Methods for Producing Improved Immune Cell Populations

Abstract

The present invention relates to methods for producing improved immune cell populations.

Description

[Technical field]

[0001] This application claims priority from AU2021 / 901496, filed May 19, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to methods for producing improved immune cell populations. [Background technology]

[0003] Chimeric antigen receptor (CAR) T-cell therapy has made remarkable progress in treating patients with hard to treat cancer, but poor persistence of CAR-T remains a significant challenge. Poor persistence of infused CAR-T cells inversely correlates with sustained clinical remission in patients with cancer. CM ) or stem cells (T SCM The frequency of CAR-T cells with the effector (TE) and effector memory (TEM) phenotypes has been shown to be an important predictor of clinical efficacy, with the ability to achieve better persistent CAR-T cells with effector (TE) and effector memory (TEM) phenotypes (McLellan and Ali Hosseini Rad, 2019). Although TE and TEM cells exhibit superior tumor killing capacity in vitro, they have low self-renewal capacity, reduced ability of niche homing and survival, and are more vulnerable to activation-induced cell death (AICD) or exhaustion (McLellan and Ali Hosseini Rad, 2019).

[0004] Thus, there is a need for improved populations of immune cells, such as CAR-T cell populations for use in CAR-T therapy. Summary of the Invention

[0005] The present inventors have shown that AKT inhibitors and / or inhibitors of PH domain proteins, when administered in vivo, can improve the efficacy of conventional CAR-T therapy in subjects in need of such treatment. The present inventors have also shown that AKT inhibitors and / or inhibitors of PH domain proteins can be used to improve the properties of cultured immune cells.

[0006] Thus, in one aspect the invention provides a method of modifying an immune response in a subject comprising administering to the subject a population of immune cells, wherein the immune cells are produced using a method comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein, preferably the immune cells are T cells, dendritic cells, natural killer cells, myeloid cells, macrophages, or a combination thereof.

[0007] In certain embodiments, the method is for modifying a T cell immune response, a dendritic cell immune response, a natural killer cell immune response, or a myeloid cell or macrophage immune response in a subject.

[0008] In a further aspect, the present invention provides the use of a population of immune cells in the preparation of a medicament for modifying an immune response in a subject, wherein the immune cells are produced using a method comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein, preferably the immune cells are T cells, dendritic cells, natural killer cells, myeloid cells, macrophages, or a combination thereof.

[0009] In a further aspect, the present invention provides a population of immune cells for use in modifying an immune response in a subject, wherein the immune cells are produced using a method comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein, preferably the immune cells are T cells, dendritic cells, natural killer cells, myeloid cells, macrophages, or a combination thereof.

[0010] In a further aspect, the invention provides a method of modifying a T cell response in a subject, comprising administering to the subject a population of T cells comprising a chimeric antigen receptor (CAR-T cells), wherein the CAR-T cells are produced using a method comprising culturing the CAR-T cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0011] In a further aspect, the present invention provides the use of a population of T cells comprising a chimeric antigen receptor (CAR-T cells) in the preparation of a medicament for modifying a T cell response in a subject, wherein the CAR-T cells are produced using a method comprising culturing the CAR-T cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0012] In a further aspect, the invention provides a population of T cells comprising a chimeric antigen receptor (CAR-T cells) for use in modifying a T cell response in a subject, wherein the CAR-T cells are produced using a method comprising culturing the CAR-T cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0013] In a further aspect, the present invention provides a method of modifying an immune response, preferably a T cell response, in a subject, comprising the steps of: a) administering to a subject an AKT inhibitor and / or an inhibitor of a PH domain protein; and b) at least about 18 hours after step a), administering to the subject a population of immune cells, preferably comprising T cells comprising a chimeric antigen receptor (CAR-T cells).

[0014] In a further aspect, the present invention relates to the use of an AKT inhibitor and / or an inhibitor of a PH domain protein in the preparation of a medicament for modifying an immune response, preferably a T cell response, in a subject, the treatment comprising: a) administering to a subject an AKT inhibitor and / or an inhibitor of a PH domain protein; b) administering to the subject a population of immune cells, preferably comprising T cells comprising a chimeric antigen receptor (CAR-T cells), at least about 18 hours after step a).

[0015] In a further aspect the present invention relates to an AKT inhibitor and / or an inhibitor of a PH domain protein for use in modifying an immune response, preferably a T cell response, in a subject, comprising: a) administering to a subject an AKT inhibitor and / or an inhibitor of a PH domain protein; and b) administering to the subject a population of immune cells, preferably comprising T cells comprising a chimeric antigen receptor (CAR-T cells), at least about 18 hours after step a).

[0016] In one embodiment, the modification of the immune response or the modification of the T cell response is achieved by the activation of central memory cells (T CM In one embodiment, the enrichment of T CM The cells are CD45RO+ CD62L+ T cells, preferably CD45RO+ CD62L hi Includes T cells.

[0017] In a further aspect, the present invention provides a method of modifying a dendritic cell and / or natural killer cell response in a subject, comprising the steps of: a) administering to a subject an AKT inhibitor and / or an inhibitor of a PH domain protein; b) administering to the subject a population of cells comprising dendritic cells and / or natural killer cells.

[0018] In some embodiments, the population of cells is administered about 18 hours to about 72 hours after the AKT inhibitor and / or the inhibitor of the PH domain protein. In some embodiments, the population of cells is administered about 24 hours to about 72 hours after the AKT inhibitor and / or the inhibitor of the PH domain protein. In some embodiments, the population of cells is administered about 24 hours to about 48 hours after the AKT inhibitor and / or the inhibitor of the PH domain protein.

[0019] The inventors have determined that an AKT inhibitor and / or an inhibitor of a PH domain protein can be used to reduce the killing activity of CAR-T cells and thus reduce the occurrence of cytokine release syndrome (CRS).Accordingly, in a further aspect, the present invention provides a method of reducing cytokine release syndrome (CRS) in a subject undergoing CAR-T cell therapy, comprising administering to the subject an AKT inhibitor and / or an inhibitor of a PH domain protein and / or CAR-T cells, wherein the CAR-T cells are cultured in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0020] In a further aspect, the present invention provides the use of an AKT inhibitor and / or an inhibitor of a PH domain protein and / or CAR-T cells in the preparation of a medicament for reducing cytokine release syndrome (CRS) in a subject undergoing CAR-T cell therapy, wherein the CAR-T cells are cultured in a medium comprising the AKT inhibitor and / or the inhibitor of a PH domain protein.

[0021] In a further aspect, the present invention provides an AKT inhibitor and / or an inhibitor of a PH domain protein and / or a CAR-T cell for use in reducing cytokine release syndrome (CRS) in a subject undergoing CAR-T cell therapy, wherein the CAR-T cell is cultured in a medium comprising the AKT inhibitor and / or the inhibitor of a PH domain protein.

[0022] In one embodiment, the chimeric antigen receptor comprises a CD28z costimulatory domain.

[0023] Examples of AKT inhibitors and / or inhibitors of PH domain proteins that can be used in the present invention include, but are not limited to, one or more selected from triciribine (TCN), triciribine 5'-monophosphate (TCN-P), AKT inhibitor VIII, MK-2206, AZD5363, GDC-0068, GSK2141795, and GSK2110183 hydrochloride.

[0024] In certain embodiments, the AKT inhibitor and / or the inhibitor of a PH domain protein is TCN or TCN-P.

[0025] In some embodiments, the immune cells or CAR-T cells are administered at a dosage of about 200,000 per kg, about 500,000 per kg, about 700,000 per kg, about 1 million per kg, about 1.2 million per kg, about 1.5 million per kg, about 1.7 million per kg, about 2 million per kg, about 2.2 million per kg, about 2.5 million per kg, about 2.7 million per kg, about 3 million per kg, or more. In another embodiment, the immune cells or CAR-T cells are administered at a dosage of about 0.2-0.5 million per kg, about 0.5-0.7 million per kg, about 0.7-1 million per kg, about 1.0-1.2 million per kg, about 1.2-1.5 million per kg, about 1.5-1.7 million per kg, about 1.7-2 million per kg, about 2.0-2.2 million per kg, about 2.2-2.5 million per kg, about 2.5-2.7 million per kg, or about 2.7-3 million per kg. In another embodiment, the immune cells or CAR-T cells are administered at a dosage of 0.5-2 million per kg.

[0026] In some embodiments, the subject is immunodepleted. Examples of methods for providing immunodepletion include, but are not limited to, lymphodepleting chemotherapy or radiation therapy.

[0027] In some embodiments, the subject has cancer, an infectious disease, or an inflammatory disease.

[0028] In some embodiments, the infection is a bacterial, fungal, protozoal, or viral infection. In some embodiments, the infection is a viral infection. In some embodiments, the viral infection is a chronic viral infection, such as an infection with Hepatitis C virus (HCV), Hepatitis B (HCB), Human Papillomavirus (HPV), Cytomegalovirus (CMV), Epstein-Barr virus (EBV), Varicella-Zoster virus, Coxsackievirus, or Human Immunodeficiency Virus (HIV).

[0029] In some embodiments, the subject has cancer, hi some embodiments, the subject has a solid tumor, such as breast cancer or colon cancer.

[0030] In some embodiments, the subject has a cancer associated with low antigen abundance. i) Acute myeloid leukemia with a predominance of low CD33+ blasts, or ii) Having diffuse large B-cell lymphoma or non-Hodgkin's lymphoma with low levels of CD19 and / or CD20.

[0031] In some embodiments, the subject is an animal, in some embodiments, the subject is a mammal, in some embodiments, the subject is a human.

[0032] There is also provided the use of an AKT inhibitor and / or an inhibitor of a PH domain protein for the manufacture of a medicament for modifying an immune response, preferably a T cell response, in a subject, wherein the subject is administered with a population of immune cells, preferably comprising T cells comprising a chimeric antigen receptor (CAR-T cells), at least 18 hours after the medicament.

[0033] There is also provided the use of a population of immune cells, preferably comprising T cells comprising a chimeric antigen receptor (CAR-T cells), for the manufacture of a medicament for modifying an immune response, preferably a T cell response, in a subject, wherein the subject is administered, or has been administered, an AKT inhibitor and / or an inhibitor of a PH domain protein at least 18 hours prior to the medicament.

[0034] There is also provided the use of an AKT inhibitor and / or an inhibitor of a PH domain protein for the manufacture of a medicament for modifying an immune response, preferably a T cell response, in a subject, wherein the medicament is a CAR-T cell.

[0035] There is also provided the use of an AKT inhibitor and / or an inhibitor of a PH domain protein for the manufacture of a medicament for modifying a dendritic cell and / or natural killer cell response in a subject.

[0036] Also provided is the use of a population of immune cells, including dendritic cells and / or natural killer cells, for the manufacture of a medicament for modifying dendritic cell and / or natural killer cell responses in a subject, wherein the subject has been or will be administered an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0037] Also provided are AKT inhibitors and / or inhibitors of PH domain proteins for modifying an immune response, preferably a T cell response, in a subject, preferably for use in generating a population of immune cells, including T cells comprising a chimeric antigen receptor (CAR-T cells).

[0038] Also provided is an AKT inhibitor and / or an inhibitor of a PH domain protein for use in modifying an immune response in a subject, preferably a T cell response in a subject, wherein the subject is administered the agent together with a population of immune cells, preferably comprising T cells comprising a chimeric antigen receptor (CAR-T cells), at least 18 hours after the agent.

[0039] In an embodiment, the methods described herein further comprise the administration of a checkpoint inhibitor, preferably an anti-PD-1 antibody. Advantageously, the administration of a checkpoint inhibitor in combination with the CAR-T cells of the invention, preferably CAR-T cells pre-treated with an AKT inhibitor and / or an inhibitor of a PH domain protein, demonstrates a synergistic effect on tumor growth and / or survival in a subject having or suspected of having cancer.

[0040] Thus, in one aspect, there is provided a method for modifying an immune response in a subject, preferably a T cell response in a subject, comprising administering to the subject a population of immune cells, preferably T cells comprising a chimeric antigen receptor (CAR-T cells), and a checkpoint inhibitor, preferably an anti-PD-1 antibody, preferably the immune cells were produced using a method comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein. Preferably, the immune cells are T cells, dendritic cells, natural killer cells, myeloid cells, macrophages, or a combination thereof. In an embodiment, the method comprises administering, optionally simultaneously or sequentially, CAR-T cells pretreated with an AKT inhibitor and / or an inhibitor of a PH domain protein and a checkpoint inhibitor. In this embodiment, the effect of the treatments (i.e., tumor growth and / or survival) is synergistic compared to the effect of each treatment individually.

[0041] In another embodiment, the method comprises administering CAR-T cells, an AKT inhibitor and / or an inhibitor of a PH domain protein, and a checkpoint inhibitor, optionally simultaneously or sequentially. In this embodiment, the effect of the treatments (on tumor growth and / or survival) is synergistic compared to the individual effect of each treatment alone. In an embodiment, the CAR-T cells are not pretreated with an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0042] In another aspect, there is provided a method for modifying an immune response in a subject, preferably a T cell response in a subject, comprising administering to the subject a checkpoint inhibitor, preferably an anti-PD-1 antibody and an AKT inhibitor and / or an inhibitor of a PH domain protein, in this embodiment, the effect of the treatments (on tumor growth and / or survival) is synergistic compared to the individual effect of each treatment alone.

[0043] In another aspect there is provided the use of a population of immune cells, preferably T cells comprising a chimeric antigen receptor (CAR-T cells), and a checkpoint inhibitor, preferably an anti-PD-1 antibody, in the preparation of a medicament for modifying an immune response in a subject, preferably a T cell response in a subject, wherein the immune cells have been produced using a method comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0044] In another aspect there is provided the use of a population of immune cells, preferably T cells comprising a chimeric antigen receptor (CAR-T cells), in the preparation of a medicament for modifying an immune response in a subject, preferably a T cell response in a subject, wherein the subject is or has been administered a checkpoint inhibitor, preferably an anti-PD-1 antibody, and the immune cells are produced using a method comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0045] In another aspect there is provided the use of a checkpoint inhibitor, preferably an anti-PD-1 antibody, in the preparation of a medicament for modifying an immune response in a subject, preferably a T cell response in a subject, wherein the subject has been administered or has been administered a population of immune cells, preferably T cells comprising a chimeric antigen receptor (CAR-T cells), and the immune cells have been produced using a method comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0046] In another aspect there is provided a population of immune cells, preferably T cells comprising a chimeric antigen receptor (CAR-T cells), and a checkpoint inhibitor, preferably an anti-PD-1 antibody, for use in modifying an immune response in a subject, preferably a T cell response in a subject, wherein the population of immune cells and the checkpoint inhibitor are produced using a method comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0047] In one embodiment, the modification of the immune response or T cell immune response increases the survival time of the subject compared to a subject not receiving the CAR-T cells and / or an AKT inhibitor and / or an inhibitor of a PH domain protein, hi one embodiment, the survival time is increased by 3, 6, 9, 12, 24, 36, 48, 60, 72, 84, 96 months or more compared to a subject not receiving the CAR-T cells and / or an AKT inhibitor and / or an inhibitor of a PH domain protein of the present invention.

