CN116615209A

CN116615209A - Methods of making regenerative T cells, compositions comprising regenerative T cells, and methods of using regenerative T cells

Info

Publication number: CN116615209A
Application number: CN202180088871.1A
Authority: CN
Inventors: R·E·维斯卡多·萨科达; N·雷斯蒂福; R·D·克劳斯纳; Y·黄; 前田拓哉; 玉木成隆; 山崎康弘
Original assignee: Lyle Immune Pharmaceutical Co ltd
Current assignee: Lyle Immune Pharmaceutical Co ltd
Priority date: 2020-11-24
Filing date: 2021-11-23
Publication date: 2023-08-18

Abstract

The present disclosure relates generally to methods of producing regenerative T cells comprising contacting T cells with at least one reprogramming factor and reactivating the contacted cells; and to compositions and methods of using the regenerative T cells. The present disclosure also describes cell populations prepared according to the methods described herein. The present disclosure also provides methods of treating a patient using the cell populations prepared by the methods described herein.

Description

Methods of making regenerative T cells, compositions comprising regenerative T cells, and methods of using regenerative T cells

Cross Reference to Related Applications

The priority of U.S. provisional patent application Ser. No. 63/117,787, U.S. provisional patent application Ser. No. 63/153,881, and U.S. provisional patent application Ser. No. 63/165,093, U.S. provisional patent application Ser. No. 2021, 3/23, and U.S. provisional patent application Ser. No. 63/117,881, respectively, filed by the patent Cooperation treaty ("PCT") is claimed, each of which is incorporated herein in its entirety.

Background

Tumor infiltrating lymphocytes ("TILs") are immune cells (e.g., T cells) found within tumors that are capable of recognizing and killing cancer cells. Adoptive transfer of autologous TIL has been widely studied, but one disadvantage of TIL-based therapies is that these cells often exhibit cell markers of extensive differentiation and aging and loss of function. See, for example, gugusamy et al, 2020,Cancer Cell 37,818-833; jiang et al, 2015,Cell Death Dis 6,e1792. Generally, TIL consists mainly of TEM or TEMRA cells and has depletion properties. Sakuishi et al 2010,J Exp Med 207,2187-2194. In addition, preclinical evidence strongly suggests that impaired T cell function at the tumor site can lead to poor clinical efficacy of the T cell product.

T cells (also known as T lymphocytes) are a blood cell with unique properties that develop from stem cells found in bone marrow; t cells prevent infection and are anticancer. T cells differ from other cell types in structure and function, such as fibroblasts, and undergo complex development, require positive and negative selection in the thymus, and involve somatic gene rearrangements of T cell receptor loci (see, e.g., kurd and Robey, immunol rev.2016, month 5; 271 (1): 114-126). Although fibroblasts are also involved in the immune response of the body, fibroblasts are cells that synthesize extracellular matrix and collagen, thereby creating a structural framework for animal tissue. Fibroblasts are mainly involved in the process of wound healing.

Tumor-reactive T cells undergo induction of cellular senescence pathways in addition to exhibiting the characteristics of depletion and increased differentiation. Aging is a process of gradual loss of potency and function that results from aging and repeated cell division cycles. Several characteristics associated with cell senescence, including epigenetic changes (Vodnala et al 2019,Cancer Cell 37,818-833e 819), telomere length reduction (Rosenberg et al 2011,Clin Cancer Res 17,4550-4557), and loss of functionality, proliferation potential and cell viability (Im et al 2016,Nature 537,417-421) are associated with poor T cell function. While TIL-based cell therapies can mediate responses in some patients, clinical evidence has demonstrated that TIL products with increased CCR7 expression and increased telomere length are associated with improved therapeutic outcomes. Rosenberg et al 2011,Clin Cancer Res 17,4550-4557. Thus, many groups have sought methods of cell dedifferentiation or reprogramming as a method of reversing T cell depletion, differentiation and senescence.

In the past, cell dedifferentiation (reprogramming) has been achieved using iPS cell technology to restore somatic cells to pluripotent stem cells, followed by subdivision into the desired cell lineages. Takahashi et al 2007,Cell 131,861-872. iPS cell technology has been demonstrated to be able to reprogram tumor antigen specific tumor-infiltrating lymphocytes into iPS cells and to generate T lineage cells in vitro. Vizcardo et al 2013,Cell Stem Cell 12,31-36. Driving cells into iPS cells has the advantage of completely resetting the biological and epigenetic clocks of the cells.

However, iPS cells derived from T cells often have abnormal biological characteristics-e.g., immature phenotype, MHC-independent killing, incorrect cd8αβ dimerization, deregulation of gene expression, and failure to produce a uniformly developing T cell population. (see, e.g., vizcardo et al 2018,Cell Rep 22,3175-3190; takada, K., kondo, K. And Takahama, Y.2017J. Immunol.198, 2215-2222; yamagata, T. Et al, (2004) Nat. Immunol.5,597-605; fink, P.J. (2013) Annu. Rev. Immunol.31,31-50; kuderer, N.M. et al, 2006Cancer 106,2258-2266.). While these limitations can be overcome by differentiating iPS cells on 3D thymus organoid cultures (see, e.g., vizcardo et al, 2018,Cell Rep 22,3175-3190), these methods are both time-and resource-intensive. More scalable methods for reversing T cell depletion, differentiation and senescence are desirable for achieving commercially viable adoptive cell therapies with T cells.

Disclosure of Invention

In view of all modes in which improvement of T cell anti-tumor function would be beneficial for cellular immunotherapy, the present disclosure relates to methods for improving therapeutic T cell product quality and anti-tumor potential, compositions and methods of treatment comprising such improved T cells, and other uses of the improved T cell compositions. Thus, the system can be applied to many other cancer treatment processes, such as TCR and CAR transduced T cells, as well as cells genetically modified with anti-aging or pro-functional genes to improve T cell function in the tumor microenvironment.

The system will also be suitable for future culture medium iterations and new methods of T cell expansion in the innovation of artificial new protein designs. In summary, regenerative T cells of the present disclosure can enhance the efficiency of all modes of cellular immunotherapy by generating T cells with enhanced expansion capacity, improved metabolic quality, and enhanced ability to persist and eliminate established solid tumors.

In various embodiments, the present disclosure relates to a method of producing regenerative T cells comprising contacting a population of T cells with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC; and optionally SV40 for at least a period of time sufficient to form T cell-derived adherent cells, and wherein the T cells are not converted to iPS or totipotent cells; and contacting the adherent cells of T cell origin with at least one T cell activator.

In various embodiments, the present disclosure provides a method of producing regenerative T cells comprising contacting T cells with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and (ii) optionally SV40 for a time sufficient to form T cell-derived adherent cells, and wherein the T cells are not converted to iPS or totipotent cells; and contacting the adherent cells of T cell origin with at least one T cell activator.

In various embodiments, the present disclosure relates to a method of producing T cells comprising contacting a population of T cells in a first medium in a culture vessel with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC; and optionally SV40 for at least a period of time sufficient for the T cells to form at least one colony attached to the surface of the culture vessel, and wherein the T cells are not converted to iPS cells; and contacting the at least one attached colony with at least one T cell activator.

In various embodiments, the present disclosure relates to a method of producing T cells comprising contacting a population of T cells isolated in a first medium with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC; and SV40 for a period of at least about 5 days to about 10 days and wherein the T cells are not transformed into iPS cells or totipotent cells; and contacting the contacted T cells with at least one T cell activator.

In various embodiments, the present disclosure relates to a method of producing at least one T cell comprising contacting an isolated population of T cells with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC; and SV40 for at least a period of time sufficient for the T cells to express at least one marker selected from the group consisting of SSEA4, CD9, and CD90, and wherein the T cells are not transformed into iPS cells or totipotent cells; and contacting the contacted T cells with at least one T cell activator.

In various embodiments, the isolated T cell is contacted with the at least one reprogramming factor for a time sufficient for at least a portion of the T cell to express CD3 and at least one marker selected from the group consisting of SSEA4, CD9, and CD90. In various embodiments, the T cell is transiently contacted with the at least one reprogramming factor for at least a period of time sufficient for at least a portion of the contacted T cell to express SSEA4 and CD3. In various embodiments, the T cell is transiently contacted with the at least one reprogramming factor for at least a period of time sufficient for at least a portion of the contacted T cell to express CD3, CD9, and CD90. In various embodiments, the T cell is contacted with the at least one reprogramming factor for at least a period of time sufficient for at least a portion of the contacted T cell to express CD3, SSEA4, CD9, and CD90.

In various embodiments, the transiently contacted T cells are contacted with IL-2 and at least one compound capable of activating the isolated T cells prior to contacting the isolated T cells with the at least one reprogramming factor. In various embodiments, the T cell is a tcra beta T cell; TCRgd T cells; cd4+cd8αβ+ biscationic cells, cd4+ single positive cells (such as Th1, th2, th17, treg), naive T cells, central memory T cells, or effector memory T cells. In various embodiments, the T cell is a TIL. In various embodiments, the isolated T cells are isolated from a mammal.

In various embodiments, the isolated T cells are transiently contacted with KLF4, OCT3/4, SOX2, and C-MYC. In various embodiments, the isolated T cells are transiently contacted with KLF4, OCT3/4, SOX2, and C-MYC for at least about 4 days to 10 days.

In various embodiments, the isolated T cells are contacted with KLF4, OCT3/4, SOX2, and C-MYC for at least about 4 days to 7 days. In various embodiments, the isolated T cells are contacted with KLF4, OCT3/4, SOX2, and C-MYC for about 5 days.

In various embodiments, KLF4, OCT3/4, SOX2, and C-MYC are transiently expressed in T cells. In various embodiments, non-integrating viral vectors are used to transiently express KLF4, OCT3/4, SOX2, and C-MYC. In this regard, T cells may be transduced with one or more viral vectors encoding reprogramming factors. In various embodiments, the Sendai virus is used to transiently express KLF4, OCT3/4, SOX2, and C-MYC. In various embodiments, KLF4, OCT3/4, SOX2 and C-MYC are constitutively expressed, where expression is later inhibited by the addition of a compound that inhibits KLF4, OCT3/4, SOX2 and C-MYC expression. In various embodiments, the compound is a small molecule inhibitor that specifically inhibits the expression of one or more of KLF4, OCT3/4, SOX2, and C-MYC expression. In various embodiments, the compound is an siRNA or shRNA molecule that specifically inhibits expression of one or more of KLF4, OCT3/4, SOX2, and C-MYC. In various embodiments, KLF4, OCT3/4, SOX2, and C-MYC are transiently expressed after delivery using nanoparticles.

In various embodiments, the present disclosure provides for further contacting the contacted T cells (i.e., partially reprogrammed T cells) with at least one T cell activating compound and optionally at least one cytokine selected from the group consisting of IL-2, IL-7, IL-15, and IL-12. In various embodiments, the partially reprogrammed cell is a T cell-derived adherent cell. In various embodiments, the at least one T cell activating compound comprises an antibody that binds CD3 or an antibody that binds CD28 or both; or wherein the at least one T cell activating compound is a tumor antigen. In various embodiments, the present disclosure provides for further engineering the T cell to express a cell surface receptor, wherein the T cell is engineered prior to contacting the T cell with the at least one reprogramming factor. In various embodiments, the present disclosure provides for further engineering the T cell to express a cell surface receptor, wherein the T cell is engineered after transiently contacting the T cell with the at least one reprogramming factor.

In various embodiments, the cell surface receptor is a chimeric antigen receptor or an engineered T cell receptor or a hybrid receptor thereof. In various embodiments, the cell surface receptor recognizes a specific antigen moiety on the surface of a target cell. In various embodiments, the antigenic moiety is MHC class I dependent. In various embodiments, the antigenic moiety is MHC class I independent.

In various embodiments, the resulting T cells comprise an incomplete set of V, D and J segments of the T cell receptor gene. In various embodiments, the disclosure provides for further measuring the epigenetic age of the resulting T cells. In various embodiments, the epigenetic age of the resulting T cells is at least 5% younger than the T cell population prior to reprogramming. In various embodiments, the partially reprogrammed T cells are capable of expanding at least 25-fold over the originally isolated cells. In various embodiments, the disclosure provides for further contacting the isolated T cells with at least one factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC; exposure to SV40 causes reduced expression of CD3 and CD 8.

In various embodiments, the present disclosure provides a method of producing a T cell comprising obtaining a plurality of isolated T cells from a source; culturing the isolated T cells in a first medium comprising IL-2 and activating the isolated T cells with at least one T cell activating compound or agent and a co-stimulatory agent (such as an antibody specific for CD3 and/or CD 28); transiently contacting the activated T cells with KLF4, OCT3/4, SOX2, and C-MYC in a second medium that does not comprise IL-2 or an antibody specific for CD3 or CD28 for a period of time ranging from about 5 days to about 10 days; wherein the isolated T cells are not fully reprogrammed to iPS cells; replacing the second medium with a third medium comprising IL-2 and at least one antibody specific for CD3 and/or CD 28; wherein the transiently contacted T cells are cultured in the third medium for at least about 5 days.

In various embodiments, the present disclosure provides for further expanding the partially reprogrammed, reactivated T cells. In various embodiments, the T cell is a tcra beta cell; TCRgd cells; cd4+cd8αβ+ biscationic cells, cd4+ single positive cells (Th 1, th2, th17, treg), naive T cells, central memory T cells, or effector memory T cells. In various embodiments, the T cell is a Tumor Infiltrating Lymphocyte (TIL).

In various embodiments, the disclosure provides a T cell population having an epigenetic age at least 5% younger than its actual age. In various embodiments, the disclosure provides a population of T cells, wherein the epigenetic age is at least 25% younger than its actual age. In various embodiments, the present disclosure provides a population of adherent cells derived from T cells, wherein at least 70% of the cells express both CD3 and SSEA 4. In various embodiments, the present disclosure provides a population of adherent cells derived from T cells, wherein at least 30% of the cells express CD9 or CD90 or both CD9 and CD 90.

In various embodiments, the invention provides a population of tumor-infiltrating lymphocytes, wherein at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the tumor-infiltrating lymphocytes express both CCR7 and CD 62L. In various embodiments, the invention provides a population of tumor-infiltrating lymphocytes, wherein at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the tumor-infiltrating lymphocytes express both CCR7 and TCF 7.

In various embodiments, the present disclosure provides a population of T cells produced by a method comprising: contacting a population of T cells with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC; and optionally, SV40 for a time sufficient to form T cell-derived adherent cells, and wherein the isolated T cells are not converted to iPS or totipotent cells; and contacting the T cell-derived adherent cells with at least one T cell activating compound.

In various embodiments, the present disclosure provides a population of T cells produced by a method comprising: contacting the isolated population of T cells in the first medium in the culture vessel with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC; and optionally SV40 for a time sufficient for the isolated T cells to form at least one colony attached to the surface of the culture vessel, and wherein the isolated T cells are not transformed into iPS cells; and contacting the at least one attached colony with at least one T cell activating compound.

In various embodiments, the present disclosure provides a population of T cells produced by a method comprising: contacting the isolated population of T cells in the first medium with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC; and SV40 for a period of at least about 5 days and not more than about 10 days and wherein the isolated T cells are not transformed into iPS cells; and contacting the transiently contacted T cells with at least one T cell activating compound.

In various embodiments, the present disclosure provides a population of T cells produced by a method comprising: contacting the isolated population of T cells with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC; contacting with SV40 for a time sufficient for the isolated T cells to express at least one marker selected from the group consisting of SSEA4, CD9, and CD90, and wherein the isolated T cells are not transformed into iPS cells; and contacting the transiently contacted T cells with at least one T cell activating compound.

In various embodiments, the isolated T cell is transiently contacted with the at least one reprogramming factor for a time sufficient for at least a portion of the isolated T cell to express CD3 and at least one marker selected from the group consisting of SSEA4, CD9, and CD90. In various embodiments, the isolated T cells are transiently contacted with the at least one reprogramming factor for a time sufficient for at least a portion of the transiently contacted T cells to express SSEA4 and CD3. In various embodiments, the isolated T cells are transiently contacted with the at least one reprogramming factor for a time sufficient for at least a portion of the transiently contacted T cells to express CD3, CD9, and CD90. In various embodiments, the isolated T cells are transiently contacted with the at least one reprogramming factor for a time sufficient for at least a portion of the transiently contacted T cells to express CD3, SSEA4, CD9, and CD90.

In various embodiments, the transiently contacted T cells are contacted with IL-2 and at least one compound capable of activating the isolated T cells prior to contacting the isolated T cells with the at least one reprogramming factor.

In various embodiments, the T cell is a tcra beta cell; tcrγδ cells; cd4+cd8αβ+ biscationic cells, cd4+ single positive cells (such as Th1, th2, th17, treg), naive T cells, central memory T cells, or effector memory T cells. In various embodiments, the T cell is a TIL. In various embodiments, the isolated T cells are isolated from a mammal. In various embodiments, the isolated T cells are transiently contacted with KLF4, OCT3/4, SOX2, and C-MYC.

In various embodiments, the isolated T cells are transiently contacted with KLF4, OCT3/4, SOX2, and C-MYC for at least about 4 days to 10 days. In various embodiments, the isolated T cells are transiently contacted with KLF4, OCT3/4, SOX2, and C-MYC for at least about 4 days to 7 days.

In various embodiments, the isolated T cells are transiently contacted with KLF4, OCT3/4, SOX2, and C-MYC for about 5 days. In various embodiments, KLF4, OCT3/4, SOX2, and C-MYC are transiently expressed in T cells. In various embodiments, non-integrating viral vectors are used to transiently express KLF4, OCT3/4, SOX2, and C-MYC. In various embodiments, the Sendai virus is used to transiently express KLF4, OCT3/4, SOX2, and C-MYC. In various embodiments, KLF4, OCT3/4, SOX2 and C-MYC are constitutively expressed, where expression is later inhibited by the addition of a compound that inhibits KLF4, OCT3/4, SOX2 and C-MYC expression. In various embodiments, the compound is a small molecule inhibitor that specifically inhibits the expression of one or more of KLF4, OCT3/4, SOX2, and C-MYC expression. In various embodiments, the compound is an siRNA or shRNA molecule that specifically inhibits expression of one or more of KLF4, OCT3/4, SOX2, and C-MYC. In various embodiments, nanoparticles are used to transiently express KLF4, OCT3/4, SOX2, and C-MYC.

In various embodiments, the present disclosure provides for further contacting the transiently contacted T cells (e.g., partially reprogrammed T cells) with at least one cytokine selected from the group consisting of IL-2, IL-7, IL-15, and IL-12; and/or with at least one T cell activator and/or at least one T cell co-stimulatory agent (e.g., anti-CD 3 and/or anti-CD 28 antibodies). In various embodiments, the at least one T cell activating compound comprises an antibody that binds CD3 or an antibody that binds CD28 or both; or wherein the at least one T cell activating compound is a tumor antigen.

In various embodiments, the present disclosure provides for further engineering the T cell to express a cell surface receptor, wherein the T cell is engineered prior to transiently contacting the T cell with the at least one reprogramming factor. In various embodiments, the present disclosure provides for further engineering the T cell to express a cell surface receptor, wherein the T cell is engineered after transiently contacting the T cell with the at least one reprogramming factor.

In various embodiments, the cell surface receptor is a chimeric antigen receptor or a T cell receptor or a hybrid receptor thereof. In various embodiments, the cell surface receptor recognizes a specific antigen moiety on the surface of a target cell. In various embodiments, the antigenic moiety is MHC class I dependent. In various embodiments, the antigenic moiety is MHC class I independent. In various embodiments, the resulting T cells comprise an incomplete set of V, D and J segments of the T cell receptor gene. In various embodiments, the disclosure provides for further measuring the epigenetic age of the resulting T cells. In various embodiments, the epigenetic age of the resulting T cells is at least 5% younger than the T cell population prior to reprogramming. In various embodiments, the partially reprogrammed T cells are capable of expanding at least 25-fold over the originally isolated cells. In various embodiments, the disclosure provides for further contacting the isolated T cells with at least one factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC; exposure to SV40 causes reduced expression of CD3 and CD 8.

In various embodiments, the disclosure relates to a population of T cells produced by a method comprising: obtaining a plurality of isolated T cells from a source; culturing the isolated T cells in a first medium comprising IL-2 and activating the isolated T cells with at least one antibody specific for CD3 and/or CD 28; transiently contacting the activated T cells with KLF4, OCT3/4, SOX2, and C-MYC in a second medium that does not comprise IL-2 or an antibody specific for CD3 and/or CD28 for a period of time ranging from about 5 days to about 10 days; wherein the isolated T cells are not fully reprogrammed to iPS cells; replacing the second medium with a third medium comprising IL-2 and at least one antibody specific for CD3 and/or CD 28; wherein the transiently contacted T cells are cultured in the third medium for at least about 5 days.

In various embodiments, the disclosure relates to further expanding the transiently contacted T cells. In various embodiments, the T cell is a tcra beta cell; tcrγδ cells; cd4+cd8αβ+ biscationic cells, cd4+ single positive cells (Th 1, th2, th17, treg), naive T cells, central memory T cells, or effector memory T cells. In various embodiments, the T cell is a Tumor Infiltrating Lymphocyte (TIL).

In various embodiments, the disclosure relates to a method of treating a patient in need thereof with or by a population of T cells produced by the methods disclosed herein. In various embodiments, the method of treatment is a method for treating cancer, a viral disorder, or an autoimmune disorder.

In various embodiments, the cancer is acute lymphocytic cancer, acute myelogenous leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal canal cancer or anal rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gall bladder cancer or pleural cancer, head and neck cancer (e.g., nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer), vulval cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, hodgkin lymphoma, hypopharynx cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omentum cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal Cell Carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer or carcinoma.

In various embodiments, the invention relates to a method of producing at least one regenerative T cell, comprising: a. isolating T cells from the tumor, wherein the T cells are tumor infiltrating lymphocytes; wherein the tumor-infiltrating lymphocytes express CD137; b. activating the tumor-infiltrating lymphocytes with at least one T cell activating compound, wherein the T cell activating compound is a tumor antigen; c. contacting the isolated population of T cells with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC; and optionally, SV40 for a time sufficient to form T cell-derived adherent cells, and wherein the isolated T cells are not converted to iPS or totipotent cells; contacting an adherent cell of T cell origin with at least one T cell activating compound.

In various embodiments, the invention relates to a method of producing at least one regenerative Tumor Infiltrating Lymphocyte (TIL), the method comprising (a) isolating TIL from a tumor, wherein TIL expresses CD137, (b) activating the TIL with at least one first tumor antigen, and (C) transiently contacting the isolated TIL population with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and (ii) optionally SV40 for a time sufficient to form adherent cells of TIL origin, and wherein the isolated TIL is not converted to iPS or totipotent cells.

In various embodiments, the invention relates to a method of producing regenerative T cells comprising contacting a population of T cells with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and (ii) optionally SV40 for at least a period of time sufficient for at least 20% of the contacted T cells to express α6β1 integrin, and wherein the contacted T cells are not converted to iPS cells; isolating the at least 20% of the contacted T cells with a binding molecule that specifically binds α6β1 integrin; contacting the isolated cells of (b) with a T cell activator and/or a T cell co-stimulator; thereby generating regenerative T cells.

In various embodiments, the invention relates to a method of producing at least one regenerative T cell, comprising contacting a population of T cells with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and (ii) optionally SV40 transiently for a time sufficient to form an adherent T cell-derived cell; wherein the T cells are not transformed into iPS or totipotent cells; isolating a subpopulation of adherent cells derived from T cells expressing a6 (CD 49 f) or b1 (CD 29) integrin or both; and contacting the isolated subpopulation with at least one T cell activating compound.

In various embodiments, the invention relates to a population of regenerative T cells produced by a method comprising: contacting a population of T cells with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC; and (ii) SV40 for a time sufficient to form T cell-derived adherent cells; wherein the T cells are not transformed into iPS or totipotent cells; isolating a subpopulation of adherent cells derived from T cells expressing a6 integrin, b1 integrin, or both; and contacting the T cell-derived adherent cell subpopulation with at least one T cell activating compound.

In various embodiments, the invention relates to a population of adherent cells derived from T cells, wherein at least 70% of the cells express integrin α6 or integrin β1.

In various embodiments, the invention relates to a population of adherent cells of T cell origin, wherein at least 50% of the cells express both integrin α6 and integrin β1.

In various embodiments, the invention relates to a population of adherent cells of T cell origin, wherein at least 70% of the cells express both integrin α6 and integrin β1.

Further features and aspects of the present invention will be appreciated by those of ordinary skill in the art in view of the following disclosure and appended claims and drawings.

Drawings

FIG. 1 shows the experimental design of partial reprogramming using Sendai virus transduction. T cells were activated with CD3, CD28 activation medium containing IL2 three days (day-2 to day 0) after isolation from Peripheral Blood Mononuclear Cells (PBMCs). On day 0, cells (i) remained undisturbed (group 1); (ii) Placed in IL 2-free stem cell medium (group 2); (iii) Placing in a stem cell culture medium without IL2 and transducing with two Sendai viruses-one Sendai virus expressing EmGFP; sendai virus expression SV40 (group 3); or (iv) placed in stem cell culture medium without IL2 and transduced with four Sendai viruses expressing KOS (KLF 4, OCT3/4, SOX 2), KLF4, cMyc and SV40 (group 4). Expression lasted five days.

FIG. 2 shows colony formation in group 4 starting on days 4 to 9 after transduction with Sendai virus expressing OSKM+SV 40.

FIGS. 3A-3F show morphological differences of T cells cultured under different conditions as described in example 1. FIG. 3A shows adherent cell colony formation for group 4, and FIG. 3B shows T cells in group 3 that do not show adherent and colony formation; fig. 3C shows the same graph as fig. 3A for comparison with fig. 3D. FIG. 3D shows T cells after 3 days of stimulation with anti-CD 3 antibody (100 ng/ml) and anti-CD 28 antibody (2. Mu.g/ml) in standard T cell media; fig. 3E is the same graph as fig. 3A for comparison with fig. 3F. Fig. 3F shows normal iPS cell colonies. The adherent cells shown in fig. 3A, 3C, and 3E are referred to herein as T cell-derived adherent cells.

Figure 4 shows CD3 expression at day 3, 4 and 5 of partial reprogramming. Notably, the reprogrammed cells in group 4 exhibited CD3 loss during partial reprogramming.

Fig. 5 shows a side scatter/forward scatter (SSC/FSC) FACS plot of adherent cells in group 4, indicating that adherent cells are larger and have a more complex structure than cells in groups 1 and 3.

FIG. 6 is a SSC/FSC FACS contour plot showing that adherent cells become larger and structurally complex on day 5.

FIG. 7 shows that adherent cells in group 4 regain CD3 and CD8 expression after two days of T cell activation after five more days of culture in T cell medium. On day 5, T cells were activated in T cell medium for 2 days using one-hundred dilutions of (1:100) T cells TRANSACTTM (Miltenyi Biotec) in 96 well plates. Cells under the 4 th set of conditions were divided into 2 wells on day 5, and floating cells and adherent cells were collected by pipetting. After activation, all cells were cultured in T cell medium for an additional 5 days.

Fig. 8 shows the phenotype of CD3 positive cells on day 12. CCR7 and CD62L are markers for detection of certain T cell types, such as naive T cells, memory stem cells and central memory T cells. Cells regain CD3 expression on day 12, indicating a return to the T cell phenotype.

Fig. 9 is a bar graph depicting cell expansion on days 0, 5 and 12. Cell expansion was measured by counting beads (123count eBeads Counting beads,Thermo) using flow cytometry. Although adherent cells in group 4 showed abnormal appearance and reduced CD3 expression on day 5, they responded to activation and expanded upon activation from day 5. Fold change between day 12 and day 5 indicates increased expansion of #4 compared to #1, especially for adherent cells. This indicates that the partial regeneration method used herein increases the T cell expansion potential, especially in adherent cells.

FIG. 10 is a FACS contour analysis of SSEA4 and CD3 expression in partially reprogrammed T cells. Sendai virus-infected T cells as described in example 2 were transferred onto iMatrix-coated dishes on day 1 and cultured in iPS cell culture medium +60IU/ml IL2 for 3 days, then replaced with IL 2-free iPS cell culture medium from day 4. FACS analysis was performed on day 8 of reprogramming. The figure shows that partially reprogrammed T cells express the hiPS cell marker SSEA4 and lose expression of the CD 3T cell marker.

Figure 11 is a schematic diagram summarizing partial reprogramming of PBMC-derived CD 8T cells (see example 4, table 3).

Figures 12A-12B show the results of flow cytometry measuring CD8a, CD8B, CD3 in cells that had been partially reprogrammed for a specified number of days prior to reactivation. Fig. 12A shows FACS plots of CD8a and CD8B expression on days 7, 9 and 10 and fig. 12B is a graph plotting the percentage of CD8a and CD3CD8ab positive cells on days 6-10. See example 4. The CD4-CD8a+ population was gated for further analysis of CD8B and CD3. By non-sendai infection is meant that CD 8T cells received the first and second stimuli but were not infected with sendai virus (no reprogramming factors).

FIG. 13 is a schematic diagram outlining partial reprogramming and reactivation of NY-ESO-1+T cells. For clarity, the different stages of the overall process are labeled.

FIGS. 14A-14D are graphs showing the results of cytokine production and degranulation assays of regenerated NY-ESO-1TCR transduced T cells. FACS results were analyzed by FlowJo software. For NY-ESO-1Tg T cells, single cell > living cells > CD3+ > CD8+CD4- > NY-ESO-1 tetramer+ cells and for mock control T cells, single cell > living cells > CD3+ > CD8+CD4- > NY-ESO-1 tetramer cells were gated. The frequencies of IFNg+ (A), TNFa+ (B), IL-2+ (C) and CD107a+ (D) were calculated and plotted as indicated.

Fig. 15 is a waterfall plot of the ratio of cell surface marker expression levels in stimulated T cells to T cell-derived iPS cells (see also table 4).

Fig. 16 is a waterfall plot of the ratio of cell surface marker expression levels in unstimulated T cells to T cell-derived iPS cells (see also table 4).

Figure 17 shows that CD9 and CD90 are potential cell surface marker indicators for early stages of T cells that are partially reprogrammed. The figure shows flow cytometry analysis of CD9 and CD90 expression in stimulated T cells, unstimulated T cells, and partially reprogrammed T cells attached on day 5 (T cell-derived adherent cells). In contrast to the expression of CD9 and CD90, the expression of the early iPS cell surface marker SSEA4 was not significantly altered in T cell-derived adherent cells on day 5.

Figures 18A-18D show that stimulation of signaling is critical for the re-acquisition of expression of the conventional T cell markers CD3 and CD8A by partially reprogrammed T cells, survival and proliferation. Fig. 18A is a FACS contour plot showing the percentage of partially reprogrammed cells expressing CD3 and CD8A on day 7 (day of shedding). FIG. 18B is a FACS contour plot showing the percentage of cells expressing CD3 and CD8a on day 20 after TRANSACT activation with 1:500 or 1:1000 dilutions or just after T cell culture (TCM+IL260 IU/ml). Fig. 18C and 18D show proliferation and viability of different groups.

Figures 19A-19B show that regenerative T cells exhibited significantly enhanced and sustained proliferation in vitro. T cells were obtained from 37 year old females, 42 year old males and 52 year old male donors.

FIGS. 20A-20C show the effect of IL-2 on regenerative T cells. On day 45 after regeneration, the regenerative T cells were co-cultured in TCM without IL-2. FACS results were analyzed by FlowJo software. For all cell groups, single cell > living cell > cd3+ cells were gated. As can be seen from fig. 20B and 20C, cells co-cultured in the absence of IL-2 died within six days.

FIG. 21 shows the results of cytokine production assays for regenerating 37yF, 42yM and 52yM T cells. FACS results were analyzed by FlowJo software. For all cell groups, single cell > living cell > CD3+ > CD8+ CD 4-cell was gated. The frequencies of il2+ and ifng+ were calculated and plotted as shown.

FIGS. 22A-22C show that regenerative T cells exhibit effector phenotypes after long-term expansion. Sendai virus-infected T cells were transferred to an iMatrix-coated petri dish on day 1 and cultured in T cell medium +60IU/ml IL2 or in iPS cell medium +60IU/ml IL2 for 3 days, and then replaced with IL 2-free iPS cell medium from day 4. FACS analysis was performed on day 7 and day 26 after reprogramming.

FIG. 23 is a FACS contour analysis of cell markers (CD 3, CD45RA, CD45RO, CD62L and CCR 7) for defining the differentiation status of T cells.

FIG. 24 shows the fold expansion of NY-ESO-1 regenerating cells or control cells from two different donors (# 3 and # 4) after stimulation with TRANSACT. In the region of 2-3 weeks of culture, regenerative T cells proliferate longer than and beyond the control.

FIG. 25 is a FACS contour analysis of NY-ESO-1-CD3, CD4-CD8b, CCR7-CD62L and CD45RO-CD45RA expression in partially reprogrammed NY-ESO-1T cells.

FIGS. 26A-26D are graphs showing the results of cytokine production and degranulation assays of regenerated NY-ESO-1TCR transduced T cells. FACS results were analyzed by FlowJo software. For NY-ESO-1Tg T cells, single cell > living cells > CD3+ > CD8+CD4- > NY-ESO-1 tetramer+ cells and for mock control T cells, single cell > living cells > CD3+ > CD8+CD4- > NY-ESO-1 tetramer cells were gated. The frequencies of IFNg+ (A), TNFa+ (B), IL-2+ (C) and CD107a+ (D) were calculated and plotted as indicated. No target (control); T2P-: peptide-free T2 target cells (-control); t2p+: t2 target cells pulsed with nyso 1 peptide; mel624: HLA-A2+ tumor cells expressing endogenous nyesao 1; PMA/I: cells activated with PMA/ionomycin (+control).

FIGS. 27A-27B show that regeneration of tumor-infiltrating lymphocytes (TILs) enhanced proliferation and produced more stem cell-like products. FIG. 27A is a graph plotting cell expansion and comparing fold change of regenerated TIL versus control cells. FIG. 27B shows a FACS diagram of CCR7-CD62L and CCR7-TCF7 expression and shows that regenerated TIL exhibits high expression of a dryness-associated marker.

FIG. 28 depicts an exemplary, non-limiting scheme of the regeneration scheme employed in example 12 described below.

Fig. 29A and 29B show phenotypic analysis performed in conjunction with the regeneration protocol of example 12. Fig. 29A shows a representative FACS plot of donor 1 showing the frequency of cd3+cd8b+. FIG. 29B is a bar graph of CD3+CD8b+ frequencies gated as in FIG. 29A; each bar shows the average value of donors 1-3. Error bar indicates +/-1SD

Figures 30A-30C show skin and blood clock results for donors 1-3 of example 12 at days 7, 13, 18 before and after Sev transduction. "Sev pre-transduction" indicates a sample prior to activation of Sendai virus transduction. Error bar display1SD.FIG

FIG. 31 illustrates the regeneration results of NY-ESO-1Tg CD4 and CD 8T cells. CD4 or CD 8T cells were stimulated by Transact and transduced with NY-ESO-1TCR prior to reprogramming.

FIGS. 32A and 32B illustrate the results of regeneration of NY-ESO-1Tg CD4 and CD 8T cells showing a low differentiation phenotype.

FIG. 33 illustrates the result of regenerating NY-ESO-1Tg CD4 and CD 8T cells to proliferate more than control NY-ESO-1Tg CD4 and CD 8T cells.

Fig. 34A and 34B are bar graphs showing eAge values from two donors: one 24 year old male (a) and one 35 year old male (B). The figure illustrates that by epigenetic age analysis, the regenerated NY-ESO-1tg CD4 and CD 8T cells show a younger phenotype than the actual age of the control NY-ESO-1tg CD4 and CD 8T cells and the donor. The figures show the mean and s.d. of age values analyzed from methylation status of certain CpG sites.

FIG. 35 depicts a bar graph showing intracellular cytokine staining from regenerative and control NY-ESO-1 cells. The graph shows that, after co-culture with T2 cells and NY-ESO-1 peptide, regenerated NY-ESO-1Tg CD4 and CD 8T cells produced more cytokines (IFNg (A), IL-2 (B) and TNFa (C)). t-test p-value statistical significance: * <0.05, <0.005, <0.0005, <0.0001, < 0.005.

FIG. 36 illustrates the results of regenerating NY-ESO-1Tg CD8T cells after repeated co-culture with NY-ESO-1 expressing target cells (A375-NLR) for prolonged duration and maintenance of their cytotoxic activity.

FIG. 37 depicts the enhanced cytotoxicity of regenerative NY-ESO-1Tg CD8T cells caused by the addition of regenerative NY-ESO-1Tg CD4T cells.

Fig. 38A and 38B show the expression levels of adaptive immune-related cytokines from regenerative cells and control cells from two different donors at the indicated time points. The y-axis represents cytokine concentration in pg/mL.

Fig. 39A and 39B show the expression levels of innate immune-related cytokines from the regenerative cells and control cells of two different donors at the indicated times. The y-axis represents cytokine concentration in pg/mL.

FIG. 40 depicts the NY-ESO-1TCR transduction efficiency in CD 8T cells using a representative FACS diagram of NY-ESO-1TCR transduced T cells: (left) CD4 x CD8b, (right) CD3 x NY-ESO-1TCR TE. See example 15.

Fig. 41 provides a schematic of the procedure of example 15.

Figure 42 provides a bar graph showing the fold change in the number of shed cells collected from each condition on day 7 as compared to day 0. See example 15.

FIG. 43 depicts the restimulation of partially reprogrammed NY-ESO-1TCR Tg T cells. Fig. 43 provides a representative FACS plot on day 14. Frequencies of NY-ESO-1TCR TE+ cells are provided. See example 15.

FIG. 44 depicts the restimulation of partially reprogrammed NY-ESO-1TCR Tg T cells. FIG. 44 provides a representative bar graph of the frequency of day 14 of NY-ESO-1TCR TE+ cells. See example 15.

FIG. 45 is a proliferation curve showing that regenerated NY-ESO-1Tg T cells have a higher proliferation capacity than non-regenerated controls (see example 15).

