Materials and methods for enhanced stem cell-like memory T cell engineering

Abstract

The present disclosure provides methods for generating stem cell-like memory T (TSCM) cells. The present disclosure also provides cells, pharmaceutical compositions, and their uses in adoptive immunotherapy for the treatment of diseases such as cancer.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 172,595, filed April 8, 2021, No. 63 / 172,601, filed April 8, 2021, No. 63 / 172,605, filed April 8, 2021, and No. 63 / 172,610, filed April 8, 2021, the disclosures of each of which are incorporated by reference in their entirety herein.

[0002] FIELD OF THEINVENTION The present invention provides stem cell-like memory T (T SCM The present invention also relates to the cells, pharmaceutical compositions, and their use in adoptive immunotherapy for the treatment of disease.

[0003] (Sequence Listing) This application contains a Sequence Listing, which has been submitted electronically in ASCII format and is incorporated by reference in its entirety. This ASCII copy, created on 2022-04-06, is named 253505_000131_SL.txt and is 21,082 bytes in size. [Background technology]

[0004] Immunotherapy offers new ways to treat solid tumors and other cancers 1,2 Biologics, including monoclonal antibodies, T cell-redirecting bispecific antibodies, checkpoint inhibitors, and most recently, chimeric antigen receptor T cells (CAR-T cells), have greatly improved the treatment of tumors. Strong evidence suggests that immunotherapy using T cell receptor (TCR) transduction and adoptive transfer of genetically modified T cells, such as CAR-engineered T cells, can result in complete regression in some patients with metastatic cancer. Currently, four CAR-T therapies have been approved by the Food and Drug Administration (FDA), with many more in the clinical pipeline. 3However, the recent success with CAR-T cell-based therapies is not without drawbacks. 4-7 . Summary of the Invention

[0005] CAR-T cells are generated by collecting a patient's blood, extracting T cells, and expressing a CAR, typically a single-chain variable fragment (scFv) that targets a tumor-associated antigen (TAA). This process reprograms the patient's T cells to specifically target and destroy tumor cells, resulting in cell death. 8 Current clinical trials utilize peripheral blood mononuclear cell (PBMC)-derived T cell subsets or unselected bulk T cell populations as the starting population for TCR engineering and CAR-T cell expansion. However, current manufacturing processes result in the generation of inconsistent and variable cell compositions of the final cell product administered to patients.

[0006] Stem cell-like memory T(T SCM ) cells are a rare population of early memory T cell subsets generated directly from naive T cells and have distinct phenotypic, transcriptional, and epigenetic states relative to other characterized memory and effector T cell subsets. SCM Cells have the ability to self-renew, thereby forming central memory (T CM ), effector memory, and effector T cell subsets. SCM Memory T cells are detected in both healthy donors and cancer patients, but at low frequencies in the latter, and display a gene signature with fewer exhaustion markers compared to other known memory T cell subsets. SCM Or T SCM Efforts to generate IL-1-like cells have proven largely ineffective, especially when currently applying conventional manufacturing approaches.

[0007] In this context, the present application provides a method for the preparation of T cells with enhanced effector function and reduced exhaustion markers to enhance antitumor immunity. SCM Methods for generating CAR-T cells are provided.

[0008] In one embodiment, the stem cell-like memory T (T SCM 1.) A method for concentrating cells, comprising: a) T SCM contacting the population of T cells with an effective amount of one or more cytokines, including interleukin 7 (IL-7), for a period of time sufficient to enrich for the cells; b) optionally, the T SCM and expanding the cells.

[0009] In some embodiments, the one or more cytokines further comprise IL-15. In some embodiments, the one or more cytokines further comprise IL-21. In some embodiments, the one or more cytokines further comprise IL-15 and IL-21.

[0010] In some embodiments, the one or more cytokines are each contacted with the T cell population at a concentration of about 1-15 ng / ml, about 2-14 ng / ml, about 3-13 ng / ml, about 4-12 ng / ml, about 5-12 ng / ml, about 6-12 ng / ml, about 7-11 ng / ml, about 8-12 ng / ml, about 8-10 ng / ml, or about 10 ng / ml. In one embodiment, the one or more cytokines are each contacted with the T cell population at a concentration of about 10 ng / ml.

[0011] In some embodiments, the one or more cytokines do not include IL-2.

[0012] In some embodiments, the T cell population is Pan-T cells, naive CD4 + T cells, naive CD8 + T cells or naive CD4 + and naive CD8 +In some embodiments, the method includes, prior to step (a), Pan-T cells, naive CD4 T cells, or any combination thereof. + Cells, naïve CD8 + T cells or naive CD4 + and naive CD8 + In some embodiments, the method further comprises isolating the T cell population, the T cell population, or any combination thereof from peripheral blood mononuclear cells (PBMCs). In some embodiments, the T cell population does not include suppressive regulatory T cells.

[0013] In some embodiments, one or more cytokines are present during the expansion step (b).

[0014] In some embodiments, the method further comprises genetically modifying the T cells to express a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). In some embodiments, the genetic modification is performed by introducing a polynucleotide encoding the CAR or engineered TCR into the cells. In some embodiments, the polynucleotide encoding the CAR or engineered TCR is introduced via viral transduction, electroporation, direct injection, magnetofection, ultrasound, ballistic or hydrodynamic methods, or a combination thereof.

[0015] In some embodiments, the CAR or engineered TCR specifically binds to a tumor antigen, an infectious antigen, or an autoimmune antigen, hi some embodiments, the tumor antigen is selected from BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, hK2, FLT3, CD20, CD22, KRASG12D, p53, BRAC1, and PSMA.

[0016] In some embodiments, the genetic modification is performed prior to the expansion step (b). In some embodiments, one or more cytokines are present during the genetic modification step.

[0017] In various embodiments, the contacting step (a) is carried out for about 5-20 days, about 10-20 days, about 5-18 days, about 8-15 days, about 10-18 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, or about 18 days. In one embodiment, the contacting step is carried out for about 14 days.

[0018] In various embodiments, the contacting step (a) and the expanding step (b) are carried out for a total of 5 to 20 days, about 10 to 20 days, about 5 to 18 days, about 8 to 15 days, about 10 to 18 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, or about 18 days. In some embodiments, the contacting step (a) and the expanding step (b) are carried out for a total of about 14 days.

[0019] In some embodiments, the contacting step (a) is carried out at a temperature of about 37 degrees.

[0020] In various embodiments, the method further comprises activating the T cell population at the start of the contacting step (a). In one embodiment, the activation step is carried out for about 24 hours with an anti-CD3 agent and / or an anti-CD28 agent.

[0021] In various embodiments, the method further comprises the step of priming the T cell population prior to the activation step (a).

[0022] In various embodiments, the method further comprises determining whether, after the expansion step (b), the T cell population is SCM In some embodiments, the method further comprises determining the percentage of T cells in the T cell population. SCM In one embodiment, the percentage of T cells in the T cell population is at least about 40%, 50%, 60%, or 70% after the expansion step (b). SCM The percentage of cells is about 60%-70% after the expansion step (b).

[0023] In some embodiments, the T SCMThe method for enriching cells may be carried out in vitro or ex vivo.

[0024] In some embodiments, genetically modified stem cell-like memory T (T SCM ) A method for generating a cell, comprising: a) Isolated Pan-T cells, naive CD4 + T cells, naive CD8 + T cells or naive CD4 + and naive CD8 + Obtaining a population of T cells, or any combination thereof; b) activating the T cell population; c) genetically modifying the cells present after step (b) to express a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR); and d) expanding the genetically modified cells. Methods are provided herein, wherein steps b), c), and d) are carried out in the presence of one or more cytokines, including interleukin-7 (IL-7).

[0025] In some embodiments, the one or more cytokines further comprise IL-15. In some embodiments, the one or more cytokines further comprise IL-21. In some embodiments, the one or more cytokines further comprise IL-15 and IL-21.

[0026] In some embodiments, the one or more cytokines are each contacted with the T cell population at a concentration of about 1-15 ng / ml, about 2-14 ng / ml, about 3-13 ng / ml, about 4-12 ng / ml, about 5-12 ng / ml, about 6-12 ng / ml, about 7-11 ng / ml, about 8-12 ng / ml, about 8-10 ng / ml, or about 10 ng / ml. In one embodiment, the one or more cytokines are each contacted with the T cell population at a concentration of about 10 ng / ml.

[0027] In various embodiments, the one or more cytokines do not include IL-2.

[0028] In various embodiments, Pan-T cells, naive CD4 + T cells, naive CD8 + T cells, or naive CD4 + and naive CD8 + The T cells, or any combination thereof, are isolated from peripheral blood mononuclear cells (PBMCs). In some embodiments, the T cells do not include suppressive regulatory T cells.

[0029] In various embodiments, the genetic modification is performed by introducing a polynucleotide encoding the CAR or engineered TCR into the cell. In some embodiments, the polynucleotide encoding the CAR or engineered TCR is introduced via viral transduction, electroporation, direct injection, magnetofection, ultrasound, ballistic or hydrodynamic methods, or a combination thereof. In some embodiments, the CAR or engineered TCR specifically binds to a tumor antigen, an infectious antigen, or an autoimmune antigen. In some embodiments, the tumor antigen is selected from BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, hK2, FLT3, CD20, CD22, KRASG12D, p53, BRAC1, and PSMA.

[0030] In some embodiments, the expansion step (d) is carried out for 5 to 20 days, about 10 to 20 days, about 5 to 18 days, about 8 to 15 days, about 10 to 18 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, or about 18 days. In some embodiments, the expansion step is carried out for about 14 days.

[0031] In some embodiments, steps (b), (c), and (d) are carried out for a total of 5-20 days, about 10-20 days, about 5-18 days, about 8-15 days, about 10-18 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, or about 18 days. In some embodiments, steps (b), (c), and (d) are carried out for a total of 14 days.

[0032] In various embodiments, steps (b), (c), and (d) are carried out at a temperature of about 37°C.

[0033] In various embodiments, the activation step is carried out with an anti-CD3 agent and / or an anti-CD28 agent, hi various embodiments, the activation step is carried out for about 12 to 48 hours (eg, 24 hours).

[0034] In various embodiments, the method further comprises the step of priming the T cell population prior to the activation step (b).

[0035] In various embodiments, the method further comprises determining whether, after the expansion step (d), the T cell population is at a high T cell population level. SCM In some embodiments, the method further comprises determining the percentage of T cells in the T cell population. SCM In one embodiment, the percentage of T cells in the T cell population is at least about 40%, 50%, 60%, or 70% after the expansion step (d). SCM The percentage of cells is about 60%-70% after the expansion step (d).

[0036] In some embodiments, the genetically modified T SCM The method for generating the cells may be carried out in vitro or ex vivo.

[0037] In another embodiment, enriched stem cell-like memory T (T SCM ) cells, a) Pan-T cells, naive CD4 + T cells, naive CD8 + T cells, or naive CD4 + and naive CD8 + T cells, or any combination thereof, in a therapeutically effective amount of one or more cytokines, including interleukin 7 (IL-7), and T SCM contacting for a period of time sufficient to enrich the cells; b) optionally, the T SCMand expanding the cells.

[0038] In another embodiment, enriched stem cell-like memory T (T SCM ) cells, a) Pan-T cells, naive CD4 + T cells, naive CD8 + T cells, or naive CD4 + and naive CD8 + T cells, or any combination thereof, in a therapeutically effective amount of one or more cytokines, including interleukin 7 (IL-7), and T SCM contacting for a period of time sufficient to enrich the cells; b) optionally, the T SCM and expanding the cells.

[0039] In some embodiments of the T cell populations described herein, the one or more cytokines further comprise IL-15. In some embodiments, the one or more cytokines further comprise IL-21. In some embodiments, the one or more cytokines further comprise IL-15 and IL-21.

[0040] In some embodiments of the T cell populations described herein, the one or more cytokines are each added at a concentration of about 1-15 ng / ml, about 2-14 ng / ml, about 3-13 ng / ml, about 4-12 ng / ml, about 5-12 ng / ml, about 6-12 ng / ml, about 7-11 ng / ml, about 8-12 ng / ml, about 8-10 ng / ml, or about 10 ng / ml. In one embodiment, the one or more cytokines are each added at a concentration of about 10 ng / ml.

[0041] In some embodiments of the T cell populations described herein, the one or more cytokines do not include IL-2.

[0042] In some embodiments of the T cell populations described herein, Pan-T cells, naive CD4 + cells, naive CD8+ T cells, or naive CD4 + and naive CD8 + In some embodiments, the cells are isolated from peripheral blood mononuclear cells (PBMCs). In some embodiments, the cells are Pan-T cells, naive CD4+ cells, naive CD8+ T cells, or naive CD4+ T cells. + and naive CD8 + The cells, or any combination thereof, do not include suppressive regulatory T cells.

[0043] In some embodiments of the T cell populations described herein, one or more cytokines are present during the expansion step (b).

[0044] In some embodiments of the T cell populations described herein, the preparation method further comprises genetically modifying the T cells to express a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). In some embodiments, the genetic modification is performed by introducing a polynucleotide encoding the CAR or engineered TCR into the cells. In some embodiments, the polynucleotide encoding the CAR or engineered TCR is introduced via viral transduction, electroporation, direct injection, magnetofection, ultrasound, ballistic or hydrodynamic methods, or a combination thereof.

[0045] In some embodiments of the T cell populations described herein, the CAR or engineered TCR specifically binds to a tumor antigen, an infectious antigen, or an autoimmune antigen.

[0046] In some embodiments of the T cell populations described herein, the tumor antigen is selected from BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, hK2, FLT3, CD20, CD22, KRASG12D, p53, BRAC1, and PSMA.

[0047] In some embodiments of the T cell populations described herein, genetic modification is performed before the expansion step (b). In some embodiments, one or more cytokines are present during the genetic modification step. In some embodiments, one or more cytokines are present during the expansion step (b).

