment, the tumor antigen is CD19.

[0118] In another aspect, the T cell therapy includes the step of administering to a patient modified T cells that express a T cell receptor (the "modified TCR T cells" "). The T cell receptor (TCR) may include a binding molecule for a tumor antigen. In some aspects, 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, tumor fetal antigen, P15, p190 minor 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. In one aspect, the TCR includes a binding molecule for a viral oncogene. In one particular

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

[0120] In yet another aspect, the TCR is a binding molecule for a testicular, placental or fetal tumor antigen and includes. In one particular aspect, 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 aspect, the TCR includes a binding molecule for a lineage-specific antigen. In one particular aspect, the lineage-specific antigen is melanoma antigen-1 (MART-1), gp100, prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), prostate stem cell antigen (PSCA), recognized by T cells, and is selected from the group consisting of any combination thereof.

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

[0123] In one aspect, the antigenic portion may also include Epstein-Barr virus (EBV) antigens (e.g g., EBNA-1, EBNA-2, EBNA-3, LMP-1, LMP-2), hepatitis A virus antigens (e.g., VP1, VP2, VP3), hepatitis B virus antigens (e.g., HBsAg, HBcAg, HBeAg), hepatitis C virus antigens ( e.g., envelope glycoproteins E1 and E2), herpes simplex virus type 1, type 2, if type 8 (HSV1, HSV2, or HSV8) viral antigens (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM, UL20, UL32, US43, UL45, UL49A), cytomegalovirus (CMV) 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 virus antigens (e.g., hemagglutinin (HA) or neuraminidase (NA)), measles or mumps virus antigens, h uman 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, among others, but not limited thereto. In such an aspect, the cell surface receptor can be any TCR or any CAR that recognizes any of the aforementioned viral antigens on a virus-infected target cell. In other aspects, the antigenic moiety is associated with cells having an immune or inflammatory dysfunction. Such antigenic moieties can include, but are not limited to, myelin basic protein (MBP), myelin prote olipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), carcinoembryonic antigen (CEA), proinsulin, glutamic acid decarboxylase (GAD65, GAD67), heat shock

[0124] protein (HSP), or any other tissue-specific antigen involved in or associated with a pathogenic autoimmune process.

[0125] The methods disclosed herein are T cell therapies that include the transfer of one or more T cells to a patient. may be accompanied. T cells can be administered in a therapeutically effective amount. For example, a therapeutically effective amount of T cells, such as modified CAR+ T cells or modified TCR+ T cells, is 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 cells, or at least about 10 10 cells. In another aspect, a therapeutically effective amount of T cells, such as modified CAR+ T cells or modified TCR+ T cells, is about 10 4 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells, or about 10 8 cells. In one particular aspect, a therapeutically effective amount of T cells, such as modified CAR+ T cells or modified TCR+ T cells, is about 2 X 10 6 cells / kg, about 3 X 10 6 cells / kg, about 4 X 10 6 cells / kg, about 5 X 10 6 cells / kg, about 6 X 10 6 cells / kg, about 7 X 10 6 cells / kg, about 8 X 10 6 cells / kg, about 9 X 10 6 cells / kg, about 1 X 10 7 cells / kg, about 2 X 10 7 cells / kg, about 3 X 10 7 cells / kg, about 4 X 10 7 cells / kg, about 5 X 10 7 cells / kg, about 6 X 10 7 cells / kg, about 7 X 10 7 cells / kg, about 8 X 10 7 cells / kg, or about 9 X 10 7 cells / kg.

[0126] In some aspects, the patient is pre-conditioned prior to administration of the T cell therapy The patient can be pre-conditioned 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, the pre-conditioning is performed prior to T cell therapy to reduce the number of endogenous lymphocytes, remove cytokine sinks, increase the serum levels of one or more homeostatic cytokines or inflammatory factors, enhance the effector function of the T cells administered after conditioning, enhance antigen-presenting cell activation and / or availability, or any combination thereof. In one embodiment, the pre-conditioning includes increasing the serum levels of one or more cytokines in the subject. In one aspect, the pre-conditioning can include any treatment that reduces the number of endogenous lymphocytes, removes cytokine sinks, increases the serum levels of one or more homeostatic cytokines or inflammatory factors, enhances the effector function of the T cells administered after conditioning, enhances antigen-presenting cell activation and / or availability, or any combination thereof.

[0127] Cancer treatment The methods of the 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 the patient, prevent tumor recurrence, prevent tumor metastasis, induce remission in the patient, or any combination thereof. In certain embodiments, the method induces a complete response. In other embodiments, the method induces a partial response.