[0048] In some embodiments, the subject has been diagnosed with or is suspected of having a disease or disorder, such as cancer, an infectious disease, or an inflammatory disease. In some embodiments, the subject has been diagnosed with or is suspected of having colon cancer or breast cancer. Thus, in some embodiments, the methods described herein include diagnosing the subject as having or being suspected of having a disease or disorder, such as cancer, preferably colon cancer or breast cancer, an infectious disease, or an inflammatory disease.

[0049] In an embodiment, the method or use may optionally further comprise administration of an additional therapeutic agent selected from the group consisting of chemotherapy, radiation therapy, surgery, bone marrow transplantation, drug therapy, cryoablation, or radiofrequency ablation.

[0050] In certain embodiments, the immune cells, CAR-T cells, AKT inhibitors and / or inhibitors of PH domain proteins, and / or checkpoint inhibitors may be administered sequentially or simultaneously.

[0051] The present inventors have also advantageously found that the efficacy of treatment against cancer can be increased by using an AKT inhibitor as an adjuvant in addition to during the manufacturing process of CAR-T cells.

[0052] Thus, in one embodiment, there is provided a method for modifying an immune response, preferably a T cell immune response, in a subject, comprising administering to the subject: (i) a population of immune cells, preferably T cells comprising a chimeric antigen receptor (CAR-T cells); (ii) an AKT inhibitor and / or an inhibitor of a PH domain protein, Methods are provided wherein the immune cells are produced using a method comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0053] In certain embodiments, the method further comprises the step of producing a cell population comprising the immune cells of the invention.

[0054] In one embodiment, the dose of the AKT inhibitor and / or the inhibitor of the PH domain protein administered to the subject is about 0.5 mg / kg, about 1 mg / kg, about 1.5 mg / kg, about 2 mg / kg, about 2.5 mg / kg, or about 3.0 mg / kg or more. In another embodiment, the dose of the AKT inhibitor and / or the inhibitor of the PH domain protein administered to the subject is about 0.5 mg / kg to 1.0 mg / kg, about 1.0 mg / kg to 1.5 mg / kg, about 1.5 mg / kg to 2.0 mg / kg, about 2.0 mg / kg to 2.5 mg / kg, about 2.5 mg / kg to 3.0 mg / kg or more. Preferably, the dose of the AKT inhibitor and / or the inhibitor of the PH domain protein administered to the subject is about 2 mg / kg.

[0055] In some embodiments, the AKT inhibitor and / or the inhibitor of the PH domain protein is administered intravenously to the subject once a week, twice a week, three times a week, four or more times a week. In other embodiments, the AKT inhibitor and / or the inhibitor of the PH domain protein, the immune cells, or the checkpoint inhibitor may be administered sequentially or simultaneously. Preferably, the first dose of the AKT inhibitor and / or the inhibitor of the PH domain protein is administered simultaneously with the administration of the immune cells and / or the checkpoint inhibitor.

[0056] In one embodiment, the method provides an expansion of CD4+ and / or CD8+ CAR-T cells in the spleen. In another embodiment, the method provides a lower percentage of modulation (T cells) compared to T cells that are not cultured in the presence of an AKT inhibitor and / or an inhibitor of a PH domain protein and are not administered with an AKT inhibitor and / or an inhibitor of a PH domain protein. REG In another embodiment, tumor T REG The T cells are reduced by approximately 50% compared to T cells that are not cultured in the presence of an AKT inhibitor and / or an inhibitor of a PH domain protein and are not administered with an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0057] In another aspect there is provided the use of a population of immune cells, preferably T cells comprising a chimeric antigen receptor (CAR-T cells), and an AKT inhibitor and / or an inhibitor of a PH domain protein, in the preparation of a medicament for modifying an immune response, preferably a T cell immune response, in a subject, wherein the immune cells have been produced using a method comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0058] In another aspect there is provided the use of a population of immune cells, preferably T cells comprising a chimeric antigen receptor (CAR-T cells), in the preparation of a medicament for modifying an immune response in a subject, preferably a T cell response in a subject, wherein the subject is or has been administered an AKT inhibitor and / or an inhibitor of a PH domain protein, and the immune cells are produced using a method comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0059] In another aspect there is provided the use of an AKT inhibitor and / or an inhibitor of a PH domain protein in the preparation of a medicament for modifying an immune response in a subject, preferably a T cell response in a subject, wherein the subject has been administered or has been administered a population of immune cells, preferably T cells comprising a chimeric antigen receptor (CAR-T cells), and the immune cells have been produced using a method comprising culturing the immune cells in a medium comprising the AKT inhibitor and / or the inhibitor of a PH domain protein.

[0060] In another aspect there is provided a population of immune cells, preferably T cells comprising a chimeric antigen receptor (CAR-T cells), and an inhibitor of AKT and / or an inhibitor of a PH domain protein, for use in modifying an immune response, preferably a T cell immune response, in a subject, wherein the population of immune cells and an inhibitor of AKT inhibitor and / or an inhibitor of a PH domain protein are produced using a method comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0061] Also provided is a population of immune cells, preferably comprising T cells comprising a chimeric antigen receptor (CAR-T cells), for use in modifying an immune response, preferably a T cell response, in a subject, where the subject is administered, or has been administered, an AKT inhibitor and / or an inhibitor of a PH domain protein at least 18 hours prior to the agent.

[0062] Also provided are AKT inhibitors and / or inhibitors of PH domain proteins for modifying dendritic cell and / or natural killer cell responses in a subject.

[0063] Also provided is the use of a population of cells, including dendritic cells and / or natural killer cells, to modify dendritic cell and / or natural killer cell responses in a subject, wherein the subject has been or will be administered an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0064] In another aspect, the invention provides a method for producing a cell population comprising immune cells, comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein, preferably wherein the immune cells are T cells, dendritic cells, natural killer cells, macrophages, myeloid cells, or a combination thereof.

[0065] In some embodiments, the immune cell is transgenic. In some embodiments, the immune cell comprises a chimeric antigen receptor. In some embodiments, the immune cell is a T cell comprising a chimeric antigen receptor (CAR-T cell).

[0066] In an embodiment, the method further comprises: a) producing a T cell enriched population from a population of immune cells isolated from a subject; b) transforming the T cell enriched cell population with a vector encoding a chimeric T cell receptor; c) culturing the cells obtained in step b) in a medium containing an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0067] In one embodiment, the CAR-T cells produced using this method are central memory (T CM ) and / or stem cells (T SCM ) including T cells.

[0068] In one embodiment, at least about 10% of the CAR-T cells produced using the method are CM and / or T SCM In one embodiment, at least about 15% of the CAR-T cells produced using the method are T CM and / or T SCM In one embodiment, at least about 20% of the CAR-T cells produced using the method are T CM and / or T SCM In one embodiment, at least about 25% of the CAR-T cells produced using the method are T CM and / or T SCM In one embodiment, at least about 25% of the CAR-T cells produced using the method are T CM and / or T SCM In one embodiment, about 10% to about 60% of the CAR-T cells produced using the method are T CM and / or T SCM In one embodiment, about 10% to about 50% of the CAR-T cells produced using the method are T CM and / or T SCM In one embodiment, about 10% to about 40% of the CAR-T cells produced using the method are T CM and / or T SCM In one embodiment, about 10% to about 30% of the CAR-T cells produced using the method are T CM and / or T SCM It is a cell.

[0069] In one embodiment, at least about 0.8% of the CD8+ T cells produced using the method are CM and / or T SCM In one embodiment, at least about 1.5% of the CD8+ T cells produced using the method are T CM and / or T SCM In one embodiment, at least about 2.5% of the CD8+ T cells produced using the method are T CM and / or T SCMIn one embodiment, at least about 3.5% of the CD8+ T cells produced using the method are T CM and / or T SCM In one embodiment, at least about 4.5% of the CD8+ T cells produced using the method are T CM and / or T SCM In one embodiment, about 0.8% to about 15% of the CD8+ T cells produced using the method are T CM and / or T SCM In one embodiment, about 0.8% to about 10% of the CD8+ T cells produced using the method are T CM and / or T SCM In one embodiment, about 0.8% to about 5% of the CD8+ T cells produced using the method are T CM and / or T SCM It is a cell.

[0070] In one embodiment, at least about 0.37% of the total lymphocytes produced using the method are T CM and / or T SCM In one embodiment, at least about 1% of the total lymphocytes produced using the method are T CM and / or T SCM In one embodiment, at least about 2% of the total lymphocytes produced using the method are T CM and / or T SCM In one embodiment, at least about 3% of the total lymphocytes produced using the method are T CM and / or T SCM In one embodiment, at least about 4% of the total lymphocytes produced using the method are T CM and / or T SCM In one embodiment, at least about 5% of the total lymphocytes produced using the method are T CM and / or T SCM In one embodiment, about 0.37% to about 15% of the total lymphocytes produced using the method are T CM and / or T SCMIn one embodiment, about 0.37% to about 10% of the total lymphocytes produced using the method are T CM and / or T SCM In one embodiment, about 0.37% to about 5% of the total lymphocytes produced using the method are T CM and / or T SCM It is a cell.

[0071] In one embodiment, T CM The cells are CD45RO+CD62L+ T cells, preferably CD45RO+CD62L hi Includes T cells.

[0072] In one embodiment, T SCM The cells were CD27 + CD95 + Includes T cells.

[0073] In one embodiment, the method further comprises: CM and / or T SCM For cells, the method further includes enriching the cultured cells. Methods for selecting such cells from a population of cells are known in the art, such as using antibody-based cell sorting.

[0074] In one embodiment, the method comprises the step of: determining whether a higher percentage of T cells are resistant to AKT stimulation than T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein. CM and / or T SCM produces.

[0075] In one embodiment, the method provides a lower percentage of modulation (T cells) than T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein. REG ) T cells.

[0076] In one embodiment, T REG The cells are CD3+ CD4+ CD25+ FoxP3+ T cells.

[0077] In one embodiment, the method produces a population of cells that express less of one or more proinflammatory cytokines than when the same cells are cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein, hi one embodiment, the one or more proinflammatory cytokines are TNFα, IFNγ, or both.

[0078] In one embodiment, the method produces a higher percentage of naive T cells than T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0079] The inventors have determined that the method of the present invention can be used to produce improved CAR-T cells that target viral infections. Thus, in one embodiment, the chimeric antigen receptor binds to a viral antigen. In one embodiment, the method produces a population of CAR-T cells that has a higher antiviral activity than a population of CAR-T cells cultured under the same conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0080] In an alternative embodiment, the T cells are not transgenic and have greater antiviral activity than a population of T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0081] In another embodiment, the method further comprises: a) producing a dendritic cell enriched population from a population of immune cells isolated from a subject; b) exposing the cells from step a) to an antigen; c) culturing the cells in a medium containing an AKT inhibitor and / or an inhibitor of a PH domain protein. Thus, the method of the present invention can be used to produce a dendritic cell vaccine. In one embodiment, the antigen is a cancer antigen or an antigen of a pathogen, such as a viral antigen.

[0082] In one embodiment, the method produces more dendritic cells than dendritic cells cultured under the same conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0083] In a further embodiment, the method further comprises: a) producing a natural killer cell (NK) enriched cell population from a population of immune cells isolated from a subject; b) culturing the cells from step a) in a medium containing an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0084] In one embodiment, the method produces a population of NK cells that has greater cytotoxic activity than a population of NK cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0085] In certain embodiments, the NK cells comprise a chimeric antigen receptor, and the method further comprises transforming the natural killer cell (NK) enriched cell population with a vector encoding the chimeric antigen receptor.

[0086] In certain embodiments, the concentration of the AKT inhibitor and / or PH domain protein inhibitor in the culture medium is about 0.5 μM to 9 μM, about 1 μM to about 7 μM, about 1 μM to about 5 μM, or about 1 μM to 3 μM. In other embodiments, the concentration of the AKT inhibitor and / or PH domain protein inhibitor in the culture medium is about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, or about 9 μM.

[0087] In some embodiments, the cell is an animal cell, in some embodiments, the cell is a mammalian cell, in some embodiments, the cell is a human cell.

[0088] In certain embodiments, the cultured cells, or a subpopulation thereof that comprises immune cells (eg, further enriched for a particular cell type of interest), are administered to a subject.

[0089] In another aspect, the invention provides a population of cells produced using the methods of the invention, preferably wherein the immune cells are produced using a method comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein.

[0090] In a further aspect, the present invention provides a cell population comprising CAR-T cells, wherein at least 10% of the CAR-T cells are CD8+ T cells. CM and / or T SCM A population of cells is provided, the population being cells.

[0091] In some embodiments, the cell population is unsorted, such as not sorted after culture.

[0092] In one embodiment, less than 25% of the CAR-T cells are REG It is.

[0093] In one embodiment, the population of immune cells, preferably a population of T cells comprising a chimeric antigen receptor of the invention (CAR-T cells), an AKT inhibitor and / or an inhibitor of a PH domain protein, and / or a checkpoint inhibitor, are administered in the form of a pharmaceutical composition. In one aspect, a pharmaceutical composition is thus provided comprising a population of immune cells of the invention.

[0094] In one embodiment, the pharmaceutical composition comprises a population of immune cells, preferably T cells comprising the chimeric antigen receptor of the present invention (CAR-T cells), and an AKT inhibitor and / or an inhibitor of a PH domain protein. In one embodiment, the pharmaceutical composition comprises a population of immune cells, preferably T cells comprising the chimeric antigen receptor of the present invention (CAR-T cells), and a checkpoint inhibitor.

[0095] Any embodiment herein is intended to apply mutatis mutandis to any other embodiment unless stated otherwise.

[0096] The present invention is not to be limited in scope by the specific embodiments described herein, which are for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the invention as described herein.

[0097] Throughout this specification, unless otherwise specified or the context requires otherwise, references to a single step, composition of matter, group of steps, or group of compositions of matter should be interpreted as encompassing one and more (i.e., one or more) of that step, composition of matter, group of steps, or group of compositions of matter.