Figure 46 illustrates CD19 CAR transduction efficiency in CD 8T cells. See example 16. Cells were gated according to lymphocyte > single cell > live/dead-, and plotted as CD3x CD8a (right two), CAR idiotype x EGFR (right most). tEGFR expression is a marker of successful transduction.

Figure 47 illustrates that regenerative CD19 CAR T cells exhibit a low differentiation phenotype. On day 13, control and regenerative CD19 CAR Tg T cells were analyzed by FACS for surface markers and Tcf1 expression. Cells were gated according to lymphocyte > single cell > live/dead- > CD19CAR+, and plotted as CD4 x CD8a (left two), CCR7 x CD62L (right two) and CCR7 x Tcf1 (rightmost)

Figure 48 shows that regenerative CD19 CAR T cells proliferated more than control CD19 CAR T cells. See example 16.CD19 CAR Tg T cells were regenerated and cultured as described in section 3, counting the number of cells every 3-4 days. The graph depicts fold changes over time in two healthy donors compared to day 7 after transduction of reprogramming factors and their corresponding control cells.

Figure 49 illustrates that regenerative CD19 CAR T cells produced considerable levels of cytokines after co-culture with target cells expressing CD 19. See example 16. On day 20, control or regenerative CD19 CAR Tg T cells were cultured alone or with PMA/ionomycin, colo205 (CD 19-) or Nalm6 (cd19+) in the presence of golgi transporter inhibitors. After staining for surface antigens, the cells are fixed, permeabilized and stained with intracellular antibodies. The frequency of each cytokine-positive car+ cell is depicted.

Figures 50A and 50B show that regenerative CD19 CAR T cells persist and retain their cytotoxic activity for longer after repeated co-culture with CD19 expressing target cells. See example 16. Fig. 50A and 50B show the results of co-culturing control or regenerative CD19 CAR Tg T cells with a Nalm6-NLR cell line. Every 3-4 days, 25% of the previous cultures (10% of the 4 th and 5 th co-cultures) were transferred to new plates with new targets. UsingThe living cell analysis system monitors the growth of the target and uses a basic software analysis module (Base Software Analysis Module) for analysis. The figure shows the Nalm6-NLR number/image for each condition of donor #1 (fig. 50A) and donor #2 (fig. 50B).

FIG. 51 is an exemplary, non-limiting experimental design of a regeneration protocol described in example 17 for illustrative purposes.

Fig. 52A and 52B are the results of phenotypic analysis of cells shed on day 11 (fig. 52A) and the same cells on day 17 (i.e., isolated on day 11) (fig. 52B) in each of Sev and mSev groups discussed in example 17. FACS plots are provided showing the frequency of cd3+cd8b+.

FIGS. 53A-53C are proliferation curves showing fold changes after various days following shedding (see example 17). Figure 53A shows fold change in cell count after shedding occurred on day 7. Figure 53B shows fold change in cell count after shedding occurred on day 9. Figure 53C shows fold change in cell count after shedding occurred on day 11. Each day of each of figures 53A-53C indicates the average of 2 donors. Error bars indicate ±1sd.

Figure 54A shows the dry phenotype exhibited 6 days after the shedding of the reprogrammed T cells of example 17. The bar graph shows the average tcf1+ccr7+ frequencies (unimodal gating, dead staining negative, lymphocyte SSC/FSC and cd3+cd8b+), in technical replicates. Error bars indicate ±1sd.

FIG. 54B shows the dry phenotype six days after the shedding of the reprogrammed T cells of example 17. The bar graph shows the average ccr7+cd62l+ frequency (unimodal gating, dead staining negative, lymphocyte SSC/FSC and cd3+cd8b+), in technical replicates. Error bar indicates + -1 SD

FIG. 55 shows the epigenetic age of T cells transiently reprogrammed with Sev or mSev and shed on days 7, 9 and 11 as described in example 17. Epigenetic age was measured using the Horvath skin blood clock.

FIG. 56 shows proliferation curves of the rejuvenated cells of example 18 and control cells.

FIGS. 57A-57D show transcriptional profiling of healthy donor CD8+ T cells from control and regenerative cells on days 7 and 13 of example 18. Fig. 57A shows single cell RNA-seq data of cd8+ T cells from control and rejuvenated cells of all four donors visualized by UMAP. FIG. 57B shows the expression levels of transcripts encoding Yamanaka factors in regenerating cells in single cell RNA-seq data from CD8+ T cells of control and regenerating cells. FIG. 57C shows the expression levels of transcripts encoding Yamanaka factors in bulk RNA-seq data from CD8+ T cells of control and regenerating cells. Fig. 57D shows genomic alignment visualization of reads aligned with endogenous cMYC genes in healthy donor cd8+ T cells from control and regenerative cells on days 7 and 13.

Fig. 58A depicts enrichment of metabolic gene sets in regenerative cells compared to control cells in 3 donors (i.e., donor numbers 11347, 12254, and 2621) of the bulk RNA-seq data of example 18 on days 7 and 13. Differences in x-axis scale show adjusted p-values on day 7 and day 13. The gray solid line shows the significance threshold (adjusted P value < 0.05).

FIG. 58B shows AddModule scores for glycolysis and oxidative phosphorylation gene sets on the same UMAP representation as shown in FIG. 59A (see example 18).

Fig. 59A is UMAP, demonstrating that the rejuvenated cells were enriched for the initial T cell gene set (Gattinoni) on day 13 compared to control cells (see example 18).

Fig. 59B is a bar graph illustrating that the rejuvenated cells were enriched for the initial gene set (Gattinoni) on day 13 compared to the control cells (see example 18).

Fig. 60A and 60B are graphs showing proliferation curves after regeneration of donor 1 and donor 2. All regenerative cells showed much higher expansion than the control group. In one donor, the TransAct stimulated group showed higher amplification than the CD137+ selection group, while in the other donor was lower. This indicates the different affinities of the tumor-reactive group for stimulation. Expansion showed a decrease after 33 days, probably due to the sub-optimal cell density of the inoculated cells of the culture for the culture plates, resulting in undernutrition and oxygen supply. See example 19.

Fig. 61A and 61B are graphs showing expression of CD62L and CCR7 in regenerative cells and control cells over time. Cd62l+ccr7+ is maintained in the early stages of regeneration. Cells were harvested on the indicated days and stained with fluorescent-labeled antibodies followed by flow cytometry analysis. The results show that in both donors, the regenerating cells highly expressed cd62l+ccr7+ at D14 and D17. See example 19.

FIG. 62 is a graph showing the proliferation curve of donor 3 after regeneration. TIL is enriched in cd45+ and then stimulated with tranact followed by regeneration. Cells were counted on the indicated days. Regenerative CD45+ showed greatly enhanced proliferation compared to control cells, while the CD 45-group showed very limited expansion. See example 19.

Fig. 63 shows FACS contour plots measuring marker expression associated with dryness in control and donor 3 on day 15. Regeneration of cd45+ TIL instead of CD 45-cells showed much higher cd62l+ ccr7+ and tcf7+ ccr7+ expression. See example 19.

FIG. 64 is a graph showing proliferation curves of control and donor 4 after regeneration. Cells were stimulated with TransAct at 1:500 (HI) or 1:2000 (LO). After redirection to T cells, the rejuvenated cells (HI and LO groups) showed similar expansion rates to the control.

Fig. 65 shows FACS contour plots measuring expression of markers associated with dryness on day 21 post-regeneration in control cells and donor 4. Regenerative cells (HI and LO groups) showed higher cd62l+ccr7+ and tcf7+ccr7+ expression than control cells. See example 19.

FIG. 66 is a graph showing the epigenetic age (eAge) of control or rejuvenated cells from donor 7 at a given time. The control cells showed an epigenetic age similar to the actual age of the donor, while the rejuvenated cells showed a much lower age at day 15 and the age gradually increased with the time of expansion. See example 19.

FIG. 67 is a graph showing the epigenetic age (eAge) of control or rejuvenated cells from donor 9 at a given time. The control group had an epigenetic age similar to the actual donor age, while the rejuvenated cells showed a much lower epigenetic age on day 15, with the epigenetic age gradually returning to the control level over time of culture proliferation. See example 19.

Fig. 68 is a graph showing proliferation of control and regenerative cells from donor 7 and donor 9. The rejuvenated cells showed a similar expansion rate to the control after redirection to T cells. See example 19.

Figure 69A shows phenotypic analysis of shed cells on days 11 and 18 from donor 6 of example 20. FACS plots show the frequency of cd3+ (unimodal gating, dead staining negative and cd3+ cells). Regenerative cells showed higher tcf7+ccr7+ on days 14 and 18. See example 20 and table 10.

FIG. 69B shows phenotypic analysis of shed cells on days 11 and 18 from donor 8 of example 20. FACS plots show the frequency of cd3+ (unimodal gating, dead staining negative and cd3+ cells). Regenerative cells showed higher tcf7+ccr7+ on days 14 and 18. See example 20 and table 10.

Figure 70 shows that adherent cells (weakly adherent cells, strongly adherent cells) showed higher integrin a6 and integrin b1 expression than activated T cell controls on day 10. Strongly adherent cells showed higher integrin a6 and integrin b1 expression than weakly adherent cells and adherent cells. The graph is obtained after a single peak and shutter control.

Figure 71 shows that integrin a6 and integrin b1 expression decreased after T cell activation on day 10. The bar graph shows the MFI (mean fluorescence intensity) of the graph of fig. 70. The values were normalized by the MFI of the activated T cell control at each time point. The control value was 1.

Figure 72 shows that adherent cells lost CD3 expression on day 10, but acquired CD3 expression on day 17.

Fig. 73 shows the high proliferation exhibited by weakly attached cells, and strongly attached cells. Strongly adherent cells showed a later onset of cell expansion.

Figures 74A and 74B show that adherent cells (particularly strongly adherent) exhibit high TCF1 and CCR7 expression on days 10 (figure 74A) and 17 (figure 74B). MFI is obtained from data after unimodal and valve control.

Figure 75 shows the simpson clonality distribution of single cell TCR sequences from control and regenerative cells on days 7 and 13 (see example 18).

Detailed Description

It is to be understood that the description herein is merely exemplary and illustrative and does not limit the claimed disclosure. In the present application, the use of the singular includes the plural unless specifically stated otherwise.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Throughout this disclosure, various patents, patent applications, and publications are cited. The disclosures of these patents, patent applications, and publications in their entireties are hereby incorporated by reference into this disclosure in order to more fully describe the state of the art as known to those skilled in the art by the date of this disclosure. In the event of any inconsistency between the cited patents, patent applications and publications and the present disclosure, the present disclosure shall control.

For convenience, certain terms employed in the description, examples, and claims are collected here. Unless defined otherwise, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

in the present application, the use of "or" means "and/or" unless stated otherwise. Furthermore, the use of the term "include" and other forms such as "include" and "include" is not limiting. In addition, unless specifically stated otherwise, terms such as "element" or "component" encompass both elements and components comprising one unit and elements and components comprising more than one sub-unit.

The present disclosure relates in part to methods of generating regenerative T cells that restore partially reprogrammed cells to regenerative functional T cells through a process of partial reprogramming and reactivation. In certain embodiments, the partial reprogramming comprises transient expression of reprogramming factors in T cells. The partial reprogramming process causes the formation of "T cell-derived adherent" cells that express one or more markers of dedifferentiated cells, but retain the phenotypic markers of the T cell lineage. Thus, adherent cells of T cell origin are partially dedifferentiated T cells that revert to functional T cells when contacted with a T cell activating compound. T cells generated by this process exhibit characteristics of cell regeneration while maintaining lineage stability and maintaining antigen specificity, as described in further detail herein.

As used herein, the term "immune cell" refers to any type of immune cell, including, for example, T cells, B cells, monocytes, macrophages, dendritic cells, and the like. In certain exemplary embodiments, the immune cells disclosed herein are T cells.

The term "polynucleotide", "nucleotide" or "nucleic acid" includes single-and double-stranded nucleotide polymers. The nucleotides making up the polynucleotide may be ribonucleotides or deoxyribonucleotides or modified forms of either type of nucleotide. The modification includes: base modifications such as bromouridine and inosine derivatives; ribose modifications such as 2',3' -dideoxyribose; and internucleotide linkage modifications such as phosphorothioate, phosphorodithioate, phosphoroselenate, phosphorodiselenate, phosphoroanilino-phosphorothioate (phospho-anilothioate), phosphoroanilino-phosphate (phospho-anilothioate), phosphoramidate, and the like.

The term "oligonucleotide" refers to a polynucleotide comprising 200 nucleotides or less. The oligonucleotides may be single-stranded or double-stranded, for example, for constructing mutant genes. The oligonucleotide may be a sense or antisense oligonucleotide. The oligonucleotides may include labels for detection assays, including radiolabels, fluorescent labels, haptens or antigenic labels. The oligonucleotides can be used, for example, as PCR primers, cloning primers or hybridization probes.

The term "control sequence" refers to a polynucleotide sequence that can affect the expression and processing of a coding sequence to which it is linked. The nature of such control sequences may depend on the host organism. In particular embodiments, control sequences for prokaryotes may include promoters, ribosome binding sites, and transcription termination sequences. For example, eukaryotic control sequences may include promoters comprising one or more transcription factor recognition sites, transcription enhancer sequences, and transcription termination sequences. "control sequences" may include leader sequences (signal peptides) and/or fusion partner sequences.

As used herein, "operably linked" means that the components to which the term is applied are in a relationship that allows them to perform their inherent functions under the appropriate conditions.

The term "vector" means any molecule or entity (e.g., nucleic acid, plasmid, phage, or virus) used to transfer protein-encoding information into a host cell. The term "expression vector" or "expression construct" refers to a vector suitable for transforming a host cell and containing a nucleic acid sequence that directs and/or controls (along with the host cell) the expression of one or more heterologous coding regions operably linked thereto. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, if present, RNA splicing of the coding region to which they are operably linked.

The term "host cell" refers to a cell that has been transformed with a nucleic acid sequence or is capable of transforming and thereby expressing a gene of interest. The term includes the progeny of a parent cell, whether or not the progeny is identical in morphology or genetic composition to the original parent cell, as long as the gene of interest is present.

The term "transformation" refers to a change in the genetic characteristics of a cell that has been transformed when the cell has been modified to contain new DNA or RNA. For example, a cell is transformed where the cell is genetically modified from its natural state via transfection, transduction, delivery using nanoparticles (e.g., lipid nanoparticles), or other techniques to introduce new genetic material. Following transfection or transduction, the transforming DNA may be recombined with the DNA of the cell by physical integration into the chromosome of the cell, or may be transiently maintained without being replicated as an episomal element, or may be replicated independently as a plasmid. A cell is considered to have been "stably transformed" when the transforming DNA replicates as the cell divides.

The term "transfection" refers to the uptake of foreign or exogenous genetic material (DNA or RNA) by a cell. Many transfection techniques are well known in the art and are disclosed herein. See, e.g., graham et al, 1973, virology,1973,52:456; sambrook et al Molecular Cloning: A Laboratory Manual,2001, supra; davis et al, basic Methods in Molecular Biology,1986, elsevier; chu et al, 1981, gene,13:197.

The term "transduction" refers to the process of introducing foreign DNA or RNA into a cell via a viral vector. See, e.g., jones et al, genetics: principles and Analysis,1998,Boston:Jones&Bartlett Publ.

The term "polypeptide" or "protein" refers to a macromolecule having the amino acid sequence of a protein, including the deletion, addition, and/or substitution of one or more amino acids of the native sequence. The terms "polypeptide" and "protein" specifically encompass antigen binding molecules, antibodies, or sequences having deletions, additions and/or substitutions of one or more amino acids of an antigen binding protein. The term "polypeptide fragment" refers to a polypeptide having an amino terminal deletion, a carboxy terminal deletion, and/or an internal deletion as compared to the full-length native protein. Such fragments may also contain modified amino acids as compared to the native protein. Useful polypeptide fragments include immunologically functional fragments of antigen-binding molecules.

The term "isolated" means (i) free of at least some other proteins that would normally be found with it, (ii) substantially free of other proteins from the same source, e.g., from the same species, (iii) from at least about 50% of polynucleotides, lipids, carbohydrates or other materials with which they are associated in nature, (iv) operably associated (by covalent or non-covalent interactions) with polypeptides with which they are not associated in nature, or (v) not occurring in nature.

A "variant" of a polypeptide (e.g., an antigen binding molecule) comprises an amino acid sequence in which one or more amino acid residues in the amino acid sequence are inserted, deleted, and/or substituted relative to another polypeptide sequence. Variants include fusion proteins.

The term "identity" refers to the relationship between sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "percent identity" means the percentage of identical residues between amino acids or nucleotides in a compared molecule and is calculated based on the size of the smallest molecule compared. For these calculations, the gaps in the alignment, if any, are preferably solved by a specific mathematical model or computer program (i.e., an "algorithm").

To calculate percent identity, the sequences compared are typically aligned in such a way that a maximum match between the sequences results. One example of a computer program that may be used to determine percent identity is the GCG package, which includes GAP (Devereux et al, nucleic acid res.,1984,12,387;Genetics Computer Group,University of Wisconsin,Madison,Wis). The computer algorithm GAP is used to align two polypeptides or polynucleotides for which percent sequence identity is to be determined. Sequences are aligned to achieve the best match of their respective amino acids or nucleotides ("match span", determined by the algorithm). In certain embodiments, standard comparison matrices (see, e.g., PAM 250 comparison matrix in Dayhoff et al, 1978,Atlas of Protein Sequence and Structure,5:345-352; henikoff et al, 1992, proc. Natl. Acad. Sci. U.S.A.,89, 10915-10919) may also be used by the algorithm.

As used herein, twenty conventional (e.g., naturally occurring) amino acids and abbreviations thereof follow conventional usage. See, e.g., immunology A Synthesis (2 nd edition, golub and Green editions, sinauer assoc., sundland, mass. (1991)), which is incorporated herein by reference for any purpose. Stereoisomers of twenty conventional amino acids (e.g., D-amino acids), unnatural amino acids such as α -, α -disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the disclosure. Examples of unconventional amino acids include: 4-hydroxyproline, gamma-carboxy-glutamic acid, epsilon-N, N-trimethyllysine, e-N-acetyllysine, 0-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,. Sigma. -N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left hand direction is the amino terminal direction and the right hand direction is the carboxy terminal direction, according to standard usage and convention.

Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other inverted or reverse forms of amino acid moieties. Naturally occurring residues can be divided into a number of categories based on common side chain characteristics:

a) Hydrophobic: norleucine, met, ala, val, leu, ile;

b) Neutral hydrophilic: cys, ser, thr, asn, gln;

c) Acidic: asp, glu;

d) Alkaline: his, lys, arg;

e) Residues that affect chain orientation: gly, pro; and

f) Aromatic: trp, tyr, phe.

For example, a non-conservative substitution may involve exchanging a member of one of these classes for a member of another class.

According to certain embodiments, the hydropathic index of amino acids may be considered when making changes to the antigen binding molecule, the costimulatory domain, or the activation domain of an engineered T cell. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge characteristics. The hydrophilic index is: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). See, e.g., kyte et al, 1982, J.mol.biol.,157,105-131. It is known that certain amino acids may be substituted for other amino acids having similar hydropathic indices or scores and still retain similar biological activity. It is also understood in the art that substitution of similar amino acids can be effectively made based on hydrophilicity, particularly where the resulting biofunctional proteins or peptides are intended for use in immunological embodiments, as in the present case. Exemplary amino acid substitutions are listed in table 1.

TABLE 1

Original residue	Exemplary substitution	Preferably substituted
			Ala	Val、Leu、Ile	Val
Arg	Lys、Gin、Asn	Lys
			Asn	Gln	Gln
Asp	Glu	Glu
			Cys	Ser、Ala	Ser
Gln	Asn	Asn
			Glu	Asp	Asp
Gly	Pro、Ala	Ala
			His	Asn、Gln、Lys、Arg	Arg
Ile	Leu, val, met, ala, phe norleucine	Leu
			Leu	Norleucine Ile, va, met, ala, phe	Ile
Lys	Arg, 1, 4-diaminobutyric acid, gin, asn	Arg
			Met	Leu、Phe、Ile	Leu
Phe	Leu、Val、Ile、Ala、Tyr	Leu
			Pro	Ala	Gly
Ser	Thr、Ala、Cys	Thr
			Thr	Ser	Ser
Trp	Tyr、Phe	Tyr
			Tyr	Trp、Phe、Thr、Ser	Phe
Val	Ile, met, leu, phe, ala norleucine	Leu

The term "derivative" refers to a molecule that includes chemical modifications other than insertions, deletions, or substitutions of amino acids (or nucleic acids). In certain embodiments, the derivatives comprise covalent modifications, including, but not limited to, chemical bonding to polymers, lipids, or other organic or inorganic moieties. In certain embodiments, the chemically modified antigen binding molecule may have a longer circulatory half-life than the non-chemically modified antigen binding molecule. In some embodiments, the derivatized antigen binding molecule is covalently modified to include one or more water-soluble polymer attachments, including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.

Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs, with properties similar to those of the template peptide. These types of non-peptide compounds are referred to as "peptidomimetics" or "mimetic peptides". Fauchere, J.L.,1986,Adv.Drug Res, 1986,15,29; veber, D.F. and Freidinger, R.M.,1985,Trends in Neuroscience,8,392-396; and Evans, B.E. et al, 1987, J.Med.chem.,30,1229-1239, incorporated herein by reference for any purpose.

The term "therapeutically effective amount" refers to an amount of an immune cell (e.g., T cell) or other therapeutic agent that produces a therapeutic response in a mammal. Such therapeutically effective amounts are readily determined by one of ordinary skill in the art.

The terms "patient" and "subject" are used interchangeably and include human and non-human animal subjects, subjects with formally diagnosed conditions, subjects with unidentified conditions, subjects receiving medical care, subjects at risk of developing conditions, and the like.

The terms "treatment" and "treatment" include therapeutic treatment, prophylactic treatment, and the use of reducing the risk of a subject developing a disorder or other risk factor. Treatment does not require complete cure of the condition and encompasses embodiments that alleviate symptoms or potential risk factors. The term "preventing" does not require 100% elimination of the possibility of an event occurring. Instead, it means that the likelihood of an event occurring in the presence of a compound or method has been reduced.

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). The enzymatic reactions and purification techniques may be carried out according to the manufacturer's instructions or as commonly done in the art or as described herein. The foregoing techniques and procedures may generally be performed according to conventional methods well known in the art and as described in many general and more specific references cited and discussed throughout this specification. See, e.g., sambrook et al Molecular Cloning: A Laboratory Manual (2 nd edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (1989)), which is incorporated herein by reference for any purpose.

As used herein, the term "substantially" or "substantially" means that the amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% compared to a reference amount, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length. In certain embodiments, the term "substantially the same" or "substantially the same" refers to a range of numbers, levels, values, numbers, frequencies, percentages, dimensions, sizes, amounts, weights, or lengths that are about the same as a reference number, level, value, number, frequency, percentage, size, amount, weight, or length.

As used herein, the terms "substantially free" and "substantially free" are used interchangeably and when used to describe a composition such as a cell population or culture medium, refer to a composition that is free of a specified substance, such as 95% free, 96% free, 97% free, 98% free, 99% free of the specified substance, or undetectable by conventional means. Similar meaning may be applied to the term "absent," where it is meant that the particular substance or component of the composition is absent.

As used herein, the term "perceptible" refers to a range or event of amounts, levels, values, numbers, frequencies, percentages, sizes, dimensions, amounts, weights, or lengths that are readily detectable by one or more standard methods. The term "imperceptible" and equivalent terms refer to a range or event of quantity, level, value, number, frequency, percentage, size, dimension, quantity, weight, or length that is not readily detectable by standard methods or that is not detectable by standard methods. In certain embodiments, an event is imperceptible if the event occurs less than 5%, 4%, 3%, 2%, 1%, 0.1%, 0.001% or less of the time.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. In particular embodiments, the terms "comprising," "having," "containing," and "including" are used synonymously.

As used herein, "consisting of … …" means including and limited to things after the phrase "consisting of … …". Thus, the phrase "consisting of" means that the listed elements are essential or mandatory and that no other elements may be present.

"consisting essentially of" means including any element listed after the phrase and is limited to other elements that do not interfere with or contribute to the activity or effect specified in the present disclosure for the listed elements. Thus, the phrase "consisting essentially of" means that the listed elements are essential or mandatory, but that no other elements are optional, may or may not be present, depending on whether they affect the activity or effect of the listed elements.

By reserving the right to remove or exclude any individual member of any such group, including any sub-ranges or combinations of sub-ranges within a group that may be claimed according to range or in any similar manner, less than all the measures of the disclosure may be claimed for any reason. Moreover, by reserving the right to remove or exclude any single substituent, analog, compound, ligand, structure or group thereof, or any member of the claimed group, less than all the measures of the present disclosure may be claimed for any reason.

Reference throughout this specification to "one embodiment," "an embodiment," "one particular embodiment," "a related embodiment," "an embodiment," "one additional embodiment," or "another embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, size, magnitude, amount, weight, or length that varies by up to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In particular embodiments, the term "about" or "approximately" when preceded by a numerical value indicates that the value is plus or minus a range of 15%, 10%, 5%, or 1%, or any intervening range thereof.

As used herein, "related to" means a relationship between two events, entities, and/or phenomena. As the term is used herein, two events, entities and/or phenomena are "related" to one another if the presence, level and/or form of one event, entity and/or phenomenon is related to another event, entity and/or phenomenon.

As used herein, the term "memory" T cell refers to a T cell that has been previously encountered and responded to its cognate antigen (e.g., in vivo, in vitro, or ex vivo) or has been stimulated (e.g., in vitro or ex vivo) with, for example, an anti-CD 3 antibody. Immune cells having a "memory-like" phenotype upon a second exposure, such memory T cells can replicate to generate a faster and stronger immune response than during the first exposure. In some aspects, the memory T cells include central memory T cells (TCM cells), effector memory T cells (TEM cells), tissue resident memory T cells (TRM cells), stem cell-like memory T cells (TSCM cells), or any combination thereof.

As used herein, the term "stem cell-like memory T cells", "T memory stem cells" or "TSCM cells" refers to memory T cells that express CD95, CD45RA, CCR7 and CD62L and are endowed with stem cell-like self-renewal capacity and multipotent capacity to reconstruct the entire memory and effector T cell subpopulation profile.

As used herein, the term "central memory T cell" or "TCM cell" refers to memory T cells expressing CD45RO, CCR7 and CD 62L. Central memory T cells are commonly found in lymph nodes and peripheral circulation.

As used herein, the term "effector memory T cell" or "TEM cell" refers to a memory T cell that expresses CD45RO but lacks CCR7 and CD62L expression. Because effector memory T cells lack lymph node homing receptors (e.g., CCR7 and CD 62L), these cells are typically present in peripheral circulation and non-lymphoid tissues.

As used herein, the term "tissue resident memory T cells" or "TRM cells" refers to memory T cells that do not circulate and remain resident in peripheral tissues such as skin, lung, and gastrointestinal tract. In some aspects, the tissue resident memory T cells are also effector memory T cells.

As used herein, the term "naive T cell" or "TN cell" refers to a T cell that expresses CD45RA, CCR7 and CD62L but does not express CD 95. TN cells represent the least differentiated cells in the T cell lineage. Interactions between TN cells and Antigen Presenting Cells (APCs) induce differentiation and immune responses of TN cells to activated TEFF cells.

As used herein, the terms "stem", "stem cell-like", "stem-like" or "hypodifferentiation" refer to immune cells (e.g., T cells or TIL) that express markers consistent with a more initial phenotype. For example, the poorly differentiated T cells may express one or more markers characteristic of TN or TSCM cells. In some aspects, "poorly differentiated" or "stem-like" T cells express CD45RA, CCR7, and CD62L. In some aspects, "poorly differentiated" or "stem-like" T cells express CD45RA, CCR7, CD62L, and TCF7. In some aspects, "poorly differentiated" or "stem-like" T cells do not express CD45RO or are low in CD45 RO. In some aspects, the methods disclosed herein promote immune cells (e.g., T cells) having a low differentiation phenotype. Without being bound by any particular mechanism, in some aspects the methods disclosed herein block, inhibit, or limit differentiation of poorly differentiated immune cells (e.g., T cells), resulting in an increase in the number of stem-like cells in culture. For example, it is generally believed that for effective control of tumors, adoptive transfer of poorly differentiated immune cells, such as T cells with stem cell-like memory or central memory phenotypes, is preferred. See Gattineni, L. et al, J.Clin. Invest.115:1616-1626 (2005), gattineni, L. et al, nat Med15 (7): 808-814 (2009), lynn, R.C. et al, nature 576 (7786): 293-300 (2019); gattinone i, L. et al, nat Rev 12:671-684 (2012), klebanonff, C. et al, J.Immunother 35 (9): 651-670 (2012) and Gattinone i, L. et al, nat Med17 (10): 1290-1297 (2011).

Dryness is characterized by self-renewing capacity, multipotency and persistence of proliferative potential. In some aspects, dryness is characterized by a specific gene signature (gene signature), such as a combined expression pattern across multiple genes. In some aspects, stem-like cells can be identified by transcriptome analysis, e.g., using the stem gene tags disclosed herein. In some aspects, the gene signature comprises one or more genes selected from the group consisting of: ACTN1, DSC1, TSHZ2, MYB, LEF1, TIMD4, MAL, KRT73, SESN3, CDCA7L, LOC283174, TCF7, SLC16a10, LASS6, UBE2E2, IL7R, GCNT4, TAF4B, SULT B1, SELP, KRT72, STXBP1, TCEA3, FCGBP, CXCR5, GPA33, NELL2, APBA2, SELL, VIPR1, FAM153B, PPFIBP2, FCER1G, GJB6, OCM2, GCET2, LRRN1, IL6ST, LRRC16A, IGSF9B, EFHA, LOC129293, APP, PKIA, ZC H12D, CHMP7, ki0748, SLC22a17, FLJ13197, NRCAM, C5orf13, GIPC3, WNT7A, FAM117B, BEND, FAM153 and combinations thereof LGMN, FAM63A, FAM153B, ARHGEF, RBM11, RIC3, LDLRAP1, PELI1, PTK2, KCTD12, LMO7, CEP68, SDK2, MCOLN3, ZNF238, EDAR, FAM153C, FAAH2, BCL9, C17orf48, MAP1D, ZSWIM1, sodb 3, IL4R, SERPINF1, C16orf45, SPTBN1, KCNQ1, LDHB, BZW2, NBEA, GAL3ST4, CRTC3, MAP3K1, HLA-DOA, RAB43, SGTB, CNN3, CWH43, KLHL3, PIM2, RGMB, C16orf74, AEBP1, SNORD115-11, GRAP and any combination thereof (see, for example Gattineni et al, nature Medicine17 (10): 1290-97 (2011)). In some aspects, the gene signature comprises one or more genes selected from the group consisting of: NOG, TIMD4, MYB, UBE2E2, FCER1G, HAVCR, FCGBP, PPFIBP2, TPST1, ACTN1, IGF1R, KRT, SLC16a10, GJB6, LRRN1, PRAGMIN, GIPC3, FLNB, arbb 1, SLC7A8, NUCB2, LRRC7, MYO15B, MAL, AEBP1, SDK2, BZW2, GAL3ST4, pitppm 2, ZNF496, FAM117B, C orf74, TDRD6, TSPAN32, C18orf22, C3orf44, LOC129293, ZC3H12D, MLXIP, C orf10, stxb 1, KCNQ1, FLJ13197, LDLRAP1, RAB43, RIN3, SLC22a17, AGBL3, TCEA3, ncn89, rna00185 FAM153B, FAM153C, VIPR1, MMP19, HBS1L, EEF2K, SNORA5C, UBASH3A, FLJ43390, RP6-213H19.1, inp 5A, PIM2, TNFRSF10D, SNRK, LOC100128288, PIGV, LOC100129858, SPTBN1, PROS1, MMP28, HES1, CACHD1, NSUN5C, LEF1, TTTY14, SNORA54, HSF2, C16orf67, NSUN5B, KIAA1257, NRG2, CAD, TARBP1, STRADB, MT1F, TMEM41B, PDHX, KDM6B, LOC100288322, UXS1, LGMN, nans 2, PYGB, rasrp 2, C14orf80, XPO6, SLC24A6, FAM113A, MRM1, FBXW8, NDUFS2, KCTD12 and any combination thereof (see for example, gattineni, L. et al, nat Med 17 (10): 1290-1297 (2011)).

As used herein, the term "monocytes" refers to cells found in blood having a single round core.

As used herein, the term "totipotent" refers to the ability of a cell to produce any cell type found in an embryo, as well as extra-embryonic (placental) cells.

As used herein, the term "multipotent" refers to the ability of a cell to form all lineages of the body or somatic cells (i.e., the embryo itself, not the placenta). For example, embryonic stem cells are a type of pluripotent stem cell that can be used to form cells from each of three germ layers: ectoderm, mesoderm and endoderm. Pluripotency can be determined in part by evaluating the pluripotency characteristics of the cells. The pluripotency characteristics may include, but are not limited to: (i) pluripotent stem cell morphology; (ii) potential for infinite self-renewal; (iii) The ability to differentiate into all three somatic lineages (ectodermal, mesodermal and endodermal); (iv) teratoma formation consisting of three cell lineages; and (v) embryoid body formation consisting of cells from three cell lineages; (vi) Expression of one or more pluripotent stem cell markers including, but not limited to, SSEA1 (mouse only), SSEA3/4, SSEA5, TRA1-60/81, TRA2-54 (ALP), TRA1-85, GCTM-2, TG343, TRA2-49, CD340, CD326, pontoplanin (Podoplanin), and TG30 (CD 9); (vii) Expression of certain other markers associated with adult stem cells or early differentiated cells from embryonic stem cells, including but not limited to integrin α6β1, CD29, CD133/prominin, CD140a, CD56, CD73, CD90, CD105, CD30, and/or LD50; (viii) Expression of certain multipotential genes (including OCT4, NANOG, SOX 2).

As used herein, the term "multipotent" refers to the ability of cells in a particular lineage to develop into a limited number of cell types.

As used herein, the term "non-pluripotent cell" refers to any cell that does not have complete multipotency, such as an incomplete or partial pluripotent stem cell, a pluripotent cell, an oligopotent cell, a monopotent cell (e.g., a progenitor cell), and a terminally differentiated cell.

As used herein, the term "introducing" is meant to include a process of contacting a cell with a polynucleotide, polypeptide, or small molecule. The introducing step may further comprise microinjection of the polynucleotide or polypeptide into the cell, delivery of the polynucleotide or polypeptide into the cell using liposomes, or fusion of the polynucleotide or polypeptide to a cell permeable moiety to introduce them into the cell.

One embodiment of the present disclosure provides a method of preparing an isolated or purified population of immune cells (e.g., T cells) in vitro. An isolated or purified population of immune cells (e.g., T cells) can be included in a pharmaceutical composition that can be used to treat or prevent a variety of different disorders as described herein.

"differentiation" is the process by which cells lose their potency and ability to self-renew and eventually become mature discrete cell types within a discrete lineage (see, e.g., crompton 2014,Trends in Immunol.35:178-185).

"dedifferentiation" refers to the process by which cells become less specialized. In certain embodiments, dedifferentiation is a loss of a specialized trait within a normal developmental pathway or within the same cell lineage. In certain embodiments, the dedifferentiation is dedifferentiation within the same cell lineage (hierarchical dedifferentiation). However, as described herein, in another embodiment, the dedifferentiation may not be within the same cell lineage. For example, in certain embodiments, partial reprogramming of T cells as described herein results in cells that dedifferentiate by obtaining expression of CD9 and/or CD90 and/or SSEA4 (which are markers in humans that are generally unrelated to conventional T cell lineages), and that begin to lose expression of the T cell markers CD3 and/or CD4 and/or CD 8. Such dedifferentiation does not meet the standard T cell lineage hierarchy. These cells can revert to normal T cells after stimulation with a T cell activator. CD90 expression is associated with rare populations of human T cells only, such as cortical thymocytes and Th17 subpopulations.

Regeneration is the process of restoring cellular functions lost by cell aging. The cell age can be described as the sum of the actual age of the donor plus an alpha factor determined by a combination of several factors that induce stress in the cellular microenvironment. These factors can be observed in vivo (e.g., heavy smoking, chronic diseases (including autoimmune diseases), infections, obesity, metabolic problems, and possibly depression), and may be induced artificially in vitro (e.g., prolonged exposure to tumor antigens or other stimuli, high levels of cytokines, hypoxia, etc.). Cells that have become older due to actual age or sustained strong stress stimuli are called senescent cells. Several cellular processes have been identified as being affected by the age of the cell (such as actual age), tumor antigen specificity, killer cell characteristics, proliferative capacity, metabolism, abrogation of the DNA repair system, telomere length, and loss of stem potential.

As used herein, "regenerative T cells" refers to T cells that: has been contacted with one or more reprogramming factors for a time sufficient to initiate attachment of T cells to the surface of the culture vessel to form T cell-derived adherent cells that revert to functional T cells upon stimulation with the T cell activator and optionally the immunomodulatory molecule.

For example, the methods described herein produce regenerative T cells that provide any one or more of increased proliferation, survival, persistence, and/or cytotoxicity in vivo as compared to terminally differentiated T cells; reduced epigenetic age, increased telomere length, improved metabolism, reduced apoptosis and other markers associated with cellular senescence, and improved antitumor activity. In certain embodiments, the regenerative T cells exhibit the ability to polyclonal TCR libraries and long term in vivo implantation. Regenerative T cells can exhibit biological and phenotypic characteristics of young T cells in terms of epigenetic signature, telomere length, metabolic activity, and functionality. In various embodiments, the partial reprogramming process as described herein may result in partial dedifferentiation of T cells, e.g., loss of certain aspects of T cell phenotype that are recovered by TCR signaling and/or T cell activation by culture in an appropriate medium.

In various embodiments, regenerative T cells can be used to target cancer, viruses, or autoimmune diseases. In various embodiments, the partially reprogrammed T cells can be used to reconstitute the entire adaptive immune system or to alleviate patients with immune dysfunction.