[0128] In one aspect, the invention relates to a method of treating a tumor in a subject in need of T cell therapy, the method 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. In another aspect, the invention relates to a method of reducing or decreasing the size of a tumor or inhibiting the growth of a tumor in a subject in need of T cell therapy, the method 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. In certain embodiments, the one or more T cells have not been contacted with exogenous IL-2. In one aspect, the invention relates to a method of reducing or decreasing the size of a tumor or inhibiting the growth of a tumor in a subject in need of T cell therapy, the method 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. In certain embodiments, the one or more T cells have not been contacted with exogenous IL-2.

[0129] The cancers that can be treated include tumors that are not forming blood vessels, not yet substantially forming blood vessels, or are forming blood vessels. The cancer can also include solid or non-solid tumors. In certain embodiments, the cancer can be selected from tumors derived from the following: acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenocortical carcinoma, AIDS-related cancers, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, central nervous system, B-cell leukemia, lymphoma or other B-cell malignancies, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brainstem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorders, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, germinoma, central nervous system, endometrial cancer, epithelioblastoma, epithelioma, esophageal cancer, neuroblastoma, Ewing sarcoma family of 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, heart cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (pancreatic endocrine), Kaposi 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 NUT gene, mouth cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell tumor, fungating polyposis, ​Myelodysplastic syndromes, myelodysplastic / myeloproliferative neoplasms, chronic myeloid leukemia (CML), acute myeloid leukemia (AML), myeloma, multiple, myeloproliferative disorders, nasal and paranasal cavity cancers, nasopharyngeal cancer, neuroblast oma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, paranasal and nasal cavity cancers, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate differentiated pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell tumor / multiple myeloma, pleuropulmonary blastoma, breast cancer during pregnancy, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, urothelial carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma of the neck, gastric cancer, supratentorial primitive neuroectodermal tumor, T cell lymphoma, skin, testicular cancer, laryngeal cancer, thymus tumor and thymic carcinoma, thyroid cancer, urothelial carcinoma of the renal pelvis and ureter, gestational trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, Wilms tumor.

[0130] In one aspect, the method can be used to treat a tumor, where the tumor is a lymphoma or leukemia. Lymphomas and leukemias are blood cancers that specifically affect lymphocytes. All white blood cells in the blood are derived 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 arising from myeloid progenitor cells include T lymphocytes (T cells), B lymphocytes (B cells), natural killer cells, and plasma cells. White blood cells arising from lymphoid progenitor cells include megakaryocytes, mast cells, basophils, neutrophils, eosinophils, monocytes, and macrophages. Lymphomas and leukemias can affect one or more of these cell types in a patient.

[0131] Generally, 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 derived 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, approximately 69,740 new cases of NHL and more than 19,000 deaths related to this disease were estimated to have occurred. Non-Hodgkin

[0132] lymphoma is the most common hematologic malignancy and the seventh most common new cancer site between men and women, accounting for 4% of all new cancer cases and 3% of cancer-related deaths.

[0133] Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of NHL, accounting for approximately 30% of NHL cases. Each year, there are approximately 22,000 new diagnoses of DLBCL in the United States. It is classified as an aggressive lymphoma, and the majority of patients are treated with conventional chemotherapy (NCCN guidelines NHL 2014). 。The standard treatments for second-line therapy for eligible patients with autologous stem cell transplantation (ASCT) include rituximab and combination chemotherapy, such as R-ICE (rituximab, ifosfamide, carboplatin, and etoposide) and R-DHAP (rituximab, dexamethasone, cytarabine, and cisplatin), which each have an objective response rate of approximately 63% and a complete response rate of approximately 26% (Gisselbrecht 2010). Patients who respond to second-line therapy and are considered suitable for transplantation receive high-dose chemotherapy and consolidation therapy with ASCT, which is curative in approximately half of the transplanted patients (Gisselbrecht 2010). Patients who fail ASCT have a very poor prognosis and have no curative options.

[0134] Primary mediastinal large B-cell lymphoma (PMBCL) has different clinical, pathological , and molecular characteristics compared to 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 a young adult population in their 30s and is slightly more common in women. Gene expression profiling suggests that the deregulated pathways in PMBCL overlap with those in Hodgkin lymphoma. The primary therapy for PMBCL generally includes anthracycline-containing regimens with or without regional radiotherapy and rituximab, such as etoposide, doxorubicin, and cyclophosphamide with adjusted infusion doses, as well as vincristine, prednisone, and rituximab (DA-EPOCH-R).

