Methods for generating primary immune cells

Abstract

The present disclosure relates to methods, cells, and compositions for preparing cell populations and compositions for adoptive cell therapy. In particular, provided herein are methods for the expansion and propagation of primary immune cells, including T cell populations.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 63 / 486,398, filed February 22, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present disclosure relates to methods, cells, and compositions for preparing cell populations and compositions for adoptive cell therapy. In particular, provided herein are methods for the expansion and propagation of primary immune cells, including T cell populations. [Background technology]

[0003] Engineered adoptive cell therapy has been transformative for patients with hematological malignancies in recent years, with the first chimeric antigen receptor (CAR)-based therapy approved by the FDA in 2017 (Larson & Maus, Nat Rev Cancer 21, 145-161 (2021); Yu, et al., Nature Reviews Drug Discovery 19, 583-584 (2020)). Since 2017, the number of clinical trials investigating adoptive cell therapies, such as CAR-T cells, CAR-Natural Killer (NK) and CAR-NKT cells, T cell receptor (TCR)-T cells, tumor-infiltrating lymphocytes (TILs), tumor-specific antigen-targeted T cells, and other cell therapies, has rapidly increased. More recently, the first CAR-macrophages (CAR-M) entered the clinic for the treatment of solid tumors (Mukhopadhyay, Nat Methods 17, 561 (2020); Klichinsky, et al., Nat Biotechnol., 8, 947-953 (2020); Villanueva, Nature Reviews Drug Discovery 19, 308 (2020); ClinicalTrials.gov Identifier: NCT04660929).

[0004] Although cell therapies have great potential to be curative for patients, many factors limit the widespread development and implementation of these drugs. Most cell therapies are currently produced in an autologous manner and are associated with variable cell product quality, cytokine release syndrome and other toxicities, long manufacturing times, high costs, and limited time periods during which these therapies can be genetically modified to enhance their efficacy (Larson & Maus, Nat Rev Cancer 21, 145-161 (2021)).

[0005] The majority of cell therapies currently being tested in the clinic utilize subsets of CAR-T or CAR-NK cells, as these immune cells exhibit potent cytotoxicity. The mature primary human T cells used in these therapies are found in human blood and secondary lymphoid organs, where they act to protect individuals from infectious diseases and cancer. T cells are composed of αβ ("classical" T cells) and γδ subsets. αβ T cells are CD4 + Helper T cells and CD8 + Consists of cytotoxic T cells. CD4 + T cells can be further subdivided into TH1, TH2, TH9, TH17, TFH, and regulatory T cells. Many αβ T cell subsets exhibit potent cytotoxic functions that have been exploited in the development of cell therapies.

[0006] Similarly, mature primary human NK cells that can be used for cell therapy are found in the blood, secondary lymphoid organs, liver, and mucosa-associated lymphoid tissues, sites where NK cells patrol for the presence of pathogens or transformed cells (Jianhua, et al., Trends in Immunology 34, 573-582 (2013)). Like T cells, NK cells exhibit potent cytotoxic functions, making them of interest for the development of cell therapies.

[0007] However, primary human immune cells, such as T cells and NK cells, also have limited potential for in vitro and in vivo expansion, limiting their ability to be used to generate a wide range of off-the-shelf cell therapies. Furthermore, this limited expansion capacity of mature primary human immune cells impairs their ability to be genetically edited to mitigate cytokine release syndrome and other potential cell therapy-associated toxicities, overcome tumor microenvironment-related challenges, and prevent rejection of allogeneic cell therapy products in patients.

[0008] Patient-derived leukemia cell lines have been studied in cell culture for decades, and their transformed state confers long-term growth capacity, enabling their use in a variety of cellular assays. This, in turn, has facilitated the development of numerous therapies. However, because these cells are often immature or derived from dysfunctional T cell clones, they generally lack the potent cytotoxic functions of mature primary human T cells and NK cells. The altered properties of these cells can be mapped to a collection of mutations that are also frequently found in patients with T-cell acute lymphoblastic leukemia. Furthermore, mature T cells from non-human primates can be transformed by herpesviruses through pathways that converge on some of the same mechanisms involved in the transformation of primary human T cells in patients (Biesinger, et al., Proc Natl Acad Sci USA 89, 3116-3119 (1992); Weber, et al., Proc Natl Acad Sci USA 90, 11049-11053 (1993); Fickenscher H, Fleckenstein B., Philos Trans R Soc Lond B Biol Sci. 356(1408):545-67 (2001); Tsygankov, J Cell Physiol. 203(2):305-18 (2005)).

[0009] Previous studies have shown that primary human T cells express telomerase-reverse transcriptase (TERT) (Barsov, Methods Mol Biol. 511, 143-58 (2009); Rufer, et al., Blood 98, 597-603 (2001); Hooijberg, et al., J Immunol. 165, 4239-45 (2000)) and the human T cell leukemia virus type 1 or human T cell leukemia virus type 2 (HTLV-1 / HTLV-2) transcriptional transactivator protein Tax (Akagi, et al., Oncogene 14, 2071-2080 (1997); Grassmann, et al., Proc Natl Acad Sci USA 86, 3351-3355 (1989); Ren, et al., J. Biol. Chem. 287, 34683-34693 (2012); or viruses such as Herpesvirus saimiri (Biesinger, et al., Proc Natl Acad Sci USA 89, 3116-3119 (1992); Weber, et al., Proc Natl Acad Sci USA 90, 11049-11053 (1993)), and overexpression of factors such as HTLV-1 / HTLV-2, but these approaches are not highly reproducible and may result in reprogramming of modified or infected cells. In addition, cells whose proliferative lifespan is enhanced through overexpression of TERT still require the use of feeder cells or extensive exogenous stimulation through their T cell receptor to drive proliferation (Rufer, et al., J. Biol. Chem. 287, 34683-34693 (2012)). al., Blood 98, 597-603 (2001); Hooijberg, et al., J Immunol. 165, 4239-45 (2000)). The use of allogeneic feeder cells and extensive repeated stimulations is undesirable when establishing banks of mature primary human T cells or NK cells because these methodologies are difficult to scale and can ultimately drive the cells into a dysfunctional state.Furthermore, the use of infectious agents capable of transforming mature primary human T cells or NK cells limits the use of these cells in the development of cell therapies, as patients are often immunocompromised.

[0010] In light of these challenges, there is a great need to establish alternative methods to extend the proliferative lifespan of primary human immune cells, allowing for the large-scale production of allogeneic cytotoxic cells. The present disclosure describes methods and cells that address this unmet need. Summary of the Invention

[0011] In one aspect, the present disclosure provides a method for generating a population of primary immune cells resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in primary immune cell populations; (b) introducing a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants into a population of primary immune cells; (c) culturing the primary immune cells in a culture medium, and culturing, wherein the culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS).

[0012] In another aspect, the present disclosure provides a method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in primary immune cell populations; (b) introducing a transgene encoding MYC into a population of primary immune cells; (c) culturing the primary immune cells in a culture medium, and culturing, wherein the culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS).

[0013] In yet another aspect, the present disclosure provides a method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in primary immune cell populations; (b) introducing a transgene encoding TERT into a population of primary immune cells; (c) culturing the primary immune cells in a culture medium, and culturing, wherein the culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS).

[0014] In yet another aspect, the present disclosure provides a method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), comprising: (a) inhibiting expression of one or more endogenous regulatory factors in a population of primary immune cells, wherein the endogenous regulatory factors are cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), or S-methyl-5'-thioadenosine phosphorylase (MTAP); (b) inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells, wherein the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant (TRAC); (c) introducing a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants into the population of primary immune cells; (d) culturing the population of primary immune cells in a culture medium, and culturing, wherein the culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS).

[0015] In yet another aspect, the present disclosure provides a method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in primary immune cell populations; (b) inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells, wherein the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC); and (c) introducing a transgene encoding MYC into a population of primary immune cells; (d) culturing the primary immune cells in a culture medium, and culturing, wherein the culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS).

[0016] In yet another aspect, the present disclosure provides a method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in primary immune cell populations; (b) inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells, wherein the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC); and (c) introducing a transgene encoding TERT into a population of primary immune cells; (d) culturing the primary immune cells in a culture medium, and culturing, wherein the culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS).

[0017] In yet another aspect, the present disclosure provides a method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), or S-methyl-5'-thioadenosine phosphorylase (MTAP) in a population of primary immune cells; (b) introducing a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants into a population of primary immune cells; (c) introducing a transgene encoding TERT into a population of primary immune cells; (d) culturing the population of primary immune cells in a culture medium, and culturing, wherein the culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS).

[0018] In yet another aspect, the present disclosure provides a method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in primary immune cell populations; (b) introducing a transgene encoding MYC into a population of primary immune cells; (c) introducing a transgene encoding TERT into a population of primary immune cells; (d) culturing the primary immune cells in a culture medium, and culturing, wherein the culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS).

[0019] In some embodiments of the methods disclosed herein, the one or more STAT5A mutants may be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants may be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

[0020] In some embodiments of the methods disclosed herein, the methods further comprise introducing a transgene encoding TERT into the population of primary immune cells.

[0021] In some embodiments of the methods disclosed herein, the method further comprises inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells, hi some embodiments, the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

[0022] In some embodiments of the methods disclosed herein, the methods include introducing one or more transgenes encoding an anti-apoptotic factor or a virus-derived factor into primary immune cells. In some embodiments, the anti-apoptotic factor is either diffuse large B-cell lymphoma-extra large (Bcl-xL) or B-cell lymphoma 2 (Bcl-2). In some embodiments, the virus-derived factor is any one of Saimiriine gammaherpesvirus2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1.

[0023] In some embodiments of the methods disclosed herein, the methods further comprise inhibiting expression of cluster of differentiation 38 (CD38) in the population of primary immune cells.

[0024] In some embodiments of the methods disclosed herein, the methods further comprise inhibiting expression of phosphatase and tensin homolog (PTEN) in the population of primary immune cells.

[0025] In some embodiments of the methods disclosed herein, the methods further comprise inhibiting expression of p53 in the population of primary immune cells.

[0026] In some embodiments of the methods disclosed herein, the methods further comprise introducing a transgene encoding MYC into the population of primary immune cells.

[0027] In some embodiments of the methods disclosed herein, the method further comprises introducing into the population of primary immune cells a transgene encoding KRAS. In some embodiments, the KRAS is mutant KRAS A146V.

[0028] In some embodiments of the methods disclosed herein, the methods further comprise introducing a polynucleotide encoding a chimeric antigen receptor (CAR) into the population of primary immune cells.

[0029] In some embodiments of the methods disclosed herein, the population of primary immune cells comprises whole T cells.

[0030] In some embodiments of the methods disclosed herein, the population of primary immune cells comprises CD8+ T cells.

[0031] In some embodiments of the methods disclosed herein, the population of primary immune cells comprises CD4+ T cells.

[0032] In some embodiments of the methods disclosed herein, the population of primary immune cells comprises gamma-delta T cells, mucosal associated invariant T (MAIT) T cells, natural killer (NK) cells, and / or natural killer T (NKT) cells.

[0033] In some embodiments of the methods disclosed herein, the population of primary immune cells is human.

[0034] In one aspect, the present disclosure provides an engineered immune cell population produced according to the methods disclosed herein.

[0035] In another aspect, the present disclosure provides a pharmaceutical composition comprising an engineered immune cell population disclosed herein and a pharmaceutically acceptable carrier.

[0036] In yet another aspect, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition disclosed herein.

[0037] In one aspect, the disclosure provides engineered T cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5′-thioadenosine phosphorylase (MTAP), and comprise an optional transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC.

[0038] In another aspect, the disclosure provides engineered T cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and that comprise a transgene encoding TERT.

[0039] In yet another aspect, the disclosure provides an engineered T cell that does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), S-methyl-5'-thioadenosine phosphorylase (MTAP), beta-2 microglobulin (B2M), and / or T cell receptor alpha constant region (TRAC), wherein the engineered T cell comprises a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants, and wherein the engineered T cell comprises a transgene encoding TERT.

[0040] In yet another aspect, the disclosure provides an engineered T cell that expresses a transgene encoding a diffuse large B-cell lymphoma (Bcl-XL), wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), S-methyl-5'-thioadenosine phosphorylase (MTAP), and / or phosphatase tensin homolog (PTEN), and the engineered T cell comprises a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants.

[0041] In some embodiments of the engineered T cells disclosed herein, the one or more STAT5A mutants can be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants can be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

[0042] In some embodiments of the engineered T cells disclosed herein, the engineered T cells further comprise a transgene encoding TERT in the population of primary immune cells.

[0043] In some embodiments of the engineered T cells disclosed herein, the engineered T cells further comprise inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells. In some embodiments, the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

[0044] In some embodiments of the engineered T cells disclosed herein, the engineered T cells comprise one or more transgenes encoding anti-apoptotic factors or virus-derived factors in primary immune cells. In some embodiments, the anti-apoptotic factor is either diffuse large B-cell lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2). In some embodiments, the virus-derived factor is any one of Saimiriine gammaherpesvirus2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1.

[0045] In some embodiments of the engineered T cells disclosed herein, the engineered T cells further comprise inhibition of expression of cluster of differentiation 38 (CD38) in a population of primary immune cells.

[0046] In some embodiments of the engineered T cells disclosed herein, the engineered T cells further comprise inhibition of expression of phosphatase and tensin homolog (PTEN) in the population of primary immune cells.

[0047] In some embodiments of the engineered T cells disclosed herein, the engineered T cells further comprise inhibition of expression of p53 in a population of primary immune cells.

[0048] In some embodiments of the engineered T cells disclosed herein, the engineered T cells further comprise a transgene encoding MYC in the population of primary immune cells.

[0049] In some embodiments of the engineered T cells disclosed herein, the engineered T cells further comprise a transgene encoding KRAS in the population of primary immune cells. In some embodiments, the KRAS is mutant KRAS A146V.

[0050] In some embodiments of the engineered T cells disclosed herein, the engineered T cells further comprise a polynucleotide encoding a chimeric antigen receptor (CAR) in the population of primary immune cells.

[0051] In one aspect, the disclosure provides use of an engineered T cell for the manufacture of a medicament for treating cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and the engineered T cell comprises a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants.

[0052] In another aspect, the disclosure provides use of an engineered T cell for the manufacture of a medicament for treating cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and the engineered T cell comprises an optional transgene encoding a diffuse large B-cell lymphoma (Bcl-xL) gene and a transgene encoding MYC.

[0053] In yet another aspect, the disclosure provides use of an engineered T cell for the manufacture of a medicament for treating cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and the engineered T cell comprises a transgene encoding TERT.

[0054] In some embodiments of the uses of the engineered T cells disclosed herein, the one or more STAT5A mutants can be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants can be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

[0055] In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells further comprise a transgene encoding TERT in the population of primary immune cells.

[0056] In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells further comprise inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells. In some embodiments, the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

[0057] In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells comprise one or more transgenes encoding anti-apoptotic factors or virus-derived factors in primary immune cells. In some embodiments, the anti-apoptotic factor is either diffuse large B-cell lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2). In some embodiments, the virus-derived factor is any one of Saimiriine gammaherpesvirus2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1.

[0058] In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells further comprise inhibition of expression of cluster of differentiation 38 (CD38) in the population of primary immune cells.

[0059] In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells further comprise inhibition of expression of phosphatase and tensin homolog (PTEN) in the population of primary immune cells.

[0060] In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells further comprise inhibition of expression of p53 in the population of primary immune cells.

[0061] In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells further comprise a transgene encoding MYC in the population of primary immune cells.

[0062] In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells further comprise a transgene encoding KRAS in the population of primary immune cells. In some embodiments, the KRAS is mutant KRAS A146V.

[0063] In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells further comprise a polynucleotide encoding a chimeric antigen receptor (CAR) in the population of primary immune cells.

[0064] In one aspect, the disclosure provides an engineered T cell for the treatment of cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and the engineered T cell comprises a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants.

[0065] In another aspect, the disclosure provides an engineered T cell for the treatment of cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5′-thioadenosine phosphorylase (MTAP), and comprises an optional transgene encoding a diffuse large B-cell lymphoma (Bcl-xL) gene and a transgene encoding MYC.

[0066] In yet another aspect, the present disclosure provides engineered T cells for the treatment of cancer in a patient, wherein the engineered T cells do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and comprise a transgene encoding TERT.

[0067] In some embodiments of the engineered T cells disclosed herein, the one or more STAT5A mutants can be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants can be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

[0068] In some embodiments of the engineered T cells disclosed herein, the engineered T cells further comprise a transgene encoding TERT in the population of primary immune cells.

[0069] In some embodiments of the engineered T cells disclosed herein, the engineered T cells further comprise inhibition of expression of one or more endogenous immune-related genes in the population of primary immune cells. In some embodiments, the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

[0070] In some embodiments of the engineered T cells disclosed herein, the engineered T cells comprise one or more transgenes encoding anti-apoptotic factors or virus-derived factors in primary immune cells. In some embodiments, the anti-apoptotic factor is either diffuse large B-cell lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2). In some embodiments, the virus-derived factor is any one of Saimiriine gammaherpesvirus2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1.

[0071] In some embodiments of the engineered T cells disclosed herein, the engineered T cells further comprise inhibition of expression of cluster of differentiation 38 (CD38) in a population of primary immune cells.

[0072] In some embodiments of the engineered T cells disclosed herein, the engineered T cells further comprise inhibition of expression of phosphatase and tensin homolog (PTEN) in the population of primary immune cells.

[0073] In some embodiments of the engineered T cells disclosed herein, the engineered T cells further comprise inhibition of expression of p53 in a population of primary immune cells.

[0074] In some embodiments of the engineered T cells disclosed herein, the engineered T cells further comprise a transgene encoding MYC in the population of primary immune cells.

[0075] In some embodiments of the engineered T cells disclosed herein, the engineered T cells further comprise a transgene encoding KRAS in the population of primary immune cells. In some embodiments, the KRAS is mutant KRAS A146V.

[0076] In some embodiments of the engineered T cells disclosed herein, the engineered T cells further comprise a polynucleotide encoding a chimeric antigen receptor (CAR) in the population of primary immune cells. [Brief explanation of the drawings]