[0098] The invention will now be described by way of the following non-limiting examples and with reference to the accompanying drawings. [Brief description of the drawings]

[0099] [Figure 1] Effect of various TCN and TCN-P concentrations on mouse T cell viability. [Diagram 2] Effect of repeated TCN exposure on the proliferation of mouse CAR-T cells after transduction. [Diagram 3] Effect of repeated TCN exposure on the central memory phenotype (CD44+CD62Lhi) in mouse CD8+ CAR-T cells. [Figure 4] Effect of TCN treatment on IFNγ and TNFα production by mouse CD8+ CAR-T cells. CAR-T cells pretreated with TCN exposure produce comparable levels of IFNγ at effector:target ratios of 2:1 and 1:1 (A). A similar trend was observed for TNFα release at the same effector:target ratios (B). [Diagram 5] Effect of repeated TCN exposure on the expression of early activation markers in mouse CAR-T cells in the absence of exposure to tumor antigens. TCN pretreatment increased the expression of PD-1 (A) and CD69 (B) on the surface of mouse CD8+ CAR-T cells. [Figure 6]Mouse CAR-T cells pretreated with TCN exert blunted tumor antigen-directed cytotoxicity (A), but TCN pretreatment does not affect the ongoing survival of CAR-T cells. [Figure 7] CAR-T transduction efficiency (Her2PE) was unchanged in CD4 and CD8 T cells. [Figure 8] Flow cytometry contour plots showing the transition of CD8+ CAR-T cells to a central memory phenotype upon treatment with either TCN or TCN-P. [Figure 9] Quantified flow cytometry data (24 hours) from Figure 8. Central memory T (TCM) cell phenotype (CD45RO+ CD62L+) increased from an average of 11% (control, vehicle) to 17% (TCN, TCN-P) in CD8+ CAR-T cells with a concomitant reduction in effector T (TE) cells (8% vs. 5%, n=3) (A). This pattern of response to TCN / TCN-P treatment was conserved across all three donors (B). [Figure 10] Brief exposure (24 h) of CAR-T cells to TCN or TCN-P led to sustained transition of effector T (TE) cells into central memory T (TCM) cells for at least 3 days, which was consistent across all three PBMC donors. [Figure 11] Quantified flow cytometry data from Figure 10 (3 days). After a 24-hour treatment period, central memory T (TCM) cell phenotype (CD45RO+ CD62L+) remained increased in CD8+ CAR-T cells (9% vs. 16%, control / vehicle vs. TCN / TCN-P), which was accompanied by a reduction in effector T (TE) cells (35% vs. 23%, n=3) (A). This pattern of response to TCN / TCN-P treatment was preserved across all three donors (B). [Figure 12] Pretreatment with TCN / TCN-P increased CCR7+ TCM from a mean of 9% to 16%, accompanied by a reduction in CCR7+ TE from 35% to 23%. [Figure 13]Pretreatment with TCN or TCN-P did not have any effect on CD4 TCM (CD45RO+ CD62L+) 24 hours after treatment. [Figure 14] Pretreatment with TCN / TCN-P did not affect CD4+ TCM or CD4+ TE, quantified flow cytometry data from FIG. 14 (24 h). [Figure 15] Pretreatment with TCN / TCN-P did not have any effect on CD4 TCM (CD45RO+ CD62L+) 24 hours after treatment. [Figure 16] Pretreatment with TCN / TCN-P did not affect CD4+ TCM or CD4+ TE, quantified flow cytometry data from FIG. 15 (3 days). [Figure 17] TCN pretreatment reduced the regulatory T (TREG) cell subpopulation (CD4+CD25+FoxP3+) in CAR-T cells. [Figure 18] Overview of the protocol to measure the in vivo effect of TCN or TCN-P pretreatment on CAR-T cell function. [Figure 19]TCN-P pretreatment during CAR-T manufacturing results in enrichment of central memory T cells. An exemplary representation of the E0771-hHer2 breast cancer model in which 200,000 tumor cells were orthotopically implanted into the mammary fat pad and 20 million CAR-T cells were administered via tail vein injection 6 days later. Animals were humanely sacrificed when tumors were >120 mm2 or reached other humane endpoints (A). Representative flow cytometry contour plots showing that TCN-P preconditioning did not affect the CD4:CD8 T cell ratio during the manufacturing process (B, upper panel) but did result in preferential enrichment for CD44+ CD62L+ central memory CD8+ T cells (B, lower panel). Animals received the same ratio of CD4:CD8 T CAR+ cells (C). Administration of untreated CAR-T cells reduced tumor volume and extended the survival of the animals by 2 days, but tumor control was most significantly reduced by TCN-P preconditioned CAR-T cells. Survival was also increased by 14 days (D). This translated into a significant improvement in the probability of survival (E, p=0.0012). [Figure 20] The reduction in tumor growth corresponded to a sustained central memory phenotype after CAR-T administration. Separate cohorts of animals were subjected to the same breast tumor model, all animals were sacrificed on day 8 after CAR-T treatment, and tumor measurements were performed every 2-3 days (A). Representative flow cytometry analysis showed that preconditioning with TCN-P was able to increase the percentage of circulating CD4+ T cells compared to CD8+ T cells (B, upper panel) and reduce CD8+ CAR-T cells (middle panel), while sustaining the central memory phenotype (lower panel). Intratumoral CD4+ and CD8+ T cells were not significantly altered (C), however, higher numbers of intrasplenic CD4+ T cells were observed in the preconditioned group (D). Changes in tumor control were not associated with significant changes in IFN-gamma and TNF-alpha production (E). [Figure 21]Effects of TCN-P preconditioning on circulating T cells. Preconditioning did not significantly affect CD4+ or CD8+ T cell numbers (A-B). No significant changes were observed in CD8+ CAR-T cells (C), but the number of CD4+ CAR-T was significantly higher in animals that received preconditioned CAR-T cells (D, p=0.0009). The percentages of CD8+ and CD4+ central memory T cells were higher in animals that received preconditioned CAR-T cells (E-F, p<0.0001). This was accompanied by a reduction in CD8+ (G, p<0.0001) and CD4+ (H, p=0.0029) effector memory T cells. [Figure 22] Effects of TCN-P preconditioning on intratumoral and intrasplenic T cells. No significant changes were observed in the intratumoral and intrasplenic percentages of CD4+ and CD8+ central memory T cells (A, C). No changes were observed in the intratumoral percentages of CD101+CD8+ T cells (B), but the intrasplenic levels of CD101+CD8+ T cells were lower in the preconditioned group, suggesting that preconditioning conferred a protective effect against exhaustion in CD8+ T cells (D). [Figure 23] TCN-P preconditioned CAR-T cells in combination with PD-1 checkpoint inhibitors improve antitumor efficacy against solid tumors. A) Her2+ transgenic mice were inoculated with 2.5e5E0771-hHer2. Mice were randomized to have an average tumor size of 20 mm2 5 days after tumor inoculation. They were left untreated or treated with the following therapies: anti-PD-1 (aPD1), CAR-T cells (CAR-T cells + 2A3), CAR-T cells with anti-PD-1 (CAR-T + aPD1), TCN-P preconditioned CAR-T cells (PTX-2 + 2A3), and TCN-P preconditioned CAR-T cells combined with anti-PD-1 checkpoint inhibitors (PTX-2 + aPD1). Tumor growth was measured every 2-3 days (mm2). Each treatment group consisted of 6 mice. B) Survival curves of mice in these treatments. [Figure 24]Overview of the protocol to measure the in vivo efficacy of TCN and TCN-P as neoadjuvants for CAR-T. [Diagram 25] Adjuvant efficacy of TCN-P. Her2+ transgenic mice were inoculated with 2.5e5MC-38-hHer2. Mice were randomized to have an average tumor size of 20mm2 5 days after tumor inoculation. Treatments included intraperitoneal injections of either DMSO (vehicle control) or TCN-P at 5, 25 or 50mg / Kg every 3-4 days. Tumor growth was measured (mm2) every 2-3 days. Each treatment group consisted of 6 mice. TCN-P has a dose-dependent antitumor effect. [Figure 26] TCN-P enhances the efficacy of CAR-T cell therapy against solid tumors. A) Her2+ transgenic mice were inoculated with 2.5e5 MC-38-hHer2. They were randomized to have an average tumor size of 20mm2 5 days after tumor inoculation. They were left untreated or subjected to treatment regimens including intraperitoneal injection of TCN-P (25mg / Kg) every 3-4 days, intravenous injection of 20e6 anti-human Her2 CAR-T cells, and combined treatment of 20x106 anti-human Her2 CAR-T cells and 25mg / Kg TCN-P intravenously (injected every 3-4 days). Tumor growth is measured every 2-3 days (mm2). Each treatment group consists of 6 mice. B) Tumor growth of individual mice (N=6 / treatment group). C) TCN-P improves survival in mice bearing solid MC-38-hHer2 tumors. Tumor-bearing mice treated with CAR-T cells in combination with TCN-P (25mg / Kg) showed a 6-day increase in median survival compared to treatment with CAR-T cells alone. [Figure 27]TCN-P as an adjuvant for CAR-T therapy. Exemplary representation of the MC-38 colon cancer model in which 250,000 MC-38 tumor cells are implanted subcutaneously approximately 6 days prior to the start of CAR-T therapy and co-administration of adjuvant TCN-P every 3 days (A). Preliminary data indicate that TCN-P adjuvant therapy significantly improved CAR-T-directed tumor reduction (*p<0.05), but the most effective regimen was the combination of TCN-P adjuvant and TCN-P preconditioned CAR-T cells. [Figure 28] A) The combination of TCN-P pretreatment and TCN-P adjuvant therapy resulted in significant accumulation and / or expansion of CD4+ and CD8+ CAR T cells in the spleen by day 14 post-treatment (p<0.0001). B) Administration of TCN-P reduced intratumoral Tregs by 50%. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0100] General Techniques and Definitions Unless specifically defined otherwise, all technical and scientific terms used herein shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., cell culture, molecular genetics, CAR-T technology, immunology, and biochemistry).

[0101] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), as well as F. M. Usubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current editions), Ed Harlow and David Lane (editors), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J. E. Coligan et al. al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all current editions), and other sources.

[0102] The term "and / or," e.g., "X and / or Y," is to be understood to mean either "X and Y" or "X or Y," and is to be interpreted as explicitly supporting both meanings or either meaning.

[0103] As used herein, unless stated to the contrary, the term about refers to + / - 10%, more preferably + / - 5% of the specified value.

[0104] Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer, or step, or group of elements, integers, or steps, but not the exclusion of any other element, integer, or step, or group of elements, integers, or steps.

[0105] As used in this application, the term "or" is intended to mean an inclusive "or" rather than an exclusive "or." That is, unless otherwise specified or clear from the context, "X employs A or B" is intended to mean any of the natural inclusive permutations. That is, if X employs A, X employs B, or X employs both A and B, then "X employs A or B" is satisfied under any of the foregoing examples. Furthermore, at least one of A and B, and / or the like, generally means A or B, or both A and B. In addition, the articles "a" and "an" used in this application and the appended claims may be construed generally to mean "one or more," unless otherwise specified or clear from the context to refer to the singular form.

[0106] As used herein, the term "subject" can be any animal. In one embodiment, the animal is a vertebrate. For example, the animal can be a mammal, a bird, a chordate, an amphibian, or a reptile. Exemplary subjects include, but are not limited to, humans, primates, livestock (e.g., sheep, cows, chickens, horses, donkeys, pigs), companion animals (e.g., dogs, cats), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, hamsters), and captive wild animals (e.g., foxes, deer). In one embodiment, the mammal is a human. In some embodiments, the methods of the invention are for veterinary use.

[0107] The term "treatment" or "treating" of a subject includes the application or administration of a population of immune cells of the present invention, or a composition thereof, for the purpose of delaying, slowing, stabilizing, curing, curing, alleviating, relieving, altering, correcting, mitigating, improving, enhancing, or affecting a disease or condition, symptoms of a disease or condition, or the risk (or susceptibility) of a disease or condition. The term "treating" refers to any indicia of successful treatment or amelioration of an injury, pathological condition, or condition, including any objective or subjective parameter, such as relief, remission, reduction in the rate of deterioration, reduction in the severity of a disease, stabilization, reduction in symptoms, or making the injury, pathological condition, or condition more tolerable to the subject, slowing the rate of degeneration or decline, making the end point of degeneration less debilitating, or improving the physical or mental well-being of the subject.

[0108] As used herein, "preventing" or "prevention" is intended to refer to at least a reduction in the likelihood of the risk of acquiring (or susceptibility to) a disease or disorder (i.e., not developing at least one of the clinical symptoms of the disease in a patient who may be exposed to or predisposed to the disease but has not yet experienced or displayed symptoms of the disease). Biological and physiological parameters for identifying such patients are provided herein and are also well known by physicians. For example, in the case of a subject suspected of having breast cancer, the subject may have a family history of cancer and have been identified as having a mutation that is likely to cause cancer but does not show obvious symptoms of the disease. In this case, it is contemplated that the immune cells of the present invention or compositions thereof have utility in preventing the development of one or more symptoms associated with the disease (e.g., breast cancer) in a subject.

[0109] As used herein, "cytokine release syndrome" (CRS) refers to an acute systemic inflammatory syndrome characterized by fever and multiple organ dysfunction that is associated with chimeric antigen receptor (CAR)-T cell therapy, therapeutic antibodies, and haploidentical allogeneic transplantation.

[0110] As used herein, "enriched population" or variations thereof refers to a population of cells that has been treated to remove or at least reduce the representation of some types of cells from a starting population of cells, such as a peripheral blood mononuclear cell (PBMC) population isolated from a subject. Methods for positively or negatively selecting for specific cell types are well known in the art, such as using magnetic beads that contain antibodies that selectively bind to cell surface proteins of the specific cell type to be enriched or removed. In an embodiment, when compared to the starting cell population (such as PBMCs), the cell type for which the population is enriched has, for example, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, or 50-fold or more representation in the enriched population compared to the starting population.

[0111] As used herein, the term "identical conditions" is a relative term meaning that the same cell population will divide, for example, into two identical subpopulations when exposed to the exact same culture procedure apart from the presence of an inhibitor for one of the subpopulations.

[0112] As used herein, terms such as "combination therapy," "combined administration," or "co-administration" are meant to encompass the administration of selected therapeutic agents to a single subject, and are intended to include therapeutic regimens in which agents are administered by the same or different routes of administration or at the same or different times.

[0113] Inhibitors As used herein, the term "PH domain protein" refers to a protein that contains a PH domain. Pleckstrin homology domain (PH domain) or (PHIP) is a protein domain of about 100-120 amino acids present in numerous proteins involved in intracellular signaling or as components of the cytoskeleton. All share the same β-sandwich fold that was first observed in the NMR structure of the N-terminal pleckstrin PH domain. The amino-terminal half of the protein forms a four-stranded β-sheet with an additional short α-helix (specific to β-spectrin PH domains) in the β3 / β4 loop. The other half of the protein forms a β-sheet meander (β5-β7 strands) that is nearly orthogonal to the first sheet. The two sheets form a "sandwich" that is filled with the hydrophobic core of the domain. In one embodiment, the PH domain protein is a small G protein. In another embodiment, the PH domain protein is a serine / threonine specific protein kinase. In another embodiment, the PH domain protein is an oxysterol binding protein (OSBP). In another embodiment, the PH domain protein is a G protein receptor kinase. Examples of PH domain proteins that can be inhibited using the methods of the present invention include, but are not limited to, oxysterol binding protein 1, oxysterol binding protein 2, spectrin beta chain, non-erythroid 1, Rho GTPase activating protein 27, phosphoinositide 3-kinase (PI3K), AKT, or one or more combinations thereof.Examples of inhibitors of PH domain proteins useful in the present invention include phosphatidylinositol ether lipid analogs (PIAs) such as D-3-deoxy-myo-inositol, e.g., D-3-deoxy-phosphatidyl-myo-inositol 1-[(R)-2-methoxy-3-octadecyloxypropyl hydrogen phosphate] (DPIEL, PX-316); alkyl phospholipids (APLs) such as edelfosine, miltefosine, and perifosine; Ins(1,3,4,5,6) phenylphospholipids (PIAs); Inositol phosphates (IPs), such as Ins(1,4,5,6) tetrakisphosphate (IP4), phytic acid (IP6), 2-O-benzyl-myo-inositol 1,3,4,5,6-pentakisphosphate (2-O-Bn-InsP5), and diphosphoinositol pentakisphosphate (5-PP-IP5); inositol phosphate-6-kinase 1 (IP6K1); diazo-sulfo-amide inhibitors, such as NSC348900 (PH-316) and 4- Sulfonamides such as dodecyl-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide (e.g., PH-427); purines / pyrimidines such as triciribine (tricyclic dinucleoside, NSC154020, TCN, AKT / PKB signaling inhibitor-2, API-2), triciribine phosphate (NSC280594; triciribine 5'-monophosphate; TCN-P), and API-1 (NSC177223-pyrido[2,3-d]pyrimidine). Other inhibitors include, but are not limited to, allosteric compounds that interact only within the PH domain via Trp80 (e.g., MK-2206, SC66), tyrucaric acid, PITenin (PIT), peptidomimetics (e.g., AKT-in such as NH2-AVTDHPDRLWAWEKF-COOH), 1,2,3-triazol-4-ylmethanol-based antagonists, and salts, esters, analogs, variants, and derivatives thereof. In one embodiment, the PH domain protein inhibitor is triciribine (TCN) or triciribine 5'-monophosphate (TCN-P).