[0135] Follicular lymphoma (FL), a B-cell lymphoma, is the most common indolent (slow-growing) ​a slow form that accounts for approximately 20% to 30% of all NHL. Some patients with FL histologically transform into DLBCL (TFL), which is more aggressive and has a poor outcome. Histological transformation to DLBCL occurs at an approximate annual rate of 3% over 15 years, and the risk of transformation continues to decline in subsequent years. The biological mechanism of histological transformation is unknown. Primary treatment of TFL is influenced by previous therapies for follicular lymphoma but generally includes anthracycline-containing regimens including rituximab to eliminate the aggressive component of this disease. The biological mechanism of histological transformation is unknown. Primary treatment of TFL is influenced by previous therapies for follicular lymphoma but generally includes anthracycline-containing regimens including rituximab to eliminate the aggressive component of this disease. Primary treatment of TFL is influenced by previous therapies for follicular lymphoma but generally includes anthracycline-containing regimens including rituximab to eliminate the aggressive component of this disease.

[0136] Treatment options for relapsed / refractory PMBCL and TFL are similar to those in DLBCL. Given the low incidence of these diseases, large-scale prospective randomized studies have not been conducted in these patient populations. Patients with chemotherapy-refractory disease have a prognosis similar to or worse than that in refractory DLBCL. Patients with chemotherapy-refractory disease have a prognosis similar to or worse than that in 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 with novel therapies is needed in these populations.

[0138] Accordingly, in some aspects, the method can be used to treat lymphoma or leukemia, where the lymphoma or leukemia is a B-cell malignancy. Examples of B-cell malignancies include non-Hodgkin lymphoma (NHL), small lymphocytic lymphoma (SLL / CLL), mantle cell lymphoma (MCL), FL, marginal zone lymphoma (MZL), extranodal (MALT lymphoma), nodal (monocytoid B-cell lymphoma), splenic, diffuse large cell lymphoma, B-cell chronic lymphocytic leukemia / lymphoma, B - Kit lymphoma and lymphoblastic lymphoma are included, but not limited to these. In some embodiments, the lymphoma or leukemia is B-cell chronic lymphocytic leukemia / small cell lymphoma, B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma (e.g., Waldenström macroglobulinemia), splenic marginal zone lymphoma, hairy cell leukemia, plasma tumor (e.g., plasmacytoma (i.e., multiple myeloma), or plasmacytosis), extranodal marginal zone B-cell lymphoma (e.g., 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, hepatosplenic T-cell lymphoma, blastic NK cell lymphoma, mycosis fungoides / Sézary 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 lymphoma. In some embodiments, the cancer is refractory to one or more prior treatments and / or is a cancer that has recurred after one or more prior treatments.

[0139] In certain 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 one or more of chemotherapy, radiotherapy, immunotherapy (including treatment with T-cell therapy and / or antibodies or antibody-drug conjugates), autologous stem cell transplantation, or any combination thereof, or is a cancer that has recurred following one or more of these 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 prior to administration to the subject to remove the AKT inhibitor, exogenous IL-7, and / or exogenous IL-15. In some embodiments, the one or more T cells include engineered CAR cells or engineered TCR cells. In one embodiment, the engineered CAR cells or engineered T cells treat tumors in the subject.

[0142] Kit A 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, e.g., a pharmaceutical kit, is further included within the scope of the present invention. The kit typically includes a label and instructions for use that indicate the intended use of the contents of the kit. The term "label" includes any descriptive or recorded material provided on or with the kit or otherwise associated with the kit.

[0143] In some aspects, 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) an AKT inhibitor, (ii) exogenous IL-7, and (iii) instructions for contacting one or more T cells intended for use in T cell therapy with the AKT inhibitor and exogenous IL-7.

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

[0145] In other aspects, 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: one or more T cells for T cell therapy for a subject in need thereof, the kit comprising: (i) an AKT inhibitor, (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 the 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 limitations. The contents of all references cited throughout this application are hereby expressly incorporated herein by reference.

[0147] The following examples are intended to illustrate various aspects of the invention. Accordingly, the specific aspects discussed are not to be construed as limitations on the scope of the invention. For example, the following examples relate to T cells transduced with an anti-CD19 chimeric antigen receptor (CAR), but one of ordinary skill 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 such equivalent aspects are understood to be included herein. Further, all references cited in this disclosure are hereby incorporated by reference in their entirety as if fully set forth herein.

[0148] Aspect E1. A method for delaying or inhibiting in vitro maturation or differentiation of T cells for T cell therapy, comprising contacting one or more T cells derived 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 have improved T cell function compared to the T cell function of T cells cultured in the absence of the AKT inhibitor. Method.

[0149] E2. A method for improving T cell function in vitro for T cell therapy, comprising contacting one or more T cells derived 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. therapy with an AKT inhibitor and at least one of exogenous interleukin-7 (IL-7) and exogenous interleukin-15 (IL-15). 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. The improved T cell function is (i) Increased T cell proliferation; (ii) Increased cytokine production; (iii) Increased cytolytic activity; and (iv) Any combination of (i) to (iii) A method of E1 or E2 selected from the group consisting of.