[0077] [Figure 1A] We show that Bcl-xL insertion conferred a selective advantage to T cell survival in long-term culture. Whole primary human T cells (Fig. 1A) or purified primary human CD8+ T cells (Fig. 1B) were isolated, stimulated, transfected, and restimulated as described in Fig. 2. [Figure 1B] We show that Bcl-xL insertion conferred a selective advantage to T cell survival in long-term culture. Whole primary human T cells (Fig. 1A) or purified primary human CD8+ T cells (Fig. 1B) were isolated, stimulated, transfected, and restimulated as described in Fig. 2. [Figure 2] 1 shows a method for identifying survival-enhancing transgenes in primary human T cells. [Figure 3]A and B show that ablation of the expression of cell cycle regulatory molecules enhanced the proliferative capacity of T cells in long-term culture. [Figure 4-1] We demonstrate that restimulation of TREX+Bcl-xL cells can enhance their proliferation in long-term culture. Figure 4A shows the total fold expansion of TREX+Bcl-xL cells over time. Figures 4B-4D show the total fold expansion of TREX+Bcl-xL cells and PTEN-deficient TREX+Bcl-xL cells restimulated with αCD3 or αCD3 / αCD28 Dynabeads for 3 days, followed by de-beading. Total fold expansion was tracked and graphed over time for resting and treated cells. Arrows indicate the period of restimulation. Black arrows indicate the time points for evaluation of additional restimulation modalities. Figures 4A-4D show logarithmic scales. [Figure 4-2] We demonstrate that restimulation of TREX+Bcl-xL cells can enhance their proliferation in long-term culture. Figure 4A shows the total fold expansion of TREX+Bcl-xL cells over time. Figures 4B-4D show the total fold expansion of TREX+Bcl-xL cells and PTEN-deficient TREX+Bcl-xL cells restimulated with αCD3 or αCD3 / αCD28 Dynabeads for 3 days, followed by de-beading. Total fold expansion was tracked and graphed over time for resting and treated cells. Arrows indicate the period of restimulation. Black arrows indicate the time points for evaluation of additional restimulation modalities. Figures 4A-4D show logarithmic scales. [Figure 5A] We demonstrate that TREX+Bcl-xL cells are dependent on IL-2 for expansion and survival in cell culture. We assessed the total fold expansion (Fig. 5A) and cell viability (Fig. 5B) of three TREX+Bcl-xL lines established as in Fig. 3 from two different donors grown for 6 days in the presence of increasing amounts of recombinant human interleukin 2 (IL-2). [Figure 5B]We demonstrate that TREX+Bcl-xL cells are dependent on IL-2 for expansion and survival in cell culture. We assessed the total fold expansion (Fig. 5A) and cell viability (Fig. 5B) of three TREX+Bcl-xL lines established as in Fig. 3 from two different donors grown for 6 days in the presence of increasing amounts of recombinant human interleukin 2 (IL-2). [Figure 6A] We demonstrate that TREX+Bcl-xL cells phenotypically resemble normal primary human CD8+ T cells. TREX+Bcl-xL cells were stained with a fixable dead cell labeling reagent and a panel of antibodies against CD3, CD4, CD8, CD28, CD45RO, CCR7, PD1, and TIGIT. TREX+Bcl-xL lines exhibited CD3 expression (Figure 6A) and contained a high frequency of CD8+ cells (Figure 6B). Regardless of Bcl-xL overexpression (GFP+ and GFP- cells), TREX+Bcl-xL lines exhibited donor- or cell line-specific attributes, as exemplified by the expression of markers such as PD1 and TIGIT (Figure 6C), CD28 (Figure 6D), and CCR7 and CD45RO (Figure 6E). TREX+Bcl-xL lines also exhibited expression of CCR2 (Figure 6F), CCR5 (Figure 6G), and CXCR3 (Figure 6J). CCR6 expression (Fig. 6H) was heterogeneous, whereas CCR7 (Fig. 6I) and CXCR5 (Fig. 6K) expression was low to absent. [Figure 6B]We demonstrate that TREX+Bcl-xL cells phenotypically resemble normal primary human CD8+ T cells. TREX+Bcl-xL cells were stained with a fixable dead cell labeling reagent and a panel of antibodies against CD3, CD4, CD8, CD28, CD45RO, CCR7, PD1, and TIGIT. TREX+Bcl-xL lines exhibited CD3 expression (Figure 6A) and contained a high frequency of CD8+ cells (Figure 6B). Regardless of Bcl-xL overexpression (GFP+ and GFP- cells), TREX+Bcl-xL lines exhibited donor- or cell line-specific attributes, as exemplified by the expression of markers such as PD1 and TIGIT (Figure 6C), CD28 (Figure 6D), and CCR7 and CD45RO (Figure 6E). TREX+Bcl-xL lines also exhibited expression of CCR2 (Figure 6F), CCR5 (Figure 6G), and CXCR3 (Figure 6J). CCR6 expression (Fig. 6H) was heterogeneous, whereas CCR7 (Fig. 6I) and CXCR5 (Fig. 6K) expression was low to absent. [Figure 6C] We demonstrate that TREX+Bcl-xL cells phenotypically resemble normal primary human CD8+ T cells. TREX+Bcl-xL cells were stained with a fixable dead cell labeling reagent and a panel of antibodies against CD3, CD4, CD8, CD28, CD45RO, CCR7, PD1, and TIGIT. TREX+Bcl-xL lines exhibited CD3 expression (Figure 6A) and contained a high frequency of CD8+ cells (Figure 6B). Regardless of Bcl-xL overexpression (GFP+ and GFP- cells), TREX+Bcl-xL lines exhibited donor- or cell line-specific attributes, as exemplified by the expression of markers such as PD1 and TIGIT (Figure 6C), CD28 (Figure 6D), and CCR7 and CD45RO (Figure 6E). TREX+Bcl-xL lines also exhibited expression of CCR2 (Figure 6F), CCR5 (Figure 6G), and CXCR3 (Figure 6J). CCR6 expression (Fig. 6H) was heterogeneous, whereas CCR7 (Fig. 6I) and CXCR5 (Fig. 6K) expression was low to absent. [Figure 6D]We demonstrate that TREX+Bcl-xL cells phenotypically resemble normal primary human CD8+ T cells. TREX+Bcl-xL cells were stained with a fixable dead cell labeling reagent and a panel of antibodies against CD3, CD4, CD8, CD28, CD45RO, CCR7, PD1, and TIGIT. TREX+Bcl-xL lines exhibited CD3 expression (Figure 6A) and contained a high frequency of CD8+ cells (Figure 6B). Regardless of Bcl-xL overexpression (GFP+ and GFP- cells), TREX+Bcl-xL lines exhibited donor- or cell line-specific attributes, as exemplified by the expression of markers such as PD1 and TIGIT (Figure 6C), CD28 (Figure 6D), and CCR7 and CD45RO (Figure 6E). TREX+Bcl-xL lines also exhibited expression of CCR2 (Figure 6F), CCR5 (Figure 6G), and CXCR3 (Figure 6J). CCR6 expression (Fig. 6H) was heterogeneous, whereas CCR7 (Fig. 6I) and CXCR5 (Fig. 6K) expression was low to absent. [Figure 6E] We demonstrate that TREX+Bcl-xL cells phenotypically resemble normal primary human CD8+ T cells. TREX+Bcl-xL cells were stained with a fixable dead cell labeling reagent and a panel of antibodies against CD3, CD4, CD8, CD28, CD45RO, CCR7, PD1, and TIGIT. TREX+Bcl-xL lines exhibited CD3 expression (Figure 6A) and contained a high frequency of CD8+ cells (Figure 6B). Regardless of Bcl-xL overexpression (GFP+ and GFP- cells), TREX+Bcl-xL lines exhibited donor- or cell line-specific attributes, as exemplified by the expression of markers such as PD1 and TIGIT (Figure 6C), CD28 (Figure 6D), and CCR7 and CD45RO (Figure 6E). TREX+Bcl-xL lines also exhibited expression of CCR2 (Figure 6F), CCR5 (Figure 6G), and CXCR3 (Figure 6J). CCR6 expression (Fig. 6H) was heterogeneous, whereas CCR7 (Fig. 6I) and CXCR5 (Fig. 6K) expression was low to absent. [Figure 6F]We demonstrate that TREX+Bcl-xL cells phenotypically resemble normal primary human CD8+ T cells. TREX+Bcl-xL cells were stained with a fixable dead cell labeling reagent and a panel of antibodies against CD3, CD4, CD8, CD28, CD45RO, CCR7, PD1, and TIGIT. TREX+Bcl-xL lines exhibited CD3 expression (Figure 6A) and contained a high frequency of CD8+ cells (Figure 6B). Regardless of Bcl-xL overexpression (GFP+ and GFP- cells), TREX+Bcl-xL lines exhibited donor- or cell line-specific attributes, as exemplified by the expression of markers such as PD1 and TIGIT (Figure 6C), CD28 (Figure 6D), and CCR7 and CD45RO (Figure 6E). TREX+Bcl-xL lines also exhibited expression of CCR2 (Figure 6F), CCR5 (Figure 6G), and CXCR3 (Figure 6J). CCR6 expression (Fig. 6H) was heterogeneous, whereas CCR7 (Fig. 6I) and CXCR5 (Fig. 6K) expression was low to absent. [Figure 6G] We demonstrate that TREX+Bcl-xL cells phenotypically resemble normal primary human CD8+ T cells. TREX+Bcl-xL cells were stained with a fixable dead cell labeling reagent and a panel of antibodies against CD3, CD4, CD8, CD28, CD45RO, CCR7, PD1, and TIGIT. TREX+Bcl-xL lines exhibited CD3 expression (Figure 6A) and contained a high frequency of CD8+ cells (Figure 6B). Regardless of Bcl-xL overexpression (GFP+ and GFP- cells), TREX+Bcl-xL lines exhibited donor- or cell line-specific attributes, as exemplified by the expression of markers such as PD1 and TIGIT (Figure 6C), CD28 (Figure 6D), and CCR7 and CD45RO (Figure 6E). TREX+Bcl-xL lines also exhibited expression of CCR2 (Figure 6F), CCR5 (Figure 6G), and CXCR3 (Figure 6J). CCR6 expression (Fig. 6H) was heterogeneous, whereas CCR7 (Fig. 6I) and CXCR5 (Fig. 6K) expression was low to absent. [Figure 6H]We demonstrate that TREX+Bcl-xL cells phenotypically resemble normal primary human CD8+ T cells. TREX+Bcl-xL cells were stained with a fixable dead cell labeling reagent and a panel of antibodies against CD3, CD4, CD8, CD28, CD45RO, CCR7, PD1, and TIGIT. TREX+Bcl-xL lines exhibited CD3 expression (Figure 6A) and contained a high frequency of CD8+ cells (Figure 6B). Regardless of Bcl-xL overexpression (GFP+ and GFP- cells), TREX+Bcl-xL lines exhibited donor- or cell line-specific attributes, as exemplified by the expression of markers such as PD1 and TIGIT (Figure 6C), CD28 (Figure 6D), and CCR7 and CD45RO (Figure 6E). TREX+Bcl-xL lines also exhibited expression of CCR2 (Figure 6F), CCR5 (Figure 6G), and CXCR3 (Figure 6J). CCR6 expression (Fig. 6H) was heterogeneous, whereas CCR7 (Fig. 6I) and CXCR5 (Fig. 6K) expression was low to absent. [Figure 6I] We demonstrate that TREX+Bcl-xL cells phenotypically resemble normal primary human CD8+ T cells. TREX+Bcl-xL cells were stained with a fixable dead cell labeling reagent and a panel of antibodies against CD3, CD4, CD8, CD28, CD45RO, CCR7, PD1, and TIGIT. TREX+Bcl-xL lines exhibited CD3 expression (Figure 6A) and contained a high frequency of CD8+ cells (Figure 6B). Regardless of Bcl-xL overexpression (GFP+ and GFP- cells), TREX+Bcl-xL lines exhibited donor- or cell line-specific attributes, as exemplified by the expression of markers such as PD1 and TIGIT (Figure 6C), CD28 (Figure 6D), and CCR7 and CD45RO (Figure 6E). TREX+Bcl-xL lines also exhibited expression of CCR2 (Figure 6F), CCR5 (Figure 6G), and CXCR3 (Figure 6J). CCR6 expression (Fig. 6H) was heterogeneous, whereas CCR7 (Fig. 6I) and CXCR5 (Fig. 6K) expression was low to absent. [Figure 6J]We demonstrate that TREX+Bcl-xL cells phenotypically resemble normal primary human CD8+ T cells. TREX+Bcl-xL cells were stained with a fixable dead cell labeling reagent and a panel of antibodies against CD3, CD4, CD8, CD28, CD45RO, CCR7, PD1, and TIGIT. TREX+Bcl-xL lines exhibited CD3 expression (Figure 6A) and contained a high frequency of CD8+ cells (Figure 6B). Regardless of Bcl-xL overexpression (GFP+ and GFP- cells), TREX+Bcl-xL lines exhibited donor- or cell line-specific attributes, as exemplified by the expression of markers such as PD1 and TIGIT (Figure 6C), CD28 (Figure 6D), and CCR7 and CD45RO (Figure 6E). TREX+Bcl-xL lines also exhibited expression of CCR2 (Figure 6F), CCR5 (Figure 6G), and CXCR3 (Figure 6J). CCR6 expression (Fig. 6H) was heterogeneous, whereas CCR7 (Fig. 6I) and CXCR5 (Fig. 6K) expression was low to absent. [Figure 6K] We demonstrate that TREX+Bcl-xL cells phenotypically resemble normal primary human CD8+ T cells. TREX+Bcl-xL cells were stained with a fixable dead cell labeling reagent and a panel of antibodies against CD3, CD4, CD8, CD28, CD45RO, CCR7, PD1, and TIGIT. TREX+Bcl-xL lines exhibited CD3 expression (Figure 6A) and contained a high frequency of CD8+ cells (Figure 6B). Regardless of Bcl-xL overexpression (GFP+ and GFP- cells), TREX+Bcl-xL lines exhibited donor- or cell line-specific attributes, as exemplified by the expression of markers such as PD1 and TIGIT (Figure 6C), CD28 (Figure 6D), and CCR7 and CD45RO (Figure 6E). TREX+Bcl-xL lines also exhibited expression of CCR2 (Figure 6F), CCR5 (Figure 6G), and CXCR3 (Figure 6J). CCR6 expression (Fig. 6H) was heterogeneous, whereas CCR7 (Fig. 6I) and CXCR5 (Fig. 6K) expression was low to absent. [Figure 7-1]We demonstrate that TREX+Bcl-xL cells are cytotoxic. Percent cytolysis was calculated 12 hours (Figure 7A) and 24 hours (Figure 7B) after addition of effector cells and T cell engagers or control antibodies. Supernatants were collected from cocultures 72 hours after addition of effector cells and T cell engagers and analyzed for the presence of interferon γ (IFN-γ) (Figure 7C), IL-2 (Figure 7D), tumor necrosis factor α (TNF-α) (Figure 7E), and granzyme B (Figure 7F). [Figure 7-2] We demonstrate that TREX+Bcl-xL cells are cytotoxic. Percent cytolysis was calculated 12 hours (Figure 7A) and 24 hours (Figure 7B) after addition of effector cells and T cell engagers or control antibodies. Supernatants were collected from cocultures 72 hours after addition of effector cells and T cell engagers and analyzed for the presence of interferon γ (IFN-γ) (Figure 7C), IL-2 (Figure 7D), tumor necrosis factor α (TNF-α) (Figure 7E), and granzyme B (Figure 7F). [Figure 7-3] We demonstrate that TREX+Bcl-xL cells are cytotoxic. Percent cytolysis was calculated 12 hours (Figure 7A) and 24 hours (Figure 7B) after addition of effector cells and T cell engagers or control antibodies. Supernatants were collected from cocultures 72 hours after addition of effector cells and T cell engagers and analyzed for the presence of interferon γ (IFN-γ) (Figure 7C), IL-2 (Figure 7D), tumor necrosis factor α (TNF-α) (Figure 7E), and granzyme B (Figure 7F). [Figure 8-1]These results demonstrate that TREX+Bcl-xL cells can generate functional CAR-TREX+Bcl-xL cells. Surface CAR expression was assessed using flow cytometry 22 days after transduction (Figure 8A). Percent cytolysis was calculated 12 hours (Figure 8B) and 24 hours (Figure 8C) after effector cell addition. CAR-TREX activity was benchmarked against the activity of CAR-T cells and CAR-CD8+ T cells. Supernatants were collected from co-cultures 72 hours after effector cell addition and analyzed for the presence of IFN-γ (Figure 8D), IL-2 (Figure 8E), TNF-α (Figure 8F), and granzyme B (Figure 8G). [Figure 8-2] These results demonstrate that TREX+Bcl-xL cells can generate functional CAR-TREX+Bcl-xL cells. Surface CAR expression was assessed using flow cytometry 22 days after transduction (Figure 8A). Percent cytolysis was calculated 12 hours (Figure 8B) and 24 hours (Figure 8C) after effector cell addition. CAR-TREX activity was benchmarked against the activity of CAR-T cells and CAR-CD8+ T cells. Supernatants were collected from co-cultures 72 hours after effector cell addition and analyzed for the presence of IFN-γ (Figure 8D), IL-2 (Figure 8E), TNF-α (Figure 8F), and granzyme B (Figure 8G). [Figure 8-3] These results demonstrate that TREX+Bcl-xL cells can generate functional CAR-TREX+Bcl-xL cells. Surface CAR expression was assessed using flow cytometry 22 days after transduction (Figure 8A). Percent cytolysis was calculated 12 hours (Figure 8B) and 24 hours (Figure 8C) after effector cell addition. CAR-TREX activity was benchmarked against the activity of CAR-T cells and CAR-CD8+ T cells. Supernatants were collected from co-cultures 72 hours after effector cell addition and analyzed for the presence of IFN-γ (Figure 8D), IL-2 (Figure 8E), TNF-α (Figure 8F), and granzyme B (Figure 8G). [Figure 8-4]These results demonstrate that TREX+Bcl-xL cells can generate functional CAR-TREX+Bcl-xL cells. Surface CAR expression was assessed using flow cytometry 22 days after transduction (Figure 8A). Percent cytolysis was calculated 12 hours (Figure 8B) and 24 hours (Figure 8C) after effector cell addition. CAR-TREX activity was benchmarked against the activity of CAR-T cells and CAR-CD8+ T cells. Supernatants were collected from co-cultures 72 hours after effector cell addition and analyzed for the presence of IFN-γ (Figure 8D), IL-2 (Figure 8E), TNF-α (Figure 8F), and granzyme B (Figure 8G). [Figure 9A] We show that TREX cells migrate to similar locations as primary CD8+ T cells and are responsive to IL-2 in vivo. [Figure 9B] We show that TREX cells migrate to similar locations as primary CD8+ T cells and are responsive to IL-2 in vivo. [Figure 9C] We show that TREX cells migrate to similar locations as primary CD8+ T cells and are responsive to IL-2 in vivo. [Figure 10A] CAR-TREX cells respond to IL-2 and IL-15 in vivo. [Figure 10B] CAR-TREX cells respond to IL-2 and IL-15 in vivo. [Figure 11A] CAR-TREX cells are shown to target solid tumors in vivo. [Figure 11B] CAR-TREX cells are shown to target solid tumors in vivo. [Figure 11C] CAR-TREX cells are shown to target solid tumors in vivo. [Figure 11D] CAR-TREX cells are shown to target solid tumors in vivo. [Figure 12]We show that the reproducibility of REX editing confers enhanced in vitro proliferation compared to unmodified donor-matched CD8+ T cells. The fold expansion of TREX cells or donor-matched primary (unedited) CD8+ T cells was followed over time in four additional healthy donors. [Figure 13A] CAR-TREX cells target BCMA+ tumor cells similarly to unmodified CAR-T cells. [Figure 13B] CAR-TREX cells target BCMA+ tumor cells similarly to unmodified CAR-T cells. [Figure 14] Figure 1 shows that anti-BCMA-TREX and anti-HER2-TREX cells produce lower levels of inflammatory cytokines than anti-BCMA-CAR-T cells and anti-HER2-CAR-T cells after CAR engagement. [Figure 15] CAR-TREX cells target BCMA+ tumor cells, persist in serial killing assays, and respond to IL-2. [Figure 16] We show that different combinations of editing can be used to generate the TREX cell phenotype. [Figure 17] Showing that TREX cells are edited at the predicted locus. [Figure 18A] Figure 1 shows that TREX cells display an enrichment of cell cycle-related gene signatures. [Figure 18B] Figure 1 shows that TREX cells display an enrichment of cell cycle-related gene signatures. [Figure 18C] Figure 1 shows that TREX cells display an enrichment of cell cycle-related gene signatures. [Figure 19] Figure 1 shows that TREX cells are dependent on IL-2 for survival and proliferation. [Figure 20A] We show that CAR-TREX cells target HER2hi tumor cells similarly to unmodified CAR-T cells with lower overall cytokine production. [Figure 20B]We show that CAR-TREX cells target HER2hi tumor cells similarly to unmodified CAR-T cells with lower overall cytokine production. [Figure 21] We show that REX editing enhances the proliferative capacity of CD4+TREX cells. [Figure 22] We show that REX editing can be used to generate γδ TREX cells. [Figure 23] We show that γδ TREX cells are active in an in vitro T cell engager (TCE) assay. [Figure 24] γδ TREX cells can be generated from multiple γδ T cell subsets, demonstrating that diversity is maintained after CAR transduction. [Figure 25A] 1 shows that γδ-TREX cells target BCMA+ tumor cells similarly to unmodified CAR-T cells. [Figure 25B] 1 shows that γδ-TREX cells target BCMA+ tumor cells similarly to unmodified CAR-T cells. [Figure 26] We show that REX editing in NK cells supports the NKREX cell phenotype. [Figure 27] Figure 1 shows that NKREX cells are dependent on cytokines for proliferation and survival. [Figure 28] NKREX cells maintain CAR expression over time. [Figure 29A] NKREX cells are cytotoxic in vitro, indicating that CAR expression can further enhance efficacy. [Figure 29B] NKREX cells are cytotoxic in vitro, indicating that CAR expression can further enhance efficacy. [Figure 29C] NKREX cells are cytotoxic in vitro, indicating that CAR expression can further enhance efficacy. [Figure 30] We show that TREX cells are sensitive to T cell depleting agents and chemotherapy. [Figure 31] We show that B2MKO TREX cells are sensitive to NK cell-mediated depletion and that this can be modulated using anti-CD38 antibodies. [Figure 32] Figure 1 shows that STAT5A and STAT5B mutants are enriched in REX-edited CD8+ T cells in vitro. STAT mutants were overexpressed with Bcl-xL in TREX cells (REX-edited CD8+ T cells) according to the indicated timeline (top). A fluorescent reporter was used to track the enrichment of STAT mutants over time (bottom). [Figure 33] We show that STAT5A and STAT5B mutants exhibit varying degrees of IL-2 independence in vitro. [Figure 34] This shows that STAT5B mutant-expressing TREX cells retain functionality in a T cell engager assay, consistent with the observed surface CD3 expression. TREX cells were cocultured with two different antigen-expressing tumor lines (left and right). TREX cells containing the STAT5B N642H mutant exhibited reduced cytotoxicity in this assay due to their low surface expression of CD3. [Figure 35] We demonstrate that STAT5 mutant CAR-TREX cells maintain cytotoxic function in a CAR-directed manner in vitro. Percent cell lysis was determined 12 hours after initiation of coculture of TREX cells with target cells at various effector:target (E:T) cell ratios. STAT5A and STAT5B mutant TREX cells retained functionality, as demonstrated by their ability to specifically lyse antigen-expressing tumor cells after CAR engagement. [Figure 36A]Figure 36 shows that additional STAT5A and STAT5B mutants are enriched in REX-edited CD8+ T cells in vitro. Two STAT5A mutants and five STAT5B mutants were overexpressed in TREX cells (REX-edited CD8+ T cells) according to the indicated timeline (Figure 36A). STAT mutants were introduced using transposons (T) or lentiviruses (L) as indicated. Fluorescent reporters were used to track the enrichment of STAT mutants over time (Figure 36B). STAT5A and STAT5B mutant-expressing TREX cells were enriched during the cell culture process. [Figure 36B] Figure 36 shows that additional STAT5A and STAT5B mutants are enriched in REX-edited CD8+ T cells in vitro. Two STAT5A mutants and five STAT5B mutants were overexpressed in TREX cells (REX-edited CD8+ T cells) according to the indicated timeline (Figure 36A). STAT mutants were introduced using transposons (T) or lentiviruses (L) as indicated. Fluorescent reporters were used to track the enrichment of STAT mutants over time (Figure 36B). STAT5A and STAT5B mutant-expressing TREX cells were enriched during the cell culture process. [Figure 37A] Expression of STAT5A and STAT5B mutants enhances the expansion of REX-edited CD8+ T cells in vitro. Growth of STAT5A mutant TREX cells, STAT5B mutant TREX cells, and control TREX cells generated in Figure 36 was tracked over time. STAT5A (Figure 37A) and STAT5B (Figure 37B) mutant TREX cells showed enhanced expansion compared to unedited control TREX cells. [Figure 37B]Expression of STAT5A and STAT5B mutants enhances the expansion of REX-edited CD8+ T cells in vitro. Growth of STAT5A mutant TREX cells, STAT5B mutant TREX cells, and control TREX cells generated in Figure 36 was tracked over time. STAT5A (Figure 37A) and STAT5B (Figure 37B) mutant TREX cells showed enhanced expansion compared to unedited control TREX cells. [Figure 38A] We demonstrate that STAT5A and STAT5B mutants are functional in TREX cells and lead to upregulation of CD25 expression. Surface expression of CD25 was assessed in control TREX cells (UT) and TREX cells containing STAT5A / STAT5B mutants over long-term cell culture. STAT mutants were introduced using transposons (Figure 38A) or lentiviruses (Figure 38B). [Figure 38B] We demonstrate that STAT5A and STAT5B mutants are functional in TREX cells and lead to upregulation of CD25 expression. Surface expression of CD25 was assessed in control TREX cells (UT) and TREX cells containing STAT5A / STAT5B mutants over long-term cell culture. STAT mutants were introduced using transposons (Figure 38A) or lentiviruses (Figure 38B). [Figure 39] 1 shows an exemplary workflow for the generation of an alternative TREX cell chassis. [Figure 40A] We show that specific editing combinations are reproducibly enriched in TREX cells. The enrichment of editing combinations was assessed over time in engineered TREX cells (Figure 40A is round 1, and Figure 40B is round 2). [Figure 40B] We show that specific editing combinations are reproducibly enriched in TREX cells. The enrichment of editing combinations was assessed over time in engineered TREX cells (Figure 40A is round 1, and Figure 40B is round 2). [Figure 41]We demonstrate that TERT-expressing TREX0, TREX3B, and TREX3C cells are enriched in culture. TREX0 (REX-edited), TREX3B (REX-edited; MYC; Bcl-xL), and TREX3C (REX-edited; KRAS A146V; MYC; Bcl-xL) cells were generated and then further engineered to overexpress TERT. The enrichment of TERT-overexpressing TREX0 (TREX0T), TREX3B (TREX3BT), and TREX3C (TREX3CT) cells was tracked over time. [Figure 42-1] We demonstrate that certain TREX variants exhibit enhanced expansion and lifespan compared to TREX-edited CD8+ T cells. TREX cell variants were generated as described above, and cell expansion was tracked over time. Certain editing combinations (3B, 3C, 3BP, 3CN, 0T, 3BT, and 3CT) enhanced the lifespan and proliferation capacity of TREX cell variants compared to TREX (TREX0) cells. However, in some cases, editing combinations (2A, 2B, 3A, and 3BN) impaired the lifespan and proliferation of TREX cells during cell culture. [Figure 42-2] We demonstrate that certain TREX variants exhibit enhanced expansion and lifespan compared to TREX-edited CD8+ T cells. TREX cell variants were generated as described above, and cell expansion was tracked over time. Certain editing combinations (3B, 3C, 3BP, 3CN, 0T, 3BT, and 3CT) enhanced the lifespan and proliferation capacity of TREX cell variants compared to TREX (TREX0) cells. However, in some cases, editing combinations (2A, 2B, 3A, and 3BN) impaired the lifespan and proliferation of TREX cells during cell culture. [Figure 42-3]We demonstrate that certain TREX variants exhibit enhanced expansion and lifespan compared to TREX-edited CD8+ T cells. TREX cell variants were generated as described above, and cell expansion was tracked over time. Certain editing combinations (3B, 3C, 3BP, 3CN, 0T, 3BT, and 3CT) enhanced the lifespan and proliferation capacity of TREX cell variants compared to TREX (TREX0) cells. However, in some cases, editing combinations (2A, 2B, 3A, and 3BN) impaired the lifespan and proliferation of TREX cells during cell culture. [Figure 42-4] We demonstrate that certain TREX variants exhibit enhanced expansion and lifespan compared to TREX-edited CD8+ T cells. TREX cell variants were generated as described above, and cell expansion was tracked over time. Certain editing combinations (3B, 3C, 3BP, 3CN, 0T, 3BT, and 3CT) enhanced the lifespan and proliferation capacity of TREX cell variants compared to TREX (TREX0) cells. However, in some cases, editing combinations (2A, 2B, 3A, and 3BN) impaired the lifespan and proliferation of TREX cells during cell culture. [Figure 43]We show that TREX cell variants exhibit increased potency compared to TREX0 in a T cell engager assay. The cytotoxic potential of TREX cells (TREX0) and TREX cell variants was assessed using the impedance-based xCELLigence platform, using control (non-targeting) T cells or activated (tumor-targeting) T cell engagers. Percent cytolysis was determined 12 and 72 hours after addition of the T cell engager. TREX0 cells (REX edited), TREX0T cells (REX edited; TERT), TREX3C cells (REX edited; KRAS A146V; MYC, Bcl-xL), TREX3C_3 cells (REX edited; KRAS A146V; MYC; Bcl-xL), TREX3CN cells (REX edited; KRAS A146V; MYC; Bcl-xL; PTEN CRISPR), TREX3B cells (REX edited; MYC; Bcl-xL), TREX3B_3 cells (REX edited; MYC; Bcl-xL), TREX3BT cells (REX edited; MYC; Bcl-xL; TERT), TREX3BP cells (REX edited; MYC; Bcl-xL; TP53 CRISPR), and TREX3BN cells (REX edited; MYC; Bcl-xL; PTEN For CRISPR, effector cells were co-cultured with target cells at different effector:target (E:T) cell ratios. The dashed line indicates the cytotoxicity of TREX (TREX0) cells at 72 hours. [Figure 44A-1] These results demonstrate that TREX cell variants can express CAR, and that CAR-TREX cell variants robustly expand during the culture process. BCMA-targeted CARs were introduced into young TREX cells (TREX0D60) and TREX cell variants (TREX0T, TREX3C_3, TREX3CN, TREX3B, TREX3B_3, TREX3BT, and TREX3BP). CAR-expressing cells were further enriched to high purity before functional evaluation (Figure 44A). The expansion of TREX cells, TREX cell variants, CAR-TREX cells, and CAR-TREX cell variants was tracked throughout the editing, transduction, and enrichment process for each group (Figure 44B). [Figure 44A-2]These results demonstrate that TREX cell variants can express CAR, and that CAR-TREX cell variants robustly expand during the culture process. BCMA-targeted CARs were introduced into young TREX cells (TREX0D60) and TREX cell variants (TREX0T, TREX3C_3, TREX3CN, TREX3B, TREX3B_3, TREX3BT, and TREX3BP). CAR-expressing cells were further enriched to high purity before functional evaluation (Figure 44A). The expansion of TREX cells, TREX cell variants, CAR-TREX cells, and CAR-TREX cell variants was tracked throughout the editing, transduction, and enrichment process for each group (Figure 44B). [Figure 44B-1] These results demonstrate that TREX cell variants can express CAR, and that CAR-TREX cell variants robustly expand during the culture process. BCMA-targeted CARs were introduced into young TREX cells (TREX0D60) and TREX cell variants (TREX0T, TREX3C_3, TREX3CN, TREX3B, TREX3B_3, TREX3BT, and TREX3BP). CAR-expressing cells were further enriched to high purity before functional evaluation (Figure 44A). The expansion of TREX cells, TREX cell variants, CAR-TREX cells, and CAR-TREX cell variants was tracked throughout the editing, transduction, and enrichment process for each group (Figure 44B). [Figure 44B-2] These results demonstrate that TREX cell variants can express CAR, and that CAR-TREX cell variants robustly expand during the culture process. BCMA-targeted CARs were introduced into young TREX cells (TREX0D60) and TREX cell variants (TREX0T, TREX3C_3, TREX3CN, TREX3B, TREX3B_3, TREX3BT, and TREX3BP). CAR-expressing cells were further enriched to high purity before functional evaluation (Figure 44A). The expansion of TREX cells, TREX cell variants, CAR-TREX cells, and CAR-TREX cell variants was tracked throughout the editing, transduction, and enrichment process for each group (Figure 44B). [Figure 44B-3]These results demonstrate that TREX cell variants can express CAR, and that CAR-TREX cell variants robustly expand during the culture process. BCMA-targeted CARs were introduced into young TREX cells (TREX0D60) and TREX cell variants (TREX0T, TREX3C_3, TREX3CN, TREX3B, TREX3B_3, TREX3BT, and TREX3BP). CAR-expressing cells were further enriched to high purity before functional evaluation (Figure 44A). The expansion of TREX cells, TREX cell variants, CAR-TREX cells, and CAR-TREX cell variants was tracked throughout the editing, transduction, and enrichment process for each group (Figure 44B). [Figure 44B-4] These results demonstrate that TREX cell variants can express CAR, and that CAR-TREX cell variants robustly expand during the culture process. BCMA-targeted CARs were introduced into young TREX cells (TREX0D60) and TREX cell variants (TREX0T, TREX3C_3, TREX3CN, TREX3B, TREX3B_3, TREX3BT, and TREX3BP). CAR-expressing cells were further enriched to high purity before functional evaluation (Figure 44A). The expansion of TREX cells, TREX cell variants, CAR-TREX cells, and CAR-TREX cell variants was tracked throughout the editing, transduction, and enrichment process for each group (Figure 44B). [Figure 45] CAR-TREX cell variants remain functional even after more than 200 days in culture. [Figure 46] We show that CAR-TREX cell variants persist as well as primary CAR-T cells in serial killing assays. 221-day-old CAR-TREX cell variants are at least as functional as the young CAR-TREX cell benchmark. [Figure 47]This shows that TERT overexpression confers an advantage to NKREX and CAR-NKREX cells. NKREX cells (NK cells containing REX editing) were generated and, in some cases, engineered to express a BCMA-targeted CAR (top). NKREX and CAR-NKREX were then further engineered to overexpress TERT. All groups were monitored over time for enrichment of TERT-expressing NKREX and CAR-NKREX cells (top). Proliferation of all groups was also measured throughout the culture process (bottom). Cells were grown under the indicated cytokine conditions (IL-2 or IL-2 + IL-15). [Figure 48] Figure 1 shows that TERT-overexpressing CAR-NKREX cells exhibit improved function in vitro. Percent cell lysis was determined 6, 12, 48, and 96 hours after initiation of co-culture of effector and target cells at various effector:target (E:T) cell ratios. [Figure 49] Figure 1 shows the workflow for the generation of alternative TREX cell chassis. TREX cells (REX edit-containing CD8+ T cells) were generated and then further engineered to overexpress specific genes of interest (transposon insertion) prior to enrichment screening. In some cases, these cells were further engineered to overexpress other genes of interest (additional editing). Different assays were used to evaluate the engineered TREX cells for long-term growth potential and functionality. [Figure 50] Additional editing combinations are shown to be enriched in TREX cells. TREX cells (REX editing-containing CD8+ T cells) were generated and then further modified as shown in Figure 49 to produce TREX0 T cells, TREX3B' cells, TREX3B' T cells, TREX3B cells, TREX3C cells, TREX3C' cells, and TREX3C' T cells. The engineered TREX cells were assessed over time for enrichment of editing combinations. [Figure 51]Figure 49 shows that TREX cell variants exhibit enhanced expansion and lifespan compared to REX-edited CD8+ T cells. TREX cell variants were generated as described in Figure 49. TREX cell variants (TREX0T, TREX3B', TREX3B'T, TREX3B, TREX3C*, TREX3C', and TREX3C'T*) exhibit enhanced lifespan and proliferation capacity compared to TREX (TREX0) cells. a: REX editing, b: transposon insertion, c: REX3B' sort, d: REX3C' sort, e: REX3B sort 1, f: REX0 sort, g: REX3B sort 2, h: REX3B re-thaw (for growth curve), i: TERT insertion. [Figure 52] We demonstrate that TREX cell variants can be single-cell cloned. TREX cells (REX-edited CD8+ T cells) were generated and then further modified as shown in Figure 49 to produce TREX3B', TREX3B, and TREX3C' cells. The single-cell clonability of engineered TREX cells was assessed using flow cytometry-based cell sorting or limiting dilution analysis. Engineered cells were seeded at 1, 10, or 100 cells per well and cultured for 2 weeks in the presence of IL-2-containing medium. In contrast to TREX0 cells, which could not be single-cell cloned, TREX3B', TREX3B, and TREX3C' cells were able to generate colonies at varying frequencies. Similar results were obtained for TREX0T, TREX3B, and TREX3CN cells. [Figure 53]We demonstrate that CAR-TREX cell variants expand robustly during the culture process. BCMA-targeted CARs were introduced into young TREX cells (TREX0) and TREX cell variants (TREX0T, TREX3B', TREX3B'T, TREX3B, TREX3C', and TREX3C'T). The expansion of CAR-TREX cells and CAR-TREX cell variants was tracked throughout the editing, transduction, and enrichment process for each group (a: REX editing, b: transposon insertion, c: REX3B' sort, d: REX3C' sort, e: REX3B sort 1, f: REX3B sort 2, g: REX3B re-thaw, 1: REX0 purification, 2: REX3C' and REX3B' purification, 3: REX0 cryopreservation, 4: REX3B purification, 5: REX3C'T purification, 6: REX3C' cryopreservation, 7: REX3C'T cryopreservation, 8: REX3B'T cryopreservation). [Figure 54A] We demonstrate that CAR-TREX cell variants are cytotoxic and persist in sequential killing assays. BCMA-targeted CARs were introduced into whole primary T cells, young TREX cells (TREX0-D88), and TREX cell variants (TREX0T, TREX3B', TREX3B'T, TREX3B, TREX3C', and TREX3C'T-D126-182). The cytotoxicity (Figure 54A) and persistence (Figure 54B) of CAR-expressing cells were measured over multiple rounds of coculture with BCMA-expressing JJN3 target cells at a 1:1 effector:target cell ratio. After each round of coculture, the percent cell lysis (Figure 54A) and effector cell expansion (Figure 54B) were assessed. [Figure 54B]We demonstrate that CAR-TREX cell variants are cytotoxic and persist in sequential killing assays. BCMA-targeted CARs were introduced into whole primary T cells, young TREX cells (TREX0-D88), and TREX cell variants (TREX0T, TREX3B', TREX3B'T, TREX3B, TREX3C', and TREX3C'T-D126-182). The cytotoxicity (Figure 54A) and persistence (Figure 54B) of CAR-expressing cells were measured over multiple rounds of coculture with BCMA-expressing JJN3 target cells at a 1:1 effector:target cell ratio. After each round of coculture, the percent cell lysis (Figure 54A) and effector cell expansion (Figure 54B) were assessed. [Figure 55] We demonstrate that CAR-TREX cell variants are functional in vivo at least as well as the young CAR-TREX cell benchmark. NSG mice were inoculated with 10E6 MM1S-luciferase tumor cells, and three days later, primary CAR-T cells (D14), CAR-TREX cells (TREX0, D99-102), or CAR-TREX cell variants (TREX0T, TREX3B', TREX3B'T, TREX3B, and TREX3C'-D137-200) were administered at 2E6, 10E6, or 20E6 cells per mouse. All CAR-TREX cell variants demonstrated in vivo functionality despite their higher degree of expansion in vitro. [Figure 56] Figure 4 shows that cryo-recovered TERT-overexpressing CAR-NKREX cells are cytotoxic and persist better in a continuous killing assay than younger cryo-recovered CAR-NKREX cells. BCMA-targeted CAR-NKREX cells and TERT-overexpressing CAR-NKREX cells were generated as described in Figure 47. The cytotoxicity (left) and persistence (right) of CAR-expressing cells were measured over multiple rounds of co-culture with BCMA-expressing JJN3 target cells at a 2:1 effector:target cell ratio in the presence of IL-2. After each round of co-culture, percent cytolysis (left) and effector cell expansion (right) were assessed. TERT-overexpressing CAR-NKREX cells showed enhanced functionality and persistence in this assay. [Figure 57] Figure 1 shows that TERT-overexpressing CAR-NKREX cells maintain cytotoxic potential after cryo-recovery and expansion for more than 400 days in culture. TERT-overexpressing CAR-NKREX cells were cryopreserved on day 256 and then cryo-recovered and expanded until day 414 before the start of the assay. Percent cell lysis was calculated at the end of co-culture. [Figure 58] Schematic diagram of the generation of TERT-overexpressing NKREX and CAR-NKREX cells. [Figure 59] We demonstrate that TERT overexpression reproducibly confers an advantage to NKREX and CAR-NKREX cells. NKREX cells were generated and engineered to express a BCMA-targeted CAR. All groups were monitored throughout the manipulation and culture process for expansion (top). Enrichment of TERT-expressing NKREX cells was also assessed over time (bottom). [Figure 60] TERT-overexpressing CAR-NKREX cells are cytotoxic and expand better in serial killing assays than younger CAR-NKREX cells. Effector cell cytotoxicity (left) and persistence (right) were measured over multiple rounds of coculture with BCMA-expressing JJN3 target cells at a 2:1 effector:target cell ratio in the presence of IL-2. [Figure 61] A timeline for the generation of STAT mutant-expressing TREX cells is shown. [Figure 62] 1 shows that STAT5A and STAT5B mutants are enriched in REX-edited CD8+ T cells upon in vitro enrichment. [Figure 63A] We show that STAT5A and STAT5B mutants are functional in TREX cells and result in upregulation of CD25 expression 5 days after transduction. Surface expression of CD25 was assessed in control TREX cells (UT) and TREX cells containing STAT5A, STAT5B, and STAT3 mutants over long-term cell culture. [Figure 63B]We show that STAT5A and STAT5B mutants are functional in TREX cells and result in upregulation of CD25 expression 20 days after transduction. Surface expression of CD25 was assessed in control TREX cells (UT) and TREX cells containing STAT5A, STAT5B, and STAT3 mutants over long-term cell culture. [Figure 63C] We show that STAT5A and STAT5B mutants are functional in TREX cells and result in upregulation of CD25 expression 35 days after transduction. Surface expression of CD25 was assessed in control TREX cells (UT) and TREX cells containing STAT5A, STAT5B, and STAT3 mutants over long-term cell culture. [Figure 63D] We show that STAT5A and STAT5B mutants are functional in TREX cells and result in upregulation of CD25 expression 42 days after transduction. Surface expression of CD25 was assessed in control TREX cells (UT) and TREX cells containing STAT5A, STAT5B, and STAT3 mutants over long-term cell culture. [Figure 64-1] We demonstrate that STAT5A and STAT5B mutant TREX cells can express CAR. BCMA-targeted CAR was introduced into unmodified TREX cells (TREX(UT)) and STAT mutant TREX cells (STAT MU1, STAT MU2, STAT MU3, STAT MU4, STAT MU5, STAT MU6, STAT MU7, STAT MU8, STAT MU9, STAT MU10, and STAT MU11). CAR-expressing cells were further enriched to high purity before functional evaluation. [Figure 64-2]We demonstrate that STAT5A and STAT5B mutant TREX cells can express CAR. BCMA-targeted CAR was introduced into unmodified TREX cells (TREX(UT)) and STAT mutant TREX cells (STAT MU1, STAT MU2, STAT MU3, STAT MU4, STAT MU5, STAT MU6, STAT MU7, STAT MU8, STAT MU9, STAT MU10, and STAT MU11). CAR-expressing cells were further enriched to high purity before functional evaluation. [Figure 64-3] We demonstrate that STAT5A and STAT5B mutant TREX cells can express CAR. BCMA-targeted CAR was introduced into unmodified TREX cells (TREX(UT)) and STAT mutant TREX cells (STAT MU1, STAT MU2, STAT MU3, STAT MU4, STAT MU5, STAT MU6, STAT MU7, STAT MU8, STAT MU9, STAT MU10, and STAT MU11). CAR-expressing cells were further enriched to high purity before functional evaluation. [Figure 65] We show that STAT mutant CAR-TREX cells expand robustly during the culture process. We followed the expansion of STAT mutant CAR-TREX cells throughout the editing, transduction, and enrichment processes. [Figure 66]STAT5A and STAT5B mutants confer varying degrees of IL-2 independence in vitro. STAT5A mutant, STAT5B mutant, and STAT3 mutant TREX cells, as well as control TREX cells, were cultured in medium without IL-2, and expansion was followed over time (top). In the absence of CAR, several groups (control TREX cells, STAT MU1, STAT MU3, STAT MU4, STAT MU6, STAT MU7, STAT MU8, STAT MU10, STAT MU12, and STAT MU13) exhibited IL-2 dependence, while other groups (STAT MU2, STAT MU5, STAT MU9, and STAT MU11) grew independently of IL-2. Cells were grown in the presence of decreasing amounts of IL-2, and expansion was followed over time (bottom). STAT MU6 and STAT MU7 CAR-TREX cells maintained their dependence on IL-2 (although these populations proliferated under low IL-2 conditions). STAT MU8 and STAT MU9 CAR-TREX cells demonstrated the ability to proliferate in the absence of IL-2, whereas the greatest degree of IL-2 independence was observed in STAT MU4, STAT MU5, STAT MU10, and STAT MU11 CAR-TREX cells. [Figure 67] We show that STAT5B mutant CAR-TREX cell variants remain functional even after more than 150 days in culture. The impedance-based xCELLigence platform was used to measure the cytotoxicity of CAR-expressing cells. Percent cytolysis was determined 12 and 72 hours after initiation of co-culture of effector and target cells at various effector:target (E:T) cell ratios. [Figure 68]These results show that STAT5B mutant CAR-TREX cells exhibit enhanced persistence and functionality compared to young CAR-TREX cell benchmarks in a continuous killing assay. The cytotoxicity and persistence of CAR-expressing cells were measured over multiple rounds of coculture with BCMA-expressing JJN3 target cells at an effector:target cell ratio of 0.3:1. After each round of coculture, percent cell lysis (top) and effector cell expansion (bottom) were assessed. All STAT5B mutants enhanced the persistence and cytotoxicity of CAR-TREX cells to varying degrees. [Figure 69] We demonstrate that STAT5B mutant CAR-TREX cells are functional in vivo at least as well as the benchmark young CAR-TREX cells. NSG mice were inoculated with 2E6 MM1S-luciferase tumor cells. Six days later, BCMA-TREX cells (TREX, day 99) or STAT mutant BCMA-TREX cells (STAT MU5, STAT MU6, STAT MU7, STAT MU9, and STAT MU11—all day 112) were administered at 2E6 or 10E6 cells per mouse. All STAT mutant TREX cells demonstrated in vivo functionality despite a higher degree of expansion in vitro (numbers indicated under group headings). [Figure 70] Schematic diagram of the generation of STAT mutant overexpressing NKREX cells. Eight STAT5B mutants and two STAT3 mutants were overexpressed in NKREX cells (REX editing-containing NK cells) according to the timeline shown. [Figure 71] We show that STAT5B and STAT3 mutants are enriched in REX-edited NK cells in vitro. A fluorescent reporter was used to track the enrichment of STAT mutants over time. STAT5B and STAT3 mutant-expressing NKREX cells were enriched during the cell culture process. [Figure 72]This shows that STAT5B and STAT3 mutant expression enhances the proliferation of NKREX cells. STAT5B mutant and STAT3 mutant were overexpressed in NKREX cells (NK cells containing REX editing) according to Figure 70. STAT5B mutant NKREX cells (top) and STAT3 mutant NKREX cells (bottom) proliferated at a faster rate than donor-matched unmodified NKREX cells, as evidenced by the steeper growth curves after introduction of the STAT mutants. [Figure 73] We demonstrate that STAT mutant-expressing NKREX cells retain functionality in in vitro cytotoxicity assays. The cytotoxic function of STAT mutant-containing NKREX cells was assessed through coculture of control (unmodified) NKREX and STAT mutant NKREX cells with K562-luciferase cells, and the percentage of cytolysis was determined 24 hours after the initiation of coculture. Effector cells were cocultured with K562 cells at two different effector:target cell ratios (1:1-upper and 2:1-lower). STAT5B mutant- and STAT3 mutant-expressing NKREX cells exhibited similar cytotoxic activity to unmodified NKREX cells. DETAILED DESCRIPTION OF THE INVENTION