In certain embodiments, T cells are partially reprogrammed and regenerated by transient expression of reprogramming factors. In various embodiments, the morphology and cell surface expression profile of the T cells begin to change. Specifically, in certain embodiments, the cells begin to express certain cell surface markers and the expression of T cell markers is reduced. In certain embodiments, the cells begin to express certain cell surface markers, and expression of T cell markers decreases from about day 5 of partial reprogramming, indicating that the dedifferentiation process has been initiated. In various embodiments, dedifferentiated T cells (also referred to herein as "T-cell-derived adherent" cells) are stimulated with one or more T cell activating molecules (such as anti-CD 3 antibodies or other CD3 agonists) and cultured in T cell culture conditions. In certain embodiments, such T cell stimulation, also referred to herein as "reactivation", may drive the restoration of dedifferentiated cells to T lineage cells. Notably, the reactivated T cells have the characteristics of analyzing much younger cells based on an epigenetic clock, have a phenotype more like stem cells and acquire high proliferation potential.

As used herein, "transient" means a period of time sufficient to effect partial reprogramming of one or more T cells, (e.g., a period of time sufficient to cause adherent cells of T cell origin to form, but insufficient to convert the one or more T cells to iPS cells or totipotent cells).

For example, "transiently contacting" with one or more reprogramming factors means contacting with one or more reprogramming factors for a period of time sufficient to form adherent cells of T cell origin or to cause the one or more T cells to form at least one colony attached to the surface of a culture vessel, but not for a period of time sufficient to allow the one or more T cells to convert to iPS cells or totipotent cells. In various embodiments, "instantaneous contact" may mean a period of contact of up to 1 day, or up to 2 days, or up to 3 days, or up to 4 days, or up to 5 days, or up to 6 days, or up to 7 days, or up to 8 days, or up to 9 days, or up to 10 days, or up to 11 days, or up to 12 days, or up to 13 days, or up to 14 days.

For example, "transiently expressing" or the like means expressing or causing expression of one or more Yamanaka factors in order to effect partial reprogramming of one or more T cells (i.e., wherein transformation into iPS cells or totipotent cells is not effected).

As used herein, "epigenetic age" (or "eAge") means the cell age determined using a known epigenetic clock, such as a Horvath clock, a Hannum clock, or a Levine clock (see, e.g., horvath et al, 2018,Aging 10,1758-1775; hannum et al, 2013, mol. Cell.49 (2), 359-367; levine et al, 2018, acting, 10 (4): 573-592). In certain embodiments, eAge is the cell age determined using the Horvath clock method that measures 353CpG methylation status (Horvath and Raj,2018Nature Reviews Genetics,19:371-385). These results indicate that the partial reprogramming and reactivation methods described herein generate regenerative T cells without going through iPS cell stages, which are both time consuming and require complex differentiation steps. Moreover, T cells differentiated from iPS cells in the absence of autologous thymus education lack true T cell function and have abnormalities including immature phenotypes, non-MHC dependent killing, inappropriate cd8αβ dimerization, deregulation of gene expression and inability to produce a developmentally homogeneous T cell population. Vizcardo et al 2018,Cell Rep 22,3175-3190. Thus, regenerative T cells generated using the partial reprogramming methods described herein provide unexpected and advantageous characteristics over T cells generated using methods known in the art.

I. Method for generating regenerative T cells

Non-limiting exemplary embodiments of the invention are described below. Other aspects of the compositions and methods of the present invention will become apparent to those of ordinary skill in the art based on the disclosure provided herein.

In various embodiments, the disclosure relates to a method of producing regenerative T cells. In various embodiments, the method comprises (i) transiently contacting a population of T cells with at least one reprogramming factor for a time sufficient to effect partial reprogramming, and wherein the T cells are not transformed into iPS cells; and (ii) contacting the partially reprogrammed T cells with at least one T cell activating compound or activator and optionally one or more immunomodulators.

In various embodiments, the disclosure relates to a method of producing regenerative T cells. In various embodiments, the method comprises (i) contacting a population of T cells with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, NANOG, LIN28, L-MYC, and C-MYC; and optionally SV40 for a time sufficient to effect partial reprogramming, wherein the T cells are not converted to iPS cells; and (ii) contacting the partially reprogrammed T cells with at least one T cell activating compound or activator and optionally one or more immunomodulators.

In various embodiments, the disclosure relates to a method of producing regenerative T cells. In various embodiments, the method comprises (i) contacting a population of T cells with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, NANOG, LIN28, L-MYC, and C-MYC; and optionally SV40 for a time sufficient to form T cell-derived adherent cells and wherein the T cells are not converted to iPS cells; and (ii) contacting the T cell-derived adherent cells with at least one T cell activating compound or activator and/or T cell co-stimulatory agent and optionally one or more immunomodulators.

As used herein, "reprogramming" refers to the process of erasing and/or reconstructing epigenetic modifications obtained during mammalian cell development or in cell culture. For example, muscle cells can be reprogrammed to neurons. Reprogramming is not inherently linked to aging and regeneration. (see, e.g., takahashi et al, cell (2007) 131, 861-872)

"complete reprogramming" refers to reprogramming of somatic cells Into Pluripotent Stem (iPS) cells or totipotent stem cells.

"partial reprogramming" refers to a reprogramming process that does not reach a totipotent stem cell state or a pluripotent stem cell state (iPS cells). Thus, a partial preprogram is any reprogramming that is not a complete reprogramming. A "partial" or "incomplete" or "transient" reprogramming is a reprogramming that is not completely reprogrammed, for example, as compared to a cell that has been completely reprogrammed to an iPS cell. In certain embodiments, the partial reprogramming is a reprogramming performed for about 1 to about 20 days. In certain embodiments, the partial reprogramming is a reprogramming of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about 25 days.

In certain embodiments, partial T cell reprogramming is a reprogramming process that proceeds until T cell reactivation is initiated. In another embodiment, partial T cell reprogramming is a reprogramming process that proceeds at least until T cells begin to lose expression of one or more T cell markers and/or begin to express markers associated with non-T cell lineages. In certain embodiments, partial T cell reprogramming is a reprogramming process that occurs for a period of time until T cells can no longer be restored to CD3, CD4, and/or CD8 expressing cells by reactivation with a T cell activator.

"partially reprogrammed T cells" refers to T cells that have been reprogrammed without reaching a totipotent or pluripotent stem cell state (iPS cells). In another embodiment, the partial reprogramming of T cells is incomplete and/or partial and/or transient reprogramming compared to iPS cells. Partially reprogrammed T cells refer to T cells that have been reprogrammed by contacting the T cells with one or more reprogramming factors for a period of time such that the T cells form adherent cells of T cell origin. In certain embodiments, the adherent cells of T cell origin are loosely attached to the surface of the culture vessel. Adherent cells of T cell origin as described herein maintain lineage stability, e.g., revert to T cell lineage upon stimulation (reactivation) with a T cell activator and optionally an immunomodulatory molecule (e.g., a cytokine). In certain embodiments, the T cell-derived adherent cells are semi-adherent, loosely adherent, or strongly adherent (strongly adherent).

T cell origin and isolation

In some embodiments, cells useful in the partial reprogramming methods described herein are derived from primary cells, such as primary human cells. Samples include tissues, fluids, and other samples taken directly from a subject, as well as samples resulting from one or more processing steps, such as isolation, centrifugation, genetic engineering (e.g., transduction with viral vectors), washing, and/or incubation. The biological sample may be a sample obtained directly from a biological source or a treated sample. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebral spinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including treated samples derived therefrom.

In some aspects, the sample from which the cells are derived or from which the cells are isolated is blood or a blood-derived sample, or is derived from an apheresis or leukopenia product. Exemplary samples include whole blood, peripheral Blood Mononuclear Cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsies, tumors, leukemias, lymphomas, lymph nodes, intestinal-related lymphoid tissue, mucosa-related lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsils, or other organs, and/or cells derived therefrom. In the case of cell therapies such as adoptive cell therapies, the samples include samples from autologous and allogeneic sources.

In some embodiments, the cells used in the partial reprogramming method are derived from a cell line, such as a T cell line. In some embodiments the cells are obtained from a heterologous source, e.g., from mice, rats, non-human primates, and pigs.

In various embodiments, the disclosure relates to isolating T cells from a source and partially reprogramming the T cells to improve aging and enhance T cell function. Examples of suitable source cells include, but are not limited to, peripheral Blood Mononuclear Cells (PBMCs). T cells for use in the methods herein may include, but are not limited to, cultured T cells, such as primary T cells or T cells from a cultured T cell line (e.g., jurkat, supT1, etc.), or T cells obtained from a mammal. If obtained from a mammal, the source cells may be obtained from a number of sources including, but not limited to, blood, bone marrow, lymph nodes, tumors, thymus, spleen, or other tissues or fluids. The source cells may also be enriched or purified. T cells may be any type of T cell and may be at any stage of development, including but not limited to cd4+cd8αβ+ double positive T cells, cd4+ helper T cells (e.g., th1 and Th2 cells), cd4+ T cells, cd8+ T cells (e.g., cytotoxic T cells), peripheral Blood Mononuclear Cells (PBMCs), peripheral Blood Leukocytes (PBLs), tumor infiltrating cells (TILs), memory T cells, naive T cells, and the like.

In various embodiments, the T cells are isolated from a tumor, and in particular the T cells used in the partial reprogramming methods described herein are TILs. As used herein, "tumor-infiltrating lymphocytes" or "TILs" means a population of cells that is initially obtained as cells that leave the subject's blood stream and migrate into a tumor. TILs include, but are not limited to, cd8+ cytotoxic T cells (lymphocytes), th1 and Th17 cd4+ T cells, natural killer cells, dendritic cells, and M1 macrophages. TIL includes primary and secondary TIL. "primary TILs" are those obtained from patient tissue samples as outlined herein (sometimes referred to as "freshly collected"). In some embodiments, TIL may be classified by expressing one or more of the following biomarkers: CD4, CD8, tcrαβ, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1 and CD25.

In various embodiments of the present disclosure, the source cells may have an initial T cell ((TN) phenotype, a central memory T Cell (TCM) phenotype, or an effector memory T cell (TEM) phenotype, a stem-like T cell (Tscm). The phenotypes of TN, TCM, and TEM cells are known in the art and described elsewhere herein.

Specific T cell subsets, such as cd3+, cd45+, cd137+, cd25+, cd28+, cd4+, cd8+, cd45ra+, gitr+ and/or cd45ro+ T cells, may be isolated by positive or negative selection techniques (e.g., using fluorescence-based or magnetic-based cell sorting). For example, T cells can be produced by conjugation to a variety of commercially available antibodies such asCELLectionTM, DETACHaBEADTM (Thermo Fisher) or +.>Any of the cell separation products (Miltenyi Biotec) are incubated together for a period of time sufficient for positive selection of desired T cells or for negative selection to remove unwanted cells.

In various embodiments herein, T cells for the isolation, pre-activation, reactivation or expansion stages are cultured in a suitable medium. T cell culture conditions are known in the art. In certain embodiments, the medium used herein to culture T cells may begin with a starter medium (referred to as a "basal" medium). Basal medium refers to any starting medium supplemented with one or more additional elements disclosed herein, e.g., glucose, one of a variety of salts, one or more cytokines (such as IL-2, IL-7, IL-15, IL-21), and any combination thereof. The basal medium may be any medium used for culturing immune cells such as T cells. In some aspects, the basal medium comprises a balanced salt solution (e.g., PBS, DPBS, HBSS, EBSS), dulbecco's Modified Eagle's Medium (DMEM), click Minimum Essential Medium (MEM), eagle's Basal Medium (BME), F-10, F-12, RPMI 1640, greenwort Minimum Essential Medium (GMEM), alpha minimum essential medium (alpha MEM), ikov's Modified Dalberk's Medium (IMDM), M199, OPTMIZERTM Pro, OPTMIZER ^TM CTS ^TM T cell expansion basal medium (ThermoFisher), OPTMIZERTM, OPTMIZER ^TM Complete medium, immunoculint ^TM XF(STEMCELL ^TM Technologies)、AIM V ^TM ,TEXMACS ^TM A culture medium,T cell CDM, X-VIVOTM 15 (Lonza), TRANSACT ^TM TIL amplification medium or any combination thereof. In some aspects, the basal medium is serum-free. In some aspects, the basal medium comprises +.>T cell CDM. In some aspects, the basal medium comprises OPTMIZERTM. In some aspects, the basal medium comprises OPTMIZERTM Pro. In some aspects, the basal medium further comprises an Immune Cell Serum Replacement (ICSR).

As used herein, the term "cytokine" refers to a small secreted protein released by a cell that has a specific effect on interactions and communication between cells. Non-limiting examples of cytokines include interleukins (e.g., interleukin (IL) -1, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, IL-6, IL-11, IL-10, IL-20, IL-14, IL-16, IL-17, IL-21, and IL-23), interferons (IFN; e.g., IFN- α, IFN- β, and IFN- γ), tumor Necrosis Factor (TNF) family members, and Transforming Growth Factor (TGF) family members. Some aspects of the disclosure relate to methods of culturing and/or expanding immune cells (e.g., T cells) or one or more engineered immune cells disclosed herein in a medium comprising cytokines. In some aspects, the cytokine is an interleukin. In some aspects, the cytokine comprises IL-2, IL-7, IL-15, IL-21, or any combination thereof.

In various embodiments of the present disclosure, the T cells used in the partial reprogramming methods herein are TCR αβ cells. In this regard, TCR αβ cells can express functional, antigen-specific T Cell Receptors (TCRs), including alpha (α) chains and beta (β) chains. TCR alpha and beta chains are known in the art. The TCR may comprise any amino acid sequence, provided that the TCR can specifically bind and immunologically recognize an antigen. The TCR may have antigen specificity for any desired antigen. As used herein, the phrases "antigen-specific" and "antigen-specific" mean that the TCR can specifically bind and immunologically recognize an antigen, e.g., a conditionally specific antigen or epitope thereof, such that binding of the TCR to the antigen or epitope thereof elicits an immune response.

In various embodiments, the antigen of the mammalian disorder from which T cells are isolated or the antigen specific for the disorder is immunized. Desirably, the mammal is immunized prior to obtaining T cells from the mammal. Thus, isolated T cells may include T cells that are induced to be specific for the condition to be treated, or include a higher proportion of cells that are specific for the condition.

Alternatively, the T cells comprising the endogenous antigen-specific TCR may be T cells in a mixed cell population isolated from a mammal. In certain embodiments, the mixed population may be exposed to an antigen recognized by an endogenous TCR while in vitro culturing. In this way, T cells comprising TCRs that recognize disease-specific antigens are expanded or proliferated in vitro, thereby increasing the number of T cells with endogenous antigen-specific receptors.

The antigen-specific TCR may be an exogenous TCR, i.e., an antigen-specific TCR that is not native to (not naturally present on) a T cell. Recombinant TCRs are TCRs that have been generated by recombinant expression of one or more exogenous tcra-, β -, γ -and/or δ -chain encoding genes. A recombinant TCR may comprise a polypeptide chain derived entirely from a single mammalian species, or an antigen-specific TCR may be a chimeric or hybrid TCR consisting of amino acid sequences derived from TCRs derived from two different mammalian species. For example, an antigen-specific TCR may comprise a variable region derived from a murine TCR and a constant region of a human TCR, such that the TCR is "humanized". Methods of preparing recombinant TCRs are known in the art. See, for example, U.S. patent nos. 7,820,174, 8,785,601, 8,216,565; and U.S. patent application publication No. 2013/0274203.

T cells comprising endogenous antigen-specific TCRs may also be transformed, e.g., transduced or transfected, with one or more nucleic acids encoding an exogenous (e.g., recombinant) TCR or other recombinant chimeric receptor. Such exogenous chimeric receptors, e.g., chimeric CR, may confer specificity to the transformed T cell for additional antigens in addition to the antigen for which the endogenous TCR has native specificity. This may, but need not, result in the production of T cells with dual antigen specificity.

One or more nucleic acids encoding a "chimeric antigen receptor" (CAR) can also be transformed, e.g., transduced or transfected, T cells comprising an endogenous antigen-specific TCR. Typically, the CAR comprises an antigen binding domain of an antibody, e.g., a single chain variable fragment (scFv), fused to the transmembrane and intracellular domains of the TCR; and in some embodiments, one or more costimulatory domains, such as the intracellular signaling region of a T cell costimulatory molecule. Thus, the antigen specificity of a TCR may be encoded by an scFv that specifically binds an antigen or epitope thereof. Methods of making such CARs are known in the art. See, for example, U.S. patent nos. 8,465,743, 10,533,055, 10,603,380; and U.S. patent application publication Nos. 2014/0037628 and 2014/0274909.

Any suitable nucleic acid encoding a CAR, engineered TCR, or TCR-like protein or polypeptide can be used. In these embodiments, the partial reprogramming process as discussed further below may occur before, after, or simultaneously with genetic modification of T cells with CARs, engineered TCRs, or TCR-like proteins or polypeptides. The CAR or engineered TCR encoded by the nucleic acid can be in any suitable form, including, for example, a single chain CAR or TCR or a fusion with other proteins or polypeptides (e.g., without limitation, a co-stimulatory molecule).

The antigen recognized by the T cells herein, whether by an endogenous antigen-specific TCR, an engineered TCR, or a CAR, can be any antigen associated with the disorder. For example, the antigen may be, but is not limited to, a cancer antigen (also referred to as a tumor antigen or tumor-associated antigen) or an infectious disease antigen (e.g., a viral antigen).

In some embodiments of the present invention, in some embodiments, the antigen is or is derived from CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B H3, KIT, IL-13Ra2, mesothelin, IL-lRa, PSCA, PRSS21, VEGFR2, lewis Y, CD24, PDGFR- β, SSEA-4, CD20, folic acid receptor α, ERBB2 (Her 2/neu), MUC1, MUC16, EGFR, NCAM prostase (proase), PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, ephA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF, CD97, CD179a, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR E2, TARP, WTl, NY-ESO-1, LAGE-la, MAGE-Al, legun, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, fos-related antigen 1, p53 mutant, prostein, survivin (survivin), telomerase, PCTA-1/galectin 8, melanA/MARTl, ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS 2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, rhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, enterocarboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3 epsilon, CD4, CD5, CD7, the extracellular portion of an APRIL protein, or any combination thereof.

In some embodiments of the present invention, in some embodiments, the CAR or engineered TCR targets AFP, CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B H3, KIT, IL-13Ra2, mesothelin, IL-lRa, PSCA, PRSS21, VEGFR2, lewis Y, CD24, PDGFR- β, SSEA-4, CD20, folic acid receptor α, ERBB2 (Her 2/neu), MUC1, MUC16, EGFR, NCAM prostase (proase), PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, ephA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF, CD97, CD179a, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR E2, TARP, WTl, NY-ESO-1, LAGE-la, MAGE-Al, legun, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, fos-related antigen 1, p53 mutant, prostein, survivin (survivin), telomerase, PCTA-1/galectin 8, melanA/MARTl, ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS 2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, rhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, enterocarboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3 epsilon, CD4, CD5, CD7, the extracellular portion of an APRIL protein, or any combination thereof.

As used herein, the term "cancer antigen" or "tumor-associated antigen" refers to any molecule (e.g., protein, polypeptide, peptide, lipid, carbohydrate, etc.) that is expressed or overexpressed by a tumor cell or cancer cell, alone or predominantly, such that the antigen is associated with a tumor or cancer. The cancer antigen may be, for example, a mutated tumor antigen or a neoantigen. The cancer antigen may additionally be expressed by normal cells, non-tumor cells, or non-cancerous cells. However, in such cases, the expression of the cancer antigen by normal, non-tumor or non-cancer cells is less robust than the expression by tumor or cancer cells. In this regard, tumor cells or cancer cells can overexpress an antigen or express an antigen at significantly higher levels than normal cells, non-tumor cells, or antigen expression by non-cancer cells. Furthermore, cancer antigens may additionally be expressed by cells of different developmental or maturation states. For example, cancer antigens may additionally be expressed by embryonic or fetal cells that are not normally present in adult mammals. Alternatively, the cancer antigen may additionally be expressed by stem cells or precursor cells, which are not normally present in adult mammals. Cancer antigens are known in the art and include, for example, the cancer antigens listed above, consensus tumor antigens such as mesothelin, CD19, CD22, CD276 (B7H 3), gp100, MART-1, epidermal growth factor receptor variant III (EGFRVIII), TRP-1, KRAS, TRP-2, tyrosinase, NY-ESO-1 (also known as CAG-3), MAGE-1, MAGE-3, and the like. In one embodiment of the present disclosure, the cancer antigen is a patient-specific neoantigen. Patient-specific neoantigens may be generated as a result of tumor-specific mutations.

The cancer antigen may be an antigen expressed by any cell of any cancer or tumor, including cancers and tumors described herein. The cancer antigen may be a cancer antigen of only one type of cancer or tumor, such that the cancer antigen is associated with or characteristic of only one type of cancer or tumor. Alternatively, the cancer antigen may be (e.g., may be characteristic of) a cancer antigen of more than one type of cancer or tumor. For example, the cancer antigen may be expressed by breast cancer and prostate cancer cells, and not by normal cells, non-tumor cells, or non-cancerous cells at all.

The disorder associated with or characterized by the antigen recognized by the TCR or CAR may be any disorder. For example, the disorder may be a cancer or an infectious trait, such as a viral trait, as discussed herein.

The cancer may be any cancer, including any of the following: sarcomas, carcinomas, acute lymphocytic carcinoma, acute myelogenous leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal canal cancer or anal rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gall bladder cancer or pleural cancer, head and neck cancer (e.g., nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer), vulval cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, hodgkin's lymphoma, hypopharynx cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omentum cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer and bladder cancer.

For purposes herein, "viral disorder" means a disorder that can be transmitted from person to person or between organisms and is caused by a virus. In one embodiment of the present disclosure, the viral disorder is caused by a virus selected from the group consisting of herpes virus, poxvirus, hepadnavirus (papilloma virus), adenovirus, coronavirus, orthomyxovirus, paramyxovirus, flavivirus and calicivirus. For example, the viral disorder may be caused by a virus selected from the group consisting of: respiratory Syncytial Virus (RSV), influenza virus, herpes simplex virus, epstein-Barr virus, varicella virus, cytomegalovirus, hepatitis a virus, hepatitis b virus, hepatitis c virus, human Immunodeficiency Virus (HIV), human T-lymphotropic virus, calicivirus, adenovirus and arenavirus.

Viral disorders may be, for example, influenza, pneumonia, herpes, hepatitis a, hepatitis b, hepatitis c, chronic fatigue syndrome, sudden Acute Respiratory Syndrome (SARS), gastroenteritis, enteritis, cardiotis, encephalitis, bronchiolitis, respiratory papillomatosis, meningitis, HIV/AIDS, and mononucleosis.

T cells for use in the partial reprogramming methods of the present disclosure may be isolated or purified from sources using any suitable technique known in the art. For example, immune cells (e.g., T cells) can be obtained from a subject. Immune cells (e.g., T cells) can be obtained from a number of sources, including Peripheral Blood Mononuclear Cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, immune cells (e.g., T cells) can use a number of techniques known to those skilled in the art, such as FICOLL ^TM Isolation, obtained from blood units collected from the subject. Cells may preferably be obtained from circulating blood of an individual by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, cells collected by apheresis may be washed to remove plasma fractions and placed in an appropriate buffer or medium for subsequent processing. Cells can be washed with PBS. It will be appreciated that a washing step may be used, such as by using a semi-automatic flow-through centrifuge, e.g., cobe ^TM 2991 cell processor and Baxter Cyto-Mate ^TM Etc. After washing, the cells may be resuspended in various biocompatible buffers or other saline solutions with or without buffers. In certain embodiments, unwanted components in the apheresis sample may be removed.

Partial reprogramming of T cells

The present disclosure relates in part to methods for partially reprogramming T cells. In certain embodiments, T cells are partially reprogrammed by: contacting T cells with one or more reprogramming factors for a number of days such that one or more T cells form colonies (forming T cell-derived adherent cell colonies) that adhere (or partially adhere) to the surface of the culture vessel, wherein such adherent cells recover to functional T cells upon stimulation (reactivation) with a T cell activator and optionally an immunomodulatory molecule (e.g., a cytokine)

As used herein, the term "reprogramming factor" refers to any protein, polypeptide, amino acid, mRNA, DNA, or small molecule capable of erasing and/or reconstructing epigenetic modifications obtained during mammalian cell development or in cell culture. Reprogramming factors can alter the differentiation state of cells. Such reprogramming factors may include, but are not limited to, transcription factors found by Yamanaka and its colleagues, OCT4 (or OCT 3/4), SOX3, KLF4, and C-MYC (see, e.g., takahashi and Yamanaka,2006, cell,136, 364-377), referred to herein as "OSKM" or "Yamanaka factor". Reprogramming factors refer to other factors that may alter the state of cell differentiation, or may enhance or alter the efficiency of cell reprogramming. Such factors are known in the art. Feng et al 2009,Cell Stem Cell Review,4:301-313 describe exemplary reprogramming factors.

In various embodiments, the reprogramming factors are at least one of the group consisting of KLF4, OCT3/4, SOX2, C-MYC, and SV 40. In various embodiments, not all four Yamanaka factors are strictly necessary (see, e.g., genome biol.2012;13 (10): 251;Cell Stem Cell,2019,25 (6): 737-753;Nature Communications (2018) 9:2865). In various embodiments, isolated immune cells (e.g., T cells) are contacted with OCT3/4 and SOX 2. In various embodiments, isolated immune cells (e.g., T cells) are contacted with OCT3/4, SOX2, and C-MYC. In various embodiments, the isolated cells are contacted with OCT3/4, SOX2, and KLF 4. In certain embodiments, T cells are contacted with OCT3/4, SOX2 and KLF4, and C-MYC or SV 40.

In various embodiments, the isolated immune cells (e.g., T cells) may be partially reprogrammed using additional reprogramming factors. Such factors include, but are not limited to, LIN28, NANOG, esrrb, pax shRNA, C/EBPa, p53 siRNA, UTF1, DNMT shRNA, wnt3a, GLIS1, DLX4, CDH1, SV40 LT (T), and hTERT. In various embodiments, the reprogramming factors may up-regulate or down-regulate certain mirnas involved in reprogramming. In certain embodiments, the reprogramming factors may up-regulate or down-regulate the expression of one or more genes upstream or downstream of one or more Yamanaka factors. In various embodiments, the one or more reprogramming factors may include, but are not limited to, a histone methyltransferase inhibitor, an L-type calcium channel agonist, a G9a methyltransferase inhibitor, a DNA methyltransferase inhibitor, a histone deacetylase inhibitor, a MEK inhibitor, a GSK3 inhibitor, or a TGF-B inhibitor. Any factor that modulates the molecular pathway upstream or downstream of the reprogramming transcription factor is contemplated for use in the partial reprogramming methods herein. Exemplary inhibitors include, but are not limited to, BIX 0194, bayK 8644, CHIR 99021, forskolin (Forskolin), and RepSox. In certain embodiments, serine/threonine protein kinase B-Raf (BRAF) inhibitors, epidermal Growth Factor Receptor (EGFR) inhibitors, vascular endothelial growth factor 1 (VEGFR 1) inhibitors, and/or fibroblast growth factor receptor 1 (FGFR 1) inhibitors are useful in the reprogramming methods herein (see, e.g., US 2017114323).

In various embodiments, the present disclosure relates to a method of producing regenerative T cells, wherein the T cells are transiently contacted with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC. In various embodiments of the present disclosure, the isolated immune cells (e.g., T cells) are transiently contacted with KLF4, OCT3/4, SOX2, and C-MYC, and optionally SV 40.

There are many strategies known in the art for delivering reprogramming factors to cells. These strategies include the use of single stranded negative sense RNA viruses (Vizcardo et al, 2013), such as the episomal plasmid system as described in Nat. Methods 2011,8 (5): 409-412, transfection of mRNA by nanoparticles (Moffett et al, 2017), and the use of small molecules that are known to induce gene expression of factors that confer pluripotency (Feng et al, 2009; hou et al, 2013).

In various embodiments, one or more reprogramming factors are transiently expressed in an isolated immune cell (e.g., T cell). In various embodiments, the one or more reprogramming factors may include one or more or all of KLF4, OCT3/4, SOX2, and C-MYC; or may additionally include one or more other reprogramming factors. In various embodiments, the reprogramming factors may be expressed in isolated T cells using gene editing techniques known in the art (e.g., TALENS, CRISPR/cas). In various embodiments, the gene editing technique delivers a set of inducible reprogramming factors for expression in isolated T cells. In various embodiments, the nucleic acid encoding a stem cell-associated gene is carried in one or more recombinant expression vectors. Recombinant expression vectors may comprise any type of nucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, synthetic or partially obtained from natural sources, and may contain natural, non-natural or altered nucleotides. Recombinant expression vectors can comprise naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. The vector may contain regulatory nucleic acid sequences that provide for expression of stem cell-related genes.

In some embodiments, the recombinant expression vector is a viral vector. Suitable viral vectors include, but are not limited to, sendai viral vectors, retroviral vectors, lentiviral vectors, alphaviral vectors, vaccinia viral vectors, adenovirus vectors, adeno-associated viral vectors, herpesviral vectors, and avipoxviral vectors, and preferably have the ability to transform immune cells (e.g., T cells), either naturally or engineered. In certain embodiments, the viral vector is pseudotyped with a heterologous viral envelope protein. In certain embodiments, the viral vector is a lentiviral or sendai viral vector pseudotyped with a suitable envelope (e.g., viral envelope) (see, e.g., blood Adv 2017, month 10, 24; 1 (23): 2088-2104;Mol Ther 2012, month 9; 20 (9): 1699-712). In certain embodiments, the viral vector is an integration-defective lentiviral vector. Suitable viral vectors are known in the art and are described, for example, in J.biol.chem. (2011) 286:4760-4771; stem Cell Research & Therapy (2019) 10:185.

In various embodiments, the recombinant expression vector is delivered in a nanoparticle. In various embodiments, the reprogramming factors are delivered using nanoparticles. Such nanoparticles can be designed to deliver specific mRNA or other macromolecules into lymphocytes in a transient and dose-controlled manner. These nanoparticles can be designed to target specific cell subtypes and upon binding to them stimulate receptor-mediated endocytosis, thereby introducing the synthetic mRNAs they carry, which can now be expressed by the cells. This process is fast and efficient because nuclear transport and transcription of transgenes are not required.

In various embodiments, one or more reprogramming vectors are delivered to immune cells (e.g., T cells) as separate vectors. In various embodiments, KLF4, OCT3/4, SOX2 (KOS) are delivered in a single vector. In various embodiments, the vector expressing the KOS is a viral vector and is delivered at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30MOI (multiplicity of infection). In various embodiments, KLF4 is delivered in a viral vector at a titer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 MOI. In various embodiments, cMyc is delivered in a viral vector at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 MOI. In various embodiments, the vector expressing SV40 is further delivered to the cell and is a viral vector delivered at a titer of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 MOI. In various embodiments, immune cells (e.g., T cells) are contacted with a sendai vector expressing KOS at 10MOI, a sendai vector expressing KLF4 at 10MOI, a sendai vector expressing C-MYC at 3MOI, and a sendai vector expressing SV40 at 5 MOI. In certain embodiments, the T cells are contacted with a polycistronic vector, such as a lentiviral vector, a sendai viral vector, or a non-viral vector, that expresses KOSM.

In certain embodiments, the T cells are stimulated with a T cell activator and optionally one or more immunomodulatory molecules for a time sufficient to stimulate and activate the T cells prior to contact with the one or more reprogramming factors. In certain embodiments, the T cells are stimulated with a tumor antigen or with autologous tumor cells or tumor cell lines. In some embodiments, the T cell is a TIL and the TIL is stimulated by tumor target cells from a cancer patient or tumor antigens derived therefrom, or one or more tumor-associated antigens.

Non-limiting examples of tumor-associated antigens include: AFP (alpha fetoprotein), αvβ6 or another integrin, BCMA, BRAF, B-H3, B7-H6, CA9 (carbonic anhydrase 9), (carcinoembryonic antigen), seal protein 18.2, seal protein 6, C-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (exonucleotide pyrophosphatase/phosphodiesterase family member 3), epCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, ephrin B2, EPHa2 (liver calpain receptor A2), ERBB dimer, estrogen receptor, ETBR (endothelin B receptor), FAP- α (fibroblast activation protein α), fetal achR (fetal acetylcholine receptor), FBP (folic acid binding protein), FCRL5, FR- α (folic acid receptor α), GCC (guanylate cyclase C), GD2, GD3, GPC2 (phosphatidylinositol proteoglycan 2), GPC3, gp100 (glycoprotein 100), NMB (glycoprotein NMB), RC5D (glycoprotein), HER2, HER3, HER4, HER 1, MAGE 1-related to human hepatitis B, MAR 1, MAR-1, human tumor antigen, HLA-A complexed with peptides derived from AFP, KRAS, NY-ESO, MAGE-A and WT 1), NCAM (neural cell adhesion molecule), nectin-4, NY-ESO, cancer embryo antigen, PRAME (melanoma preferential expression antigen), progesterone receptor, PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate specific membrane antigen), ROR1, ROR2, sirpa (signal regulatory protein α), SLIT, SLITRK6 (NTRK-like protein 6), survivin, TAG72 (tumor associated glycoprotein 72), TPBG (trophoblast glycoprotein), trop-2, VEGFR1 (vascular endothelial growth factor receptor 1), p53 mutant, prostaglandin (prostein), survivin, telomerase, PCTA-1/galectin 8, melanA/MARTl, ras mutant hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS 2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, rhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, enterocarboxyesterase, muthsp 70-2, CD79a, CD79B, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3 epsilon, CD4, CD5, CD7, APRIL protein extracellular portion, neoantigen, or any combination thereof.

In this way, polyclonal antigen-specific (e.g., tumor-specific) T cells from a cancer patient can be partially reprogrammed and rejuvenated. In certain embodiments, T cells are selected, sorted, or otherwise enriched after stimulation with an antigen, such as a tumor antigen, tumor organoid, autologous tumor cells, or tumor cell line. In certain embodiments, T cells are enriched, selected, or sorted by selecting cells that express one or more cell surface markers, such as 4-1BB (CD 137), PD1, LAG3, CD45, CD39, TIGIT, TIM3, CD69, OX40, CD28, CD25, CD49d, and CTLA4. In certain embodiments, the T cells are enriched, selected, or sorted prior to activation with any of the T cell activators described herein. In certain embodiments, the cd45+ T cells are selected or enriched prior to activation with any of the activators described herein.

In some embodiments, the method comprises activating the cells before, during, and/or after incubating the cells with the viral vector particles. In some embodiments, the stimulation conditions may include incubating the cells in the presence of an agent capable of activating one or more intracellular signaling domains of one or more components of the TCR complex, such as a primary agent that specifically binds one member of the TCR complex (e.g., CD 3), and a secondary agent that specifically binds a T cell costimulatory molecule, e.g., CD28, CD27, CD137 (4-1 BB), OX40, or ICOS, including antibodies such as those present on the surface of a solid support such as a bead (e.g., tranact, miltenyi). Without being bound by theory, stimulating T cells in this manner (the pre-activation phase) increases the efficiency of viral transduction and reprogramming factor delivery. Similarly, pre-stimulation of T cells increases the efficiency of gene delivery if nucleic acid transfection is used for delivery of reprogramming factors.

In certain embodiments, T cells are pre-activated, re-activated and/or expanded with one or more T cell activating and/or co-stimulatory and/or immunomodulatory compounds.

In certain embodiments, T cells are contacted with the one or more reprogramming factors for a period of time of about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days, or about 21 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of about 7 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of about 8 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of about 9 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of about 10 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of about 11 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of about 12 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of about 13 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of about 14 days.

In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of at least about 5 days and no more than about 10 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of at least about 5 days and no more than about 11 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of at least about 5 days and no more than about 12 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of at least about 5 days and no more than about 12 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of at least about 5 days and no more than about 13 days.

In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of at least about 6 days and no more than about 10 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of at least about 6 days and no more than about 11 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of at least about 6 days and no more than about 12 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of at least about 6 days and no more than about 12 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of at least about 6 days and no more than about 13 days.

The period of time that the T cells are contacted with the one or more reprogramming factors may vary depending on the amount or level of expression of the reprogramming factors. For example, in embodiments where T cells are transduced with viral vectors that express high levels of one or more reprogramming factors, less time may be required to achieve the desired partial reprogramming (forming adherent cells from which the T cells are derived). In another embodiment, T cells are transduced with viral vectors that express low levels of one or more reprogramming factors, and may take longer to achieve the desired partial reprogramming (forming adherent cells of T cell origin). In this regard, in certain embodiments, an expression vector encoding the one or more reprogramming factors, such as a viral vector, may comprise an inducible promoter, wherein expression of the partial reprogramming factors may be modulated to a desired expression level.

In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of about 5 to 30 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of about 5 to 25 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of about 5 to 20 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of about 5 to 15 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of about 5 to 13 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of about 5 to 12 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of about 6 to 12 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of about 6 to 13 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of about 7-12 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of about 7-13 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of about 7-14 days. In certain embodiments, the T cells are contacted with the one or more reprogramming factors for a period of time of about 6-20, 7-20, 8-20, 9-20, 10-20, 5-18, 6-18, 7-18, 8-18, 9-18, or 10-18 days.

During reprogramming of somatic cells into totipotent or pluripotent stem cells (TSCs or PSCs), somatic cells will begin to dedifferentiate and lose their lineage-specific epigenetic status, while they also begin to acquire a PSC phenotype. These changes proceed gradually, so the longer the somatic cells remain in the reprogramming process, the more PSC phenotypes they acquire. In various embodiments of the present disclosure, the reprogramming process is stopped before the somatic cells dedifferentiate so much to prevent them from losing their source cell function. In various embodiments, the partial reprogramming will be sufficient to cause immune cells (e.g., T cells) to form at least one cell that attaches to the surface of the culture vessel and convert to an intermediate cell type referred to herein as "T cell-derived adherent cells.