[0151] A method for increasing T cell proliferation in vitro before E4 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] A method for increasing T cell cytokine production in vitro before E5 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 cytokine production of T cells cultured in the absence of the AKT inhibitor.

[0153] A 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 tumor necrosis factor alpha (TNFa) production, and (iii) increased production of both IFNg and TNFa.

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

[0155] A method for delaying or inhibiting the maturation or differentiation of T cells in vitro for adoptive T cell therapy, the method comprising contacting one or more T cells derived from a subject in need of adoptive 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. The method according to any one of E1 to E8, wherein the contacting step comprises culturing one or more T cells in a medium comprising (i) an AKT inhibitor and (ii) exogenous IL-7 and / or exogenous IL-15. - A method.

[0157] E10. The method according to any one of E1 to E9, wherein one or more T cells are not contacted with exogenous interleukin-2 (IL-2).

[0158] E11. The method according to any one of E1 to E10, wherein the T cells are washed and the AKT inhibitor, exogenous IL-7, and / or exogenous IL-15 are removed. - A method.

[0159] E12. The AKT inhibitor is A6730, B2311, 124018, GSK2110183 (afuresertib), pe Rhosine (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, elsylphosphocholine, 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 # 1191951-57-1), 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-041164 (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, a method of any of E1 to E11 selected from the group consisting of.

[0160] E13. The AKT inhibitor is (i) 3-[1-[[4-(7-phenyl-3H-imidazo[4,5-g]quinoxali n-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]me Tanimine; or (iii) a compound selected from the group consisting of 1-{1-[4-(3-phenylbenzo[g]quinoxalin-2-yl)benzyl]piperidin-4-yl}-1,3-dihydro-2H-benzimidazol-2-one A method according to any one of E1 to E12, comprising a compound selected therefrom.

[0161] E14. The AKT inhibitor is (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 Tanimine; or (iii) a compound selected from the group consisting of 1-{1-[4-(3-phenylbenzo[g]quinoxalin-2-yl)benzyl]piperidin-4-yl}-1,3-dihydro-2H-benzimidazol-2-one Tanimine; or (iii) a compound selected from the group consisting of 1-{1-[4-(3-phenylbenzo[g]quinoxalin-2-yl)benzyl]piperidin-4-yl}-1,3-dihydro-2H-benzimidazol-2-one A method according to any one of E1 to E13, wherein the compound is selected therefrom.

[0162] E15. 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, and the method is according to any one of E1 to E14.

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

[0164] The E17. AKT inhibitor is in an amount selected from the group consisting of 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 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 The method of E15, which is in an amount so selected.

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

[0166] The method according to any one of E1 to E18, wherein the exogenous IL-7 is in an amount of about 0.001 to about 500 ng / ml IL-7 .

[0167] The method according to any one of E1 to E18, wherein the exogenous IL-7 is in an amount of about 1 to about 10 ng / ml IL-7.

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

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

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

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

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

[0173] E26. Any method of E25, wherein 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 method of E1 to E26, wherein one or more T cells are collected from a subject in need of anti-cancer treatment.

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

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

[0177] E30. Any method of E1 to E29, wherein the T cells are activated.

[0178] E31. Any method of E30, wherein the activation of the T cells is performed in a closed system.

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

[0180] A method according to any one of E1 to E32 for proliferating E33. T cells.

[0181] A method according to E32 for proliferating E34. T cells in vitro.

[0182] A method according to E32 for proliferating E35. T cells in vivo.

[0183] E36. Contacting one or more T cells with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15 prolongs the persistence of the T cells in vivo, according to any one of E1 to E35 of the methods.

[0184] E37. Contacting one or more T cells with an AKT inhibitor and at least one of exogenous IL-7 and exogenous IL-15, followed by the resulting T cells expressing one or more genes that are markers of undifferentiated or immature T cells, according to any one of E1 to E36 or immature T cells, according to any one of E1 to E36 of the methods. of the methods.

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

[0186] E39. The method according to any one of E1 to E38, further comprising the step of transducing the T cells with a retrovirus. of the methods.

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

[0188] E41. The method of E40, wherein the cell surface receptor can bind 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). or the method of E42.