[0114] AKT, also known as protein kinase B (PKB), is a serine / threonine-specific protein kinase that plays an important role in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration. AKT1 is involved in the PI3K / AKT / mTOR pathway and other signaling pathways. Examples of AKT inhibitors for use in the present invention include MK-2206 2HCl (8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl[1,2,4]triazolo[3,4]-f][1,6]naphtho-pyridin-3(2H)-one dihydrochloride); Perifosine (1,1-dimethyl-4[(octadecyloxy)hydroxyphosphinyl]oxy]-piperidinium inner salt, KRX-0401); GSK690693 (4-[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl -7-[[(3S)-piperidin-3-yl]methoxy]imidazo[4,5-c]pyridin-4-yl]-2-methylbut-3-yn-2-ol; Ipatasertib ((2S)-2-(4-chlorophenyl)-1-{4-[(5R,7R)-7-hydroxy-5-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-yl]-1-piperazinyl}-3-(isopropylamino)-1-propanone-, GDC-00 68;AZD5363(4-amino-N-[(1S)-1-(4-chlorophenyl)-3-hydroxypropyl]-1-(7H-pyrrolo[2,3-d]-pyrimidin-4-yl)piperidine-4-carboxamide;PF-04691502(2-amino-8-[4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxypyridin-3-yl)-4-methylpyrido[2,3-d]pyrimidin-7-one);AT7867( 4-(4-chlorophenyl)-4-[4-(1H-pyrazol-4-yl)phenyl]piperidine;Triciribine (5-methyl-1-(β-D-ribofuranosyl)-1,5-dihydro-1,4,5,6,8-pentaazaacenaphthylene-3-amine);Triciribine 5'-monophosphate;CCT128930 (4-(4-chlorobenzyl)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4-piperidinamine)-;A-674563 ((2S)-1-[5-(3-methyl-2H-indazol-5-yl)pyridin-3-yl]oxy-3-phenylpropan-2-amine); PHT-427 (4-dodecyl-N-(1,3,4-thiadiazol-2-yl)benzenesulfonamide); AKTi-1 / 2 (3-[1-[[4-(7-phenyl-3H-imidazo[4,5-g]quinoxalin-6-yl)phenyl]methyl]piperidin-4-yl]-1H-benzamide) N-imidazol-2-one; afuresertib (GSK2110183, N-[(2S)-1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-2-methylpyrazol-3-yl))thiophene-2-carboxamide; AT13148 ((1S)-2-amino-1-(4-chlorophenyl)-1-[4-(1H-pyrazol-4-yl)phenyl]ethanol);

[0189] Miltefosine ( Hexadecyl 2-(trimethylazaniummyl)ethyl phosphate; Honokiol (2-(4-hydroxy-3-prop-2-enylphenyl)-4-prop-2-enylphenol); TIC10 analogs (2,6,7,8,9,10-hexahydro-10-[(2-methylphenyl)methyl]-7-(phenylmethyl)-imidazo[1,2-a]pyrido[4,3-d]pyrimidin-5(3H)-one); AKT inhibitor VIII (1,3-di Hydro-1-(1-((4-(6-phenyl-1H-imidazo[4,5-g]quinoxalin-7-yl)phenyl)methyl)-4-piperidinyl)-2H-benzimidazol-2-one; Uprosertib (GSK2141795); TIC10 (2,4,6,7,8,9-hexahydro-4-[(2-methylphenyl)methyl]-7-(phenylmethyl)-imidazo-o[1,2-a]pyrido[3,4-e]pyrimidin-5(1H)-one);Examples of AKT inhibitors include, but are not limited to, capivasertib (AZD5363) and MS-222 (ethyl-3-aminobenzoic acid methanesulfonate), and salts, esters, analogs, variants, and derivatives thereof. In some embodiments, the AKT inhibitor is triciribine, triciribine 5'-monophosphate, AKT inhibitor VIII, MK-2206, AZD5363, GDC-0068, GSK2141795, and GSK2110183 hydrochloride, and salts, esters, analogs, variants, and derivatives thereof. In some embodiments, the AKT inhibitor is triciribine (TCN) or triciribine 5'-monophosphate (TCN-P);

[0115] Inhibition of AKT and / or PH domain protein activity can be less than 100%, e.g., about 10% to about 95%, e.g., about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or another percent inhibition of activity, e.g., about 10% to about 95%. The inhibitor can be, for example, a small molecule, a peptide, a protein (such as an antibody), a nucleic acid, or a combination thereof.

[0116] immune cells As used herein, the phrase "immune cell" refers to a cell capable of influencing or inducing an immune response upon recognition of an antigen. In some embodiments, the immune cell is a T cell, a natural killer (NK) cell, a macrophage, a myeloid cell, or a dendritic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. The cells can be autologous or allogeneic to the subject to which they are administered. In certain embodiments, the invention provides a population of CAR-expressing cells, such as CAR-T cells.

[0117] As used herein, the phrases "modifying an immune response" or "modifying a T cell immune response" refer to the ability of an immune cell or T cell to induce or increase an immune response upon recognition of an antigen. It will be understood that such modification of the immune response or T cell response is sufficient to treat cancer, infectious diseases, or inflammatory diseases as described herein.

[0118] As used herein, the phrase "cytotoxic activity" refers to the ability of immune cells, such as NK cells, to destroy living cells.

[0119] As used herein, the term "immune response" has its ordinary meaning in the art and includes both humoral and cellular immunity. An immune response can be manifested as one or more of the following: development of anti-antigen antibodies, expansion of antigen-specific T cells, increase in tumor infiltrating lymphocytes (TILs), development of anti-tumor or anti-tumor antigen delayed-type hypersensitivity (DTH) responses, clearance of pathogens, inhibition of pathogen and / or tumor growth and / or spread, tumor reduction, reduction or elimination of metastasis, increase in time to recurrence, increase in pathogen or tumor-free survival, and increase in survival time. An immune response can be mediated by one or more of B cell activation, T cell activation, natural killer cell activation, activation of antigen-presenting cells (e.g., B cells, DCs, monocytes, and / or macrophages), cytokine production, chemokine production, specific cell surface marker expression, especially expression of costimulatory molecules. An immune response can be characterized as a humoral, cellular, Th1 or Th2 response, or a combination thereof. In certain embodiments, the immune response is an innate immune response.

[0120] T cells In some embodiments, the immune cell is a T cell, e.g., a CAR-T cell. T cells or T lymphocytes are a type of lymphocyte that plays a central role in cell-mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T cell receptor (TCR) on the cell surface. There are several subsets of T cells, each with different functions.

[0121] In one embodiment, the T cells are central memory (T CM ) T cells or comprising them. CMThe cells patrol lymph nodes and provide central immune surveillance against known pathogens, but have not been described as performing primary tissue immune surveillance. In one embodiment, the T cells produced using the methods of the invention CM The cells are CD45RO+ CD62L+ T cells, preferably CD45RO+ CD62L hi Such cells may also be CCR7+.

[0122] In one embodiment, the T cells are central memory stem cells (T SCM ) T cells or comprising them. SCM The cells are a rare subset of memory lymphocytes with stem cell-like self-renewal capacity and the pluripotent ability to reconstitute the full spectrum of memory and effector subsets. In one embodiment, T SCM The cells were CD27 + CD95 + Includes T cells.

[0123] In one embodiment, the method comprises the step of: determining whether a higher percentage of T cells are resistant to AKT stimulation than T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein. CM and / or T SCM In one embodiment, the method produces at least about 25% more central memory (T) T cells than T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein. CM ) and / or stem cells (T SCM In one embodiment, the method produces central memory (T) T cells that are at least about 50% more abundant than T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein. CM ) and / or stem cells (T SCM In one embodiment, the method produces central memory (T) T cells that are at least about 75% more abundant than T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein. CM ) and / or stem cells (T SCMIn one embodiment, the method produces about 25% to about 90% more central memory (T) T cells than T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein. CM ) and / or stem cells (T SCM ) T cells.

[0124] As used herein, regulatory T cells (T REG ) or variants thereof refer to a population of T cells that are important in the maintenance of immune tolerance. Their main role is to shut down T cell-mediated immunity towards the end of an immune response and to suppress autoreactive T cells that have escaped the process of negative selection in the thymus. REG Two major classes of cells have been described - Foxp3+ and Foxp3-.

[0125] In one embodiment, the method provides a lower percentage of modulation (T cells) than T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein. REG In one embodiment, the method produces regulatory (T) T cells that are at least about 5% less abundant than T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein. REG In one embodiment, the method produces regulatory (T) T cells that are at least about 10% less abundant than T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein. REG In one embodiment, the method produces regulatory (T) T cells that are at least about 15% less abundant than T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein. REG In one embodiment, the method produces regulatory (T) T cells that are at least about 20% less abundant than T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein. REGIn one embodiment, the method produces regulatory (T) T cells that are at least about 25% less abundant than T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein. REG In one embodiment, the method produces regulatory (T) T cells that are about 5% to about 30% less abundant than T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein. REG In one embodiment, the method produces regulatory (T) T cells that are about 5% to about 25% less abundant than T cells cultured under identical conditions in the absence of an AKT inhibitor and / or an inhibitor of a PH domain protein. REG ) T cells. In one embodiment, T REG The cells are CD25+ FoxP3+ T cells.

[0126] As used herein, the term "naive T cells" refers to a population of T cells that have matured and been released by the thymus, but have not yet encountered the corresponding antigen. In other words, naive T cells are in a stage between maturation and activation. Naive T cells are generally characterized by surface expression of L-selectin (CD62L) and CC chemokine receptor type 7 (CCR7), the absence of activation markers CD25, CD44, or CD69, and the absence of memory CD45RO isoforms. They also express a functional IL-7 receptor, consisting of the subunits IL-7 receptor-α, CD127, and the common-γ chain, CD132.

[0127] A T cell that lacks a functional endogenous T cell receptor (TCR) may, for example, be engineered to not express any functional TCR on its surface, may be engineered to not express one or more subunits that comprise a functional TCR, or may be engineered to produce little to no functional TCR on its surface. Alternatively, the T cell may express a substantially impaired TCR, for example, by expression of a mutated or truncated form of one or more of the subunits of the TCR. The term "substantially impaired TCR" means that the TCR does not elicit a deleterious immune response in the host.

[0128] The T cells described herein can be engineered, for example, to not express a functional HLA on their surface. For example, the T cells described herein can be engineered to downregulate T cell surface expressed HLA, e.g., HLA class I and / or HLA class II. In some embodiments, the T cells can lack a functional TCR and a functional HLA, e.g., HLA class I and / or HLA class II.

[0129] Modified T cells lacking expression of a functional TCR and / or HLA can be obtained by any suitable means, including knocking out or knocking down one or more subunits of the TCR or HLA. For example, the T cells can include knockdown of the TCR and / or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR) transcription activator-like effector nuclease (TALEN), or zinc finger endonuclease (ZFN).

[0130] Natural killer cells In some embodiments, the immune cells are natural killer cells. Natural killer (NK) cells are CD56 CD3 large granular lymphocytes capable of killing infected and transformed cells and constitute an important cell subset of the innate immune system. Unlike cytotoxic CD8+ T lymphocytes, NK cells exert cytotoxicity against tumor cells without the need for prior sensitization and can also eradicate MHC-I-negative cells. In some embodiments, the NK cells are CD3-CD56+ CD7+CD127-NKp46+T-bet+Eomes+. In some embodiments, the cytotoxic NK cells CD56 dim CD16+.

[0131] Dendritic cells In some embodiments, the immune cells are dendritic cells. Dendritic cells are a heterogeneous group of specialized antigen-presenting cells that originate in bone marrow from CD34+ stem cells and express major histocompatibility complex (MHC) class II molecules. Mature dendritic cells can prime, activate, and expand effector immune cells, such as T cells and NK cells. Dendritic cell therapy is known in the art (see, for example, Sabado et al., 2017). Briefly, dendritic cells can be isolated from a patient, exposed to a disease-specific antigen, e.g., a cancer-specific antigen, or genetically modified to express a CAR or disease-specific antigen, and then infused back into the patient to prime, activate, and expand effector immune cells, e.g., T cells.

[0132] Bone marrow cells In some embodiments, the immune cells are myeloid cells. Granulocytes, monocytes, macrophages, and dendritic cells represent a subgroup of white blood cells collectively referred to as myeloid cells. They circulate through the blood and lymphatic system and are rapidly recruited to sites of tissue damage and infection via various chemokine receptors. Within tissues, they are activated for phagocytosis and secretion of inflammatory cytokines, thereby playing a major role in protective immunity. Bone marrow cell therapy is known in the art and may be useful for the treatment of cancer, infection, or disease. For example, myeloid cells are known to be abundant in the tumor stroma, and the presence of these cells may affect patient outcomes in many cancer types. Briefly, bone marrow cells may be isolated from a patient, exposed to disease-specific antigens, e.g., cancer-specific antigens, or genetically modified to express CARs or disease-specific antigens, and then infused back into the patient to prime, activate, and expand effector immune cells, e.g., T cells.

[0133] Macrophages In some embodiments, the immune cells are macrophages. Macrophages are myeloid cells that arise from bone marrow-derived monocyte precursors that differentiate into tissue macrophages, antigen-presenting dendritic cells, and bone-resorbing osteoclasts. Macrophage cell therapy is known in the art and can be useful for treating cancer, infection, or disease. Briefly, macrophages can be isolated from a patient, exposed to disease-specific antigens, such as cancer-specific antigens, or genetically modified to express CARs or disease-specific antigens, and then infused back into the patient to prime, activate, and expand effector immune cells, such as T cells.

[0134] Chimeric Antigen Receptor The term "chimeric antigen receptor" or alternatively "CAR" refers to a polypeptide or set of polypeptides which, when present in an immune cell, provides the cell with specificity for a target T cell, e.g., a cancer cell, and intracellular signal generation.