[0078] The present disclosure relates to methods, cells, and compositions for preparing cell populations and compositions for adoptive cell therapy. In particular, provided herein are methods for the expansion and propagation of primary immune cells, including T cell populations.

[0079] As utilized in accordance with this disclosure, unless otherwise indicated, all technical and scientific terms shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0080] As used herein, the terms "comprise" and "include," and variations thereof (e.g., "comprises," "comprising," "includes," and "including") are understood to indicate the inclusion of a stated component, feature, element, or step, or group of components, features, elements, or steps, but not the exclusion of any other component, feature, element, or step, or group of components, features, elements, or steps. The terms "comprising," "consisting essentially of," and "consisting of" may be substituted for either of the other two terms while retaining their ordinary meaning.

[0081] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0082] The percentages disclosed herein can vary from the disclosed values ​​by ±10, 20, or 30% amounts and still be within the contemplated range of the disclosure.

[0083] Unless otherwise indicated or apparent from the context and the understanding of one of ordinary skill in the art, values ​​herein expressed as ranges can contemplate in different aspects of this disclosure any specific value or subrange within the stated range, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0084] As used herein, ranges and amounts can be expressed as "about" a particular value or range. The term "about" also includes the exact amount. For example, "about 5%" means "about 5%" and also "5%." The term "about" can also refer to ±10% of a given value or range of values. Thus, about 5% also means, for example, 4.5% to 5.5%. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about."

[0085] As used herein, the terms "or" and "and / or" may describe multiple components in combination or exclusively with each other. For example, "x, y, and / or z" may refer to "x" alone, "y" alone, "z" alone, "x, y, and z," "(x and y) or z," "x or (y and z)," or "x or y or z." Resistant to replicative senescence (RRS) refers to primary immune cells that are resistant to replicative senescence (RS), which results in a finite number of population doublings. As a result, the populations of primary immune cells described herein advantageously have extended proliferative capacity.

[0086] In one aspect, the disclosure provides a method for generating a population of primary immune cells resistant to replicative senescence (RRS), the method comprising: (a) inhibiting expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5′-thioadenosine phosphorylase (MTAP) in the population of primary immune cells; (b) introducing a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants into the population of primary immune cells; and (c) culturing the primary immune cells in a culture medium, wherein culturing induces proliferation of the primary immune cells to obtain the population of primary immune cells resistant to replicative senescence (RRS).

[0087] In one aspect, the disclosure provides a method for generating a population of primary immune cells resistant to replicative senescence (RRS), the method comprising: (a) inhibiting expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5′-thioadenosine phosphorylase (MTAP) in the population of primary immune cells; (b) introducing a transgene encoding MYC into the population of primary immune cells; and (c) culturing the primary immune cells in a culture medium, wherein culturing induces proliferation of the primary immune cells to obtain the population of primary immune cells resistant to replicative senescence (RRS).

[0088] In one aspect, the disclosure provides a method for generating a population of primary immune cells resistant to replicative senescence (RRS), the method comprising: (a) inhibiting expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5′-thioadenosine phosphorylase (MTAP) in the population of primary immune cells; (b) introducing a transgene encoding TERT into the population of primary immune cells; and (c) culturing the primary immune cells in a culture medium, wherein culturing induces proliferation of the primary immune cells to obtain the population of primary immune cells resistant to replicative senescence (RRS).

[0089] In one aspect, the disclosure provides a method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), the method comprising: (a) inhibiting expression of one or more endogenous regulatory factors in the population of primary immune cells, wherein the endogenous regulatory factors are cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), or S-methyl-5'-thioadenosine phosphorylase (MTAP); and (b) inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells, wherein the endogenous immune-related genes are cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), or S-methyl-5'-thioadenosine phosphorylase (MTAP). (c) inhibiting expression of one or more endogenous immune-related genes, wherein the immune-related genes are beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC); (c) introducing a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants into a population of primary immune cells; and (d) culturing the population of primary immune cells in a culture medium, wherein culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS).

[0090] In one aspect, the disclosure provides a method for generating a population of primary immune cells resistant to replicative senescence (RRS), the method comprising: (a) inhibiting expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5′-thioadenosine phosphorylase (MTAP) in the population of primary immune cells; (b) inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells, wherein the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC); (c) introducing a transgene encoding MYC into the population of primary immune cells; and (d) culturing the primary immune cells in a culture medium, wherein the culturing induces proliferation of the primary immune cells to obtain the population of primary immune cells resistant to replicative senescence (RRS).

[0091] In one aspect, the disclosure provides a method of generating a population of primary immune cells resistant to replicative senescence (RRS), the method comprising: (a) inhibiting expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5′-thioadenosine phosphorylase (MTAP) in the population of primary immune cells; (b) inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells, wherein the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC); (c) introducing a transgene encoding TERT into the population of primary immune cells; and (d) culturing the primary immune cells in a culture medium, wherein the culturing induces proliferation of the primary immune cells to obtain the population of primary immune cells resistant to replicative senescence (RRS).

[0092] In one aspect, the disclosure provides a method for generating a population of primary immune cells resistant to replicative senescence (RRS), the method comprising: (a) inhibiting expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), or S-methyl-5′-thioadenosine phosphorylase (MTAP) in a population of primary immune cells; (b) introducing a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants into the population of primary immune cells; (c) introducing a transgene encoding TERT into the population of primary immune cells; and (d) culturing the population of primary immune cells in a culture medium, wherein culturing induces proliferation of the primary immune cells to obtain the population of primary immune cells resistant to replicative senescence (RRS).

[0093] In one aspect, the disclosure provides a method for generating a population of primary immune cells resistant to replicative senescence (RRS), the method comprising: (a) inhibiting expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5′-thioadenosine phosphorylase (MTAP) in the population of primary immune cells; (b) introducing a transgene encoding MYC into the population of primary immune cells; (c) introducing a transgene encoding TERT into the population of primary immune cells; and (d) culturing the primary immune cells in a culture medium, wherein culturing induces proliferation of the primary immune cells to obtain the population of primary immune cells resistant to replicative senescence (RRS).

[0094] In some embodiments of the methods disclosed herein, the one or more STAT5A mutants may be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants may be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

[0095] In some embodiments of the methods disclosed herein, the method further comprises introducing a transgene encoding TERT into the population of primary immune cells. In some embodiments of the methods disclosed herein, the method further comprises inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells. In some embodiments, the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC). In some embodiments of the methods disclosed herein, the method comprises introducing one or more transgenes encoding an anti-apoptotic factor or a virus-derived factor into the primary immune cells. In some embodiments, the anti-apoptotic factor is either diffuse large B-cell lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2). In some embodiments, the anti-apoptotic factor is diffuse large B-cell lymphoma (Bcl-xL). In some embodiments, the virus-derived agent is any one of Saimiriine gammaherpesvirus2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1.

[0096] In some embodiments of the methods disclosed herein, the methods further comprise inhibiting expression of cluster of differentiation 38 (CD38) in the population of primary immune cells. In some embodiments of the methods disclosed herein, the methods further comprise inhibiting expression of phosphatase and tensin homolog (PTEN) in the population of primary immune cells. In some embodiments of the methods disclosed herein, the methods further comprise inhibiting expression of p53 in the population of primary immune cells.

[0097] In some embodiments of the methods disclosed herein, the methods further comprise introducing a transgene encoding MYC into the population of primary immune cells.

[0098] In some embodiments of the methods disclosed herein, the method further comprises introducing into the population of primary immune cells a transgene encoding KRAS. In some embodiments, the KRAS is mutant KRAS A146V.

[0099] In some embodiments of the methods disclosed herein, the methods further comprise introducing a polynucleotide encoding a chimeric antigen receptor (CAR) into the population of primary immune cells.

[0100] In some embodiments of the methods disclosed herein, the population of primary immune cells comprises total T cells. In some embodiments of the methods disclosed herein, the population of primary immune cells comprises CD8+ T cells. In some embodiments of the methods disclosed herein, the population of primary immune cells comprises CD4+ T cells.

[0101] In some embodiments of the methods disclosed herein, the population of primary immune cells comprises gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells, and / or natural killer T (NKT) cells. In some embodiments of the methods disclosed herein, the population of primary immune cells is human.

[0102] Gene editing refers to changes to the genetic material of primary immune cells. Gene editing includes adding, removing, or altering genetic material. In certain embodiments, gene editing includes introducing a transgene into the primary immune cells and / or inhibiting expression of a gene in the primary immune cells. In certain embodiments, introducing the one or more gene edits includes introducing one or more transgenes. In some embodiments, the one or more transgenes encode an anti-apoptotic factor, a virus-derived factor, or an oncogene or proto-oncogene into the primary immune cells. In some embodiments, gene editing may refer to one or more of: (1) inhibiting expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP); (2) introducing a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants; (3) introducing a transgene encoding diffuse large B-cell lymphoma (Bcl-xL); (4) introducing a transgene encoding MYC; (5) introducing a transgene encoding TERT; (6) inhibiting expression of phosphatase and tensin homolog (PTEN); (7) introducing a transgene encoding KRAS or a mutant thereof; (8) inhibiting expression of p53; (9) inhibiting expression of cluster of differentiation 38 (CD38); and / or (10) inhibiting expression of beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC).

[0103] The term "primary immune cells" can refer to any cell involved in a primary immune response, such as T cells, B cells and NK cells, neutrophils, and monocytes / macrophages / dendritic cells. In some embodiments, primary immune cells can include total T cells, CD4+ T cells, CD8+ T cells, regulatory T cells, gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells, or natural killer T (NKT) cells.

[0104] The term "transgene" refers to any nucleic acid sequence introduced into a cell by experimental manipulation. A transgene may be an "endogenous DNA sequence" or a "heterologous DNA sequence." Transgenes can be isolated and obtained in suitable quantities using one or more methods well known in the art. These methods and other methods useful for isolating transgenes are described, for example, in Sambrook et al., supra, and Berger and Kimmel (Methods in Enzymology: Guide to Molecular Cloning Techniques, vol. 152, Academic Press, Inc., San Diego, CA (1987)).

[0105] A transgene can be incorporated into a "transgene construct" that contains the gene of interest along with other regulatory DNA sequences required for either transient, cell-specific, or enhanced expression of the transgene of interest.

[0106] Transgene can be introduced into cell by any suitable method or technique known in the art.In certain embodiments, transgene is introduced using plasmid-based DNA transposon, lentivirus platform or CRISPR-mediated site-specific integration.Transgene expression in cell can be constitutive or inducible.

[0107] In certain embodiments, the transgene encodes an anti-apoptotic factor. "Anti-apoptotic factor" refers to a protein or oligonucleotide (which may be an oligonucleotide encoding a protein or a silencing nucleotide) that acts to prevent apoptosis in cells, particularly cells undergoing stress, cells signaled to undergo apoptosis, or cells undergoing abnormal cell proliferation. In certain embodiments, the anti-apoptotic factor is diffuse large B-cell lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0108] In certain embodiments, the transgene encodes one or more oncogenes or proto-oncogenes selected from MYC, KRAS, mutant KRAS, NRAS, and mutant NRAS. In certain embodiments, the oncogene is a mutant KRAS selected from G12C and / or A146V. In certain embodiments, the anti-apoptotic factor is a mutant NRAS selected from G12D.

[0109] In certain embodiments, the transgene encodes a virus-derived factor. "Virus-derived factor" refers to both naturally occurring viral peptides, polypeptides, or proteins, as well as peptides, polypeptides, or proteins that exhibit a degree of sequence identity and / or similarity to a viral protein and / or maintain one or more structural, mechanistic, or antigenic qualities of the viral protein. In certain embodiments, the virus-derived factor is from Saimiriine gammaherpesvirus2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1.

[0110] In other embodiments, the transgene encodes a protein associated with an activation signal in a cell.

[0111] In some embodiments, the methods of the present disclosure further comprise inhibiting the expression of one or more endogenous regulatory factors in primary immune cells such that the activity of the endogenous regulatory factors is eliminated or reduced. As used herein, "regulator" refers to a gene encoding a protein involved in regulating cell cycle arrest, cell death, or signal suppression. Endogenous regulatory factors can be downregulated or blocked by any suitable method or technique known in the art. Known methods for downregulating gene expression or reducing the activity of a factor include, but are not limited to, CRISPR / Cas (including cytosine and adenine base editors), microRNA, shRNA, RNAi, TALEN, zinc finger nucleases, meganucleases, neutralizing antibodies, small molecule inhibitors, chemical inhibitors that block downstream signaling pathways, and the like. Inhibition of endogenous regulatory factors can be complete inhibition, partial inhibition, downregulation, or reduction of gene expression or the activity of the factor. In some embodiments, endogenous regulator activity or gene expression is reduced by 1% to 100% (i.e., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%). Regulators include genes encoding proteins involved in regulating cell cycle arrest, cell death, or signal suppression. In certain embodiments, the one or more endogenous regulators are cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP).In particular aspects, the one or more endogenous regulators are RB Transcriptional Corepressor 1 (RB1), TP53, Autophagy and Beclin 1 Regulator 1 (AMBRA1), Neurofibromatosis type 1 (NF1), Tyrosine-protein phosphatase non-receptor type 2 (PTPN2), or Suppressor of Cytokine Signaling 1 (SOCS1).

[0112] In some embodiments, the methods of the present disclosure further comprise introducing one or more transgenes encoding one or more regulatory factors into the primary immune cells. In some embodiments, the methods of the present disclosure further comprise introducing one or more transgenes encoding one or more regulatory factors into the primary immune cells, wherein the one or more regulatory factors are overexpressed in the primary immune cells. In certain embodiments, the transgenes are introduced using a plasmid-based DNA transposon, a lentiviral platform, or CRISPR-mediated site-specific integration. In certain embodiments, the one or more regulatory factors may include a signal transducer and activator of transcription 5A (STAT5A) mutant, a signal transducer and activator of transcription 5B (STA5B) mutant, or a MYC proto-oncogene bHLH transcription factor (c-MYC, MYC).

[0113] In certain aspects, the transgene encodes a signal transducer and activator of transcription 5A (STAT5A) mutant and / or a signal transducer and activator of transcription 5B (STA5B) mutant. STAT5A and STAT5B are members of the STAT family of transcription factors. STAT family members act as transcriptional activators that mediate signal transduction pathways triggered by various cellular ligands, such as IL2, IL4, CSF1, and different growth hormones. In certain aspects, the transgene is introduced using a plasmid-based DNA transposon, a lentiviral platform, or CRISPR-mediated site-specific integration. In some embodiments, STAT5A mutants include, but are not limited to, H299R, N642H, Y665F, S711F, and combinations thereof. In some embodiments, STAT5B mutants include, but are not limited to, H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

[0114] In a particular embodiment, the transgene encodes telomerase reverse transcriptase (TERT).

[0115] The term "endogenous" refers to occurring within or originating from a cell, tissue, or organism, or part of a cell, tissue, or organism.

[0116] In some aspects, the methods of the present disclosure further comprise inhibiting the expression of one or more endogenous immune-related genes in the primary immune cells such that the activity of the immune-related genes is eliminated or reduced. As used herein, "immune-related gene" refers to a gene encoding a protein involved in bringing about an immune response. In certain aspects, the immune-related gene encodes a protein involved in host-versus-graft (HvG) and graft-versus-host (GvH) alloimmune responses. Immune-related genes can be downregulated or blocked by any suitable method or technique known in the art. Known methods for downregulating gene expression or reducing the activity of immune-related genes include, but are not limited to, CRISPR / Cas (including cytosine and adenine base editors), microRNA, shRNA, RNAi, TALEN, zinc finger nucleases, meganucleases, neutralizing antibodies, small molecule inhibitors, chemical inhibitors that block downstream signaling pathways, etc. Inhibition of endogenous immune-related genes can be complete inhibition, partial inhibition, downregulation of gene expression, or reduction of factor activity. In some embodiments, endogenous immune-related gene activity or expression is reduced by 1% to 100% (i.e., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%). Immune-related genes include genes encoding proteins involved in effecting an immune response. Immune-related genes can encode proteins involved in host-versus-graft (HvG) and graft-versus-host (GvH) alloimmune responses. In certain embodiments, the one or more endogenous immune-related genes are beta-2 microglobulin (B2M) or T-cell receptor alpha constant region (TRAC).In certain aspects, the one or more endogenous immune-related genes are selected from the group consisting of major histocompatibility complex (MHC), human leukocyte antigen class I genes (e.g., HLA-A, HLA-B, HLA-C), human leukocyte antigen class II genes (HLA-DR, HLA-DQ, and HLA-DP), T cell receptors (e.g., αβ T cell receptor), interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 23 (IL-23), interferon-γ (IFNγ), CCL2, CCL3, CCL4, CCL5, CXCL2, CXCL9-11, CCL17, CCL27, programmed death 1 (PD-1), and / or PD-L1. death-1, PD-1), TIM3, or TIGIT genes.

[0117] In a further aspect, the methods disclosed herein include inhibiting expression of cluster of differentiation 38 (CD38) in primary immune cells such that CD38 activity is eliminated or reduced. CD38 can be downregulated or blocked by any suitable method or technique known in the art. Known methods for downregulating gene expression or reducing activity of CD38 include, but are not limited to, CRISPR / Cas (including cytosine and adenine base editors), microRNA, shRNA, RNAi, TALEN, zinc finger nucleases, meganucleases, neutralizing antibodies, small molecule inhibitors, chemical inhibitors that block downstream signaling pathways, and the like. In some embodiments, CD38 activity or gene expression is reduced by 1% to 100% (i.e., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%).

[0118] In a further aspect, the methods disclosed herein include inhibiting the expression of p53 in primary immune cells such that p53 activity is eliminated or reduced. In some embodiments, p53 can be downregulated or blocked by any suitable method or technique known in the art. Known methods for downregulating gene expression or reducing the activity of p53 include, but are not limited to, CRISPR / Cas (including cytosine and adenine base editors), microRNA, shRNA, RNAi, TALEN, zinc finger nucleases, meganucleases, neutralizing antibodies, small molecule inhibitors, chemical inhibitors that block downstream signaling pathways, and the like. In some embodiments, p53 activity or gene expression is reduced by 1% to 100% (i.e., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%).

[0119] In a further aspect, the method disclosed herein comprises inhibiting the expression of phosphatase and tensin homolog (PTEN) in primary immune cells such that PTEN activity is eliminated or reduced. PTEN can be downregulated or blocked by any suitable method or technique known in the art. Known methods for downregulating PTEN gene expression or reducing its activity include, but are not limited to, CRISPR / Cas (including cytosine and adenine base editors), microRNA, shRNA, RNAi, TALEN, zinc finger nucleases, meganucleases, neutralizing antibodies, small molecule inhibitors, chemical inhibitors that block downstream signaling pathways, and the like. In some embodiments, PTEN activity or gene expression is reduced by 1% to 100% (i.e., 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, 100%).

[0120] "T REXThe term "regeneratively expandable T cells" refers to "T cells that are regeneratively expandable," e.g., using the techniques and genetic modifications provided herein. More specifically, T REX The cells refer to cells in which expression of some or all of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B CDKN2B, and S-methyl-5'-thioadenosine phosphorylase (MTAP) is reduced or ablated.

[0121] In some embodiments, inhibiting expression of one or more endogenous regulators (e.g., CDKN2A, CDKN2B, or MTAP) occurs after introducing one or more transgenes into the cells. In some embodiments, the primary immune cells into which one or more transgenes have been introduced are cultured for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days before inhibiting one or more endogenous regulators is performed. In a further embodiment, inhibiting expression of PTEN occurs after introducing one or more transgenes into the cells. In a further embodiment, inhibiting expression of P53 occurs after introducing one or more transgenes into the cells. In a further embodiment, inhibiting expression of CD38 occurs after introducing one or more transgenes into the cells. In a further embodiment, introducing a transgene encoding TERT occurs after introducing one or more transgenes into the cells. In some embodiments, the method comprises the sequential steps of: i) introducing one or more transgenes into immune cells and then culturing the cells for at least 2 days, 5 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days; ii) inhibiting one or more endogenous regulatory factors and culturing the cells for at least 2 days, 5 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days; and iii) inhibiting PTEN expression, and / or P53 expression, and / or CD38 expression, and / or introducing TERT.

[0122] In some embodiments, inhibiting expression of one or more endogenous regulators (e.g., CDKN2A, CDKN2B, or MTAP) occurs before introducing one or more transgenes into the cells. In some embodiments, the primary immune cells in which one or more endogenous regulators (e.g., CDKN2A, CDKN2B, MTAP) have been inhibited or ablated are cultured for at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days prior to introducing the one or more transgenes. In certain embodiments, one or more transgenes encoding Bcl-xL, MYC, KRAS, STAT5A, STAT5B, or any combination or variant thereof, may be introduced at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days after inhibiting expression of one or more endogenous regulators (e.g., CDKN2A, CDKN2B, or MTAP). In a further aspect, inhibiting expression of PTEN occurs after introducing one or more transgenes into the cell. In a further aspect, inhibiting expression of P53 occurs after introducing one or more transgenes into the cell. In a further aspect, inhibiting expression of CD38 occurs after introducing one or more transgenes into the cell. In a further embodiment, introducing a transgene encoding TERT occurs after introducing one or more transgenes into the cell.In some embodiments, the method comprises the following sequential steps: i) inhibiting one or more endogenous regulatory factors and culturing the cells for at least 2 days, 5 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days; ii) introducing one or more transgenes into immune cells and then culturing the cells for at least 2 days, 5 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days; and iii) inhibiting PTEN expression, and / or P53 expression, and / or CD38 expression, and / or introducing TERT.

[0123] The primary immune cells are cultured under appropriate conditions to promote growth and expansion. In vitro expansion using a culture process activates and induces proliferation of the primary immune cells to obtain an expanded population containing sufficient numbers of primary immune cells for use in therapy.

[0124] The methods disclosed herein are performed ex vivo, meaning that the methods are performed outside of an organism. Treating immune cells ex vivo means exposing the cells to certain biological molecules in vitro, preferably under sterile conditions. In some cases, ex vivo methods additionally include culturing immune cells isolated from a human before administering them back to the same or a different human subject.

[0125] The primary immune cells and / or engineered T cells comprising the expanded populations of the present disclosure may include total T cells, CD4+ T cells, CD8+ T cells, regulatory T cells, gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells, or natural killer T (NKT) cells. T cells are broadly classified into cells expressing CD4 on their surface (also referred to as CD4+ cells) and cells expressing CD8 on their surface (also referred to as CD8+ cells). T cells suitable for use in accordance with the methods provided herein are mononuclear lymphocytes derived from bone marrow (BM), peripheral blood (PB), or cord blood (CB) of a human donor. These cells can be collected directly from the BM, PB, or CB, or after mobilization or stimulation via administration of growth factors and / or cytokines, such as granulocyte-colony stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF), to allogeneic or autologous donors. Those skilled in the art will appreciate that there are many established protocols for isolating peripheral blood mononuclear cells (PBMCs) from peripheral blood. PBMC isolation can be assisted by density gradient separation protocols, typically using density gradient centrifugation techniques using Ficoll®-Hypaque or Histopaque® to separate lymphocytes from other elements in the blood. Preferably, PBMC isolation is performed under sterile conditions. PBMC isolation can also utilize negative selection kits. Alternatively, cell elutriation methods can be used to separate mononuclear cell populations. In some aspects, the primary immune cells are human.

[0126] In some cases, the methods of the present disclosure further include introducing an engineered or chimeric antigen receptor into the activated T cells, thereby generating an expanded population comprising T cells expressing the engineered or chimeric antigen receptor. Chimeric antigen receptors (CARs), also known as chimeric T cell receptors, artificial T cell receptors, and chimeric immune receptors, are engineered receptors that transfer specificity to immune effector cells. Generally, chimeric antigen receptors are transmembrane proteins with a target-antigen binding domain fused to a signaling endodomain via a spacer and a transmembrane domain. When the CAR binds to its target antigen, an activation signal is transmitted to the T cell. In one embodiment, a polynucleotide encoding the chimeric antigen receptor is introduced into primary cells. In one embodiment, a nucleic acid vector encoding the chimeric antigen receptor or an engineered receptor is introduced into T cells, thereby causing the T cells to express the chimeric antigen receptor. In some embodiments, the CAR binds to glypican 3 (GPC3), human epidermal growth factor receptor 2 (HER2), also known as Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2), or B-cell maturation antigen (BCMA). In certain embodiments, CARs for use in immunotherapy can bind to any target.

[0127] CAR Construct Design: The CAR constructs of the present disclosure may have several components, many of which may be selected based on the desired or improved function of the resulting CAR construct. In addition to an antigen-binding domain, a CAR construct may have a spacer domain, a hinge domain, a signal peptide domain, a transmembrane domain, and one or more costimulatory domains. Selecting one component over another (i.e., selecting a particular costimulatory domain from one receptor versus a costimulatory domain from a different receptor) may affect clinical efficacy and safety profile.

[0128] Antigen-binding domain: Antigen-binding domains contemplated herein may comprise an antibody or one or more antigen-binding fragments thereof. In one embodiment, the CAR construct targets GPC3. In one embodiment, the CAR construct targets BCMA. In one embodiment, the CAR construct targets HER2. In one embodiment, the CAR construct targets any molecule useful in immunotherapy. In certain aspects, the antigen-binding domain comprises a single chain variable fragment (scFv) containing light and heavy chain variable regions from one or more antibodies specific for GPC3, BCMA, or HER2, either linked together directly or via a flexible linker (e.g., G4S repeats with one, two, three, or more repeats).

[0129] Spacer domain: CAR constructs can have a spacer domain to provide conformational freedom to facilitate binding to target antigens on target cells. The optimal length of the spacer domain can depend on the proximity of the binding epitope to the target cell surface. For example, proximal epitopes may require longer spacers, while distal epitopes may require shorter spacers. In addition to promoting CAR binding to target antigens, achieving an optimal distance between the CAR cell and the cancer cell can also help sterically block large inhibitory molecules from the immune synapse formed between the CAR cell and the target cancer cell. CARs can have long, medium, or short spacers. Long spacers can include the CH2CH3 domains (approximately 220 amino acids) of immunoglobulin G1 (IgG1) or IgG4 (either native or with modifications common in therapeutic antibodies, such as the S228P mutation), while the CH3 region itself can be used to construct intermediate spacers (approximately 120 amino acids). Shorter spacers can be derived from segments (<60 amino acids) of CD28, CD8α, CD3, or CD4. Short spacers can also be derived from the hinge region of an IgG molecule. These hinge regions can be derived from any IgG isotype and may or may not contain mutations common in therapeutic antibodies, such as the S228P mutation described above.

[0130] Hinge domain: CARs may also have a hinge domain. A flexible hinge domain is a short peptide fragment that provides conformational freedom to facilitate binding to target antigens on tumor cells. It can be used alone or in combination with a spacer sequence. The terms "hinge" and "spacer" are often used interchangeably; for example, an IgG4 sequence can be considered both a "hinge" and a "spacer" sequence (i.e., a hinge / spacer sequence).