[0048] In some embodiments of the T cell populations described herein, the contacting step (a) is carried out for about 5-20 days, about 10-20 days, about 5-18 days, about 8-15 days, about 10-18 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, or about 18 days. In one embodiment, the contacting step is carried out for about 14 days.

[0049] In some embodiments of the T cell populations described herein, the contacting step (a) and the expanding step (b) are carried out for a total of about 5-20 days, about 10-20 days, about 5-18 days, about 8-15 days, about 10-18 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, or about 18 days. In one embodiment, the contacting step (a) and the expanding step (b) are carried out for a total of 14 days.

[0050] In some embodiments of the T cell populations described herein, the contacting step (a) is carried out at a temperature of about 37°C.

[0051] In some embodiments of the T cell populations described herein, the preparation method further comprises activating the T cell population at the start of the contacting step (a). In one embodiment, the activation step is carried out for about 24 hours with an anti-CD3 agent and / or an anti-CD28 agent.

[0052] In some embodiments of the T cell populations described herein, the methods further comprise the step of priming the T cell population prior to the activation step (a).

[0053] In some embodiments of the T cell population described herein, the method further comprises the step of determining whether, after the expansion step (b), the T SCMIn some embodiments, the method further comprises determining the percentage of T cells in the T cell population. SCM In one embodiment, the percentage of T cells in the T cell population is at least about 40%, 50%, 60%, or 70% after the expansion step (b). SCM The percentage of cells is about 60%-70% after the expansion step (b).

[0054] In another embodiment, enriched stem cell-like memory T (T SCM A T cell population comprising: a) Isolated Pan-T cells, naive CD4 + T cells, naive CD8 + T cells or naive CD4 + and naive CD8 + Obtaining a population of T cells, or any combination thereof; b) activating the T cell population; c) genetically modifying the cells present after step (b) to express a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR); and d) expanding the genetically modified cells. Provided herein is a T cell population prepared (or obtainable) by the method, wherein steps b), c), and d) are carried out in the presence of one or more cytokines, including interleukin-7 (IL-7).

[0055] In some embodiments of the T cell populations described herein, isolated Pan-T cells, naive CD4 + T cells, naive CD8 + T cells, or naive CD4 + and naive CD8 + Obtaining a population of T cells, or any combination thereof, is performed on a sample obtained from a subject.

[0056] In some embodiments of the T cell populations described herein, the one or more cytokines further comprise IL-15. In some embodiments, the one or more cytokines further comprise IL-21. In some embodiments, the one or more cytokines further comprise IL-15 and IL-21.

[0057] In some embodiments of the T cell populations described herein, the one or more cytokines are each added at a concentration of about 1-15 ng / ml, about 2-14 ng / ml, about 3-13 ng / ml, about 4-12 ng / ml, about 5-12 ng / ml, about 6-12 ng / ml, about 7-11 ng / ml, about 8-12 ng / ml, about 8-10 ng / ml, or about 10 ng / ml. In one embodiment, the one or more cytokines are each added at a concentration of about 10 ng / ml.

[0058] In various embodiments of the T cell populations described herein, the one or more cytokines do not include IL-2.

[0059] In various embodiments of the T cell populations described herein, Pan-T cells, naive CD4 + T cells, naive CD8 + T cells, or naive CD4 + and naive CD8 + The T cells, or any combination thereof, are isolated from peripheral blood mononuclear cells (PBMCs). In some embodiments, the T cells do not include suppressive regulatory T cells.

[0060] In various embodiments of the T cell populations described herein, genetic modification is performed by introducing a polynucleotide encoding the CAR or engineered TCR into the cells. In some embodiments, the polynucleotide encoding the CAR or engineered TCR is introduced via viral transduction, electroporation, direct injection, magnetofection, ultrasound, ballistic or hydrodynamic methods, or a combination thereof. In some embodiments, the CAR or engineered TCR specifically binds to a tumor antigen, an infectious antigen, or an autoimmune antigen. In some embodiments, the tumor antigen is selected from BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, hK2, FLT3, CD20, CD22, KRASG12D, p53, BRAC1, and PSMA.

[0061] In various embodiments of the T cell populations described herein, the expansion step (d) is carried out for about 5-20 days, about 10-20 days, about 5-18 days, about 8-15 days, about 10-18 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, or about 18 days. In some embodiments, the expansion step is carried out for about 14 days.

[0062] In various embodiments of the T cell populations described herein, steps (b), (c), and (d) are carried out for a total of about 5-20 days, about 10-20 days, about 5-18 days, about 8-15 days, about 10-18 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, or about 18 days. In some embodiments, steps (b), (c), and (d) are carried out for a total of 14 days.

[0063] In various embodiments of the T cell populations described herein, steps (b), (c), and (d) are carried out at a temperature of about 37°C.

[0064] In various embodiments of the T cell populations described herein, the activation step is carried out with an anti-CD3 agent and / or an anti-CD28 agent, hi various embodiments, the activation step is carried out for about 24 hours.

[0065] In various embodiments of the T cell populations described herein, the methods further comprise priming the T cell population prior to the activation step (b).

[0066] In various embodiments of the T cell populations described herein, the method further comprises the step of determining whether, after the expansion step (d), the T SCM In some embodiments, the method further comprises determining the percentage of T cells in the T cell population. SCM In some embodiments, the percentage of T cells in the T cell population is at least about 40%, 50%, 60%, or 70% after the expansion step (d). SCM The percentage of cells is approximately 60%-70% after the expansion step (d).

[0067] In another aspect, provided herein is a pharmaceutical composition comprising a population of T cells described herein and a pharma- ceutical acceptable carrier or excipient.

[0068] In another aspect, a method of treating a disease or disorder in a subject in need thereof includes administering to the subject a therapeutically effective amount of enriched stem cell-like memory T (T SCM ) cells, or enriched T SCM and administering to the subject a pharmaceutical composition comprising the T cell population comprising enriched T cells and a pharma- ceutical carrier or excipient, wherein the T cell population comprising enriched T cells comprises a) Isolated Pan-T cells, naive CD4 + T cells, naive CD8 + T cells or naive CD4 + and naive CD8 + Obtaining a population of T cells, or any combination thereof; b) activating the T cell population; c) genetically modifying the cells present after step (b) to express a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR); and d) expanding the genetically modified cells. Provided herein is a method prepared (or obtainable) by a method, wherein steps b), c), and d) are carried out in the presence of one or more cytokines, including interleukin-7 (IL-7).

[0069] In some embodiments of the therapeutic methods described herein, isolated Pan-T cells, naive CD4 + T cells, naive CD8 + T cells or naive CD4 + and naive CD8 + Obtaining a population of T cells, or any combination thereof, is performed on a sample obtained from the subject.

[0070] In some embodiments of the methods of treatment described herein, the one or more cytokines further comprise IL-15. In some embodiments, the one or more cytokines further comprise IL-21. In some embodiments, the one or more cytokines further comprise IL-15 and IL-21.

[0071] In some embodiments of the methods of treatment described herein, the one or more cytokines are each added at a concentration of about 1-15 ng / ml, about 2-14 ng / ml, about 3-13 ng / ml, about 4-12 ng / ml, about 5-12 ng / ml, about 6-12 ng / ml, about 7-11 ng / ml, about 8-12 ng / ml, about 8-10 ng / ml, or about 10 ng / ml. In one embodiment, the one or more cytokines are each added at a concentration of about 10 ng / ml.

[0072] In some embodiments of the methods of treatment described herein, the one or more cytokines do not include IL-2.

[0073] In some embodiments of the methods of treatment described herein, Pan-T cells, naive CD4 + T cells, naive CD8 + T cells, or naive CD4 + and naive CD8 + The T cells, or any combination thereof, are isolated from peripheral blood mononuclear cells (PBMCs). In some embodiments, the T cells do not include suppressive regulatory T cells.

[0074] In some embodiments of the therapeutic methods described herein, the genetic modification is performed by introducing a polynucleotide encoding the CAR or engineered TCR into the cell. In some embodiments, the polynucleotide encoding the CAR or engineered TCR is introduced via viral transduction, electroporation, direct injection, magnetofection, ultrasound, ballistic or hydrodynamic methods, or a combination thereof. In some embodiments, the CAR or engineered TCR specifically binds to a tumor antigen, an infectious antigen, or an autoimmune antigen. In some embodiments, the tumor antigen is selected from BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, hK2, FLT3, CD20, CD22, KRASG12D, p53, BRAC1, and PSMA.

[0075] In some embodiments of the methods of treatment described herein, the expanding step (d) is carried out for about 5-20 days, about 10-20 days, about 5-18 days, about 8-15 days, about 10-18 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, or about 18 days. In some embodiments, the expanding step (d) is carried out for about 14 days.

[0076] In some embodiments of the methods of treatment described herein, steps (b), (c), and (d) are carried out for a total of about 5-20 days, about 10-20 days, about 5-18 days, about 8-15 days, about 10-18 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, or about 18 days. In some embodiments, steps (b), (c), and (d) are carried out for a total of 14 days.

[0077] In some embodiments of the methods of treatment described herein, steps (b), (c), and (d) are carried out at a temperature of about 37°C.

[0078] In some embodiments of the methods of treatment described herein, the activation step is carried out for about 24 hours using an anti-CD3 agent and / or an anti-CD28 agent.

[0079] In some embodiments of the therapeutic methods described herein, the methods further comprise the step of priming the T cell population prior to the activation step (b).

[0080] In some embodiments of the methods of treatment described herein, the method further comprises, after the expansion step (d), determining whether the T SCM In some embodiments, the method further comprises determining the percentage of T cells in the T cell population. SCM The percentage of T cells after expansion step (d) is at least about 40%, 50%, 60%, or 70%. In some embodiments, the percentage of T cells in the T cell population is at least about 40%, 50%, 60%, or 70%. SCM The percentage of cells is approximately 60%-70% after the expansion step (d).

[0081] In some embodiments of the therapeutic methods described herein, the T cell population is allogeneic to the subject. In some embodiments, the T cell population is autologous to the subject.

[0082] In some embodiments of the methods of treatment described herein, the disease or disorder is cancer, an infectious disease, or an autoimmune disease. In some embodiments, the cancer is a hematological malignancy. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a squamous cell carcinoma, adenosquamous cell carcinoma, lung cancer, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer or gastric cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, skin cancer, multiple myeloma and acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and Diffuse chronic lymphocytic leukemia (CLL), lymphomas such as Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal B-cell lymphoma, mediastinal plasma cell lymphoma, lymphoplasmocytic lymphoma, immunoblastic large cell lymphoma, hairy cell leukemia (HCL), precursor B-lymphoblastic lymphoma and primary central nervous system Nervous system (CNS) lymphomas, T-cell NHL, e.g. precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous sebaceous adenitis-like T-cell lymphoma, anaplastic large cell lymphoma, mixtures of one or more of the above leukemia / lymphomas, brain and head and neck cancer, cholangiocarcinoma, bronchial carcinoma, chordoma, choriocarcinoma, epithelial carcinoma, endothelial sarcoma, esophageal carcinoma , Ewing's sarcoma, heavy chain disease, hematopoietic cancer, immune amyloidosis, monoclonal gammopathy of undetermined significance, myelodysplastic syndrome, myeloproliferative disorder, idiopathic myeloid metaplasia (AMM) or myelofibrosis (MF), chronic idiopathic myelofibrosis, myeloproliferative neoplasm, polycythemia vera, rectal adenocarcinoma, essential thrombocytosis, chronic neutrophilic leukemia, hypereosinophilic syndrome, or soft tissue sarcoma, or a combination or metastasis thereof.

[0083] In some embodiments, the cancer is a BCMA-expressing cancer. In some embodiments, the BCMA-expressing cancer is acute myeloid leukemia (AML) or multiple myeloma (MM), or smoldering multiple myeloma (SMM).

[0084] In another embodiment, the stem cell-like memory T( SCM 1.) A system for concentrating cells, comprising: a) T SCM a means for contacting the population of T cells with an effective amount of one or more cytokines, including interleukin 7 (IL-7), for a period of time sufficient to enrich for the cells; b) optionally, the T SCM and a means for expanding the cells.

[0085] In some embodiments of the systems described herein, the T cell population is Pan-T cells, naive CD4 + T cells, naive CD8+ T cells, or naive CD4 + and naive CD8 + T cells, or any combination thereof.

[0086] In some embodiments of the systems described herein, the systems detect and measure Pan-T cells, naive CD4 + Cells, naïve CD8 + T cells, or naive CD4 + and CD8 + In some embodiments, the system further comprises a means for isolating T cells, naive cells, or any combination thereof from peripheral blood mononuclear cells (PBMCs). In some embodiments, the system further comprises a means for genetically modifying the T cells to express a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). In some embodiments, the system further comprises a means for activating the T cell population at the start of contacting the T cell population with one or more cytokines, including interleukin 7 (IL-7). In some embodiments, the system further comprises a means for priming the T cell population prior to activation. In some embodiments, the system further comprises a means for determining the T cell activity in the T cell population after expansion. SCM Further included is a means for determining the percentage of cells.

[0087] In another embodiment, genetically modified stem cell-like memory T (T SCM ) A system for generating cells comprising the following elements: a) Isolated Pan-T cells, naive CD4 + T cells, naive CD8+ T cells, or naive CD4 + and naive CD8 + means for obtaining a population of T cells, or any combination thereof; b) a means for activating said T cell population; c) means for genetically modifying the cells following activation to express a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR); d) means for expanding the genetically modified cells; Isolated Pan-T cells, naive CD4 + T cells, naive CD8 + T cells or naive CD4 + and naive CD8 + Provided herein is a system, wherein obtaining a population of T cells, or any combination thereof, activating the T cell population, and genetically modifying the T cell population are performed in the presence of one or more cytokines, including interleukin-7 (IL-7).

[0088] In various embodiments of the systems described herein, the one or more cytokines further comprise IL-15 and / or IL-21.

[0089] In another embodiment, the stem cell-like memory T (T SCM ) A composition for concentrating cells, comprising: aT cell population, bT SCM one or more cytokines, including interleukin 7 (IL-7), for enriching the cells; c. Optionally, the T SCM and a means for expanding the cells.