As used herein, "T cell-derived adherent cells" refers to intermediate cells in the process of T cell partial reprogramming. Adherent cells of T cell origin attach (at least in part) to the surface of the culture vessel and form colonies during partial reprogramming. In certain embodiments, the adherent cells of T cell origin are loosely attached to the surface of the culture vessel. T cell-derived adherent cells differ from iPS cells (and ittsc) in that they have not yet been reprogrammed. In certain embodiments, the T cell-derived adherent cell colonies are loosely attached to the surface of the culture vessel. T cell-derived adherent cells as described herein maintain lineage stability, e.g., the ability of T cell-derived adherent cells of the present disclosure to recover to the T cell lineage after culturing under T cell activation/stimulation conditions (e.g., with T cell culture media in the presence of activating and/or immunoregulatory molecules such as anti-CD 3, anti-CD 28, anti-CD 27 antibodies, and cytokines such as IL2, IL7, IL15, etc.). In certain embodiments, the adherent cells of T cell origin are not pluripotent. In certain embodiments, the adherent cells of T cell origin are larger than the non-partially reprogrammed T cells or activated T cells. In certain embodiments, the T cell-derived adherent cells are larger and have a more complex structure, as measured by FACS analysis. In certain embodiments, the adherent cells from which the T cells are derived are ssea4+cd3+. In another embodiment, the T cell-derived adherent cells express one or more of the following cell surface markers as determined using flow cytometry or transcriptome analysis: CD50, CD352, CD31, integrin beta 7, CD49e, CD122, CD314, HLA-DR, CD134, CD245, CD105 (endoglin), CD366, CD39, integrin alpha 6 beta 1, integrin beta 5, CD71, CD164, CD10, CD63, XCR1, CD298, CD201, CD151, CD325, CD324, CD147, SSEA-3, TRA-1-81, TRA-2-54, pontoplanin, CD9, CD340, CD24, CD90, CD326, SSEA-5, SSEA4, TRA-1-60-R.

As one of ordinary skill in the art readily appreciates, iPS cells can be differentiated from T cell-derived adherent cells by morphology. Specifically, iPS cell colonies are much larger and have a more defined colony structure, as shown in fig. 3F. iPS cells are well known in the art and are, for example, nishimura 2013,Cell Stem Cell 12,114-126; vizcardo et al 2013,Cell Stem Cell 12,31-36).

In certain embodiments, at least 10% of the adherent cells of T cell origin express one or more of the following cell surface markers as determined using flow cytometry or transcriptome analysis: CD50, CD352, CD31, integrin alpha 6 beta 1, integrin beta 7, CD49e, CD122, CD314, HLA-DR, CD134, CD245, CD105 (endoglin), CD366, CD39, integrin beta 5, CD71, CD164, CD10, CD63, XCR1, CD298, CD201, CD151, CD325, CD324, CD147, SSEA-3, TRA-1-81, TRA-2-54, pontoplanin, CD9, CD340, CD24, CD90, CD326, SSEA-5, SSEA4, TRA-1-60-R.

In certain embodiments, at least about 10% to about 80% of the adherent cells of T cell origin express one or more cell surface markers of the following as determined using flow cytometry or transcriptome analysis: CD50, CD352, CD31, integrin alpha 6 beta 1, integrin beta 7, CD49e, CD122, CD314, HLA-DR, CD134, CD245, CD105 (endoglin), CD366, CD39, integrin beta 5, CD71, CD164, CD10, CD63, XCR1, CD298, CD201, CD151, CD325, CD324, CD147, SSEA-3, TRA-1-81, TRA-2-54, pontoplanin, CD9, CD340, CD24, CD90, CD326, SSEA-5, SSEA4, TRA-1-60-R. In another embodiment, at least about 15% to about 80%, at least about 20% to about 75%, at least about 25% to about 75%, at least about 30% to about 50%, at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75% or about 80% of the adherent cells of T cell origin express one or more of the above cell surface markers as determined using assays known in the art, such as flow cytometry, gene expression or transcriptome analysis. The expression levels of these markers and% of positive cells will vary depending on the number of days of partial reprogramming.

In certain embodiments, partial reprogramming may result in partial loss of certain markers characteristic of immune cells (e.g., T cells). For example, in certain embodiments, partial reprogramming of T cells may result in loss or reduction of expression of any one or more of CD3, CD4, CD8 (e.g., CD8 alpha and/or CD8 alpha beta). In certain embodiments, reduced expression may mean reduced expression of the marker by about 10% to about 95% as measured by flow cytometry. In certain embodiments, the reduced expression is a reduction in the Mean Fluorescence Intensity (MFI) of at least a portion of the T cell-derived adherent cells by about 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, or 2log.

In certain embodiments, the T cells are transiently contacted with one or more reprogramming factors for a time sufficient to form adherent cells of T cell origin. In certain embodiments, the T cells are transiently contacted with one or more reprogramming factors for a time sufficient to form an adherent cell colony, wherein at least a portion of the adherent cells express one or more of the following cell surface markers: CD50, CD352, CD31, integrin alpha 6 beta 1, integrin beta 7, CD49e, CD122, CD314, HLA-DR, CD134, CD245, CD105 (endoglin), CD366, CD39, integrin beta 5, CD71, CD164, CD10, CD63, XCR1, CD298, CD201, CD151, CD325, CD324, CD147, SSEA-3, TRA-1-81, TRA-2-54, pontoplanin, CD9, CD340, CD24, CD90, CD326, SSEA-5, SSEA4, TRA-1-60-R. Expression of such markers may be determined using flow cytometry, gene expression analysis, RNAseq, or other techniques known in the art.

In certain embodiments, the T cells are transiently contacted with one or more reprogramming factors for a time sufficient to form a colony of adherent cells, wherein at least a portion of the adherent cells express integrin α6β1, CD9, CD90, and/or SSEA4.

In certain embodiments, at least a portion of the T cell adherent cells express the integrin α6β1, CD9, CD90, or SSEA4, or any combination thereof. In certain embodiments, at least a portion of the T cell adherent cells express integrin α6β1; at least a portion of the T cell adherent cells express integrins α6β1 and SSEA4, and at least a portion of the T cell adherent cells express integrins α6β1, SSEA4 and CD9; or at least a portion of the T cell adherent cells express integrins α6β1, SSEA4, CD9 and CD90. In certain embodiments, at least a portion of the T cell adherent cells express SSEA4; SSEA4 and CD9; or SSEA4, CD9 and CD90.

In certain embodiments, at least a portion of the T cell-derived adherent cells express CD9 and/or CD90, which are early markers of the partial reprogramming process. CD9 is also known as four transmembrane protein-29, having four transmembrane domains. It is involved in cell adhesion, signal transduction and cell differentiation. CD90 is also known as THY-1. It is an immunoglobulin superfamily surface glycoprotein, a marker associated with mesenchymal stem cells. It is also involved in cell adhesion and communication. Although Thy-1 is typically expressed in murine T cells, expression of T cells Thy-1 in the human T cell lineage is limited to the thymus cortex and a specialized population in the CD4 Th 17T cell subset.

In certain embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more of the adherent cells of T cell origin express CD9 and/or CD90.

In certain embodiments, T cell-derived adherent cells express one or more of the integrins α6β1, CD164, CD9, CD63, CD90, CD71, CD326, TRA-1-81, and TRA-1-60-R in certain embodiments as compared to stimulated and unstimulated T cells. In another embodiment, the adherent cells of T cell origin have reduced expression of CD352 and CD31 as compared to stimulated or unstimulated T cells.

In certain embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more of the T cell-derived adherent cells express one or more of CD164, CD9, CD63, CD90, CD71, CD326, TRA-1-81 and TRA-1-60-R.

In certain embodiments, the present disclosure provides an adherent population of T cells that has an epigenetic age that is at least 5% less than the actual age of the relevant control T cells or the starting isolated T cell population. In another embodiment, the present disclosure provides an adherent cell population derived from T cells that has an epigenetic age that is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80% younger than the actual age of the T cells from which they were derived (i.e., the isolated T cells that were contacted with the reprogramming factors).

In certain embodiments, when a T cell-derived adherent cell is contacted with a T cell activator (e.g., anti-CD 3, anti-CD 28, one or more cytokines such as IL2, IL7, IL 15), the cell exhibits increased expansion potential as compared to a control T cell. In certain embodiments, the partially reprogrammed T cells expand at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100, 500, 100, 1500, or 2000 fold over control T cells that have not been partially reprogrammed. In certain embodiments, the partially reprogrammed T cells expand at least about 50-2000 fold, at least about 50-1000 fold, at least 50-900 fold, at least 50-800 fold, at least 50-700 fold, at least 50-600 fold, at least 50-500 fold, at least 100-400 fold, at least 150-500 fold, at least 200-500 fold, or at least 300-500 fold over control T cells that have not been partially reprogrammed. In certain embodiments, the partially reprogrammed T cells expand faster than T cells that have not been partially reprogrammed. In certain embodiments of the present disclosure, the partial reprogramming method is performed under conditions suitable to maintain isolated T cells and/or partially reprogrammed T cells (T cell-derived adherent cells). Conditions suitable for the maintenance and proliferation of a particular cell type will be apparent to the skilled artisan. Specialized media may be obtained from commercial sources or factors necessary or desired to enhance proliferation may be added to standard media. Additional factors and agents may also be added to the medium, for example, to induce expression of the inducible elements in the cells or to inhibit growth of cells that are sensitive to the particular agent.

In certain embodiments, the isolated T cells are cultured in a T cell culture medium (TCM) comprising a commercially available medium optimized for T cell culture, such as TexMACS medium or OpTmizer basal medium, with the addition of one or more supplements, such as OpTmizer cell supplements, in certain embodiments with the addition of immune cell serum substitutes (e.g., T cell serum substitutes) and/or other supplements, such as L-glutamine; glutamine; and/or cytokines such as IL-2, IL-7, and/or IL-15.

In certain embodiments, the partial reprogramming phase of the methods herein is performed using a medium optimized for cell reprogramming, such as, but not limited to, STEMFIT medium (Amsbio, abington, UK), mTESR2 (Stem Cell Technologies), and escential 6 or 8 (Life Technologies); STEMPRO hESC SFM (Gibco); TESR, clone-R, dMEM F12, KSR.

In another embodiment, the partial reprogramming is performed in a medium consisting of 50% T cell medium and 50% stem cell medium (e.g., STEMFIT or Essential 8, mTESR, TESR, clone-R, dMEM F12, KSR), optionally with the addition of one or more cytokines such as IL2, IL7, and/or IL15. In certain embodiments, the partial reprogramming is performed in a first medium for a first period of time and then in a second medium for a second period of time. For example, in certain embodiments, the first medium is a 50/50 medium as described, optionally supplemented with one or more cytokines, for a period of 1, 2, 3, 4, 5, 6 days or more, followed by culturing the cells 1, 2, 3, 4, 5, 6, 7, 8, 9 days or more in a second medium that is a stem cell medium (e.g., STEMFIT or Essential 8). In certain embodiments, the first medium is 50/50 and is used for 1, 2, or 3 days, and the second medium is a stem cell medium (e.g., STEMFIT or Essential 8) and is used from day 2, 3, or 4 until T cell-derived adherent cells are formed and reactivated as further described herein. In certain embodiments of the partial reprogramming method, the T cells are cultured in a first medium for a first period of time and then in a second medium for a second period of time. In certain embodiments of the partial reprogramming phase of the methods herein, T cells contacted with the one or more reprogramming factors (e.g., one or more of KLF4, OCT3/4, SOX2, C-MYC, and SV 40) are cultured in a suitable medium in a culture vessel coated with a recombinant human laminin 511-E8 fragment (such as iMatrix).

3. Reactivation of partially reprogrammed T cell derived adherent cells

In various embodiments, the partially reprogrammed, T cell-derived adherent cells are reactivated in an activation medium that enables the reprogrammed immune cells (e.g., T cells) to regain T cell characteristics and functionality.

In various embodiments of the present disclosure, T cell-derived adherent cells are re-activated immediately after partial reprogramming using one or more T cell stimulators or activators and/or co-stimulators in the appropriate T cell media.

In certain embodiments of the present disclosure, T cell-derived adherent cells are reactivated after partial reprogramming in T cell culture medium supplemented with one or more cytokines such as IL2, IL7, IL15, IL21, with or without one or more T cell activators and/or one or more costimulators.

In certain embodiments, T cell-derived adherent cells are re-activated with one or more T cell activators and/or co-stimulators and/or immunomodulators as described elsewhere herein in an appropriate medium for T cell culture, such as T cell culture medium (TCM), including commercially available media optimized for T cell culture, such as TexMACS medium or OpTmizer basal medium, with the addition of one or more supplements, such as OpTmizer cell supplements, in certain embodiments with immune cell serum substitutes (e.g., T cell serum substitutes) and other supplements, such as L-glutamine; glutaMAX; cytokines such as IL-2, IL7 and/or IL15.

In some embodiments, the stimulus or activation conditions or agents used herein can be used in the pre-activation, reactivation and/or amplification stages of the methods herein. T cells can generally be activated and expanded using methods as described, for example, in the following U.S. patents: 5,858,358; 5,883,223; 6,352,694; 6,534,055; 6,797,514; 6,867,041; 6,692,964; 6,887,466; 6,905,680; 6,905,681; 6,905,874; 7,067,318; 7,144,575; 7,172,869; 7,175,843; 7,232,566; 7,572,631; and 10,786,533. In certain embodiments, the T cells are contacted with a T cell activator and/or costimulatory agent that provides a primary activation signal and an agent that provides a costimulatory signal. Agents that provide a primary activation signal are known in the art and include, for example, antibodies or antigen-binding fragments or ligands thereof that bind to CD3 cell surface receptors expressed on T cells or target-binding fragments thereof (e.g., anti-CD 3 antibodies). Agents that provide co-stimulatory signals are known in the art and include, but are not limited to, antibodies or ligands that bind CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, etc. In certain embodiments, the activator comprises one or more agents, e.g., ligands, capable of activating the intracellular signaling domain of the TCR complex. In some aspects, the agent turns on or initiates a primary TCR/CD3 intracellular signaling cascade in the T cell, such as an agent suitable for delivering a primary signal, e.g., initiating activation of an ITAM-induced signal, e.g., an agent specific for a TCR component. In another embodiment, the activator is provided in combination with or simultaneously with the facilitation of the co-stimulatory signal. Suitable agents that promote the co-stimulatory signal are known in the art and include antibodies or ligands that promote signaling of CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, inducible T cell co-stimulatory factor (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD 1 1a/CD 18), CD247, CD276 (B7-H3), igalpha (CD 79 a), DAP-10, fc gamma receptor, or any combination thereof in certain embodiments, the stimulatory agents and co-stimulatory agents used in the activation step of the methods herein include, for example, anti-CD 3, anti-CD 28, anti-41-BB, or anti-CD 27 antibodies bound to a solid support such as beads, and/or one or more cytokines in certain embodiments, the agents include ligands that activate a receptor or co-stimulatory receptor, such as peptide/MHC complex and/or CD27 ligand (e.g., CD70 or trisomy pattern), CD80, CD86, and the like stimulatory agents such as ADM 3 and anti-CD 7, and/or anti-IL 15, including anti-CD 28, and/or anti-IL-bead 15, in certain embodiments, and the like, expansion of the agents may include, such as anti-CD 15 and/or anti-IL-bead media.

In various embodiments, the activator is a tumor antigen, particularly a tumor antigen as disclosed herein.

In some embodiments, the activators and/or co-stimulators herein may be coated or adsorbed onto one or more surfaces. Such surfaces include, for example, solid surfaces, porous surfaces, semi-porous surfaces, spherical surfaces, non-spherical surfaces, rod-like surfaces, and polymeric surfaces.

In certain embodiments, a commercially available agent such as TRANSACT (Miltenyi Biotec) is used to activate T cells (e.g., during the pre-activation, reactivation, or expansion stages of the methods herein). In various embodiments, TRANSACT is used to activate partially reprogrammed T cells at a rate that allows T cell-derived adherent cells to regain their T cell characteristics. In various embodiments, the partially reprogrammed T cells are activated with TRANSACT at 1:10, 1:50, 1:100, 1:150, 1:200, 1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700, 1:750, 1:800, 1:850, 1:900, 1:1,000, 1:2000, 1:3000, 1:4000, or 1:10,000. In various embodiments, the T cell-derived adherent cells are activated in an appropriate T cell activation medium and cultured for a time sufficient to allow the T cell-derived adherent cells to regain their properties. In various embodiments, the partially reprogrammed cells are activated and cultured in T cell activation medium for a period of about 1, 2, 3, 4, 5, 6, or 7 days or more. In certain embodiments, the partially reprogrammed cells are activated and cultured for about 1-20 days, about 2-20 days, about 3-20 days, about 4-20 days, about 5-20 days, about 6-20 days, about 7-20 days, 8-20 days, about 9-20 days, or about 10-20 days. In certain embodiments, the partially reprogrammed cells are activated and then cultured for about 1-15 days, about 2-15 days, about 3-15 days, about 4-15 days, about 5-15 days, about 6-15 days, about 7-15 days, 8-15 days, about 9-15 days, or about 10-15 days.

In certain embodiments, the T cell-derived adherent cells are redirected or reactivated by an antigen or antigen-expressing cell, such as a tumor antigen-expressing autologous cell or tumor antigen. In certain embodiments, the T cell-derived adherent cells are reactivated by co-culture with APCs expressing the tandem minigene (i.e., the fragments of the tumor re-expressed from the tandem minigene expressed and presented by the APCs may be sequenced). Any tumor antigen known to be associated with cancer is contemplated for use herein. Non-limiting examples of antigens include: AFP (alpha fetoprotein), αvβ6 or another integrin, BCMA, BRAF, B-H3, B7-H6, CA9 (carbonic anhydrase 9), (carcinoembryonic antigen), seal protein 18.2, seal protein 6, C-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (exonucleotide pyrophosphatase/phosphodiesterase family member 3), epCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, ephrin B2, EPHa2 (liver calpain receptor A2), ERBB dimer, estrogen receptor, ETBR (endothelin B receptor), FAP- α (fibroblast activation protein α), fetal achR (fetal acetylcholine receptor), FBP (folic acid binding protein), FCRL5, FR- α (folic acid receptor α), GCC (guanylate cyclase C), GD2, GD3, GPC2 (phosphatidylinositol proteoglycan 2), GPC3, gp100 (glycoprotein 100), NMB (glycoprotein NMB), RC5D (glycoprotein), HER2, HER3, HER4, HER 1, MAGE 1-related to human hepatitis B, MAR 1, MAR-1, human tumor antigen, HLA-A complexed with peptides derived from AFP, KRAS, NY-ESO, MAGE-A and WT 1), NCAM (neural cell adhesion molecule), nectin-4, NY-ESO, cancer embryo antigen, PRAME (melanoma preferential expression antigen), progesterone receptor, PSA (prostate specific antigen), PSCA (prostate stem cell antigen), PSMA (prostate specific membrane antigen), ROR1, ROR2, sirpa (signal regulatory protein α), SLIT, SLITRK6 (NTRK-like protein 6), survivin, TAG72 (tumor associated glycoprotein 72), TPBG (trophoblast glycoprotein), trop-2, VEGFR1 (vascular endothelial growth factor receptor 1), p53 mutant, prostaglandin (prostein), survivin, telomerase, PCTA-1/galectin 8, melanA/MARTl, ras mutant hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS 2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, rhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, enterocarboxyesterase, muthsp 70-2, CD79a, CD79B, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3 epsilon, CD4, CD5, CD7, APRIL protein extracellular portion, neoantigen, or any combination thereof.

In various embodiments, the activation medium used to reactivate T cell-derived adherent cells may also comprise an immunomodulatory molecule such as a cytokine. Examples of immunoregulatory molecules are lymphokines, monokines and traditional polypeptide hormones. Included among cytokines are: growth hormone such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; a relaxin source; glycoprotein hormones such as Follicle Stimulating Hormone (FSH), thyroid Stimulating Hormone (TSH) and Luteinizing Hormone (LH); liver growth factor (HGF); fibroblast Growth Factor (FGF); prolactin; placental lactogen; a miller tube inhibiting substance; a mouse gonadotrophin-related peptide; inhibin; an activin; vascular endothelial growth factor; integrins; thrombopoietin (TPO); nerve Growth Factor (NGF) such as NGF- β; platelet growth factors; transforming Growth Factors (TGFs) such as TGF- α and TGF- β; insulin-like growth factor-I and insulin-like growth factor-II; erythropoietin (EPO); an osteoinductive factor; interferons such as interferon- α, interferon- β and interferon- γ; colony Stimulating Factors (CSF) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (IL) such as IL-1, IL-lα, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15; tumor necrosis factors such as TNF- α or TNF- β; and other polypeptide factors include LIF and Kit Ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell cultures, as well as biologically active equivalents of native sequence cytokines.

In certain embodiments, a partially reprogrammed and reactivated T cell herein may be further stimulated and expanded using the same reactivation method as described herein. Such methods of activating and expanding T cells are known in the art.

In various embodiments, including methods of partial reprogramming followed by reactivation and optionally further expansion herein, will be sufficient for regenerative T cells to acquire any one or more of the following characteristics:

in certain embodiments, the regenerative T cells have one or more stem properties, including high epigenetic plasticity. Other advantageous phenotypic markers available to regenerative cells include the expression of L-selectin (CD 62L), IL-7Ra, CD132, CCR7, CD45RA, CD45RO, CD27, CD28, CD95, CXCR3, TCF7 and LFA-1. In certain embodiments, regenerative T cells obtain T memory stem cell expression markers, e.g., memory T cells that express CD95, CD45RA, CCR7, and CD62L and are endowed with stem cell-like self-renewal capacity and multipotent capacity to reconstruct the entire memory and effector T cell subpopulation profile. In certain embodiments, the regenerative T cells are "central memory T cells" or "TCM cells," which are memory T cells expressing CD45RO, CCR7, and CD 62L. In another embodiment, the regenerative T cells have expression of effector memory T cell markers, such as CD45RO, but lack expression of CCR7 and CD 62L. In certain embodiments, the regenerative T cells herein have a stem-like phenotype and have increased proliferative capacity.

In certain embodiments, the regenerative T cells are characterized as naive T cells, i.e., "TN cells," as T cells that express CD45RA, CCR7, and CD62L, but do not express CD 95.

In certain embodiments, regenerative T cells may exhibit biological and phenotypic characteristics of young T cells in terms of epigenetic signature, telomere length, and functionality.

In various embodiments, partial reprogramming and reactivation as described herein produces regenerative T cells, as can be measured by assessing the methylation status of individual CpG sites in cellular DNA. Such techniques are known in the art. See, e.g., horvath and Raj,2018,Nature Reviews Genetics,19:371-375. Each team has built a model or "epigenetic clock" that uses the methylation status of a cell to provide an estimate of the "biological age" of that cell or tissue using a mathematical algorithm that uses the value assigned to the methylation status of a particular CpG in the genome. In various embodiments, the methylation status of each CpG site is used to determine whether partial reprogramming has occurred. In various embodiments, the methylation status of each CpG site is used to predict the count of depleted CD 8T cells. In various embodiments, the methylation status of each CpG site is used to determine whether a population of immune cells (e.g., T cells) is suitable for administration to a patient.

In various embodiments, the "Horvath clock" described in Horvath and Raj (2018,Nature Reviews Genetics,19:371-375) is used to determine the epigenetic age of cells described herein, including regenerative immune cells (e.g., T cells). In various embodiments, the Horvath clock is used to determine whether partial reprogramming has occurred. In various embodiments, the horvat clock is used to predict the count of depleted CD 8T cells. In various embodiments, the Horvath clock is used to determine whether a population of immune cells (e.g., T cells) is suitable for patient administration. In various embodiments, other epigenetic clocks or algorithms (e.g., hannum clocks or Levine clocks) are used. See, e.g., hannum et al, 2013, mol. Cell.,49:359-369; levine et al, 2018, aging,10 (4): 573-591.

In certain embodiments, the regenerative T cells (partially reprogrammed and reactivated T cells as described herein) exhibit a reduced epigenetic age (eAge) as compared to control T cells (e.g., T cells that have been cultured without contact with one or more reprogramming factors). eAge may be determined using known epigenetic clock determination methods, such as the Horvath clock described elsewhere herein. In certain embodiments, the eAge of a regenerative T cell as described herein is reduced by about 5% -75%, 10% -50%, 15% -75%, 15% -50%, 20% -75%, or 20% -50% as compared to eAge of an appropriate control T cell (e.g., a T cell from an original donor sample or an actual age-matched T cell prior to partial reprogramming and reactivation). In certain embodiments, the eAge of a regenerative T cell as described herein is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more compared to an appropriate control T cell (e.g., a T cell from an original donor sample prior to partial reprogramming or an actual age-matched T cell).

In certain embodiments, the disclosure provides a T cell population having an epigenetic age at least 5% younger than its actual age. In another embodiment, the disclosure provides a population of T cells that has an epigenetic age that is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% >, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80% younger than its actual age. In certain embodiments, the present disclosure provides a population of T cells that has an epigenetic age that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 years old or older than its actual age. In this regard, the epigenetic age may be measured using methods known in the art, such as by measuring the horvat epigenetic clock.

In certain embodiments, regenerative T cells (partially reprogrammed and reactivated T cells as described herein) exhibit an epigenetic age (eAge) of about 7-16 years old. In certain embodiments, the regenerative T cells exhibit a reduction to an epigenetic age (eAge) of about the adolescent equivalent age as compared to control T cells (e.g., T cells cultured without contact with one or more reprogramming factors; e.g., as compared to the actual age of the starting cells). In certain embodiments, the regenerative T cells have increased telomere length as compared to control T cells. Telomere length can be measured using methods known in the art (see, e.g., rosenberg et al, 2011,Clin Cancer Res 17,4550-4557) and commercially available kits (e.g., TELOTAGG, sigma-Aldrich). In certain embodiments, the telomeres are elongated by about 0.1-3kb in length. In certain embodiments, the telomere length is elongated by about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or about 1kb. In another embodiment, the telomeres are elongated by about 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2kb.

In certain embodiments, regenerative T cells exhibit increased expansion potential. In certain embodiments, the regenerative T cells are expanded to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50-fold that of control T cells that have not been partially reprogrammed or that are actually age matched. In certain embodiments, the regenerative T cells expand 25-1000 fold over actual age-matched control T cells or control T cells that have not been partially reprogrammed. In certain embodiments, the regenerative T cells are expanded to 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500-fold that of an actual age-matched control T cell or a control T cell that has not been partially reprogrammed. In certain embodiments, regenerative T cells expand faster than T cells that have not been partially reprogrammed.

In certain embodiments, the disclosure provides a population of T cells that are at least 5% younger than their actual age in epigenetic age, wherein the T cells do not express an aberrant NK, T, or B cell marker.

In another embodiment, the present disclosure provides a population of T cells that has an epigenetic age that is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% >, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or at least 80% younger than the actual age of the source T cell or an appropriate control T cell, and wherein the T cell does not express an aberrant NK, T or B cell marker.

In another embodiment, the present disclosure provides a population of T cells that has an epigenetic age that is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% >, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or at least 80% younger than the actual age of the source T cell or an appropriate control T cell, and wherein the T cells have a stem-like phenotype.

In another embodiment, the present disclosure provides a population of T cells that has an epigenetic age that is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% >, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80% younger than the actual age of the source T cell or an appropriate control T cell, and wherein the T cells express CCR7, CD62L, and TCF7.

In another embodiment, the present disclosure provides a population of T cells that has an epigenetic age that is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% >, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or at least 80% younger than the actual age of the source T cell or an appropriate control T cell, and wherein the T cells express CCR7, CD62L, and TCF7; and has increased killing capacity compared to an appropriate control T cell. Killing capacity may be measured using assays known in the art, such as those described in the examples herein.

In certain embodiments, the present disclosure provides a population of T cells that has an epigenetic age that is at least 5% younger than the actual age of the source T cell, wherein the T cell does not express NCAM1, NCR2, FCGR3A, KIR DL4, or KIR2DS4.

In certain embodiments, the disclosure provides a population of T cells that has an epigenetic age that is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80% younger than its actual age, and wherein the T cells do not express NCAM1, NCR2, FCGR3A, KIR DL4, or KIR2DS4.

In certain embodiments, the present disclosure provides a T cell that is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or at least 80% younger than its actual age (or a suitable control T cell), and wherein the T cell is enriched for oxidative phosphorylation, fatty acid metabolism, glycolysis, and hypoxia gene set as determined by transcriptome analysis.

In certain embodiments, the disclosure provides a T cell or population of such T cells that is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% >, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or at least 80% younger than its actual age (or a suitable control T cell), and wherein the T cells are enriched for oxidative phosphorylation and glycolytic gene sets as determined by transcriptome analysis.

In certain embodiments, the disclosure provides T cells having an epigenetic age that is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or at least 80% younger than the actual age of an appropriate control T cell, and wherein the T cells have increased TCR repertoire diversity as compared to the control T cell.

In various embodiments, the disclosure provides a population of T cells that are at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% >, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or at least 80% younger than their actual age, and wherein the T cells comprise a set of incomplete V, D and J segments of a T cell receptor gene.

In certain embodiments, the present disclosure provides a population of T cells that has an epigenetic age that is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or at least 80% younger than the actual age of the source T cell; wherein the T cell does not express NCAM1, NCR2, FCGR3A, KIR2DL4 or KIR2DS4; and wherein the T cells are enriched for oxidative phosphorylation, fatty acid metabolism, glycolysis, and hypoxia gene sets as determined by transcriptome analysis.

In certain embodiments, the present disclosure provides a population of T cells having an epigenetic age that is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% >, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or at least 80% younger than the actual age of the source T cell; wherein the T cell does not express NCAM1, NCR2, FCGR3A, KIR2DL4 or KIR2DS4; and wherein the T cells are enriched for oxidative phosphorylation and glycolytic gene sets compared to control T cells, as determined by transcriptome analysis.

In certain embodiments, the present disclosure provides a population of T cells having an epigenetic age that is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% >, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or at least 80% younger than the actual age of the source T cell; wherein the T cell does not express NCAM1, NCR2, FCGR3A, KIR2DL4 or KIR2DS4; wherein the T cells are enriched for oxidative phosphorylation and glycolytic gene sets compared to control T cells, as determined by transcriptome analysis; and wherein the T cells have increased TCR repertoire diversity as compared to control T cells.

In certain embodiments, the present disclosure provides a population of T cells that has an epigenetic age that is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or at least 80% younger than the actual age of the source T cell; wherein the T cell does not express NCAM1, NCR2, FCGR3A, KIR2DL4 or KIR2DS4; wherein the T cells are enriched for oxidative phosphorylation and glycolytic gene sets compared to control T cells, as determined by transcriptome analysis; wherein the T cells have increased TCR repertoire diversity as compared to control T cells; and wherein the T cell comprises a set of incomplete V, D and J segments of a T cell receptor gene.

As will be appreciated by those skilled in the art, T cell receptor V, D and J gene segments rearrange during T cell development to form complete variable domain exons. These gene rearrangements occur in the thymus. The regenerative T cells produced herein are produced by isolated T cells that have rearranged TCR loci and express TCRs. Thus, in certain embodiments, the regenerative T cells produced herein are epigenetically younger than the T cells from which they were derived, but have a functional TCR (i.e., a set of incomplete V, D and J segments of the T cell receptor gene), which distinguishes them from isolated T cells.

In certain embodiments, the metabolic state of the regenerative T cells is improved (see, e.g., cell Metabolism 14, 264-271). In certain embodiments, the metabolic gene sets corresponding to oxidative phosphorylation, fatty acid metabolism, glycolysis, and hypoxia are enriched in the regenerative T cells described herein compared to an appropriate control (see, e.g., nishimura 2019int.j.mol. Sci.20:2254). In certain embodiments, the regenerative T cells described herein have an up-regulated glycolytic enzyme and a down-regulated electron transport chain subunit. In certain embodiments, the regenerative T cells herein exhibit one or more of the following characteristics: metabolic switches that convert somatic oxidative metabolism to a glycolytic flux-dependent, mitochondrial-independent state; expression of age-related stress response genes (including p16.sup.INK4a, p21.sup.CIP 1, atf3 and Gadd 45B) in the p53 tumor suppressor pathway is down-regulated; down-regulation of the senescence-associated metalloproteinases MMP13 and interleukin 6; a decrease in aging-related beta-galactosidase activity; reduced production of mitochondrial Reactive Oxygen Species (ROS); H3K9me3 and H4K20me3 levels are restored (epigenetic modifications involved in heterochromatin maintenance) (see, e.g., ocampo, cell.2016,167 (7): volumes 1719-1733;Benayoun BA,Nat Rev Mol Cell Biol.2015;16:593-610;Liu B,Nat Commun.2013,4:1868;Database,2016, baw, 100; doi.org/10.1093/database/baw, 100).

In certain embodiments, the regenerative T cells herein are enriched for stem gene tags (see, e.g., gattinone et al 2011Nature Medicine 17 (10): 1290-1297) as compared to non-regenerative control T cells.

II therapeutic methods

In various embodiments, the present disclosure provides a method of treating a patient in need thereof with a population of regenerative T cells produced by the partial reprogramming and reactivation methods disclosed herein. Methods of treating diseases or disorders (including cancer) by administering compositions comprising regenerative T cells as described herein are provided. In various embodiments, the disclosure relates to a set of methods of treating a patient in need thereof with a population of T cells produced by a method comprising: (a) Transiently contacting T cells with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and optionally SV40, for a time sufficient to form T cell-derived adherent cell colonies; and (c) contacting the T cell-derived adherent cells with a medium comprising a T cell activator and/or co-stimulatory agent such as anti-CD 3, anti-CD 28 and or one or more cytokines such as IL-2. In some embodiments, T cells are engineered to express cell surface receptors that recognize specific antigen moieties on the surface of target cells. In various embodiments of the present disclosure, the target cell is a cancer cell.

In certain embodiments, the disclosure relates to a method of treating a patient in need thereof with a population of regenerative T cells as described herein.

In various embodiments, the disclosure relates to a method of treating a patient in need thereof with a population of regenerative immune cells (e.g., T cells) produced by the methods disclosed herein. Methods for treating diseases or disorders, including cancer, infectious diseases, or autoimmune diseases, are provided. In various embodiments, the disclosure relates to a method of treating a patient in need thereof with a population of immune cells (e.g., T cells) produced by a method comprising: (a) Isolating a plurality of immune cells (e.g., T cells) from a source; (b) Transiently contacting the plurality of immune cells (e.g., T cells) with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC; and (c) contacting the immune cells (e.g., T cells) with a medium comprising IL-2, wherein the immune cells (e.g., T cells) are contacted with at least one reprogramming factor for a period of at least about four days. In some embodiments, the plurality of immune cells (e.g., T cells) are engineered to express a cell surface receptor that recognizes a specific antigen on the surface of a target cell. In various embodiments of the present disclosure, the target cell is a cancer cell.

In various embodiments, the disclosure includes a pharmaceutical composition comprising a plurality of regenerative T cells produced by the methods herein. In certain embodiments, the pharmaceutical composition comprises regenerative T cells produced by a method comprising: (a) Transiently contacting a plurality of T cells with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC for a time sufficient to allow attachment of partially reprogrammed cells to a culture vessel surface; and (b) contacting the attached reprogrammed T cells with a medium comprising a T cell activator. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

In some aspects, the disclosure includes a pharmaceutical composition comprising at least one regenerative T cell as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

It will be appreciated that the target dosage of regenerative T cells may range from about 1X106 to about 2X1010 cells/kg, preferably 2X106 cells/kg. It will be appreciated that dosages above and below this range may be appropriate for certain subjects, and that appropriate dosage levels may be determined by the healthcare provider as desired. In various embodiments, the target dose is 1x105. In various embodiments, the target dose is 1x106. In various embodiments, the target dose is 1x107. In various embodiments, the target dose is 1x108. In various embodiments, the target dose is 1x109. In various embodiments, the target dose is 1x1010. In various embodiments, the target dose is 2x1010. In addition, multiple doses of cells may be provided according to the present disclosure. In various embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of cells are administered to a patient in need thereof.

Also provided are methods for reducing tumor size in a subject comprising administering to a subject the regenerative T cells of the present disclosure. In some embodiments, the subject has a solid tumor or hematological malignancy such as lymphoma or leukemia. In some embodiments, regenerative immune cells (e.g., T cells) are delivered to the tumor bed. In some embodiments, the cancer is present in bone marrow of the subject.

As used herein, the term "subject" or "patient" means an individual. In some aspects, the subject is a mammal, such as a human. In some aspects, the subject can be a non-human primate. For example, non-human primates include marmosets, monkeys, chimpanzees, gorillas, orangutans, and gibbons. The term "subject" also includes domestic animals (such as cats, dogs, etc.), domestic animals (e.g., llamas, horses, cattle), wild animals (e.g., sika deer, elk, moose, etc.), laboratory animals (e.g., mice, rabbits, rats, gerbils, guinea pigs, etc.), and birds (e.g., chickens, turkeys, ducks, etc.). Preferably, the subject is a human subject. More preferably, the subject is a human patient.

The method may further comprise administering one or more chemotherapeutic agents. In certain embodiments, the chemotherapeutic agent is a lymphocyte-clearing (pretreatment) chemotherapeutic agent. Beneficial pretreatment regimens and related beneficial biomarkers are described in U.S. provisional patent applications 62/262,143 and 62/167,750. For example, these describe methods of modulating a patient in need of T cell therapy comprising administering to the patient a prescribed beneficial dose of cyclophosphamide (200 mg/m 2/day to 2000mg/m 2/day) and a prescribed dose of fludarabine (20 mg/m 2/day to 900mg/m 2/day). A preferred dosage regimen involves treating a patient comprising administering to the patient about 500mg/m 2/day cyclophosphamide and about 60mg/m 2/day fludarabine daily for three days, followed by administering to the patient a therapeutically effective amount of regenerative T cells.

In other embodiments, the regenerative T cells and the chemotherapeutic agent are each administered in an amount effective to treat the disease or disorder in the subject.