[0191] E44. The cell surface receptor is 707-AP (707 alanine proline), AFP (alpha (α)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen; β-catenin / m, β-catenin / variant), BCMA (B cell maturation antigen), Bcr-abl (breakpoint cluster region-Abelson), CAIX (carbonic anhydrase IX), CD19 (cluster of differentiation 19), CD20 (cluster of differentiation class 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 (Cancer Embryonic 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 (Helicase Antigen), HER-2 / neu (Human Epidermal Receptor-2 / Neurological; also known as EGFR2), HLA-A (Human Leukocyte Antigen-A), HPV (Human Papillomavirus), HSP70-2M (Heat Shock Protein 70 - 2 mutant type), HST-2 (Human Stamp 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 Lipoprotein Receptor / GDP-L-Fucose:b-D-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 (Recognized by T cells Recognized melanoma antigen-1 / melanoma antigen A), MC1R (melanocortin 1 receptor), Myosin / m (myosin variant), MUC1 (mucin 1), MUM-1, -2, -3 (melanoma ubiquitous variant 1, 2, 3), NA88-A (NA cDNA clone of patient M88), NKG2D (natural killer group 2, member D) ligand, NY-BR-1 (New York breast differentiation antigen 1), NY-ESO-1 (New York esophageal squamous cell carcinoma-1), tumor fetal antigen (h5T4), P15 (protein 15), p190 minor bcr-abl (190KD bcr-abl protein), Pml / RARa (promyelocytic leukemia / retinoic acid receptor a), PRAME (antigen preferentially expressed 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 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 ( translocated Ets-family leukemia / acute myeloid leukemia 1), TPI / m (triose phosphate isomerase variant), 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, a method of any of E41 to E43 that can bind to an antigen selected from the group consisting of .

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

[0193] A subject in need of T cell therapy, comprising the step of 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 A method for treating a tumor in the subject.

[0194] A subject in need of T cell therapy, comprising the step of 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 A method for reducing or decreasing the size of a tumor in the subject or inhibiting the growth of the tumor.

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

[0196] E49. The method according to any one of E46 to 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. The method according to any one of E46 to 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. The method according to any one of E46 to E48, wherein the T cells show 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 as compared to the expression of CCR7, CD45RO, CD45RA by T cells that have not been contacted with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15 The method of.

[0199] E52. Any of the methods of 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. Following contact of T cells with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15, expression of CD62L, CD28 or both is reduced 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. Any of the methods E46 to E52, showing increased expression.

[0201] E54. Following contact of T cells with an AKT inhibitor and exogenous IL-7, exogenous IL-15, or both, T cells exhibit 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 were not contacted with an AKT inhibitor and exogenous IL-7 and / or exogenous IL-15. One of the following methods, E46 to E53, indicating the current status.

[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 a 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, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) Type 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 cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia (ALL) (including non-T cell ALL), chronic lymphocytic leukemia (CLL), solid tumors in childhood, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal pelvic cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brainstem glioma, pituitary adenoma, Kaposi sarcoma, epidermoid carcinoma, squamous cell carcinoma, T Cell lymphoma, environmentally induced cancers including those induced by asbestos, other B cell malignancies Tumors, and methods of E57 selected from any combination thereof.

[0206] E59. A method of any of E1 to E58, wherein the T cell therapy comprises a modified CAR cell therapy or a modified TCR cell therapy. Of these methods.

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

Example

[0208] Example 1 Donor T cells were cultured for 10 days at equal plate culture concentrations in the presence of IL-2, IL-7, IL-15, and / or an AKT inhibitor. The T cell phenotype of the cultured T cells was determined by comparing the CD4 of T cells cultured for 7 and 14 days in IL-2 alone (i) compared to IL-7 and IL-15, and (ii) cultured in IL-7 and IL-15 compared to IL-7, IL-15, and an AKT inhibitor. + T Cells and CD8 + T cells were determined (Figures 1A - 1F). When cells were grown in the presence of IL - 7 and IL - 15 a trend towards more naive T cells was observed. In particular, a significantly higher (p = 0.03, n = 6) percentage of naive and Tcm cells CD4 + was observed in IL - 7 / IL - 15 - treated cells compared to IL - 2 - treated cells (Figure 1A), but no difference was observed in the percentage of more mature effector T cells (Figure 1B). This effect was not maintained after long - term culture in the CD4 + compartment (data not shown). A similar effect was observed in the CD8 + compartment, with a significantly higher (p = 0.03, n = 6) percentage of naive and Tcm cells (Figure 1C) and a significantly lower (p = 0.03, n = 6) percentage of effector T cells (Figure 1D) in IL - 7 / IL - 15 - treated cell cultures compared to IL - 2 - treated cell cultures. However, unlike in the CD4 + compartment, this effect was observed after long - term culture in the CD8 + compartment (Figure 1E, p = 0.03, n = 6; and Figure 1F, p = 0.03, n = 6).