[0090] In another embodiment, the stem cell-like memory T (T SCM ) A composition for concentrating cells, comprising: aT cell population, b. an effective amount of one or more cytokines, including interleukin 7 (IL-7); (i) contacting a population of T cells with an effective amount of one or more cytokines, including IL-7, to induce T cell proliferation and / or proliferation; SCM To concentrate cells, (ii) the concentrated T SCM To activate cells, and (iii) optionally, the T SCM Provided herein are compositions comprising a means for expanding the cells.

[0091] In another embodiment, genetically modified stem cell-like memory T (T SCM ) A composition for generating cells, comprising: aT cell population, b. an effective amount of one or more cytokines, including interleukin 7 (IL-7); (i) contacting a population of T cells with an effective amount of one or more cytokines, including IL-7, to induce T cell proliferation and / or proliferation; SCM To concentrate cells, (ii) the concentrated T SCM To activate cells, (iii) the concentrated T SCM To genetically modify cells to express chimeric antigen receptors (CARs) or engineered T cell receptors (TCRs), and (iv) optionally, the T SCM and a means for expanding the cells.

[0092] In various embodiments, T SCM The cells are T SCM The cells are enriched by contacting them with an effective amount of one or more cytokines, including IL-7, for a period of time sufficient to enrich the cells. [Brief description of the drawings]

[0093] [Figure 1A] Exemplary second generation CAR-T cell designs are shown. Figure 1A is a diagram of a second generation CAR-T comprising a hinge and transmembrane sequence derived from human CD8A and a scFv targeting a TAA of interest, e.g., B-cell maturation antigen (BCMA), fused with 4-1BB (CD137) and CD3 zeta intracellular domains. Figure 1B shows the amino acid sequence of an exemplary anti-BCMA CAR construct described herein. [Figure 1B] Exemplary second generation CAR-T cell designs are shown. Figure 1A is a diagram of a second generation CAR-T comprising a hinge and transmembrane sequence derived from human CD8A and a scFv targeting a TAA of interest, e.g., B-cell maturation antigen (BCMA), fused with 4-1BB (CD137) and CD3 zeta intracellular domains. Figure 1B shows the amino acid sequence of an exemplary anti-BCMA CAR construct described herein. [Figure 2A] Figure 2 shows that cytokine modulation enhances TSCM cell phenotype in CD4+ T cell subsets. Pan-T cells were activated in the presence or absence of the indicated cytokines. For fluorescence-activated cell sorting (FACS) analysis, cells were gated on CD4+CAR+ T cells. The frequency levels of expression of CD62L (Figure 2A), CCR7 (Figure 2B), and CD27 (Figure 2C) in CD4+BCMA-HL CAR-transduced cells were determined. Shown here is the phenotypic characterization 14 days after CAR-T cell transduction. [Figure 2B] Figure 2 shows that cytokine modulation enhances TSCM cell phenotype in CD4+ T cell subsets. Pan-T cells were activated in the presence or absence of the indicated cytokines. For fluorescence-activated cell sorting (FACS) analysis, cells were gated on CD4+CAR+ T cells. The frequency levels of expression of CD62L (Figure 2A), CCR7 (Figure 2B), and CD27 (Figure 2C) in CD4+BCMA-HL CAR-transduced cells were determined. Shown here is the phenotypic characterization 14 days after CAR-T cell transduction. [Figure 2C]Figure 2 shows that cytokine modulation enhances TSCM cell phenotype in CD4+ T cell subsets. Pan-T cells were activated in the presence or absence of the indicated cytokines. For fluorescence-activated cell sorting (FACS) analysis, cells were gated on CD4+CAR+ T cells. The frequency levels of expression of CD62L (Figure 2A), CCR7 (Figure 2B), and CD27 (Figure 2C) in CD4+BCMA-HL CAR-transduced cells were determined. Shown here is the phenotypic characterization 14 days after CAR-T cell transduction. [Diagram 3] A-B show that cytokine modulation enhances TSCM cell phenotype in CD8+ T cell subsets. Pan-T cells were activated in the presence or absence of the indicated cytokines. For FACS analysis, cells were gated on CD8+ CAR+ T cells. The frequency levels of expression of CD62L (Figure 3A), CCR7 (Figure 3B), and CD27 (Figure 3C) in CD8+ BCMA-HL CAR-transduced cells were determined. Shown here is the phenotypic characterization 14 days after CAR-T cell transduction. [Figure 4A] Figure 4 shows that cytokine modulation enhances CD45RO- / CD45RA+ TSCM cell phenotype in both CD4+ and CD8+ T cell subsets. Pan-T cells were activated in the presence or absence of the indicated cytokines. For FACS analysis, cells were gated on CAR+ T cells. Frequency levels of expression of CD45RO- / CD45RA+ in CD4+CAR+ T cell subsets (Figure 4A). Frequency levels of expression of CD45RO- / CD45RA+ in CD8+CAR+ T cell subsets (Figure 4B). Shown here is the phenotypic characterization 14 days after CAR-T cell transduction. [Figure 4B]Figure 4 shows that cytokine modulation enhances CD45RO- / CD45RA+ TSCM cell phenotype in both CD4+ and CD8+ T cell subsets. Pan-T cells were activated in the presence or absence of the indicated cytokines. For FACS analysis, cells were gated on CAR+ T cells. Frequency levels of expression of CD45RO- / CD45RA+ in CD4+CAR+ T cell subsets (Figure 4A). Frequency levels of expression of CD45RO- / CD45RA+ in CD8+CAR+ T cell subsets (Figure 4B). Shown here is the phenotypic characterization 14 days after CAR-T cell transduction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0094] definition The terms "T cell" and "T lymphocyte" are interchangeable and are used synonymously herein. As used herein, T cells include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Th1), T helper 2 (Th2) cells, T helper 17 (Th17), or regulatory T (Treg) cells. T cells can be T helper T cells (Th; CD4 + T cells), CD4 + T cells, CD8 + T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), tumor-infiltrating cytotoxic T cells (TIL, CD8 + T cells), CD4 + CD8 + T cells, stem cell-like memory T (T SCM ) cells, central memory T cells (T CM ), effector memory T cells (T EM ), terminal effector T cells (T eff ), or any other subset of T cells. Exemplary T cell populations suitable for use in certain embodiments include stem central memory T cells (T SCM ) are mentioned.

[0095] Naive T cells can have the following expression pattern of cell surface markers: CCR7 + , CD62L + , CD45RA + , CD45RO - , CD95 - Stem cell-like memory T cells (T SCM ) can have the following expression pattern of cell surface markers: CCR7 + , CD62L + , CD45RA + , CD45RO - , CD95 + Central memory T cells (T CM ) can have the following expression pattern of cell surface markers: CCR7 + , CD62L + , CD45RA - , CD45RO + , CD95 + Effector memory T cells (T EM ) can have the following expression pattern of cell surface markers: CCR7 - , CD62L - , CD45RA - , CD45RO + , CD95 + Terminal effector T cells (T eff ) can have the following expression pattern of cell surface markers: CCR7 - , CD62L - , CD45RO - , CD95 + See, e.g., Gattinoni et al. Nat. Med. 17(2011):1290-7, and Flynn et al. Clin. Translat. Immunol. 3(2014):e20, which are incorporated by reference in their entireties for all purposes.

[0096] The terms "express" and "expression" mean to allow or cause the information of a gene or DNA sequence to be produced, e.g., to produce an RNA or protein, by activating cellular functions involved in the transcription and / or translation of the corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an "expression product," such as an RNA or protein. The expression product itself, e.g., the resulting protein, can also be said to be "expressed" by the cell. Expression products can be characterized as intracellular, extracellular, or transmembrane.

[0097] The terms "vector," "cloning vector," and "expression vector" refer to a vehicle capable of introducing DNA or RNA sequences (e.g., foreign genes) into a host cell to genetically modify the cell and promote expression (e.g., transcription and translation) of the introduced sequences. Vectors include plasmids, synthetic RNA and DNA molecules, phages, viruses, and the like. In certain embodiments, the vector is a viral vector, for example, but not limited to, an adenovirus, adeno-associated, alphavirus, herpes, lentivirus, retrovirus, or vaccinia vector.

[0098] As used herein, the terms "specifically bind," "specifically recognize," or "specifically for" refer to a measurable and reproducible interaction, such as binding between a target and an antigen binding protein (such as a CAR or engineered TCR), that determines the presence of the target in the presence of a heterogeneous population of molecules, including biological molecules.

[0099] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term "protein" encompasses all types of modified proteins, including natural and synthetic proteins, including protein fragments of all lengths, fusion proteins, and glycoproteins, as well as all other types of modified proteins, including, but not limited to, proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, and the like.

[0100] The terms "nucleic acid," "nucleotide," and "polynucleotide" encompass both DNA and RNA unless otherwise specified. "Nucleic acid sequence" or "nucleotide sequence" means a nucleic acid sequence that encodes an amino acid, and the term can also refer to a nucleic acid sequence that includes portions that encode any amino acids added as an artifact of cloning, including any amino acids encoded by linkers.

[0101] The term "treating" or "treatment" of a condition, disorder, or condition includes: (1) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or preclinical symptom of the condition, disorder, or condition developing in a subject who may be afflicted with or susceptible to the condition, disorder, or condition, but who has not yet undergone or exhibited a clinical or preclinical symptom of the condition, disorder, or condition, or (2) inhibiting the condition, disorder, or condition, i.e., arresting, reducing, or delaying the onset or recurrence of the disease, or at least one clinical or preclinical symptom thereof, or (3) relieving the disease, i.e., causing regression of the condition, disorder, or condition, or at least one clinical or preclinical symptom thereof. The benefit to a subject being treated is either statistically significant or at least perceptible to the patient or physician.

[0102] The term "effective" as applied to a dose or amount refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity upon administration to a subject in need thereof. It should be noted that when administering a combination of active ingredients, the effective amount of the combination may or may not include the amount of each ingredient that would be effective when administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition, the severity of the condition being treated, the particular drug used, the mode of administration, and the like.

[0103] The phrase "pharmaceutical acceptable," when used in connection with the compositions described herein, refers to molecular entities and other components of such compositions that are physiologically tolerable and do not normally produce adverse reactions when administered to a mammal (e.g., a human). Preferably, the term "pharmaceutical acceptable" means approved by a federal or state regulatory agency, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias, for use in mammals, more specifically, humans.

[0104] The terms "patient", "individual", "subject" and "animal" are used interchangeably herein and refer to mammals, including but not limited to humans, non-human primates, and veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models. Examples of mammals include mammals of the order Rodentia (e.g., mice and hamsters), mammals of the order Lagomorpha (e.g., rabbits), mammals of the order Carnivora, including Felidae (cats) and Canidae (dogs), mammals of the order Artiodactyla, including Bovidae (cattle) and Porcineae (pigs), mammals of the order Perissodactyla, including Equidae (horses), or mammals of the order Primates, Ceboids or Simoids (monkeys) or Anthropoids (humans and apes). In a preferred embodiment, the subject is a human.

[0105] The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous solutions, saline solutions, and aqueous dextrose and glycerin solutions are preferably used as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier, including, but not limited to, one or more of binders (for compressed pills), glidants, encapsulating agents, flavoring agents, and coloring agents. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0106] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the type described herein that will become apparent to those skilled in the art upon reading this disclosure.

[0107] The term "about" or "approximately" includes being within a statistically meaningful range of values. Such ranges may be within an order of magnitude, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5% of a given value or range. The acceptable variation encompassed by the term "about" or "approximately" depends on the specific system under study and can be readily understood by one of ordinary skill in the art.

[0108] Throughout the disclosure, various aspects of the disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes those with 95%, 96%, 97%, 98%, or 99% identity, including subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the breadth of the scope.

[0109] All embodiments of any aspect of the invention can be used in combination unless the context clearly dictates otherwise.

[0110] Methods of the Invention Cells suitable for use in the methods of the present disclosure may be derived from any cell and tissue, particularly mammalian cells and tissues. Suitable cells may be derived from humans, apes, monkeys, pigs, or rodents, and may be primary or cultured cells. In some embodiments, the cells modified using the methods of the present disclosure are human cells.

[0111] Donor cell isolation / enrichment In some embodiments, the cells used in the methods of the present disclosure are obtained from a donor. In some embodiments, the cells may be allogeneic or non-autologous ("non-autologous") to the recipient to whom the cells are administered. In alternative embodiments, the cells may be autologous to the recipient to whom the cells are administered. In some embodiments, the cells are obtained from a mammalian subject. In other embodiments, the cells are obtained from a primate subject. In some embodiments, the cells are obtained from a human subject.

[0112] In some embodiments, the cells used in the methods of the present disclosure are lymphocytes (e.g., T cells). Lymphocytes can be obtained from sources such as, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. Lymphocytes can also be generated by differentiation of stem cells. In some embodiments, lymphocytes can be obtained from blood drawn from a subject using techniques generally known to those skilled in the art, such as sedimentation, e.g., FICOLL™ separation.

[0113] Cells from the circulating blood of a subject can be obtained by apheresis. Apheresis devices typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing. The cells can be washed with PBS or another suitable solution lacking calcium, magnesium, and most, if not all, other divalent cations. The washing step can be accomplished by methods known to those skilled in the art, such as, for example, using a semi-automated flow-through centrifuge, such as, but not limited to, a Cobe 2991 cell processing machine or a Baxter CytoMate. After washing, the cells can be resuspended in a variety of biocompatible buffers, cell culture media, or other saline solutions with or without buffers.

[0114] T cells can be isolated from peripheral blood mononuclear cells (PBMCs) by lysing red blood cells and depleting monocytes. As an example, T cells can be sorted by centrifugation through a PERCOLL™ gradient. In some embodiments, after isolation of PBMCs, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations, either before or after activation, expansion, and / or genetic modification.