In certain embodiments, compositions comprising regenerative immune cells (e.g., T cells) as disclosed herein can be administered in conjunction with a number of chemotherapeutic agents. Examples of chemotherapeutic agents include: alkylating agents, e.g. thiotepa and Cyclophosphamide (CYTOXAN) ^TM ) The method comprises the steps of carrying out a first treatment on the surface of the Alkyl sulfonates such as busulfan (busulfan), imperoshu (imposulfan) and piposulfan (piposulfan); aziridines such as benzotepa (benzodopa), carboquinone (carboquone), mettussidine (meturedopa) and uratepa (uredopa); ethyleneimines and methyltriamines, including altretamine, triamcinolone acetoamide, triethylenephosphoramide, triethylenethiophosphamide and trimethylol melamine (trimethylolomelamine resume); nitrogen mustards such as chlorambucil (chloramabilin), napthalamus (chloraphanizine), cholesteryl phosphoramide (chlorophosphoramide), estramustine (estramustine), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), chlorambucil hydrochloride (mechlorethamine oxide hydrochloride), melphalan (melphalan), novembril (novemblichin), bennethol (phenaestine), prednisolone (prednisone), triamcinolone (trofosfamide), uracil mustard (uracilstard); nitroureas, e.g. Carmustine (carmustine), chlorozotocin (chlorozotocin), fotemustine (fotemustine), lomustine (lomustine), nimustine (nimustine), and ranimustine (ranimustine); antibiotics such as aclacinomycin (aclacinomycin), actinomycin (actinomycin), anthramycin (automycin), azaserine (azaserine), bleomycin (bleomycin), actinomycin C (cactinomycin), calicheamicin (calicheamicin), cartriamycin (carbicin), carminomycin (carminomycin), acidophilicin (carzinophilin), chromomycins (chromomycins), actinomycin D (dactinomycin), daunorubicin (daunorubicin), dithizomycin (deoxymycin), 6-diazo-5-oxo-norubicin, doxorubicin (doxorubicin), epirubicin (epirubicin), epothilone (epothilin), idarubicin (idarubicin), doxorubicin (mitomycin), streptomycin (35), streptomycin (streptomycin), and the like; antimetabolites, for example, methotrexate (methotrexate) and 5-fluorouracil (5-FU); folic acid analogs such as, for example, dimethyl folic acid (denopterin), methotrexate, pteroyltri-glutamic acid (pteroplegin), trimetric sand (trimetricate); purine analogs, e.g., fludarabine (fludarabine), 6-mercaptopurine, thioazane (thiamiprine), thioguanine (thioguanine); pyrimidine analogues, for example, ancitabine, azacitidine, 6-azauridine, carmofur (carmofur), cytarabine, dideoxyuridine (dideoxyuridine), doxifluridine, enocitabine (enocitidine), floxuridine (floxuridine), 5-FU; androgens, for example, card Lu Gaotong (calasterone), droxidone propionate (dromostanolone propionate), epithioandrosterol (epiostanol), melandrane (mepistostane), testolactone (testolactone); anti-adrenal, e.g. aminoglutethimide (aminogl) utaethimide), mitotane (mitotane), trilostane (trilostane); folic acid supplements, for example, folinic acid (folinic acid); acetoglucurolactone (aceglatone); aldehyde phosphoramidate glycoside (aldophosphamide glycoside); aminolevulinic acid (aminolevulinic acid); amsacrine (amacrine); betabacib (bestabuic); bisantrene (bisantrene); edatraxate (edatraxate); ground phosphoramide (defofame); dimecoxine (demecolcine); deaquinone (diaziquone); eformitine (elfomithin); ammonium elide (elliptinium acetate); etodolac (etoglucid); gallium nitrate; hydroxyurea; lentinan (lentinan); lonidamine (lonidamine); mitoguazone (mitoguazone); mitoxantrone (mitoxantrone); mo Pai darol (mopidamol); diamine nitroacridine (nitroane); penstatin (penstatin); egg ammonia nitrogen mustard (phenol); pirarubicin (pirarubicin); podophylloic acid (podophyllinic acid); 2-ethyl hydrazide; procarbazine (procarbazine);raschig (razoxane); sisofilan (silzofuran); spiral germanium (spiral); tenuazonic acid (tenuazonic acid); triiminoquinone (triaziquone); 2,2',2 "-trichlorotriethylamine; uratam (urethan); vindesine (vindeline); dacarbazine (dacarbazine); mannomustine (mannomustine); dibromomannitol (mitobronitol); dibromodulcitol (mitolactol); pipobromine (pipobroman); metropolicine (tetracytine); cytarabine (arabinoside) ("Ara-C"); cyclophosphamide; thiotepa; taxanes, e.g. taxol () >Bristol-Myers Squibb) and docetaxel (++>Rhone-Poulenc Rorer); chlorambucil (chloranil); gemcitabine (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, such as cisplatin (cispratin) and carboplatin (carboplatin); vinblastine (vinblastine); platinum (platinum); etoposide(etoposide) (VP-16); ifosfamide (ifosfamide); mitomycin C (mitomycin C); mitoxantrone (mitoxantrone); vincristine (vincristine); vinorelbine (vinorelbine); novelline (naveldine); norubin (novantrone); teniposide (teniposide); daunomycin (daunomycin); aminopterin (aminopterin); hilded (xeloda); ibandronate (ibandronate); CPT-11; topoisomerase inhibitor RFS2000; difluoromethyl ornithine (DMFO); retinoic acid (retinoic acid) derivatives such as targetretin (bexarotene), panretin (alisretinin); ontaktM (dinium interleukin (denileukin diftitox)); epothilones (esperamicins); capecitabine (capecitabine); and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to modulate or inhibit the effect of hormones on tumors, such as antiestrogens including, for example, tamoxifen, raloxifene, (raloxifene), aromatase-inhibiting 4 (5) -imidazole, 4-hydroxy tamoxifen, troxifene (trioxifene), raloxifene hydrochloride (keoxifene), LY117018, onapristone (onapristone) and toremifene (toremifene) (farston) and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprorelin (leuprorelin) and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Combinations of chemotherapeutic agents are also administered where appropriate, including but not limited to CHOP, i.e., cyclophosphamide +. >Doxorubicin (hydroxy doxorubicin), fludarabine, vincristine +.>And prednisone.

In some embodiments, the chemotherapeutic agent is administered simultaneously with or within one week after the engineered cell or nucleic acid is administered. In other embodiments, the chemotherapeutic agent is administered 1 to 4 weeks or 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months or 1 week to 12 months after administration of the engineered cell or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least 1 month prior to administration of the cell or nucleic acid. In some embodiments, the method further comprises administering two or more chemotherapeutic agents.

A variety of additional therapeutic agents may be used in conjunction with the compositions described herein. For example, additional therapeutic agents that may be useful include PD-1 (or PD-L1) inhibitors, such as nivolumab (nivolumab)Pembrolizumab (pembrolizumab)>Pembrolizumab (pidilizumab) and atuzumab (atezolizumab)

Other therapeutic agents suitable for use in combination with the present disclosure include, but are not limited to ibrutinib (ibrutinib)Offatumumab (ofatumumab)>Rituximab (rituximab)Bevacizumab (bevacizumab) is added to the composition >Trastuzumab (trastuzumab)Enmetrastuzumab (trastuzumab emtansine)/(E)>Imatinib (imatinib)>Cetuximab (cetuximab)>Panitumumab (panitumumab)Katuzumab (catumaxmab), ibritumomab (ibritimomab), ofatumumab (ofatumumab), tositumomab (tositumomab), bentuximab (brentuximab), alemtuzumab (alemtuzumab), gemtuzumab (gemtuzumab), erlotinib (erlotinib), gefitinib (gefitinib), vandetanib (vanretanib), afatinib (afatinib), lapatinib (lapatinib), lenatinib (necatinib), axitinib (axitinib), maritiminib (masitinib), pazopanib (pazopanib), sunitinib (sunitanib), sorafenib (sorafenib), toletanib (toxanib), lentinib (leutinib), afatinib (aletinib), aletinib (dioxitinib), and dioxitinib (dio) lenvatinib (lenvatinib), nilvadanib (nintedanib), pazopanib, regorafenib (regorafenib), semaxanib (semaxanib), sorafenib, sunitinib, tivozanib (tivozanib), torseminib (toceranib), vandetanib (vanretanib), emtrictinib (entrectinib), cabozantininib (cabozantinib), imatinib (imatinib), dasatinib (dasatinib), nilotinib (nilotinib), panatinib (ponatinib), radatinib (radaninib), bosutinib (bosutinib), letatinib (lesatinib), ruxotinib (ruxolitinib), panitinib (panitinib), coltinib (cobitinib), coltinib (coltinib), mestinib (mestinib), trametinib (trametinib), bimetainib (bimetainib), aletinib (aletinib), ceritinib (ceritinib), crizotinib (crizotinib), aflibercept (aflibercept), adioteide, dimesleukin, mTOR inhibitors such as Everolimus (Everolimus) and sirolimus (Temsirolimus), hedgehog inhibitors such as sonidegini (sonidegini) and valmonide gin (vismod) egib)), CDK inhibitors such as CDK inhibitors (palbociclib).

In further embodiments, the composition comprising regenerative T cells may be administered with an anti-inflammatory agent. Anti-inflammatory agents or agents include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDS) (including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF agents, cyclophosphamide and mycophenolate esters). Exemplary NSAIDs include ibuprofen, naproxen sodium, cox-2 inhibitors, and sialic acid. Exemplary analgesics include acetaminophen, oxycodone, propoxyphene tramadol hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept) Adalimumab->And infliximab) Chemokine inhibitors and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies and recombinant forms of the molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular injection), and minocycline.

In certain embodiments, the compositions described herein are administered in conjunction with a cytokine. In some embodiments, the cytokine may include proteins from natural sources or from recombinant cell cultures, as well as biologically active equivalents of the native sequence cytokine.

III formulations and pharmaceutical preparations

A variety of known techniques may be used to prepare polynucleotides, polypeptides, vectors, antigen binding molecules, immune cells (e.g., T cells), compositions, and the like according to the present disclosure.

The isolated T cells may be genetically modified after isolation using known methods, or the T cells may be partially reprogrammed, activated and expanded in vitro prior to being genetically modified. In another embodiment, the T cells are genetically modified with a recombinant TCR or CAR and then regenerated using the partial reprogramming and reactivation methods described herein. In certain embodiments, the regenerative cells described herein are further activated and/or expanded in vitro. Methods for activating and expanding immune cells (e.g., T cells) are known in the art and are described, for example, in U.S. patent nos. 6,905,874, 6,867,041, 6,797,514; described in WO 2012/079000. Typically, such methods comprise contacting T cells with a stimulating (e.g., activating) agent (in certain embodiments, referred to as a T cell activating compound) (e.g., an agent that stimulates the CD3/TCR complex (e.g., an anti-CD 3 antibody or CD3 agonist) and a costimulatory agent (an antibody or ligand that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof, etc.).

In other embodiments, regenerative T cells can be activated and stimulated to proliferate with feeder cells and appropriate antibodies and cytokines using methods such as those described in U.S. Pat. Nos. 6,040,177, 5,827,642 and WO/2012129514.

Certain methods for preparing the constructs and engineered T cells of the present disclosure are described in PCT application PCT/US 2015/14520.

In certain embodiments, the present disclosure provides a method of storing regenerative T cells described herein. This involves cryopreserving T cells so that the cells remain viable after thawing. Some T cells may be cryopreserved by methods known in the art to provide a permanent source of such cells for future treatment of patients with malignancy. When desired, cryopreserved cells can be thawed, grown, and expanded to obtain more such cells.

As used herein, "cryopreservation" refers to preservation of cells by cooling to a sub-zero temperature, such as (typically) 77 ° or 196 ℃ (the boiling point of liquid nitrogen). Cryoprotectants are often used at sub-zero temperatures to prevent damage to cell preservation due to freezing at low temperatures or warming to room temperature. Cryoprotectants and optimal cooling rates may prevent cell damage. Cryoprotectants that may be used in accordance with the present disclosure include, but are not limited to: dimethyl sulfoxide (DMSO) (Lovelock and Bishop, nature,1959,183,1394-1395; ashwood-Smith, nature,1961,190,1204-1205), glycerol, polyvinylpyrrolidone (Rinfret, ann.N.Y. Acad.Sci.,1960,85,576) and polyethylene glycol (Sloviter and Ravdin, nature,1962,196,48). The preferred cooling rate is 1 deg. to 3 deg. per minute.

The term "substantially pure" is used to indicate that a given component is present at high levels. This component is desirably the major component present in the composition. Preferably, the component is present at a level of more than 30%, more than 50%, more than 75%, more than 90% or even more than 95%, said level being determined on a dry weight/dry weight basis relative to the total composition considered. At very high levels (e.g., levels in excess of 90%, in excess of 95%, or in excess of 99%), the component may be considered to be in "pure form". The biologically active substances (including polypeptides, nucleic acid molecules, antigen binding molecules, moieties) of the present disclosure may be provided in a form that is substantially free of one or more contaminants that might otherwise associate with the substance. When the composition is substantially free of a given contaminant, the contaminant will be at a low level (e.g., a level of less than 10%, less than 5%, or less than 1% on a dry weight/dry weight basis as described above).

In some embodiments, the cells herein are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a therapeutically effective amount in a medium and container system suitable for administration ("pharmaceutically acceptable" carrier). Suitable infusion media may be any isotonic media formulation, typically normal saline, normosol R (Abbott) or Plasma-Lytet A (Baxter), but 5% dextrose in water or ringer's lactic acid solution may also be used. The infusion medium may be supplemented with human serum albumin.

The desired therapeutic amount of cells in the composition is typically at least 2 cells or more typically greater than 102 cells, and up to 106, up to and including 108 or 109 cells and can exceed 1010 cells. The number of cells will depend on the intended use of the composition, and the type of cells contained therein. The desired cell density is typically greater than 106 cells/ml, typically greater than 107 cells/ml, typically 108 cells/ml or higher. A clinically relevant number of immune cells (e.g., T cells) can be dispensed into multiple infusions, accumulating equal to or exceeding 105, 106, 107, 108, 109, 1010, 1011, or 1012 cells. In some aspects of the disclosure, lower numbers of cells in the range of 106 per kilogram (106-1011 per patient) may be administered, particularly because the infused cells will be redirected throughout to a particular target antigen. Regenerative T cell therapy may be administered multiple times at doses within these ranges. The cells may be autologous, allogenic or xenogenic to the patient being treated.

The regenerative T cells of the present disclosure can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations. The pharmaceutical compositions of the present disclosure may comprise a population of regenerative T cells as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such pharmaceutical compositions may comprise buffers, such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids, such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The compositions of the present disclosure are preferably formulated for intravenous administration. Treatment may also include one or more corticosteroid treatments, such as dexamethasone and/or methylprednisolone.

The compositions of the present application may comprise, consist essentially of, or consist of the disclosed components.

The pharmaceutical compositions (solutions, suspensions, etc.) of the present disclosure may include one or more of the following: sterile diluents such as water for injection, saline solutions (preferably physiological saline), ringer's solution, isotonic sodium chloride, fixed oils which can act as solvents or suspending media (such as synthetic mono-or diglycerides), polyethylene glycols, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediamine tetraacetic acid; buffers such as acetates, citrates or phosphates and agents for adjusting tonicity such as sodium chloride or dextrose. Parenteral formulations may be presented in ampules, disposable syringes or multiple dose vials made of glass or plastic. The injectable pharmaceutical composition is preferably sterile.

It is understood that adverse events can be minimized by transduction of immune cells (e.g., T cells) with suicide genes. Suitable "kill switches" are described, for example, in WO 2021/189008. It may also be desirable to incorporate an inducible "on" or "accelerator" switch into immune cells (e.g., T cells). These techniques may employ dimerization domains and optional activators of dimerization of such domains. These techniques include, for example, those described by Wu et al, science 2014,350 (6258), which utilize the FKBP/Rapalog dimerization system in certain cells. Additional dimerization techniques are described, for example, in Fegan et al chem. Rev.2010,110,3315-3336 and U.S. Pat. Nos. 5,830,462, 5,834,266, 5,869,337 and 6,165,787. Additional dimerization pairs may include cyclosporin-a/cyclophilin receptors, estrogen/estrogen receptors (optionally using tamoxifen), glucocorticoid/glucocorticoid receptors, tetracycline/tetracycline receptors, vitamin D/vitamin D receptors. Other examples of dimerization techniques can be found in, for example, WO 2014/127261, WO 2015/090229, US2014/0286987, US 2015/0266973, US 2016/0046700, US patent No. 8,486,693, US 2014/0171649 and US 2012/013076.

Suitable techniques for genetically modifying the regenerative cells herein include the use of inducible caspase 9 (U.S. application publication No. 2011/0286980) or thymidine kinase, either before, after, or simultaneously with the genetic modification of the cells to express CARs or other engineered TCRs. Other methods of introducing suicide genes and/or "turning on" the switch include CRISPR, TALENS, MEGATALEN, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other techniques known in the art.

IV. kit

Also included within the scope of the present disclosure are kits, e.g., pharmaceutical kits, comprising at least one reprogramming factor for contacting one or more T cells in vitro. The kit typically includes a label indicating the intended use of the kit contents and instructions for use. The term "label" includes any written or recorded material on or with the kit or otherwise accompanying the kit.

In various embodiments, the present disclosure provides a kit for preparing one or more T cells for T cell therapy for a subject in need thereof, the kit comprising at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and optionally SV40. In various embodiments, the present disclosure provides a kit for preparing one or more T cells for T cell therapy for a subject in need thereof, the kit comprising at least one expression vector capable of expressing at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC. In various embodiments, the present disclosure provides a kit for preparing one or more T cells for T cell therapy for a subject in need thereof, the kit comprising at least one sendai virus vector capable of expressing at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2 and C-MYC. In various embodiments, the kit further comprises a sendai virus vector capable of expressing SV40.

In various embodiments, the present disclosure provides a kit for preparing one or more T cell carriers for T cell therapy for a subject in need thereof, the kit comprising:

expression vectors encoding KLF4, OCT3/4 and SOX 2;

an expression vector encoding KLF 4;

an expression vector encoding C-MYC; and

an expression vector encoding SV 40.

sendai virus encoding KLF4, OCT3/4 and SOX 2;

a sendai virus encoding KLF 4;

sendai virus encoding C-MYC; and

sendai virus encoding SV 40.

In various embodiments, the present disclosure provides a kit for preparing one or more T cell vectors for T cell therapy for a subject in need thereof, the kit comprising a polycistronic sendai virus vector encoding KLF4, OCT3/4, SOX2 and C-MYC, and optionally SV 40.

In various embodiments, the kit may further comprise a T cell activating compound or activator. In various embodiments, the T cell activating compound or activator is an anti-CD 3 antibody. In various embodiments, the kit comprises a co-stimulatory agent, such as an anti-CD 28 antibody. In various embodiments, the kit comprises both an anti-CD 3 antibody and an anti-CD 28 antibody. In various embodiments, the kit may further comprise one or more suitable media for culturing and/or partially reprogramming T cells. In various embodiments, the kit may further comprise one or more cytokines. Such cytokines include, but are not limited to, IL-2, IL-7, IL-15, and IL-12.

V. further embodiments

The invention may be characterized by defining one or more of the following clauses of the various embodiments as described herein.

In a first embodiment, the invention relates to [1]:

[1] an in vitro method of producing at least one regenerative T cell comprising (a) contacting a population of T cells with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, C-MYC, and SV40 for a time sufficient to form an adherent T cell-derived cell; wherein the T cells are not transformed into iPS or totipotent cells; and (b) contacting said adherent cells of T cell origin with at least one T cell activating compound.

In a second embodiment, the invention relates to [2]:

[2] an in vitro method of producing at least one T cell comprising (a) contacting a population of T cells in a first medium in a culture vessel with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, C-MYC, and SV40 for a time sufficient for the T cells to form at least one colony attached to the surface of the culture vessel; wherein the T cells are not transformed into iPS cells or totipotent cells; and (b) contacting the at least one attached colony with at least one T cell activating compound.

In a third embodiment, the invention relates to [3]:

[3] an in vitro method of producing at least one T cell comprising (a) contacting a population of T cells in a first medium with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, C-MYC and SV40 for at least about 5 days and no more than about 10 days; wherein the T cells are not transformed into iPS or totipotent cells; and (b) contacting the T cells with at least one T cell activating compound.

In a fourth embodiment, the invention relates to [4]:

[4] an in vitro method of producing at least one T cell comprising (a) contacting a population of T cells with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, C-MYC and SV40 for a time sufficient for the T cells to express at least one marker selected from the group consisting of integrin α6β1, SSEA4, CD9 and CD 90; wherein the T cells are not transformed into iPS cells or totipotent cells; and (b) contacting the T cells with at least one T cell activating compound.

In other embodiments, the disclosure relates to:

[5] the method of any one of [1] to [4], wherein the T cell is contacted with the at least one reprogramming factor for a time sufficient for at least a portion of the T cell to express CD3 and at least one marker selected from the group consisting of: integrins α6β1, SSEA4, CD9 and CD90, preferably SSEA4 and CD3, more preferably CD3, CD9 and CD90, even more preferably CD3, SSEA4, CD9 and CD90.

[6] The method of any one of [1] to [5], wherein prior to contacting the T cell with the at least one reprogramming factor, the T cell is contacted with IL-2 and/or at least one agent capable of activating the T cell, preferably the agent capable of activating the T cell is a tumor antigen.

[7] The method of any one of [1] to [6], wherein the T cell is a tcra+tcrβ+ cell; TCRg (γ) +tcrd (δ) cells; cd4+cd8αβ+ biscationic cells, cd4+ single positive cells (such as Th1, th2, th17, treg), naive T cells, central memory T cells, or effector memory T cells, or wherein the T cells express an activation marker after co-culture with autologous tumor cells, wherein the activation marker is 4-1BB (CD 137), PD1, LAG3, CD45, CD39, TIGIT, TIM3, CD69, OX40, CD28, CD25, CD49d, and CTLA4.

[8] The method of any one of [1] to [7], wherein the T cells are isolated from a mammal, preferably a human.

[9] The method of any one of [1] to [8], wherein the T cell is contacted with KLF4, OCT3/4, SOX2, and C-MYC.

[10] The method of any one of [1] to [9], wherein the T cells are contacted with at least one reprogramming factor, preferably KLF4, OCT3/4, SOX2, and C-MYC, for at least about 4 to 10 days, preferably at least about 4 to 7 days, more preferably about 5 days.

[11] The method of any one of [1] to [10], wherein the at least one reprogramming factor is expressed in the T cell, preferably using a non-integrating viral vector or nucleic acid delivered to the cell with nanoparticles, more preferably the vector is sendai virus, even more preferably the at least one reprogramming factor is KLF4, OCT3/4, SOX2 and C-MYC.

[11a] The method of any one of [1] to [11], wherein the at least one reprogramming factor is expressed in T cells: a) A time sufficient to form an adherent cell of T cell origin, and wherein the isolated T cell is not converted to an iPS or totipotent cell; b) About 4 days to about 10 days; c) About 4 days to about 11 days; d) About 4 days to about 12 days; e) About 4 days to about 13 days; or f) from about 4 days to about 14 days.

[12] The method of any one of [1] to [11a ], wherein the at least one reprogramming factor is constitutively expressed, wherein expression is later inhibited by addition of a T cell activator or a compound that inhibits expression of the at least one reprogramming factor, preferably the compound is a small molecule inhibitor that specifically inhibits expression of the at least one reprogramming factor, more preferably the compound is an siRNA or shRNA molecule that specifically inhibits expression of the at least one reprogramming factor, even more preferably the at least one reprogramming factor is KLF4, OCT3/4, SOX2, and C-MYC.

[13] The method of any one of [1] to [12], further comprising contacting the T cell with at least one cytokine selected from the group consisting of IL-2, IL-7, IL-15, and IL-12.

[14] The method of any one of [1] to [13], wherein the at least one T cell activating compound comprises an antibody that binds CD3 or an antibody that binds CD28 or both; and/or wherein the at least one T cell activating compound is a tumor antigen.

[15] The method of any one of [1] to [14], further comprising engineering the T cell to express a cell surface receptor, wherein the T cell is engineered before or after contacting the T cell with the at least one reprogramming factor, preferably the cell surface receptor is a chimeric antigen receptor or a T cell receptor or a hybrid receptor thereof, more preferably the cell surface receptor recognizes a specific antigen moiety on the surface of a target cell.

[16] The method of [15], wherein the antigen moiety is MHC class I-dependent or MHC class I-independent.

[17] The method of any one of [1] to [16], wherein the resulting T cell comprises a set of incomplete V, D and J segments of a T cell receptor gene, and/or the method further comprises measuring the epigenetic age of the resulting T cell, preferably the epigenetic age of the resulting T cell is at least 5% younger than the T cell population prior to programming. The method of any one of [1] to [16], wherein the resulting T cell comprises a set of incomplete V, D and J segments of a T cell receptor gene, and/or the method further comprises measuring the epigenetic age of the resulting T cell, preferably the epigenetic age of the resulting T cell is at least 5% younger than the T cell population prior to programming and the T cell does not express NCAM1, NCR2, FCGR3A, KIR DL4 or KIR2DS4.

[18] The method of any one of [1] to [17], wherein the partially reprogrammed T cell is capable of expanding at least 25-fold as compared to a T cell prior to contact with the at least one reprogramming factor.

[19] The method of any one of [1] to [18], wherein contacting the isolated T cell with the at least one reprogramming factor results in reduced expression of CD3 and CD 8.

In another embodiment, the invention relates to [20]:

[20] an in vitro method of producing T cells comprising culturing T cells in a first medium comprising IL-2 and activating the T cells with at least one antibody specific for CD3 or CD28 or both; transiently contacting the activated T cells with KLF4, OCT3/4, SOX2, and C-MYC in a second medium that does not comprise IL-2 or an antibody specific for CD3 or CD28 for a period of about five days to about 10 days; wherein the T cells are not fully reprogrammed to iPSC cells; replacing the second medium with a third medium comprising IL-2 and at least one antibody specific for CD3 and/or CD 28; wherein the T cells are cultured in the third medium for at least about 5 days.

In other embodiments, the disclosure relates to one or more of the following:

[21] The method of [20], further comprising expanding the T cells.

[22] The method of [20] or [21], wherein the T cell is a tcrα+tcrβ+ cell; TCRg (γ) +tcrd (δ) cells; cd4+cd8αβ+ biscationic cells, cd4+ single positive cells (Th 1, th2, th17, treg), naive T cells, central memory T cells, or effector memory T cells.

[23] The method of any one of [20] to [22], wherein the contacting further comprises contacting the activated T cells with SV 40.

[24] The method of any one of [20] to [23], wherein the T cell expresses an activation marker after co-culture with an autologous tumor cell, wherein the activation marker is 4-1BB (CD 137), PD1, LAG3, CD45, CD39, TIGIT, TIM3, CD69, OX40, CD28, CD25, CD49d, and CTLA4.

[25] The method of any one of [1] to [24], wherein the T cells are Tumor Infiltrating Lymphocytes (TILs) that have been obtained from a tumor.

In another embodiment, the invention relates to [26]:

[26] a population of T cells having an epigenetic age at least 5% younger than its actual age, preferably at least 25% younger than its actual age.

In another embodiment, the invention relates to [27]:

[27] A population of adherent cells of T cell origin, wherein at least 70% of the cells express both CD3 and SSEA 4.

In another embodiment, the invention relates to:

[28] the population of [27], wherein at least 30% of the cells express CD9 and/or wherein at least 30% of the cells express CD90, preferably at least 30% of the cells express both CD9 and CD 90.

[29] A population of T cells produced by any one of the methods of [1] to [25 ].

[30] The population of T cells of any one of [26] to [29], wherein contacting the T cells with at least one factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC and SV40 causes a decrease in CD3 and CD8 expression.

[31] The population of any one of [26] to [30], wherein the T cells are TILs, wherein at least 50% of the TILs express CCR7 and CD62L, or wherein at least 50% of the TILs express CCR7 and TCF7.

[32] The population of any one of [26] to [31], wherein the T cells are regenerative TILs.

[33] The T cell population of any one of [26] to [32], for use in therapy.

[34] The population of T cells of any one of [26] to [32], for use in a method for treating cancer, a viral disorder, or an autoimmune disorder.

[35] The population of T cells for use of [34], wherein the cancer is acute lymphocytic cancer, acute myelogenous leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal canal cancer or rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gall bladder cancer or pleural cancer, head and neck cancer (e.g., nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer), vulval cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, hodgkin's lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal Cell Carcinoma (RCC)), small intestine cancer, soft tissue cancer, gastric cancer, testicular cancer, ureteral cancer or bladder cancer.

In another embodiment, the invention relates to:

[37] a method of producing regenerative T cells comprising contacting a population of T cells with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and (ii) optionally SV40 for at least a period of time sufficient for at least 20% of the contacted T cells to express α6β1 integrin, and wherein the contacted T cells are not converted to iPS cells; isolating the at least 20% of the contacted T cells with a binding molecule that specifically binds α6β1 integrin; contacting the isolated cells of (b) with a T cell activator and/or a T cell co-stimulator; thereby generating regenerative T cells.

In another embodiment, the invention relates to:

[38] a method of producing at least one regenerative T cell comprising contacting a T cell with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and (ii) optionally SV40 transiently for a time sufficient to form a T cell-derived adherent cell; wherein the T cells are not transformed into iPS or totipotent cells; isolating a subpopulation of adherent cells derived from T cells expressing a6 (CD 49 f) or b1 (CD 29) integrin or both; contacting the isolated subpopulation with at least one T cell activating compound.

In another embodiment, the invention relates to:

[39] a population of regenerative T cells produced by a method comprising: contacting a population of T cells with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC; and (ii) SV40 for a time sufficient to form T cell-derived adherent cells; wherein the T cells are not transformed into iPS or totipotent cells; isolating a subpopulation of adherent cells derived from T cells expressing a6 integrin, b1 integrin, or both; and contacting the T cell-derived adherent cell subpopulation with at least one T cell activating compound.

In another embodiment, the invention relates to:

[40] a population of adherent cells of T cell origin, wherein at least 70% of said cells express integrin α6 or integrin β1, or

[41] A population of adherent cells of T cell origin, wherein at least 50% of the cells express both integrin α6 and integrin β1.

In another embodiment, the invention relates to:

[42] a population of adherent cells of T cell origin, wherein at least 70% of the cells express both integrin α6 and integrin β1.

In another embodiment, the invention relates to:

[43] a T cell whose epigenetic age is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or at least 80% younger than its actual age (or a suitable control T cell), and wherein the T cell is enriched for oxidative phosphorylation, fatty acid metabolism, glycolysis, and hypoxia gene set as determined by transcriptome analysis.

[44] A T cell whose epigenetic age is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30% >, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or at least 80% younger than its actual age (or a suitable control T cell), and wherein the T cell is enriched for oxidative phosphorylation and glycolytic gene sets as determined by transcriptome analysis.

[45] A T cell whose epigenetic age is at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or at least 80% younger than its actual age (or a suitable control T cell), and wherein the T cell does not express an unconventional NK, T or B cell marker (e.g., NCAM1, NCR2, FCGR3A, KIR DL4 or KIR2DS 4).

In another embodiment, the invention relates to

[46] A method of producing regenerative T cells, comprising: a) Contacting a population of T cells with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and (ii) optionally SV40 for at least a period of time sufficient for at least 20% of the contacted T cells to express α6β1 integrin, and wherein the contacted T cells are not converted to iPS cells; b) Isolating the at least 20% of the contacted T cells with a binding molecule that specifically binds α6β1 integrin; c) Contacting the cells of (b) with a T cell activator and/or a T cell co-stimulator; thereby producing regenerative T cells; [47] wherein the binding molecule that specifically binds to α6β1 integrin is selected from the group consisting of laminin-511, laminin-511E 8, anti-CD 29 antibodies, and anti-CD 49f antibodies.

In another embodiment, the disclosure relates to

[48] A method of producing regenerative T cells comprising a) contacting a population of T cells with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and (ii) optionally SV40 transiently for a time sufficient to form T cell-derived adherent cells; wherein the T cells are not transformed into iPS or totipotent cells; b) Isolating a subpopulation of adherent cells derived from T cells expressing a6 (CD 49 f) or b1 (CD 29) integrin or both; and c) contacting the subpopulation with at least one T cell activating compound.

In another embodiment, the disclosure relates to

[48] A population of regenerative T cells produced by a method comprising: a) Contacting a population of T cells with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC; and (ii) SV40 for a time sufficient to form T cell-derived adherent cells; wherein the T cells are not transformed into iPS or totipotent cells; b) Isolating a subpopulation of adherent cells derived from T cells expressing a6 integrin, b1 integrin, or both; and c) contacting the T cell-derived adherent cell subpopulation with at least one T cell activating compound.

[49] The population of regenerative T cells of any one of [46] to [48], wherein at least 20% of the cells in a subpopulation express integrin α6 or integrin β1; or [50] wherein in the subpopulation, at least 50% of the cells express integrin α6 or integrin β1; or [51] wherein in the subpopulation, at least 20% of the cells express both integrin α6 and integrin β1; or [52] wherein in the subpopulation, at least 50% of the cells express both integrin α6 and integrin β1.

[50] A population of adherent cells of T cell origin, wherein at least 70% of the cells express integrin α6 or integrin β1; or [51] wherein at least 50% of the cells express both integrin α6 and integrin β1; or [52] wherein at least 70% of the cells express both integrin α6 and integrin β1.

[53] A method of treating a patient in need thereof with the T cell population of any one of embodiments [1] to [52 ].

Examples

The following examples are not meant to be limiting, but rather are presented to provide further information and support to the invention. The following examples demonstrate that transient exposure of T cells to cell reprogramming conditions (in which case sendai viruses carrying four Yamanaka factors are used) results in partially reprogrammed T cells of reduced epigenetic age, which have high proliferative capacity and retain antigen specificity. The treated T cells begin to dedifferentiate, express a more common marker in stem cells (e.g., SSEA 4), and attach to the surface of the culture vessel to form epithelial-type colonies. At the same time, the treated cells begin to lose T cell characteristics (loss of CD3 and CD8 expression). Subsequent activation can direct these "T cell-derived adherent cells" to restore their T cell characteristics and exhibit a surprisingly increased expansion potential (250-fold expansion within 6 days).

Example 1: partial reprogramming conditions of T cells

In this experiment, reprogramming conditions for regenerating and improving the biological properties of T cells were explored. CD8 positive T cells isolated from PBMC of a 21 year old male (donor 11360; cells purchased from Allcels, alameda, calif.) with MACS beads (Miltenyi Biotec) were stimulated with soluble CD3 (catalog number 317325, bioLegend) and CD28 in T Cell Medium (TCM) with 60IU/mL IL2; optmizer cell supplement (supplement # 02), immune cell serum replacement (CTS SR), L-glutamine 200mM (100 x), glutaMAX 200mM (100 x), IL-2 (1 ug = 2.1x10e4 IU) in RS 50ug in 105ul, (1 ug = 4.5x10e5 IU) in RS 25ug in 1125ul for 3 days (TCM: optmizer basal medium). All T cell media described in this experiment contained 60IU/ml IL2. This activation process is performed in order to increase transduction efficiency of Sendai virus. Sendai virus (Cytotune iPS 2.0 sendai reprogramming kit), consists of three vectors: 1) Encoding KLF4-OCT3/4-SOX2 (KOS), 2) encoding KLF4; 3) Encoding C-MYC. These are collectively referred to as "Yamanaka factors" and are depicted in the figures as "4 factors" or "4F". In addition, sendai virus vectors encoding SV40 were used to increase reprogramming efficiency.

Activated CD 8T cells are divided into four groups. Group 1 is a control group in which T cells are cultured in T cell medium throughout the process. Group 2 was also not transduced with sendai virus and was cultured from day 1 in IL 2-free stem cell culture medium (SCM) (bFGF-containing StemFit Basic02, ajinomoto). The cells of groups 1 and 2 were plated in triplicate in 96-well plates at 50,000 cells per well. Group 3 is transduced with 23 multiplicity of infection (MOI) EmGFP Sendai virus (Cytotune EmGFP Sendai fluorescent reporter, thermo) and 5MOI SV40 Sendai virus. Group 3 cells were seeded at a density of 50,000 per well in 24 well plates coated in triplicate with recombinant human Laminin511-E8 fragment (iMatrix-511, catalog number T304, takara) according to the manufacturer's protocol. Group 4 was transduced with the 10MOI KOS Sendai vector, the 10MOI KLF4 vector, the 3MOI cMyc vector and the 5MOI SV40 vector. Cells were cultured from day 1 in SCM without IL2 and plated in quadruplicate at a density of 80,000 cells per well in 24-well plates coated with iMatrix-511. See fig. 1 and table 2 for a complete experimental design.

T cells in group 4 form colonies starting on day 3 or day 4. See fig. 2. No cell attachment to the bottom of the coated plate was seen to form colonies in normal T cell culture. T cells under other conditions did not show any colony formation and did not attach to the bottom of the culture plate. FIG. 3 compares the appearance of the adherent cells of group 4 (expressing four Yamanaka factors) with the T cells of group 3 (not expressing Yamanaka factors; but rather GFP). The adherent cells showed colony formation and were larger than T cells in group 3. This indicates that adherent cells of T cell origin are losing T cell properties. The T cell-derived adherent cells of group 4 were also larger than standard activated T cells (fig. 3D). Figures 3A-3F are diagrams showing T cells cultured under various conditions: 3A, 3C, 3E: attached colonies of group 4; 3B: group 3 cells; 3D: t cells cultured under standard T cell activation conditions; 3F: standard iPS cell colonies. As is apparent from fig. 3, the partially reprogrammed cells exhibited a unique morphology. Furthermore, flow cytometry analysis showed reduced expression of CD3 markers in group 2, 3, and 4 cells, all of which were in IL 2-free stem cell medium from day 1. See, for example, fig. 4.CD3 is an important marker of T cell characteristics between lymphocytes. The reduction was evident in T cell-derived adherent cells of group 4. This also indicates that adherent cells in group 4 are losing T cell characteristics. Notably, this is in contrast to other groups of work that have demonstrated that the transient expression of Yamanaka factors does not interfere with cell fate, but can still reverse aging and enhance the efficacy of certain cell types. See, e.g., sarkar et al, 2020,Nat Commun 11,1545.Sarkar and colleagues demonstrated that "transient reprogramming does not interfere with myogenic fate, but can enhance myogenic potential" based on the results of no change in myogenic marker MyoD expression after transient reprogramming. Here, T cell characteristics are at least partially transformed. In addition, as shown by FSC/SSC FACS analysis, the adherent cells of group 4 were slightly larger and more complex in structure (see FIGS. 5 and 6).