[0135] CAR can be used to generate immune cells, such as T cells, dendritic cells, or natural killer (NK) cells, specific to selected targets. Suitable constructs for generating CAR are described in US5,843,728, US5,851,828, US5,912,170, US6,004,811, US6,284,240, US6,392,013, US6,410,014, US6,753,162, US8,211,422, and WO9215322. Alternative CAR constructs can be characterized as belonging to successive generations. First generation CARs typically consist of a single chain variable fragment of an antigen-specific antibody, e.g., comprising a VL linked to the VH of a particular antibody, linked by a flexible linker, e.g., the CD8a hinge domain and the CD8a transmembrane domain, to the transmembrane and intracellular signaling domains of either CD3C or FcRy or scFv-FcRy (see, e.g., US 7,741,465, US 5,912,172, and US 5,906,936). Second generation CARs incorporate the intracellular domain of one or more costimulatory molecules, such as CD28, CD28z, OX40 (CD134), or 4-1BB (CD137), within the endodomain, e.g., scFv-CD28 / OX40 / 4BB-CD3 (see, e.g., US8,911,993, US8,916,381, US8,975,071, US9,101,584, US9,102,760, US9,102,761). Third generation CARs include combinations of costimulatory endodomains such as CD3C chains, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, or CD28 signaling domains, e.g., scFv-CD28-4BB-CD3C or scFv-CD28-OX40-CD3Q (see, e.g., US8,906,682, US8,399,645, US5,686,281, WO2014 / 134165, and WO2012 / 079000). In some embodiments, costimulation can be coordinated by expressing the CAR in antigen-specific T cells selected for activation and expansion, for example, following interaction with an antigen on a professional antigen presenting cell, along with the costimulation.For example, immune cells can be provided with additional engineered receptors to improve targeting of T cell attack and / or minimize side effects.

[0136] Methods for preparing CAR-expressing cells Cell Source Prior to expansion and possible genetic or other modification, a cell population comprising or consisting of immune cells, such as T cells, dendritic cells, natural killer (NK) cells, or combinations thereof, can be obtained from a subject. Immune cells can be obtained from a number of sources, such as peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors.

[0137] In certain embodiments of the present disclosure, immune cells, e.g., T cells, can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation. In a preferred embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, dendritic cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and, optionally, to place the cells in an appropriate buffer or medium for subsequent processing steps. In one embodiment, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the washing solution can lack calcium, lack magnesium, or lack many, but not all, divalent cations.

[0138] An initial activation step in the absence of calcium may lead to amplified activation. As will be readily understood by those skilled in the art, the washing step may be accomplished by methods known to those skilled in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) following the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0139] It is recognized that the methods of the present application can utilize culture medium conditions comprising 5% or less, e.g., 2% human AB serum, and can employ known culture medium conditions and compositions, e.g., those described in Smith et al. (2015).

[0140] In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation through a PERCOLL™ gradient or counterflow centrifugal elutriation.

[0141] The methods described herein can include, for example, the selection of a particular subpopulation of immune cells, e.g., T cells, that is a T regulatory cell depleted population. For example, a CD25+ depleted cell population can be obtained, for example, using negative selection techniques described herein. Preferably, the population of T regulatory depleted cells contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells. However, as discussed herein, AKT inhibitors and / or inhibitors of PH domain proteins used in the methods of the invention alone do not necessarily result in the depletion of T regulatory cells during culture. REG The cells can be reduced.

[0142] In one embodiment, T regulation (T REG) cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody or fragment thereof, or a CD25-binding ligand, IL-2. In one embodiment, the anti-CD25 antibody or fragment thereof, or the CD25-binding ligand, is conjugated to or otherwise coated on a substrate, e.g., a bead. In one embodiment, the anti-CD25 antibody or fragment thereof is conjugated to a substrate as described herein.

[0143] Without wishing to be bound by a particular theory, it is contemplated that reducing the levels of negative regulators of immune cells in a subject prior to apheresis or during manufacturing of the CAR-expressing cell product (e.g., reducing the levels of undesirable immune cells, e.g., T REG For example, reducing the number of T REG Methods for depleting cells are known in the art. REG Methods for reducing cells include, but are not limited to, cyclophosphamide, anti-GITR antibodies (anti-GITR antibodies described herein), CD25-depletion, and combinations thereof.

[0144] In some embodiments, the manufacturing method includes the step of: REG For example, the manufacturing method may include contacting a sample, e.g., an apheresis sample, with an anti-GITR antibody and / or an anti-CD25 antibody (or fragment thereof, or a CD25-binding ligand) to, e.g., reduce the number of cells (e.g., deplete) prior to manufacturing a CAR-expressing cell (e.g., T cell, NK cell) product. REG This involves depleting the cells.

[0145] In one embodiment, the methods of the invention do not include sorting the cultured cells to isolate CD45RO- CCR7- CD62L- T memory cells.

[0146] T cells for stimulation can also be frozen after a washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more homogenous product by removing granulocytes and to some extent monocytes within the cell population. After a washing step that removes plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and useful in this context, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A, and then the cells are frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing can also be used, along with uncontrolled freezing at close to -20°C or in liquid nitrogen.

[0147] In certain embodiments, cryopreserved cells are thawed as described herein, washed, and allowed to rest at room temperature for 1 hour before being activated using the methods of the invention.

[0148] In the context of the present invention, collection of blood samples or apheresis products from a subject at a time period before the expanded cells described herein may be needed is also contemplated. Thus, a source of expanded cells can be collected at any time needed, and desired cells, such as T cells, can be isolated and frozen for later use in immune cell therapy for any number of diseases or conditions that would benefit from immune cell therapy, such as those described herein. In one embodiment, a blood sample or apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or apheresis is taken from a generally healthy subject who is at risk of developing a disease but has not yet developed the disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, T cells can be expanded, frozen, and later used. In certain embodiments, a sample is collected from a patient immediately after diagnosis of a particular disease described herein and prior to any treatment. In further embodiments, the cells are isolated from a blood sample or apheresis from the subject prior to any number of relevant therapeutic modalities, including, but not limited to, treatment with drugs such as natalizumab, efalizumab, antivirals, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation.

[0149] Methods for generating CAR-expressing cells In an embodiment, the method of the present invention includes generating a CAR-expressing cell by introducing a vector or nucleic acid encoding a CAR into a cell. Methods for introducing and expressing genes into cells are known in the art. In the context of an expression vector, the vector can be easily introduced into a host T cell, e.g., a mammalian, bacterial, yeast, or insect T cell, by any method in the art. For example, the expression vector can be transferred into the host T cell by physical, chemical, or biological means.

[0150] Physical methods for introducing a polynucleotide into a host T cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art (see, e.g., Sambrook Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press). A preferred method for introducing a polynucleotide into a host T cell is calcium phosphate transfection.

[0151] Biological methods for introducing the polynucleotide of interest into host T cells include the use of DNA and RNA vectors.Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells.Other viral vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus, and adeno-associated virus, etc. (see, for example, US5,350,674 and US5,585,362).

[0152] Chemical means for introducing polynucleotides into host T cells include macromolecular complexes, nanocapsules, microspheres, beads, and colloidal dispersion systems, such as lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides by targeted nanoparticles or other suitable submicron-sized delivery systems.

[0153] An exemplary non-viral delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acids into host T cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. The nucleic acid associated with the lipid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, contained as a suspension in lipid, contained in or complexed with a micelle, or otherwise associated with lipid. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure, as micelles, or in a "collapsed" structure. They may also simply be interspersed in the solution, and in some cases may form aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include the naturally occurring lipid droplets in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma Aldrich, dicetyl phosphate ("DCP") can be obtained from K&K Laboratories, cholesterol ("Choi") can be obtained from Calbiochem-Behring, and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained, for example, from Avanti Polar Lipids, Inc. Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol."Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium.

[0154] Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components self-rearrange before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991). However, compositions that have structures different from normal vesicular structures in solution are also encompassed. For example, lipids may assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine nucleic acid complexes are also contemplated. Regardless of the method used to introduce exogenous nucleic acid into host T cells or otherwise expose the cells to the inhibitors of the present invention, various assays may be performed to confirm the presence of recombinant DNA sequences in the host T cells. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of particular peptides by immunological means (ELISA and Western blot) or by the assays described herein to identify agents within the scope of the invention.

[0155] Methods for culturing and expanding immune cells Immune cells, such as T cells, can generally be activated and expanded using methods described, for example, in US6,352,694, US6,534,055, US6,905,680, US6,692,964, US5,858,358, US6,887,466, US6,905,681, US7,144,575, US7,067,318, US7,172,869, US7,232,566, US7,175,843, US5,883,223, US6,905,874, US6,797,514, US6,867,041, and US2006 / 0121005.

[0156] Expanding T cells by the methods disclosed herein can expand the cells by about 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 200x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x, 2000x, 3000x, 4000x, 5000x, 6000x, 7000x, 8000x, 9000x, 10,000x, 100,000x, 1,000,000x, 10,000,000x or more, and all integers or sub-integers therebetween. In one embodiment, the T cells are expanded in the range of about 20x to about 50x.

[0157] In one embodiment, the cells are cultured for about 7 to about 14 days, or about 7 to about 10 days.

[0158] In general, a population of immune cells, e.g., T regulatory cell depleted cells, can be expanded by contacting the surface bound thereto with an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells. Specifically, the T cell population can be stimulated as described herein, such as by contacting with an anti-CD3 antibody or an antigen-binding fragment thereof or an anti-CD2 antibody immobilized on the surface, or by contacting with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. For costimulation of accessory molecules on the surface of the T cells, a ligand that binds to the accessory molecule is used. For example, the population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate proliferation of the T cells. Anti-CD3 antibody and anti-CD28 antibody can be used to stimulate proliferation of either CD4+ T cells or CD8+ T cells. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), which may be used as well as others commonly known in the art (Berg et al., 1998; Haanen et al., 1999; Garland et al., 1999).

[0159] Suitable conditions for immune cell culture include an appropriate medium (e.g., Minimal Essential Medium or RPMI Media 1640, or X-vivo 15 (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF, and TNF-a, or any other additives for cell growth known to those of skill in the art. Other additives for cell growth include, but are not limited to, detergents, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Media may include RPMI1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, supplemented with amino acids, sodium pyruvate, and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokines in sufficient amounts for T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells injected into subjects. Target T cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2).

[0160] CAR-expressing cell therapy CAR-T cell therapy CAR-T cell therapy is a type of cell therapy in which immune cells (e.g., T cells) are genetically modified to express a CAR, and the CAR-expressing cells (e.g., CAR-T cells) are infused into a recipient in need thereof. The infused cells can kill diseased cells expressing the target of the CAR in the recipient. Unlike antibody therapy, CAR-modified immune cells (e.g., CAR-T cells) can replicate in vivo, resulting in long-term persistence that can result in sustained tumor control. In various embodiments, the CAR-T cells are administered to a patient, and the CAR-T cells or their progeny persist in the patient for at least 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 2 years, 3 years, 4 years, or 5 years after administration of the CAR-T cells to the patient.

[0161] The present invention also includes a type of cell therapy in which immune cells (e.g., T cells) are modified to transiently express a chimeric antigen receptor (CAR), for example, by in vivo transcribed RNA, and the CAR-T cells are infused into a recipient in need thereof. The infused cells can kill tumor cells in the recipient. Thus, in various embodiments, the immune cells (e.g., CAR-T cells) administered to the patient are present for less than one month, for example, three weeks, two weeks, one week, after the CAR-T cells are administered to the patient. Without wishing to be bound by any particular theory, the anti-tumor immune response elicited by the CAR-T cells may be an active or passive immune response, or alternatively, may result from a direct versus indirect immune response.

[0162] As mentioned above, ex vivo procedures are well known in the art and are described above. Briefly, cells are isolated from a mammal (e.g., human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR. The CAR-expressing cells (e.g., CAR-T cells) can be administered to a mammalian recipient to provide a therapeutic effect. The mammalian recipient can be a human, and the CAR-expressing cells can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic, or xenogeneic with respect to the recipient.

[0163] Procedures for ex vivo expansion of hematopoietic stem and progenitor cells are described in US 5,199,942 and can be applied to the cells of the present invention. Other suitable methods are known in the art, and therefore the present invention is not limited to any particular method of ex vivo expansion of cells. Briefly, ex vivo culture and expansion of immune cells (e.g., T cells) includes (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammal from a peripheral blood draw or bone marrow explant, and (2) expanding such cells ex vivo. In addition to the cell growth factors described in US 5,199,942, other factors such as flt3-L, IL-1, IL-3, and c-kit ligand can be used for cell culture and expansion.

[0164] The CAR-T cells of the present invention can be administered as described herein either alone or as a pharmaceutical composition in combination with a diluent and / or in combination with other components such as IL-2 or other cytokines or cell populations. The immune cells can be administered either alone or as a pharmaceutical composition in combination with a diluent and / or in combination with other components such as IL-2, IL-15 or other cytokines or cell populations. Briefly, a pharmaceutical composition can include the immune cells described herein in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers such as neutral buffered saline, phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; amino acids such as polypeptides or glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions for use in the disclosed methods are, in some embodiments, formulated for intravenous administration.

[0165] The pharmaceutical composition comprising the cells described herein may be administered in a dose of 10 5 ~10 6 10 cells / kg body weight etc. 4 ~10 9 The cell composition may be administered at a dosage of 1000 to 10000 cells / kg body weight (including all integer values ​​within these ranges). The cell composition may be administered multiple times at these dosages. The cells may be administered by using injection techniques commonly known in immunotherapy (see, for example, Rosenberg et al., 1988). Optimal dosages and treatment regimens for a particular patient can be readily determined by one of ordinary skill in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0166] In certain embodiments, it may be desirable to administer activated immune cells to a subject, then subsequently redraw blood (or perform apheresis), activate and expand immune cells from the blood, and reinfuse these activated and expanded cells into the patient. This process can be performed multiple times every few weeks. In certain embodiments, immune cells can be activated from a blood draw of 10cc to 400cc. In certain embodiments, immune cells are activated from a blood draw of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 100cc. Using this multiple blood draw / multiple reinfusion protocol can function to select for certain populations of immune cells.