[0115] In some embodiments, the T cell populations used in the methods described herein include Pan-T cells, naive CD4 + T cells, naive CD8 + T cells or naive CD4 + and CD8 + In some embodiments, the T cell population is isolated from a sample obtained from a subject.

[0116] In some embodiments, the T cell populations used in the methods described herein do not include suppressive regulatory T cells.

[0117] In some embodiments, T lymphocytes can be enriched, for example, stem central memory T cells (T cells) expressing one or more markers, including, but not limited to, CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD27, CD28, CD34, CD36, CD45RA, CD45RO, CD56, CD62, CD62L, CD122, CD123, CD127, CD235a, CCR7, HLA-DR, or combinations thereof. SCM Specific subpopulations of T lymphocytes, such as stem cell-like memory T cells (T), can be enriched using either positive or negative selection techniques. In some embodiments, stem cell-like memory T cells (T SCM ) are enriched using the methods detailed herein.

[0118] In some embodiments, T lymphocytes can also be differentiated from stem cells, such as umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, hematopoietic stem cells (HSCs), and induced pluripotent stem cells (iPSCs).

[0119] Genetic modification T cells can be genetically modified to express a high affinity T cell receptor (engineered TCR) or a chimeric antigen receptor (CAR). In some embodiments, the methods described herein include introducing into the cell an exogenous nucleic acid molecule that includes a nucleotide sequence encoding a CAR or engineered TCR. In some embodiments, the T cells are genetically modified to express one or more engineered TCRs or CARs.

[0120] In some embodiments, the genetic modification may be performed in the presence of one or more cytokines, including IL-7, IL-15, and / or IL-21. In some embodiments, the genetic modification may be performed in the presence of IL-7. In some embodiments, the genetic modification may be performed in the presence of IL-7 and IL-15. In some embodiments, the genetic modification may be performed in the presence of IL-7 and IL-21. In some embodiments, the genetic modification may be performed in the presence of IL-7, IL-15, and IL-21.

[0121] In some embodiments, the genetic modification may be performed in the absence of IL-2.

[0122] In some embodiments, genetic modification can be performed prior to expansion of T cells. For example, Pan-T cells isolated from PBMCs, naive CD4 + T cells, naive CD8 + T cells or naive CD4 + and CD8 + The T cells, T cells, or any combination thereof may be genetically modified.

[0123] In some embodiments, genetic modification may be performed after expansion of T cells. In some embodiments, enriched stem cell-like memory T cells (TSCM ) are genetically modified.

[0124] As used herein, "chimeric antigen receptor" or "CAR" refers to a cell surface receptor that comprises an extracellular target binding domain, a transmembrane domain, and a cytoplasmic domain that includes a lymphocyte activation domain and optionally at least one costimulatory signaling domain, all in a combination not found together in a single protein in nature. This specifically includes receptors in which the extracellular and cytoplasmic domains are not found together in a single receptor protein in nature.

[0125] Naturally occurring T cell receptors contain two subunits, an α subunit and a β subunit, each of which is a unique protein produced by a recombination event in the genome of each T cell. Libraries of TCRs can be screened for their selectivity for a particular target antigen. In this way, natural TCRs with high avidity and reactivity to the target antigen can be selected, cloned, and subsequently introduced into a T cell population to be used for adoptive immunotherapy.

[0126] In one embodiment, T cells are modified by introducing a polynucleotide encoding a subunit of a TCR capable of forming a TCR that confers specificity to the T cell for tumor cells expressing a target antigen. In a particular embodiment, the subunit has one or more amino acid substitutions, deletions, insertions, or modifications compared to a naturally occurring subunit, so long as the subunit retains the ability to form a TCR that confers the transfected T cell the ability to home to the target cell and participate in immunologically relevant cytokine signaling. The engineered TCR also preferably binds with high avidity to target cells presenting the relevant tumor associated peptide, and optionally mediates efficient killing of target cells presenting the relevant peptide in vivo.

[0127] The exogenous nucleic acid molecule comprising a nucleotide sequence encoding a CAR or engineered TCR may be expressed episomally. Alternatively, the exogenous nucleic acid molecule comprising a nucleotide sequence encoding a CAR or engineered TCR may be knocked into a locus via homology directed repair (HDR) (e.g., by using a genome editing nuclease such as CRISPR / Cas). For example, but not limited to, an exogenous nucleic acid molecule comprising a nucleotide sequence encoding a CAR or engineered TCR may be knocked into a TCRα, TCRβ, or B2M locus to replace the endogenous gene. For knock-in, the nucleic acid molecule comprising a nucleotide sequence encoding a CAR or engineered TCR may be provided as double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), or in a viral vector (e.g., AAV). In either embodiment, the gene is operably linked (i.e., under transcriptional control) to a promoter active in the cell.

[0128] The CAR or engineered TCR can be directed against an antigen expressed on the surface of a malignant or infected cell, such as a tumor antigen or an infectious antigen.

[0129] Non-limiting examples of tumor antigens that can be targeted by the engineered cells described herein include B-cell maturation antigen (BCMA), human epidermal growth factor receptor 2 (HER2), kallikrein-related peptidase 2 (KLK2), hexokinase 2 (hK2), interleukin-13 receptor subunit alpha-2 (IL-13Ra2), ephrin type A receptor 2 (EphA2), kinase anchor protein 4 (AKAP-4), adrenergic receptor beta 3 (ADRB3), anaplastic lymphoma kinase (ALK), immunoglobulin lambda-like polypeptide 1 (IGLL1), and IL-13R1 (IL-13R2). gen receptor, angiopoietin-binding cell surface receptor 2 (Tie2), B7H3 (CD276), bone marrow stromal cell antigen 2 (BST2), carbonic anhydrase IX (CAIX), CCCTC-binding factor (ZFinger)-like (BORIS), CD171, CD179a, CD24, CD300 molecule-like family member f (CD300LF), CD38, CD44v6, CD72, CD79a, CD79b, CD97, chromosome X open reading frame 61 (CXORF61), claudin 6 (CLDN6), CS-1 (CD2 subset 1, CRACC, SLAMF7, CD319 or 19A24), C-type lectin domain family 12 member A (CLEC12A), C-type lectin-like molecule-1 (CLL-1), cyclin B1, cytochrome P4501B1 (CYP1B1), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), epidermal growth factor receptor (EGFR), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), ETS translocation variant gene 6, located on chromosome 12p (ETV6-AML), Fc fragment of IgA receptor FCAR, Fc receptor-like 5 (FCRL5), Fms-like tyrosine kinase 3 (FLT3), folate receptor β, Fos-related antigen 1, fucosyl GM1, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), ganglioside GD3, ganglioside GM3, glycoceramide (GloboH), glypican-3 (GPC3), hepatitis A virus cellular receptor 1 (HAVCR1), hexasaccharide portion of globe H, high molecular weight melanoma-associated antigen (HMWMAA), human telomerase reverse transcriptase (hTERT),Interleukin-11 receptor alpha (IL-11Ra), KIT (CD117), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), leukocyte immunoglobulin-like receptor sub-A family member 2 (LILRA2), Lewis (Y) antigen, lymphocyte antigen 6 complex, locus K9 (LY6K), lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), mammary differentiation antigen (NY-BR-1), melanoma cancer testis antigen 1 (MAD-CT-1), melanoma cancer testis antigen 2 (MAD-CT-2), apoptosis Inhibitor (ML-IAP), mucin 1, cell surface-associated (MUC1), N-acetylglucosaminyltransferase V (NA17), neural cell adhesion molecule (NCAM), o-acetylglucosamine-glucosamine complex (OAcGD2), olfactory receptor 51E2 (OR51E2), p53 mutant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), pannexin 3 (PANX3), placenta-specific 1 (PLAC1), platelet-derived growth factor receptor β (PDGFR-β), polysialic acid, proac oleicin-binding protein sp32 (OY-TES1), prostate stem cell antigen (PSCA), protease serine 21 (PRSS21), proteasome (macropain) subunit, beta type, 9 (LMP2), Ras homolog C (RhoC), sarcoma translocation breakpoint, sialyl Lewis adhesion molecule (sLe), sperm protein 17 (SPA17), squamous cell carcinoma antigen 3 recognized by T cells (SART3), stage-specific embryonic antigen 4 (SSEA-4), synovial sarcoma, X breakpoint 2 (SSX2 ), TCR gamma alternative reading frame protein (BCMA), TGS5, thyrotropin receptor (ALK), Tn antigen (TAg), tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), uroplakin 2 (UPK2), vascular endothelial growth factor receptor 2 (VEGFR2), v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (CAIX), Wilms tumor protein (WT1), and X antigen family, member 1A (XAGE1), or fragments or variants thereof.

[0130] Additional antigens that may be targeted by the modified cells described herein include carbonic anhydrase EX, alpha-fetoprotein, A3, antigen specific for the A33 antibody, Ba733, BrE3-antigen, CA125, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD80, CD123, CD1 38, Fms-related receptor tyrosine kinase 3 (FLT3) or CD135, colon-specific antigen-p (CSAp), CEA (CEACAM5), CEACAM6, CSAp, EGFR, EGP-I, EGP-2, Ep-CAM, EphA1, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA10, EphB1, EphB2, EphB3, EphB4, EphB6, FIt-I, Fl t-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, hypoxia-inducible factor (HIF-1), Ia, IL-2, IL-6, IL-8, insulin growth factor-1 (IGF-I), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophage inhibitory factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, PAM Examples of antigens that may be used include, but are not limited to, an antigen specific for -4ll antibody, placenta growth factor, p53, prostatic acid phosphatase, PSA, PSMA, RS5, S100, TAC, TAG-72, tenascin, TRAIL receptor, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGF, ED-B fibronectin, 17-1A-antigen, angiogenesis markers, oncogene markers or oncogene products.

[0131] In certain embodiments, the tumor antigen targeted by the modified cells described herein is BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, hk2, FLT3, CD20, CD22, KRASG12D, p53, BRAC1, or PSMA.

[0132] In one embodiment, a CAR that specifically binds to BCMA comprises an extracellular target binding domain comprising the amino acid sequence of SEQ ID NO:2, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2.

[0133] In one embodiment, a CAR that specifically binds to BCMA comprises an extracellular target binding domain comprising the amino acid sequence of SEQ ID NO:3, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:3.

[0134] In one embodiment, a CAR that specifically binds to BCMA comprises the amino acid sequence of SEQ ID NO:11, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:11.

[0135] In one embodiment, a CAR that specifically binds to BCMA comprises an extracellular target binding domain comprising the amino acid sequence of SEQ ID NO:12, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:12.

[0136] The infectious antigen may be a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, or a prion antigen, etc. Infectious antigens include intact microorganisms (e.g., viruses, bacteria, fungi) and natural isolates and fragments or derivatives thereof, and also synthetic or recombinant compounds that are identical or similar to natural microbial antigens and induce specific immune responses to the microorganisms (e.g., viruses, bacteria, fungi). A compound is similar to a natural microbial antigen if it induces an immune response (humoral and / or cellular) against the natural microbial antigen. Such antigens are routinely used in the art and are well known to those skilled in the art.

[0137] The infectious antigen may be or be derived from an infectious virus. Non-limiting examples of infectious viruses found in humans include Adenoviridae (most adenoviruses); Arenaviridae (hemorrhagic fever viruses); Birnaviridae; Bunyaviruses (e.g., Hantavirus, Bungavirus, Phlebovirus, and Nairovirus); Calciviruses (e.g., strains that cause gastroenteritis); Coronoviridae (e.g., coronaviruses); Filoviridae (e.g., Ebola virus); Flaviviridae (e.g., Dengue virus, encephalitis virus, yellow fever virus); Hepadnaviridae (Hepatitis B virus); Herpesviridae (Herpes simplex virus (HSV) types 1 and 2, Varicella zoster virus, Cytomegalovirus (CMV), Herpes virus); Iridoviridae (e.g., African swine fever virus); Norwalk and related viruses, and Astrovirus; Orthomyxoviridae (e.g., influenza virus); Papovaviridae (papillomavirus, polyomavirus); Paramyxoviridae (e.g., Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, human coxsackievirus, rhinovirus, echovirus); Poxviridae (variola virus, vaccinia virus, poxvirus); Reoviridae (e.g., reovirus, orbivirus, and rotavirus); Retroviridae (e.g., human immunodeficiency viruses such as HIV-1 (also called HTLV-III, LAV, or HTLV-III / LAV, or HIV-III); and other isolates such as HIV-LP); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Togaviridae (e.g., equine encephalitis virus, rubella virus); and unclassified viruses (e.g., pathogens of spongiform encephalopathy, pathogens of delta hepatitis, pathogens of non-A non-B hepatitis (i.e., hepatitis C)).

[0138] The infectious antigen may be or may be derived from an infectious bacterium. Both gram-negative and gram-positive bacteria can function as antigens in vertebrates. Such gram-positive bacteria include, but are not limited to, Pasteurella species, Staphylococcus species, and Streptococcus species. Grain-negative bacteria include, but are not limited to, Escherichia coli, Pseudomonas species, and Salmonella species. Non-limiting examples of infectious bacteria include Actinomyces israelli, Bacillus anthracis, Bacteroides sp., Borrelia burgdorferi, Chlamydia, Clostridium perfringers, Corynebacterium diphtheriae, Corynebacterium sp., Enterobacter aerogenes, Enterococcus sp., Erysipelothrix rhusiopathiae, Fusobacterium nucleatum, Haemophilus influenzae, Helicobacter pylori, pylori, Klebsiella pneumoniae, Legionella pneumophilia, Leptospira, Listeria monocytogenes, Mycobacteria sps. (e.g., M. tuberculosis, M. avium, M gordonae, M.M intracellulare, M kansaii, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, pathogenic Campylobacter sp., Rickettsia, Staphylococcus aureus, Streptobacillus monihformis, Streptococcus (anaerobic genera), Streptococcus (viridans group), Streptococcus agalactiae (group B streptococcus), Streptococcus bovis, Streptococcus faecalis faecalis, Streptococcus pneumoniae, Streptococcus pyogenes (Group A streptococcus), Treponema pallidium, and Treponema pertenue.