[0209] CD4 + and CD8 + In both the CD4 and CD8 compartments, the addition of an AKT inhibitor further increased the trend towards more immature T cells. On day 7, a slightly higher percentage of naive and Tcm CD4 + cells was observed in IL - 7 / IL - 15 / AKTi - treated cells compared to IL - 7 / IL - 15 - treated cells (Figure 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 (Figure 2B). No significant difference was observed after 14 days of culture ​ Not observed in cultured cells (data not shown). However, CD8 + A significant difference was observed on day 7 in the compartment. In particular, on day 7, the percentages of naive and Tcm CD4 + cells were significantly higher in IL-7 / IL-15 / AKTi-treated cells compared to IL-7 / IL-15-treated cells (p = 0.03, n = 6) (Figure 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) (Figure 2D). However, this effect was not observed on day 14 (data not shown). 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 class I TCR two days after stimulation in OriGen PERMALIFE™ PL30 bags. The transduced

[0210] 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. The T cells were then analyzed for CD3 expression and positive soluble MHC-tetramer staining (Tet ) as an indicator of successful transduction. No significant differences in transduction efficiency were observed among the culture conditions ( Figure 3A), and no significant differences in tetramer mean fluorescence intensity (MFI) were observed across the culture conditions (Figure 3B). + ) was analyzed. No large differences in transduction efficiency were observed among the culture conditions ( Figure 3A), and no significant differences in tetramer mean fluorescence intensity (MFI) were observed across the culture conditions (Figure 3B).

[0211] Example 2 The effect of the AKTi inhibitor on cell proliferation was examined under various conditions. First, the effect of AKTi culture conditions on various sources of donor cells was evaluated as follows. Four healthy donors Apheresis products from [donors] were processed using density gradient centrifugation to obtain peripheral blood mononuclear cells (PBMCs) (Figs. 4A-4D). Cells from 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) (Figs. 4A-4D). When cell proliferation was observed for each donor cell line under each culture condition, AKTi had no negative effect on cell proliferation (Figs. 4A-4D). Next, (HPV-E6)-transduced PBMCs expressing class I TCR were evaluated. Apheresis products from three healthy donors were again processed using density gradient centrifugation to obtain PBMCs, which were counted and stimulated with OKT3. The 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) (Figs. 5A-5C). On day 2, the cells were transduced with class I TCR (HPV-E6). When cell proliferation was observed for each donor cell line under each culture condition, AKTi had no negative effect on cell proliferation (Figs. 5A-5C). Next, the effect of AKTi culture conditions on CD4 / CD8

[0212] T cells was evaluated. Apheresis products from three healthy donors were again processed using density gradient centrifugation to obtain PBMCs. The PBMCs were then cultured with anti-CD4 and anti-CD8 Ab beads, and CD4 and CD8

[0213] cells were selected using the CLINIMACS® system (Miltenyi Biotec). CD4 + / CD8 + T cells from three donors + and CD8 + were selected using the CLINIMACS® system (Miltenyi Biotec). CD4 + and CD8+ Cells were counted and stimulated using OKT3 and anti-CD28 Ab. Next, the cells were 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) (Figs. 6A - 6C). On day 2, the cells were transduced with class II TCR (MAGE-A3). When cell proliferation was observed for each donor cell line under each culture condition, AKTi had no negative effect on cell proliferation (Figs. 6A - 6C).

[0214] Next, CD4+ and CD+ T cells were evaluated separately. Apheresis products from three healthy donors were processed again using high-density centrifugation to obtain PBMC. Next, PBMC were cultured with either anti-CD4 beads (Figs. 7A - 7C) or anti-CD8 beads (Figs. 8A - 8C), and the target cells were selected using the CLINIMACS® system (Miltenyi Biotec). Next, the cells from three donors were counted and stimulated using OKT3 and anti-CD28 Ab. Next, the cells were cultured in the presence of IL-2 (circles); IL-2 and AKTi (squares); IL-7 and IL-15 (triangles); or also IL-7, IL-15, and AKTi (inverted triangles). On day 2, the cells were transduced with class II TCR (MAGE-A3). Cell proliferation was observed for CD4 + (Figs. 7A - 7C) and CD8 + cells (Figs. 8A - 8C) from each donor cell line under each culture condition, and AKTi had no ma inus effect on cell proliferation.