[0131] Signal peptide: The CAR construct may further comprise a sequence containing a signal peptide. The signal peptide functions to prompt the cell to translocate the CAR to the cell membrane. Examples include an IgG1 heavy chain signal polypeptide, an Ig kappa or lambda light chain signal peptide, a granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2 or CSFR2) signal peptide, a CD8a signal polypeptide, or a CD33 signal peptide.

[0132] Transmembrane domain: The CAR construct may further comprise a sequence comprising a transmembrane domain. The transmembrane domain may comprise a hydrophobic alpha helix that spans the cell membrane. The properties of the transmembrane domain have not been as thoroughly studied as other aspects of the CAR construct, but they may potentially affect CAR expression and association with endogenous membrane proteins. The transmembrane domain may be derived from, for example, CD4, CD8α, or CD28.

[0133] Costimulatory domain: The CAR construct may further comprise one or more sequences forming a costimulatory domain. A costimulatory domain is a domain capable of enhancing or modulating the response of immune effector cells. The costimulatory domain may comprise, for example, sequences from one or more of CD3 zeta (or CD3z), CD28, 4-1BB, OX-40, ICOS, CD27, GITR, CD2, IL-2Rβ, and MyD88 / CD40. The choice of costimulatory domain influences the phenotype and metabolic signature of the CAR cell. For example, CD28 costimulation results in a potent but short-lived effector-like phenotype with high levels of cytolytic capacity, interleukin-2 (IL-2) secretion, and glycolysis. In contrast, T cells engineered with CARs containing a 4-1BB costimulatory domain tend to expand and persist longer in vivo, have increased oxidative metabolism, are less susceptible to exhaustion, and have an increased ability to generate central memory T cells.

[0134] In certain embodiments, the methods disclosed herein impair the early stimulation of primary immune cells to ensure that cells are in a cycle before introducing one or more gene edits into the cells. In other embodiments, the methods disclosed herein impair the late stimulation (also referred to as "re-stimulation") of primary immune cells. When primary immune cells exit the cell cycle, the cells are re-stimulated to cause the cells to re-enter the cell cycle (i.e., proliferate).

[0135] In certain aspects, the methods disclosed herein further comprise stimulating the population of primary immune cells prior to introducing the one or more gene edits into the population of primary immune cells. In some embodiments, the one or more gene edits may refer to one or more of: (1) inhibiting expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP); (2) introducing a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants; (3) introducing a transgene encoding diffuse large B-cell lymphoma (Bcl-xL); (4) introducing a transgene encoding MYC; (5) introducing a transgene encoding TERT; (6) inhibiting expression of phosphatase and tensin homolog (PTEN); (7) introducing a transgene encoding KRAS or a mutant thereof; (8) inhibiting expression of p53; (9) inhibiting expression of cluster of differentiation 38 (CD38); and / or (10) inhibiting expression of beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC). In certain aspects, the population of primary immune cells is stimulated at least 1 day, at least 2 days, at least 5 days, at least 10 days, at least 15 days, at least 20 days, or at least 30 days before introducing one or more gene edits into the population of primary immune cells. Accordingly, in certain aspects, provided herein are methods of generating a population of primary immune cells resistant to replicative senescence (RRS), comprising: i) stimulating a population of primary immune cells; ii) introducing one or more gene edits into the population of primary immune cells; and iii) culturing the population of primary immune cells in a culture medium, wherein culturing induces proliferation of the primary immune cells to obtain the population of primary immune cells resistant to replicative senescence (RRS).

[0136] In certain aspects, the methods disclosed herein further comprise stimulating the population of primary immune cells after introducing one or more gene edits into the primary immune cells. In some embodiments, the one or more gene edits may refer to one or more of: (1) inhibiting expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP); (2) introducing a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants; (3) introducing a transgene encoding diffuse large B-cell lymphoma (Bcl-xL); (4) introducing a transgene encoding MYC; (5) introducing a transgene encoding TERT; (6) inhibiting expression of phosphatase and tensin homolog (PTEN); (7) introducing a transgene encoding KRAS or a mutant thereof; (8) inhibiting expression of p53; (9) inhibiting expression of cluster of differentiation 38 (CD38); and / or (10) inhibiting expression of beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC). In certain aspects, the population of primary immune cells is stimulated at least 1 day, at least 2 days, at least 5 days, at least 10 days, at least 15 days, at least 20 days, or at least 30 days after the introduction of one or more gene edits into the population of primary immune cells. Accordingly, in certain aspects, provided herein are methods of generating a population of primary immune cells resistant to replicative senescence (RRS), comprising: i) introducing one or more gene edits into a population of primary immune cells; ii) culturing the population of primary immune cells in a culture medium; iii) stimulating the population of primary immune cells; and iv) culturing the population of primary immune cells in a culture medium, wherein culturing induces proliferation of the primary immune cells to obtain the population of primary immune cells resistant to replicative senescence (RRS).

[0137] In further aspects, the primary immune cells are restimulated at least once, at least twice, at least three times, at least four times, or at least five times. Accordingly, in certain aspects, provided herein are methods of generating a population of primary immune cells resistant to replicative senescence (RRS), comprising: i) introducing one or more gene edits into a population of primary immune cells; ii) culturing the population of primary immune cells in a culture medium; iii) stimulating the population of primary immune cells; iv) culturing the population of primary immune cells in a culture medium; v) restimulating the population of primary immune cells; and vi) culturing the population of primary immune cells in a culture medium, wherein culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells resistant to replicative senescence (RRS). Any suitable stimulus known in the art can be used to stimulate the immune cells.

[0138] In certain embodiments, the primary immune cells undergo at least about a 50-fold expansion, at least about a 500-fold expansion, at least about a 5000-fold expansion, at least about a 250,000-fold expansion, at least about a 500,000-fold expansion, at least about a 100,000-fold expansion, at least about a 100,000-fold expansion, at least about a 100,000-fold expansion, at least about a 2 ... 6 Magnification of at least about 10 times 7 Magnification of at least about 10 times 8 Magnification of at least about 10 times 9 fold magnification, or at least about 10 10 In certain aspects, the expanded primary immune cell population is resistant to replicative senescence. Furthermore, these cells are not functionally exhausted after long-term expansion and can be directed to carry out cytotoxic functions through engagement of their TCR by T cell engager antibodies or through engagement of chimeric antigen receptors (CARs) (or through natural or transgenic TCRs).

[0139] In certain embodiments, primary immune cells are cultured in culture medium containing supportive cytokines but lacking primary immune cell stimuli. In certain embodiments, primary immune cells undergo expansion during culture in the absence of feeder cells or stimulation through CD3 and / or their antigen receptors. The ability of the disclosed methods to generate immune cells without extensive T cell restimulation or feeder cells advantageously eliminates the problems of scaling up the methods and producing dysfunctional populations of immune cells.

[0140] The methods disclosed herein advantageously allow for the production of human CD8 T cells that have the ability to proliferate for substantial periods in the absence of restimulation through the T cell receptor (TCR) and expand millions of fold during long-term culture. + T cells, human CD4 + Provided are expanded populations of primary immune cells comprising T cells, human regulatory T cells, human gamma-delta T cells, or human natural killer T cells. In certain embodiments, the population of primary immune cells is cultured for at least 20 days, at least 30 days, at least 40 days, at least 50 days, at least 60 days, at least 70 days, at least 80 days, at least 90 days, at least 100 days, at least 150 days, at least 200 days, at least 300 days, or at least 400 days.

[0141] In a further aspect, provided herein are engineered T cells that express a transgene encoding cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or diffuse large B-cell lymphoma that does not express S-methyl-5'-thioadenosine phosphorylase (MTAP) (Bcl-xL).

[0142] In a further aspect, provided herein are engineered T cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP).

[0143] In a further aspect, provided herein are engineered T cells that express a transgene encoding cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), S-methyl-5'-thioadenosine phosphorylase (MTAP), and / or diffuse large B-cell lymphoma that does not express phosphatase and tensin homolog (PTEN) (Bcl-XL).

[0144] In a further aspect, provided herein are engineered T cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and comprise a transgene encoding a signal transducer and activator of transcription 5A (STAT5A) mutant and / or a signal transducer and activator of transcription 5B (STA5B) mutant. In some embodiments, STAT5A mutants can include, but are not limited to, H299R, N642H, Y665F, S711F, and combinations thereof. In some embodiments, STAT5B mutants can include, but are not limited to, H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

[0145] In a further aspect, provided herein are engineered T cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and comprise a transgene encoding a signal transducer and activator of transcription 5A (STAT5A) mutant and / or a signal transducer and activator of transcription 5B (STA5B) mutant, and TERT. In some embodiments, STAT5A mutants can include, but are not limited to, H299R, N642H, Y665F, S711F, and combinations thereof. In some embodiments, STAT5B mutants can include, but are not limited to, H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

[0146] In a further aspect, provided herein are engineered T cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), S-methyl-5'-thioadenosine phosphorylase (MTAP), and / or express a transgene encoding a diffuse large B-cell lymphoma (Bcl-XL) gene, including a transgene encoding a signal transducer and activator of transcription 5A (STAT5A) mutant and / or a signal transducer and activator of transcription 5B (STA5B) mutant, and TERT. In some embodiments, STAT5A mutants may include, but are not limited to, H299R, N642H, Y665F, S711F, and combinations thereof. In some embodiments, STAT5B mutants may include, but are not limited to, H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

[0147] In a further aspect, provided herein are engineered T cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), S-methyl-5'-thioadenosine phosphorylase (MTAP), and / or express a transgene encoding a diffuse large B-cell lymphoma (Bcl-XL) gene, including a transgene encoding a signal transducer and activator of transcription 5A (STAT5A) mutant and / or a signal transducer and activator of transcription 5B (STA5B) mutant, and MYC. In some embodiments, STAT5A mutants may include, but are not limited to, H299R, N642H, Y665F, S711F, and combinations thereof. In some embodiments, STAT5B mutants may include, but are not limited to, H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

[0148] In a further aspect, provided herein are engineered T cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), S-methyl-5'-thioadenosine phosphorylase (MTAP), and / or express a transgene encoding a diffuse large B-cell lymphoma (Bcl-XL) gene, including transgenes encoding signal transducer and activator of transcription 5A (STAT5A) mutants and / or signal transducer and activator of transcription 5B (STA5B) mutants, TERT, and MYC. In some embodiments, STAT5A mutants may include, but are not limited to, H299R, N642H, Y665F, S711F, and combinations thereof. In some embodiments, STAT5B mutants may include, but are not limited to, H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

[0149] In some aspects, provided herein are engineered T cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP), and that contain an optional transgene encoding diffuse large B-cell lymphoma (Bcl-XL) and a transgene encoding MYC.

[0150] In some embodiments, the engineered T cells further comprise a transgene encoding KRAS. In some embodiments, the KRAS is a mutant KRAS selected from G12C and A146V. In one embodiment, the mutant KRAS is A146V. In some embodiments, the engineered T cells further comprise a knockout or ablation of p53. In some embodiments, the engineered T cells further comprise a knockout or ablation of PTEN. In some embodiments, the engineered T cells further comprise a transgene encoding TERT.

[0151] In some aspects, provided herein are engineered T cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), S-methyl-5'-thioadenosine phosphorylase (MTAP), do not express p53, and contain an optional transgene encoding diffuse large B-cell lymphoma (Bcl-XL) and a transgene encoding MYC.

[0152] In some aspects, provided herein are engineered T cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), S-methyl-5'-thioadenosine phosphorylase (MTAP), do not express PTEN, and comprise an optional transgene encoding diffuse large B-cell lymphoma (Bcl-XL) and a transgene encoding MYC.

[0153] In some aspects, provided herein are engineered T cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP), and that contain a transgene encoding TERT.

[0154] In a further aspect, provided herein is an engineered T cell that does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP), and that comprises a transgene encoding TERT, an optional transgene encoding diffuse large B-cell lymphoma (Bcl-XL), and a transgene encoding MYC.

[0155] In some embodiments, the engineered T cells further comprise a transgene encoding KRAS. In some embodiments, the KRAS is a mutant KRAS selected from G12C and A146V. In one embodiment, the mutant KRAS is A146V. In some embodiments, the engineered T cells further comprise a knockout or ablation of p53. In some embodiments, the engineered T cells further comprise a knockout or ablation of PTEN.

[0156] In some embodiments, any of the engineered T cells described herein further comprise a transgene encoding a signal transducer and activator of transcription 5A (STAT5A) mutant and / or a signal transducer and activator of transcription 5B (STA5B) mutant. In some embodiments, STAT5A mutants may include, but are not limited to, H299R, N642H, Y665F, S711F, and combinations thereof. In some embodiments, STAT5B mutants may include, but are not limited to, H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

[0157] In some aspects, provided herein are engineered NK cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP), and that contain a transgene encoding TERT.

[0158] In a further aspect, provided herein are engineered NK cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP), and that contain a transgene encoding TERT, an optional transgene encoding diffuse large B-cell lymphoma (Bcl-XL), and a transgene encoding MYC.

[0159] In some embodiments, the engineered NK cells further comprise a transgene encoding KRAS. In some embodiments, the KRAS is a mutant KRAS selected from G12C and A146V. In one embodiment, the mutant KRAS is A146V. In some embodiments, the engineered NK cells further comprise a knockout or ablation of p53. In some embodiments, the engineered NK cells further comprise a knockout or ablation of PTEN.

[0160] In some embodiments, any of the engineered NK cells described herein further comprise a transgene encoding a signal transducer and activator of transcription 5A (STAT5A) mutant and / or a signal transducer and activator of transcription 5B (STA5B) mutant. In some embodiments, STAT5A mutants may include, but are not limited to, H299R, N642H, Y665F, S711F, and combinations thereof. In some embodiments, STAT5B mutants may include, but are not limited to, H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

[0161] In certain aspects, the engineered T cells disclosed herein do not express one or more endogenous immune-related genes that are present in primary immune cells. In some aspects, the endogenous immune-related gene is beta-2 microglobulin (B2M) or T cell receptor alpha constant region (TRAC).

[0162] In certain embodiments, the engineered T cells disclosed herein do not express p53.

[0163] In certain aspects, the engineered T cells disclosed herein do not express cluster of differentiation 38 (CD38).

[0164] In a further aspect, the disclosure provides engineered T cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), S-methyl-5'-thioadenosine phosphorylase (MTAP), beta-2 microglobulin (B2M), T cell receptor alpha constant region (TRAC), cluster of differentiation 38 (CD38), and / or phosphatase and tensin homolog (PTEN).

[0165] In certain embodiments, the disclosed engineered T cells comprise a polynucleotide encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR binds to glypican 3 (GPC3), B-cell maturation antigen (BCMA), or human epidermal growth factor receptor 2 (HER2, also known as Erb-B2 receptor tyrosine kinase 2 (ERBB2)).

[0166] In certain aspects, the engineered T cells disclosed herein are CD8+ T cells, CD4+ T cells, gamma delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells, natural killer T (NKT) cells, or combinations thereof.

[0167] In some embodiments, the engineered T cells are resistant to replicative senescence (RRS). In some embodiments, the engineered T cells are CD8 + In some embodiments, the engineered T cells are CD4 + T cells. In some embodiments, the engineered T cells are human.

[0168] The expanded T cell populations disclosed herein are useful for cellular immunotherapy, including but not limited to T cell therapy, adoptive cell therapy (ACT), and CAR T cell therapy.

[0169] The expanded T cell populations disclosed herein are useful for treating or preventing a variety of disorders, such as cancer (e.g., a hematological malignancy such as lymphoma or leukemia, or a solid tumor such as melanoma or renal cancer), an autoimmune disease, or an infectious disease such as HIV.

[0170] In one aspect, the disclosure provides use of an engineered T cell for the manufacture of a medicament for treating cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and the engineered T cell comprises a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants.

[0171] In one aspect, the disclosure provides use of an engineered T cell for the manufacture of a medicament for treating cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and the engineered T cell comprises an optional transgene encoding a diffuse large B-cell lymphoma (Bcl-xL) gene and a transgene encoding MYC.

[0172] In one aspect, the disclosure provides use of an engineered T cell for the manufacture of a medicament for treating cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and the engineered T cell comprises a transgene encoding TERT.

[0173] In some embodiments of the uses of the engineered T cells disclosed herein, the one or more STAT5A mutants can be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants can be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

[0174] In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells further comprise a transgene encoding TERT in the population of primary immune cells.

[0175] In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells further comprise inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells. In some embodiments, the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

[0176] In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells comprise one or more transgenes encoding anti-apoptotic factors or virus-derived factors in primary immune cells. In some embodiments, the anti-apoptotic factor is either diffuse large B-cell lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2). In some embodiments, the virus-derived factor is any one of Saimiriine gammaherpesvirus2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1.

[0177] In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells further comprise inhibition of expression of cluster of differentiation 38 (CD38) in the population of primary immune cells. In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells further comprise inhibition of expression of phosphatase and tensin homolog (PTEN) in the population of primary immune cells. In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells further comprise inhibition of expression of p53 in the population of primary immune cells. In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells further comprise a transgene encoding MYC in the population of primary immune cells.

[0178] In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells further comprise a transgene encoding KRAS in the population of primary immune cells. In some embodiments, the KRAS is mutant KRAS A146V.

[0179] In some embodiments of the uses of the engineered T cells disclosed herein, the engineered T cells further comprise a polynucleotide encoding a chimeric antigen receptor (CAR) in the population of primary immune cells.

[0180] In one aspect, the disclosure provides an engineered T cell for the treatment of cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and the engineered T cell comprises a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants.

[0181] In one aspect, the disclosure provides an engineered T cell for the treatment of cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5′-thioadenosine phosphorylase (MTAP), and comprises an optional transgene encoding a diffuse large B-cell lymphoma (Bcl-xL) gene and a transgene encoding MYC.

[0182] In one aspect, the present disclosure provides engineered T cells for the treatment of cancer in a patient, wherein the engineered T cells do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and comprise a transgene encoding TERT.

[0183] As used herein, the term "treatment" or "treating" refers to both therapeutic treatment and prophylactic or preventative measures. Subjects in need of treatment include subjects with cancer, as well as subjects prone to have cancer, or subjects in which cancer is to be prevented. In some aspects, the methods, compositions, and combinations disclosed herein can be used for the treatment of cancer. In other aspects, subjects in need of treatment include subjects with a tumor, as well as subjects prone to have a tumor, or subjects in which a tumor is to be prevented. In certain aspects, the methods, compositions, and combinations disclosed herein can be used for the treatment of tumors. In other aspects, treating tumors includes inhibiting tumor growth, promoting tumor reduction, or both inhibiting tumor growth and promoting tumor reduction.

[0184] In some cases, the T cells obtained according to the methods provided herein can be administered as a therapeutic agent (i.e., for therapeutic use) in a pharmaceutical composition comprising a therapeutically effective amount of the T cells.

[0185] As used herein, the term "pharmaceutical composition" or "therapeutic composition" refers to a compound or composition that can induce a desired therapeutic effect when properly administered to a subject. In some aspects, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one immune cell of the present disclosure.

[0186] As used herein, the term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" refers to one or more formulation materials suitable for achieving or enhancing delivery of one or more immune cells of the present disclosure.

[0187] The term "subject" is intended to include human and non-human animals, particularly mammals. In certain embodiments, the subject is a human patient.

[0188] As used herein, the term "administration" or "administering" refers to providing, contacting, and / or delivering one or more compounds by any suitable route to achieve a desired effect. Administration may include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intra-articular, intra-arterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection), transdermal, topical, buccal, rectal, vaginal, nasal, ocular, inhalation, and implant.

[0189] Without limiting the disclosure, several aspects of the disclosure are described herein for purposes of illustration.