Example 2: reactivation of partially reprogrammed T cells

Next, partially reprogrammed T cells were re-activated in T cell medium for 2 days on day five in 96 well plates using one-to-one hundred dilutions of T cells TRANSACTTM (Miltenyi Biotec). On day 5, the cells under the 4 th set of conditions were split into two wells-floating cells were transferred into one well, and the attached cells after exposure to reprogramming conditions were detached from the vessel surface, washed and transferred into the other well. A large number of cells from group 3 die, probably due to the virulence of sendai virus. It is assumed that Yamanaka factors expressed in group 4 are able to rescue cells from sendai virus virulence. After activation, all cells were cultured in T cell medium for five more days. As shown in fig. 5, cells that were losing CD3 and CD8 expression on day 5 of reprogramming regain expression of these genes on day 12. See, for example, fig. 7.

In addition to regaining CD3 expression on day 12, reactivated T cells also exhibited CCR7 and CD62L expression, CCR7 and CD62L being T cell homing markers indicative of the initial T cell population. See, for example, fig. 8.

To assess T cell expansion, cells were counted by counting beads (123 count eBeads count beads, thermo) using flow cytometry on days 0, 5 and 12. Although the adherent cells in group 4 showed abnormal appearance and reduced CD3 expression on day 5, they showed robust cell expansion after activation. As shown in fig. 9, fold change in cell number at day 12 versus day 5 demonstrated an increase in cell expansion in group 4 compared to group 1. This indicates that the partial reprogramming methods described herein increase the T cell expansion potential, especially in adherent cells.

In a similar experiment, PBMC-derived CD 8T cells were stimulated with tranact for 1 day as described above. The next day, reprogramming factors (Yamanaka factor+sv 40) were also introduced into T cells by sendai virus transduction as described above. Infected T cells were transferred to iMatrix coated dishes on day 1 and cultured in either (a) T cell medium +60IU/ml IL2 or (b) iPS cell medium 60IU/ml IL2 for 3 days, then replaced with IL 2-free iPS cell medium from day 4. On day 8, floating cells and adherent cells were harvested for flow cytometry analysis. Specifically, SSEA4 and CD3 expression were analyzed in order to more accurately monitor T cell reprogramming. SSEA4 is a carbohydrate epitope and is considered a broad range of stem cell markers, ranging from partially reprogrammed somatic cells to multipotent stem cell expression. Notably, no attached colonies were detected in condition (a), indicating that culture conditions can have an effect on T cell reprogramming. As shown in fig. 10, day 8 FACS analysis showed 71.9% of adherent cells were positive for CD3 and SSEA4, indicating that adherent cells expressed both T cell lineage markers as well as stem cell markers. Cell populations with high SSEA4 and negative CD3 were observed. Few floating cells in condition (b) expressed SSEA4 compared to adherent cells, and no high CD 3-population of SSEA4 was observed. As expected, SSEA4+ cells were not found after culture condition (a) (standard T cell culture conditions). This data strongly suggests that partially reprogrammed T cells (T cell-derived adherent cells) that form adherent colonies are dedifferentiated (i.e., partially dedifferentiated) and express SSEA4. Our observations are consistent with previous reports of reprogramming into iPS cells using fibroblasts (see, e.g., biol open.2017, 1 month 15; 6 (1): 100-108).

Example 3: measurement of epigenetic age

Horvath and colleagues have demonstrated that methods of analyzing the methylation status of various DNA CpG sites in the genome of cells can be used to estimate the "epigenetic age" (eAge) of cells. (Horvath et al, aging,10 (7): 1758-1775). In this example, eAge of T cells before and after partial reprogramming was calculated using the techniques described by Horvath and colleagues. Prior to partial reprogramming, eAge was estimated to be 21.5 years old, consistent with the true (actual) age of the donor for all experimental groups as described in example 1 and example 2 and summarized in table 1. After reprogramming and subsequent T cell activation, the calculated eAge for group 1 cells is 20.8 years old, while the calculated eAge for adherent cells from group 4 is 10.0 years old (note that cells that adhere to the surface of a blood vessel during the partial reprogramming process fall off the surface upon or shortly after activation. Such cells are referred to as "shedding" cells to distinguish them from floating cells). This indicates that the partial reprogramming process results in regeneration of cells from the measured eAge of 21.5 years old to about 10 years old. Although none of the four Yamanaka factors were used for transduction, the calculated eAge for floating cells in group 4 was 14.5 years old and the calculated eAge for T cells in group 2 was also 14.6 years old. This suggests that the stem cell culture medium itself (StemFit, ajinomoto) contains components that offer some potential for cell regeneration, but its contents are not known. The results are summarized in table 3 below.

Example 4: partial reprogramming beyond day 10 causes the generation of non-conventional T cells

In this example, the time point range of the second stimulus (reactivation) was studied. The experiments are summarized in table 3 below. Sendai virus transduction is as follows: KOS 10MOI, KLF4 10MOI, cMyc 3MOI, SV40 5MOI, and were performed in TCM containing 60IU/ml IL 2. The reprogramming culture conditions were 50/50TCM/iPS medium (Stemfit) containing 60IU/ml IL2 for 3 days followed by culture in Stemfit without IL2 until second activation. After reprogramming for the indicated period, floating cells and adherent cells are harvested. Cells were then activated in TCM+60IU/ml IL2 with TRANSACT at 1:100. 7 days after the second stimulation, the cells were analyzed by flow cytometry for expression of the following markers: CD3, CD4, CD8 a, CD8 β. Fig. 11 is a schematic diagram summarizing the process of PBMC-derived CD 8T cells (see also table 4).

Typically, conventional cytotoxic T cells express CD8 a-CD 8 β heterodimers. Non-conventional T cells, such as intraepithelial lymphocytes (IEL) and γδ T cells express cd8αα homodimers. Thus, in this experiment FACS was used to determine whether T cells generated using the partial regeneration process were able to express canonical CD 8T cell markers. The lack of CD8 a expression after stimulation may indicate that partially rejuvenated T cells are not conventional cytotoxic T cells.

As shown in fig. 12A and 12B, T cell-derived adherent cell colonies re-activated with TCM from day 6-10 expressed CD8 a and CD8 β, indicating a return to normal CD 8T cell phenotype. The delay in reactivation after day 10 of the current reprogramming method to restore dedifferentiated T cells to T cell phenotype resulted in an increase in CD4-CD 8-population (see fig. 12A, upper and lower panels, day 10, where CD8 a expression is moving to the left (lower MFI.) as described above, CD8 a negative cells are not mature T cells after day 10, when CD8 a positive populations are gated, more CD8 a negative cells (cd8a+ non-conventional T cells) are observed (see FACS plot at day 13 in fig. 12B).

Example 5: cytokine production assays and degranulation assays of regenerated NY-ESO-1TCR transduced T cells demonstrated T cell function.

To test whether the partially reprogrammed T cells retain antigen specificity, cd8+ T cells genetically modified to express NY-ESO-1 specific TCRs were partially reprogrammed, stimulated with T cell activating molecules, and measured for response to target cells pulsed with NY-ESO-1 peptides.

Cd8+ T cells from peripheral blood sources from a 42 year old male were purchased from alcels (Alameda, CA). Cells were thawed and stimulated with TRANSACT (1/100) in TCM with IL-2 (60 IU/ml) as described in example 4. The following day, T cells were transduced with NY-ESO-1TCR by lentiviral vector transduction as follows:

For lentiviral transduction, T cells were stimulated with T cells TRANSACT (Miltenyi) diluted 1:100 for 30 hours. The virus was then added to T cells for 24 hours. Stimulation and viral infection was then terminated by adding 7 volumes of fresh medium without TRANSACT and the cells were cultured in Grex-24 plates (Wilson Wolf) for additional 7 days and then cryopreserved at 3X 107 cells/ml in CryoStor CS10 (STEMCELL Technologies) (see also Robbins 2008J Immunol,180 (9) 6116-6131). Control T cells were not transduced and stimulated similarly.

For partial reprogramming and reactivation, NY-ESO-1 specific T cells were thawed and stimulated in TCM with TRANSACT (1/100) containing IL-2 (60 IU/ml). On day 2, T cells were infected with sendai virus (moi=3) containing four Yamanaka factors (KOSM moi=10) and SV40 large T antigen. Cells were then stimulated with TRANSACT on day 9 as described above to obtain regenerated NY-ESO-1 specific T cells (second stimulation). For functional assays, regenerated NY-ESO-1 specific T cells were re-stimulated again with tranact on day 16 to expand them (3 rd stimulation) (see fig. 13).

Cytokine production and degranulation assays were performed on day 26 as follows. Control non-transduced T cells and NY-ESO-1 specific TCR transduced T cells were thawed and stimulated with TRANSACT (1/500) for one week in TCM with IL-2 (60 IU/ml) for expansion. Non-transduced T cells, NY-ESO-1 specific TCR transduced T cells, and regenerative NY-ESO-1 specific TCR T cells were compared to effector cells. Target cells T2 were cultured in T2 medium (RPMI, 20% FCS and P/SM). T2 cells were pre-incubated for 1 hour with or without NY-ESO-1 peptide (SLLMWITQC) (10 nM), washed and counted. One well contained 5x104 effector cells and 1x105 target cells (E: T ratio of 2:1). As a positive control, PMA/ionomycin (Biolegend's cell activation mixture) was added according to the manufacturer's instructions. GolgiPlug and GolgiStop (BD Biosciences) were added according to manufacturer's instructions. In addition, anti-CD 107a Ab-BV421 (1 ul/well) was added to the medium. Cells were cultured in TCM without cytokines for six hours. The co-cultured cells were then subjected to surface antigen staining (PE-NY-ESO-1 tetramer, BUV395-CD8, BUV496-CD4 and BUV805-CD 3). Live/dead eFluor 780 was also added to exclude dead cells. Cells were washed after 20 minutes. Cells were fixed and permeabilized using the BD cytofix/cytoperm kit according to the manufacturer's instructions. Cells were then stained for intracellular cytokine expression (FITC-IFNg, APC-IL-2 and BV 785-TNFa). After 30 minutes incubation, the cells were washed twice and analyzed by flow cytometry using a ZE5 instrument. FACS results were analyzed by FlowJo software. For NY-ESO-1Tg T cells, single cell > living cells > CD3+ > CD8+CD4- > NY-ESO-1 tetramer+ cells and for mock control T cells, single cell > living cells > CD3+ > CD8+CD4- > NY-ESO-1 tetramer cells were gated. As shown in FIG. 14, the frequencies of IFNg+, TNFa+, IL-2+ and CD107a+ were calculated. The results show that the regenerated NY-ESO-1+ transduced T cells retain function and are antigen specific.

Example 6: surface protein profiling of T cells, iPS and partially reprogrammed T cells

In this example, cell surface protein profiles in activated T cells, unstimulated T cells, iPS cells, and day 5 attached partially reprogrammed T cells (T cell-derived adherent cells) were analyzed. The purpose of the screen is to identify markers expressed in the partially reprogrammed cells, which can be used to identify intermediate cells and characterize the partially reprogrammed process and cells produced using this method.

T cell culture medium (TCM) was prepared as described previously. For initial screening, cd4+ and cd8+ T cells (cell ID 306087i & k from alcels) were thawed. T cells were stimulated with 100ng/ml anti-CD 3 and 2ug/ml anti-CD 28 (anti-human CD3 OKT3 and CD28 CD28.2 antibodies from BioLegend, san Diego, calif.) plus 60IU IL-2 for three days. The umbilical cord blood-derived iPS cell line NL5GFP and the tumor-infiltrating lymphocyte (TIL) -derived iPS cell line hi4095#8 were cultured and harvested in Stemfit. T cells and iPS cells were stained with BioLegend LEGENDScreen (BioLegend, san Diego, CA) plus anti-CD 8, CD4, CD3, SSEA4, live/dead stain and expression levels of 371 markers were determined using flow cytometry.

The results of the primary screening identified 39 candidate markers listed below, which were selected based on their expression levels in T cells and iPS cells. The selected markers show significantly higher levels in stimulated or unstimulated T cells than iPS cells or show different expression patterns between iPS cells and T-iPS cells. The ranking of these markers and their relative expression levels in stimulated or unstimulated T cells versus iPS cells is summarized in table 5 below. The data are also graphically displayed in a waterfall plot in fig. 15 (stimulated T cells versus NL5iPS cells) and fig. 16 (unstimulated T cells versus T-iPS cells).

In the second screen, CD4+ and CD8+ T cells (cell ID 306087I & K from Allcels) were thawed and stimulated with 100ng/ml anti-CD 3 and 2ug/ml anti-CD 28 plus 60IU IL-2. Three days later, T cells were transduced with the 10MOI KOS sendai vector, the 10MOI KLF4 vector, the 3MOI cMyc vector and the 5MOI SV40 vector as described in the previous examples. After 5 days, adherent cells were harvested and screened as described above for surface expression of the markers identified in the first screen. Unstimulated T cells, stimulated T cells, NL5iPS cells, and hi4095 iPS cells were used as controls.

The expression of CD164, CD9, CD63, CD90, CD71, CD326, TRA-1-81 and TRA-1-60-R was found to be increased in the partially reprogrammed adherent cells on day 5, while CD352 and CD31 were down-regulated, as compared to stimulated and unstimulated T cells. As shown in fig. 17, this second screen identified CD9 and CD90 as potential indicators of early transition during partial reprogramming, whereas on day 5, the expression levels of iPS cell markers SSEA3 and SSEA4 were known to have not changed significantly. CD9 is also known as four transmembrane protein-29, having four transmembrane domains. It is involved in cell adhesion, signal transduction and cell differentiation. CD90 is also known as THY-1. It is an immunoglobulin superfamily surface glycoprotein, a marker associated with mesenchymal stem cells. It is also involved in cell adhesion and communication. Thus, the expression of these two markers may explain the adhesion properties obtained in dedifferentiated T cells. Expression of these two markers also indicated that partially reprogrammed T cells dedifferentiated into an unknown type of cell that, to the inventors' knowledge, had not been identified as a differentiated cell stage corresponding to the T cell lineage grade.

Example 7: stimulation of signaling is critical for regenerative T cell survival and proliferation

To determine whether stimulation of signaling is critical for enhanced survival and proliferation, T cell adherent cells were subjected to different concentrations of TRANSACTTM (Miltenyi Biotec) activation.

First, T cells were partially reprogrammed using the method described in example 1. Briefly, CD8 positive T cells were stimulated with soluble CD3 and CD28TRANSACTTM (Miltenyi Biotec) for 3 days in T cell medium containing 60IU/ml IL 2. After 3 days, T cells were transduced with 10MOI KOS sendai vector, 10MOI KLF4 vector, 3MOI cMyc vector and 5MOI SV40 vector. Cells were cultured from day 1 in SCM without IL2 and plated in quadruplicate at a density of 80,000 cells per well in 24-well plates coated with iMatrix-511.

On day 7, adherent cells were detached from the surface of the vessel, washed and transferred to another well. Adherent T-cell-derived cells were then re-activated with diluted T-cells TRANSACTTM (Miltenyi Biotec) at a concentration of 1:500 or 1:1000, or incubated in medium without stimulation at all. After activation, all cells were cultured for another nine days in T cell medium (TCM plus 60IU/ml IL 2). As shown in fig. 18A, T cell-derived adherent cells largely lost CD3 and CD8A expression. However, by day 22, expression of the conventional T cell markers CD3 and CD8a was re-acquired with 93% -94% of the TRANSACT-activated cells at either concentration, whereas only 8.4% of the cells cultured in TCM without TRANSACT expressed CD3 and CD8a. In addition, as shown in fig. 18C and 18D, activated T cells showed significantly stronger viability and proliferation capacity compared to partially reprogrammed T cells cultured in T cell medium without TRANSACTTM.

Example 8: regenerative T cells show an effector phenotype after prolonged expansion in the presence of IL-2

In this example, regenerative T cells from different donors were examined for their ability to survive and proliferate over time. Cd8+ T cells from peripheral blood sources from three donors (one 42 years old male, one 37 years old female and one 52 years old male) were purchased from allcels, alameda, CA. Cells were thawed and stimulated with TRANSACTTM (Miltenyi Biotec) at a concentration of 1:500 in TCM with IL-2 (60 IU/ml).

On day 2, T cells were infected with sendai virus (moi=3) containing four Yamanaka factors (KOSM moi=10) and SV40 large T antigen. Cells were then stimulated with TRANSACTTM (1:500) on day 9. Another aliquot of CD8+ T cells from each donor was thawed and stimulated with TRANSACTTM (1:500) in TCM containing IL-2 (60 IU/mL) on day 9 ("control 1") or thawed and stimulated with TRANSACTTM (1:500) on day 0 in TCM containing IL-2 (60 IU/mL) and cultured in TCM containing IL-2 (60 IU/mL) ("control 2"). On day 9, cells of control 2 were stimulated with TRANSACTTM (1:500). Regenerative T cells were allowed to proliferate in vitro for 42 days. After about 3 weeks of in vitro expansion, expansion of non-regenerative T cells reached a steady level. See fig. 19A. In culture, regenerative T cells continued to show significant expansion for up to 42 days. See fig. 19B. This expansion is cytokine dependent and regenerative T cells begin to die within 6 days after IL-2 inactivation. See fig. 20B and 20C.

Cytokine production and degranulation assays were performed as follows on day 36. Cells were cultured in TCM without cytokines, with or without PMA/ionomycin (cell activation mixture of Biolegend). GolgiPlug and GolgiStop (BD Biosciences) are also added according to manufacturer's instructions. The co-cultured cells were then subjected to surface antigen staining (BUV 395-CD8, BUV496-CD4 and BUV805-CD 3). Live/dead eFluor 780 was also added to exclude dead cells. Cells were washed after 20 minutes. Cells were fixed and permeabilized using the BD cytofix/cytoperm kit according to the manufacturer's instructions. Cells were then stained for intracellular cytokine expression (FITC-IFNg and APC-IL-2). After 30 minutes incubation, the cells were washed twice and analyzed by flow cytometry using a ZE5 instrument. FACS results were analyzed by FlowJo software. For all cells, single cell > living cell > CD3+ > CD8+ CD 4-cell was gated. As shown in FIG. 21, the frequencies of IFNg+ and IL-2+ were calculated. The results show that regenerative T cells retain function. Typically, effector T cells express IFNg but do not express substantial amounts of IL2. Thus, these results indicate that the regenerative cells still did not reach the full effector phenotype after 36 days of culture.

Cell surface expression of CD3, CD8b, CD45RA, CCR7 and CD62L was measured by flow cytometry on days 7 and 26. See fig. 22 and 23. Shed T cells that have undergone reprogramming exhibit reduced cd3+cd8b+ expression, but elevated levels of CD45RA and ccr7+cd62l+ expression. By day 26, regenerative T cells showed normal cd3+cd8b+ expression levels, as well as reduced cd45ra+ cells and ccr7+cd62l+ cells levels. See fig. 22 and 23.

Taken together, these data indicate (FIGS. 19-23) that the regenerative cells have high proliferation capacity and are multifunctional after prolonged in vitro culture. The regenerating cells continued to express IL2 (an indication of dryness), but had an effector phenotype as indicated by cell surface markers. Thus, the regenerative cells appear to have dry properties despite prolonged culture and expansion.

Example 9: regenerated NY-ESO-1TCR transduced T cells recognize tumor cells expressing NY-ESO-1

To test whether regenerative T cells retain antigen specificity for tumor cells, CD8+ T cells genetically modified to express a NY-ESO-1 specific TCR were partially reprogrammed, stimulated with T cell activating molecules, and responses to target cells pulsed with the NY-ESO-1 peptide were measured.

Cd8+ T cells derived from peripheral blood from two different donors (# 3 42 years old male and #4 37 years old female) were purchased from alcels (Alameda, CA). 2X106 cells were thawed for each donor and stimulated in TCM with IL-2 (60 IU/mL) in 1 well of a 24 well plate with TRANSACTTM (1:100) as described in example 4. The following day, T cells were transduced with NY-ESO-1TCR by lentiviral vector transduction as follows: first, T cells were stimulated with 1:100 diluted T cells TRANSACT (Miltenyi) for 30 hours. Viruses (MOI: 5) and LentiBOOSTTM A and B (Sirion Biotech) were then added to the T cells for 24 hours. Stimulation and viral infection were then terminated by adding 7ml of TCM containing IL-2 (60 IU/L). On day 2, T cells were infected with sendai virus (moi=3) containing four Yamanaka factors (KOS 10MOI, KLF 10MOI, cMyc 3MOI, SV40 5 MOI) and SV40 large T antigen. Control cells were maintained in TCM with IL-2 (60 IU/mL).

After 16 hours, the infected T cells were washed and transferred to iMatrix coated dishes on day 1 and the medium was replaced with Stemfit without IL-2 (2 mL/well). Sample #3 had a density of 2.6x105 cells/well and #4 had a density of 1.76x105 cells/well in triplicate. On day 3, stemfit (2 mL/well, 4mL total) was added.

On day 5, floating cells were removed and adherent cells were harvested according to the following protocol. The medium was aspirated and washed twice with 2mL PBS per well. 1mL of EDTA was added and the mixture incubated at 37℃for 5 minutes. Cells were collected by P1000 pipetting, with a final density of 6.1x105 in 3 wells of #3 and 1 in 1 well of #4 of 1.9x106. This resulted in about 2x105 cells/well in a 96 well U-shaped bottom microplate. Cells were then activated in TCM with IL-2 (60 IU/mL) at 1:500 using TRANSACT. Control non-Sendai infected NY-ESO-1Tg cells were restimulated in the same manner.

On day 6, the cell densities were too high, so they were split into two wells in a 96-well microplate. TCM with IL-2 (60 IU/mL) was added. On day 8, cells from two wells of a 96-well microplate were pooled into one well of a 48-well plate and TCM containing IL-2 (60 IU/mL) was added. On day 12, cells were transferred to 24-well plates such that the cell density was 2x106 cells/well. TCM containing IL-2 (60 IU/mL) was added to each well. Cells were counted and fold changes calculated every three to four days. See fig. 24. The cell number was adjusted to-2 x106 cells/well and TCM containing IL-2 (60 IU/mL) was added to make the volume 2 mL/well.

On day 19 (14 days after the second stimulation), target cells T2 expressing endogenous nyso 1 and Mel624HLA-A 02:01 positive tumor cells were cultured in T2 medium (RPMI, 20% FCS and P/SM). T2 cells were pre-incubated for 1 hour with or without NY-ESO-1 peptide (SLLMWITOC) (10 nM), washed and counted. One well contained 5x104 effector cells and 1x105 target cells (E: T ratio 1:2). As a positive control, PMA/ionomycin (Biolegend's cell activation mixture) was added according to the manufacturer's instructions. GolgiPlug and GolgiStop (BD Biosciences) were added according to manufacturer's instructions. In addition, anti-CD 107a Ab-BV421 (1. Mu.L/well) was added to the medium. Cells were cultured in TCM without cytokines for six hours. The co-cultured cells were then subjected to surface antigen staining (PE-NY-ESO-1 tetramer, BUV395-CD8, BUV496-CD4 and BUV805-CD 3). Live/dead eFluor 780 was also added to exclude dead cells. After 20 minutes, the cells were washed. Cells were fixed and permeabilized using the BD cytofix/cytoperm kit according to the manufacturer's instructions. Cells were then stained for intracellular cytokine expression (FITC-IFNg, APC-IL-2 and BV 785-TNFa). After 30 minutes incubation, the cells were washed twice and analyzed by flow cytometry for expression of CD45RA, CD45RO, CCR7 and CD62L cell surface markers using a ZE5 instrument. FACS results were analyzed by FlowJo software. For NY-ESO-1Tg T cells, single cell > living cells > CD3+ > CD8+CD4- > NY-ESO-1 tetramer+ cells and for mock control T cells, single cell > living cells > CD3+ > CD8+CD4- > NY-ESO-1 tetramer cells were gated. Fig. 25. As shown in FIGS. 26A-26D, the frequencies of IFNg+, TNFa+, IL-2+ and CD107a+ were calculated. This experiment demonstrated that regenerative nyso 1T cells were antigen specific and recognized nyso 1 positive T2 and Mel624 target cells (fig. 24-26) (see also fig. 14). These data also confirm that regenerating cells continue to express IL2 (an indication of dryness), but have an effector phenotype as shown by cell surface markers.

Example 10: NY-ESO-1TCR transduced T cells regenerated by antigen specific stimulation

To test whether antigen-specific T cells can be regenerated using antigen-specific stimulation, cd8+ T cells are isolated from one or more donors genetically modified to express NY-ESO-1 specific TCRs. For each donor, donor cells were thawed and stimulated with TRANSACTTM in TCM containing IL-2 (60 IU/mL) in 1 well of a 24 well plate. One day later, T cells were transduced with lentiviruses expressing the NY-ESO-1T cell receptor. NY-ESO-1 specific T cells are then co-cultured with T2 cells (T2+NY-ESO-1 cells) that have been pulsed with the NY-ESO-1 peptide. Next, the cells were enriched for NY-ESO-1 specific T cells (group 1) or maintained as a heterogeneous mixture of NY-ESO-1 specific T cells and untransduced T cells (group 2). During reprogramming, the cells were again co-cultured with T2+NY-ESO-1 cells. The cells were then infected with sendai virus (moi=3) containing four Yamanaka factors (KOS 10MOI, KLF 10MOI, cMyc 3MOI, SV40 5 MOI) and SV40 large T antigens. Control cells were maintained in TCM with IL-2 (60 IU/mL) and co-cultured with T2+NY-ESO-1 cells.

In the absence of any further stimulation, NY-ESO-1 specific T cells are capable of forming T cell-derived adherent cells in the presence of T2+NY-ESO-1 cells. Adherent cells were harvested according to the protocol described in example 9 and then re-activated by co-culture with T2+ NY-ESO-1 cells.

Example 11: regeneration enhances T cell stem properties in tumor infiltrating lymphocytes

In this example, tumor Infiltrating Lymphocytes (TILs) were isolated from a 66 year old donor and regenerated as follows. TIL was enriched by culturing the tumor fragments in T cell medium for 2 weeks. T cells were activated by overnight co-culture with autologous tumor-derived antigen presenting cells. T cells were then FACS sorted and CD137 (4-1 BB) positive cells isolated. After one day (day 0), cells were not disturbed or transduced with four Sendai viruses expressing KOS (KLF 4, OCT3/4, SOX 2), KLF4, cMyc and SV40 as described previously. The cells were then transferred to stem cell medium (StemFit) and cultured for 7 days. T cell-derived adherent cells were harvested on day 7 and re-activated with TransAct (1:500) in T cell medium for two more days. From day 9, cells were cultured in normal T cell medium. On day 21, regenerated cells or control cells (which underwent the same procedure except that the cells were not transduced with the four sendai viruses) were evaluated for CD62L, CCR7 and TCF7 expression using flow cytometry. Regenerated TIL showed enhanced proliferation (fig. 27A) and enhanced expression of dry phenotypic markers (fig. 27B) relative to the non-regenerated control. In this experiment 73.0% of regenerated TIL co-expressed CCR7 and CD62L,64.5% of regenerated TIL co-expressed CCR7 and TCF7, which is significantly higher than the non-regenerated TIL population, which only showed 2.94% CCR7 and CD62L co-expression and 3.07% CCR7 and TCF7 co-expression. These data indicate that TIL, which is generally characterized by low proliferation capacity and a exhaustion phenotype marker, can be successfully regenerated to enhance proliferation and dryness.

Example 12: dynamic analysis of epigenetic clock during T cell regeneration

In this example, CD8+ T cells from a 53 year old male, a 55 year old male, and a 50 year old male were human T cells at a cell density of 100 ten thousand cells/ml in 48 well plates(Miltenyi Biotec) was stimulated in T Cell Medium (TCM) containing 60IU/ml IL-2 at 1:500 dilution. All TCM's described below contain 60IU/ml IL-2. After 26.5 hours of activation, the cells were divided into two groups. A set of regeneration protocols; the other group was used as a control, without any viral manipulation and throughout the culture in TCM.

Sendai virus (SeV) containing Yamanaka factor and SV40 was transduced with KLF4-OCT3/4-SOX2 at 10MOI, KLF4 at 10MOI, cMyc at 3MOI and SV40 at 5MOI into regenerated samples (day 0) and then incubated at 37 ℃. After 16.5 hours, the cells were washed and suspended with stem cell medium. Cells were seeded at 50,000 cells/well (corresponding to a count of 50,000 on day 0) onto iMatrix-511 coated 24-well plates and cultured at 37 degrees celsius (day 1). 500ul of SCM was added on day 3 and day 5.

On day 7, to shed adherent cells, 1ml of TrypLE Express (Thermo catalog number 12604013) was added and the cells incubated at 37 degrees celsius for 10 minutes. The exfoliated cells were then harvested by pipetting. Floating cells in the supernatant were also maintained and mixed with shed cells. A portion of the cells was preserved to obtain DNA to determine eAge. Human T cell tranact diluted 1:500 in 500ul TCM at a density of 100 ten thousand cells/ml activates 50 ten thousand control and regenerative sample cells in 48 well plates. On day 9, cells were transferred to 12-well plates and 1ml TCM was added per well. On day 11, cells were transferred to 6-well plates. Cells were then cultured in 6-well plates.

Cell samples were collected prior to the pre-activation step of SeV transduction and collected on days 7, 13 and 18 for epigenetic clock analysis. Cells from each time point were pelleted and kept at-20 degrees celsius until DNA extraction. DNA was extracted from frozen cell pellets using PureLink genomic DNA mini kit (Invitrogen, K182002). Each extracted DNA was split into 3 branches and the technique for methylation set analysis was repeated. Control conditions for donor 1 on day 18 and donor 3 on days 7 and 18 are present for only 2 technical replicates due to the lack of sufficient DNA. Samples were prepared for epigenetic analysis by Illumina Infinium array. CpG methylation status data was analyzed using the Horvath method as described in Horvath et al 2018,Aging 10,1758-1775; horvath and Raj,2018,Nature Reviews Genetics,19:371-375 to obtain skin and blood clock values.

Cells were stained with fluorescent conjugated antibodies and reactive dyes and cell phenotypes were obtained by flow cytometry in Cytek Aurora.

CD8 positive T cells from three male donors (53 years, 55 years and 50 years) were transduced with Yamanaka factor and SV40 according to the regeneration protocol depicted in fig. 28 and compared to controls in non-transduced condition at each time point. The regenerative cells showed reduced CD3 and CD8b expression on day 7. T cell normal marker expression was completely restored in the regenerative cells at day 13 and day 18 after they were shed and stimulated (fig. 29A and 29B). Thus, partially reprogrammed cells regain expression of conventional T cell markers only 6 days after reactivation.

Skin and blood epigenetic clocks (highly accurate age estimators) were performed and analyzed in DNA samples of 3 male donors to check the epigenetic age (eAGE) at 4 time points of the regeneration protocol (fig. 30A-30C).

eAge of donor 1, an actual age of 53 years (y/o), was shown to be 45.1y/o prior to Sev transduction pre-activation. Although eAge in the control cells that did not regenerate after the activation of the tranact showed no change, [ ranging from 43.3 to 45.7y/o (change 2.4 years) ], eAge of the 7 th astrovirus-transduced cells (regenerative cells) showed the least age, i.e., 30.9 years of age, gradually increased and reached 46.2 years of age at day 18. The difference between the control and regenerated eAGE on day 7 was 12.9y/o. This is an age reduction of about 24.3% as measured by horvat clock analysis.

eAge ranges from 43.8 to 50.0y/o (change 6.2 years) in control cells of donor 2 at an actual age of 55 y/o. Likewise, the minimum age, 34.4y/o (12.9 years less than the control eAGE on day 7) was achieved on day 7 by regeneration. Consistent with the results of donor 1, eAge gradually increased.

The actual age of donor 3 was 50y/o. eAge of non-regenerative control cells varied from 46.0 to 51.4 (5.4 years of change), while the minimum age in regenerative cells on day 7 was 31.5y/o, 19.9 years less than control eAge on day 7. In summary, among all three donors, the rejuvenated cells showed the smallest eAge on day 7, and then the eAge gradually increased. Donor 1 rejuvenated cells reached the control level at day 18, while in the case of the other two donors, rejuvenated cells continued to be younger than the control.

As demonstrated by the above results, partially reprogrammed T cells lost common T cell markers (e.g., CD3 and CD8 b) as measured on the day of shedding (i.e., day 7). However, after re-stimulation on day 7 and culture in T cell medium, T cell adherent cells completely re-acquired T cell markers by day 13 or at day 13. The epigenetic clock shows the smallest epigenetic age (50 years, 53 years and 55 years, respectively) among the three donors tested on day 7. The results of this example demonstrate that, although a gradual increase in eAge was observed after day 7, the rejuvenated cells were still younger than the control when they displayed a complete T cell phenotype on day 13.

Example 13: regeneration of NY-ESO-1 transduced T cells

This example demonstrates that T cells can be genetically engineered to express TCRs specific for a desired target (e.g., nyso 1) and regenerated using the methods herein.

Healthy donor CD4/CD 8T cell activation: on day 1, healthy human donor CD4 and CD 8T cells from two male donors (24 and 35 years old) were thawed, washed once and resuspended in pre-warmed complete TCM+IL-2 (60 IU/ml) (day 3). The cells were diluted to 2e6 cells/ml. 10ul/ml of Transact (1/100 dilution) was added to stimulate CD4 and CD 8T cells (see FIG. 31). On day 2, stimulated cd8+ and cd4+ T cells were counted and 7.5e+5 cells were suspended in 0.75ml tcm+il-2. Lentiviral vectors encoding nyso 1-specific TCRs were prepared using standard protocols. Lentiviral supernatants encoding NYESO1 (7.5 ul, MOI; 10) and LentiBoost A and B (7.5 ul each) were added. The next day, 5.25ml of fresh TCM+IL-2 (6 ml total) was added.

Reprogramming of NY-ESO-1 transgenic (Tg) T cells and collection thereof: on day 0, 4e+5 cells were collected and resuspended in TCM+IL-2 in 48-well plates.The sendai reprogramming kit was used for reprogramming (KOS: moi=10, klf4: moi=10, c-MYC: moi=3, and SV40: moi=5). The following day (i.e., day 1), cells were collected, resuspended in stem cell medium and seeded in iMatrix coated 6-well plates (two wells per condition). On day 3, new stem cell medium was added and on day 5, half of the medium was replaced with new stem cell medium. Control cells were kept in TCM+IL-2 culture. On day 7, floating cells were collected, wells were washed with PBS, trypLE was added, and adherent cells were shed. The cell numbers of all collected cells (i.e., floating cells + shed cells) were counted. The shed cells were restimulated with Transact (1/500 dilution) in 500ul of TCM+IL2 (in 48 well plates). Harvested on day 11Cells were pooled, resuspended in 1ml of new TCM+IL-2 and transferred to 6-well plates. Cell counts and medium changes were performed every 3-4 days. On day 13, 1/20 cells were surface expression stained with antibodies to CD3, CD62L, CD RO, HLA-A02:01NYESO1 MHC tetramer, cd197, CD4, CD8a, CD45RA, and TCF1 was stained intracellular, fixed and permeabilized using the Foxp3 staining kit as described according to the manufacturer's protocol, stained with Tcf7 antibody and analyzed by FACS. See, for example, fig. 32. Genomic DNA was extracted from 1e+6 cells per condition using the PureLink genomic DNA mini kit as described. The DNA was treated with rnase and evaluated for epigenetic age analysis as previously described.

One vial of stock T2 cells was thawed and washed once and cultured in complete RP10 medium for one week. In a 5% CO2 incubator, 5e+6 cells were resuspended in full RP10 with or without 10nM NY-ESO1 peptide for 2 hours. After 2 hours, cells were washed and resuspended in TCM.

On day 19, control and regenerated NY-ESO-1TCR Tg T cells were analyzed for their ability to produce cytokines when co-cultured with target cells. 5 e+4T cells were co-cultured with 1e+5T 2 cells with or without the NY-ESO-1 peptide. Wells containing no target cells and wells containing PMA/ionomycin (cell activation mixture) were added as negative and positive controls, respectively. CD107a antibodies were added to the medium. After 6 hours of co-culture, the cells were stained with a surface antibody mixture, fixed and permeabilized as described in the protocol of the manufacturer by BD kit, stained with an intracellular antibody mixture and analyzed by FACS (Cytek Aurora). The data was analyzed using Flowjo software. The gating strategy is as follows: lymphocytes > single cells > live/dead- > cd3+ny-ESO-1 tetramer+.

On day 19, control and regenerative NY-ESO-1TCR Tg T cells were assayed for their ability to kill target cells using sequential stimulation. Specifically, control and regenerative T cells were counted. 50,000 NY-ESO-1 tetramer+ T cells were co-cultured with 20,0000 NY-ESO-1+ HLA A02:01+ target cells (A375-nucleic Red (NLR) or H1703-NLR) at 1:4E:T in 24 well plates. Every 3-4 days, 25% of the culture was transferred to new plates and fresh target cells were seeded at the initial seeding density. Target clearance was quantified using Incucyte.

The results showed that the regenerated NY-ESO-1TCR Tg T cells had a low differentiation phenotype. Specifically, on day 13, regenerated NY-ESO-1 TCR-transduced T cells (detected by binding of NY-ESO-1 tetramers) contained a higher percentage of ccr7+cd62l+ and tcf1+ populations, indicating a low differentiation phenotype. See, for example, fig. 32A and 32B. Control and regenerated NY-ESO-1TCR Tg T cells were maintained in TCM+IL-2 for prolonged culture. The medium was changed every 3-4 days and the cells were kept at a concentration of 1-2e+6/ml. Regeneration of NY-ESO-1TCR Tg T cells takes longer to begin to proliferate, possibly due to recovery from reprogramming. However, over time they proliferate more than 100-fold over control non-regenerative cells. See, for example, fig. 33. The epigenetic age of control and regenerated NY-ESO-1TCR Tg T cells was analyzed as depicted in FIG. 34. The regenerative cells showed a phenotype of 8-18 years old at day 19 compared to control T cells. More specifically, for the first donor (fig. 34A), the epigenetic age of the regenerative CD 4T cells (T cell-derived adherent cells) at day 7 showed a 68% decrease in age compared to eAge of control cells (eAge at day 19). On day 19, regenerative cd4+ T cells showed a 33% decrease in age compared to control cells. The epigenetic age of the regenerative CD8T cells showed a 70% decrease in age on day 7 compared to control cells. On day 19, regenerative CD8T cells showed a 45% decrease in age compared to control cells. In the second donor (fig. 34B), the epigenetic age of the regenerative CD 4T cells (T cell-derived adherent cells) on day 7 showed a 62% decrease in age compared to control cells. On day 19, regenerative cd4+ T cells showed a 27% decrease in age compared to control cells. The epigenetic age of regenerative CD8T cells (T cell-derived adherent cells) showed a 68% decrease in age compared to control cells at day 7. On day 19, regenerative CD8T cells showed a 67% decrease in age compared to control cells.