[0167] Combination therapy Immune cells, such as the CAR-T cells of the invention, or produced by the methods of the invention, can be administered a variety of therapeutic options, including, but not limited to, a PRLR antagonist (e.g., an anti-PRLR antibody or a small molecule inhibitor of PRLR), an EGFR antagonist (e.g., an anti-EGFR antibody [e.g., cetuximab or panitumumab] or a small molecule inhibitor of EGFR [e.g., gefitinib or erlotinib]), an antagonist of another EGFR family member, such as Her2 / ErbB2, ErbB3, or ErbB4 (e.g., an anti-ErbB2 [e.g., trastuzumab or T-DM1], anti-ErbB3, or anti-ErbB4 antibody, or a small molecule inhibitor of ErbB2, ErbB3, or ErbB4 activity), a cMET antagonist, agonists (e.g., anti-cMET antibodies), IGF1R antagonists (e.g., anti-IGF1R antibodies), B-raf inhibitors (e.g., vemurafenib, sorafenib, GDC-0879, PLX-4720), PDGFR-alpha inhibitors (e.g., anti-PDGFR-alpha antibodies), PDGFR-beta inhibitors (e.g., anti-PDGFR-beta antibodies, or small molecule kinase inhibitors such as imatinib mesylate or sunitinib malate), PDGF ligand inhibitors (e.g., anti-PDGF-A, -B, -C, or -D antibodies, aptamers, siRNA, etc.), VEGF antagonists (e.g., VEGF-Trap such as aflibercept, e.g., US 7,087,See US 2009 / 0142354, such as REGN421), Ang2 antagonists (e.g., anti-Ang2 antibodies disclosed in US 2011 / 0027286, such as H1H685P), FOLH1 antagonists (e.g., anti-FOLH1 antibodies), STEAP1 or STEAP2 antagonists (e.g., anti-STEAP1 antibodies or anti-STEAP2 antibodies), TMPRSS2 antagonists (e.g., anti-TMPRSS2 antibodies), MSLN antagonists (e.g., anti-VEGF antibodies), anti-VEGF antibodies (e.g., anti-VEGF antibodies), anti-VEGF receptor kinase inhibitors (e.g., sunitinib, sorafenib, or pazopanib), DLL4 antagonists (e.g., anti-DLL4 antibodies disclosed in US 20009 / 0142354, such as REGN421), Ang2 antagonists (e.g., anti-Ang2 antibodies disclosed in US 2011 / 0027286, such as H1H685P), FOLH1 antagonists (e.g., anti-FOLH1 antibodies), STEAP1 or STEAP2 antagonists (e.g., anti-STEAP1 antibodies or anti-STEAP2 antibodies), TMPRSS2 antagonists (e.g., anti-TMPRSS2 antibodies), MSLN antagonists (e.g., anti-VEGF receptor kinase inhibitors), anti-VEGF receptor kinase inhibitor ... For example, the therapeutically active agent may be co-formulated with and / or administered in combination with one or more additional therapeutically active ingredients selected from the group consisting of an anti-MSLN antibody), a CA9 antagonist (e.g., an anti-CA9 antibody), a uroplakin antagonist (e.g., an anti-uroplakin [e.g., anti-UPK3A] antibody), a MUC16 antagonist (e.g., an anti-MUC16 antibody), a Tn antigen antagonist (e.g., an anti-Tn antibody), a CLEC12A antagonist (e.g., an anti-CLEC12A antibody), a TNFRSF17 antagonist (e.g., an anti-TNFRSF17 antibody), an LGR5 antagonist (e.g., an anti-LGR5 antibody), a monovalent CD20 antagonist (e.g., a monovalent anti-CD20 antibody such as rituximab), a PD-1 antibody, a PD-L1 antibody, a CD3 antibody, a CTLA-4 antibody, and the like. Other agents that may be beneficially administered in combination with the CAR-T cells of the invention include, for example, tamoxifen, aromatase inhibitors, and cytokine inhibitors, including small molecule cytokine inhibitors and antibodies that bind to cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-11, IL-12, IL-13, IL-17, IL-18, or their respective receptors.

[0168] Immune cells, such as the CAR-T cells of the present invention, optionally produced by the methods of the present invention, may be used in combination with checkpoint inhibitors. In another example, an AKT inhibitor and / or an inhibitor of a PH domain protein may be administered with the checkpoint inhibitor. Two known inhibitory checkpoint pathways involve signaling through the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death 1 (PD-1) receptors. These proteins are members of the CD28-B7 family of co-signaling molecules that play important roles throughout all stages of T-cell function. The PD-1 receptor (also known as CD279) is expressed on the surface of activated T cells. Its ligands, PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273), are expressed on the surface of APCs, such as dendritic cells or macrophages. While PD-L1 is the predominant ligand, PD-L2 has a much more restricted expression pattern. When the ligand binds to PD-1, an inhibitory signal is delivered to the T cell, reducing cytokine production and suppressing T cell proliferation. Checkpoint inhibitors include, but are not limited to, antibodies that block PD-1 (nivolumab (BMS-936558 or MDX1106), CT-011, MK-3475), PD-L1 (MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (rHlgM12B7), CTLA-4 (ipilimumab (MDX-010), tremelimumab (CP-675,206)), IDO, B7-H3 (MGA271), B7-H4, TIM3, LAG-3 (BMS-986016).

[0169] In some embodiments, the PD-L1 inhibitor comprises an antibody that specifically binds to PD-L1, such as BMS-936559 (Bristol-Myers Squibb) or MPDL3280A (Roche). In some embodiments, the PD1 inhibitor comprises an antibody that specifically binds to PD1, such as lambrolizumab (Merck), nivolumab (Bristol-Myers Squibb), or MED14736 (AstraZeneca). Human monoclonal antibodies against PD-1 and methods for treating cancer using anti-PD-1 antibodies alone or in combination with other immunotherapeutic agents are disclosed in U.S. Pat. No. 8,008,449, which are incorporated by reference as to these antibodies. Anti-PD-L1 antibodies and their uses are thus described in U.S. Pat. No. 8,552,154, which are incorporated by reference as to these antibodies. Anti-cancer agents comprising anti-PD-1 or anti-PD-L1 antibodies are described in U.S. Pat. No. 8,617,546, which are incorporated by reference as to these antibodies.

[0170] The present invention includes compositions and therapeutic formulations comprising any of the immune cells, such as CAR-T cells, described herein in combination with one or more chemotherapeutic agents. Exemplary chemotherapeutic agents include alkylating agents, such as thiotepa and cyclosphosphamide (Cytoxan™); alkylsulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, metholedopa, and uredopa; ethyleneimines and methylamelanamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenephosphoramide, and trimethylmelamine; chlorambucil, ... Nitrogen mustards such as nafazine, colofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide, melphalan, nobemitin, phenesterine, prednimustine, trophosphamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, and calicheamaycin. isin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potofilomycin, puromycin, keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubeni Antibiotics such as mexamex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluoracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;Androgens such as calcineurin, drostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamon; ol; nitracrine; pentostatin; phenameth; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g., paclitaxel (Taxol™, Bristol-Myers Squibb Oncology, Princeton, NJ) and docetaxel (Taxotere™, Aventis Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid; esperamycin; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition includes, for example, antiestrogens, including tamoxifen, raloxifene, aromatase-inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxyfene, keoxyfene, LY117018, onapristone, and toremifene (Fareston);and anti-hormonal agents that act to regulate or inhibit hormone action on tumors, such as anti-androgens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, as well as pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0171] Administration of any of the disclosed therapeutic agents may be performed in any convenient manner, including injection, transfusion, or implantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramuscularly, intravenously (iv) injection, or intraperitoneally. In some embodiments, the disclosed compositions are administered by iv injection. The compositions may also be injected directly into a tumor, lymph node, or site of infection.

[0172] As will be understood by those skilled in the art, the cells are administered to a subject in a therapeutically effective amount. As used herein, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a therapeutic agent administered to alleviate to some extent one or more of the symptoms of the disease or condition being treated, or to prevent the worsening. The result may be a reduction in the signs, symptoms, or causes of a disease, or prevention of progression, or any other desired change in a biological system. For example, an "effective amount" for therapeutic use is the amount of a therapeutic agent necessary to produce a clinically significant reduction in disease symptoms without undue adverse side effects.

[0173] The term "therapeutically effective amount" includes, for example, a prophylactically effective amount. An "effective amount" of a therapeutic agent is an amount effective to achieve a desired pharmacological effect or therapeutic improvement without undue adverse side effects. It is understood that an "effective amount" or "therapeutically effective amount" may vary from subject to subject due to variations in the subject's age, weight, general condition, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician, as well as variations in metabolism of any compound.

[0174] It is believed to be within the skill of the art to determine such therapeutically effective amounts by routine experimentation, including but not limited to, a dose escalation clinical trial. An appropriate "effective amount" in any individual case may be determined using techniques such as a dose escalation study.

[0175] When one or more therapeutic agents are used in combination, a "therapeutically effective amount" of each therapeutic agent can refer to the amount of that therapeutic agent that would be therapeutically effective when used by itself, or can refer to a reduced amount that is therapeutically effective in combination with one or more additional therapeutic agents.

[0176] Treatment methods Immune cells, e.g., CAR-T cells, of the invention or produced using the invention are useful, inter alia, for the treatment, prevention, and / or amelioration of diseases or disorders. For example, the CAR-T cells of the invention are useful for the treatment of cancer, infectious diseases, or inflammatory diseases. As another example, dendritic cells produced by the methods of the invention can be used as dendritic cell vaccines (see, e.g., Datta et al., 2014) for treating, e.g., cancer, infectious diseases (such as bacterial or viral infections), or autoimmune diseases (such as diabetes). As a further example, NK cells, such as NK-CAR cells, can be used to treat cancer (see, e.g., Liu et al., 2021).

[0177] CAR-T cells may be used to treat primary and / or metastatic tumors arising in the brain and meninges, oropharynx, lungs and bronchial tree, gastrointestinal tract, male and female reproductive tract, muscle, bone, skin and adnexa, connective tissue, spleen, immune system, blood forming cells and bone marrow, liver and urinary tract, and special sensory organs such as the eye. In certain embodiments, the CAR-T cells of the present invention are used to treat one or more of renal cell carcinoma, pancreatic cancer, head and neck cancer, prostate cancer, malignant glioma, osteosarcoma, colorectal cancer, gastric cancer (e.g., gastric cancer with MET amplification), malignant mesothelioma, multiple myeloma, ovarian cancer, small cell lung cancer, non-small cell lung cancer, synovial sarcoma, thyroid cancer, breast cancer, melanoma, leukemia, or lymphoma.

[0178] In one embodiment, the CAR-T cells of the present invention are used to treat leukemia, such as acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, or chronic lymphocytic leukemia. In one embodiment, the leukemia is acute myeloid leukemia with a predominance of low CD33+ blasts.

[0179] In another embodiment, the CAR-T cells of the present invention are used to treat lymphoma, such as Hodgkin's lymphoma or non-Hodgkin's lymphoma. Types of non-Hodgkin's lymphoma include diffuse large B-cell lymphoma, anaplastic large cell lymphoma, Burkitt's lymphoma, lymphoblastic lymphoma, mantle cell lymphoma, or peripheral T-cell lymphoma. In one embodiment, the lymphoma is diffuse large B-cell lymphoma or non-Hodgkin's lymphoma with low levels of CD19 and / or CD20.

[0180] In the context of the methods of treatment described herein, immune cells such as CAR-T cells may be administered as a monotherapy (i.e., as the only therapeutic agent) or in combination (combination therapy) with one or more additional therapeutic agents (examples of which are described elsewhere herein).

[0181] In one embodiment, a subject is at risk for developing cancer (e.g., cancer). A subject is at risk if the subject has a higher risk of developing cancer than a control population. The control population may include one or more subjects randomly selected (e.g., matched by age, sex, race, and / or ethnicity) from the general population who are cancer-free or have a family history of cancer. A subject can be considered at risk for cancer if a "risk factor" associated with cancer is found to be associated with the subject. Risk factors may include any activity, trait, event, or characteristic associated with a given disorder, for example, through statistical or epidemiological studies of a population of subjects. Thus, a subject can be classified as at risk for cancer even if the subject has not been specifically included in a study that identifies the underlying risk factor.

[0182] In one embodiment, the subject is at risk for developing cancer and the cells or compositions are administered prior to or after the onset of symptoms of the cancer. In one embodiment, the cells or compositions are administered prior to the onset of symptoms of the cancer. In one embodiment, the cells or compositions are administered after the onset of symptoms of the cancer. In one embodiment, the cells or compositions of the invention are administered in a dose that alleviates or reduces one or more of the symptoms of the cancer in the at-risk subject.

[0183] In some embodiments, the subject has been diagnosed with or is suspected of having a disease or disorder, such as cancer, an infectious disease, or an inflammatory disease. In some embodiments, the subject has been diagnosed with or is suspected of having colon cancer or breast cancer. In some embodiments, the methods described herein include diagnosing the subject as having or being suspected of having a disease or disorder, such as cancer, an infectious disease, or an inflammatory disease, preferably colon cancer or breast cancer.

[0184] As used herein, diagnosis refers to the determination that a subject or patient is in need of treatment with the CAR-T cells of the present invention and / or an AKT inhibitor and / or an inhibitor of a PH domain protein. The type of disease or disorder diagnosed according to the methods described herein can be any type known in the art or described herein.

[0185] In one embodiment, the step of identifying or diagnosing a subject in need of treatment with the CAR-T cells of the present invention and / or an AKT inhibitor and / or an inhibitor of a PH domain protein comprises determining that the subject has cancer, -Blood profiling; - cytological biopsy aspirate; -imaging such as computed tomography (CT) scans, bone scans, magnetic resonance imaging (MRI), positron emission tomography (PET) scans, ultrasound and x-rays; - May include one or more or all of the following evaluations: physical examination.

[0186] Examples of diseases that can be treated with NK cells include, but are not limited to, cancer (e.g., melanoma, prostate cancer, breast cancer, and liver cancer), as well as viral infections (e.g., infections with HSV, hepatitis viruses, human cytomegalovirus, influenza viruses, flaviviruses, and HIV-1), bacterial infections (e.g., infections with mycobacteria, listeria, and staphylococci), and protozoal infections (e.g., infections with malaria parasites), and fungal infections (e.g., infections with Aspergillus).

[0187] As will be clear to those skilled in the art, the "reduction" of cancer symptoms in a subject will be compared with another subject that also suffers from cancer but is not treated by the methods described herein.This does not necessarily require a control comparison of two subjects.Rather, population data can be relied upon.For example, a population of subjects with cancer that are not treated by the methods described herein (optionally a population of subjects similar to the treated subjects, e.g., age, weight, race) is evaluated, and the average value is compared with the results of a subject or a population of subjects that are treated by the methods described herein.

[0188] In one embodiment, the CAR-T cells and methods of the present invention are used to improve survival of subjects suffering from a disease or disorder, such as cancer, an infectious disease, or an inflammatory disease. When survival is contemplated, survival analysis can be performed using the Kaplan-Meier method (as shown in Figure 26C). The Kaplan-Meier method can be used to estimate a survival function from survival data and measure the proportion of patients who survive a certain period of time after treatment. A Kaplan-Meier plot of a survival function is a series of horizontal steps of decreasing magnitude that, when a large enough sample is taken, approaches the true survival function of the population. The value of the survival function between successive distinct sampled observations ("clicks") is assumed to be constant.

[0189] An important advantage of Kaplan-Meier curves is that the method can account for "censored" data loss from samples before the final outcome is observed (e.g., when a patient drops out of the study). In the plots, small vertical check marks indicate censored losses of patient data. When no truncation or censoring occurs, Kaplan-Meier curves are equivalent to the empirical distribution.

[0190] In statistics, the log-rank test (also known as the Mantel-Cox test) is a hypothesis test that compares the survival distributions of two groups of patients. It is a nonparametric test and is suitable for use when the data are correctly censored. It is widely used in clinical trials to establish the efficacy of new drugs compared to a control group when the measurement is the time to event. The log-rank test statistic compares estimates of the hazard function of two groups at each observed event time. It is constructed by calculating the number of observed and expected events in one of the groups at each observed event time, and then adding these together to obtain an overall overview across all time points where there are events. The log-rank statistic can be derived as a score test of the Cox proportional hazards model that compares the two groups. It is therefore asymptotically equivalent to the likelihood ratio test statistic based on that model. EXAMPLES

[0191] Example 1 - Materials and Methods Mouse CAR-T Manufacturing Protocol Mouse splenocytes were activated using CD3 / CD28 antibodies and cultured in the presence of recombinant IL-2 and IL-7 for 24 hours prior to transduction with CAR-T vectors. TCN (triciribine) or TCN-P (triciribine phosphate) was added immediately after transduction, and CAR-T cells were then exposed to TCN / TCN-P for either 24, 48, or 72 hours.