[0190] Embodiments: Embodiment 1. A method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in primary immune cell populations; (b) introducing a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants into a population of primary immune cells; (c) culturing the primary immune cells in a culture medium, and culturing, wherein the culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS). Embodiment 2. The method of embodiment 1, further comprising stimulating the population of primary immune cells prior to performing step (a) and / or step (b) and / or step (c) on the population of primary immune cells. Embodiment 3. The method of either embodiment 1 or embodiment 2, further comprising stimulating the population of primary immune cells after performing step (a) and / or step (b) and / or step (c) on the population of primary immune cells. Embodiment 4. The method of any one of embodiments 1-3, wherein the one or more STAT5A mutants can be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants can be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof. Embodiment 5. The method of any one of embodiments 1-4, further comprising introducing a transgene encoding TERT into the population of primary immune cells. Embodiment 6 The method of any one of embodiments 1-5, further comprising inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells. Embodiment 7. The method of embodiment 6, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC). Embodiment 8 The method of any one of embodiments 1-7, further comprising introducing into the population of primary immune cells one or more transgenes encoding anti-apoptotic factors or virus-derived factors. Embodiment 9. The method of embodiment 8, wherein the anti-apoptotic factor is either diffuse large B-cell lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2). Embodiment 10. The method of embodiment 8, wherein the virus-derived agent is any one of Saimiriine gammaherpesvirus2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1. Embodiment 11. The method of any one of embodiments 1-10, further comprising inhibiting expression of cluster of differentiation 38 (CD38), inhibiting expression of phosphatase and tensin homolog (PTEN), and / or inhibiting expression of p53 in the population of primary immune cells. Embodiment 12. The method of any one of embodiments 1 to 11, further comprising introducing a transgene encoding MYC and / or introducing a transgene encoding KRAS into the population of primary immune cells. Embodiment 13. A method for generating a population of primary immune cells resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in primary immune cell populations; (b) introducing a transgene encoding MYC into a population of primary immune cells; (c) culturing the primary immune cells in a culture medium, and culturing, wherein the culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS). Embodiment 14 The method of embodiment 13, further comprising introducing a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) into the population of primary immune cells. Embodiment 15 The method of either embodiment 13 or embodiment 14, further comprising inhibiting expression of p53 in the population of primary immune cells. Embodiment 16 The method of any one of embodiments 13-15, further comprising introducing a transgene encoding KRAS into the population of primary immune cells. Embodiment 17 The method of embodiment 16, wherein KRAS comprises a KRAS A146V mutation. Embodiment 18 The method of any one of embodiments 13-17, further comprising inhibiting expression of phosphatase and tensin homolog (PTEN) in the population of primary immune cells. Embodiment 19 The method of embodiment 18, wherein PTEN expression is inhibited by a CRISPR / Cas system. Embodiment 20. A method for generating a population of primary immune cells resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in primary immune cell populations; (b) introducing a transgene encoding TERT into a population of primary immune cells; (c) culturing the primary immune cells in a culture medium, and culturing, wherein the culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS). Embodiment 21. The method of embodiment 20, further comprising introducing into the population of primary immune cells a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC. Embodiment 22 The method of either embodiment 20 or embodiment 21, further comprising introducing a transgene encoding KRAS into the population of primary immune cells. Embodiment 23 The method of embodiment 22, wherein KRAS comprises a KRAS A146V mutation. Embodiment 24. The method of any one of embodiments 13 to 23, further comprising stimulating the population of primary immune cells prior to performing step (a) and / or step (b) and / or step (c) on the population of primary immune cells. Embodiment 25. The method of any one of embodiments 13 to 23, further comprising stimulating the population of primary immune cells after performing step (a) and / or step (b) and / or step (c) on the population of primary immune cells. Embodiment 26 The method of any one of embodiments 13 to 25, further comprising inhibiting expression of one or more endogenous immune-related genes in the primary immune cells in the population of primary immune cells. Embodiment 27. The method of embodiment 26, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC). Embodiment 28. The method of any one of embodiments 13 to 27, further comprising introducing into the primary immune cells one or more transgenes encoding any one of Saimiriine gammaherpesvirus2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1. Embodiment 29. The method of any one of embodiments 13-28, further comprising inhibiting expression of cluster of differentiation 38 (CD38) in the population of primary immune cells. Embodiment 30 The method of any one of embodiments 1-29, wherein the population of primary immune cells comprises whole T cells. Embodiment 31 The method of any one of embodiments 1-29, wherein the population of primary immune cells comprises CD8+ T cells. Embodiment 32 The method of any one of embodiments 1 to 29, wherein the population of primary immune cells comprises CD4+ cells. Embodiment 33. The method of any one of embodiments 1-32, wherein the population of primary immune cells comprises gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells, and / or natural killer T (NKT) cells. Embodiment 34 The method of any one of embodiments 1 to 33, wherein the population of primary immune cells is human. Embodiment 35 The method of any one of embodiments 1 to 34, further comprising introducing a polynucleotide encoding a chimeric antigen receptor (CAR) into the population of primary immune cells. Embodiment 36 The method of any one of embodiments 1 to 35, wherein the population of primary immune cells may be cultured with or without TCR stimulation for at least 100 days. Embodiment 37. The population of primary immune cells is cultured at least about 10 6 37. The method of any one of embodiments 1 to 36, wherein the image is subjected to a 2x magnification. Embodiment 38 The method of any one of embodiments 1 to 37, wherein the population of primary immune cells is cultured in a culture medium that does not contain a primary immune cell stimulus. Embodiment 39. The method of any one of embodiments 1 to 38, further comprising: (d) restimulating the population of primary immune cells. Embodiment 40. The population of primary immune cells is cultured at least about 10 8 39. The method of claim 39, wherein the image is subjected to a 2x magnification. Embodiment 41 The method of any one of embodiments 1 to 40, wherein the transgene is introduced using a plasmid-based DNA transposon. Embodiment 42 The method of any one of embodiments 1 to 40, wherein the transgene is introduced using a lentiviral platform. Embodiment 43 The method of any one of embodiments 1 to 40, wherein the transgene is introduced using CRISPR-mediated site-specific integration. Embodiment 44. A method for generating a population of primary immune cells resistant to replicative senescence (RRS), comprising: (a) detecting in a population of primary immune cells one or more endogenous regulatory factors, inhibiting expression of one or more endogenous regulatory factors, wherein the endogenous regulatory factors are cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), or S-methyl-5'-thioadenosine phosphorylase (MTAP); (b) one or more endogenous immune-related genes in the population of primary immune cells, inhibiting expression of one or more endogenous immune-related genes, wherein the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC); (c) introducing a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants into the population of primary immune cells; (d) culturing the population of primary immune cells in a culture medium, and culturing, wherein the culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS). Embodiment 45. The method of embodiment 44, further comprising introducing a transgene encoding either diffuse large B-cell lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2) into the population of primary immune cells. Embodiment 46. The method of any of embodiment 44 or embodiment 45, further comprising stimulating the population of primary immune cells prior to performing step (a) and / or step (b) and / or step (c) and / or step (d) on the population of primary immune cells. Embodiment 47. The method of any one of embodiments 44 to 46, further comprising stimulating the population of primary immune cells after performing step (a) and / or step (b) and / or step (c) and / or step (d) on the population of primary immune cells. Embodiment 48. The method of any one of embodiments 44-47, wherein the one or more STAT5A mutants can be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants can be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof. Embodiment 49. The method of any one of embodiments 44 to 48, further comprising introducing a transgene encoding TERT into the population of primary immune cells. Embodiment 50. The method of any one of embodiments 44-49, further comprising inhibiting expression of cluster of differentiation 38 (CD38), inhibiting expression of phosphatase and tensin homolog (PTEN), and / or inhibiting expression of p53 in the population of primary immune cells. Embodiment 51. The method of any one of embodiments 44 to 50, further comprising introducing into the population of primary immune cells a transgene encoding MYC and / or a transgene encoding KRAS. Embodiment 52. A method for generating a population of primary immune cells resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in primary immune cell populations; (b) inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells, wherein the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC); and (c) introducing a transgene encoding MYC into a population of primary immune cells; (d) culturing the primary immune cells in a culture medium, and culturing, wherein the culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS). Embodiment 53 The method of embodiment 52, further comprising introducing a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) into the population of primary immune cells. Embodiment 54 The method of either embodiment 52 or embodiment 53, further comprising inhibiting expression of p53 in the population of primary immune cells. Embodiment 55 The method of any one of embodiments 52-54, further comprising introducing a transgene encoding KRAS into the population of primary immune cells. Embodiment 56 The method of embodiment 55, wherein KRAS comprises a KRAS A146V mutation. Embodiment 57. The method of any one of embodiments 52-56, further comprising inhibiting expression of phosphatase and tensin homolog (PTEN) in the population of primary immune cells. Embodiment 58 The method of embodiment 57, wherein PTEN expression is inhibited by a CRISPR / Cas system. Embodiment 59. A method for generating a population of primary immune cells resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in primary immune cell populations; (b) inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells, wherein the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC); and (c) introducing a transgene encoding TERT into a population of primary immune cells; (d) culturing the primary immune cells in a culture medium, and culturing, wherein the culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS). Embodiment 60. The method of embodiment 59, further comprising introducing into the population of primary immune cells a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC. Embodiment 61 The method of any of embodiment 59 or embodiment 60, further comprising introducing a transgene encoding KRAS into the population of primary immune cells. Embodiment 62 The method of embodiment 61, wherein KRAS comprises a KRAS A146V mutation. Embodiment 63. The method of any one of embodiments 52 to 62, further comprising stimulating the population of primary immune cells prior to performing step (a) and / or step (b) and / or step (c) and / or step (d) on the population of primary immune cells. Embodiment 64. The method of any one of embodiments 52 to 62, further comprising stimulating the population of primary immune cells after performing step (a) and / or step (b) and / or step (c) and / or step (d) on the population of primary immune cells. Embodiment 65. The method of any one of embodiments 52 to 64, further comprising introducing into the primary immune cells one or more transgenes encoding any one of Saimiriine gammaherpesvirus2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1. Embodiment 66 The method of any one of embodiments 52-65, further comprising inhibiting expression of cluster of differentiation 38 (CD38) in the population of primary immune cells. Embodiment 67. The method of any one of embodiments 44-66, wherein the population of primary immune cells comprises whole T cells. Embodiment 68 The method of any one of embodiments 44-66, wherein the population of primary immune cells comprises CD8+ T cells. Embodiment 69. The method of any one of embodiments 44-66, wherein the population of primary immune cells comprises CD4+ cells. Embodiment 70. The method of any one of embodiments 44-66, wherein the population of primary immune cells comprises gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells, and / or natural killer T (NKT) cells. Embodiment 71 The method of any one of embodiments 44-70, wherein the population of primary immune cells is human. Embodiment 72 The method of any one of embodiments 44 to 71, further comprising introducing a polynucleotide encoding a chimeric antigen receptor (CAR) into the population of primary immune cells. Embodiment 73 The method of any one of embodiments 44 to 72, wherein the population of primary immune cells can be cultured for at least 100 days. Embodiment 74. The population of primary immune cells is cultured at least about 10 6 The method of any one of embodiments 44 to 73, wherein the image is subjected to a 2x magnification. Embodiment 75 The method of any one of embodiments 44-74, wherein the population of primary immune cells is cultured in a culture medium that does not contain a primary immune cell stimulus. Embodiment 76. The method of any one of embodiments 44-75, further comprising: (e) restimulating the population of primary immune cells. Embodiment 77. The population of primary immune cells is cultured at least about 10 8 77. The method of embodiment 76, wherein the image is subjected to a 2x magnification. Embodiment 78. The method of any one of embodiments 44 to 77, wherein the transgene is introduced using a plasmid-based DNA transposon. Embodiment 79. The method of any one of embodiments 44 to 78, wherein the transgene is introduced using a lentiviral platform. Embodiment 80. The method of any one of embodiments 44 to 79, wherein the transgene is introduced using CRISPR-mediated site-specific integration. Embodiment 81. A method for generating a population of primary immune cells resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), or S-methyl-5'-thioadenosine phosphorylase (MTAP) in a population of primary immune cells; (b) introducing a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants into a population of primary immune cells; (c) introducing a transgene encoding TERT into a population of primary immune cells; (d) culturing the population of primary immune cells in a culture medium, and culturing, wherein the culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS). Embodiment 82. The method of embodiment 81, further comprising stimulating the primary immune cells before performing step (a) and / or step (b) and / or step (c) and / or step (d) in the population of primary immune cells. Embodiment 83. The method of either embodiment 81 or embodiment 82, further comprising stimulating the primary immune cells after performing step (a) and / or step (b) and / or step (c) and / or step (d) in the population of primary immune cells. Embodiment 84 The method of any one of embodiments 1 to 83, further comprising inhibiting expression of 81 or more endogenous immune-related genes in the population of primary immune cells. Embodiment 85. The method of any one of embodiments 81-84, wherein the one or more STAT5A mutants can be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants can be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof. Embodiment 86. The method of embodiment 84, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC). Embodiment 87. The method of any one of embodiments 81-86, further comprising inhibiting expression of cluster of differentiation 38 (CD38), inhibiting expression of phosphatase and tensin homolog (PTEN), and / or inhibiting expression of p53 in the population of primary immune cells. Embodiment 88. The method of any one of embodiments 81 to 87, further comprising introducing into the population of primary immune cells a transgene encoding MYC and / or a transgene encoding KRAS. Embodiment 89. A method for generating a population of primary immune cells resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in primary immune cell populations; (b) introducing a transgene encoding MYC into a population of primary immune cells; (c) introducing a transgene encoding TERT into a population of primary immune cells; (d) culturing the primary immune cells in a culture medium, and culturing, wherein the culturing induces proliferation of the primary immune cells to obtain a population of primary immune cells that are resistant to replicative senescence (RRS). Embodiment 90 The method of embodiment 89, further comprising introducing a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) into the population of primary immune cells. Embodiment 91 The method of either embodiment 89 or embodiment 90, further comprising inhibiting expression of p53 in the population of primary immune cells. Embodiment 92 The method of any one of embodiments 89-91, further comprising introducing a transgene encoding KRAS into the population of primary immune cells. Embodiment 93 The method of embodiment 92, wherein KRAS comprises a KRAS A146V mutation. Embodiment 94 The method of any one of embodiments 89-93, further comprising inhibiting expression of phosphatase and tensin homolog (PTEN) in the population of primary immune cells. Embodiment 95 The method of embodiment 94, wherein PTEN expression is inhibited by a CRISPR / Cas system. Embodiment 96. The method of any one of embodiments 89 to 95, further comprising stimulating the population of primary immune cells prior to performing step (a) and / or step (b) and / or step (c) and / or step (d) on the population of primary immune cells. Embodiment 97. The method of any one of embodiments 89 to 96, further comprising stimulating the population of primary immune cells after performing step (a) and / or step (b) and / or step (c) and / or step (d) on the population of primary immune cells. Embodiment 98 The method of any one of embodiments 89 to 97, further comprising inhibiting expression of one or more endogenous immune-related genes in the primary immune cells in the population of primary immune cells. Embodiment 99. The method of embodiment 98, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC). Embodiment 100. The method of any one of embodiments 89 to 99, further comprising introducing into the primary immune cells one or more transgenes encoding any one of Saimiriine gammaherpesvirus2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1. Embodiment 101. The method of any one of embodiments 89-100, further comprising inhibiting expression of cluster of differentiation 38 (CD38) in the population of primary immune cells. Embodiment 102. The method of any one of embodiments 81-101, wherein the population of primary immune cells comprises whole T cells. Embodiment 103. The method of any one of embodiments 81-101, wherein the population of primary immune cells comprises CD8+ T cells. Embodiment 104. The method of any one of embodiments 81-101, wherein the population of primary immune cells comprises CD4+ cells. Embodiment 105. The method of any one of embodiments 81-101, wherein the population of primary immune cells comprises gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells, and / or natural killer T (NKT) cells. Embodiment 106 The method of any one of embodiments 81-105, wherein the population of primary immune cells is human. Embodiment 107 The method of any one of embodiments 81 to 106, further comprising introducing a polynucleotide encoding a chimeric antigen receptor (CAR) into the population of primary immune cells. Embodiment 108 The method of any one of embodiments 81 to 107, wherein the population of primary immune cells can be cultured for at least 100 days. Embodiment 109. The population of primary immune cells is cultured at least about 10 6 The method of any one of embodiments 81 to 108, wherein the image is subjected to a 2x magnification. Embodiment 110. The method of any one of embodiments 81 to 109, wherein the population of primary immune cells is cultured in a culture medium that does not contain a primary immune cell stimulus. Embodiment 111. The method of any one of embodiments 81 to 110, further comprising: (e) stimulating the population of primary immune cells. Embodiment 112. The population of primary immune cells is cultured at least about 10 8 The method of embodiment 111, wherein the image is magnified by 2x. Embodiment 113. The method of any one of embodiments 81 to 112, wherein the transgene is introduced using a plasmid-based DNA transposon. Embodiment 114. The method of any one of embodiments 81 to 112, wherein the transgene is introduced using a lentiviral platform. Embodiment 115. The method of any one of embodiments 81 to 112, wherein the transgene is introduced using CRISPR-mediated site-specific integration. Embodiment 116. An engineered immune cell population produced according to the method of any one of embodiments 1 to 115. Embodiment 117. A pharmaceutical composition comprising the engineered immune cell population of embodiment 116 and a pharmaceutically acceptable carrier. Embodiment 118. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 117. Embodiment 119. An engineered T cell that does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), wherein the engineered T cell comprises a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants. Embodiment 120. The engineered T cell of embodiment 119, wherein the one or more STAT5A mutants can be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants can be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof. Embodiment 121. The engineered T cell of any of embodiment 119 or embodiment 120, further comprising introducing a transgene encoding TERT. Embodiment 122. The engineered T cell of embodiment 119, wherein the engineered T cell further comprises a transgene encoding either diffuse large B-cell lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2). Embodiment 123. The engineered T cell of either embodiment 119 or embodiment 120, wherein the engineered T cell does not express one or more endogenous immune-related genes. Embodiment 124. The engineered T cell of embodiment 123, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC). Embodiment 125. The engineered T cell of any one of embodiments 119 to 124, wherein the engineered T cell does not express cluster of differentiation 38 (CD38), phosphatase and tensin homolog (PTEN), and / or p53. Embodiment 126. The engineered T cell of any one of embodiments 119 to 125, further comprising a transgene encoding MYC and / or a transgene encoding KRAS. Embodiment 127. Engineered T cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP). Embodiment 128. The engineered T cell of embodiment 127, further comprising a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC. Embodiment 129. The engineered T cell of either embodiment 127 or embodiment 128, wherein the engineered T cell does not express p53. Embodiment 130. The engineered T cell of any one of embodiments 127-129, further comprising a transgene encoding KRAS. Embodiment 131. The engineered T cell of embodiment 130, wherein KRAS comprises a KRAS A146V mutation. Embodiment 132. The engineered T cell of any one of embodiments 127 to 131, wherein the engineered T cell does not express phosphatase tensin homolog (PTEN). Embodiment 133. The engineered T cell of embodiment 132, wherein PTEN expression is inhibited by a CRISPR / Cas system. Embodiment 134. An engineered T cell that does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and comprises a transgene encoding TERT. Embodiment 135. The engineered T cell of embodiment 134, wherein the engineered T cell comprises a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC. Embodiment 136 The engineered T cell of any of embodiment 134 or embodiment 135, further comprising a transgene encoding KRAS. Embodiment 137. The engineered T cell of embodiment 136, wherein KRAS comprises a KRAS A146V mutation. Embodiment 138. The engineered T cell of any one of embodiments 127 to 138, wherein the engineered T cell does not express one or more endogenous immune-related genes in primary immune cells in the population of primary immune cells. Embodiment 139. The engineered T cell of embodiment 138, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC). Embodiment 140. The engineered T cell of any one of embodiments 127 to 139, wherein the engineered T cell does not express cluster of differentiation 38 (CD38). Embodiment 141. The engineered T cell of any one of embodiments 119 to 140, further comprising a polynucleotide encoding a chimeric antigen receptor (CAR). Embodiment 142. The engineered T cells of any one of embodiments 119 to 141, wherein the engineered T cells are CD8+ T cells, CD4+ T cells, gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells, natural killer T (NKT) cells, or a combination thereof. Embodiment 143. The engineered T cell of any one of embodiments 119 to 141, wherein the engineered T cell is a CD8+ T cell. Embodiment 144. The engineered T cell of any one of embodiments 119 to 141, wherein the engineered T cell is a CD4+ T cell. Embodiment 145. The engineered T cell of any one of embodiments 119 to 144, wherein the engineered T cell is human. Embodiment 146. An engineered T cell that does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), S-methyl-5'-thioadenosine phosphorylase (MTAP), beta-2 microglobulin (B2M), and / or T cell receptor alpha constant region (TRAC), wherein the engineered T cell comprises a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants, and the engineered T cell comprises a transgene encoding TERT. Embodiment 147. The engineered T cell of embodiment 146, wherein the engineered T cell does not express cluster of differentiation 38 (CD38), phosphatase tensin homolog (PTEN), and / or p53. Embodiment 148. The engineered T cell of either embodiment 146 or embodiment 147, wherein the engineered T cell further comprises a transgene encoding MYC and / or a transgene encoding KRAS. Embodiment 149. The engineered T cell of any one of embodiments 146 to 148, further comprising a polynucleotide encoding a chimeric antigen receptor (CAR). Embodiment 150. The engineered T cell of any one of embodiments 146 to 149, wherein the engineered T cell is a gamma-delta T cell, a mucosal-associated invariant T (MAIT) T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, or a combination thereof. Embodiment 151. The engineered T cell of any one of embodiments 146 to 149, wherein the engineered T cell is a CD8+ T cell. Embodiment 152. The engineered T cell of any one of embodiments 146 to 149, wherein the engineered T cell is a CD4+ T cell. Embodiment 153. The engineered T cell of any one of embodiments 146 to 152, wherein the engineered T cell is human. Embodiment 154. An engineered T cell expressing a transgene encoding a diffuse large B-cell lymphoma (Bcl-XL), wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), S-methyl-5'-thioadenosine phosphorylase (MTAP), and / or phosphatase tensin homolog (PTEN), and the engineered T cell comprises a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants. Embodiment 155. The engineered T cell of embodiment 154, wherein the one or more STAT5A mutants can be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants can be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof. Embodiment 156. The engineered T cell of either embodiment 154 or embodiment 155, wherein the engineered T cell does not express one or more endogenous immune-related genes. Embodiment 157. The engineered T cell of any one of embodiments 154 to 156, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) or T cell receptor alpha constant region (TRAC). Embodiment 158. The engineered T cell of any one of embodiments 154 to 157, wherein the engineered T cell does not express cluster of differentiation 38 (CD38), phosphatase and tensin homolog (PTEN), and / or p53. Embodiment 159. The engineered T cell of any one of embodiments 154 to 158, wherein the engineered T cell comprises a transgene encoding MYC and / or a transgene encoding KRAS. Embodiment 160. The engineered T cell of any one of embodiments 154 to 159, further comprising a polynucleotide encoding a chimeric antigen receptor (CAR). Embodiment 161. The engineered T cells of any one of embodiments 154 to 160, wherein the engineered T cells are CD8+ T cells, CD4+ T cells, delta gamma T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) T cells, or a combination thereof. Embodiment 162. The engineered T cell of any one of embodiments 154 to 160, wherein the engineered T cell is a CD8+ T cell. Embodiment 163. The engineered T cell of any one of embodiments 154 to 160, wherein the engineered T cell is a CD4+ T cell. Embodiment 164. The engineered T cell of any one of embodiments 154 to 163, wherein the engineered T cell is human. Embodiment 165. Use of engineered T cells for the manufacture of a medicament for treating cancer in a patient, wherein the engineered T cells do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and the engineered T cells comprise a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants. Embodiment 166. The use of embodiment 165, wherein the one or more STAT5A mutants can be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants can be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof. Embodiment 167. The use of either embodiment 165 or embodiment 166, wherein the engineered T cells further comprise a polynucleotide encoding a chimeric antigen receptor (CAR). Embodiment 168. The use of any one of embodiments 165 to 167, wherein the engineered T cells further comprise a transgene encoding either diffuse large B-cell lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2). Embodiment 169. The use of any one of embodiments 165 to 168, wherein the engineered T cells further comprise a transgene encoding TERT. Embodiment 170. The use of any one of embodiments 165 to 169, wherein the engineered T cells do not express one or more endogenous immune-related genes. Embodiment 171. The use of embodiment 166, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC). Embodiment 172. The use of any one of embodiments 165 to 171, wherein the engineered T cells do not express cluster of differentiation 38 (CD38), phosphatase and tensin homolog (PTEN), and / or p53. Embodiment 173. The use of any one of embodiments 165 to 172, wherein the engineered T cells comprise a transgene encoding MYC and / or a transgene encoding KRAS. Embodiment 174. Use of engineered T cells for the manufacture of a medicament for treating cancer in a patient, wherein the engineered T cells do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP). Embodiment 175. The use of embodiment 174, wherein the engineered T cells comprise a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC. Embodiment 176. The use of either embodiment 174 or embodiment 175, wherein the engineered T cells do not express p53. Embodiment 177. The use of any one of embodiments 174 to 176, wherein the engineered T cells comprise a transgene encoding KRAS. Embodiment 178. The use of embodiment 177, wherein KRAS comprises a KRAS A146V mutation. Embodiment 179. The use of any one of embodiments 174 to 178, wherein the engineered T cells do not express phosphatase tensin homolog (PTEN). Embodiment 180. The use of embodiment 179, wherein PTEN expression is inhibited by a CRISPR / Cas system. Embodiment 181. Use of engineered T cells for the manufacture of a medicament for treating cancer in a patient, wherein the engineered T cells do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and the engineered T cells comprise a transgene encoding TERT. Embodiment 182. The use of embodiment 181, wherein the engineered T cells comprise a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC. Embodiment 183. The use of either embodiment 181 or embodiment 182, wherein the engineered T cells comprise a transgene encoding KRAS. Embodiment 184. The use of embodiment 183, wherein KRAS comprises a KRAS A146V mutation. Embodiment 185. The use of any one of embodiments 181 to 184, wherein the engineered T cells further comprise a polynucleotide encoding a chimeric antigen receptor (CAR). Embodiment 186. The use of any one of embodiments 174 to 185, wherein the engineered T cells do not express one or more endogenous immune-related genes in the primary immune cells in the population of primary immune cells. Embodiment 187. The use of embodiment 186, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC). Embodiment 188. The use of any one of embodiments 174 to 187, wherein the engineered T cells do not express cluster of differentiation 38 (CD38). Embodiment 189. The use of any one of embodiments 165 to 188, wherein the engineered T cells are CD8+ T cells, CD4+ T cells, gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells, natural killer T (NKT) cells, or a combination thereof. Embodiment 190. The use of any one of embodiments 165 to 188, wherein the engineered T cells are CD8+ T cells. Embodiment 191. The use of any one of embodiments 165 to 188, wherein the engineered T cells are CD4+ T cells. Embodiment 192. The use of any one of embodiments 165 to 191, wherein the engineered T cells are human. Embodiment 193. An engineered T cell for the treatment of cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and the engineered T cell comprises a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants. Embodiment 194. The engineered T cell of embodiment 193, wherein the one or more STAT5A mutants can be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants can be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof. Embodiment 195. The engineered T cell of either embodiment 193 or embodiment 194, wherein the engineered T cell further comprises a transgene encoding either diffuse large B-cell lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2). Embodiment 196. The engineered T cell of any one of embodiments 193 to 195, wherein the engineered T cell further comprises a transgene encoding TERT. Embodiment 197. The engineered T cell of any one of embodiments 193 to 196, wherein the engineered T cell does not express one or more endogenous immune-related genes. Embodiment 198. The engineered T cell of embodiment 197, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC). Embodiment 199. The engineered T cell of any one of embodiments 193 to 198, wherein the engineered T cell does not express cluster of differentiation 38 (CD38), PTEN, and / or p53. Embodiment 200. The engineered T cell of any one of embodiments 193 to 199, wherein the engineered T cell comprises a transgene encoding MYC and / or a transgene encoding KRAS. Embodiment 201. The engineered T cell of any one of embodiments 193 to 200, wherein the engineered T cell further comprises a polynucleotide encoding a chimeric antigen receptor (CAR). Embodiment 202. An engineered T cell for the treatment of cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP). Embodiment 203. The engineered T cell of embodiment 202, further comprising a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC. Embodiment 204. The engineered T cell of either embodiment 202 or embodiment 203, wherein the engineered T cell does not express p53. Embodiment 205. The engineered T cell of any one of embodiments 202 to 204, further comprising a transgene encoding KRAS. Embodiment 206. The engineered T cell of embodiment 205, wherein KRAS comprises a KRAS A146V mutation. Embodiment 207. The engineered T cell of any one of embodiments 202 to 206, wherein the engineered T cell does not express phosphatase tensin homolog (PTEN). Embodiment 208. The engineered T cell of embodiment 207, wherein PTEN expression is inhibited by a CRISPR / Cas system. Embodiment 209. An engineered T cell for the treatment of cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and comprises a transgene encoding TERT. Embodiment 210. The engineered T cell of embodiment 209, wherein the engineered T cell comprises a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC. Embodiment 211 The engineered T cell of any of embodiment 209 or embodiment 210, further comprising a transgene encoding KRAS. Embodiment 212. The engineered T cell of embodiment 211, wherein KRAS comprises a KRAS A146V mutation. Embodiment 213. The engineered T cell of any one of embodiments 202 to 212, wherein the engineered T cell does not express one or more endogenous immune-related genes in primary immune cells in the population of primary immune cells. Embodiment 214. The engineered T cell of embodiment 213, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC). Embodiment 215. The engineered T cell of any one of embodiments 202 to 214, wherein the engineered T cell does not express cluster of differentiation 38 (CD38). Embodiment 216. The engineered T cell of any one of embodiments 202 to 215, wherein the engineered T cell further comprises a polynucleotide encoding a chimeric antigen receptor (CAR). Embodiment 217. The engineered T cells of any one of embodiments 193 to 216, wherein the engineered T cells are CD8+ T cells, CD4+ T cells, gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells, natural killer T (NKT) cells, or a combination thereof. Embodiment 218. The engineered T cell of any one of embodiments 193 to 216, wherein the engineered T cell is a CD8+ T cell. Embodiment 219. The engineered T cell of any one of embodiments 193 to 216, wherein the engineered T cell is a CD4+ T cell. Embodiment 220. The engineered T cell of any one of embodiments 193 to 219, wherein the engineered T cell is human. [Example]

[0191] The following examples illustrate certain aspects of the present disclosure and various uses thereof. They are provided for illustrative purposes only and should not be construed as limiting the scope of the disclosure in any way.

[0192] Example 1: Overexpression of anti-apoptotic or virus-derived factors can provide a selective survival advantage to transfected T cells in long-term culture. Transposon frequencies were assessed in T cell subsets over a 66- to 137-day period using flow cytometry. Total primary human T cells were isolated from the blood of healthy donors, activated with Dynabeads (Human T-Activator CD3 / CD28) for 72 hours, and then transfected with plasmids containing transposons encoding anti-apoptotic factors, virus-derived factors, mutant cytokine receptors, mutant signaling molecules, and / or mutant cell cycle regulatory molecules in addition to fluorescent reporters. mRNA encoding the transposase was simultaneously transfected into the cells to allow chromosomal integration of the transposase. A total of 52 transposon constructs were tested across various screens. Four to eight days after transfection, cells were assessed for baseline transposon integration using flow cytometry. Cells were periodically restimulated with Dynabeads to drive them through proliferation, and transposon enrichment was assessed using flow cytometry. Molecules that enhance the survival of expanding T cells include CD8 + and CD4 + As shown in T cells, they are expected to be enriched above their starting frequency within the total T cell pool.

[0193] These screens revealed that the anti-apoptotic factor Bcl-xL (Bcl-xL) in diffuse large B-cell lymphoma cells was driven through multiple rounds of proliferation over long-term in vitro culture. + and CD8 + We found consistent enrichment in both T cell subsets and in mature T cells, suggesting that this factor may act to enhance the survival of mature T cells that overexpress it (Figure 1). Other factors, such as Bcl-2 and the virus-derived proteins StpA A11 (Saimiriine gammaherpesvirus 2), StpC and Tip (Herpesvirus saimiri), and modified Tio-LMP1 (Herpesvirus Ateles, Epstein-Barr virus), also showed enrichment in T cell subsets (Figure 1).

[0194] Cells expressing transgenes encoding endogenous antiapoptotic factors (Bcl-2 and Bcl-xL) or virus-derived factors (StpA A11, StpC and Tip, and modified Tio-LMP1) showed an enhanced ability to survive in long-term culture with repeated stimulation through their TCR compared with untransfected control cells in the same wells, but these cells did not exhibit a large, sustained increase in their proliferative capacity. These data support the conclusion that T REX This suggests that additional editing may be required to confer the desired phenotype of the cell.

[0195] Example 2: Ablation of CDKN2A, CDKN2B, and MTAP expression substantially increases the proliferative capacity of primary human T cells in long-term culture. Patient-derived leukemia cell lines have been used in laboratories for many years to perform a variety of cellular assays. The transformed nature of these cells can be mapped to a collection of mutations that are also frequently found in patients with T-cell acute lymphoblastic leukemia (T-ALL) (Table 1). Mutations in T-ALL patients can be categorized into several broad classes, each of which likely contributes to the phenotype and generation of T-ALL cells: gain of activating signals, loss of signal suppressors, loss of cell cycle arrest regulators, and alterations of pleiotropic factors such as transcription factors, epigenetic regulators, and other cellular mechanisms.

[0196] [Table 1]

[0197] As mentioned above, we hypothesized that in addition to providing survival factors such as Bcl-XL, it may be necessary to modify T cell expression of the collection of genes previously described to recreate the desired phenotype (Table 1). +T cells were isolated from the blood of healthy donors and activated using αCD3 / αCD28 Dynabeads, and after 72 hours, purified CD8 + A transgene encoding Bcl-XL was inserted into a collective pool of T cells. These cells were then expanded for a period of 17 days before being reactivated using αCD3 / αCD28 Dynabeads, followed by ablation of the expression of factors identified in leukemic T cell lines and patients with T-ALL. The increased proliferative capacity was observed in T REX The effect of ablation of expression of molecules from the "cell cycle arrest" bin: cyclin-dependent kinase inhibitor 2A (CDKN2A) and CDKN2B, one of the key characteristics to be engineered into cells, was therefore tested in these cells (Figure 3A). S-methyl-5'-thioadenosine phosphorylase (MTAP) is chromosomally adjacent to CDKN2A and CDKN2B and is also frequently lost in patients with CDKN2A and CDKN2B deletions. Therefore, the effect of ablation of MTAP expression in conjunction with CDKN2A and CDKN2B (Figure 3A) was tested. These Bcl-XL and CDKN2A / CDKN2B / MTAP-edited cells were then designated "T REX +Bcl-xL” cells (Table 2), and CDKN2A / CDKN2B / MTAP-edited (without Bcl-xL) cells were designated “T REX " cells.

[0198] [Table 2]

[0199] Bcl-xL editing, Bcl-xL and CDKN2A / CDKN2B editing, and T REX +Bcl-xL cells were maintained in culture for approximately 100 days without additional stimulation through their TCR, and the total fold expansion of each population was assessed (Figure 3A). REX The proliferation of +Bcl-xL cells diverged from the other populations approximately 31 days after introduction of these edits, indicating that T REX+Bcl-xL cell expansion persisted in the absence of additional TCR stimulation, achieving a >400,000-fold expansion during this period. In contrast, Bcl-xL-edited and Bcl-xL / CDKN2A / CDKN2B-edited CD8 + T cells achieved a 73- to 286-fold lower level of expansion at this time point (Figure 3A, Table 3). Furthermore, even when unedited or Bcl-xL-edited cells were repeatedly restimulated through their TCR using αCD3 / αCD28 Dynabeads to drive proliferation, they remained stable as T REX They achieved only low levels of total fold expansion, well below that of +Bcl-xL cells (Fig. 1A, Table 3).

[0200] [Table 3]

[0201] T REX +Bcl-xL cells were compared with control CD8 + Although these edits showed substantially enhanced proliferative capacity compared to T cells, it remains to be seen whether these edits can confer a similar phenotype in other donors and whether they can enhance T cell proliferation by ablat- ing the expression of signaling suppressors that are frequently mutated in patients with T-ALL. REX Further experiments were performed to test whether the +Bcl-xL phenotype could be further enhanced (Table 1). The phosphatase and tensin homolog (PTEN) gene locus exhibits frequent loss-of-function mutations in patient-derived leukemia cell lines and patients with T-ALL and is known to negatively regulate cell cycle progression (Table 1). REX +Bcl-xL cells were generated as above from two different donors (40A30 and 40B30), and PTEN expression was measured in these T cells approximately 2 weeks after "triple" editing. REX +Bcl-xL was ablated in one of the lines (40B32) (Figure 3B). REXBoth sets of cells (lacking CDKN2A / CDKN2B / MTAP) exhibited substantial proliferative capacity in the absence of additional TCR stimulation, achieving total fold expansions of >3.7e8 and >1.8e7 by day 118 of culture. Furthermore, consistent with its established role in negatively regulating cell cycle progression through control of AKT signaling, T REX Ablation of PTEN expression in +Bcl-xL cells was observed in donor B-2T REX +Bcl-xL further enhanced the proliferative capacity of cells, allowing these cells to reach a total expansion of >2.0e8 by day 118 in culture (Figure 3B). The additive effect of ablation of PTEN expression took 49 days to appear, reflecting either a low initial editing efficiency or a slow competitive advantage of this editing after cells had expanded >3e6-fold.

[0202] T with full PTEN expression REX +Bcl-xL cells expanded dramatically in the absence of additional TCR stimulation, whereas their proliferation was not affected by T cells lacking PTEN expression. REX The final growth rate was slower compared with +Bcl-xL cells (Fig. 3B). REX Even +Bcl-xL cells eventually showed a decreased proliferation rate (Fig. 3B). REX To determine whether restimulation of +Bcl-xL cells could serve as a viable alternative or complementary approach to ablation of PTEN expression, we tested whether αCD3 or αCD3 / αCD28 Dynabeads could jump-start cells back into the cell cycle (Figure 4). REX + Bcl-xL or PTEN-deficient T REX +Bcl-xL cells were left untreated or restimulated with Dynabeads as above, de-beaded, and the total fold expansion of each population was followed (Figure 4). REX +Bcl-xL and PTEN-deficient T REX +Bcl-xL substantially enhanced the ability of the line to expand.

[0203] Example 3: T REX+Bcl-xL cells resemble primary human T cells in terms of cytokine dependency and cell phenotype. Primary T cells depend on cytokines such as IL-2 for survival and proliferation in vitro and in vivo; however, some leukemia cell lines grow independently of IL-2. REX +Bcl-xL cells and PTEN-deficient T REX +Bcl-xL cells were generated in medium containing IL-2. Whether these cells still resembled normal primary human T cells in terms of cytokine dependency was investigated by tracking cell proliferation and survival over a range of IL-2 concentrations over a 6-day period in culture (Figure 5). Consistent with normal T cells, T REX +Bcl-xL cells and PTEN-deficient T REX +Bcl-xL cells were highly dependent on IL-2 for both growth and survival.

[0204] T REX +Bcl-xL cells and PTEN-deficient T REX Whether Bcl-xL+ cells maintain a phenotype similar to normal T cells after modification and long-term in vitro culture, or whether these conditions contribute to the development of T REX We also tested whether +Bcl-xL drives cells toward an exhausted phenotype (Figure 6). REX +Bcl-xL lines maintained cell surface CD3 and CD8 expression (Figures 6A and 6B). Furthermore, they expressed activation markers such as PD1 and TIGIT at various levels (Figure 6C) and maintained CD28 expression in a donor-dependent manner (Figure 6D). Finally, these T REX +Bcl-xL lines exhibited a differentiated phenotype defined by surface expression of CD45RO and CCR7, which was tracked in a donor-dependent manner (Figure 6E). These data support the conclusion that despite substantial expansion and extended duration of in vitro culture, T REX +Bcl-xL cells resemble normal T cells, suggesting that they do not display a surface phenotype associated with dysfunctional states.

[0205] Chemokine receptors are important for the trafficking of immune cells to sites of inflammation. REX +Bcl-xL cells, PTEN-deficient T REX +Bcl-xL cells, restimulated T REX +Bcl-xL cells and restimulated PTEN-deficient T REX +Bcl-xL cells were analyzed for expression of the chemokine receptors CCR2, CCR5, CCR6, CCR7, CXCR3, and CXCR5 using flow cytometry (Figures 6F-6K). REX +Bcl-xL strain and PTEN-deficient T REX +Bcl-xL lines showed expression of CCR2 (Fig. 6F), CCR5 (Fig. 6G), and CXCR3 (Fig. 6J). Expression of CCR6 (Fig. 6H) was heterogeneous, whereas expression of CCR7 (Fig. 6I) and CXCR5 (Fig. 6K) was low to absent. Therefore, T REX +Bcl-xL cells and PTEN-deficient T REX +Bcl-xL cells maintain expression of key chemokine receptors that enable their trafficking to sites of inflammation.

[0206] Example 4: T REX +Bcl-xL cells are cytotoxic. T REX +Bcl-xL lines were established to resemble normal primary human T cells, REX We determined whether +Bcl-xL cells maintain potent cytotoxic function after long-term culture and expansion. T cell engagers were used in the presence of target tumor cells and the impedance-based xCELLigence platform to detect T REX The cytotoxic function of +Bcl-xL cells was quantified (Figure 7). REX +Bcl-xL line inhibited the proliferation of unmodified primary total T cells and unmodified primary CD8 +These co-cultures demonstrated comparable ability to lyse target tumor cells as effector cells (Figures 7A and 7B). Supernatants from these co-cultures were collected 72 hours after addition of effector cells and active T cell engagers or control T cell engager molecules and analyzed for the presence of interferon-γ (IFN-γ), IL-2, tumor necrosis factor-α (TNF-α), and granzyme B (Figures 7C-7F). REX +Bcl-xL lines produced these cytokines at lower levels compared to unmodified primary T cells, despite a similar ability to lyse target cells in an antigen-dependent manner. These data support the conclusion that even after 80 days of culture and substantial expansion, T REX +Bcl-xL cells are not functionally exhausted and maintain their cytotoxic potential.

[0207] Example 5: T REX +Bcl-xL cells have functional CAR-T REX The cells can be produced. T REX To develop +Bcl-xL cells into potential cell therapies, these cells must be capable of expressing targeting molecules, such as chimeric antigen receptors (CARs), to direct their cytotoxic function. REX +Bcl-xL cell lines were transduced with a lentivirus encoding a CAR that recognizes glypican 3 (GPC3). Surface expression of the GPC3 CAR was then measured using flow cytometry (Figure 8A). REX +Bcl-xL lines are normal total primary T cells and normal primary CD8 + They were found to successfully express the GPC3 CAR at levels similar to T cells (Figure 8A).