In addition, it was observed that regenerated NY-ESO-1TCR Tg T cells produced more cytokines (IL-2, IFNg and TNFa) after co-culture with target cells in the presence of the NY-ESO-1 peptide (FIG. 35). Control and regenerated NY-ESO-1TCR Tg T cells were analyzed for production of cytokines (IL-2, TNFa, IFNg) by co-culturing them with T2 cells (HLA A02:01+) with or without the NY-ESO-1 peptide as described above. Neither control nor regenerated NY-ESO-1TCR Tg T cells were co-cultured with T2 cells without peptide to produce cytokines. A higher percentage of regenerative NY-ESO-1TCR Tg T cells did produce IL-2, IFNg and TNFa when co-cultured with T2 cells in the presence of the NY-ESO-1 peptide. See, for example, fig. 35.

See generally, FIGS. 31-37. FIG. 31 is a graphical representation of NY-ESO-1Tg CD4 and CD8T cell regeneration experiments. CD4 or CD8T cells were stimulated by Transact and transduced with NY-ESO-1TCR prior to reprogramming. From day 1 to day 7, cells were cultured under iPS cell culture conditions. On day 7, cells were collected, counted, re-stimulated by Transact and maintained in TCM+IL-2 culture. On day 19, control and regenerative cells were analyzed for cytokine production and cytotoxic activity. Regenerative CD4 and CD8T cells were mixed under conditions of sequential killing assay to examine their synergistic effect.

FIGS. 32A and 32B illustrate that regenerated NY-ESO-1Tg CD4 (FIG. 32A) and CD 8T cells (FIG. 32B) exhibit a low differentiation phenotype. On day 13, control and regenerated NY-ESO-1tg CD4 and CD 8T cells were analyzed by FACS for surface markers and Tcf1 intracellular expression. Cells were gated according to lymphocyte > single cell > live/dead- > NY-ESO-1 tetramer+ and plotted as CD4 x CD8a, CCR7 x CD62L and CCR7 x Tcf1.

FIG. 33 illustrates that regenerative NY-ESO-1Tg CD4 and CD 8T cells proliferated more than control NY-ESO-1Tg CD4 and CD 8T cells. NY-ESO-1Tg CD4 and CD 8T cells were regenerated and cultured as described, and the cell numbers were counted every 3-4 days. The graph depicts fold changes over time compared to day 7 after transduction of reprogramming factors and their corresponding control cells. Representative data for four different donors.

FIG. 34 illustrates that by epigenetic age analysis, regenerated NY-ESO-1Tg CD4 and CD 8T cells showed a younger phenotype than the actual age of control NY-ESO-1Tg CD4 and CD 8T cells and the donor. The figures show the mean and s.d. of age values analyzed from methylation status of certain CpG sites.

FIG. 35 illustrates that regenerative NY-ESO-1Tg CD4 and CD 8T cells produced more cytokines (IFNg, IL-2 and TNFa) when co-cultured with T2 cells in the presence of the NY-ESO-1 peptide. On day 19, control or regenerative NY-ESO-1Tg CD4 and CD 8T cells were cultured alone or with PMA/ionomycin, T2 cells in the presence or absence of the NY-ESO-1 peptide in the presence of a Golgi transporter inhibitor. After staining for surface antigens, cells were fixed, permeabilized and stained by intracellular antibodies. The frequency of each cytokine-positive NY-ESO-1 tetramer+ cell is depicted.

FIG. 36 illustrates that, following repeated co-culture with NY-ESO-1 expressing target cells (A375-NLR), regenerative NY-ESO-1Tg CD8T cells persist and retain their cytotoxic activity longer than control cells. Control or regenerative NY-ESO-1Tg CD8T cells were co-cultured with the A375-NLR cell line. Every 3-4 days, 25% of the previous culture was transferred to new plates with fresh targets. UsingThe living cell analysis system monitors the growth of the target and uses a basic software analysis module (Base Software Analysis Module) for analysis. The graph shows the number of a375-NLR per image for each condition. Representative data for two different donors. NLR: nubright Red.

FIG. 37 depicts the enhanced cytotoxicity of regenerative NY-ESO-1Tg CD8T cells caused by the addition of regenerative NY-ESO-1Tg CD4T cells. Regenerated NY-ESO-1tg CD4 and CD 8T cells were co-cultured with the a375-NLR cell line at E: t=1:4. Under one condition, exogenous IL-2 (10 IU/ml) was added. Under another condition, regenerative CD4 and CD 8T cells were mixed at a ratio of 1:1 and co-cultured with an a375-NLR cell line at E: t=1:2. Every 3-4 days, 25% of the previous culture was transferred to new plates with fresh targets. UsingThe living cell analysis system monitors the growth of target cells and uses a basic software analysis module for analysis. The graph shows the number of a375-NLR per image for each condition. Representative of Sex data were from two different donors.

From this example, it can be concluded that the regenerated NY-ESO-1TCR Tg T cells show a low differentiation phenotype and proliferate more than the control NY-ESO-1TCR Tg T cells. In addition, the epigenetic age of the regenerated NY-ESO-1TCR Tg T cells was lower than that of the control NY-ESO-1TCR Tg T cells. Regenerated NY-ESO-1TCR Tg T cells also produced more cytokines (IL-2, IFNg and TNFa) when co-cultured with target cells pulsed with relevant nyso 1 peptide. In addition, the regenerative nyso 1 tcr+ cells retain cytotoxic activity for a longer period of time after repeated exposure to target antigen cells than non-regenerative control cells. In addition, regenerative NY-ESO-1TCR Tg CD8T cell activity was enhanced by the addition of IL-2 or regenerative NY-ESO-1TCR Tg CD4T cells.

Example 14: cytokine identification

This example was performed to evaluate cytokine production by regenerative T cells.

Regeneration of PBMC. On day-2, 1X106 donor cells were thawed and incubated in 1mL of TCM+IL-2 (60 IU/ml) per well of a 24-well plate for 24 hours. The next day (i.e., day-1) cells were counted and T cells were activated with Cell TransAct. Human (Miltenyi) REA 107:500 followed by incubation for 24 hours. On day 0, sendai vector transduction (MOI: KOS 10, KLF4 10, c-MYC 3, SV40 5) was performed, and matrix coated (iMatrix 511) plates were prepared by diluting iMatrix 511 (57.6ul iMatrix+9ml PBS). 500ul of diluted iMatrix was added to each well of a 24-well plate and kept at 37 ℃ for >1 hour. On day 1, cells were pelleted and dispensed into iMatrix coated plates containing 500uL of stem cell medium+bfgf per well. On day 3, 500ul of fresh stem cell medium and bFGF were added to each well of the 24-well plate. On day 7, all floating cells were harvested without suspension and 1ml of ltryple Express was added to shed cells. Cells were incubated at 37℃for 10 min, and then diluted by adding 500ul PBS per well. Cells were harvested and 1ml of PBS was added thereto. Cells were suspended and harvested, and floating cells were combined with shed cells. Cells were counted, pelleted and resuspended with TCM+IL-2 (60 IU/ml) and reactivated with T Cell Transact. Human (Miltenyi) (1:500). The supernatant was harvested and frozen. On days 11, 14 and 18, cells were pelleted, harvested and resuspended with fresh TCM+IL-2 (60 IU/ml).

Quantitative analysis of adaptive immune-related cytokines. Supernatants from control and regenerative cells on days 7, 11, 14 and 18 were analyzed by Isoplexis Codeplex adaptive immune secretory proteome chip. The results are depicted in fig. 38A and 38B. In both donors, D7T cell-derived adherent cells showed relatively low levels of GM-CSF, IFN-g, IL-5, IL-13, MIP-1a, and MIP-1b expression. The expression of these cytokines gradually increased at D18 to levels comparable to control cells. The expression patterns of IL-6, IL-8 and TNF-. Beta.in T-cell derived adherent cells (D7 cells) were different from control cells. These results indicate that adherent cells of T cell origin are of a different cell type than the control cells.

Quantitative analysis of innate immunity-related cytokines. Supernatants from control and regenerative cells on days 7, 11, 14 and 18 were analyzed by Isoplexis Codeplex innate immune secreted proteome chips. The results are depicted in fig. 39A and 39B. T cell-derived adherent cells (D7 cells) showed relatively low IFN-g, MIP-1a expression levels in both donors.

Example 15: selective regeneration of antigen-specific T cells

This example was performed to investigate whether antigen stimulation of specific T cells would cause selective regeneration of those cells.

Establishment of autologous LCL. PBMCs depleted of healthy human donors CD4 and CD8 were thawed, washed once and resuspended in pre-warmed complete RP10 medium. 1e+6 cells were cultured in 1ml of complete RP10 medium in 24-well plates. The supernatant of human gamma herpes virus 4 (HHV-4) (ATCC) was thawed and 250ul was added to the culture. After 4 days, 500ul Chen Peiyang base was aspirated and new full RP10 (500 ul) was added. The medium was changed every three or four days until the cells began to proliferate. After three weeks, the cells began to proliferate and transferred to a culture flask. 2e+6 LCLs were frozen per bottle.

Step A: healthy donor CD 8T cell activation. Healthy human donor CD 8T cells were thawed, washed once and resuspended in pre-warmed complete TCM+IL-2 (60 IU/ml). The cells were diluted to 2e6 cells/ml. 10ul/ml of Transact (1/100 dilution) was added to stimulate CD 8T cells.

And (B) step (B): transduction of NY-ESO-1 TCR. The next day, stimulated CD 8T cells were counted and 1e+6 cells were suspended in 1ml tcm+il-2. Supernatants from lentiviral vectors encoding NYESO1-TCR (10 ul, MOI; 9.7) and LentiBoost A and B (12.5 ul each) were added to suspension cells. The following day, transduction efficiency was determined by FACS. Specifically, 7ml of TCM+IL-2 was added to dilute the virus. On day 7, cells were stained using the antibody mixtures described in Table 6 below and analyzed for NY-ESO-1TCR expression. See fig. 40.

Table 6: antibody cocktail staining of NY-ESO-1TCR transduced regenerative T cells

Material	ul/test
		CD3-BUV805(SK7)	0.5
CD4-PECy7(RPA-T4)	0.5
		CD8b-BV421(2ST8.5H7)	0.5
PE-iTAg MHC tetramer HLA-A02:01NY-ESO-1	1
		Fixable reactive dye eFluor 780
BD Horizon Brilliant staining buffer Plus	5

Step C: reprogramming.

Mitomycin C (MMC) treated autologous LCL and pulsed with NY-ESO-1 peptide. Autologous LCLs were thawed, washed once and cultured in complete RP10 medium for one week. 1e+7 cells were resuspended in 2ml of pre-warmed complete RP10 medium supplemented with MMC (final concentration 50 ug/ml) and incubated in a 5% CO2 incubator for 1 hour. After one hour, cells were collected in a conical tube to which 10ml of PBS was added. The cells were centrifuged and the supernatant aspirated. The cells were then suspended in full RP 10. Cells were cultured in a 5% CO2 incubator for 2 hours in full RP10 with or without 10nM NY-ESO1 peptide. After 2 hours, cells were washed and resuspended in TCM+IL-2.

NY-ESO-1TCR transduced T cells were activated by transactor or autologous LCL pulsed with or without the NY-ESO-1 peptide. On day-2, NY-ESO-1TCR Tg CD8T cells from step B (transduction of NY-ESO-1 TCR) were counted, resuspended in tcm+il-2, and seeded into 24-well plates with 5e+5 cells/well. Four different conditions were tested; non-stimulated, transact (1/500 dilution), autologous LCL without NY-ESO-1 peptide (1e+6 cells (T cells: lcl=1:2)), and autologous LCL pulsed with NY-ESO-1 peptide (1e+6 cells (T cells: lcl=1:2)). The following day (day-1) Transact activated T cells were collected, washed with 10ml PBS, resuspended in 1ml TCM+IL-2 and kept in culture. T cells co-cultured with autologous LCL were collected and isolated using EasySep ^TM Human T cell isolation kits enrich for T cells, are resuspended in TCM+IL-2 and remain cultured. See fig. 41.

Ny-ESO-1TCR Tg T cells were reprogrammed using the Cytotune reprogramming kit. On day 0, 2.4e+5 cells were collected from each condition and resuspended in TCM+IL-2 in 96U-shaped bottom plates. Reprogramming was performed using the Cytotune sendout reprogramming kit (KOS: moi=10, klf4: moi=10, c-myc: moi=3, and SV40: moi=5). The next day (day 1), cells were collected, resuspended in stem cell medium and seeded in iMatrix coated 6-well plates (2 wells per condition). On day 3, new stem cell medium was added and on day 5, half of the medium was replaced with new stem cell medium (fig. 3A).

Step D: the shedding and restimulation of partially reprogrammed NY-ESO-1TCR Tg T cells.

Collection of partially reprogrammed cells. On day 7, floating cells were collected, wells were washed with PBS, trypLE was added, and adherent cells were shed. The cell numbers of all collected cells (floating cells + shed cells) were counted. All cells were then used for reactivation.

Restimulation of partially reprogrammed cells. Cells previously stimulated with either Transact or autologous LCL containing the NY-ESO-1 peptide were re-stimulated with either Transact or autologous LCL containing the NY-ESO-1 peptide. Autologous LCLs were treated with MMC and pulsed with ny_eso-1 peptide as previously described. 1e+5 cells/well were restimulated in 200ul TCM+IL2 (96U-shaped bottom plate) by no stimulation/Transact (1/500)/LCL P-/LCL P+ (E: T=1:1). On day 10, half of the medium was replaced with fresh TCM+IL-2. Cells were collected on day 14, resuspended in 1ml of new tcm+il-2 and transferred into 24-well plates. Cell counts and medium changes were performed on days 18 and 21.

Surface phenotyping of regenerative T cells. On day 14, 1/20 of the cells were stained with the antibodies described in Table 6, and then the frequency of NY-ESO-1TCR tetramer positive (TE+) cells was analyzed by FACS.

The results show that stimulation of TCR by autologous lcl+ny-ESO-1 peptide prior to reprogramming produced more partially reprogrammed cells on day 7. NY-ESO-1TCR transduced CD 8T cells containing about 60% NY-ESO-1TCR positive cells (as shown in FIG. 40) were stimulated by either Transact or autologous LCL with or without the NY-ESO-1 peptide as described above. On day 7, cells (floating cells and adherent cells) were collected and counted (see fig. 41 and 42). The conditions stimulated with Transact and autologous LCL with NY-ESO-1 peptide showed 5.5-fold and 8.7-fold increase in cell number, respectively, compared to the cell number on day 0 (day of sendai virus infection), whereas fold changes of 0.62 and 1.6 were observed in the conditions without stimulation and autologous LCL without peptide, respectively. The autologous LCL expresses epstein barr virus antigen and therefore epstein barr virus specific T cells can receive TCR stimulation when co-cultured with autologous LCL without peptide.

The restimulation of partially reprogrammed NY-ESO-1Tg T cells resulted in preferential regeneration of NY-ESO-1TCR+ cells. On day 7, partially reprogrammed cells, including adherent cells and floating cells, were collected and used as described Or autologous LCL with or without NY-ESO-1 peptide. On day 14, 1/20 of the cells were harvested and analyzed for expression of NY-ESO-1TCR using the NY-ESO-1 tetramer as described above.

T cells stimulated with antigen (lcl+nyesao 1 peptide) at either step had a higher frequency of NY-ESO-1 tetramer + cells (see fig. 43 and 44, 78.6% versus 97% or 97.1%) compared to cells stimulated with tranact non-specificity at the pre-activation and reactivation steps (i.e. day-2 and day 7), whether primary stimulation was non-specific (with tranact) or antigen-specific (with LCL + peptide) (see fig. 43).

Regenerated NY-ESO-1Tg T cells proliferate. Partially reprogrammed T cells re-stimulated with Transact or autologous LCL containing peptide (first stimulation with Transact) were cultured as described until day 21. Proliferation curves are shown in fig. 45. Regenerated T cells re-stimulated with autologous LCL containing the NY-ESO-1 peptide proliferate those re-stimulated with Transact.

Example 16: regeneration of CD19-CAR T cells

This example generated CD19 CAR-T cells and underwent a partial reprogramming process, followed by assessment of proliferation and cytotoxicity. Regenerative CD19 CAR T cells showed higher proliferation compared to controls, as previously shown with NY-ESO-1tg CD8T cells. The cytotoxicity of regenerative CD19 CAR T cells was tested by repeated co-culture with target cells expressing CD19 (Nalm 6). After repeated co-culture, regenerative CAR T cells showed higher persistence and cytotoxicity.

Materials and methods

Step A: healthy donor CD 8T cell activation. Healthy human donor CD 8T cells were thawed, washed once and resuspended in pre-warmed complete TCM+IL-2 (60 IU/ml) (day-3). The cells were diluted to 2e6 cells/ml. 10ul/ml of Transact (1/100 dilution) was added to stimulate CD 8T cells.

And (B) step (B): transduction of CD 19-CAR: one day after stimulation with TRANSACT (day-2), stimulated CD 8T cells were counted and 3e+5 cells were suspended in 1ml of TCM+IL-2. Lentiviral vectors encoding CD19 CAR and truncated EGFR were generated using conventional methods. Viral supernatants (6 ul, MOI;) and LentiBoost A and B (10 ul each) were added to the activated cells. The next day, 0.7ml of virus-containing medium was removed and 1.8ml of fresh TCM+IL-2 was added. On day 5 post CAR transduction, cells were stained with the antibody mixture in table 7 below and CAR expression was analyzed by idiotype antibody and EGFR antibody. (FIG. 46)

Table 7: antibody mixture surface staining of CD19 CAR transduced regenerative T cells

CD19 CAR Tg T cells were reprogrammed with the Cytotune reprogramming kit. On day 0, 3e+5 cells (donor # 1) or 4e+5 cells (donor # 2) were collected and resuspended in tcm+il-2 in 48 well plates. Reprogramming was performed using the Cytotune sendout reprogramming kit (KOS: moi=10, klf4: moi=10, c-myc: moi=3, and SV40: moi=5). The following day cells were collected, resuspended in stem cell medium (StemFit) and seeded onto iMatrix coated 6-well plates (2 wells/condition). Fresh stem cell medium was added on day 3 and half of the medium was replaced on day 5. Control cells were kept in TCM+IL-2 culture.

Step D: restimulation of partially reprogrammed CD19 CAR Tg T cells. On day 7, floating cells were collected, wells were washed with PBS, trypLE enzyme was added, and adherent cells were shed. The collected cells (floating cells + shed cells) were counted. See fig. 47. 5e+5 cells were stored at-80C for DNA age examination. Harvested cells (1.7e+6 and 2.3e+6 for donor #1 and #2, respectively) were restimulated with Transact (1/500 dilution) in TCM+IL-2. 1e+6 control cells were stimulated in the same manner. On day 10, half of the medium was replaced with fresh TCM+IL-2. Cell counts and medium changes were performed every three or four days on and after day 13. Cells were cultured at a concentration of 1-2e+6 cells/ml.

FACS analysis of regenerative T cells. On day 13, 1/20 of the cells were stained with surface antibody (TCF 7-PB (C63D 9), commercially available from Cell Signaling Technology), fixed and permeabilized using Foxp3 staining kit as described in the manufacturer's protocol, stained with TCF7 antibody and analyzed by FACS.

Intracellular cytokine production assay of regenerative CD19 CAR Tg T cells. On day 20, control and regenerative CD19 CAR Tg T cells were analyzed for their ability to produce cytokines when co-cultured with CD19 antigen specific target cells. 5 e+4T cells were co-cultured with 1e+5 Colo205 (CD 19-) or Nalm6 (CD19+) cells at a 1:2E:T ratio (see FIG. 49). Wells containing no target cells and wells containing PMA/ionomycin (cell activation mixture) were added as negative and positive controls, respectively. After 5 hours of co-culture, the cells were stained with surface antibodies, fixed and permeabilized as described in the manufacturer's protocol using BD kit, stained with intracellular antibody mixtures (table 7) and analyzed by FACS (Cytek Aurora). The data was analyzed using Flowjo software. The gating strategy used is as follows. Lymphocytes > single cells > live/dead- > cd3+egfr+.

Step F: the ability of CAR-T cells to repeatedly clear target cells was determined using sequential stimulation.

Sequential killing assay of regenerative CD19CAR Tg T cells. On day 20, control and regenerative CD19CAR Tg T cells were analyzed for their ability to kill target cells using sequential stimulation. Control and regenerative T cells were counted. 50,000 EGFR+CAR T cells were coated with 20,0000 CD19+ targets (Nalm 6-NLR) at 1:4E:T with Poly-D-LysiCo-culturing in 24-well plates of n. Every 3-4 days, 25% of the previous culture (10% co-culture at 4 and 5) was transferred to new plates, which were inoculated with fresh target cells at the initial inoculation density. UsingThe living cell analysis system quantifies target clearance and uses a basic software analysis module for analysis.

Results

Regenerative CD19CAR T cells showed a low differentiation phenotype. Healthy donor cd8+ T cells were activated and transduced with CD19 CARs, reprogrammed for 7 days, cells shed and re-stimulated with Transact as described above. On day 13, regenerated CD19CAR transduced T cells (distinguished by expression of EGFRt) had a higher percentage of ccr7+cd62l+ and tcf1+ populations, indicating that the regenerated CAR-T cells had a less differentiated, more stem-like phenotype. (see FIG. 47)

Regenerative CD19CAR T cells proliferated more than control CD19CAR T cells. Control and regenerative CD19CAR Tg T cells were cultured in tcm+il-2 and cell counts were monitored over time. The medium was changed every 3-4 days and the cell concentration was adjusted to be kept at 1-2e+6/ml. Although regenerative CD19CAR Tg T cells initially showed a delay in proliferation rate, day 25 marked an inflection point, after which by day 55, regenerative cells showed more than 100-fold expansion over control. (see FIG. 48)

Regenerative CD19 CAR T cells produce considerable levels of cytokines after co-culture with target cells expressing CD 19. The production of their cytokines (IL-2, TNFa, IFNg) was analyzed by co-culturing control and regenerative CD19 CAR T cells with CD19 expressing target cells (Nalm 6) or CD19 free target cells (Colo 205) as described above. Neither control nor regenerative CD19 CAR Tg T cells produced cytokines when co-cultured with control Colo205 cells. However, both groups produced considerable levels of cytokines when co-cultured with Nalm6, except IFNg. (see FIG. 49). Notably, IFNg production was higher in the rejuvenated cells compared to the control.

Regenerative CD19 CAR T cells persist and retain their ability to kill target cells longer than control cells after repeated stimulation (co-culture) with CD19 expressing target cells. In both donors, regenerative and control CAR T cells effectively kill and clear target cells until round 4 or round 5 co-culture. However, on the 6 th and 7 th rounds of restimulation, the rejuvenated cells maintained excellent cytotoxicity and persistence, whereas the control cells did not. Overall (see fig. 50A and 50B), these studies demonstrate that regeneration processes utilizing partial reprogramming produce CD19 CAR T cells with a more stem-like phenotype, higher proliferative capacity, higher persistence and cytotoxicity than control cells.

Example 17: mSev contrast Sev

In this example, T cells were regenerated using sendai virus vectors modified to express four Yamanaka factors from a single polycistronic expression (i.e., "mSev") vector and compared to regeneration using the Cytotune kit as described in the previous examples.

As previously described, the Cytotune-iPS 2.0 sendai reprogramming kit (i.e., "Sev") comprises 3 separate viral vectors: (KOS encoding Klf4, OCT4 and SOX 2; cMyc; klf4 for reprogramming in general, another Sendai virus (Sev) expressing SV40 was also used to inhibit apoptosis and increase reprogramming efficiency. Using the cytotune kit, four separate vectors must be transduced into T cells. Transduction with four separate viruses was found to result in uneven co-transduction, inefficient reprogramming, and significant variation in post-production, as discussed in further detail below.

Thus, T cells were subjected to the regeneration process described herein using either (a) a polycistronic sendai vector expressing four Yamanaka factors (but not SV 40) or (b) Cytotune Sev (+sv 40).

Both experimental groups showed similar proliferation curves 23 days after shedding. Polycistronic immortal desk groups showed a slightly higher stem T cell phenotype (tcf1+ccr7+). These data indicate that polycistronic sendai vectors, even without SV40, which increase reprogramming efficiency, can be used for partial reprogramming and regeneration of T cells.

Step A: t cell regeneration. Cd8+ T cells from a 53 year old male (donor 1) and a 55 year old male (donor 2) were stimulated with human T cell tranact (Miltenyi Biotec, catalogue No. 130-111-160) at a cell density of 100 ten thousand cells/mL in 48 well plates at a 1:500 dilution in T cell medium containing 60IU/mL of IL-2. In this example, the inventors found that 24 hours of activation resulted in the most efficient transduction using polycistronic sendai virus vector (mSev). After 26 hours of stimulation with TransAct, the cells were divided into two groups (i.e., day 0). One group of cells underwent a regeneration process using the sendai virus vector (Sev or "comparative example 17") from the CytoTune kit, while the other group was regenerated using the polycistronic sendai virus vector (mSev). Sev regeneration groups were transduced with 10MOI Klf4-Oct3/4-Sox2, 10MOI Klf4, 3MOI cMyc (Cytotune iPS 2.0 sendai reprogramming kit), and 5MOI SV40, whereas the MSev group was transduced with 5MOI mSev expressing all four Yamanaka factors but not SV 40. The cells were then cultured at 37 ℃. After 16.5 hours, the cells were washed and suspended in stem cell culture medium (SCM; bFGF-containing Stemfit Basic 04). Cells were seeded at 50,000 cells/well (corresponding to day 0 count) on an iMatrix-511 (PEPROTEC catalog No. RL 511S) coated 24-well plate and cultured at 37 ℃ (i.e. day 1). 500ul of SCM was added on days 3, 5, 7, 9. On day 7 1ml of SCM was aspirated. For restimulation, two groups were detached and incubated at 37 ℃ for 10 min using 1ml of TrypLE Express (Thermo catalog number 12604013) on days 7, 9 and 11. The exfoliated cells were collected by pipetting. In a 96-well round bottom plate, 1x105 cells were activated with a 1:500 dilution of human T cell tranact in 200uL of T cell medium at a density of 5x105 cells/ml. Two days after shedding, cells were transferred to a 12-well plate and 1ml of T cell medium was added to each well. Four days after shedding, cells were transferred to 6-well plates. Thereafter, the cells were cultured in a six-well plate with a T cell medium containing 60IU/ml IL 2.

And (B) step (B): flow cytometry analysis. Cells were stained with fluorescent conjugated antibodies and reactive dyes (see table 8 below). Cell phenotypes were obtained by flow cytometry in Cytek Aurora.

Table 8: antibodies and reactive dyes for staining

Step C: DNA extraction and epigenetic analysis

DNA was extracted from frozen cell pellets using PureLink genomic DNA mini kit (Invitrogen, K182002). Each extracted DNA was split into 3 branches and the technique for methylation set analysis was repeated. Samples were sent to AKESOgen, inc for epigenetic analysis by Illumina Infinium array. CpG methylation status data was analyzed using the Horvath method as described in (AGING 2018, vol.10, no. 7, 1758-1774) to obtain skin and blood clock values.

Step D: analysis

Cd8+ T cells from 2 male donors aged 53 and 55 were transduced with Sev or mSev, shed and restimulated on days 7, 9 and 11 (see fig. 51). After shedding and restimulation, the conventional T cell markers CD3 and CD8B were completely restored in both sets of regenerative cells 6 days after shedding and reactivation (see fig. 52A and 52B).

Cells were cultured for 23 days after shedding.

Both Sev and mSev transduced groups showed similar proliferation curves for cells shed on day 7 (fig. 53A) and day 9 (fig. 53B). For cells shed on day 11 (fig. 53C), the mSev group showed higher proliferation. It was observed that Sev reprogramming resulted in larger colonies when shed and in cell yields that were 3-4 times the yields measured in the mSev group. more colonies were found in the mSev group, but smaller colonies were found. Without being bound by a single theory, it is believed that the difference in cell count between Sev and mSev groups is probably caused by the lower expression level of reprogramming factors of the mSev vector and the absence of SV40, SV40 increasing reprogramming efficiency. Overall, the proliferation curves of Sev and mSev groups were similar, indicating that mSev reprogrammed cells acquired proliferation capacity similar to regeneration.

Sev and mSev appear to have different reprogramming rates. Specifically, on day 11 shedding (see fig. 52A and 52B), cells in group Sev lost more CD3 and CD8B cell surface expression than mSev group (see fig. 52A). This suggests that more cells in group Sev were reprogrammed to an unrecoverable extent compared to group mSev, resulting in less cells in group Sev responding to TranAct and proliferating. These data also indicate that reprogramming with the mSev vector is slower than reprogramming with Sev, and that mSev reprogramming requires more time to form enough T cell-derived adherent cells and regenerate partially reprogrammed cells. Without wishing to be bound by a single theory, the observed differences may be caused by the expression levels of reprogramming factors in the polycistronic vector and/or the absence of SV40, as discussed above. Modification of the vector to increase expression of reprogramming factors and/or addition of SV40 may reduce the number of days required to achieve the desired reprogramming.

Regarding cell phenotype, mSev showed higher tcf1+ccr7+ and higher ccr7+cd62l+ populations in the cd3+cd8b+ population, indicating a more stem-like population in the mSev group (see fig. 54A and 54 BA).

Determination of epigenetic age by Horvath clock analysis showed that mSev transient reprogrammed T cells had reduced epigenetic age when shed on day 7, day 9 and day 11 compared to control cells (see figure 55). As shown in fig. 55, the decrease in eAge for each group is summarized in table 9 below:

as shown in table 9, the decrease in eAge in the mSev group was lower than that observed in the Sev group. This is not surprising given that the mSev vector requires a longer time to reprogram. As discussed above, modifying the vector to increase expression of the reprogramming factors may cause further decrease in eAge of the transiently reprogrammed T cells.

Thus, this example and the accompanying data indicate that polycistronic sendai vector can be used for T cell regeneration.

Example 18: transcriptome characterization of regenerative cd8+ T cells

In this example, a high-dimensional transcriptional analysis of single cells and bulk RNAseq was used to characterize regenerative T cells as compared to control T cells. Overall, analysis of the clustered single cell RNA-seq data showed that, seven days after reprogramming, the regenerative T cells had an overall phenotype that was distinct from the control T cells. Most of the reprogrammed cells expressed four Yamanaka factors on day 7, but by day 13, the expression of the transduced Yamanaka factors was at background levels. Expression of one of the Yamanaka factors-C-MYC-was observed on day 13, but was confirmed to be endogenous (i.e., non-transduced) C-MYC. By day 13, the regenerative cells recovered as T cells expressing classical T cell markers, but did not express lymphocyte/bone marrow lineage markers in iPSC-derived T cells (Maeda et al, 2016). Single cell RNA-seq results were confirmed by batch RNA-seq analysis. On days 7 and 13, enrichment of several metabolic gene sets was seen in the regenerating cells. Overall, the transcriptional analysis emphasized that T cell adherent cells were distinct from corresponding control T cells on day 7 after Yamanaka factor transduction. After D13 reactivation, the regenerative cells express T cell markers and genes rich in metabolic gene sets, with loss of Yamanaka factor expression.

T cell regeneration

In this example, two independent experiments were performed using four donors.

In the first experiment, CD8 positive T cells from a 54 year old male donor (donor number 18698) were subjected to the regeneration process described below. Cells were divided into two groups, one group regenerated as described below and the other group used as a control.

In a second experiment, CD8 positive T cells from 53 year old male donor, 55 year old male donor and 50 year old male donor (donor numbers 11347, 12254 and 2621, respectively) were subjected to the same protocol.

For both experiments, CD8 positive T cells from each donor were stimulated with human T cells TransAct (Miltenyi Biotec) at a cell density of 100 ten thousand cells/mL in a 48 well plate at a 1:500 dilution in T cell medium containing 60IU/mL IL-2. After 24 hours of activation with tranact, cells from each donor were divided into 2 groups, one group was subjected to the regeneration process as described herein, and the other group was used as a control (not transduced with sendai vector) and cultured in T cell medium. The control group was activated with TRANSACT in the pre-activation step, cultured in TCM with IL2, and then re-activated with TRANSACT at the same time as the partially reprogrammed cells were re-activated (after the 7 th day of de-watering).

In the regeneration group, sendai virus (SeV) containing four Yamanaka factors and SV40 was transduced (i.e., day 0) with Klf4-Oct3/4-Sox2 at 10MOI, klf4 at 10MOI, cMyc at 3MOI (Cytotune iPS 2.0 Sendai reprogramming kit, thermo) and SV40 at 5MOI (ID Pharma, tsukuba, japan) and then cultured at 37 ℃. After 16 hours, the cells were washed and suspended with stem cell culture medium (bFGF-containing Stemfit Basic 02, ajinomoto Co., tokyo, japan). Cells were seeded at 50,000 cells/well (corresponding to a count of 50,000 on day 0) onto an iMatrix-511 (PEPROTEC) coated 24-well plate and incubated at 37 ℃ (day 1). On days 3 and 5, 500ul of SCM was added to the culture. On day 7, T cell-derived adherent cells were incubated with 0.5ml of TrypLE Express (Thermo) for 10 minutes at 37 ℃ to detach the cells from the culture dish. The exfoliated cells were then harvested by pipetting. Floating cells in the supernatant were also harvested and mixed with shed cells. Floating cells in the first experiment were discarded due to insufficient cell numbers.

In the second experiment, 50 ten thousand control and regeneration samples of cells were activated with human T cell TransAct diluted 1:500 in 500ul of T cell medium at a density of 100 ten thousand cells/ml in 48 well plates, whereas the number of cells in 48 well plates in the first experiment was 364,050 cells/well due to the small number of cells. Both control cells and regenerative cells were seeded at the same density. Cells were cultured to day 13 (beginning with day 7 shedding) and samples were taken at 2 time points (day 7 and day 13) and subjected to dead cell removal for transcriptome analysis as described below. Cells were allowed to continue to culture to day 17 of experiment 1 and to day 29 of experiment 2. Cells were counted on days 7 and 13 for the first and second experiments. Cells were also counted on day 18, day 21 and day 29 for the second experiment.

Dead cells were removed using the EasySep dead cell removal (annexin V) kit (STEMCELL technique) according to the manufacturer's instructions. Samples were treated twice with EasySep to increase viable cell purity.

Transcription profiling of T cells by batch RNA-seq

Cells from regenerated samples and control samples from four male donors were also collected on days 7 and 13 for batch RNA-seq.

At each collection time point, cells were subjected to dead cell removal (as described above) and approximately 50,000 cells per sample were collected in the lysis-binding mixture prepared according to MagMAX mirVana total RNA isolation kit protocol (Thermo Fisher Scientific) and stored at-80 ℃ until treatment.

Total RNA was extracted from cells using the MagMAX mirVana RNA kit on a Kingfisher Flex system and stored at-80 ℃. RNA quality and quantity were assessed on a 2100BioAnalyzer using the Agilent RNA 6000Pico kit (Agilent). Libraries for mRNA sequencing were prepared with 5ng total RNA for cDNA generation using the SMART-Seq v4 ultra low input RNA kit (Takara Bio USA) using the manufacturer's protocol automated on the Biomek i7 workstation. cDNA evaluations were performed on a 2100BioAnalyzer using a high sensitivity DNA kit (Agilent) and a 150pg full-length cDNA was used to prepare a barcoded library for sequencing using a Nextera XT DNA library preparation kit (Illumina) using the manufacturer's protocol automated on the Biomek i7 workstation. Quality assessment was performed using the DNA1000 Screen Tape assay (Agilent) with 4200TapeStation system. The barcoded library across the sample was then multiplexed, purified in equimolar libraries, and sequenced using the NovaSeq 6000 system.

Single cell CITE-Seq analysis

Single Cell CITE-Seq data was processed using 10X Cell range software version 5.0.1 (10X Genomics) with genome reference co-worker construct 38 (GRCh 38) as the reference genome and default parameters. The reference genome is supplemented with transduced Yamanaka factor sequences and control sequences from the sendai virus backbone. The cell gene matrix was further processed using the setup package in R (see Hao et al 2021). Cell level quality control and outlier removal were performed by visual selection of thresholds for mitochondrial percentage, ncount_rna, nfeature_rna, ncount_adt, and tag doublet. Cells of the regenerated samples and control samples collected from four male donors were pooled for single cell transcriptome analysis on days 7 and 13. The filtered cell gene matrix was globally scaled with a "normazedata" function, scale.factor parameter set to default value 10000. For each donor sample, the effects of cell cycle heterogeneity were corrected by calculating the cell cycle phase fraction (g 2m. Score, s. Score) in the course of the "cellcycle" function, followed by regression on the cell cycle phase fraction and mitochondrial read percentage in the function. Genes associated with either of the two cell cycle phase scores (Pearson correlation coefficient greater than 0.25) were excluded from the selected features to further minimize the effects of cell cycle heterogeneity. Mitochondrial, ribosomal, TCR and IG complex related genes were also excluded from the selected profile.