[0192] Human PBMCs were extracted from buffy packs (Australian Red Cross Blood Service). They were activated using anti-CD3 antibody (OKT3) for 2 days prior to transduction for 48 hours, all in the presence of IL-2. TCN or TCN-P was immediately added to these CAR-T cells cultured in IL-2 for up to 3 days.

[0193] Tumor killing assay 100,000 E0771-Her2 tumor cells were seeded into each well of a 96-well plate and maintained at 37°C and 5% CO2. After 2 hours, mouse CAR-T cells were seeded into the same wells at effector-to-target T cell ratios of 2:1, 1:1, and 0.5:1 and incubated for up to 16 hours. Culture supernatants were collected and interferon gamma (IFNγ) and tumor necrosis factor alpha (TNFα) levels were measured.

[0194] Flow cytometry phenotyping To phenotype mouse CAR-T cells post-manufacture, cells were stained using fluorochrome-conjugated antibodies against CD4, CD8, CD44, and CD62L. Human CAR-T cells were phenotyped using fluorochrome-conjugated antibodies against CD4, CD8, CD45RA, CD45RO, CD44, CD62L, CCR7, CD27, PD-1, and CD69. Fixable live / dead dyes were used to distinguish live or dead cells.

[0195] mass spectrometry Human CAR-T cells were harvested after treatment with either TCN or TCN-P for 24 or 3 days. At each time point, cells were harvested and washed three times with cold DPBS before cell lysis for global proteomic analysis. Specifically, for phosphoproteomic analysis, human CAR-T cells were left untreated or washed with DPBS and then treated with TCN or TCN-P for 0, 5, and 15 minutes before cell lysis.

[0196] Mass spectrometry of cellular proteomes and phosphoproteomes Cells containing protease and phosphatase inhibitors (HALT) were homogenized / solubilized by tip-probe sonication, quantified, normalized, and reduced (dithiothreitol, DTT) and alkylated (iodoacetamide) prior to Sera-Mag Speed ​​Bead-based protein digestion with LysC (enzyme:substrate 1:100, Wako Pure Chemical Industries) trypsin (enzyme:substrate 1:50). Tandem mass tag (TMT) multiplexing was performed on the normalized peptide mixture (9-plex TMT, reference 131C isobaric labeling). Peptides were desalted (SDB-RPS stage chip) and analyzed for the global cellular proteome. For phosphoproteome analysis, phosphopeptides were enriched from each TMT set using highly selective titanium dioxide (TiO2) bead capture.

[0197] Spectra acquired by data-dependent acquisition on an Orbitrap Q-Exactive HF-X mass spectrometer coupled to an Easy-nLC1200 (Thermo Fisher Scientific) ultra-high pressure liquid chromatography (UHPLC) pump. Peptides were separated by direct injection at 55 °C with a gradient 5-100% buffer B (80% ACN, 0.1% FA) over 240 min at a flow rate of 300 nL / min (1.9 μm particle size C18, 0.075 × 250 mm, Nikkyo Technos Co. Ltd). The scan sequence included MS1 spectra (resolution 60,000; mass range 300-1650 m / z; automatic gain control (AGC) target 3e6, maximum injection time 128 ms, isolation window 0.8 Th). The most intense MS1 ions were selected for MS2 analysis and fragmented by high-energy collisional dissociation with a normalized collision energy of 33. Precursors were filtered according to charge state ≧2, and for MS / MS the AGC was set to 1e5 and monoisotopic peaks were used.

[0198] Data processing and bioinformatics pipeline Mass spectra were pre-processed and processed using MaxQuant (1.6.14). Spectra were searched against the entire set of human protein sequences annotated in UniProt (sequence database Jan-2021) using Andromeda. Data were searched for fixed modifications, cysteine ​​carbamidomethylation and variable modifications, N-acetylation and methionine oxidation (as well as phosphorylation (STY)). Searches were performed using 20 ppm precursor ion tolerance for total protein / phosphoprotein level analysis, and an internal reference label channel to normalize for batch effects. Further modifications included a TMT tag on the peptide N-terminus / lysine residue (+229.16293 Da), which was set as a static modification. A stringent 1% false discovery rate was applied to filter poor identifications at the peptide and protein level. The obtained p-values ​​were corrected by the Benjamini-Hochberg multiple testing correction method for multiple comparisons.

[0199] For further data analysis, normalized intensities were converted to log2 ratios of intensities over the median intensity of each protein measured across each sample group, and statistical analysis was performed using Student's T-test or ANOVA (p-values ​​< 0.05 were considered significant). Data analysis was performed using Microsoft Office Excel, R (ggPlot2), and Perseus (Max-Planck Institute of Biochemistry, Department of Proteomics and Signal Transduction, Munich) software. Gene enrichment functional annotation clustering analysis was performed using Gprofiler / Reactome bioinformatics. All data were normalized to the internal reference TMT channel and comparisons were made to either DMSO and untreated controls (proteome analysis), and DMSO control at time 0 (phosphoproteome analysis). PH-containing domains were obtained from the SMART online software tool (http: / / smart.embl-heidelberg.de / ).

[0200] Example 2 - Results Addition of 50 μM TCN or TCN-P either during or after the transduction process resulted in at least 80% CAR-T cell death (Figure 1). However, lower concentrations of TCN or TCN-P (up to 10 μM, less than 50 μM) maintained high levels of viable CAR-T cells (approximately 60%) even up to 3 days of treatment. TCN-pretreated mouse CAR-Ts showed a lower proliferation rate (Figure 2) while retaining a central memory phenotype (CD44 hi CD62L hi ), while producing slightly reduced levels of IFNγ and TNFα, retained cytotoxicity (FIGS. 4A-B).

[0201] When measuring activation markers in mouse CAR-T cells, we observed that TCN pretreatment increased the expression of classical early activation markers PD-1 and CD69 (Figure 5A-B). Tumor antigen-directed cytotoxicity, albeit blunted, is preserved after TCN pretreatment (Figure 6A). In contrast, survival of mouse CD8+ CAR-T cells was only slightly improved with TCN pretreatment (Figure 6B).

[0202] Based on these outcomes in mouse CAR-T cells, the same protocol was used to generate CAR-T cells from human PBMCs. Buffy coats from three donors were processed to isolate PBMCs, and 50 million PBMCs were cultured for 2 days in the presence of medium containing anti-CD3 antibody (OKT3, 1.5 μg / mL) and recombinant IL-2 (600 U / mL). Activated T cells were then subjected to a 2-day retroviral transduction protocol with a Her2-targeted CAR construct before being treated with 5 μM TCN or vehicle (DMSO) in the presence of 600 U / mL recombinant IL-2. Samples were collected after either 24 or 72 hours. Here, it was observed that TCN treatment did not affect the transduction efficiency of CD4+ or CD8+ T cells (Figure 7).

[0203] Interestingly, TCN treatment consistently reduced central memory T (T CM ) cells (Figure 8). When quantified across three PBMC donors, this T CM The increase in effector T cells (T E Note that the increase in T was accompanied by a reduction in T (8% vs. 5%) (FIG. 9, 11% vs. 17%). FIG. 9A shows quantification data where the data are representative of each biological donor (i.e., individual PBMC donors), and FIG. 9B shows the T in response to vehicle (DMSO), TCN, or TCN-P. CM This short exposure of CAR-T cells to TCN or TCN-P also resulted in the development of effector T (T E ) cells (35% vs. 23%) for at least 3 days. CM Note that the phenotype of the T cells was significantly higher than that of the control cells (Figures 10-11, 9% vs. 16%). CM Additional markers of T cells (i.e., CD45RO and CCR7) were included to demonstrate T cell proliferation in CD8+ CAR-T after conditioning with TCN or TCN-P. CM A similar pattern was observed with an increase in T cell proliferation and T cell proliferation in CD4+ CAR-T cells (Figure 12). Interestingly, pretreatment with TCN or TCN-P significantly increased T cell proliferation and T cell proliferation in CD4+ CAR-T cells. CM Or T E The results showed that T CM This could have been due to the relatively high starting abundance of CD4+ CAR-T cells (45–60% CD62L+CD45RO+ CD4+ CAR-T cells, Figure 14A).

[0204] Regulating immune tolerance in the tumor microenvironment (T REG ) cells, T in transduced human CAR-T cells REG The relative percentage of T cells was also evaluated, where TCN pretreatment increased the T cell proliferation rate in CD4+ transduced CAR-T cells. REGIt was observed that TCN or TCN-P treatment of PBMCs increased the enrichment of proteins associated with interferon signaling and antiviral activity (Figure 17). These proteins included MX1-interferon-inducible GTP-binding protein Mx1, guanylate-binding protein-1 and -2, and DnaJ homology subfamily B member 1. Antiviral activity is also suggested by the upregulation of LRRC59, which is essential for nucleic acid sensing and TLR-3, 8, and -9 signaling. TCN or TCN-P treatment of PBMCs enriched for proteins associated with metabolism and RNA processing.

[0205] Significant dephosphorylation of multiple PH domain-containing and non-PH domain-containing proteins, likely acted upstream of them by TCN or TCN-P, including serine / threonine-protein kinase PRP4 homolog, spectrin beta chain, and non-erythroid 1, which are important for regulatory T cell function via inhibition of TGFβ signaling. Data showed that the following proteins, oxysterol binding protein 1, oxysterol binding protein 2, spectrin beta chain, non-erythroid 1, and Rho GTPase-activating protein 27, were significantly inhibited after TCN administration.

[0206] conclusion Pretreatment with TCN or TCN-P allowed enrichment in T cell phenotypes that have been shown to persist in vivo and correspond to clinical responses following CAR-T therapy. Given previous reports of toxicity to T cells when used as AKT inhibitors (see, e.g., Mousset et al., 2018), the findings of the current study were surprising. Very low concentrations of both TCN and TCN-P were well tolerated by transduced T cells and enriched for T cell phenotypes, including CCR7+CD45RO+CD8+ T cells. CM Furthermore, pretreatment with low concentrations of TCN or TCN-P was effective in enriching T cells with the CAR-T phenotype. REG The cells were reduced.

[0207] Taken together, these findings demonstrate that TCN or TCN-P pretreatment is an effective method to enhance the efficacy of CAR-T therapy by enriching for T cell phenotypes known to persist in vivo and associated with partial or complete clinical responses. TCN and TCN-P pretreatment appear to have a major effect on CD8+ CAR-T cells and little or no effect on CD4+ CAR-T cells. The data further showed that the inhibitors can be used to increase dendritic cell numbers and NK cell cytotoxic activity.

[0208] Example 3 - Materials and methods for testing the efficacy of TCN and TCN-P as pretreatments or neoadjuvants in vivo We next investigated the in vivo T CM and CAR-T SCM We sought to determine the effect of AKT inhibitors on phenotypic enhancement. The first approach was to induce CAR-T activation in either endogenous CAR-T or adoptively transferred CAR-T according to the schematic illustrated in FIG. CM and CAR-T SCM The second approach was to test the effect of AKT inhibitors when used as manufacturing reagents to enrich for phenotypes. The second approach was to test the effect of AKT inhibitors when used as manufacturing reagents to enrich for phenotypes in either endogenous CAR-T or adoptively transferred CAR-T according to the schematic illustrated in FIG. CM and CAR-T SCM The aim of this study was to test the effect of AKT inhibitors when used as adjuvants to enrich for the phenotype.

[0209] Cell lines and mice Mouse colon adenocarcinoma MC-38-hHer2 and breast cancer E0771-hHer2 cancer cell lines were used in in vitro and in vivo experiments. The GP+e86 cell line used to produce retroviral vectors was obtained from ATCC (VA, USA). All tumor and packaging cell lines were cultured in 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 10 mM HEPES, 100 U mL-1 penicillin, and 100 ug mL -1 They were maintained at 37° C. with 5% CO in RPMI medium supplemented with streptomycin. All cell lines were tested negative for mycoplasma.

[0210] All C57BL / 6J wild-type (WT) and human Her2 (hHer2) transgenic mouse strains were bred and maintained at the Peter MacCallum Cancer Centre (Victoria, Australia). Mice aged 6–16 weeks were sex-matched and randomized into different treatment groups. All animal experiments were approved by the Animal Experimental Ethics Committee (Protocol E678).

[0211] Retroviral transduction of mouse CAR-T cells Splenocytes from C57BL / 6 donor mice were cultured at 1 μg mL -1 After activation with anti-CD3, 1 μg mL-1 anti-CD28 in the presence of IL-2 and 200 pg mL-1 IL-7 for 1 day, Ficoll gradient processing was performed. Cells were transduced onto 1ug mL of Retronectin-coated plates using supernatant produced from the GP+86 LXSN-anti-Her2 CAR packaging cell line. Transduced cells were then expanded in complete RPMI with the same concentrations of IL-2 and IL-7 with or without 5 μM PTX-200. Fresh IL-2 and IL-7 in complete medium were added to all CAR-T cells 2 days after transduction.

[0212] Human Her2+ syngeneic mouse tumor model. Her2 transgenic recipients, 2.5 × 10 5 MC38-hHer2 or 2.5 × 10 5 Either E0771-hHer2 or E0771-hHer2 was injected subcutaneously into the mammary fat pad. Tumors became palpable and were measured using manual calipers, and tumor area was calculated in square millimeters (mm 2 On the 6th to 7th day after tumor inoculation, tumor-bearing Her2+ mice were cultured at 20 mm 2 The patients were randomized to have a mean tumor size of 20 x 10 6 CAR-transduced T cells were adoptively transferred into these recipients. 50,000 U of IL-2 was administered by intraperitoneal injection five times over the next two days. Tumor size was measured and tumor size was 120 mm 2 Tumor-bearing mice were monitored every 2-3 days until the tumor-bearing mice reached the designated experimental time point.

[0213] Intraperitoneal injection of PTX-200 PTX-200 diluted in DPBS was administered at 5, 25, or 50 mg / Kg per mouse via intraperitoneal injection every 3-4 days.

[0214] Analysis of immune subsets in the tumor site, draining lymph nodes, spleen, and blood of mice Mice's blood was collected directly into EDTA-containing tubes via submandibular bleeding on days 8–9 after treatment. For experimental endpoints, various organs including tumors, draining lymph nodes (dLNs), and spleens were collected immediately after euthanasia. Single cell suspensions from dLNs and spleens were achieved by processing these organs through 70 μm filters. For splenocytes, red blood cells were lysed with ACK lysis buffer. Solid tumors were manually sliced ​​and aliquoted at 1 mg mL -1 Type IV collagenase and 0.02 mg mL -1The cells were digested in DMEM containing DNAse for 30 min at 37°C with rocking incubation at 120 rpm. The digestion was neutralized with DMEM, processed through a 70 μm filter, and resuspended in either FACS buffer or complete RPMI medium. To restimulate the cells ex vivo, single cell suspensions from these different tissues were resuspended in supplemented RPMI medium and incubated with phorbol 12-myristate 12-acetate (PMA; 10 ng mL ) supplemented with Golgi Plug (BD Biosciences) and STOP (BD Biosciences) for 4 h at 37°C before analysis for flow cytometry analysis. -1 ), calcium ionophore (1 μg mL -1 ) was activated.