[0208] T was determined by performing an impedance-based xCELLigence assay using target tumor cells with varying degrees of antigen expression: OE21 (antigen negative), HuH-7 (antigen intermediate), and Hep3B (antigen high). REX The CAR-directed cytotoxic function of +Bcl-xL cells was evaluated (Figures 8B and 8C). REX+Bcl-xL cells were compared with normal CAR-T cells and normal CAR-CD8 + They rapidly lysed target tumor cells in a CAR-specific and antigen-specific manner at levels similar to those of T cells (Figures 8B and 8C). Supernatants from these co-cultures were collected 72 hours after T cell addition and then analyzed for secretion of IFN-γ, IL-2, TNF-α, and granzyme B (Figures 8D-G). Consistent with their potent cytotoxic function, CAR-T REX +Bcl-xL cells were compared with normal CAR-T cells and normal CAR-CD8 + CAR-T cells showed the ability to secrete effector cytokines comparable to T cells (Figures 8D-8G). However, in general, IFN-γ and TNF-α levels were significantly higher than those of CAR-T cells. REX +Bcl-xL cells were found to be lower.

[0209] These data are REX +Bcl-xL cells and PTEN-deficient T REX This demonstrates that +Bcl-xL cells are able to express CAR and carry out CAR-directed cytotoxic function in an antigen-dependent manner, even after significant in vitro expansion.

[0210] Example 6: T REX The cells were primary CD8 + They migrate to locations similar to T cells and are responsive to IL-2 in vivo. Cytokine cues can be used to regulate the activity and expansion of human and mouse T cells (Zhang et al., Science Translational Medicine, 22 Dec 2021, Vol 13, Issue 625; Aspuria et al., Science Translational Medicine, 22 Dec 2021, Vol 13, Issue 625), and therefore, T cells have been studied for their ability to respond to different human cytokines in vivo. REX Briefly, primary human CD8 + T cells or 278 day old T REXCells were labeled with a luciferase reporter, and 3E6 luciferase-expressing cells were injected into NSG mice supplemented with or without recombinant human IL-2 fusion protein. Mice were imaged using an IVIS optical imaging system to detect luciferase-expressing T cells (Figures 9A and 9B). As shown in Figure 9A, imaging at 216 hours revealed primary human CD8+ T cells and T cells in the mice. REX Furthermore, mice supplemented with recombinant human IL-2 fusion protein showed similar localization of T REX Ventral radiance was plotted over time (Fig. 9B), demonstrating enhanced T cell proliferation in response to exogenously supplemented IL-2 in vivo. REX Ten days after adoptive cell transfer, mice were sacrificed and primary CD8+ T cells or T REX Their blood, spleen, and bone marrow were evaluated for the presence of T cells (Figure 9C). REX Although CD8+ T cells were found in similar organs as primary CD8+ T cells (Fig. 9C), they exhibited slower decay kinetics, and administration of recombinant human IL-2 fusion protein resulted in the proliferation of T cells in the blood and bone marrow of treated mice. REX These data suggest that T REX The cells reside in similar locations as primary CD8+ T cells, suggesting that they maintain responsiveness to exogenous cytokine cues.

[0211] Example 7: CAR-T REX Cells respond to IL-2 and IL-15 in vivo GPC3-targeted CAR-Ts for their ability to respond to different human cytokines in vivo REX NSG, hIL-2NOG, or hIL-15NOG mice were inoculated with GPC3-expressing Hep3B tumor cells. After tumor establishment, mice were either left untreated or injected with 10E6 GPC3-targeting CAR-T cells. REX +Bcl-xL cells. CAR-T REX +Bcl-xL cells were 121 days old at the time of infusion. REXMice were sacrificed and weighed 8 days after +Bcl-xL cell injection (Figure 10A), and no discernible differences were observed, indicating a lack of toxicity. Tumors, blood, and spleens were collected and analyzed for CAR-T REX +Bcl-xL cells were analyzed (Figure 10B). REX +Bcl-xL cell numbers were enhanced in tumor-bearing hIL-2NOG and hIL-15NOG mice, and CAR-T REX These results demonstrate that +Bcl-xL cells are capable of responding to exogenous cytokine cues in vivo. The expansion profile was specific to the particular cytokine support provided (Figure 10B).

[0212] Example 8: CAR-T REX Cells target solid tumors in vivo GPC3-targeted CAR-T REX The ability of the cells to control solid tumors was determined. Hep3B tumors were established in NSG mice, and then 92-day-old purified CAR-T REX Cells (Figure 11A) were injected into mice. 10E6 CAR-T REX 2E6 CAR-T cells or 2E6 CAR-T cells were injected, and tumor volume was measured over time and graphed (Figure 11B, left). CAR-TREX cells exhibited tumor growth inhibition and control of Hep3B tumors. 18 days after CAR-TREX cell transfer, mice were sacrificed, and tumors, blood, and spleens were collected for further analysis (Figure 11B, Figure 11C, and Figure 11D). In these different tissues, intratumoral CAR-T REX The cell phenotype was examined (Figure 11B), and CAR-T REX The cell number was determined (Figure 11C). REX T cells were found in the highest numbers in mouse tumors, and these cells displayed an activated phenotype, actively secreting effector cytokines and degranulating (Fig. 11D). REX The cells exhibit an enhanced ability to proliferate in vitro, can be expanded millions of fold, and can be maintained in culture for over 100 days without additional stimulation through their TCR (Figure 12). Furthermore, CRISPR / Cas9 was used to transfect healthy donor CD8+ When the aforementioned target genes were simultaneously edited in T cells, these edits reproducibly conferred the REX phenotype across different donors (Figure 12).

[0213] Example 9: Additional gene editing for clinical profiling REX Editing is T REX The invention confers enhanced proliferation of T cell products, but additional edits at the B2M and CD38 loci are performed to inhibit T cell proliferation by the patient's immune system. REX B2M is a 119-amino acid protein encoded by a gene on chromosome 15 in humans. It is also a component of major histocompatibility class (MHC) I molecules and associates with non-classical MHC I-like molecules such as CD1, MR1, neonatal Fc receptor, and Qa-1. B2M is located outside the MHC locus but is required for the successful expression of classical and non-classical MHC I molecules on the surface of nucleated cells. Using CRISPR / Cas9, we have demonstrated that B2M inhibits the expression of T cells. REX By eliminating B2M expression on the cells, the cells are shielded from patient CD8+ T cells. REX Ablation of B2M expression and consequent MHC-I expression by cell products sensitizes patients to rejection by NK cells. REX This was performed in conjunction with targeted knock-in of the CAR (e.g., GPC3, HER2, BCMA) in the cell product.

[0214] NK cells express high levels of CD38 and are depleted in certain cancer patients, e.g., multiple myeloma patients, who are receiving anti-CD38 monoclonal antibodies such as daratumumab and isatuximab. To prolong the persistence of this allogeneic cell population in patients, CRISPR / Cas9 has been used to transduce CD38 to T cells. REX knocked out from cells and administered daratumumab or isatuximab to T REX It can be co-administered with cells (see Figures 30 and 31).

[0215] Allogeneic CD8+ As a T cell population, T REX These cells may target healthy cells of HLA-mismatched patients through their TCRs, leading to GvHD. REX The cell population was edited at the T cell receptor alpha constant region (TRAC) locus, which encodes the TCR α chain. CRISPR / Cas9 editing of TRAC results in loss of expression of the TCR α chain, which in turn results in T REX Prevents surface expression of TCR by cells.

[0216] [Table 4]

[0217] Example 10: Anti-BCMA-T REX Allogeneic cell therapy. BCMA-targeting CARs were used in T REX Anti-BCMA-T cells (i.e., cells lacking CDKN2A / CDKN2B / MTAP) were expressed (Figure 13A). REX The cells' genomes were further edited to ablate expression of human leukocyte antigen (HLA) class I and αβ T cell receptor (TCR) by inactivating the B2M and TRAC genes, respectively, to minimize host-versus-graft (HvG) and graft-versus-host (GvH) allogeneic responses, respectively. Additionally, CRISPR / Cas9 was used to express anti-BCMA-T REX The CD38 gene was inactivated in cells, rendering the cells resistant to anti-CD38 depleting monoclonal antibodies. Inactivation of these three genes reduced peripheral blood CD8 + The total in vitro expansion potential of T cells was enhanced, and downstream cell populations were assessed using unedited peripheral blood CD8 + far exceeding what is achievable with T cells. The cells retain the hallmark proliferation characteristics of primary T cells (dependent on both anti-CD3 stimulation and IL-2 prior to TRAC inactivation for expansion / survival) but have a much higher expansion potential. REX The cells maintain cytotoxic function but are mixed CD4 + and CD8+ Shows reduced cytokine release compared to conventional CAR-T cell preparations composed of T cell populations.

[0218] anti-BCMA-T REX Evaluation of the cells indicates that these cells have a similar potential to control BCMA-expressing tumors as primary anti-BCMA-CAR-T cells, but offer a potentially improved safety profile in the form of reduced cytokine release and a potentially reduced risk of CRS (Figure 13B). REX Cells and anti-BCMA-CAR-T cells were cultured with BCMA-expressing tumor cells. Tumor cell lysis was measured at varying effector:target cell ratios at different time points after the initiation of co-culture (Figure 13B, top row). Supernatants were collected 72 hours after the initiation of co-culture, and levels of IFN-γ, TNF-α, and IL-2 were determined by MSD (Figure 13B, bottom row).

[0219] anti-BCMA-T REX BCMA-expressing tumor cells (day 82 of culture) or anti-BCMA-CAR-T cells were cultured with BCMA-expressing tumor cells. Supernatants were collected 72 hours after the initiation of co-culture and assessed for IFN-γ levels using the MSD kit (Figure 14, left). The data show that despite similar control of tumor cells, anti-BCMA-T cells (day 83 of culture) showed significantly higher levels of IFN-γ than co-cultures with anti-BCMA-CAR-T cells. REX These data demonstrated a 90% reduction in IFN-γ levels in co-cultures with CAR-T cells. REX We show that the cells exhibit a cytokine secretion profile that may confer a lower risk of CRS than CAR-T cells.

[0220] Anti-BCMA-T cells were assayed for their ability to persist in a continuous killing assay with or without IL-2 support. REX Cells were evaluated (culture day 112). Briefly, anti-BCMA-T REXAnti-BCMA-CAR-T cells or anti-BCMA-CAR-T cells were serially cultured with BCMA-expressing JJN3 cells at an effector:target cell ratio of 1:1. Tumor cell control (% cytolysis), effector cell number, and effector cytokine secretion were measured and graphed after each round of co-culture (Figure 15). In this serial killing assay, anti-BCMA-T cells REX The cells persisted for a comparable number of rounds as anti-BCMA-CAR-T cells, and the inclusion of IL-2 in the cell culture medium significantly enhanced the anti-BCMA-T REX The number of rounds in which the anti-BCMA CAR-T cells could control tumor cell growth was further increased. REX Both anti-BCMA-CAR-T cells and anti-BCMA-CAR-T cells showed enhanced proliferation in response to IL-2, and effector cytokine secretion was sustained for a longer period in co-cultures in which IL-2 was included in the culture medium (Figure 15, top vs. bottom rows). These data support the conclusion that anti-BCMA-T REX We demonstrate that the cells exhibit similar cytotoxicity as anti-BCMA-CAR-T cells in vitro and a similar ability to respond to exogenous IL-2. REX The cells secreted lower levels of effector cytokines than anti-BCMA-CAR-T cells after CAR engagement, despite comparable tumor control.

[0221] Example 11: Anti-HER2-T REX Allogeneic cell therapy. T REX HER2-targeting CAR was expressed in CAR-T cells or primary T cells (Figure 20A). REX We generated anti-HER2 T cells and CAR-T cells. REX Anti-HER2-CAR-T cells and anti-HER2-CAR-T cells were evaluated for their ability to target HER2-overexpressing OE21 cells at varying effector:target cell ratios (Figure 20B, left). REXThe cells showed comparable or improved control of HER2-expressing tumor cells compared to anti-HER2-CAR-T cells generated from three different primary T cell donors. Supernatants were collected 72 hours after the initiation of co-culture and then examined for the presence of effector cytokines (Figure 20B, right). As previously observed, despite comparable or improved tumor cell control, anti-HER2-T REX The cells secreted lower levels of cytokines (IFN-γ, TNF-α, and IL-2) than anti-HER2-CAR-T cells, suggesting that CAR-T REX This suggests that anti-BCMA-T cells may be less prone to causing CRS in patients. REX As shown above for cells, reduced secretion of IFN-γ was observed in supernatants from co-cultures of HER2-expressing tumor cells with anti-HER2-CAR-T cells compared to supernatants from co-cultures with anti-HER2-CAR-T cells. REX These data were also observed in supernatants obtained from co-cultures with CAR-T cells (Figure 14, right). REX We show that the cells exhibit a cytokine secretion profile that may confer a lower risk of CRS than CAR-T cells.

[0222] Example 12: Different combinations of editing are used to REX A cell phenotype can be generated. The requirement for overexpression of Bcl-xL and various REX target genes to confer the REX phenotype was demonstrated in CD8 cells isolated from two donors (designated G and H). + The REX phenotype was evaluated in T cells. Briefly, CD8+ T cells were negatively selected and then activated with αCD3 / αCD28 Dynabeads for 3 days. Some cells were transduced with Bcl-xL, while others were cultured and knocked out various combinations of REX target genes using CRISPR / Cas9 (Figure 16). Cell expansion was monitored over time and graphed. (CDKN2A and CDKN2A' reflect targeting of a single isoform versus multiple isoforms.) While Bcl-xL was found to be dispensable for the REX phenotype, the three REX target genes produced consistent phenotypes across donors (Figure 16, right).

[0223] Example 13: T REX The cells are edited at the target locus. Western blot analysis revealed ablation of REX target gene expression in T REX cells and γδ T REX We examined the cells (Figure 17, left and center panels). As expected, these cells showed loss of expression of MTAP, CDKN2A(p14), CDKN2A(p16), and CDKN2B(p15). In contrast, expression of these genes was maintained in donor-matched, non-edited control cells. Furthermore, Sanger sequencing data show a high prevalence of indels at these three loci in edited TREX cells (Figure 17, right).

[0224] Example 14: T REX The cells show enrichment of cell cycle-associated gene signatures. Bcl-xL-overexpressing T REX Cell and donor-matched unedited control CD8 + T cells were cultured over time. RNAseq analysis was performed on the resulting cell pellets at various time points to identify the gene signature of Bcl-xL T cells. REX As expected, TREX cells showed enrichment of gene signatures associated with the cell cycle, including E2F target genes and G2M checkpoint target genes, as assessed in both TREX and control cells (Figure 18A). REX The cells also showed higher levels of expression of MYC target genes, consistent with the observed proliferation rate of these cells (Fig. 18B). REX The cells showed regulated expression of multiple cell cycle-related genes (Figure 22C). These data confirm that the REX phenotype is associated with increased cell cycle progression and proliferation.

[0225] Example 15: T REX The cells depend on IL-2 for survival and proliferation. T REXCells were cultured with varying amounts of IL-2 for a period of 12-14 days. Cell expansion was followed and graphed over this time (Figure 19). Bcl-xL T REX As shown above for cells (see, e.g., Figure 5), T REX T cells are highly dependent on IL-2 for proliferation and survival in vitro. REX T cells exhibited dose-dependent proliferation in response to IL-2, and in the absence of IL-2, REX Cells showed a rapid decline in survival and T REX Over 60% of the cells were eliminated within the first 4 days.

[0226] Example 17: REX editing is a novel method for the treatment of CD4+ T REX Enhances cell proliferation ability. REX editing is CD8 + Reproducibly confers enhanced resistance to replicative senescence in T cells. We determined the ability of ablation of REX target gene expression (CDKN2A, CDKN2B, and MTAP) to enhance resistance to replicative senescence in CD4+ T cells. + T cells were isolated from three healthy donors, stimulated using αCD3 / αCD28 Dynabeads, and then edited at these loci. + T REX Cell and donor matched unedited CD4 + The proliferation of T cell controls was tracked over time and graphed (Figure 21). CD8 + As previously shown for T cells, CD4 + Targeting the REX gene in T cells reproducibly reinforced the proliferative capacity of these cells and rendered them resistant to replicative senescence.

[0227] Example 18: Using REX editing to generate γδ T REX The cells can be generated. γδ T cells are another cytotoxic subset of T cells. γδ T cells from eight different donors were used to investigate the T activity of γδ T cells. REXThe ability of REX editing to confer a cell phenotype was investigated (Figure 22). γδ T cells were isolated, stimulated with αCD3 / αCD28 Dynabeads or αCD3 antibody, and then edited at the REX locus using CRISPR / Cas9. γδ T cells and γδ T REX Cell proliferation was monitored and graphed over time. REX editing reproducibly enhanced the resistance of γδ T cells to replicative senescence, and γδ T REX This resulted in the generation of a cellular phenotype.

[0228] γδ T REX It was established that γδ T cell lines exhibit enhanced resistance to replicative senescence. REX We determined whether the cells maintain potent cytotoxic function after long-term culture and expansion. T cell engagers were used in the presence of target tumor cells and the impedance-based xCELLigence platform to target γδ T cells. REX The cytotoxic function of the cells was quantified (Figure 23). REX The cell line was engineered to express unmodified primary CD8 T cells in the presence of T cell engagers. + These co-cultures demonstrated comparable ability to lyse target tumor cells as effector cells (Figure 23, top). Supernatants from these co-cultures were collected 72 hours after addition of effector cells and active T cell engagers or control T cell engager molecules and analyzed for the presence of IFN-γ, IL-2, and TNF-α (Figure 23, bottom). REX The cell lines were significantly more potent than unmodified primary CD8 despite their similar ability to lyse target cells in an antigen-dependent manner. + These data suggest that even after 79 days of culture and substantial expansion, γδ T cells produced lower levels of these cytokines than γδ T cells. REX It is shown that the cells are not functionally exhausted and maintain their cytotoxic potential.

[0229] γδ T cells are typically composed of multiple subsets, including Vδ1, Vδ2, Vδ3, and Vδ5, among others (Lawand et al., Front. Immunol., 30 June 2017). In humans, Vδ1 and Vδ2 constitute the majority of γδ T cells, with Vδ2 cells found primarily in the blood and Vδ1 cells found in tissues.

[0230] γδ T REX cells, γδ CAR-T REX Vδ1 and Vδ2 expression were analyzed by FACS analysis (Figure 24). REX The cells are composed of multiple γδ T cell subtypes (Vδ1, Vδ2, and Vδ1 - Vδ2 - ) and demonstrate that REX editing can enhance the resistance of multiple γδ T cell subtypes to replicative senescence. REX The cells maintained the diversity of γδ T cell subtypes (FIG. 28, bottom).

[0231] Next, γδ T REX The cells were investigated for their ability to receive instruction from a tumor-targeting moiety, such as a BCMA-targeted CAR (Figure 25). REX Cells were transduced to express a BCMA-targeted CAR (Figure 25A), and these cells were co-cultured with BCMA-expressing tumor cells at various effector:target cell ratios. Tumor cell lysis was monitored over time using the xCELLigence platform (Figure 25B), and supernatants were harvested 72 hours after the initiation of co-culture. γδ T REX The cells exhibited a similar ability to control BCMA-expressing tumor cells as primary CAR-T cell controls, however, generally secreted lower levels of effector cytokines, including IFN-γ, TNF-α, and IL-2 (Figure 25B, bottom). These data support the conclusion that γδ CAR-T REX We show that the cells can receive instructions from tumor-targeting CARs and may be less likely to cause CRS than primary CAR-T cells.

[0232] Example 19: REX editing in NK cells enhances NK REX Supports cell phenotype. REX editing enhances the resistance of T cells to replicative senescence, however, they are not associated with NK REX It was unclear whether this would support the cell phenotype. Therefore, NK cells were isolated from three different donors and cultured in medium containing IL-2 or a combination of IL-2 and IL-15. NK cells were then edited at the REX locus using CRISPR / Cas9 to enhance NK cell proliferation. REX The proliferation of donor-matched unedited NK cells was monitored over time (Figure 26). Across all donor and cytokine conditions, REX editing significantly increased the proliferation of NK cells. REX It was possible to reproducibly enhance the resistance of cells to replicative senescence (Figure 26). NK REX Cells can be cultured for more than 90 days and 6 Super~10 10 The unedited NK cells expanded more than 2-fold, whereas the unedited NK cells failed to expand and died within 80 days.

[0233] Considering the enhanced resistance to replicative senescence, NK REX It was important to determine whether NK cells maintained their dependence on cytokine support. REX Cells were generated in medium containing IL-2 or a combination of IL-2 and IL-15. Cytokines were derived from the growth medium and NK REX In experiments in which cell numbers were monitored over a 37-day period, NK cell responses to these cytokines were REX The cell dependency was determined. REX The cells were unable to proliferate after cytokine withdrawal, and despite editing of the REX gene, these cells showed a rapid decline in cell viability and viable cell diameter, and an increased dependence on cytokine support (Figure 27).

[0234] NK REXCells were transduced to express a BCMA-targeting CAR to determine whether these cells were capable of stably expressing a tumor-targeting CAR (Figure 28). CAR expression was determined by CAR-NK REX The expression levels (mean fluorescence intensity, MFI) were maintained over time in purified CAR-T cells, and were similar to those in purified CAR-NK cells. REX We demonstrate that the cells are capable of stably expressing CAR and that the level of expression is comparable to that of standard CAR-T cells.

[0235] NK REX Although the cells could be expanded in culture for long periods of time, it was unclear 1) whether their cytotoxic potential was maintained after sustained expansion, and 2) whether they could receive instruction from tumor-targeted CARs. REX The cells were then cultured in two different NK REX These CAR-NK REX One of the lines was purified based on CAR expression to yield >95% CAR + CAR-NK REX NK strains from donors 50-1 and 47-1 were generated (Figure 29A, bottom right). REX and CAR-NK REX The lines were tested for their ability to lyse BCMA-expressing tumor cells in the xCELLigence assay (Figure 29B). Even after 78 and 86 days of culture, NK REX Cells and CAR-NK REX The cells were potently cytotoxic. These lines rapidly lysed BCMA-expressing target cells and inhibited CAR-T REX NK cells achieved higher levels of regulation more rapidly than NK cells (Fig. 29B). REX NK cells were able to lyse tumor cells independently of CAR expression, but at lower effector:target cell ratios. REX and CAR-NK REXA contribution of CAR-directed cytotoxicity, likely due to engagement of activating receptors on the cells, could be observed for both donors 50-1 and 47-1. Supernatants were collected 48 hours after co-culture, and levels of IFN-γ, IL-2, and TNF-α were determined using MSD (Figure 29C). NK REX The cells are CAR-NK REX These CAR-T cells secrete lower levels of these cytokines than REX Cells secreted the highest levels of these factors (Figure 29C). These data support the conclusion that CAR-NK REX We demonstrate that these cells can stably express and receive instructions from tumor-targeted CARs. Furthermore, these cells rapidly lyse tumor cells and accumulate low levels of IFN-γ, IL-2, and TNF-α in the coculture supernatant.

[0236] Example 20: T REX The cells are sensitive to T cell depleting agents and chemotherapy. T REX The cells were modified to increase their resistance to replicative senescence, however, these cells displayed normal T cell characteristics. REX To better understand their ability to regulate T cells, we determined their sensitivity to standard T cell depletion agents and chemotherapy compared to non-edited whole T cells activated to enter the cell cycle (Figure 30). REX Cells were incubated with 10 μg / mL anti-CD52 and 10% human complement (Figure 30, top left) or 10% rabbit complement (Figure 30, bottom left). After 3 hours, cell viability was assessed using the Cell Titer Glo assay. Non-edited, recently activated total T cells or T REX Cells were incubated with the indicated amounts of melphalan (Figure 30, top right) or chlorambucil (Figure 30, bottom right), and cell viability was measured after 2 days using the Cell Titer Glo assay. REX The cells showed sensitivity to these agents comparable to that of non-edited, recently activated whole T cells.

[0237] Example 21: B2M KO T REX The cells are susceptible to NK cell-mediated depletion, which can be modulated using anti-CD38 antibodies. As an allogeneic cell product, T REX T cells are modified at the B2M locus to increase their susceptibility to NK cell-mediated depletion. These cells can be further modified at the CD38 locus to limit their depletion by anti-CD38 antibodies. REX Cell lines and CD38 KO B2M KO T REX The cell lines were generated using CRISPR / Cas9. REX Cells and CD38 KO B2M KO T REX The cells were co-cultured with PBMCs isolated from healthy donors. REX The cells did not exhibit a decrease in numbers when co-cultured with PBMCs, but CD38 KO B2M KO T REX The cells were susceptible to NK cell-mediated lysis, as expected (Fig. 31, top). NK cells express high levels of CD38 and CD38 KO B2M KO Total T cells or CD38 KO B2M KO T REX When NK cells were preincubated with the CD38-targeting antibody daratumamab (Dara) prior to co-culture with CD38 cells, this conferred a greater than 50% reduction in cell lysis. These data support the conclusion that CD38 KO B2M KO T REX We show that the cells are susceptible to NK cell-mediated lysis and that this susceptibility to depletion can be modulated through the administration of anti-CD38 antibodies.

[0238] Example 22: STAT5A and STAT5B mutants inhibit REX-edited CD8 in vitro +Enriched in T cells. T REX Cells (REX-containing CD8 + T cells) were further engineered to modulate their dependence on exogenous cytokines. REX T cells are highly dependent on IL-2 for growth and survival and can respond to both IL-2 and IL-15 in vivo. The IL-2 and IL-15 pathways signal through STAT3 and STAT5 family members, and mutant forms of these molecules have been identified that extend the duration of signaling through these pathways. Overexpression of mutant forms of STAT5 and STAT3 increases the proliferation and proliferation of T cells. REX To test whether STAT5A mutants, two STAT5B mutants, and one STAT3 mutant could confer a selective advantage to cells, we transfected them with T cells according to the timeline shown. REX Bcl-xL was overexpressed in T cells (Figure 32, top). A fluorescent reporter was used to track the enrichment of STAT mutants over time (Figure 32, bottom). For these experiments, cells were cultured in the presence of 300 IU / mL IL-2 and monitored for reporter positivity at the indicated time points. STAT5A and STAT5B mutant-containing T cells were cultured in the presence of IL-2 at 300 IU / mL and monitored for reporter positivity at the indicated time points. REX Cells showed enrichment throughout the culture process, whereas the STAT3 mutant and Bcl-xL alone groups showed little or no selective advantage. These results support the results of T cells overexpressing STAT5A H299R / S711F, STAT5B N642H, or STAT5B R430C / P702A. REX Even when cells were cultured in the presence of IL-2, T REX Cells and T REX +Bcl-xL cells.

[0239] [Table 5]

[0240] Example 23: STAT5A and STAT5B mutants exhibit varying degrees of IL-2 independence in vitro. T cells overexpressing STAT5A H299R / S711F, STAT5B N642H, or STAT5B R430C / P702A REX Even when cells were cultured in the presence of IL-2, T REX Cells and T REX +Bcl-xL cells (Figure 32). Not all STAT5A and STAT5B mutants are functionally equivalent, and therefore overexpression of STAT5A and STAT5B mutants may subsequently enhance T cell response to exogenous IL-2. REX One STAT5A mutant and two STAT5B mutants were tested in T cells according to Figure 32. REX STAT5 mutant T cells were overexpressed with Bcl-xL. REX cells and control cells (T REX Cells ("without transposon") and Bcl-xL-overexpressing T REX STAT5A H299R / S711F mutant and STAT5B R430C / P702A mutants were cultured in decreasing amounts of IL-2 and cell expansion was followed over time. Both control groups, the STAT5A H299R / S711F mutant group, and the STAT5B R430C / P702A group exhibited dependence on IL-2, whereas the STAT5B N642H mutant group grew independently of IL-2 over an 18-day period in culture (Figure 33, white bar). As shown in Figure 33, the STAT5B R430C / P702A mutant exhibited IL-2 dependence, but this mutation was able to inhibit T cell proliferation even in the presence of low amounts of IL-2. REX These data demonstrate that all STAT5A and STAT5B mutants investigated in this example dramatically enhance cell expansion. REX Although they provide a selective advantage to T cells, they are sensitive to varying degrees of T cell proliferation, independent of exogenous IL-2. REX It indicates that cells are provided.

[0241] Example 24: STAT mutant-expressing T cells in a T cell engager assay REX The cells retain functionality according to the observed surface CD3 expression. STAT5A mutant and STAT5B mutant overexpression T REX The cells are standard TREX Having established that these cells exhibit enhanced growth potential and cytokine independence compared to STAT5A, STAT5B, and STAT3 cells, we next determined whether these cells maintain potent cytotoxic function. One STAT5A mutant, two STAT5B mutants, and one STAT3 mutant were cultured in T cells according to Figure 32. REX STAT mutant-containing T cells were then isolated using the impedance-based xCELLigence platform, using control (non-targeting) or activated (tumor-targeting) T cell engagers. REX The cytotoxic function of the cells was assessed. The percentage of cytolysis was determined 6 hours after the addition of T cell engagers. REX Cells were co-cultured with two different antigen-expressing tumor lines at an effector:target cell ratio (E:T) of 3:1 (left and right). As shown in Figure 34, STAT5A H299R / S711F, STAT5B R430C / P702A, and STAT3 Y640F transfected T cells were co-cultured with two different antigen-expressing tumor lines at an effector:target cell ratio (E:T) of 3:1 (left and right). REX Cells were control T REX +Bcl-xL cells exhibited cytotoxicity equivalent to that of T cells containing the STAT5B N642H mutant. REX Cells showed low cytotoxicity in this assay due to low surface expression of CD3. STAT mutant overexpression did not promote nonspecific cytotoxicity in this assay, and STAT mutant overexpressing T cells showed low cytotoxicity in this assay due to low surface expression of CD3. REX This suggests that the cells maintain normal functionality.

[0242] Example 25: STAT mutant CAR-T REX The cells maintain cytotoxic function in vitro in a CAR-directed manner. To serve as a potential chassis, STAT mutants overexpressing T REX The cells must be capable of expressing a targeting molecule, such as a chimeric antigen receptor (CAR), to direct their cytotoxic function. Therefore, one STAT5A mutant and two STAT5B mutants were transfected into T cells according to Figure 32. REXBcl-xL was overexpressed in these cells. After these cells were enriched to 100% STAT mutant expression, a GPC3-targeting CAR was introduced. The impedance-based xCELLigence platform was used to transfect STAT mutant-containing CAR-T cells. REX The cytotoxic function of the cells was evaluated. T cells were cultured at various effector:target (E:T) cell ratios. REX The percentage of cell lysis was determined 12 hours after the initiation of co-culture of the cells with the target cells. As shown in Figure 35, the STAT5A and STAT5B mutant T cells REX The cells retained functionality as demonstrated by their ability to specifically lyse antigen-expressing tumor cells after engagement of the CAR.