[0215] Next, the effect of culture conditions during large-scale production culture was evaluated. Apheresis products from four healthy donors were processed again using high-density centrifugation to obtain peripheral blood mononuclear cells PBMC (Figs. 9A - 9D). Next, PBMC were cultured with anti-CD4 and anti-CD8 beads, and CD4 ​+ / CD8 + Thin The cells were selected using the CLINIMACS® system. + / CD8 + Cells were counted and stimulated using OKT3 and anti-CD28. Cells were then cultured at large scale in the XURI™ Cell Expansion System (GE Healthcare Life Sciences) 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) for 8 days. On the second day of culture, cells were transduced with class II TCR (MAGE-A3). Cell proliferation was observed 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. 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 cultured in T-75 tissue culture flasks. Class I TCR (HPV-E6) in a PERMALIFE™ bag (Figure 10) or in an OriGen PERMALIFE™ bag T cells were transduced with class II TCR (MAGE-A3) (Figures 11A-11F) on day 2 after stimulation. Transduction efficiency was measured by anti-mTCRb antibody staining on day 10 (Figures 10 and 11A-11F). Anti-mTCRb staining MFI was significantly higher in cells cultured in the presence of IL-7, IL-15, and AKTi compared to IL-7 and IL-15 alone. Although showing slightly greater overall intensity for transduced cells (FIGS. 11C and 11F), AKTi had no negative effect on transduction efficiency (FIGS. 10, 11A, 11B, 11D, and 11E).

[0217] Similar results were observed for T cells cultured at manufacturing scale (Figure 12). Cryopreserved 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 . On day 2 post-stimulation, cells were transduced with class II TCR (MAGE-A3). Cells were then expanded in an XURI™ Bioreactor Cell Expansion System. T cell transduction efficiency was determined on day 8 by anti-mTCRb (mC TCR PE) antibody staining. The percent of CD3 cells expressing the transduced TCR for each culture condition for each run + is shown (Figure 12). Under large scale manufacturing conditions, cells grown in the presence of IL-7, IL-15, and AKTi have a 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 from three donors were transduced with 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 the early stages of differentiation. The percent of cells staining positive for CD62L expression was determined for cells from each culture condition for each of donors 1, 2, and 3 (Figure 13A, 13B, and 13C, respectively). The mean fluorescence intensity (MFI) indicated that cells cultured in the presence of AKTi had a greater level of CD62L on the surface of positive cells compared to cells cultured in the absence of AKTi (Figure 13D-13E).

[0219] Example 5​ To determine the effect of AKTi on T cell function, cytokine production and T cell proliferation were evaluated 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, the T cells were transduced with class II TCR (MAGE-A3) and then expanded in an XURI™ Bioreactor Cell Expansion System in the presence of IL-7 and IL-15, or IL-7, IL-15, and AKTi. The T cells were stimulated with PMA + ionomycin + Brefaldin A + monensin for 5.5 hours. Intracellular flow cytometry showed increased T cell activity for cells cultured in the presence of AKTi, as demonstrated by increased production of the cytokines IFNg (Figure 14A) and TNFa (Figure 14B). 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 co-cultured overnight with positive (H1299, HT1197, and HT1367) as well as 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 have greater potency in response to stimulation than cells cultured in the absence of AKTi. Similar results were observed for small-scale cultures of donor T cells. Donor 1, Donor 2, and

[0220]

[0221] ​​​​​​Stimulate cells from donor 3 and culture them in the presence of IL-2; IL-2 and AKTi; IL-7 and IL-15; or IL-7, IL-15, and AKTi, and then, as described above, transduce class I TCR on the second day after stimulation. Co-culture the T cells overnight with a tumor cell line (Caski cells; Figure 16A) or T2 cells loaded with titrated amounts of TCR-specific peptide (Figure 16B - 16D). As observed in the large-scale manufacturing experiments described above, cells cultured in the presence of AKTi produced higher levels of IFNγ than cells cultured in the absence of AKTi (Figure 16A - 16D). Titration of the TCR-specific peptide showed that, at almost all levels, AKTi culture conditions induced greater IFNγ production.

[0222] Following culture in the presence of AKTi, it was also found that T cell proliferation increased. Transduce class II TCR on T cells from donor 1, donor 2, and donor 3 on the second day after stimulation as described above. Stain the T cells with CFSE and co-culture them with a tumor cell line (positive control) for 4 days. Increased T cell proliferation was observed in cells cultured in the presence of AKTi (Figure 17B and 17D) compared to cells proliferated without AKTi (Figure 17A and 17C). Figures 17A - 17D show representative data from donor 3, where a greater percentage of cells were characterized as being in late (L) or mid (M) proliferation for cells cultured in IL-2 and AKTi (Figure 17B) and in IL-7, IL-15, and AKTi (Figure 17D) compared to cells cultured in the absence of AKTi (Figure 17A and 17C). Increased T cell proliferation was also observed under large-scale manufacturing culture conditions. Two large-scale manufacturing runs (21

[0223] Increased T cell proliferation was also observed under large-scale manufacturing culture conditions. Two large-scale manufacturing runs (21 T cells from (0) and 22) were transduced with class II TCR on day 2 after 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 each of Runs 21A / 21B (Figure 18A) and 22A / 22B (Figure 18B).