To correct for possible batch effects and account for donor heterogeneity, datasets from each donor on days 7 and 13 were combined with "findsegregatennas" and "IntegrateData" function calls using the first 30 typical correlation analysis dimensions. The integrated data is then scaled and the first 30 PCs are calculated by the "RunPCA" function using the filtered features. Cells were then mapped to two-dimensional space for visualization using Unified Manifold Approximation and Projection (UMAP) by the "RunUMAP" function in semat, with each point representing one cell. The cells were subjected to a cluster analysis using the "FindClusters" function in Seurat. The CITE-Seq assay is also referred to in the report as single cell RNA-Seq assay, because the assay (UMAP, clustering) is done in CD8+ T cells using RNA expression in the absence of protein expression.

Single cell phenotype assessment

After single cell cluster analysis, the expression level of the marker gene encoding Yamanaka factor was visualized using the "featurelets" function. Expression of conventional and non-conventional T or B cell markers observed on human induced pluripotent stem cell-derived cells (Themeli et al 2013, maeda et al 2016) was assessed by thermal map visualization implemented in the pHeatmap package (pHeatmap CRAN). Pseudo-bulk gene markers for regenerative cells were identified using "findwarks" in semoat. Cells positive for the significant gene set were visualized with the "AddModuleScore" function in semat.

Single cell read alignment was visualized with an integrated genomics browser (see, e.g., thorvaldsd hatir et al, 2013).

Batch RNA sequence analysis

Gene expression was generated from sequencing data using standard processing tubing. Splice trimming was performed using trim_gain (Krueger-giloub) and alignment was performed using STAR (see, e.g., dobin et al, 2013) with a reference genome compiled from GRCh38 sequences supplemented with over-expressed Yamanaka factor sequences and control sequences from sendai virus backbone. Quantification of gene expression was performed by RSEM (see, e.g., li and dewey.2011) with default parameters. Differential analysis was performed using the DESeq2 (Love et al 2014) package from which statistics such as log2 fold change of expression (log 2 FC) and adjusted p-value (padj) were calculated. Enrichment of metabolic gene sets (MSigDB version 7, sulbrannian et al 2005) was performed with the "enricher" function implemented in the clusterif iotaler package (see, e.g., yu et al 2012).

Phenotype evaluation of regenerative cells.

CD8 positive T cells from four male donors were transduced with Yamanaka factor and SV40 (i.e., regenerative cells) according to a regenerative protocol and compared to corresponding controls at each time point under non-transduced conditions. In experiments 1 and 2, cells were grown to day 17 and day 29, respectively. As shown in fig. 56, consistent with the previous examples, the rejuvenated cells showed increased proliferation compared to the control cells.

Single cell RNA-seq analysis was performed on cd8+ T cells from control and regenerative cells on days 7 and 13 using cells from all four donors (donor numbers 18698, 11347, 12254 and 2621). Bulk RNA-seq was performed from the same samples used in the second experiment described above (i.e., 53 years old, 55 years old and 50 years old men in the second experiment).

Cells at the end of seven-day partial reprogramming showed a phenotype different from the corresponding control T cells. Single cell RNA-seq UMAP plots show that regenerative cells clustered individually with control cells (see figure 57A). On day 7 there were two regenerative cell clusters, one containing most of the cells expressing Yamanaka factors (oct4.sox2, KLF4 and C-MYC) and the other cell cluster negative for these factors (see fig. 57A). Downregulation of exogenous reprogramming factors after day 7 was observed in both single cell (see fig. 57B) and bulk (see fig. 57C) RNA-seq data. Notably, some single cells expressed cMYC on day 13 after redirecting to T cell state (see figure 57B, graph labeled Myc). We have demonstrated that C-MYC expression is primarily endogenous after reprogramming, rather than from transduced C-MYC (fig. 57D). From day 7, the major alignment peak for single cell RNA-seq reads was exon 1 (data range, 0-66547), while the alignment peak on day 13 was from the 5' UTR of C-MYC (data range 0-2596). Transduced C-MYC started from exon 1, while endogenous cMC was expected to start from the 5' UTR, confirming that C-MYC seen on day 7 was transduced, while that seen on day 13 was endogenous. These data indicate that regenerative T cells are still expressing four Yamanaka factors prior to shedding, resuspension and activation (day 7). Exogenous expression of these factors was not detected on day 13. Although regenerative T cells are still expressing C-MYC, the source of expression is endogenous.

The expression of lineage specific lymphoid/myeloid marker genes in regenerative and control T cells was then assessed in single cell and bulk RNA-seq data (thermeli et al 2013, maeda et al 2016). Regenerative cells (e.g., T cell adherent cells) shed on day 7 showed reduced expression of CD3 and CD8 b. T cell lineage markers completely recovered in regenerative cells on day 13 after shedding and stimulating them. Abnormal expression of non-conventional, NK, T or B cell markers (e.g., NCAM1, NCR2, FCGR3A, KIR DL4, KIR2DS 4), such as those observed on human iPSC-derived T cell products (Themeli et al 2013, maeda et al 2016), is not seen in regenerative T cells. Thus, regenerative T cells generated using the partial reprogramming methods described herein do not aberrantly express the non-conventional markers NCAM1, NCR2, FCGR3A, KIR DL4, KIR2DS4.

Differentiating human ipscs into traditional mature T cells has been challenging, involving the use of 3D organoid cultures to provide a suitable environment for the orderly typing (commit) and differentiation required for T cell differentiation (Montel-Hagen et al 2019). In addition, T cells differentiate from ipscs to produce T cells expressing aberrant NK, T or B cell markers (thermeli et al 2013, maeda et al 2016). The data demonstrate that not only does the partial T cell reprogramming process avoid time consuming reprogramming to ipscs and the use of a complex T cell re-differentiation system, but surprisingly, the process yields T cells that do not express abnormal markers, have reduced epigenetic age compared to control and starting T cells, and have increased proliferation capacity after T cell activation.

The rejuvenated cells were then evaluated to determine if they were metabolically different from the corresponding control T cells on days 7 and 13. The metabolic gene sets corresponding to oxidative phosphorylation, fatty acid metabolism, glycolysis and hypoxia were significantly enriched in regenerative cells on days 7 and 13 (see fig. 58A). Projection of these metabolic gene sets onto single cell RNA-seq data indicated that on day 7, the regenerating cells were enriched in glycolysis and oxidative phosphorylation. Surprisingly, by day 13, the regenerating cells were transformed into either hyperphosphorylation or hyperglycolysis (see fig. 58B), but the cells were still significantly enriched for both gene sets compared to the control cells.

In addition, the initial gene set in the regenerative cells and corresponding control T cells was evaluated on day 13 (Gattinone et al 2011Nature Medicine 17 (10): 1290-1297). The gene set corresponding to the initial T cell phenotype was enriched in the regenerative cells on day 13 (see fig. 59A and 59B). In addition, relative T cell receptor pool diversity was estimated by Simpson clonality (Wong et al, J.Immunology V:197. Pages 1642-1649). T cell pool diversity was estimated using Simpson clonality indicators ranging from 0 to 1, where 0 represents a completely homogeneous sample and 1 represents a monoclonal sample. Lower Simpson clonality was observed in the regenerated samples on days 7 and 13 compared to control T cells, indicating increased TCR pool diversity. (see FIG. 75).

Thus, the regenerative T cells of this example are enriched for expression of genes associated with higher metabolic activity compared to control cells. The data in this example also shows that regenerative T cells are better suited for using energy and synthesizing nucleotides required for proliferation, and further illustrates the significantly increased proliferation capacity observed in regenerative cells compared to controls. Oxidative phosphorylation and enrichment of glycolytic gene sets is related to metabolic transitions during reprogramming of somatic cells to ipscs (see, e.g., nishimura 2019int.j. Mol. Sci.20:2254; doi:10.3390/ijms 20092254) and indicates that regenerative cells may have acquired some stem cell-related qualities without reprogramming to the fully pluripotent iPSC stage. Furthermore, regenerative T cells are enriched for the gene set corresponding to the initial T cell phenotype and have increased polyclonality. Taken together, the data demonstrate that the regenerative process described herein produces T cells with improved and advantageous characteristics for use in the treatment of adoptive cell therapies.

Example 19: regeneration enhances T cell stem properties in tumor infiltrating lymphocytes

In this example, TIL was regenerated from many different tumor types (lung adenocarcinoma, colorectal carcinoma, liver carcinoma, melanoma, colorectal carcinoma metastasis to the liver) using the procedure described in example 11, with the differences shown in table 10 below.

Tumor samples were purchased from synthetic human tissue networks (CHTN). TIL is freshly isolated and 1) cd45+ immune cells (e.g., T cells) are enriched by magnetic bead isolation and used directly; 2) Enrichment of cd45+ immune cells (e.g., T cells) by magnetic bead isolation followed by activation with tranact (1:500) for 1 or 2 days; or 3) isolation and amplification in a medium containing 6000IU/ml IL-2 for 5 to 17 days. Because tumor samples are small, enrichment and/or expansion is required to generate enough cells for the regeneration process. For the pre-activation step, the TIL is activated by one of the following outlined in table 10 below:

1) TransAct (1:500 dilution, unless otherwise indicated; in some experiments, a dilution of 1:2000 was used. There was no significant difference between the two dilutions);

2) Co-culture with autologous tumor organoids prepared essentially as follows: tumor tabletThe segments were finely chopped on ice, washed with PBS and embedded with Matrigel on ice, forming domes in 6-well plates, each of about 20ul. After curing at 37℃for 20 minutes, 2mL of Intersticult was covered on the dome ^TM Organoid growth medium (human) (Stemcell Technologies). In the case where the tumor fragments cannot be finely minced, the tissues were digested with collagenase IV (200 IU/mL; (Worthington Biochemical Corporation) at 37℃for 30 minutes, then autologous tumor cell lines were washed and embedded, followed by sorting of 4-1BB+ cells by FACS.

The TIL is then regenerated as follows: TIL from the pre-activation step was transduced with Klf4-Oct3/4-Sox2 at 10MOI, klf4 at 10MOI, cMyc at 3MOI (Cytotune iPS 2.0 sendai reprogramming kit, thermo) and SV40 at 5 MOI. The cells were then cultured at 37 ℃. After about 16 hours, the cells were washed and suspended in stem cell culture medium (SCM; bFGF-containing Stemfit Basic 02). Cells were seeded at about 50,000 cells/well (corresponding to day 0 cell count) onto an iMatrix-511 (PEPROTEC) coated 24-well plate and cultured at 37 ℃ (day 1). TIL was detached on day 7 with enzyme (1 ml of TryplE Express (Thermo)) and incubated for 10 min at 37 ℃. The exfoliated cells were harvested by pipetting. 1E5 cells were activated with a 1:500 dilution of human T cell TransAct in a 96 well round bottom plate at a density of 5X105 cells/mL in 200ul of TCM supplemented with 60IU/mL IL 2. As shown in the correlation graphs (fig. 60, 62, 64, and 68), cell counts were performed at various time points until day 30. The expression of the surface markers associated with dryness (CD 62L, CCR, TCF 7) was measured by flow cytometry on the days marked in fig. 61, 63 and 65. Note that at an earlier point in time there is often insufficient cell number to assess phenotype.

For experiment 6, the epigenetic age determination was performed as follows: DNA was extracted from frozen cell pellets using PureLink genomic DNA mini kit (Invitrogen, K182002). Each extracted DNA was split into 3 branches and the technique for methylation set analysis was repeated. Samples were sent to AKESOgen, inc for epigenetic analysis by Illumina Infinium array. CpG methylation status data was analyzed using the Horvath method as described in ((1) Horvath Genome Biology 2013,14:R115; 2) doi: 10.1371/journ.fine.0014821) to obtain skin and blood epigenetic clock values (see FIGS. 66 and 67)

As shown in fig. 60-68, the data from this example demonstrates the results and conclusions previously outlined in example 11 and shown in fig. 27, TIL can be regenerated using the methods herein and the data expanded to several different tumor types, including lung adenocarcinoma, colorectal cancer, liver cancer, melanoma, and colorectal cancer metastasis to the liver. The results show that regenerated TIL exhibits enhanced proliferative capacity, increased expression of dry-associated phenotypic markers, and reduced epigenetic age.

While over time, the enhanced expression of dry phenotypic markers decreases and the epigenetic age gradually increases with amplification, TIL is typically depleted, terminally differentiated and difficult to amplify. The ability of TIL to acquire enhanced proliferative capacity, increased expression of stem-associated phenotypic markers, and reduced epigenetic age following a regenerative process suggests that the regenerative process may be useful in improving the therapeutic potential of TIL in adoptive cell therapy applications.

^a ++ + is remarkable in superior to the control; ++ was better than control; + is comparable to control; no proliferation

^b Dry phenotype analysis;

^c horvath clock epigenetic age (eAge) analysis

Example 20: regeneration of tumor infiltrating lymphocytes using mSev

In this example, TIL was regenerated using polycistronic sendai vector (mSev) encoding four Yamanaka factors (without SV 40) as described in example 17.

TIL from 52 and 83 year old female donors (donor 6 and donor 8; see table 10) was isolated from tumor samples by mechanical dissociation on ice followed by enzymatic digestion for about 30 to about 90 minutes, either directly (i.e., "batch") or enriched for cd45+ immune cells (e.g., T cells) by magnetic bead separation followed by expansion with a tranact.

For donor 8 (83 year old female), TIL was amplified by culturing in T cell medium containing 6000IU/ml IL-2 and for 1 month. For the pre-activation step prior to transduction, TIL was activated with human T cells TransAct (Miltenyi Biotec) at 1:500 dilution for 1 day. Pre-activated TIL was then transduced with mSev expressing 4 Yamanaka factors at 5MOI without SV 40. The cells were then cultured at 37 ℃.

After about 16 hours, the cells were washed and suspended in stem cell culture medium (bFGF-containing Stemfit Basic 02) and seeded at 50,000 cells/well (corresponding to day 0 count) on an iMatrix-511 (PEPROTEC) coated 24-well plate and cultured at 37 ℃ (i.e. day 1).

On day 11, T cell-derived adherent cells were detached using 1mL of Tryple Express (Thermo), and incubated at 37℃for 10 minutes. The exfoliated cells were then harvested by pipetting. Human T cell tranact was activated by 1:500 dilution of 1x105 cells in 200ul T cell medium at a density of 5x105 cells/mL in 96 well round bottom plate. Two days after shedding, cells were transferred to a 12-well plate and 1mL of T cell medium was added to each well. Four days after shedding, cells were transferred to 6-well plates. Thereafter, the cells were cultured in a 6-well plate with T cell medium. On days 14 and 18, cells were stained with fluorescent conjugated antibodies and reactive dyes and cell phenotypes were obtained by flow cytometry in Cytek Aurora.

On days 14 and 18, mSev regenerating cells showed a higher tcf7+ccr7+ population in the cd3+ population, indicating a more stem-like population in the mSev group compared to the control (see fig. 69A and 69B).

Thus, this example indicates that the mSev vector can be used for regeneration of TIL.

Example 21: integrin alpha 6 and integrin beta 1 as markers for regenerative T cells

In this example, it was assessed whether integrin α6β1 could act as a marker for T cell adherent cells, which might contribute to cell adhesion and the low differentiation phenotype.

Integrins are one of the most important proteins involved in cell adhesion. The heterodimeric transmembrane proteins bind the actin cytoskeleton to the extracellular matrix, thereby linking the cell to the extracellular matrix. They act not only as cell adhesion molecules, but also as receptors for the transmission of intracellular signaling that regulate cell proliferation and survival. An important feature of the T cell partial reprogramming described herein is the attachment of cells to the matrix coating plate at an early stage, which suggests that the partially reprogrammed T cells express cell adhesion molecules, i.e. integrins (see, e.g., www (dot) ncbi (dot) n lm (dot) ni (dot) gov/books/NBK26867 /). In iPS cells, the predominant integrin complex is integrin α6β1 (see, e.g., nishiuchi et al, matrix Biology 2006,25 (3): 189-197). Integrin α6β1 is known to bind to laminin-511, and laminin-511 is used as an extracellular matrix in this example, and has been used for the culture of human pluripotent stem cells (see, for example, miyazaki t. Et al Nat Commun (2012) 3,1236).

Materials and methods

Step A: t cell regeneration

CD8 positive T cells from a 49 year old female donor were stimulated in 48 well plates at 100 tens of thousands of cells per 1ml of human T cell TransAct (Miltenyi Biotec, catalog No. 130-111-160) in T Cell Medium (TCM) containing 60IU/ml of IL2 for 1 day. All TCM's used in this example contained 60IU/ml IL2. Transduction was performed 24 hours after activation.

Sendai virus was transduced with 10MOI Klf4-Oct3/4-Sox2, 10MOI Klf4, 3MOI cMyc and 5MOI SV40 (Cytotune iPS 2.0 Sendai reprogramming kit, thermo) for 17 hours, then washed and suspended in IL 2-free Stem Cell Medium (SCM) (bFGF-containing StemFit Basic02, ajinomoto).

Cells were seeded at 5 ten thousand cells/well (corresponding to 5 ten thousand counts on day 0) onto an iMatrix-511 (PEPROTEC catalog No. RL 511S) coated 24-well plate.

And (B) step (B): partial reprogrammed T cell shedding

On day 10, partially reprogrammed T cells were divided into 3 groups. After SCM removal, 1ml TCM was added and resuspended twice with a P1000 pipette, then harvested and called weakly adherent cells. The detached cells harvested by vigorous pipetting with 1ml TCM are called adherent cells. Strongly adherent cells were detached by incubation with the recombinase TrypLE Express (Thermo cat # 12604013) for 10 min at 37 ℃. Cells were stained for viability and stained with fluorescent conjugated antibodies to detect CCR7, CD3, CD45RA, CD45RO, CD8b, CD4, CD62L, CD f, CD29, TCF1/7. Integrin α6 and integrin β1 were detected with CD49f antibody and CD29 antibody, respectively. For intracellular staining of TCF1, cells were fixed and permeabilized with Foxp3 staining buffer set (Thermo, 00-5523-00). Cell phenotypes were obtained by Cytek Aurora.

Analysis and results

Integrin α6 and integrin β1 are expressed in regenerative cells higher than activated T cells (i.e., controls). On day 10, the shed cells were compared to activated T cells activated on day-1 and cultured in TCM for 10 days. Integrin α6 and integrin b1 were expressed in adherent cells of T cell origin higher than activated T cell controls (figure 70). Among the shed cells, strongly adherent cells showed a higher proportion of integrin α6 (CD 49 f) and integrin β1 (CD 29) double positive cells than adherent cells, and in particular weakly adherent cells, indicating that the integrin complex expression level was correlated with adhesion strength.

T cells were activated on day 10, followed by culturing in TCM, and integrin expression levels decreased to that in activated control cells (fig. 71).

T cell phenotype and proliferation. CD3 is the most common T cell marker, and CD8 is a marker of cytotoxic T cells. Cd3+cd8+ populations decreased in weakly adherent cells, and strongly adherent cells on day 10, but increased from day 17 (fig. 72). Whereas strongly adherent cells almost completely lost cd3+cd8+ expression on day 10, the results on day 17 showed the reappearance of the cd3+cd8+ population, confirming that CD 3-or CD 8-populations (T cell-derived adherent cell populations) regained expression of T cell markers by T cell activation and growth under T cell culture conditions. It is unclear from this experiment whether the cd3+cd8-population observed on day 10 (see fig. 72) in strongly adherent cells corresponds to the population that regained CD8 expression. Weakly adherent cells, adherent cells and strongly adherent cells showed more proliferation after T cell activation on day 10 (fig. 73). Strongly adherent cells proliferate slowly directly after shedding, but eventually rebound and expand more than control cells.

A low differentiation phenotype. TCF1 and CCR7 are well known markers for poorly differentiated T cells. On days 10 and 17, strongly adherent cells showed higher TCF1 and CCR7 expression than the activated control, whereas adherent cells and weakly adherent cells showed TCF1 expression and higher CCR7 expression at both time points comparable to the activated control (fig. 74).

Summarizing: it was observed that on day 10, shed T cell adherent (i.e. partially reprogrammed) cells showed higher integrin α6 and integrin β1 expression than activated T cells. Activation on day 10 followed by a decrease in expression levels after culture under T cell conditions. The expression levels are higher in strongly adherent cells that require enzymatic digestion to be detached from the plate. Expression is lower in weakly attached cells that can be detached from the plate by pipetting without the need for enzymes. This suggests that integrin expression levels are correlated with cell adhesion strength. All adherent cell populations (weakly adherent cells, and strongly adherent cells) showed high proliferation after activation on day 10 (see figure 73). Strongly adherent cells proliferate slowly directly after shedding, but eventually rebound and exhibit robust proliferation. Strongly adherent cells also had high TCF1 and CCR7 expression on days 10 and 17, indicating a stem-like phenotype. High expression of integrin β1 can cause up-regulation of β -catenin expression by signaling (see, e.g., yuzurilha et al, stem Cell res.2021May; 53:102287), which can potentially contribute to the low differentiation phenotype of regenerative T cells (see, e.g., clin Cancer Res, 10 th 2010; 16 (19): 4695-701).

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Claims

1. A method of generating regenerative T cells comprising

a. Contacting T cells with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and (ii) optionally SV40 for a time sufficient to form T cell-derived adherent cells, and wherein the T cells are not converted to iPS or totipotent cells; and

b. contacting said adherent cells of T cell origin with at least one T cell activating compound.

2. A method of producing a T cell comprising

a. Contacting a population of T cells in a first medium in a culture vessel with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and (ii) optionally SV40 for a time sufficient for the T cells to form at least one colony attached to the surface of the culture vessel, and wherein the T cells are not converted to iPS or totipotent cells; and

b. contacting the at least one attached colony with at least one T cell activating compound.

3. A method of producing a T cell comprising

a. Contacting a population of T cells in a first medium with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and (ii) SV40 for a period of at least about 5 days to about 10 days, and wherein the T cells are not converted to iPS or totipotent cells;

b. Contacting the T cells of (a) with at least one T cell activating compound.

4. A method of producing a T cell comprising

a. Contacting a population of T cells with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and (ii) SV40 for a time sufficient for the T cells to express at least one marker selected from the group consisting of integrin α6β1, SSEA4, CD9, and CD90, and wherein the T cells are not converted to iPS or totipotent cells; and

b. contacting the T cells of (a) with at least one T cell activating compound.

5. The method of claim 4, wherein the T cell is contacted with the at least one reprogramming factor for a time sufficient for at least a portion of the T cell to express CD3 and at least one marker selected from the group consisting of integrin α6β1, SSEA4, CD9, and CD90.

6. The method of claim 5, wherein the T cells are contacted with the at least one reprogramming factor for a time sufficient for at least a portion of the contacted T cells to express SSEA4 and CD3.

7. The method of claim 5, wherein the T cells are contacted with the at least one reprogramming factor for a time sufficient for at least a portion of the contacted T cells to express CD3 and CD9, or CD3 and CD90, or CD3, CD9, and CD90.

8. The method of claim 5, wherein the T cell is contacted with the at least one reprogramming factor for a time sufficient for at least a portion of the contacted T cell to express CD3, SSEA4, CD9, and CD90.

9. The method of any one of claims 1-8, wherein the T cell is activated with IL-2 and at least one agent capable of activating the T cell prior to contacting the T cell with the at least one reprogramming factor.

10. The method of any one of claims 1-9, wherein the T cell is a tcra beta cell; tcrγδ cells; cd4+cd8αβ+ biscationic cells, cd4+ single positive cells (such as Th1, th2, th17, treg), naive T cells, central memory T cells, or effector memory T cells.

11. The method of claim 9, wherein the activated T cells are enriched by selecting cells expressing CD137, PD1, or LAG 3.

12. The method of any one of claims 1-11, wherein the T cells are from a mammal.

13. The method of any one of claims 1-11, wherein the T cells are from a human.

14. The method of any one of claims 1-13, wherein the T cells are contacted with KLF4, OCT3/4, SOX2, and C-MYC.

15. The method of claim 14, wherein the T cells are contacted with KLF4, OCT3/4, SOX2, and C-MYC for at least about 4 days to about 10 days.

16. The method of claim 15, wherein the T cells are contacted with KLF4, OCT3/4, SOX2, and C-MYC for at least about 4 days to about 7 days.

17. The method of claim 16, wherein the T cells are contacted with KLF4, OCT3/4, SOX2, and C-MYC for about 7 days.

18. The method of any one of claims 1-17, wherein the at least one reprogramming factor is expressed in the T cells.

19. The method of claim 18, wherein the at least one reprogramming factor is expressed using a non-integrating viral vector.

20. The method of claim 19, wherein the at least one reprogramming factor is expressed using sendai virus.

21. The method of claim 18, wherein the at least one reprogramming factor is expressed and wherein expression is later inhibited by adding a compound that inhibits expression of the at least one reprogramming factor.

22. The method of claim 21, wherein the compound is a small molecule inhibitor that specifically inhibits the expression of one or more of KLF4, OCT3/4, SOX2, and C-MYC expression.

23. The method of claim 18, wherein expression of the at least one reprogramming factor is inhibited by contacting the T cell-derived adherent cells with an agent that activates T cells.

24. The method of claim 18, wherein the at least one reprogramming factor is delivered with nanoparticles.

25. The method of any one of claims 1-24, further comprising contacting the T cell with a cytokine.

26. The method of any one of claims 1-25, wherein the at least one T cell activator comprises an antibody that binds CD3 or an antibody that binds CD28 or both; or wherein the at least one T cell activator is a tumor antigen.

27. The method of any one of claims 1-26, further comprising engineering the T cell to express a cell surface receptor, wherein the T cell is engineered prior to contacting the T cell with the at least one reprogramming factor.

28. The method of any one of claims 1-26, further comprising engineering the T cell to express a cell surface receptor, wherein the T cell is engineered after contacting the T cell with the at least one reprogramming factor.

29. The method of claim 27 or claim 28, wherein the cell surface receptor is a Chimeric Antigen Receptor (CAR) or a T cell receptor or a hybrid receptor thereof.

30. The method of claim 27 or claim 28, wherein the cell surface receptor recognizes a specific antigen on the surface of a target cell.

31. The method of claim 30, wherein the antigen is MHC class I dependent.

32. The method of claim 31, wherein the antigen is MHC class I independent.

33. The method of any one of claims 1-32, wherein the resulting T cell comprises an incomplete set of V, D and J segments of a T cell receptor gene.

34. The method of any one of claims 1-32, further comprising measuring the epigenetic age of the resulting T cells.

35. The method of any one of claims 1-34, wherein the epigenetic age of the resulting T cells is at least 5% younger than the T cell population prior to reprogramming.

36. The method of any one of claims 1-35, wherein the partially reprogrammed T cell is capable of expanding at least 25-fold over a control T cell.

37. The method of any one of claims 1-36, wherein the T cells are contacted with (i) at least one factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC; and (ii) SV40, resulting in reduced expression of CD3 and CD 8.

38. A method of producing a T cell comprising

a. Culturing T cells in a first medium comprising IL-2 and using at least one antibody specific for CD3 or CD28 or both; or a tumor antigen, a tumor organoid or a tumor cell line activates said T cells;

b. contacting the activated T cells with KLF4, OCT3/4, SOX2 and C-MYC in a second medium that does not comprise IL-2 or an antibody specific for CD3 or CD28 for a period of time; or a tumor antigen, a tumor organoid or a tumor cell line; wherein the T cells are not fully reprogrammed to iPS or totipotent cells;

c. replacing the second medium with a third medium comprising IL-2 and at least one antibody specific for CD3 and/or CD 28; wherein the contacted T cells are cultured in the third medium for at least about 5 days.

39. The method of claim 38, further comprising expanding the contacted T cells.

40. The method of claim 38, wherein the T cell is a tcra beta cell; tcrγδ cells; CD4 ⁺ CD8αβ ⁺ Double positive cells, CD4 ⁺ Single positive cells (Th 1, th2, th17, treg), naive T cells, central memory T cells or effector memory T cells.

41. The method of any one of claims 38-40, wherein the contacting further comprises contacting the activated T cells with SV 40.

42. The method of any one of claims 38-41, wherein the T cells express an activation marker after co-culture with autologous tumor cells, wherein the activation marker is CD137, PD1, or LAG3.

43. A population of T cells, the population of T cells having an epigenetic age at least 5% younger than their actual age.

44. The population of T cells of claim 43, wherein the epigenetic age is at least 25% younger than its actual age.

45. A population of adherent cells of T cell origin, wherein at least 70% of the cells express CD3 and SSEA4.

46. The population of claim 45, wherein at least 30% of the cells express CD9.

47. The population of claim 45, wherein at least 30% of the cells express CD90.

48. The population of claim 45, wherein at least 30% of the cells express CD9 and CD90.

49. A population of adherent cells of T cell origin, wherein at least 30% of the cells express CD9 or CD90.

50. A population of adherent cells of T cell origin, wherein at least 30% of the cells express both CD9 and CD90.

51. A population of T cells produced by a method comprising:

a. contacting a population of T cells with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and (ii) optionally SV40 for a time sufficient to form T cell-derived adherent cells, and wherein the T cells are not converted to iPS or totipotent cells; and

b. contacting said adherent cells of T cell origin with at least one T cell activator.

52. A population of T cells produced by a method comprising:

b. contacting the at least one attached colony with at least one T cell activator.

53. A population of T cells produced by a method comprising:

b. Contacting the T cells of (a) with at least one T cell activator.

54. A population of T cells produced by a method comprising:

b. contacting the T cells of (a) with at least one T cell activator.

55. The population of T cells of claim 54, wherein the T cells are contacted with the at least one reprogramming factor for a time sufficient for at least a portion of the T cells to express CD3 and at least one marker selected from the group consisting of integrin α6β1, SSEA4, CD9, and CD90.

56. The population of T cells of claim 55, wherein the T cells are contacted with the at least one reprogramming factor for a time sufficient for at least a portion of the contacted T cells to express SSEA4 and CD3.

57. The population of T cells of claim 55, wherein the T cells are contacted with the at least one reprogramming factor for a time sufficient for at least a portion of the contacted T cells to express CD3, CD9, and CD90.

58. The population of T cells of claim 55, wherein the T cells are contacted with the at least one reprogramming factor for a time sufficient for at least a portion of the contacted T cells to express CD3, SSEA4, CD9, and CD90.

59. The population of T cells of any one of claims 51-58, wherein the T cells are activated by contacting the T cells with a cytokine and at least one T cell activator prior to contacting the T cells with the at least one reprogramming factor.

60. The population of T cells of any one of claims 51-58, wherein the T cells are TCR αβ cells; tcrγδ cells; cd4+cd8αβ+ biscationic cells, cd4+ single positive cells (such as Th1, th2, th17, treg), naive T cells, central memory T cells, or effector memory T cells.

61. The method of claim 59, wherein the T cell activator is an autologous tumor cell.

62. The method of claim 61, wherein the activated T cells are enriched by selecting cells that express CD137, PD1, or LAG 3.

63. The population of T cells of any one of claims 51-58, wherein the T cells are from a mammal.

64. The population of T cells of any one of claims 51-58, wherein the T cells are contacted with KLF4, OCT3/4, SOX2, and C-MYC.

65. The population of T cells of claim 64, wherein the T cells are contacted with KLF4, OCT3/4, SOX2 and C-MYC for at least about 4 days to about 11 days.

66. The population of T cells of claim 65, wherein the T cells are contacted with KLF4, OCT3/4, SOX2 and C-MYC for at least about 4 days to about 7 days.

67. The population of T cells of claim 66, wherein the T cells are contacted with KLF4, OCT3/4, SOX2 and C-MYC for about 7 days.

68. The T cell population of any one of claims 51-67, wherein KLF4, OCT3/4, SOX2, and C-MYC are expressed in the T cells.

69. The T cell population of claim 68, wherein KLF4, OCT3/4, SOX2, and C-MYC are expressed using non-integrating viral vectors.

70. The population of T cells of claim 69, wherein KLF4, OCT3/4, SOX2 and C-MYC are expressed using sendai virus.

71. The T cell population of claim 69, wherein KLF4, OCT3/4, SOX2, and C-MYC are expressed and wherein expression is later inhibited by adding a compound that inhibits expression of KLF4, OCT3/4, SOX2, and C-MYC.

72. The population of T cells of claim 69, wherein the compound is a small molecule inhibitor that specifically inhibits the expression of one or more of KLF4, OCT3/4, SOX2 and C-MYC expression.

73. The population of T cells of claim 66, wherein expression of said at least one reprogramming factor is inhibited by contacting said T cell-derived adherent cells with a T cell activator.

74. The population of T cells of claim 68, wherein the KLF4, OCT3/4, SOX2 and C-MYC are expressed by one or more expression vectors delivered to the T cells in nanoparticles.

75. The population of T cells of any one of claims 51-74, further comprising contacting the T cells with at least one cytokine selected from the group consisting of IL-2, IL-7, IL-15 and IL-12.

76. The population of T cells of any one of claims 1-74, wherein the at least one T cell activator comprises an antibody that binds CD3 or an antibody that binds CD28 or both; or wherein at least one T cell activator is a tumor antigen.

77. The population of T cells of any one of claims 51-76, further comprising engineering the T cells to express a cell surface receptor, wherein the T cells are engineered prior to contacting the T cells with the at least one reprogramming factor.

78. The population of T cells of any one of claims 51-76, further comprising engineering the T cells to express a cell surface receptor, wherein the T cells are engineered after contacting the T cells with the at least one reprogramming factor.

79. The population of claim 77 or claim 78, wherein the cell surface receptor is a chimeric antigen receptor or a T cell receptor or a hybrid receptor thereof.

80. The population of claim 77 or claim 78, wherein the cell surface receptor recognizes a specific antigen on the surface of a target cell.

81. The population of claim 80, wherein the antigen is MHC class I-dependent.

82. The T cell population of claim 80, wherein the antigen is MHC class I independent.

83. The population of T cells of any one of claims 51-82, wherein the resulting T cells comprise a set of incomplete V, D and J segments of a T cell receptor gene.

84. The population of T cells of any one of claims 51-82, further comprising measuring the epigenetic age of the resulting T cells.

85. The population of claim 84, wherein the epigenetic age of the resulting T cells is at least 5% younger than the population of T cells prior to reprogramming.

86. The population of T cells of any one of claims 51-85, wherein the partially reprogrammed T cells are capable of expanding at least 25-fold over control T cells.

87. The population of T cells of any one of claims 51-86, wherein the T cells are contacted with at least one factor selected from the group consisting of klf4, OCT3/4, SOX2, and C-MYC; exposure to SV40 causes reduced expression of CD3 and CD 8.

88. A population of T cells produced by a method comprising:

a. culturing T cells in a first medium comprising IL-2 and activating the T cells with at least one antibody specific for CD3 or CD 28;

b. contacting the activated T cells with KLF4, OCT3/4, SOX2, and C-MYC in a second medium that does not comprise IL-2 or an antibody specific for CD3 or CD28 for a period of time ranging from about 5 days to about 10 days; wherein the T cells are not fully reprogrammed to iPS or totipotent cells;

89. The population of T cells of claim 88, further comprising expanding the T cells.

90. The population of claim 89, wherein the T cells are TCR αβ cells; tcrγδ cells; CD4 ⁺ CD8αβ ⁺ Double positive cells, CD4 ⁺ Single positive cells (Th 1, th2, th17, treg), naive T cells, central memory T cells or effector memory T cells.

91. A method of treating a subject in need thereof with a population of T cells produced by the method of any one of claims 1-42.

92. A method of treating a patient in need thereof with the population of any one of claims 43-50.

93. A method of treating a patient in need thereof with the T cell population of any one of claims 51-91.

94. The method of claim 92 or claim 93, wherein the method of treatment is a method for treating cancer, a viral disorder, or an autoimmune disorder.

95. The method of claim 94, wherein the cancer is acute lymphocytic cancer, acute myelogenous leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal canal cancer or anal rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gall bladder cancer or pleural cancer, head and neck cancer (e.g., nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer), vulval cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal Cell Carcinoma (RCC)), sarcoma, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, carcinoma, ureteral cancer or bladder cancer.

96. A method of producing regenerative Tumor Infiltrating Lymphocytes (TILs), comprising:

a. contacting a population of TILs with at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and optionally SV40, for a time sufficient to form T cell-derived adherent cells, and wherein the TILs is not converted to iPS or totipotent cells; and

97. A population of TILs prepared according to the method of claim 96.

98. A method of producing regenerative Tumor Infiltrating Lymphocytes (TILs), comprising:

a. activating a population of TILs with a first tumor antigen, such that the activated TILs express CD137; optionally, enriching the cd137+ TIL;

b. contacting the activated population of TILs with (i) at least one reprogramming factor selected from the group consisting of KLF4, OCT3/4, SOX2, and C-MYC, and (ii) optionally SV40 for a time sufficient to form TIL-derived adherent cells; wherein the TIL is not converted to iPS or totipotent cells.

99. The method of claim 98, further comprising contacting the TIL-derived adherent cells with at least one T-cell activating compound.

100. The method of claim 99, wherein the at least one T cell activating compound is the first tumor antigen.

101. The method of claim 100, wherein the first tumor antigen is an autologous tumor cell, an autologous tumor cell line, a tumor organoid, or an antigen derived from any of the foregoing.

102. The method of claim 98, wherein the population of TILs is contacted with KLF4, OCT3/4, SOX2, and C-MYC.

103. The method of claim 102, wherein the TIL population is contacted with SV 40.

104. The method of claim 98, wherein the population of TILs is contacted with KLF4, OCT3/4, SOX2, C-MYC, and SV 40.

105. A population of regenerated TILs prepared according to the method of any of claims 98-104.

106. The population of claim 105, wherein at least 50% of the regenerated TILs express both CCR7 and CD 62L.

107. The population of claim 105, wherein at least 50% of the regenerated TILs express both CCR7 and CD 62L.

108. The population of claim 105, wherein at least 50% of the regenerated TILs express both CCR7 and TCF 7.

109. The population of claim 105, wherein at least 50% of the regenerated TILs express both CCR7 and TCF 7.

110. A method of treating a patient in need thereof with a population of regenerated TILs produced by the method of any of claims 98-109.

111. The method of claim 110, wherein the method of treatment is a method for treating cancer, a viral disorder, or an autoimmune disorder.

112. The method of claim 111, wherein the cancer is acute lymphocytic cancer, acute myelogenous leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal canal cancer or anal rectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gall bladder cancer or pleural cancer, head and neck cancer (e.g., nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer), vulval cancer, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal Cell Carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer or carcinoma.