[0243] Example 26: Additional STAT5A and STAT5B mutants inhibit REX-edited CD8 in vitro + Enriched in T cells. The above study was conducted by T REX Although we established the functional enhancement resulting from overexpression of STAT5A H299R / S711F, STAT5B N642H, and STAT5B R430C / P702A in T cells, these studies also incorporated overexpression of the anti-apoptotic factor Bcl-xL. REX We expanded the list of STAT5 mutants characterized in cells and demonstrated enhanced T REX The role of the Bcl-xL transgene in conferring a cellular phenotype was evaluated. To that end, T (in the absence of the Bcl-xL transgene) was cultured according to the timeline shown. REX Two STAT5A mutants and five STAT5B mutants were overexpressed in cells (Figure 36A). STAT mutants were introduced using transposons (T) or lentiviruses (L) as indicated. Again, fluorescent reporters were used to track and graph the enrichment of STAT mutants over time (Figure 36B). As shown in Figure 36, STAT5A and STAT5B mutant expression T REX Cells were enriched during the cell culture process regardless of the mode of transgene introduction. These data support the conclusion that T REXOverexpression of mutant STAT5A and STAT5B in T cells inhibits T cell proliferation without the need for a Bcl-xL transgene. REX These studies further expand the collection of STAT5A and STAT5B mutants that promote this enhanced phenotype.

[0244] Example 27: Expression of STAT5A and STAT5B mutants in vitro using REX-edited CD8 + Enhances T-cell expansion. STAT5A mutant T generated in Figure 36 REX cells, STAT5B mutant T REX cells, and control T REX Cell proliferation was followed over time. As shown in Figure 37, STAT5A and STAT5B mutant T cells were significantly increased in the STAT5A and STAT5B mutant T cells. REX Cells were non-edited control T REX Most STAT5A and STAT5B mutants exhibited enhanced proliferation compared to control T cells under standard in vitro cell culture conditions. REX Although STAT5A R430C / P702A overexpression resulted in a similar enhancement in proliferation capacity compared to T cells, REX The cells again REX The greatest degree of enhancement of cell expansion was observed (Fig. 37B, top). REX Overexpression of STAT5A and STAT5B mutants in cells enhances the expansion capacity of these cells under standard cell culture conditions.

[0245] Example 28: STAT5A and STAT5B mutants REX It is functional in cells and leads to upregulation of CD25 expression. The preliminary experiment was T REXThese results suggest that overexpression of STAT5A and STAT5B mutants in T cells enhances the phenotype of these cells and modulates their dependence on exogenous cytokines such as IL-2. To further verify the functionality of the overexpressed STAT5A and STAT5B mutants in these cells, two STAT5A mutants and five STAT5B mutants were transfected into T cells (in the absence of the Bcl-xL transgene) according to the timeline shown. REX These STAT5A and STAT5B mutants were transfected into T cells (Figure 36A). Signaling through the IL-2 pathway in T cells leads to the upregulation of CD25 (IL-2Rα) and the formation of the high-affinity IL-2 receptor composed of CD25, IL-2Rβ (CD122), and IL-2Rγ (CD132), enhancing the ability of these cells to utilize IL-2. REX If functional in the cell, the reporter + Cells were control T REX Over long-term cell culture, the T cells should show increased cell surface CD25 expression compared to control T cells. REX cells (UT), and STAT5A and STAT5B mutant-containing T REX The surface expression of CD25 was assessed in the cells. STAT mutants were introduced using transposons (Figure 38A) or lentiviruses (Figure 38B). As shown in Figure 38, STAT5A and STAT5B mutant-expressing T cells were significantly increased in the STAT5A and STAT5B mutant-expressing T cells. REX The cells showed enhanced expression of cell surface CD25 and CD25 + STAT5A mutants and CD25 + STAT5B mutant T REX The frequency of STAT5A and STAT5B cells increased throughout the culture process. These data demonstrate that the STAT5A and STAT5B mutants investigated in these studies are functional and REX This supports the prediction that overexpression of these molecules in cells enhances downstream signaling, leading to upregulation of cell surface CD25, which in turn would further sensitize these cells to IL-2.

[0246] Example 29: Alternative TREX and N.K. REX Cell generation. T REX Cells and NK REX To determine whether it is possible to further enhance the resistance of cells to replicative senescence, T REX Cellular variants and NK REX Additional combinations of edits were investigated in cell variants (Table 6). In some cases, the cells were additionally engineered to express a CAR. REX Cell variants were generated, screened, and functionally evaluated according to the workflow described in Figure 39. Briefly, T REX Cells were generated and then further modified to overexpress specific genes of interest (transposon insertion) prior to enrichment screening. In some cases, these cells were further modified to overexpress or ablate the expression of other genes of interest (further editing). A variety of assays were used to characterize the engineered T cells for long-term growth potential and functionality. REX The cells were evaluated.

[0247] [Table 6]

[0248] T REX Additional manipulation of cells results in T REX To determine whether cells could be further enhanced to resist replicative senescence, T REX Cells were generated and then further modified as in Figure 39. Enrichment of the editing combinations was analyzed using flow cytometry to identify the engineered T REX Cellular variants were evaluated over time (Figure 40A, Round 1, Figure 40B, Round 2). Five editing combinations were re-evaluated at later time points to confirm the reproducibility of previous results (Figure 40B, Round 2). As shown in Figure 40, certain editing combinations were associated with T REX These T REX The cellular variant is basal T REXshowed that the T REX Not all editing combinations examined in cells conferred this selective advantage, highlighting the importance of specific modifications.

[0249] Cellular senescence can occur as a result of telomere shortening. Telomere reverse transcriptase (TERT) functions as part of a complex that elongates telomeres. Therefore, this study investigated the T cells generated according to Figure 39. REX 0 cells (REX edited), T REX 3B cells (REX edited; MYC; Bcl-xL), and T REX We investigated whether overexpression of TERT in 3C cells (REX-edited; KRAS A146V; MYC; Bcl-xL) could further confer resistance to replicative senescence to these cells. REX 0 cells (T REX 0T), T REX 3B cells (T REX 3BT), and T REX 3C cells (T REX The enrichment of TERT-expressing T cells was followed over time (Figure 41). As shown in Figure 41, the enrichment of TERT-expressing T cells was REX 0, T REX 3B and T REX 3C cells enrich in culture and exhibit the selective advantage conferred by overexpression of this transgene.

[0250] The results in Figures 40 and 41 show that T REX Further manipulation of the cells allows the identification of these T REX We show that combinations of these edits can confer a selective advantage, as evidenced by enrichment of cellular variants. REX To better understand whether this also enhances the resistance of cells to replicative senescence, we generated T REX Cell (T REX 0) and T REXThe long-term expansion of cellular variants was tracked (Figure 42). Specific editing combinations (3B, 3C, 3BP, 3CN, 0T, 3BT, and 3CT) were associated with T REX (T REX 0) cells, compared to T REX Substantially enhance the lifespan and proliferation capacity of cell variants, and REX Cell variants were allowed to persist in culture for over 245 days, and expansion of these cells was achieved with a maximum T at the end of this experiment. REX Cell expansion 6.14 x 10 11 times ~9.82×10 17 However, in some cases, the editing combination (2A, 2B, 3A, 3BN) was found to enhance T REX These data suggest that T REX We show that further manipulation of the cells can enhance their resistance to replicative senescence, and that the specific combination of these edits is important for this phenotypic enhancement.

[0251] T REX Having established that further manipulation of cells can increase their resistance to replicative senescence, these T REX The cytotoxic potential of the cell variants was investigated after long-term cell culture. In particular, the impedance-based xCELLigence platform was used to investigate the cytotoxic potential of T cells using control (non-targeting) T cells or activated (tumor-targeting) T cell engagers. REX Cell (T REX 0) and T REX The cytotoxic potential of the cell variants was assessed. Percent cell lysis was determined 12 and 72 hours after addition of the T cell engager (Figure 43). REX 0 cells (REX edited), T REX 0T cells (REX-edited; TERT), T REX 3C cells (REX edited; KRAS A146V; MYC, Bcl-xL), T REX 3C_3 cells (REX edited; KRAS A146V; MYC; Bcl-xL), T REX3CN cells (REX edited; KRAS A146V; MYC; Bcl-xL; PTEN CRISPR), T REX 3B cells (REX edited; MYC; Bcl-xL), T REX 3B_3 cells (REX edited; MYC; Bcl-xL), T REX 3BT cells (REX edited; MYC; Bcl-xL; TERT), T REX 3BP cells (REX edited; MYC; Bcl-xL; TP53 CRISPR), and T REX For 3BN cells (REX-edited; MYC; Bcl-xL; PTEN CRISPR), REX Effector cells were co-cultured with target cells at different effector:target (E:T) cell ratios. The dashed line indicates the T at 72 h. REX (T REX 0) means the cytotoxicity of the cells. As shown in Figure 43, most of the T REX The cell variants not only maintained cytotoxic function but also exhibited enhanced T cell activation in this T cell engager assay, as indicated by accelerated killing kinetics and increased tumor cell lysis at low E:T ratios. REX It also exhibits increased efficacy compared to 0.

[0252] T REX Having confirmed that the cell variants maintained cytotoxic potential in T cell engager assays, the ability of these cells to express CARs and expand after CAR expression and purification was then assessed. BCMA-targeted CARs were used in young (day 21) T cells as a benchmark. REX 0 cells or long-term expanded T REX Cellular variants (T REX 0T, T REX 3C_3, T REX 3CN, T REX 3B, T REX 3B_3, T REX 3BT and T REX CAR-T cells were transduced into the CAR-T cells at day 60 (Figure 44A). CAR expression was measured by flow cytometry 39-43 days after CAR transduction (Figure 44B). REX 0 cells and CAR-T from day 158 to day 214 REXThe cell variants were further enriched to high purity (Figure 44A). REX cell, T REX Cellular variants, CAR-T REX Cells and CAR-T REX The expansion of cell variants was tracked throughout the editing, transduction, and enrichment process for each group (Figure 44B). REX We show that cell variants expand robustly during the culture process.

[0253] Example 30: CAR-T REX The cell variant remains functional even after more than 200 days in culture. T REX Having confirmed the ability of the cell variants to express BCMA-targeted CARs, we then investigated the efficacy of these CAR-T cells to lyse tumor cells in a CAR-directed and antigen-directed manner. REX The ability of cell variants to differentiate was investigated. As shown in Figure 44, BCMA-targeted CARs were administered to whole primary T cells, young T REX Cells (T REX 0D60) and T REX Cellular variants (T REX 0T, T REX 3C_3, T REX 3CN, T REX 3B, T REX 3B_3, T REX 3BT and T REX 3BP). The impedance-based xCELLigence platform was used to measure the cytotoxicity of CAR-expressing cells (Figure 45). Percent cytolysis was determined 12 and 72 hours after the initiation of co-culture of effector and target cells at various effector:target (E:T) cell ratios. Antigen-negative (HuH-7) and antigen-positive (HuH-7 BCMA) target cells were used in this experiment. The results, shown in Figure 45, demonstrate the efficacy of CAR-T REX This confirms that the cell variant remains functional even after more than 200 days in culture.

[0254] Long-term expansion CAR-T REXHaving established that the cell variants generally retained cytotoxic function through one round of tumor cell lysis (Figure 45), we then assessed the persistence and cytotoxic capacity of these cells through multiple rounds of tumor cell lysis. BCMA-targeted CARs were administered to whole primary T cells, young T cells, and guinea pig T cells. REX Cells (T REX 0D67) and T REX Cellular variants (T REX 0T, T REX 3C_3, T REX 3CN, T REX 3B, T REX 3B_3, T REX 3BT and T REX 3BP). The cytotoxicity and persistence of CAR-expressing cells were measured over multiple rounds of coculture with BCMA-expressing JJN3 target cells at a 1:1 effector:target cell ratio. After each round of coculture, the percentage of cell lysis (top) and effector cell expansion (bottom) were assessed (Figure 46). As shown in Figure 46, most CAR-T REX The cell variants persisted similarly to primary CAR-T cells in serial killing assays, and these 221-day-old CAR-T REX The cell variants, at least, are young CAR-T REX Functionality comparable to cell benchmarks. CAR-T REX 3C_3 and CAR-T REX The 3CN variant was shown to be superior in this assay to primary CAR-T cells, young CAR-T REX Cell benchmarks and other CAR-T REX persisted substantially longer than the cellular variant.

[0255] Example 31: TERT overexpression inhibits NK REX and CAR-NK REX confers an advantage to the cells. T REX Cells and T REX Having established that overexpression of TERT in cell variants can further enhance their resistance to replicative senescence, we compared these studies with NK REX Cells (REX-edited NK cells) and CAR-NKREX It has been further expanded to include NK cells. REX NK cells were generated and, in some cases, engineered to express a BCMA-targeted CAR. REX and CAR-NK REX Cells were further engineered to overexpress TERT (Figure 47). All groups were compared with TERT-expressing NK cells. REX and CAR-NK REX Cell enrichment was monitored over time (Figure 47, top). Proliferation of all groups was also measured throughout the culture process (Figure 47, bottom). Cells were grown under the indicated cytokine conditions (IL-2 or IL-2 + IL-15). As shown in Figure 47, TERT-expressing NK cells were enriched in TERT-positive cells. REX Cells and TERT-expressing CAR-NK REX The cells showed enrichment throughout the cell culture process (Figure 47, top). Furthermore, these TERT expression variants were enriched for NK REX and CAR-NK REX Compared to controls, CAR-NK cells exhibited enhanced resistance to replicative senescence as measured by increased lifespan and expansion in cell culture (Figure 47, bottom). REX TERT IL-2 IL-15 cells also continued to expand after cryopreservation and reculture (ultimately reaching 10^30 cells).

[0256] NK REX Cells and CAR-NK REX Although overexpression of TERT in CAR-NK cells enhanced the resistance of these cells to replicative senescence, it was unclear whether these variants maintained their cytotoxic function. REX Cells (CAR-NK REX B) and TERT-overexpressing CAR-NK REX Cells (CAR-NK REXB TERT) were generated as described above, and the cytotoxicity of CAR-expressing cells was measured using the impedance-based xCELLigence platform. Percent cytolysis was determined 6, 12, 48, and 96 hours after the initiation of co-culture of effector and target cells at various effector:target (E:T) cell ratios (Figure 48). Antigen-negative and antigen-positive target cells were used in this experiment. Prior to the co-culture experiment in which no cytokine support was provided, CAR-NK cells were co-cultured under the indicated cytokine conditions (IL-2 + IL-15). REX The cells were grown. As shown in Figure 48, TERT-overexpressing CAR-NK REX Cells (CAR-NK REX B TERT) inhibited CAR-NK activation, as evidenced by the accelerated killing kinetics and maximal cytotoxicity observed at each E:T ratio. REX Cells (CAR-NK REX B) shows improved function compared to B. Furthermore, these results indicate that the enhanced cytotoxicity was CAR-mediated, as no increased lysis of antigen-negative tumor cells was observed.

[0257] Example 32: T REX Cells show enrichment in combinatorial editing. T REX Cells (REX-containing CD8 + T cells) were generated and then further modified to overexpress specific genes of interest (transposon insertion) prior to enrichment screening. In some cases, these cells were further modified to overexpress other genes of interest (further editing) (see Figure 49). Different assays were used to characterize the engineered T cells for long-term growth potential and functionality. REX The cells were evaluated.

[0258] As shown in Figure 50, for the enrichment of editing combinations, T REX 0T cells, T REX 3B' cells, T REX 3B'T cells, T REX 3B cells, T REX 3C cells, T REX 3C' cells, and TREX 3C' T cells were evaluated over time. Each of these additional editing combinations detailed in Table 7 resulted in REX-edited CD8 + T cell pool (T REX ), indicating that they conferred an additional selective advantage throughout the cell culture process beyond that observed with REX editing.

[0259] [Table 7]

[0260] T REX Cell variants were generated as described in Figure 49. Cell expansion was followed over time and T REX Cellular variants (T REX 0T, T REX 3B', T REX 3B'T, T REX 3B, T REX 3C * , T REX 3C' and T REX 3C'T * ) is T REX (T REX 0) cells exhibited enhanced lifespan and proliferation capacity (see Figure 51). REX 0 is unedited donor matched CD8 + showed improved proliferation (max expansion = 9.92E+024, max culture = 203 days) compared to T cells (max expansion = 1.05E+013, max expansion = 130 days). REX The T0 reached a plateau by approximately 190 days of culture. REX In contrast to 0, the T investigated in this example REX The cell variants continued to grow in a log-linear fashion comparable to the cell lines. REX Cell variants were generally cultured for approximately 240 days, at which point they were cryopreserved for further use and characterization. In some cases, due to prioritization, T REX Cellular variants (T REX 3C, T REX3C'T) were cryopreserved early. However, these cells exhibited enhanced cell line-like growth. With the exception of the two groups that were cryopreserved early due to prioritization, T REX Cell variants achieved 4.08E+027 to 5.17E+035-fold expansion before cryopreservation. In some cases, Bcl-xL was not required for the cells to exhibit advantageous enrichment and growth characteristics (e.g., T in Figure 51). REX 3B' vs T REX 3B and T REX 3C' vs. T REX (Compare 3C).

[0261] Example 33: T REX The cell variants can be single cell cloned. T REX Cells (REX-containing CD8 + T cells) and then further modified as shown in Figure 49 to produce T REX 3B' cells, T REX 3B cells and T REX 3C' cells were generated. The engineered T cells were analyzed using flow cytometry-based cell sorting or limiting dilution analysis. REX The single-cell clonability of the cells was evaluated. The engineered cells were seeded at 1, 10, or 100 cells per well and cultured for 2 weeks in the presence of IL-2-containing medium. The plates were imaged to assess colony formation. A maximum of 60 colonies were obtained per plate. T cells that could not be single-cell cloned were REX In contrast to 0 cells, T REX 3B' cells, T REX 3B cells and T REX 3C' cells were able to produce colonies at variable frequencies. REX 0T, T REX 3B and T REX Similar results were obtained with 3CN cells (see Figure 52).

[0262] Example 34: T REX Cell variants expand robustly during the culture process. BCMA-targeted CARs were administered to young TREX Cell (T REX 0) and T REX Cellular variants (T REX 0T, T REX 3B', T REX 3B'T, T REX 3B, T REX 3C' and T REX CAR-T cells were further enriched for high purity before functional evaluation. REX Cells and CAR-T REX The expansion of cell variants was tracked throughout the editing, transduction, and enrichment processes for each group. REX 0 CAR-T cells were cryopreserved at the peak of their expansion and functionality (4.30E+015, day 117) for use in subsequent studies, but T REX The cell variant CAR-T cells demonstrated the ability to continue proliferation throughout the culture process, achieving the following degrees of expansion at the indicated time points of cryopreservation: T REX 0T(1.05E+028, 277th day), T REX 3B'(1.17E+032, 221st day), T REX 3B'T(5.86E+034, 239th day), T REX 3B (7.48E+032, 239th day), T REX 3C' (3.06E+020, 171st day), and T REX 3C'T(8.35E+021, 204). Even the early cryopreserved group showed a consistent growth pattern and did not begin to plateau during cell culture. The results showed that T REX We demonstrate that cell variants expand robustly during the culture process (see Figure 53). In some cases, Bcl-xL was not required for the cells to exhibit advantageous enrichment and growth characteristics (e.g., T REX 3B' and T REX 3B has similar growth characteristics).

[0263] Example 35: CAR-T REX The cell variants are cytotoxic and persist in continuous killing assays. BCMA-targeted CAR, whole primary T cells, young T cells REX Cell (T REX 0-D88) and T REX Cellular variants (T REX 0T, T REX 3B', T REX 3B'T, T REX 3B, T REX 3C' and T REX 3C'T-D126-182). The cytotoxicity (Figure 54A) and persistence (Figure 54B) of CAR-expressing cells were measured over multiple rounds of co-culture with BCMA-expressing JJN3 target cells at a 1:1 effector:target cell ratio. After each round of co-culture, percent cytolysis (Figure 54A) and effector cell expansion (Figure 54B) were assessed. Unmodified CAR-T at peak function REX Cell (T REX 0 (young)) completed five rounds of sequential killing and achieved over 30% cell lysis. REX The cell variants completed 1 to 5 rounds of sequential killing with over 30% cell lysis, as seen in Figure 54A. REX 0T and CAR-T REX The activity of CAR-T 3B cells was the most potent in this assay, with the cells completing four to five rounds of sequential killing. REX 0 T cells (maximum expansion: 7.13 times) and CAR-T REX 3B cells (maximum expansion: 4.00x) also demonstrated CAR-T proliferation in this assay, as seen in Figure 54B. REX 0 cells (maximum expansion: 2.19-fold).

[0264] Example 36: CAR-T REX The cell variants are at least as potent as young CAR-Ts in vivo. REX It is functional to the extent of the cellular benchmark. NSG mice were inoculated with 10E6 MM1S-luciferase tumor cells. Three days later, primary CAR-T cells (D14), CAR-T REX Cell (T REX 0, D99-102), or CAR-T REX Cellular variants (TREX 0T, T REX 3B', T REX 3B'T, T REX 3B and T REX 3C'-D137-200) were administered at 2E6, 10E6, or 20E6 cells per mouse. Tumor burden was monitored twice weekly using IVIS imaging. All CAR-T REX Cellular variants demonstrated in vivo functionality despite a higher degree of expansion in vitro (see Table 8 below and Figure 55). REX Cellular variants are more effective at lower doses and with younger CAR-T REX Cell (T REX 0) was more effective than (T REX 3B and T REX 0T was able to control tumors in vivo at a cell dose of 2E6, whereas T REX 0 cells failed to control disease under these conditions. At a cell dose of 2E6, CAR-T REX 0 cells failed to eliminate tumors, as evidenced by tumor growth of 246 ± 116.6% at day 3 after effector injection. In contrast, at this time point, 2E6 CAR-T REX 3B cells eliminated 47.3% ± 20.2% and 94.3% ± 0.58% of tumors depending on the donor line, whereas 2E6 CAR-T REX 0 cells eliminated 93% ± 1.7% of tumors in mice).

[0265] [Table 8]

[0266] Example 37: Cryo-recovered TERT-overexpressing CAR-NK REX The cells are cytotoxic and inhibit younger cryo-recovered CAR-NK REX They persist better in continuous killing assays than cells. BCMA-targeted CAR-NK REX Cells and TERT-overexpressing CAR-NK REXCells were generated as described in Figure 47. Cells were cryopreserved on days 84 and 256, respectively. Cells were cryopreserved and cultured until days 89 and 304, respectively, at the start of the sequential killing assay. The cytotoxicity and persistence of CAR-expressing cells were measured over multiple rounds of co-culture with BCMA-expressing JJN3 target cells at a 2:1 effector:target cell ratio in the presence of IL-2. After each round of co-culture, percent cell lysis and effector cell expansion were assessed (see Figure 56). TERT-overexpressing CAR-NK REX The cells expanded in culture for over 450 days, reaching a >10^30-fold expansion. Despite prolonged additional culture (at the start of the assay, CAR-NK REX = 10^9-fold expansion, TERT-overexpressing CAR-NK at the start of the assay REX = 10^26-fold expansion), TERT-overexpressing CAR-NK REX The cells showed enhanced functionality and persistence in this assay, completing 16 rounds of sequential killing with at least 30% cell lysis. In contrast, despite being younger and less expanded, CAR-NK REX Cells only completed 11 rounds under these conditions. REX Cells only achieved a maximum of 69-fold expansion in this study, whereas TERT-overexpressing CAR-NK REX Cells reached a 3236-fold expansion in this assay.

[0267] TERT-overexpressing CAR-NK REX Cells were generated as in Figure 47. Cells were cryopreserved on day 256 and then cryo-recovered and expanded until day 414 before the start of the assay. TERT-overexpressing CAR-NK cells were cultured at various effector:target cell ratios for a 3-day period. REX The cells were co-cultured with BCMA-expressing JJN3 cells. The percentage of cell lysis was calculated at the end of the co-culture. TERT-overexpressing CAR-NK REX Cells maintain their cytotoxic potential after cryo-recovery and expansion for over 400 days in culture (see Figure 57).

[0268] Example 38: TERT-overexpressing NK REX and CAR-NK REX Cell generation. In previous studies, CAR-NK REX The cells were engineered to overexpress TERT. TERT inhibits NK REX Cells and CAR-NK REX To investigate its ability to enhance cell expansion and functionality, NK REX The cells were first generated and then engineered to overexpress TERT. TERT-overexpressing NK cells were then generated. REX The cells were engineered to express a BCMA-targeted CAR (see Figure 58).

[0269] NK REX Cells (REX-edited NK cells) were generated from two additional donors and further engineered to overexpress TERT. In some cases, these cells were modified to express a BCMA-targeted CAR. All groups were monitored throughout the engineering and culture process for expansion. TERT-expressing NK cells REX The enrichment of NK cells was also assessed over time. Consistent with previous results, REX Cell donors C and D showed enrichment of the TERT transgene over time, with cells reaching nearly 100% transgene expression, as seen in the bottom panel of Figure 59. Furthermore, NK cells from donors C and D REX The cells plateaued on days 96 and 103 of culture, achieving a maximum expansion of 2.88E+008-fold and 4.36E+008-fold, respectively. REX TERT cells continued to proliferate through 225 days of cryopreservation, achieving a 1.47E+019-fold (donor C) and 8.53E+016-fold (donor D) expansion. REX TERT cells also continued to proliferate throughout cryopreservation at day 225, achieving a 5.65E+017-fold expansion (donor C) and a 1.7E+014-fold expansion (donor D). These results demonstrate that TERT overexpression enhances the proliferation and proliferation of NK cells. REX and CAR-NK REX The results show that the ATP-dependent agonist agonist reproducibly confers a cellular benefit (see Figure 59).

[0270] NK REX cells, TERT-overexpressing NK REX cells, BCMA-targeted CAR-NK REX cells, and TERT-overexpressing CAR-NK REX The cytotoxicity and persistence of effector cells were measured over multiple rounds of co-culture with BCMA-expressing JJN3 target cells at a 2:1 effector:target cell ratio in the presence of IL-2. After each round of co-culture, the percentage of cytolysis and effector cell expansion were assessed. At the start of the assay, NK REX and CAR-NK REX The cells were cultured for 119 days, but TERT-overexpressing NK cells were REX and TERT-overexpressing CAR-NK REX The CAR expression was measured on day 139 of culture. REX cells and TERT-overexpressing NK cells REX The CAR-negative group received only two rounds (donor E NK) of CAR-positive cells. REX ), 2 rounds only (donor C NK REX TERT), and 5 rounds only (donor D NK REX TERT) sustained, 0 rounds of continuous killing (donor E NK REX ), 0 rounds of continuous killing (donor C NK REX TERT), and three rounds of sequential killing (donor D NK REX TERT), they achieved at least 30% cell lysis. Consistent with the poor function and persistence of these CAR-deficient populations, they showed limited maximal expansion in this assay: 1.1-fold (donor E NK REX ), 4.6 times (donor C NK REX TERT), and 3.8 times (donor D NK REX TERT). In contrast, CAR-NK REX Cells (donor E) completed 10 rounds of sequential killing, achieved at least 30% cell lysis, and reached a maximum expansion of 3.07-fold. CAR NK from donor C REXTERT cells completed 8 rounds of sequential killing with 30% cytolysis but continued to kill tumor cells for an additional 2 rounds at a level of approximately 25%. CAR NK from donor C REX TERT cells achieved a maximum expansion of 45.8-fold. Finally, CAR NK from donor D REX TERT cells completed 10 rounds of sequential killing with 30% cell lysis and reached a maximum expansion of 27.7-fold. REX cells and TERT-overexpressing NK cells REX Although the cells completed a similar number of rounds of tumor cell lysis, TERT overexpression significantly outnumbered CAR-NK REX The results showed that TERT-overexpressing CAR-NK cells enhanced overall cell expansion. REX The cells are cytotoxic and younger than CAR-NK REX These results demonstrate that the IL-16 / ...

[0271] Example 39: STAT5A and STAT5B mutants inhibit REX-edited CD8 in vitro + Enriched in T cells. According to the timeline shown in Figure 61, REX Cells (REX-containing CD8 + Three STAT5A mutants, eight STAT5B mutants, and two STAT3 mutants were overexpressed in T cells (T cells). Table 9 below lists all STAT mutants tested in this study, as well as a description of the signaling pathways regulated by these molecules. Fluorescent reporters were used to track the enrichment of STAT mutants over time. STAT5A and STAT5B mutant-expressing T cells were overexpressed in T cells (T cells). REX The cells were enriched during the cell culture process, but the STAT3 mutant alone group showed no selective advantage. Figure 62 shows the enrichment of STAT mutants.

[0272] [Table 9]

[0273] Example 40: STAT5A and STAT5B mutants REX It is functional in cells. Over long-term cell culture, control T REX cells (UT), and STAT5A, STAT5B, and STAT3 mutant-containing T cells REX Surface expression of CD25 was assessed in T cells. STAT5A and STAT5B mutants were REX It is functional in cells and leads to upregulation of CD25 expression 5 days after transduction (see Figure 63A).

[0274] Over long-term cell culture, control T REX cells (UT), and STAT5A, STAT5B, and STAT3 mutant-containing T cells REX Surface expression of CD25 was assessed in T cells. STAT5A and STAT5B mutants were REX It is functional in cells and leads to upregulation of CD25 expression 20 days after transduction (see Figure 63B).

[0275] Over long-term cell culture, control T REX cells (UT), and STAT5A, STAT5B, and STAT3 mutant-containing T cells REX Surface expression of CD25 was assessed in T cells. STAT5A and STAT5B mutants were REX It is functional in cells and leads to upregulation of CD25 expression 35 days after transduction (see Figure 63C).

[0276] Over long-term cell culture, control T REX cells (UT), and STAT5A, STAT5B, and STAT3 mutant-containing T cells REX Surface expression of CD25 was assessed in T cells. STAT5A and STAT5B mutants were REX It is functional in cells and leads to upregulation of CD25 expression 42 days after transduction (see Figure 63D).

[0277] Example 41: STAT5A and STAT5B mutant T REXThe cells express the CAR and can be expanded robustly during the culture process. BCMA-targeting CARs were administered to unmodified T REX Cell (T REX (UT)) and STAT mutant T REX STAT mutant CAR-T cells (STAT MU1, STAT MU2, STAT MU3, STAT MU4, STAT MU5, STAT MU6, STAT MU7, STAT MU8, STAT MU9, STAT MU10, and STAT MU11) were transfected with CAR-T cells. CAR-expressing cells were further enriched to high purity prior to functional assessment, and STAT mutant CAR-T cells were transfected with CAR-T cells throughout the editing, transduction, and enrichment process. REX Cell expansion was followed (see Figure 64).

[0278] Throughout the editing, transduction, and enrichment process, STAT mutant CAR-T REX The expansion of the cells was tracked and, as shown in Figure 65, the STAT mutant CAR-T REX The cells expand robustly during the culture process.