[0224] Example 6 To determine the effect of AKTi on the cytolytic activity of T cells, target cells expressing luciferase were co-cultured 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 over a period ranging from 16 to 96 hours. The T cells were then co-cultured with target cells expressing luciferase. Target cell viability was measured by luciferase intensity, and a decrease in luciferase intensity indicates T cell recognition and target-specific killing. Thus, a reduction in luciferase level 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

**Claim 1** A method for preparing T cells, comprising contacting T cells with an AKT inhibitor, exogenous interleukin-7 (IL-7), and exogenous interleukin-15 (IL-15) in vitro, wherein the contacting is carried out for 1 to 14 days, and the T cells include naive CD4+ T cells and / or central memory CD4+ T cells. **Claim 2** The method according to claim 1, wherein the T cells further include CD8+ T cells. **Claim 3** The method according to claim 1 or 2, wherein the contacting is carried out for 1 to 10 days. **Claim 4** The method according to any one of claims 1 to 3, wherein the contacting is carried out in the absence of exogenous interleukin-2 (IL-2). **Claim 5** The method according to any one of claims 1 to 4, wherein each of the IL-7 and IL-15 is present at a concentration of at least 7 ng / mL. **Claim 6** The method according to any one of claims 1 to 4, wherein each of the IL-7 and IL-15 is present at a concentration of at least 10 ng / mL. **Claim 7** ​ The method according to any one of claims 1 to 6, wherein the AKT inhibitor is selected from the group consisting of A6730, B2311, 124018, afuresertib, perifosine, 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, elsylphosphocholine, GSK-2141795, KP372-1, L-418, NL-71-101, PBI-05204, PIA5, PX-316, SR13668, triciribine, CAS #937174-76-0, CAS #902779-59-3, miltefosine, CAS #1191951-57-1, 10-DEBC hydrochloride, Akt inhibitor III, Akt inhibitor VIII, CAS #1032350-13-2, SC79, CAS #857531-00-1, CAS #885499-61-6, CAS #552325-73-2, AGL 2263, 5-benzo[1,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione, CAS #32387-96-5, XL-418, CAS #612847-09-3, CAS #98510-80-6, 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, 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.

8. The method according to claim 7, 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.

9. The method according to any one of claims 1 to 8, wherein the T cell is 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.

10. The method according to any one of claims 1 to 9, wherein the T cell is transformed with a retrovirus.

11. The method according to claim 10, wherein the retrovirus contains a heterologous gene encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

12. The TCR or CAR is 707-AP (707 alanine proline), AFP (alpha (α)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen; β-catenin / m, β-catenin / variant), BCMA (B cell maturation antigen), Bcr-abl (breakpoint cluster region-Ablelson), 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 recognition antigen on melanoma), CAP-1 (cancer fetal antigen peptide-1), CASP-8 (caspase-8), CDC27m (cell division cycle 27 variant), CDK4 / m (cyclin-dependent kinase 4 variant), CEA (cancer fetal 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 variant), 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 papillomavirus), HSP70-2M (heat shock protein 70-2 variant), 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 lipoprotein receptor / GDP-L-fucose:b-D-galactosidase 2-a-L-fucosyltransferase), 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 recognized by T cells-1 / melanoma antigen A), MC1R (melanocortin 1 receptor), Myosin / m (myosin variant), MUC1 (mucin 1), MUM-1,-2,-3 (melanoma ubiquitous variant 1, 2, 3), NA88-A (NA cDNA clone of patient M88), NKG2D (natural killer group 2,Member D) Ligand, NY-BR-1 (New York breast differentiation antigen 1), NY-ESO-1 (New York esophageal squamous cell carcinoma-1), tumor fetal antigen (h5T4), P15 (protein 15), p190 minor bcr-ab l (protein of 190KD bcr-ab l), Pml / RARa (promyelocytic leukemia / retinoic acid receptor a), PRAME (antigen preferentially expressed 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 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 (triose phosphate isomerase variant), TRP-1 (tyrosinase related protein 1, or gp75), TRP-2 (tyrosinase related protein 2), TRP-2 / INT2 (TRP-2 / intron 2), VEG F-R2 (vascular endothelial growth factor receptor 2), WT1 (Wilms tumor gene), and the method according to claim 11, which can bind to an antigen selected from the group consisting of any combination thereof.,

13. The method according to claim 12, wherein the TCR or CAR binds to CD19 or BCMA.

14. The method according to any one of claims 1 to 13, wherein the cell is autologous.

15. The method according to any one of claims 1 to 13, wherein the cell is allogeneic.

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