[0139] Infectious antigens may be or be derived from other infectious microorganisms. Non-limiting examples of infectious fungi include Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dernatitidis, Chlamydia trachomatis, and Candida albicans. Other infectious organisms (i.e., protozoa) include, for example, Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax, Toxoplasma, and plasmodium such as Shistosoma. Other medically relevant microorganisms have been extensively described in the literature, see, for example, CGA Tomas, "Medical Microbiology", Bailliere Tindall, Great Britain 1983, which is incorporated herein by reference in its entirety.

[0140] Other non-limiting examples of infectious antigens include viral antigens such as HIV antigens (e.g., gp120, gp160, p18, Tat, Gag, Pol, Env, Nef), glycoproteins from herpesviruses, and surface and core antigens from Hepatitis B virus; bacterial antigens such as OspA, OspB and OspC antigens from Borrelia spp.; fungal and parasitic antigens such as MP65 from Candida albicans and CS proteins from Plasmodium family.

[0141] In some embodiments, the CAR or engineered TCR can be directed against an autoantigen, such as, for example, an antigen associated with an autoimmune disease, such as rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease, and ulcerative colitis.

[0142] In further embodiments, the methods of the present disclosure may also involve reducing or inhibiting expression of one or more endogenous T cell receptors (TCRs).

[0143] Various embodiments of the above methods involve introducing one or more polynucleotide / polypeptide agents (e.g., CAR or engineered TCR) into cells. The polynucleotides and / or polypeptides described in the present invention can be introduced into cells via viral, non-viral gene delivery methods, or physical methods. Suitable methods for polynucleotide and / or polypeptide delivery for use in the methods of the present invention include any method known to those skilled in the art that can introduce polynucleotides and / or polypeptides into organelles, cells, tissues, or organisms. The introduction of polynucleotides and / or polypeptides can be performed in vitro, ex vivo, or in vivo.

[0144] In various embodiments, the polypeptide or polynucleotide is introduced into the cell using physical methods. Suitable physical methods include, but are not limited to, electroporation, direct injection (e.g., microinjection), magnetofection, ultrasound, ballistic or hydrodynamic methods, or a combination thereof.

[0145] Electroporation is a method for polynucleotide and / or polypeptide delivery. See, e.g., Potter et al., (1984) Proc. Nat'l Acad. Sci. USA, 81, 7161-7165 and Tur-Kaspa et al., (1986) Mol. Cell Biol. 6, 716-718, both of which are incorporated herein in their entirety for all purposes. Electroporation involves exposing a suspension of cells and DNA to a high-voltage electrical discharge. In some embodiments, cell wall degrading enzymes, such as pectin degrading enzymes, can be used to make cells more susceptible to genetic modification by electroporation than untreated cells. See, e.g., U.S. Patent No. 5,384,253, which is incorporated herein in its entirety for all purposes.

[0146] When CRISPR / Cas nucleases are used, one or more CRISPR / Cas nucleases and one or more gRNAs can be assembled to form one or more ribonucleoprotein (RNP) complexes, which can then be introduced into a cell by electroporation.

[0147] Methods of electroporation for use in the present invention include, for example, Sardesai, NY and Weiner, DB, Current Opinion in Immunotherapy 23:421-9 (2011) and Ferraro, B. et al., Human Vaccines 7:120-127 (2011), both of which are incorporated by reference in their entirety for all purposes.

[0148] Another physical method for polynucleotide and / or polypeptide transfer includes injection. In some embodiments, the polypeptide, polynucleotide, or vector can be delivered to a cell, tissue, or organism via one or more injections (e.g., needle injection). Non-limiting methods of injection include injection of a composition (e.g., a saline-based composition). The polynucleotide and / or polynucleotide can also be introduced by direct microinjection. Non-limiting sites of injection include subcutaneous, intradermal, intramuscular, intranodal (allowing for direct delivery of antigens to lymphatic tissues), intravenous, intraprostatic, intratumoral, intralymphatic (allowing for direct administration of dendritic cells), and intraperitoneal. It is understood that proper injection site preparation (e.g., scraping the injection site to observe proper needle placement) is required.

[0149] In some embodiments, the polynucleotides and / or polypeptides described in the present invention are introduced into cells by pinocytosis induced by hypertonicity or hypotonicity. For example, cells can be placed in a buffer with a salt concentration that is either higher or lower than normal saline. This can activate an active uptake mechanism in cells that takes up the extracellular environment. Various chemicals can be used to enhance and modify this process. It does not require special machinery. Exemplary methods in which pinocytosis can be used for transduction are described in the art. See, for example, WO2017093326(A1), which is incorporated herein by reference in its entirety for all purposes.

[0150] In various embodiments, the polynucleotides and / or polypeptides described in the present invention are introduced into the cell via a vector, which may be a viral or non-viral vector.

[0151] In some embodiments, the vector is a viral vector. Suitable viral vectors that can be used in the present invention include, but are not limited to, retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, alphavirus vectors, vaccinia virus vectors, herpes simplex viral vectors, or baculovirus vectors. In one particular embodiment, the viral vector is a lentiviral vector. In one particular embodiment, the viral vector is a retroviral vector. In some embodiments, the cells are transduced via retroviral transduction. References describing retroviral transduction of genes are Anderson et al., U.S. Pat. No. 5,399,346; Mann et al., Cell, 33:153 (1983); Temin et al., U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289; Markowitz et al. J. Virol. 62:1120 (1988); Temin et al., U.S. Pat. No. 5,124,263; WO 95 / 07358 by Dougherty et al., published March 16, 1995; and Kuo et al., Blood 82:845 (1993), each of which is incorporated herein by reference in its entirety.

[0152] In some embodiments, the vector is a non-viral vector. Non-limiting examples of non-viral vectors useful in the methods of the present invention include plasmids or transposons.

[0153] Nucleic acid vaccines can also be used to transfer polynucleotides into cells. Such vaccines include, but are not limited to, non-viral polynucleotide vectors, "naked" DNA and RNA, and viral vectors. Methods for genetically modifying cells with these vaccines and for optimizing the expression of genes contained in these vaccines are known to those skilled in the art.

[0154] In some embodiments, polynucleotides and / or polypeptides may be introduced into cells using nanoparticles, polymers, dendrimers, liposomes, and polyethyleneimine (PEI) particles. In some embodiments, polypeptides (e.g., CRISPR / Cas nucleases) are introduced into cells as soluble proteins or ribonucleoproteins.

[0155] Additional methods of polynucleotide and / or polypeptide transfer include liposome-mediated transfection (e.g., polynucleotides are entrapped in lipid complexes suspended in excess aqueous solution). See, e.g., Ghosh and Bachhawat, (1991) In:Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands. pp.87-104. Polynucleotides and / or polypeptides complexed with Lipofectamine or Superfect; DEAE-dextran (e.g., polynucleotides are delivered to cells using DEAE-dextran followed by polyethylene glycol) are also contemplated. See, e.g., Gopal, TV, Mol Cell Biol. 1985 May; 5(5):1188-90); calcium phosphate (e.g., polynucleotides are introduced into cells using calcium phosphate precipitation). See, e.g., Graham and van der Eb, (1973) Virology, 52, 456-467; Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987), and Rippe et al., Mol. Cell Biol., 10:689-695, 1990); sonication loading (introduction of polynucleotides by direct sonication loading); see, e.g., Fechheimer et al., (1987) Proc. Nat'l Acad. Sci. USA, 84, 8463:8467); biolistic bombardment (e.g., one or more particles can be coated with at least one polynucleotide and / or polypeptide and delivered into cells by a propelling force).See, e.g., U.S. Pat. No. 5,550,318; U.S. Pat. No. 5,538,880, U.S. Pat. No. 5,610,042; and WO 94 / 09699; Klein et al., (1987) Nature, 327, 70-73; Yang et al., (1990) Proc. Nat'l Acad. Sci. USA, 87, 9568:9572; and receptor-mediated transfection (e.g., selective uptake of macromolecules by receptor-mediated endocytosis occurs in target cells using cell-type specific distribution of various receptors). See, e.g., Wu and Wu, (1987) J. Biol. Chem., 262, 4429-4432; Wagner et al., Proc. Natl. Acad. Sci. USA, 87(9):3410-3414, 1990; Perales et al., Proc. Natl. Acad. Sci. USA 91:4086-4090(1994); Myers, EPO 0273085; Wu and Wu, Adv. Drug Delivery Rev., 12:159-167, 1993; Nicolau et al., (1987) Methods Enzymol., 149, 157-176), each of which is incorporated by reference in its entirety for all purposes.

[0156] It should be appreciated that in the case of CRISPR / Cas nucleases, the Cas protein (e.g., Cas9, Cas12a) and gRNA do not have to be delivered using the same method. In some embodiments, the Cas protein (e.g., Cas9, Cas12a) and gRNA are delivered using the same method. For example, both the Cas protein (e.g., Cas9, Cas12a) and gRNA can be introduced into the cell via electroporation or in the same vector. In some embodiments, the Cas protein (e.g., Cas9, Cas12a) and gRNA are delivered using different methods. For example, the Cas protein (e.g., Cas9, Cas12a) is introduced into the cell via electroporation and the gRNA is delivered in a viral vector. As another example, the Cas protein (e.g., Cas9, Cas12a) and gRNA are delivered in separate vectors.

[0157] T cell stimulation / activation To reach a sufficient therapeutic dose of the cell composition, the T cells may be subjected to one or more rounds of stimulation / activation. The cells may be activated and / or expanded ex vivo before, after, and / or during the genetic modification process.

[0158] In some embodiments, stimulation / activation of T cells may be performed in the presence of one or more cytokines including IL-7, IL-15, and / or IL-21. In some embodiments, stimulation / activation of T cells may be performed in the presence of IL-7. In some embodiments, stimulation / activation of T cells may be performed in the presence of IL-7 and IL-15. In some embodiments, stimulation / activation of T cells may be performed in the presence of IL-7 and IL-21. In some embodiments, stimulation / activation of T cells may be performed in the presence of IL-7, IL-15 and IL-21.

[0159] In some embodiments, stimulation / activation of T cells may be carried out in the absence of IL-2.

[0160] In some embodiments, the methods described herein include stimulating T cells to become activated in the presence of one or more stimulatory signals or agents (e.g., compounds, small molecules, such as small organic molecules, nucleic acids, polypeptides, or fragments, isoforms, variants, analogs, or derivatives thereof). In some embodiments, the methods described herein include stimulating T cells to become activated and proliferate in the presence of one or more stimulatory signals or agents.

[0161] T cells can be activated by inducing changes in their biological state in which the cells express activation markers, produce cytokines, proliferate, and / or become cytotoxic to target cells. All of these changes can be caused by a primary stimulatory signal. Costimulatory signals amplify the magnitude of the primary signal and suppress cell death after the initial stimulation, resulting in a more durable activation state and therefore a higher cytotoxic potential.

[0162] T cells can generally be activated using methods such as those described in, for example, U.S. Patent Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, and 6,867,041, each of which is incorporated herein by reference in its entirety.

[0163] In some embodiments, α-β T cells can be activated using CD3 / CD28 stimulation. As an example, α-β T cells can be stimulated using anti-CD3 and anti-CD28 antibodies.

[0164] In some embodiments, gamma-delta T cells can be activated by zoledronate and / or an agent (e.g., an antibody) that binds to the gamma-delta TCR, and IL-2 and IL-15 can be used to expand gamma-delta T cells.

[0165] In some embodiments, T cells can be activated by binding to an agent that activates CD3ζ.

[0166] In other embodiments, CD2 binding agents can be used to provide a primary stimulatory signal to T cells. For example, but not limited to, CD2 agents include CD2 ligands and anti-CD2 antibodies, such as Tl1.3 antibody combined with Tl1.1 or Tl1.2 antibody (Meuer, SC et al. (1984) Cell 36:897-906, which is incorporated herein by reference in its entirety), and 9.6 antibody (which recognizes the same epitope as TI1.1) combined with 9-1 antibody (Yang, SY et al. (1986) J. Immunol. 137:1097-1100, which is incorporated herein by reference in its entirety). Other antibodies that bind to the same epitope as any of the above antibodies can also be used.

[0167] In some embodiments, T cells are activated by administering phorbol myristate acetate (PMA) and ionomycin. In some embodiments, T cells are activated by administering an appropriate antigen that induces activation and subsequent expansion. In some embodiments, PMA, ionomycin, and / or an appropriate antigen are administered in conjunction with CD3-induced activation and / or expansion.

[0168] Generally, the activating agent used in the present invention includes, but is not limited to, antibodies, fragments thereof, and proteinaceous binding molecules with antibody-like functions. Examples of (recombinant) antibody fragments are bivalent antibody fragments such as Fab fragments, Fv fragments, single chain Fv fragments (scFv), (Fab)2'-fragments, diabodies, triabodies (Iliades, P., et al., FEBS Lett (1997) 409, 437-441, incorporated herein in its entirety by reference), decabodies (Stone, E., et al., Journal of Immunological Methods (2007) 318, 88-94, incorporated herein in its entirety by reference), and other domain antibodies (Holt, LJ, et al., Trends Biotechnol. (2003), 21, 11, 484-490, incorporated herein in its entirety by reference). The bivalent antibody fragment may be a (Fab)2'-fragment, or a bivalent single chain Fv fragment, while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single chain Fv fragment (scFv).

[0169] In some embodiments, one or more binding sites of the CD3ζ agent may be a bivalent proteinaceous artificial binding molecule, such as a dimeric lipocalin mutein (i.e., duocalin). In some embodiments, the receptor binding reagent may have a single second binding site (i.e., monovalent). Examples of monovalent agents (monovalent agents) include, but are not limited to, monovalent antibody fragments, proteinaceous binding molecules with antibody-like binding properties, or MHC molecules. Examples of monovalent antibody fragments include, but are not limited to, Fab fragments, Fv fragments, and single chain Fv fragments (scFv), including bivalent single chain Fv fragments.