[0215] In vitro chronic tumor restimulation CAR-T cells were treated with IL-7 (200 pgmL -1 ) and IL-15 (10 ng mL -1 ) at the indicated effector:target ratios with either MC-38 or E0771-hHer2 cancer cells. After one day, the CAR-T cell suspension was collected and co-cultured with a fresh layer of cancer cells. This was repeated three times with IL-7 and IL-15. Supernatants were collected daily and analyzed by cytokine bead array, and cells after the third tumor restimulation were subjected to flow cytometry analysis.

[0216] Analysis of live cancer cells over time Untreated or PTX-200 pretreated CAR-T cells were co-cultured with either MC-38 or E0771-hHer2 at the indicated effector:target ratios in 384-well plates. Caspase 3 / 7 dye was dispensed into the cell suspension. Images were taken every 4 hours using an Incucyte SX5. Cell numbers and associated caspase 3 / 7 activity were quantified using IncuCyte Zoom software.

[0217] Flow cytometry Cells were blocked with Fc receptor block (clone 2.4G2 of hybridoma supernatant diluted 1:50 in FACS buffer) for 15 min at room temperature. Cells were washed with FACS buffer and then stained with fluorochrome-conjugated antibodies for 30 min on ice. Stained cells were washed twice with FACS buffer before resuspending in FACS buffer with 20,000 counting beads. For intracellular or intranuclear staining, stained cells were then fixed and permeabilized with either BD Biosciences or eBioscience kits, respectively, according to the manufacturer's instructions. Fixed and permeabilized cells were stained with fluorochrome-conjugated antibodies for 30 min at room temperature, followed by washing twice with perm / wash buffer and finally resuspending in FACS buffer with counting beads. Stained samples were acquired on a BD FACSymphony® (BD Biosciences). Numbers of targeted T cell populations were calculated using the number of samples / bead events of targeted population x the number of input beads in the events.

[0218] statistical analysis FACS analysis was performed using Flowjo vs. 10. Figures and statistical analysis were generated using GraphPad PRISM software, version 9. Data are presented as mean ± standard error, statistical analysis was performed using one-way / two-way analysis of variance (ANOVA), and statistical significance in survival analyses was determined using the log-rank (two-tailed Mantel-Cox) test.

[0219] Example 4 - Efficacy results of TCN and TCN-P as pretreatments in vivo We first performed a CAR-T assay in either endogenous CAR-T or adoptively transferred CAR-T according to the schematic diagram illustrated in FIG. CM and CAR-T SCM We sought to determine the effect of AKT inhibitors when used as manufacturing reagents to enrich for a phenotype.

[0220] A representative example of a tumor model in immune-competent syngeneic hHer2 mice is shown in Figure 19A. Prior to administration of CAR-T cells, flow cytometry was employed to determine the ratio of CD4:CD8 T cells, which was observed to be unchanged by TCN-P conditioning during CAR-T manufacturing (Figure 19B, top panel, and Figure 19C). However, TCN-P preconditioning conferred a significant enrichment for central memory CD8+ CAR-T cells (Figure 19B, bottom panel). The impact of TCN-P preconditioning on CAR-T cells translated into improved tumor control in vivo. When left uncontrolled, the E0771 breast cancer tumor model reached the experimental endpoint by day 14 (i.e., tumors >120 mm 2 ) CAR-T administration could extend this to day 16, and preconditioned CAR-T cells could extend this to day 24 (Figure 19D), with a significant improvement in the probability of survival (Figure 19E, p=0.0012).

[0221] Using the same syngeneic hHer2 breast cancer model, another cohort of animals was evaluated for tumor growth up to 8 days after treatment (Figure 20A). Analysis of circulating T cells showed that preconditioning of CAR-T cells resulted in a skewed CD4:CD8 ratio in vivo, such that CD4+ CAR-T cells were more prevalent in animals receiving TCN-P preconditioned CAR-T cells compared to those receiving untreated CAR-T cells (Figure 20B, top panel). It was also noted that preconditioning resulted in a decrease in CD8+ CAR-T cells in vivo (Figure 20B, middle panel). These observations were consistent with a near doubling of central memory T cells (Figure 20B, bottom panel).

[0222] Flow cytometry analysis further revealed that preconditioning of CAR-T cells did not significantly affect the CD4+ or CD8+ composition within the tumor (Figure 20C), but appeared to increase CD4+ cells within the secondary lymphoid organ, the spleen (Figure 20D). Interestingly, the improved tumor killing efficiency by preconditioned CAR-T cells was not coincident with any significant changes in the production of the cytokines, TNF-alpha, or IFN-gamma (Figure 20E).

[0223] Circulating numbers of host CD4+ and CD8+ T cells were not significant between groups (Figure 21A-B). CD8+ CAR-T cells were similar between groups, but circulating CD4+ CAR-T cells were increased an average of 3-fold in animals that received preconditioned CAR-T cells (Figure 21D, p=0.0009). Phenotyping of circulating CAR-T cells suggested that preconditioning during the manufacturing step enabled CAR-T cells to retain a central memory phenotype in vivo. The percentage of CD8+ CAR-T cells with a central memory phenotype was nearly doubled in the preconditioned group (Figure 21E, p<0.0001). The percentage of CD4+ CAR-T cells with a central memory phenotype was approximately 20% higher in the preconditioned group (Figure 21F, p<0.0001). These findings were consistent with a halving of CD8+ and CD4+ CAR T cells with an effector memory phenotype (Figures 21G-H, p<0.001 and p=0.0029, respectively).

[0224] When analyzing immune cells in tumors and spleens, we observed that preconditioning had no significant effect on CD8+ or CD4+ CAR-T cells carrying a central memory phenotype (Figures 22A and C). Although no significant differences were observed in the expression of early exhaustion markers in CD8+ CAR-T cells in tumors (Figure 22B), preconditioning appeared to protect CD8+ CAR-T cells from exhaustion when mobilized to the spleen (Figure 22D).

[0225] We then sought to determine the effect of CAR-T cells pretreated with TCN-P in combination with a PD-1 checkpoint inhibitor to determine whether this treatment would improve anti-tumor efficacy against solid tumors. As shown in Figures 23A-B, a synergistic reduction in tumor area was observed when CAR-T cells were pretreated with TCN-P in combination with a PD-1 checkpoint inhibitor.

[0226] Example 5 - Results of efficacy of TCN and TCN-P as neoadjuvants in vivo In the second approach, CAR-T is expressed either in endogenous CAR-T or adoptively transferred CAR-T according to the schematic illustrated in FIG. CM and CAR-T SCM The effect of AKT inhibitors when used as adjuvants to enrich for the phenotype was examined.

[0227] As shown in FIG. 25, Her2+ transgenic mice were e5 When MC-38-hHer2 was inoculated and then injected intraperitoneally every 3-4 days with either 5, 25, or 50 mg / Kg of DMSO (vehicle control) or TCN-P, TCN-P induced a strong antitumor effect demonstrating its ability to affect tumor growth in vivo. The effect of TNC-p was then tested in combination with CAR-T cell therapy. As shown in Figure 26A-B, 20×10 6 Mice receiving combined treatment with anti-human Her2 CAR-T cells and 25 mg / Kg of TCN-P intravenously demonstrated a significant reduction in tumor size compared to single treatment alone. Consistently, 20×10 6 Mice receiving combined treatment with anti-human Her2 CAR-T cells and 25 mg / Kg TCN-P intravenously also demonstrated increased survival compared to single treatment alone (Figure 26C). Notably, the median survival time in the control group was 15 days and 20 days for mice treated with 25 mg / Kg TNC-p. In the presence of CAR-T cells, survival time increased to 26 days, whereas in the presence of both CAR-T cells and TCN-P, survival time increased to 32.5 days.

[0228] Finally, the utility of TCN-P as an adjuvant for CAR-T therapy was also evaluated using the MC-38 hHer2 model of colon cancer in which mice received either vehicle, untreated CAR-T cells, CAR-T cells in combination with 25 mg / kg TCN-P adjuvant every 3 days after CAR-T administration, or a combination of pre-treated CAR-T cells and TCN-P adjuvant (Figure 27A). Tumor size was assessed by day 14. Here, we observed that administration of TCN-P as an adjuvant significantly improved tumor control by Her2-targeted CAR-T cells (Figure 27B, *p<0.05), where the combination of pre-conditioned CAR-T cells with TCN-P adjuvant resulted in the most significant tumor killing in vivo (p<0.01). Unexpectedly, it was determined that the spleen contained significant accumulation and / or expansion of CD4+ and CD8+ CAR T cells (p<0.0001) in response to the combination of TCN-P pretreatment and TCN-P adjuvant therapy (Figure 28A). Furthermore, administration of TCN-P as an adjuvant in the presence of CAR-T cells resulted in a 50% reduction in intratumoral Tregs (Figure 28B).

[0229] Findings from this study demonstrate that TCN-P preconditioning can enhance the efficacy of conventional CAR-T therapy by enriching for T cell phenotypes known to persist in vivo and associated with partial or complete clinical responses. Given the clinical safety data regarding the use of TCN-P in oncology indications, it is plausible that TCN-P and similar compounds could be used in combination with CAR-T therapy to enhance the in vivo persistence of central memory CAR-T cells and, in so doing, improve clinical durability and responses.

[0230] Those skilled in the art will appreciate that numerous variations and / or modifications may be made to the invention as illustrated in the specific embodiments without departing from the spirit or scope of the invention as broadly described, and the present embodiments are therefore to be considered in all respects as illustrative and not restrictive.

[0231] All publications discussed and / or referenced herein are incorporated herein in their entirety.

[0232] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention and is not to be construed as an admission that any or all of such matters form part of the prior art base or were common general knowledge in the art relevant to the present invention prior to the priority date of each claim of this application.

[0233] References Berg et al. (1998) Transplant Proc.30:3975-3977. Datta et al. (2014)Yale J Biol Med 87:491-518. Garland et al. (1999) J. Immunol Meth. 227:53-63. Ghosh et al. (1991) Glycobiology 5:505-510. Haanen et al. (1999) J. Exp. Med. 190:1319-1328. Lui et al. (2021) J Hemat Oncol 14:7. McLellan and Ali Hosseini Rad (2019) Immunol Cell Biol 97:664-674. Mousset et al. (2018) Oncoimmunology 7:e1488565. Rosenberg et al. (1988) New Eng. J. of Med 319:1676. Sabado et al.(2017)Cell Res 27:74-95. Smith et al.(2015)Clinical & Translational Immunology(2015)4:e31.

Claims

1. A pharmaceutical composition for use in a method of modifying an immune response in a subject, wherein the pharmaceutical composition comprises a population of immune cells, and the immune cells are produced using a method comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein.

2. The pharmaceutical composition according to claim 1, wherein the immune cells are dendritic cells, T cells, natural killer cells, myeloid cells, macrophages, or a combination thereof.

3. A pharmaceutical composition for use in a method of modifying dendritic cell and / or natural killer cell responses in a subject, wherein the pharmaceutical composition comprises an AKT inhibitor and / or an inhibitor of a PH domain protein, and the method comprises: a) administering to the subject an AKT inhibitor and / or an inhibitor of a PH domain protein; and b) administering to the subject a population of immune cells comprising dendritic cells and / or natural killer cells.

4. A pharmaceutical composition for use in a method of modifying dendritic cell and / or natural killer cell responses in a subject, wherein the pharmaceutical composition comprises dendritic cells and / or natural killer cells, and the method comprises: a) administering to the subject an AKT inhibitor and / or an inhibitor of a PH domain protein; and b) administering to the subject a population of immune cells.

5. A pharmaceutical composition for use in a method of reducing cytokine release syndrome (CRS) in a subject undergoing CAR-T cell therapy, wherein the pharmaceutical composition comprises an AKT inhibitor and / or an inhibitor of a PH domain protein.

6. A pharmaceutical composition for use in a method of modifying immunotherapy in a subject, wherein the pharmaceutical composition comprises a population of CAR-T cells, and the CAR-T cells are cultured in a medium comprising the AKT inhibitor and / or an inhibitor of a PH domain protein.

7. A pharmaceutical composition for use in modifying an immune response, preferably a T cell response, in a subject, wherein the pharmaceutical composition comprises an AKT inhibitor and / or an inhibitor of a PH domain protein. ​ ​ ​ A pharmaceutical composition, wherein the subject is administered, preferably together with a population of immune cells, including T cells comprising a chimeric antigen receptor (CAR-T cells), at least 18 hours after an AKT inhibitor and / or an inhibitor of a PH domain protein. [

8. ] A pharmaceutical composition for use in modifying an immune response, preferably a T cell response, in a subject, wherein the pharmaceutical composition comprises a population of immune cells, including T cells comprising a chimeric antigen receptor (CAR-T cells), and the subject is administered an AKT inhibitor and / or an inhibitor of a PH domain protein at least 18 hours prior to the pharmaceutical. [

9. ] A pharmaceutical composition for use in a method for modifying an immune response in a subject, wherein the pharmaceutical composition comprises a population of immune cells, and the method comprises: administering to the subject a population of immune cells and a checkpoint inhibitor, wherein the immune cells are produced using a method comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein, and preferably, the immune cells are T cells, dendritic cells, natural killer cells, myeloid cells, macrophages, or a combination thereof. [

10. ] A pharmaceutical composition for use in a method for modifying an immune response in a subject, wherein the pharmaceutical composition comprises a checkpoint inhibitor, and the method comprises: administering to the subject a checkpoint inhibitor and a population of immune cells, wherein the immune cells are produced using a method comprising culturing the immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein, and preferably, the immune cells are T cells, dendritic cells, natural killer cells, myeloid cells, macrophages, or a combination thereof. [

11. ] A pharmaceutical composition for use in a method for modifying an immune response, preferably a T cell immune response, in a subject, wherein the pharmaceutical composition comprises a population of immune cells, and the method comprises: administering to the subject (i) a population of immune cells, wherein the immune cells are T cells, dendritic cells, natural killer cells, or a combination thereof, (ii) an AKT inhibitor and / or an inhibitor of a PH domain protein, thereby. A pharmaceutical composition for use in a method for modifying an immune response, preferably a T cell immune response, in a subject, wherein the pharmaceutical composition comprises an AKT inhibitor and / or an inhibitor of a PH domain protein, wherein the method comprises administering to the subject (i) an AKT inhibitor and / or an inhibitor of a PH domain protein, and (ii) a population of immune cells, preferably a population of immune cells produced using a method comprising culturing immune cells in a medium comprising an AKT inhibitor and / or an inhibitor of a PH domain protein, wherein the immune cells are T cells, dendritic cells, natural killer cells, or a combination thereof, A pharmaceutical composition comprising.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the subject is immune depleted.

14. The pharmaceutical composition according to any one of claims 1 to 12, wherein the subject has cancer associated with a low antigen abundance.

15. The subject is i) acute myeloid leukemia in which low CD33+ blasts are dominant, or ii) diffuse large B cell lymphoma or non-Hodgkin lymphoma having low levels of CD19 and / or CD20, The pharmaceutical composition according to claim 14.

16. The pharmaceutical composition according to any one of claims 1 to 12, wherein the AKT inhibitor and / or the inhibitor of the PH domain protein is selected from triciribine (TCN), triciribine 5'-monophosphate (TCN-P), AKT inhibitor VIII, MK-2206, AZD5363, GDC-0068, GSK2141795, and GSK2110183 hydrochloride.

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