[0170] Agents that specifically bind to CD3 include, but are not limited to, anti-CD3 antibodies, bivalent antibody fragments of anti-CD3 antibodies, monovalent antibody fragments of anti-CD3 antibodies, and proteinaceous CD3 binding molecules with antibody-like binding properties. Proteinaceous CD3 binding molecules with antibody-like binding properties can be aptamers, muteins based on the polypeptides of the lipocalin family, glubodies, proteins based on ankyrin scaffolds, proteins based on crystalline scaffolds, adnectins, and avimers. They can also be bound to beads.

[0171] In some embodiments, the activating agent (e.g., CD3 binding agent) can be present at a concentration of about 0.1 to about 10 μg / ml. In some embodiments, the activating agent (e.g., CD3 binding agent) can be present at a concentration of about 0.2 μg / ml to about 9 μg / ml, about 0.3 μg / ml to about 8 μg / ml, about 0.4 μg / ml to about 7 μg / ml, about 0.5 μg / ml to about 6 μg / ml, about 0.6 μg / ml to about 5 μg / ml, about 0.7 μg / ml to about 4 μg / ml, about 0.8 μg / ml to about 3 μg / ml, or about 0.9 μg / ml to about 2 μg / ml. In some embodiments, the activating agent (e.g., CD3 binding agent) is administered at a concentration of about 0.1 μg / ml, about 0.2 μg / ml, about 0.3 μg / ml, about 0.4 μg / ml, about 0.5 μg / ml, about 0.6 μg / ml, about 0.7 μg / ml, about 0.8 μg / ml, about 0.9 μg / ml, about 1 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, or about 10 μg / ml. In some embodiments, the CD3 binding agent can be present at a concentration of 1 μg / ml.

[0172] In some embodiments, the activator may be attached to a solid support such as, but not limited to, beads, absorbent polymers present in a culture plate or well, or other matrices such as, but not limited to, sepharose or glass, and expressed (e.g., in native or recombinant form) on the cell surface of a native or recombinant cell line by means known to those of skill in the art.

[0173] T cell expansion / proliferation After the T cells are activated and transduced, the cells can be cultured and expanded. The T cells can be cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, at least 1 or 2 weeks, at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or more, for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more expansion times. In some embodiments, the T cells are cultured for 1-20 days, 1-18 days, 1-14 days, 3-20 days, 3-18 days, 3-14 days, 5-20 days, 5-18 days, 5-14 days, 7-20 days, 7-18 days, 7-16 days, 7-15 days, 7-14 days, 8-20 days, 8-18 days, 8-16 days, 8-15 days, 8-14 days, 8-13 days, 8-12 days, 9-20 days, 9-18 days, 9-16 days, 9-15 days, 9-14 days, 9-13 days, 9-12 days, 10-20 days, 10-18 days, 10-16 days, 10-15 days, or 10-14 days. In some embodiments, the T cells are cultured for 8-14 days. In one embodiment, the T cells are cultured for 14 days.

[0174] According to the present disclosure, stem cell-like memory T (T SCM ) cells can be concentrated.

[0175] In some embodiments, stem cell-like memory T (T SCM ) cells can be enriched by culturing T cells in the presence of IL-7. In some embodiments, stem cell-like memory T (T SCM ) cells can be enriched by culturing T cells in the presence of IL-7 and IL-15. In some embodiments, stem cell-like memory T (T SCM) cells can be enriched by culturing T cells in the presence of IL-7 and IL-21. In some embodiments, stem cell-like memory T (T SCM ) cells can be enriched by culturing T cells in the presence of IL-7, IL-15, and IL-21. In some embodiments, stem cell-like memory T (T SCM ) cells can be enriched by culturing T cells in the presence of IL-7, IL-15, and IL-21.

[0176] In some embodiments, stem cell-like memory T (T SCM ) The interleukins used to enrich the cells do not include IL-2.

[0177] In some embodiments, the agent used for augmentation (e.g., IL-7, IL-15, IL-21) is administered at about 1 ng / ml to about 20 ng / ml. In some embodiments, the agent used for augmentation (e.g., IL-7, IL-15, IL-21) is administered at about 2 ng / ml to about 20 ng / ml, 5 ng / ml to about 20 ng / ml, 5 ng / ml to about 18 ng / ml, 5 ng / ml to about 15 ng / ml, 5 ng / ml to about 12 ng / ml, 7 ng / ml to about 20 ng / ml, 7 ng / ml to about 18 ng / ml, 7 ng / ml to about 15 ng / ml, 5.5 ng / ml to about 9.5 ng / ml, about 6 ng / ml to about 9 ng / ml, about 6.5 ng / ml to about 12 ng / ml, or about 9 ng / ml to about 12 ng / ml. In some embodiments, the agent used for augmentation (e.g., IL-7, IL-15, IL-21) is administered at about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml or about 15 ng / ml.

[0178] Other examples of agents that can be used to expand T cells are agents that bind to CD8, CD45, or CD90, such as αCD8, αCD45, or αCD90 antibodies. Examples of T cell populations include antigen-specific T cells, T helper cells, cytotoxic T cells, memory T cells (an example of a memory T cell is CD62L + CD8 + specific central memory T cells) or regulatory T cells (Tregs are an example of CD4 + CD25 + CD45RA + Treg cells).

[0179] Additional agents that may be used to expand T lymphocytes include, for example, methods as described in U.S. Pat. Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, and 6,867,041, each of which is incorporated herein by reference in its entirety.

[0180] Suitable conditions for T cell culture include an appropriate medium (e.g., Minimum Essential Medium (MEM), RPMI Medium 1640, Lonza RPMI 1640, Advanced RPMI, Clicks, AIM-V, DMEM, alpha-MEM, F-12, TexMACS, X-Vivo 15, and X-Vivo 20, Optimizer supplemented with amino acids, sodium pyruvate, and vitamins, and either 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 growth and expansion).

[0181] Examples of other additives for T cell expansion include, but are not limited to, detergents, plasmin, pH buffers such as HEPES, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Antibiotics (e.g., penicillin and streptomycin) are included only in the experimental cultures, not in the cultures of cells injected into subjects. Target cells are maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).

[0182] In some embodiments where the cells are iPSC-derived cells, the modified cells are selected for individual clones to create a master cell bank.

[0183] In some embodiments, T SCM The percentage of T cells is determined in the T cell population after cell expansion. SCM The percentage of cells is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% after cell expansion. In one embodiment, the percentage of cells in the T cell population is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. SCM The percentage of cells after cell expansion is about 30%-90%, 40%-80%, 50%-60%, 50%-70%, 50%-80%, 60%-70%, or 60%-80%.

[0184] Compositions of the Invention In one embodiment, the present disclosure provides a method for the preparation of stem cell-like memory T (T SCM The present invention provides a T cell population comprising T-cells comprising:

[0185] In another embodiment, the present disclosure also provides a method for the treatment of stem cell-like memory T (T SCMThe present invention provides a pharmaceutical composition comprising a T cell population comprising IL-16 (IL-16) cells and, optionally, a pharma- ceutical acceptable carrier and / or excipient. Examples of pharmaceutical carriers include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous solutions, saline solutions, and aqueous dextrose and glycerin solutions are preferably used as carriers, particularly for injectable solutions.

[0186] Compositions comprising the modified cells described herein may include a buffer such as neutral buffered saline, phosphate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol, etc.; a protein; a polypeptide or an amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative.

[0187] The compositions comprising the modified cells described herein may include one or more of the following: sterile diluents, such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils, synthetic mono- or diglycerides, polyethylene glycols, glycerin, propylene glycol or other solvents that may serve as solvents or suspending media; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for adjusting tonicity, such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Pharmaceutical compositions for injection are preferably sterile.

[0188] In some embodiments, the compositions are formulated for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal, intratumoral, intraventricular, intrapleural, or intramuscular administration. In some embodiments, the compositions are reconstituted from a lyophilized preparation prior to administration.

[0189] In some embodiments, the modified cells may be mixed with an adhesive or penetrating substance, such as, but not limited to, nanoparticles, prior to their administration.

[0190] Treatment method In another embodiment, the present disclosure provides a method of transplantation in a subject in need thereof, comprising administering to the subject an effective amount of a stem cell-like memory T (T SCM In yet another aspect, methods are provided that include administering a T cell population comprising the T cell (s) or a pharmaceutical composition described herein.

[0191] In another embodiment, the present disclosure provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a stem cell-like memory T (T SCM In yet another aspect, methods are provided that include administering a cell population comprising the IL-15 (IL-15) cells or a pharmaceutical composition described herein.

[0192] In another embodiment, the disclosure provides a T cell population or pharmaceutical composition described herein for use in medicine, in some embodiments, the disclosure provides a T cell population or pharmaceutical composition described herein for use in treating a disease or disorder, such as, but not limited to, cancer, an autoimmune disease, or an infectious disease.

[0193] In some embodiments, the methods for producing a T cell population or pharmaceutical composition are carried out in vitro or ex vivo.

[0194] In some embodiments, the cells in the cell population or pharmaceutical composition are allogeneic to the subject to which the cell population or pharmaceutical composition is administered, hi some embodiments, the cells in the cell population or pharmaceutical composition are autologous to the subject to which the cell population or pharmaceutical composition is administered.

[0195] Diseases or disorders that can be treated using the methods and / or compositions of the present disclosure include, but are not limited to, cancer, an autoimmune disease, or an infectious disease.

[0196] In some embodiments, the disease or disorder that can be treated by the methods described herein is cancer. The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell growth. The term "cancer" includes, for example, soft tissue tumors (e.g., lymphomas), as well as tumors of the blood and blood-forming organs (e.g., leukemias), and solid tumors that grow at anatomical sites outside the bloodstream (e.g., carcinomas). Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas (e.g., osteosarcoma or rhabdomyosarcoma), and leukemia or malignant lymphomas. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), adenosquamous carcinoma, lung cancer (including, for example, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bronchogenic carcinoma, Lewis lung carcinoma, lung neuroendocrine tumor, typical carcinoid, atypical carcinoid, and large cell neuroendocrine carcinoma), cancer of the peritoneum, hepatocellular carcinoma, gastric cancer or gastric cancer (including, for example, gastrointestinal cancer, gastrointestinal stromal tumor, pancreatic cancer, pancreatic adenocarcinoma, intraductal papillary mucinous tumor, liquid neoplasms (IPMN), pancreatic islet cell tumors), cervical cancer (including but not limited to cervical adenocarcinoma), ovarian cancer (including but not limited to cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma, ovarian clear cell carcinoma, ovarian serous cystadenoma), liver cancer, bladder cancer, cancer of the urinary tract, liver cancer, breast cancer (including but not limited to adenocarcinoma of the breast, papillary carcinoma of the breast, medullary carcinoma of the breast), colon cancer (including but not limited to colon adenocarcinoma), colorectal cancer (including but not limited to rectal cancer, colorectal adenocarcinoma), endometrial cancer (including but not limited to uterine cancer, uterine sarcoma), salivary gland cancer, kidney or renal cancer (including but not limited to nephroblastoma or Wilms' tumor, renal cell carcinoma), prostate cancer (including but not limited to prostate adenocarcinoma), vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, skin cancer (including but not limited to primary or metastatic melanoma, squamous cell carcinoma, keratoacanthoma, basal cell carcinoma, cell carcinoma), multiple myeloma (including but not limited to smoldering multiple myeloma) and acute lymphoblastic leukemia (ALL) (including but not limited to B cell ALL, T cell ALL), acute myeloid leukemia (AML) (e.g., B cell AML, T cell AML), chronic myelogenous leukemia (CML) (e.g., B cell CML, T cell CML) and chronic lymphocytic leukemia (CLL) (e.g., B cell CLL, T cell CLL),Lymphomas such as Hodgkin's lymphoma (HL) (including, but not limited to, B-cell HL, T-cell HL), and non-Hodgkin's lymphoma (NHL) (e.g., diffuse large B-cell lymphoma (DLBCL)), B-cell NHL, such as follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma (including, but not limited to, nephroblastoma or Wilms' tumor, renal cell carcinoma), primary mediastinal B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma (e.g., supraglottic lymphoma, thyroid cancer ... cutaneous squamous cell carcinoma), immunoblastic large cell lymphoma, hairy cell leukemia (IPMN), precursor B-lymphoblastic lymphoma and primary central nervous system (ALL) lymphoma, T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (AML) (including, for example, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, bronchogenic carcinoma, Lewis lung carcinoma, pulmonary neuroendocrine tumor, typical carcinoid, atypical carcinoid, and large cell neuroendocrine carcinoma), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous sebaceous T-cell lymphoma, and / or follicular lymphoma, anaplastic large cell lymphoma, mixed leukemia / lymphomas such as one or more of the above, brain (e.g., high-grade glioma, diffuse pontine glioma, ependymoma, neuroblastoma, meningioma, astrocytoma, oligodendroglioma, oligodendroglioma; medulloblastoma, or glioblastoma) as well as head and neck cancer (including but not limited to head and neck squamous cell carcinoma), bile duct carcinoma (including but not limited to cholangiocarcinoma), bronchial carcinoma, chordoma, choriocarcinoma, epithelial carcinoma, endothelial sarcoma (including but not limited to Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), esophageal cancer (including but not limited to adenocarcinoma of the esophagus, Barrett's adenocarcinoma), Ewing's sarcoma. , heavy chain disease (including but not limited to alpha chain disease, gamma chain disease, mu chain disease), hematopoietic cancer, immunocytic amyloidosis, monoclonal gammopathy of undetermined significance, myelodysplastic syndrome, myeloproliferative disorder, idiopathic myeloid metaplasia (AMM) or myelofibrosis (MF), chronic idiopathic myelofibrosis, myeloproliferative neoplasms, polycythemia vera, rectal adenocarcinoma, essential thrombocytosis, chronic neutrophilic leukemia, hypereosinophilic syndrome, soft tissue sarcoma (e.g. malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma) and associated metastases. Further examples of cancer include:The Merck Manual of Diagnosis and Therapy, 19th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); The Merck Manual of Diagnosis and Therapy, 20th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2018 (ISBN 978-0-911-91042-1) (2018 digital online edition at internet website of Merck Manuals); and SEER Program Coding and Staging Manual 2016, each of which is incorporated by reference in its entirety for all purposes.

[0197] In some embodiments, the cancer is a BCMA-expressing cancer or disorder, which in some embodiments includes a hematological cancer such as acute myeloid leukemia (AML), or a lymphoma (e.g., multiple myeloma (MM), smoldering multiple myeloma (SMM)).

[0198] The compositions and methods described in the present disclosure can be used to treat infectious diseases. Infectious diseases are well known to those skilled in the art, and non-limiting examples include, but are not limited to, infectious diseases of viral etiology such as human immunodeficiency virus (HIV), influenza, herpes, viral hepatitis, Epstein-Barr, polio, viral encephalitis, measles, chickenpox, papilloma virus, cytomegalovirus, rabies virus, chickenpox virus, yellow fever virus, West Nile virus, Ebola, etc.; infectious diseases of bacterial etiology such as pneumonia, tuberculosis, syphilis, Lyme disease, babesiosis, etc.; or infectious diseases of parasitic etiology such as malaria, trypanosomiasis, leishmaniasis, trichomoniasis, amoebic dysentery, etc.

[0199] The compositions and methods described in the present disclosure can be used to treat autoimmune diseases, including but not limited to rheumatoid arthritis (RA), multiple sclerosis (MS), Sjogren's syndrome, sarcoidosis, insulin-dependent diabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis, ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis, psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease, and ulcerative colitis.

[0200] In some embodiments, the combination is administered in a therapeutically effective amount. The dosage of the composition administered in the method of the present invention varies widely depending on the subject's physical parameters, frequency of administration, mode of administration, clearance rate, etc. The initial dosage may be higher, followed by a lower maintenance dosage. The dosage may be administered infrequently, such as once a week or every other week, or may be divided into smaller dosages and administered daily, semi-weekly, etc., to maintain an effective dosage level. It is contemplated that various dosages are effective for achieving in vivo persistence of modified cells. It is also contemplated that various dosages are effective for improving in vivo effector function of modified cells.

[0201] In some embodiments, a composition comprising cells produced by the methods described herein comprises 10 2 ~10 10 cells / kg body weight, 10 5 ~10 9 cells / kg body weight, 10 5 ~10 8 cells / kg body weight, 10 5 ~10 7 cells / kg body weight, 10 7 ~10 9 cells / kg body weight, or 10 7 ~10 8 The composition may be administered at a dosage of cells / kg body weight (including all integer values ​​within these ranges). The number of cells will vary depending on the therapeutic use for which the composition is intended. Therapeutic cells may be administered multiple times at the dosages listed above.

[0202] The compositions and methods described in the present disclosure may be used in combination with other types of cancer therapy, such as chemotherapy, surgery, radiation, gene therapy, etc. It is also contemplated that when used to treat various diseases / disorders, the compositions and methods of the present disclosure may be used with other therapeutic methods / drugs suitable for the same or similar disease / disorder. Such other therapeutic methods / drugs may be co-administered (simultaneously or sequentially) to produce additive or synergistic effects. The suitable therapeutically effective dosage of each agent may be reduced due to additive or synergistic effects.

[0203] In some embodiments of any of the above methods of treatment, the method further comprises administering to the subject one or more additional compounds selected from the group consisting of immunosuppressants, biologics, probiotics, prebiotics, and cytokines (e.g., IFN or IL-2).

[0204] As a non-limiting example, the present invention can be combined with other therapies that block inflammation (eg, via blockade of IL1, INFα / β, IL6, TNF, IL23, etc.).

[0205] The methods and compositions of the present disclosure can be combined with other immunomodulatory therapies, such as, for example, therapeutic vaccines (including but not limited to, GVAX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to, agents that block CTLA4, PD1, LAG3, TIM3, etc.), or activators (including but not limited to, agents that enhance 4-1BB, OX40, etc.). The methods of the present disclosure can also be combined with other therapeutics capable of modulating NKT function or stability, including but not limited to, CD1d, CD1d fusion proteins, CD1d dimers or larger polymers of CD1d unloaded or loaded with antigen, CD1d chimeric antigen receptor (CD1d-CAR), or any other of the five known CD1 isomers present in humans (CD1a, CD1b, CD1c, CD1e). The methods of the present invention can also be combined with other therapeutics, such as midostaurin, enasidenib, or combinations thereof.

[0206] The therapeutic methods of the present disclosure can be combined with additional cell therapy, immunotherapy and therapy. For example, when used to treat cancer, the compositions of the present invention can be used in combination with conventional cancer therapy, such as surgery, radiation therapy, chemotherapy, or a combination thereof, depending on the type of tumor, the patient's condition, other health problems, and various factors. In certain aspects, other therapeutic agents useful for combined cancer treatment with the inhibitors of the present invention include anti-angiogenic agents. Many anti-angiogenic agents have been identified and are known in the art, including, for example, TNP-470, platelet factor 4, thrombospondin-1, tissue inhibitor of metalloproteases (TIMP1 and TIMP2), prolactin (16Kd fragment), angiostatin (38Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, placental proliferin-related protein, and those listed by Carmeliet and Jain (2000). In one embodiment, the T cells of the invention can be used in combination with a VEGF antagonist or a VEGF receptor antagonist, such as an anti-VEGF antibody, a VEGF variant, a soluble VEGF receptor fragment, an aptamer capable of blocking VEGF or VEGFR, a neutralizing anti-VEGFR antibody, an inhibitor of VEGFR tyrosine kinase, and any combination thereof (e.g., the anti-hVEGF antibody A4.6.1, bevacizumab or ranibizumab).

[0207] Non-limiting examples of chemotherapeutic compounds that may be used in the combination therapy of the present invention include, for example, aminoglutethimide, amsacrine, anastrozole, asparaginase, azacytidine, bcg, bicalutamide, bleomycin, buserelin, busulfan, campotothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide. , cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, zenigesin, genistein, goserelin, Hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxalate These include platins, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.

[0208] These chemotherapeutic compounds can be classified according to their mechanism of action, for example, into the following groups: antimetabolites / anticancer drugs such as pyrimidine analogues (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogues, antifolates and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); natural products, for example, the vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disrupting agents, for example, the taxanes (paclitaxel). , docetaxel), vincristine, vinblastine, nocodazole, epothilones and navelbine, epipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylenetetramine oxaliplatin, ifosfamide, melphalan, mercury, ethyl amyloid phosphate, methyl methacrylate ... asparagine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide, and etoposide (VP16); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; enzymes (the ability to metabolize L-asparagine systemically and synthesize their own asparagine) L-asparaginase, which deprives cells of L-asparaginase; antiplatelet agents; antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazene-dacarbazine (DTIC); antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methotrexate);Platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts, and other thrombin inhibitors); fibrinolytic agents (e.g., tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (bleverdin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (e.g., TNP-470, genistein , bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, etoposide, epirubicin, etoposide, idarubicin, and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers and caspase activators; and chromatin disruptors.

[0209] In various embodiments of the methods described herein, the subject is a human. The subject may be a juvenile or adult of any age or sex. EXAMPLES

[0210] The following examples are provided to further describe some of the embodiments disclosed herein and are intended to be illustrative and not limiting of the embodiments of the present disclosure.

[0211] Example 1. Design and engineering of CAR-T The second generation CAR-T of this example contained a single chain variable fragment (scFv) targeting a tumor-associated antigen (TAA) of interest, e.g., B-cell maturation antigen (BCMA), fused with hinge and transmembrane sequences derived from human CD8A and intracellular domains, e.g., 4-1BB and CD3ζ, as shown in FIG. 1A. The scFv of the CAR-T construct was specifically designed to contain 5' and 3' overlaps corresponding to EcoRI and SpeI restriction sites in a viral vector, e.g., a lentiviral vector. The designed DNA insert was codon-optimized for Homo sapiens and then synthesized. Cloning of the construct was performed using the In-Fusion® cloning method. All constructs were sequence-verified prior to transfection. The sequence of the BCMA CAR-T construct used in the experiments presented herein is shown in FIG. 1B.

[0212] Below is a list of reagents useful for any of the CAR-T cell assay methods disclosed herein.

[0213] reagent: -TransAct(trademark) -Naive Pan-T cells -Costar® 24-well Clear TC-treated multiwell plates -Mouse anti-human CD4 antibody -Mouse anti-human CD8 antibody -Mouse anti-human CD62L antibody -Mouse anti-human CCR7 antibody -Mouse anti-human CD27 antibody -Anti-human CD45RO antibody -Anti-human CD45RA antibody -Anti-human CD279 (PD-1) -Anti-human CD233 (LAG-3) -Anti-human CD366 (Tim-3) -Anti-human TIGIT

[0214] Example 2. Culture and electroporation of human T cells Human Pan-T cells were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors and cultured in complete T cell medium / RPMI medium containing 10% fetal calf serum (FCS), 2 mM GlutaMax, 1 mM sodium pyruvate, 55 μM β-mercaptoethanol, and 100 U penicillin / streptomycin.

[0215] Pan-T cells were expanded ex vivo using anti-CD3 / CD28 Dynabeads magnetic beads for approximately 12–14 days according to the manufacturer's protocol. These cells were cultured at 1 × 10 6 The samples were frozen at a density of one piece per vial and stored in liquid nitrogen.

[0216] Example 3. T cell activation and chimeric antigen receptor (CAR) transduction On day 0, naive Pan-T cells from three donors were thawed and cultured at 1×10 in complete T cell medium / RPMI medium (see, e.g., Example 2). 6 Prior to T cell activation, thawed naive T cells were immunophenotyped. The immunophenotypic panel consisted of, for example, CD4 + and CD8 + For T cell activation, 10 μl of TransAct™ / ml was added to each well of a 24-well plate at 1×10 6 T cells / well were added at a density of 100-200 T cells / well. Plates were incubated overnight at 37° C. / 5% CO2. On day 1, cells were transduced with B cell maturation antigen (BCMA)-HL CAR lentiviral particles at a multiplicity of infection (MOI) equal to 5.

[0217] Example 4. Phenotypic characterization of BCMA-HL CAR-transduced T cells CAR-transduced T cells were then transduced into CD4 + and CD8 + Stem cell-like memory T cells (T SCM ) phenotype was augmented for 14 days using cytokine regulation.SCM -like cells were generated using cytokines, for example, either in the presence of IL-7 alone, or in the presence of IL-7 in combination with IL-15 (IL-7+IL-15) or IL-15 and IL-21 (IL7+IL-15 and IL-21).

[0218] For phenotypic characterization, CAR T cells were evaluated for memory and effector T cell markers using fluorescence-activated cell sorting (FACS) flow cytometry analysis 14 days after transduction. For FACS staining, 100 μl cells / well from BCMA-HL CAR-transduced cells were washed twice with phosphate-buffered saline (PBS) and then fluorescently conjugated cell marker-specific antibodies were used. Antibodies used in this experiment were each diluted 1:200 and included, for example, CD4, CD8, FVD (live / dead), CD27, CD62L, CD45RA, CD45RO, CCR7, CD69, and CAR+ (1 μg / ml), as well as Alexa Fluor™ 647 (AF647) secondary antibodies. All samples were evaluated using a Fortessa cell sorter system.

[0219] CD4 on day 14 post-transduction + CAR-T cell subsets and CD8 + T in CAR-T cell subsets SCM Representative cytokine enhancement of cell phenotypes is shown in Figures 2A-2C and 3A-3C, respectively. CD4 + For phenotypic characterization of CAR-T cells (Figures 2A-2C), pan-T cells were activated in the presence or absence of cytokines according to the cytokine regulation paradigm described above. For FACS analysis, cells were cloned into CD4 + CAR + Gating on T cells, CD4 + The frequency levels of expression (parental frequency, %) for CD62L (Figure 2A), CCR7 (Figure 2B), and CD27 (Figure 2C) in BCMA-HL CAR-transduced cells were determined. +For phenotypic characterization of CAR-T cells (Figures 3A-3C), Pan-T cells were similarly activated in the presence or absence of cytokines according to the cytokine modulation paradigm described above. For FACS analysis, cells were cloned into CD8 + CAR + Gating on T cells, the frequency levels of expression (parental frequency, %) for CD62L (Figure 3A), CCR7 (Figure 3B), and CD27 (Figure 3C) in CD8+BCMA-HL CAR-transduced cells were determined. As shown in Figures 4A-B, CD4 + CAR-T cells (Figure 4A) and CD8 + CD45RO in both subsets of CAR-T cells (Figure 4B) - / CD45RA + Phenotypic characterization was performed following the same method as described above for quantification of the frequency levels of expression (parental frequency, %) of CD4 + and CD8 + CD45RO in both CAR-T cell subsets - / CD45RA + T SCM Enhanced cell phenotype.

[0220] Based on the above results, a single cytokine (IL-7) was shown to mediate the expression of CD4 + and CD8 + T in both subsets SCM When IL-15 and IL-21 were added to the IL-7 cytokine regimen, CD4 + and CD8 + T in both T cells SCM It was further observed that the phenotype was enhanced. When IL-7+IL-15+IL-21 cytokine-regulated CAR T cells were co-cultured with tumor targets, the IL-7+IL-15+IL-21 CAR T cells had enhanced cytokine production. Without wishing to be bound by theory, it is believed that the addition of IL-15 and IL-21 activates STAT5 and STAT3 signaling, respectively, which may be advantageous compared to single cytokine regulation.

[0221] The above approach to generate cytokine-regulated CAR-T cells with enhanced effector function and reduced exhaustion markers was developed using CD4 + and CD8 + CAR-T SCM They generated both types of cells to support the proof-of-concept of enhancing antitumor immunity.

[0222] References 1. June CH, O'Connor RS, Kawalekar OU, Ghassemi S, Milone MC. CAR T cell immunotherapy for human cancer. Science(New York, NY)2018;359(6382):1361-5.doi:10.1126 / science.aar6711. 2. Mirzaei HR, Rodriguez A, Shepphird J, Brown CE, Badie B. Chimeric Antigen Receptors T Cell Therapy in Solid Tumor: Challenges and Clinical Applications. Front Immunol 2017;8-1850.doi:10.3389 / fimmu.2017.01850. 3.Anderson JK,Mehta AA review of chimeric antigen receptor T-cells in lymphoma.Expert Rev Hematol 2019;12(7):551-61.doi:10.1080 / 17474086.2019-1629901. 4. Minutolo NG, Hollander EE, Powell DJ, Jr. The Emergence of Universal Immune Receptor T Cell Therapy for Cancer. Front Oncol 2019;9-176.doi:10.3389 / fonc.2019.00176. 5.Kloss CC,Condomines M,Cartellieri M,Bachmann M,Sadelain M.Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells.Nat Biotechnol 2013;31(1):71-5 doi 10.1038 / nbt.2459。 6.Brudno JN,Kochenderfer JN.Toxicities of chimeric antigen receptor T cells:recognition and management.Blood 2016;127(26):3321-30 doi 10.1182 / blood-2016-04-703751。 7.Porter DL,Hwang WT,Frey NV,Lacey SF,Shaw PA,Loren AW,et al.Chimeric antigen receptor T cells persist and induce sustained remissions in relapsed refractory chronic lymphocytic leukemia.Sci Transl Med 2015;7(303):303ra139.doi:10.1126 / scitranslmed.aac5415。 8.Eshar Z, Waks T, Gross G, Schindler DG.Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors.Proceedings of the National Academy of Sciences of the United States of America 1993;90(2):720-4.doi:10.1073 / pnas.90.2.720. * * *

[0223] The present invention is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims.

[0224] All patents, applications, publications, test methods, literature, and other materials cited herein are incorporated by reference in their entirety as if physically present herein.

[0225] Sequence Listing Sequence number 1 Leader MAWVWTLLFLMAAAQSIQA

[0226] SEQ ID NO:2 BCMA scFv-HL QLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSSG TEGKSSGSGSESKSTSYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEAVYYCQVWDSSSDHVVFGGGTKLTVL

[0227] SEQ ID NO: 3 BCMA scFv-LH SYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEAVYYCQVWDSSSDHVVFGGGTKLTVLGTEGKSSGSGSESK STQLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSS

[0228] SEQ ID NO:4 Linker GTEGKSSGSGSESKST

[0229] SEQ ID NO:5 CD8a hinge TSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0230] SEQ ID NO:6 CD8a-TM IYIWAPLAGTCGVLLLSLVITLYC

[0231] SEQ ID NO: 7 CD137 strong stimulation KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

[0232] SEQ ID NO:8 Zeta RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0233] SEQ ID NO: 9 Extracellular domain MAWVWTLLFLMAAAQSIQAQLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSSGTEGKSSGSGSESK STSYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEAVYYCQVWDSSSDHVVFGGGTKLTVLTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0234] SEQ ID NO:10 Cytoplasmic domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0235] SEQ ID NO: 11 BCMA CAR-HL MAWVWTLLFLMAAAQSIQAQLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYCARHDGAVAGLFDYWGQGTLVTVSSGTEGKSSGSGSESKSTSYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSRPSGIPERFSGSNSGNTATLTISRVEAGDEAVYC QVWDSSSDHVVFGGGTKLTVLTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIIYWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0236] SEQ ID NO:12 BCMA CAR-LH MAWVWTLLFLMAAAQSIQASYVLTQPPSVSVAPGQTARITCGGNNIGSKSVHWYQQPPGQAPVVVVYDDSDRPSGIPERFSGSNSGNTATLTISRVEAGDEAVYYCQVWDSSSDHVVFGGGTKLTVLGTEGKSSGSGSESKSTQLQLQESGPGLVKPSETLSLTCTVSGGSISSGSYFWGWIRQPPGKGLEWIGSIYYSGITYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARHDGAVAGLFDYWGQGTLVTVSSTSTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Claims

1. Stem cell-like memory T (T ＳＣＭ 2.) A method for concentrating cells, comprising: I)T ＳＣＭ contacting the population of T cells with an effective amount of one or more cytokines, including interleukin 7 (IL-7), for a period of time sufficient to enrich for the cells; II) Optionally, the T ＳＣＭ and expanding the cells.

2. a) The T cell population is selected from the group consisting of Pan-T cells, naive CD4 ＋ T cells, naive CD8 ＋ T cells or naive CD4 ＋ and naive CD8 ＋ T cells, or any combination thereof, optionally further comprising, prior to step (I), isolating Pan-T cells, naive CD4 + cells, naive CD8 + T cells, or naive CD4 + and naive CD8 + cells from peripheral blood mononuclear cells (PBMCs); b) the one or more cytokines are present during the expansion step (II); c) genetically modifying the T cells to express a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR); and / or d) the method further comprises activating the population of T cells at the start of the contacting step (I), optionally wherein the activating step is carried out for about 24 hours with an anti-CD3 agent and / or an anti-CD28 agent; The method of claim 1.

3. Genetically modified stem cell-like memory T (T ＳＣＭ 2.) A method for generating a cell, comprising: W) Isolated Pan-T cells, naive CD4 ＋ T cells, naive CD8 ＋ T cells or naive CD4 ＋ and naive CD8 ＋ Obtaining a population of T cells, or any combination thereof, optionally wherein Pan-T cells, naive CD4 + cells, naive CD8 + T cells, or naive CD4 + and naive CD8 + cells, or a combination thereof, are isolated from peripheral blood mononuclear cells (PBMCs); X) activating said T cell population, optionally wherein said activating step is carried out for about 24 hours using an anti-CD3 agent and / or an anti-CD28 agent; Y) genetically modifying the cells present after step (X) to express a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR); Z) expanding the genetically modified cells; The method, wherein steps X), Y), and Z) are carried out in the presence of one or more cytokines, including interleukin-7 (IL-7).

4. a) the genetic modification is effected by introducing into the cell a polynucleotide encoding the CAR or the engineered TCR, optionally wherein the polynucleotide encoding the CAR or the engineered TCR is introduced via viral transduction, electroporation, direct injection, magnetofection, ultrasound, ballistic or hydrodynamic methods, or a combination thereof; b) the CAR or the engineered TCR specifically binds to a tumor antigen, an infectious antigen, or an autoimmune antigen, optionally wherein the tumor antigen is selected from BCMA, GPRC5D, CD79, KLK2, CD19, CD30, CD33, CD123, hK2, FLT3, CD20, CD22, KRASG12D, p53, BRAC1, and PSMA; c) the genetic modification is performed before the expansion step (I), and optionally the one or more cytokines are present during the genetic modification step; and / or d) the method further comprises the step of priming the T cell population prior to the activation step 2(d) or (X); The method of claim 2.

5. The method of claim 1, wherein the one or more cytokines further comprise IL-15, IL-21, or IL-15 and IL-21. b) contacting said T cell population with each of said one or more cytokines at a concentration of about 1 to about 15 ng / ml, e.g., about 5 to 12 ng / ml, e.g., about 10 ng / ml; c) the one or more cytokines do not include IL-2; d) the T cell population does not include suppressive regulatory T cells; e) the contacting step (I) or the increasing step (Z) is carried out for a period of 10 to 20 days, for example about 14 days; f) the contacting step (I) and the increasing step (II), or the steps (X), (Y), and (Z) are carried out for a total of 10 to 20 days, for example a total of 14 days; g) the contacting step (I), or the steps (X), (Y), and (Z) are carried out at a temperature of about 37° C.; and / or h) the method further comprises determining the percentage of T sCM cells in the T cell population after the expansion step (II) or (Z), optionally wherein the percentage of T sCM cells in the T cell population after the expansion step (II) or (Z) is at least about 40%, 50%, 60%, or 70%, e.g., about 60% to 70%; The method of claim 1.

6. The enriched stem cell-like memory T (T ＳＣＭ ) cells.

7. 7. A pharmaceutical composition comprising the population of T cells according to claim 6 and a pharma- ceutically acceptable carrier or excipient.

8. 8. The population of T cells of claim 6 or the pharmaceutical composition of claim 7 for use in a method of treating a disease or disorder in a subject in need thereof, said method comprising administering a therapeutically effective amount of said population or pharmaceutical composition to said subject. a) the T cell population is allogeneic to the subject or autologous to the subject; and / or b) the disease or disorder is cancer, an infectious disease, or an autoimmune disease, and optionally the cancer is i) is a hematological malignancy; ii) is a solid tumor; iii) squamous cell carcinoma, adenosquamous carcinoma, lung cancer, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer, uterine cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the ureter, liver cancer, breast cancer, colon cancer, colorectal cancer, endometrial cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, skin cancer, multiple myeloma and acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and chronic lymphocytic leukemia (CL). leukemia (CLL), lymphomas such as Hodgkin's lymphoma (HL) and non-Hodgkin's lymphoma (NHL), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphoma, primary mediastinal B-cell lymphoma, Burkitt's lymphoma, lymphoplasmacytic lymphoma, immunoblastic large cell lymphoma, hairy cell leukemia (HCL), precursor B-lymphoblastic Lymphomas and primary central nervous system (CNS) lymphomas, T-cell NHLs such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy-type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma, brain and head and neck cancer, bile duct cancer, bronchial cancer, chordoma, choriocarcinoma, epithelial carcinoma, endothelial sarcoma, esophageal cancer, Eugene's disease, sarcoma heavy chain disease, hematopoietic cancer, immune cell amyloidosis, monoclonal gammopathy of undetermined significance, myelodysplastic syndrome, myeloproliferative disease, myeloid metaplasia of unknown etiology (AMM) or myelofibrosis (MF), chronic idiopathic myelofibrosis, myeloproliferative neoplasm, polycythemia vera, rectal adenocarcinoma, essential thrombocytosis, chronic neutrophilic leukemia, hypereosinophilic syndrome, or soft tissue sarcoma, or a combination or metastasis thereof; or iv) a BCMA-expressing cancer, optionally wherein the BCMA-expressing cancer is acute myeloid leukemia (AML), multiple myeloma (MM), or smoldering multiple myeloma (SMM); A population or pharmaceutical composition for use as claimed in claim 8.

10. Stem cell-like memory T (T ＳＣＭ 1.) A system for concentrating cells, comprising: a) T ＳＣＭ a means for contacting said population of T cells with an effective amount of one or more cytokines, including interleukin 7 (IL-7), for a period of time sufficient to enrich for the cells; b) Optionally, the T ＳＣＭ and a means for expanding the cells.

11. a) The T cell population is selected from the group consisting of Pan-T cells, naive CD4 ＋ T cells, naive CD8 ＋ T cells or naive CD4 ＋ and naive CD8 ＋ T cells, or any combination thereof, optionally wherein the system further comprises a means for isolating Pan-T cells, naive CD4 + cells, naive CD8 + T cells, or naive CD4 + and naive CD8 + cells, or any combination thereof, from peripheral blood mononuclear cells (PBMCs). b) the system further comprises means for genetically modifying the T cells to express a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR); c) the system further comprises means for activating the T cell population at the initiation of the step of contacting the T cell population with the one or more cytokines, including interleukin 7 (IL-7), optionally the system further comprises means for priming the T cell population prior to said activation; and / or d) the system further comprises means for determining the percentage of T SCM cells in the T cell population after said expansion; The system of claim 10.

12. Genetically modified stem cell-like memory T (T ＳＣＭ 2.) A system for generating cells, comprising: a) Isolated Pan-T cells, naive CD4 ＋ T cells, naive CD8 ＋ T cells or naive CD4 ＋ and naive CD8 ＋ a means for obtaining a population of T cells, or any combination thereof; b) a means for activating said population of T cells; and c) means for genetically modifying said cells after activation to express a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR); d) a means for expanding the genetically modified cells; Isolated Pan-T cells, naive CD4 ＋ T cells, naive CD8 ＋ T cells or naive CD4 ＋ and naive CD8 ＋ A system, wherein obtaining a population of T cells, or any combination thereof, activating the T cell population, and genetically modifying the T cell population are performed in the presence of one or more cytokines, including interleukin-7 (IL-7).

13. The system of any one of claims 10 to 12, wherein the one or more cytokines further comprise IL-15 and / or IL-21.

14. a) Stem cell-like memory T (T ＳＣＭ 2.) A composition for concentrating cells, comprising: a.T ＳＣＭ one or more cytokines, including interleukin 7 (IL-7), for enriching the cells; b. A T cell population that is a means for enriching for TSCM cells; c. Optionally, said T ＳＣＭ and a means for expanding the cells. b) a composition for enriching stem cell-like memory T (TSCM) cells in a T cell population, comprising: a. a population of T cells; b. an effective amount of one or more cytokines, including interleukin 7 (IL-7); (i) enriching for TSCM cells by contacting said T cell population with an effective amount of one or more cytokines, including IL-7; (ii) to activate the enriched TSCM cells; and (iii) optionally, a means for expanding said TSCM cells; or c) A composition for generating genetically modified stem cell-like memory T (TSCM) cells, comprising: a. a population of T cells; b. an effective amount of one or more cytokines, including interleukin 7 (IL-7); (i) enriching for TSCM cells by contacting said T cell population with an effective amount of one or more cytokines, including IL-7; (ii) to activate the enriched TSCM cells; (iii) means for genetically modifying the enriched TSCM cells to express a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR); (iv) optionally, a means for expanding said TSCM cells.

15. Said T ＳＣＭ The T cell population is treated with an effective amount of one or more cytokines, including IL-7, and ＳＣＭ 15. The composition of claim 14, which is enriched by contacting the cells for a period of time sufficient to enrich the cells.

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