[0279] Example 42: STAT5A and STAT5B mutants confer varying degrees of IL-2 independence in vitro. According to Figure 61, T REX Cells (REX-containing CD8 + Three STAT5A mutants, eight STAT5B mutants, and two STAT3 mutants were overexpressed in T cells. REX cells, as well as control T REX Cells were cultured in medium in the absence of IL-2 and expansion was followed over time. In the absence of CAR, multiple groups (control T REXCells (STAT MU1, STAT MU3, STAT MU4, STAT MU6, STAT MU7, STAT MU8, STAT MU10, STAT MU12, STAT MU13) exhibited IL-2 dependence, while the other groups (STAT MU2, STAT MU5, STAT MU9, and STAT MU11) grew independently of IL-2. Despite exhibiting IL-2 dependence, the following STAT mutants: STAT MU6, STAT MU8, showed growth rates comparable to baseline (control T) at early time points after IL-2 withdrawal. REX )T REX Several STAT mutant T cells expanded more rapidly than REX Cells were further engineered to express CAR according to Figure 61. Cells were grown in the presence of decreasing amounts of IL-2 and expansion was followed over time. The combination of CAR and STAT variant expression enhanced expansion of multiple populations. STAT MU6 and STAT MU7 CAR-T REX The cells remained dependent on IL-2 (although these populations proliferated under low IL-2 conditions). STAT MU8 and STAT MU9 CAR-T REX Although the cells demonstrated the ability to proliferate in the absence of IL-2, the greatest degree of IL-2 independence was observed for STAT MU4, STAT MU5, STAT MU10, and STAT MU11 CAR-T REX This was observed in cells (see Figure 66).

[0280] BCMA-targeted CARs were administered to young T REX Cells (BCMA-T REX (UT, day 99 of culture) and STAT mutant T REXCells (STAT MU4, STAT MU5, STAT MU6, STAT MU7, STAT MU8, STAT MU9, STAT MU10, STAT MU11, all at day 155 of culture) were transfected with STAT5B mutant CAR-T cells. The impedance-based xCELLigence platform was used to measure the cytotoxicity of CAR-expressing cells. Percent cytolysis was determined 12 and 72 hours after the initiation of co-culture of effector and target cells at various effector:target (E:T) cell ratios. Antigen-negative (HuH-7) and antigen-positive (HuH-7 BCMA) target cells were used in this experiment. STAT5B mutant CAR-T cells were transfected with STAT5B mutant CAR-T cells. REX The cell variants remain functional even after more than 150 days in culture (see Figure 67).

[0281] Example 43: STAT5B mutant CAR-TREX cells show enhanced persistence and functionality compared to young CAR-TREX cell benchmarks in sequential killing assays. BCMA-targeting CARs were used in T REX Cells (BCMA-T REX ), and STAT5B mutant-expressing T REX STAT5B mutants were introduced into CAR-T cells (STAT MU4, STAT MU5, STAT MU6, STAT MU7, STAT MU8, STAT MU9, STAT MU10, STAT MU11). The cytotoxicity and persistence of CAR-expressing cells were measured over multiple rounds of co-culture with BCMA-expressing JJN3 target cells at an effector:target cell ratio of 0.3:1. After each round of co-culture, percent cell lysis and effector cell expansion were assessed. All STAT5B mutants were expressed in CAR-T cells. REX The persistence and cytotoxicity of the cells were enhanced to varying degrees (see Figure 68). REX Cells were only able to complete two rounds of this assay, achieving at least 30% cytolysis, and after three rounds were effectively killed and unable to expand and persist. REXThe cells completed at least five rounds of sequential killing and exhibited at least 30% cytolysis. Furthermore, several STAT5B mutant-containing BCMA-T REX Cells achieved >80% cytolysis at the end of the assay (STAT MU9 BCMA-T REX Cells and STAT MU11 BCMA-T REX cells), others even achieved over 95% cell lysis at the end of 7 rounds (STAT MU4 BCMA-T REX Cells and STAT MU5 BCMA-T REX All STAT5B mutant-containing CAR-T REX Cell(STAT MU5 BCMA-T REX The maximum expansion observed in cells (=2.45E+05) was BCMA-T REX expanded more robustly than cells (maximum expansion = 1x).

[0282] NSG mice were inoculated with 2E6 MM1S-luciferase tumor cells. Six days later, BCMA-T REX Cell (T REX , day 99) or STAT mutant BCMA-TREX cells (STAT MU5, STAT MU6, STAT MU7, STAT MU9, and STAT MU11 - all day 112) were administered at 2E6 or 10E6 cells per mouse. Tumor burden was monitored twice weekly using IVIS imaging. All STAT mutant T REX Cells showed in vivo functionality despite a higher degree of expansion in vitro (numbers shown under group headings). In some cases, STAT mutant BCMA-T REX The cells were treated with BCMA-T at low doses and in control REX In these groups, STAT5B mutant BCMA-T cells administered at 2E6 cells were more effective than STAT5B mutant BCMA-T cells (STAT MU5, STAT MU6, and STAT MU9). REX The cells were BCMA-T administered at 10E6 cells. REX STAT5B mutant CAR-T cells eliminated tumors as effectively as or more effectively than STAT5B mutant CAR-T cells. REX The cells are then injected into the CAR-T cells, at least in vivo.REX It is as functional as the cellular benchmark (see Figure 69).

[0283] Example 44: STAT5B and STAT3 mutants are enriched in REX-edited NK cells in vitro.

[0284] According to the timeline shown, NK REX Eight STAT5B mutants and two STAT3 mutants were overexpressed in REX-edited NK cells (NK cells) (see Figure 70). Table 10 below lists all STAT mutants tested in this study, as well as a description of the signaling pathways regulated by these molecules. Fluorescent reporters were used to track the enrichment of STAT mutants over time. STAT5B and STAT3 mutants were found to be enriched in REX-edited NK cells in vitro, and overexpression of these mutant proteins significantly increased the NK REX This suggests that the cell phenotype can be enhanced by the addition of ribosomal RNA (see Figure 71).

[0285] [Table 10]

[0286] Example 45: STAT5B and STAT3 mutant expression in NK cells REX Enhances cell proliferation. According to Figure 70, NK REX Eight STAT5B mutants and two STAT3 mutants were overexpressed in REX-edited NK cells. REX cells (REX-edited NK cells) and STAT mutant-containing NK cells REX Cell expansion was assessed. STAT5B mutant NK REX cells and STAT3 mutant NK REX The cells outgrow donor-matched unmodified NK cells, as evidenced by the steeper growth curve after introduction of the STAT mutant. REX STAT5B and STAT3 mutant expression was significantly higher in NK cells than in NK cells.REX Enhances cell proliferation (see Figure 72).

[0287] Example 46: STAT mutant-expressing NK REX The cells remain functional in in vitro cytotoxicity assays. According to Figure 70, NK REX Eight STAT5B mutants and two STAT3 mutants were overexpressed in control (unmodified) NK cells (REX-edited NK cells). REX and STAT mutant NK REX STAT mutant-containing NK cells through co-culture with K562-luciferase cells REX The cytotoxic function of the cells was evaluated. Primary T cells were used as a control because they do not exhibit cytotoxicity against K562 cells in this assay. 24 hours after the initiation of co-culture, the percentage of cytolysis was determined. Effector cells were co-cultured with K562 cells at two different effector:target cell ratios (1:1-upper and 2:1-lower). STAT5B mutant and STAT3 mutant expressing NK cells were used. REX The cells were unmodified NK REX In this assay, primary T cell control did not lyse K562-luciferase cells. Therefore, STAT mutant-expressing NK cells REX Cells remain functional in in vitro cytotoxicity assays (see Figure 73).

[0288] The embodiments described herein can be practiced in the absence of any one or more elements or limitations not specifically disclosed herein. The terms and expressions used are used as terms of description and not of limitation, and the use of such terms and expressions is not intended to exclude the features shown and described or equivalents thereof, but it is recognized that various modifications are possible within the scope of the claimed embodiments. Thus, while the present specification is specifically disclosed by embodiments, it should be understood that any features, modifications, and variations of the concepts disclosed herein may be employed by those skilled in the art, and such modifications and variations are deemed to be within the scope of these embodiments as defined by the description and the appended claims. While several aspects of the present disclosure may be identified herein as particularly advantageous, it is intended that the present disclosure is not limited to these particular aspects of the disclosure.

[0289] A claim or description including "or" between one or more members of a group is considered to be satisfied when one, more than one, or all group members are present in, used in, or relevant to a given product or process, unless stated to the contrary or clear from context. The present disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which two or more or all group members are present in, used in, or relevant to a given product or process.

[0290] Furthermore, this disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as a list, for example, in Markush group format, each subgroup of the elements is also disclosed, and any element may be removed from the group.

[0291] Generally, when the present disclosure or aspects of the present disclosure are referred to as including particular elements and / or features, it is to be understood that certain embodiments of the present disclosure or aspects of the present disclosure consist of or consist essentially of such elements and / or features, and for simplicity, these embodiments are not specifically set forth herein.

[0292] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each individual patent and publication was specifically and individually indicated to be incorporated by reference. Citation or identification of any reference in any section of this application shall not be construed as an admission that such reference is available as prior art to the present disclosure.

Claims

1. 1. A method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in the population of primary immune cells; (b) introducing a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants into said population of primary immune cells; (c) culturing the primary immune cells in a culture medium, wherein said culturing comprises inducing proliferation of said primary immune cells to obtain a population of said primary immune cells that are resistant to replicative senescence (RRS).

2. 2. The method of claim 1, further comprising stimulating the population of primary immune cells prior to performing step (a) and / or step (b) and / or step (c) on the population of primary immune cells.

3. 3. The method of claim 1 or 2, further comprising stimulating the population of primary immune cells after performing step (a) and / or step (b) and / or step (c) on the population of primary immune cells.

4. 4. The method of any one of claims 1 to 3, wherein the one or more STAT5A mutants may be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants may be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

5. The method of any one of claims 1 to 4, further comprising introducing a transgene encoding TERT into the population of primary immune cells.

6. The method of any one of claims 1 to 5, further comprising inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells.

7. The method of claim 6, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC).

8. 8. The method of any one of claims 1 to 7, further comprising introducing one or more transgenes encoding anti-apoptotic factors or virus-derived factors into the population of primary immune cells.

9. 9. The method of claim 8, wherein the anti-apoptotic factor is either diffuse large B-cell lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

10. 9. The method of claim 8, wherein the virus-derived agent is any one of Saimiriine gammaherpesvirus2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1.

11. 11. The method of any one of claims 1 to 10, further comprising inhibiting expression of cluster of differentiation 38 (CD38), inhibiting expression of phosphatase tensin homolog (PTEN), and / or inhibiting expression of p53 in the population of primary immune cells.

12. 12. The method of any one of claims 1 to 11, further comprising introducing a transgene encoding MYC and / or introducing a transgene encoding KRAS into the population of primary immune cells.

13. 1. A method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in the population of primary immune cells; (b) introducing a transgene encoding MYC into said population of primary immune cells; (c) culturing the primary immune cells in a culture medium, wherein said culturing comprises inducing proliferation of said primary immune cells to obtain a population of said primary immune cells that are resistant to replicative senescence (RRS).

14. 14. The method of claim 13, further comprising introducing a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) into the population of primary immune cells.

15. 15. The method of claim 13 or 14, further comprising inhibiting expression of p53 in the population of primary immune cells.

16. The method of any one of claims 13 to 15, further comprising introducing a transgene encoding KRAS into said population of primary immune cells.

17. 17. The method of claim 16, wherein the KRAS comprises a KRAS A146V mutation.

18. 18. The method of any one of claims 13 to 17, further comprising inhibiting expression of phosphatase and tensin homolog (PTEN) in the population of primary immune cells.

19. 19. The method of claim 18, wherein PTEN expression is inhibited by a CRISPR / Cas system.

20. 1. A method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in the population of primary immune cells; (b) introducing a transgene encoding TERT into said population of primary immune cells; (c) culturing the primary immune cells in a culture medium, wherein said culturing comprises inducing proliferation of said primary immune cells to obtain a population of said primary immune cells that are resistant to replicative senescence (RRS).

21. 21. The method of claim 20, further comprising introducing into the population of primary immune cells a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC.

22. 22. The method of claim 20 or 21, further comprising introducing a transgene encoding KRAS into the population of primary immune cells.

23. 23. The method of claim 22, wherein the KRAS comprises a KRAS A146V mutation.

24. 24. The method of any one of claims 13 to 23, further comprising stimulating the population of primary immune cells prior to performing step (a) and / or step (b) and / or step (c) on the population of primary immune cells.

25. 24. The method of any one of claims 13 to 23, further comprising stimulating the population of primary immune cells after performing step (a) and / or step (b) and / or step (c) on the population of primary immune cells.

26. 26. The method of any one of claims 13 to 25, further comprising inhibiting expression of one or more endogenous immune-related genes in the primary immune cells in the population of primary immune cells.

27. 27. The method of claim 26, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC).

28. 28. The method of any one of claims 13 to 27, further comprising introducing into the primary immune cells one or more transgenes encoding any one of: Saimiriine gammaherpesvirus2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1.

29. 29. The method of any one of claims 13 to 28, further comprising inhibiting expression of cluster of differentiation 38 (CD38) in the population of primary immune cells.

30. 30. The method of any one of claims 1 to 29, wherein the population of primary immune cells comprises total T cells.

31. The population of primary immune cells is CD8 ＋ 30. The method of any one of claims 1 to 29, comprising T cells.

32. The population of primary immune cells is CD4 ＋ 30. The method of any one of claims 1 to 29, comprising T cells.

33. 33. The method of any one of claims 1 to 32, wherein the population of primary immune cells comprises gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells, and / or natural killer T (NKT) cells.

34. 34. The method of any one of claims 1 to 33, wherein the population of primary immune cells is human.

35. 35. The method of any one of claims 1 to 34, further comprising introducing a polynucleotide encoding a chimeric antigen receptor (CAR) into the population of primary immune cells.

36. 36. The method of any one of claims 1 to 35, wherein the population of primary immune cells can be cultured with or without TCR stimulation for at least 100 days.

37. The population of primary immune cells is cultured for at least about 10 ６ 37. The method of any one of claims 1 to 36, wherein the image is subjected to a 2x magnification.

38. 38. The method of any one of claims 1 to 37, wherein the population of primary immune cells is cultured in a culture medium that does not contain a primary immune cell stimulus.

39. 39. The method of any one of claims 1 to 38, further comprising (d) restimulating the population of primary immune cells.

40. The population of primary immune cells is cultured for at least about 10 ８ 40. The method of claim 39, wherein the image is subjected to a 2x magnification.

41. 41. The method of any one of claims 1 to 40, wherein the transgene is introduced using a plasmid-based DNA transposon.

42. The method of any one of claims 1 to 40, wherein the transgene is introduced using a lentiviral platform.

43. 41. The method of any one of claims 1 to 40, wherein the transgene is introduced using CRISPR-mediated site-specific integration.

44. 1. A method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), comprising: (a) in said population of primary immune cells, one or more endogenous regulatory factors, inhibiting expression of one or more endogenous regulatory factors, wherein the endogenous regulatory factors are cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), or S-methyl-5'-thioadenosine phosphorylase (MTAP); (b) detecting in said population of primary immune cells one or more endogenous immune-related genes, inhibiting expression of one or more endogenous immune-related genes, wherein the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC); (c) introducing a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants into said population of primary immune cells; (d) culturing the population of primary immune cells in a culture medium, wherein said culturing comprises inducing proliferation of said primary immune cells to obtain a population of said primary immune cells that are resistant to replicative senescence (RRS).

45. 45. The method of claim 44, further comprising introducing a transgene encoding either diffuse large B-cell lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2) into the population of primary immune cells.

46. 46. ​​The method of claim 44 or 45, further comprising stimulating the population of primary immune cells prior to performing step (a) and / or step (b) and / or step (c) and / or step (d) on the population of primary immune cells.

47. 47. The method of any one of claims 44 to 46, further comprising stimulating the population of primary immune cells after performing step (a) and / or step (b) and / or step (c) and / or step (d) on the population of primary immune cells.

48. 48. The method of any one of claims 44 to 47, wherein the one or more STAT5A mutants may be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants may be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

49. 49. The method of any one of claims 44 to 48, further comprising introducing a transgene encoding TERT into said population of primary immune cells.

50. 50. The method of any one of claims 44-49, further comprising inhibiting expression of cluster of differentiation 38 (CD38), inhibiting expression of phosphatase and tensin homolog (PTEN), and / or inhibiting expression of p53 in the population of primary immune cells.

51. 51. The method of any one of claims 44 to 50, further comprising introducing a transgene encoding MYC and / or introducing a transgene encoding KRAS into the population of primary immune cells.

52. 1. A method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in the population of primary immune cells; (b) inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells, wherein the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC); and (c) introducing a transgene encoding MYC into said population of primary immune cells; (d) culturing the primary immune cells in a culture medium, wherein said culturing comprises inducing proliferation of said primary immune cells to obtain a population of said primary immune cells that are resistant to replicative senescence (RRS).

53. 53. The method of claim 52, further comprising introducing a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) into said population of primary immune cells.

54. 54. The method of claim 52 or 53, further comprising inhibiting expression of p53 in said population of primary immune cells.

55. 55. The method of any one of claims 52 to 54, further comprising introducing a transgene encoding KRAS into said population of primary immune cells.

56. 56. The method of claim 55, wherein the KRAS comprises a KRAS A146V mutation.

57. 57. The method of any one of claims 52 to 56, further comprising inhibiting expression of phosphatase and tensin homolog (PTEN) in the population of primary immune cells.

58. 58. The method of claim 57, wherein PTEN expression is inhibited by a CRISPR / Cas system.

59. 1. A method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in the population of primary immune cells; (b) inhibiting expression of one or more endogenous immune-related genes in the population of primary immune cells, wherein the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC); and (c) introducing a transgene encoding TERT into the population of primary immune cells; (d) culturing the primary immune cells in a culture medium, wherein said culturing comprises inducing proliferation of said primary immune cells to obtain a population of said primary immune cells that are resistant to replicative senescence (RRS).

60. 60. The method of claim 59, further comprising introducing into the population of primary immune cells a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC.

61. 61. The method of claim 59 or 60, further comprising introducing a transgene encoding KRAS into said population of primary immune cells.

62. 62. The method of claim 61, wherein the KRAS comprises a KRAS A146V mutation.

63. 63. The method of any one of claims 52 to 62, further comprising stimulating the population of primary immune cells prior to performing step (a) and / or step (b) and / or step (c) and / or step (d) on the population of primary immune cells.

64. 63. The method of any one of claims 52 to 62, further comprising stimulating the population of primary immune cells after performing step (a) and / or step (b) and / or step (c) and / or step (d) on the population of primary immune cells.

65. 65. The method of any one of claims 52-64, further comprising introducing into the primary immune cells one or more transgenes encoding any one of: Saimiriine gammaherpesvirus2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1.

66. 66. The method of any one of claims 52-65, further comprising inhibiting expression of cluster of differentiation 38 (CD38) in the population of primary immune cells.

67. 67. The method of any one of claims 44 to 66, wherein the population of primary immune cells comprises whole T cells.

68. The population of primary immune cells is CD8 ＋ 67. The method of any one of claims 44 to 66, comprising T cells.

69. The population of primary immune cells is CD4 ＋ 67. The method of any one of claims 44 to 66, comprising T cells.

70. 67. The method of any one of claims 44 to 66, wherein the population of primary immune cells comprises gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells, and / or natural killer T (NKT) cells.

71. 71. The method of any one of claims 44 to 70, wherein the population of primary immune cells is human.

72. 72. The method of any one of claims 44-71, further comprising introducing a polynucleotide encoding a chimeric antigen receptor (CAR) into the population of primary immune cells.

73. 73. The method of any one of claims 44 to 72, wherein the population of primary immune cells can be cultured for at least 100 days.

74. The population of primary immune cells is cultured for at least about 10 ６ 74. A method according to any one of claims 44 to 73, wherein the method is subjected to a 2-fold magnification.

75. 75. The method of any one of claims 44 to 74, wherein the population of primary immune cells is cultured in a culture medium that does not contain a primary immune cell stimulus.

76. 76. The method of any one of claims 44-75, further comprising (e) restimulating the population of primary immune cells.

77. The population of primary immune cells is cultured for at least about 10 ８ 77. The method of claim 76, wherein the image is subjected to a 2x magnification.

78. 78. The method of any one of claims 44 to 77, wherein the transgene is introduced using a plasmid-based DNA transposon.

79. 79. The method of any one of claims 44 to 78, wherein the transgene is introduced using a lentiviral platform.

80. 80. The method of any one of claims 44 to 79, wherein the transgene is introduced using CRISPR-mediated site-specific integration.

81. 1. A method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), comprising: (a) inhibiting expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), or S-methyl-5'-thioadenosine phosphorylase (MTAP) in the population of primary immune cells; (b) introducing a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants into said population of primary immune cells; (c) introducing a transgene encoding TERT into the population of primary immune cells; (d) culturing the population of primary immune cells in a culture medium, wherein said culturing comprises inducing proliferation of said primary immune cells to obtain a population of said primary immune cells that are resistant to replicative senescence (RRS).

82. 82. The method of claim 81, further comprising stimulating the primary immune cells prior to performing step (a) and / or step (b) and / or step (c) and / or step (d) on the population of primary immune cells.

83. 83. The method of claim 81 or 82, further comprising stimulating the primary immune cells after performing step (a) and / or step (b) and / or step (c) and / or step (d) on the population of primary immune cells.

84. 84. The method of any one of claims 81 to 83, further comprising inhibiting expression of one or more endogenous immune-related genes in said population of primary immune cells.

85. 85. The method of any one of claims 81 to 84, wherein the one or more STAT5A mutants may be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants may be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

86. 85. The method of claim 84, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC).

87. 87. The method of any one of claims 81-86, further comprising inhibiting expression of cluster of differentiation 38 (CD38), inhibiting expression of phosphatase and tensin homolog (PTEN), and / or inhibiting expression of p53 in the population of primary immune cells.

88. 88. The method of any one of claims 81 to 87, further comprising introducing a transgene encoding MYC and / or introducing a transgene encoding KRAS into the population of primary immune cells.

89. 1. A method for generating a population of primary immune cells that are resistant to replicative senescence (RRS), comprising: (a) inhibiting the expression of cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and S-methyl-5'-thioadenosine phosphorylase (MTAP) in the population of primary immune cells; (b) introducing a transgene encoding MYC into said population of primary immune cells; (c) introducing a transgene encoding TERT into the population of primary immune cells; (d) culturing the primary immune cells in a culture medium, wherein said culturing comprises inducing proliferation of said primary immune cells to obtain a population of said primary immune cells that are resistant to replicative senescence (RRS).

90. 90. The method of claim 89, further comprising introducing a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) into said population of primary immune cells.

91. 91. The method of claim 89 or 90, further comprising inhibiting expression of p53 in said population of primary immune cells.

92. 92. The method of any one of claims 89 to 91, further comprising introducing a transgene encoding KRAS into said population of primary immune cells.

93. 93. The method of claim 92, wherein the KRAS comprises a KRAS A146V mutation.

94. 94. The method of any one of claims 89-93, further comprising inhibiting expression of phosphatase and tensin homolog (PTEN) in said population of primary immune cells.

95. 95. The method of claim 94, wherein PTEN expression is inhibited by a CRISPR / Cas system.

96. 96. The method of any one of claims 89 to 95, further comprising stimulating the population of primary immune cells prior to performing step (a) and / or step (b) and / or step (c) and / or step (d) on the population of primary immune cells.

97. 97. The method of any one of claims 89 to 96, further comprising stimulating the population of primary immune cells after performing step (a) and / or step (b) and / or step (c) and / or step (d) on the population of primary immune cells.

98. 98. The method of any one of claims 89-97, further comprising inhibiting expression of one or more endogenous immune-related genes in said primary immune cells in said population of primary immune cells.

99. 99. The method of claim 98, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC).

100. 100. The method of any one of claims 89-99, further comprising introducing into the primary immune cells one or more transgenes encoding any one of: Saimiriine gammaherpesvirus2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1.

101. 101. The method of any one of claims 89-100, further comprising inhibiting expression of cluster of differentiation 38 (CD38) in the population of primary immune cells.

102. 102. The method of any one of claims 81 to 101, wherein the population of primary immune cells comprises whole T cells.

103. The population of primary immune cells is CD8 ＋ 102. The method of any one of claims 81 to 101, comprising T cells.

104. The population of primary immune cells is CD4 ＋ 102. The method of any one of claims 81 to 101, comprising T cells.

105. 102. The method of any one of claims 81-101, wherein the population of primary immune cells comprises gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells, and / or natural killer T (NKT) cells.

106. 106. The method of any one of claims 81 to 105, wherein the population of primary immune cells is human.

107. 107. The method of any one of claims 81-106, further comprising introducing a polynucleotide encoding a chimeric antigen receptor (CAR) into said population of primary immune cells.

108. 108. The method of any one of claims 81 to 107, wherein the population of primary immune cells is capable of being cultured for at least 100 days.

109. The population of primary immune cells is cultured for at least about 10 ６ 109. A method according to any one of claims 81 to 108, wherein the method is subjected to a 2x magnification.

110. 110. The method of any one of claims 81 to 109, wherein the population of primary immune cells is cultured in a culture medium that does not contain a primary immune cell stimulus.

111. 111. The method of any one of claims 81-110, further comprising (e) stimulating the population of primary immune cells.

112. The population of primary immune cells is cultured for at least about 10 ８ 112. The method of claim 111, wherein the image is subjected to a 2x magnification.

113. 113. The method of any one of claims 81 to 112, wherein the transgene is introduced using a plasmid-based DNA transposon.

114. 113. The method of any one of claims 81 to 112, wherein the transgene is introduced using a lentiviral platform.

115. 113. The method of any one of claims 81 to 112, wherein the transgene is introduced using CRISPR-mediated site-specific integration.

116. 116. An engineered immune cell population produced according to the method of any one of claims 1 to 115.

117. 117. A pharmaceutical composition comprising the engineered immune cell population of claim 116 and a pharmaceutically acceptable carrier.

118. 118. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 117.

119. 1. An engineered T cell that does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), wherein the engineered T cell comprises a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants.

120. 120. The engineered T cell of claim 119, wherein the one or more STAT5A mutants can be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants can be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

121. 121. The engineered T cell of claim 119 or 120, further comprising introducing a transgene encoding TERT.

122. 120. The engineered T cell of claim 119, wherein the engineered T cell further comprises a transgene encoding either diffuse large B-cell lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

123. 121. The engineered T cell of claim 119 or 120, wherein the engineered T cell does not express one or more endogenous immune-related genes.

124. 124. The engineered T cell of claim 123, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

125. 125. The engineered T cell of any one of claims 119-124, wherein the engineered T cell does not express cluster of differentiation 38 (CD38), phosphatase tensin homolog (PTEN), and / or p53.

126. 126. The engineered T cell of any one of claims 119 to 125, further comprising a transgene encoding MYC and / or a transgene encoding KRAS.

127. Engineered T cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP).

128. 128. The engineered T cell of claim 127, further comprising a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC.

129. 129. The engineered T cell of claim 127 or 128, wherein the engineered T cell does not express p53.

130. 130. The engineered T cell of any one of claims 127 to 129, further comprising a transgene encoding KRAS.

131. 131. The engineered T cell of claim 130, wherein KRAS comprises a KRAS A146V mutation.

132. 132. The engineered T cell of any one of claims 127-131, wherein the engineered T cell does not express phosphatase tensin homolog (PTEN).

133. 133. The engineered T cell of claim 132, wherein PTEN expression is inhibited by a CRISPR / Cas system.

134. Engineered T cells that do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and contain a transgene encoding TERT.

135. 135. The engineered T cell of claim 134, wherein the engineered T cell comprises a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC.

136. 136. The engineered T cell of claim 134 or 135, further comprising a transgene encoding KRAS.

137. 137. The engineered T cell of claim 136, wherein KRAS comprises a KRAS A146V mutation.

138. 139. The engineered T cell of any one of claims 127-138, wherein the engineered T cell does not express one or more endogenous immune-related genes in the primary immune cells in the population of primary immune cells.

139. 139. The engineered T cell of claim 138, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

140. 140. The engineered T cell of any one of claims 127-139, wherein the engineered T cell does not express cluster of differentiation 38 (CD38).

141. 141. The engineered T cell of any one of claims 119 to 140, further comprising a polynucleotide encoding a chimeric antigen receptor (CAR).

142. The engineered T cells are CD8 ＋ T cells, CD4 ＋ 142. The engineered T cell of any one of claims 119-141, which is a T cell, a gamma-delta T cell, a mucosal-associated invariant T (MAIT) T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, or a combination thereof.

143. The engineered T cells are CD8 ＋ 142. The engineered T cell of any one of claims 119 to 141, which is a T cell.

144. The engineered T cells are CD4 ＋ 142. The engineered T cell of any one of claims 119 to 141, which is a T cell.

145. 145. The engineered T cell of any one of claims 119 to 144, wherein the engineered T cell is human.

146. 1. An engineered T cell that does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), S-methyl-5'-thioadenosine phosphorylase (MTAP), beta-2 microglobulin (B2M), and / or T cell receptor alpha constant region (TRAC), wherein the engineered T cell comprises a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants, and wherein the engineered T cell comprises a transgene encoding TERT.

147. 147. The engineered T cell of claim 146, wherein the engineered T cell does not express cluster of differentiation 38 (CD38), phosphatase tensin homolog (PTEN), and / or p53.

148. 148. The engineered T cell of claim 146 or 147, wherein the engineered T cell further comprises a transgene encoding MYC and / or a transgene encoding KRAS.

149. 149. The engineered T cell of any one of claims 146-148, further comprising a polynucleotide encoding a chimeric antigen receptor (CAR).

150. 150. The engineered T cell of any one of claims 146-149, wherein the engineered T cell is a gamma-delta T cell, a mucosal-associated invariant T (MAIT) T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, or a combination thereof.

151. The engineered T cells are CD8 ＋ 150. The engineered T cell of any one of claims 146 to 149, which is a T cell.

152. The engineered T cells are CD4 ＋ 150. The engineered T cell of any one of claims 146 to 149, which is a T cell.

153. 153. The engineered T cell of any one of claims 146-152, wherein the engineered T cell is human.

154. 1. An engineered T cell that expresses a transgene encoding diffuse large B-cell lymphoma (Bcl-XL), wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), S-methyl-5'-thioadenosine phosphorylase (MTAP), and / or phosphatase tensin homolog (PTEN), and wherein the engineered T cell comprises a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants.

155. 155. The engineered T cell of claim 154, wherein the one or more STAT5A mutants can be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants can be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

156. 156. The engineered T cell of claim 154 or 155, wherein the engineered T cell does not express one or more endogenous immune-related genes.

157. 157. The engineered T cell of any one of claims 154-156, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) or T cell receptor alpha constant region (TRAC).

158. 158. The engineered T cell of any one of claims 154-157, wherein the engineered T cell does not express cluster of differentiation 38 (CD38), phosphatase tensin homolog (PTEN), and / or p53.

159. 159. The engineered T cell of any one of claims 154 to 158, wherein the engineered T cell comprises a transgene encoding MYC and / or a transgene encoding KRAS.

160. 160. The engineered T cell of any one of claims 154-159, further comprising a polynucleotide encoding a chimeric antigen receptor (CAR).

161. The engineered T cells are CD8 ＋ T cells, CD4 ＋ 161. The engineered T cell of any one of claims 154-160, which is a T cell, a delta gamma T cell, a mucosal-associated invariant T (MAIT) T cell, a natural killer (NK) T cell, or a combination thereof.

162. The engineered T cells are CD8 ＋ 161. The engineered T cell of any one of claims 154 to 160, which is a T cell.

163. The engineered T cells are CD4 ＋ 161. The engineered T cell of any one of claims 154 to 160, which is a T cell.

164. 164. The engineered T cell of any one of claims 154 to 163, wherein the engineered T cell is human.

165. 1. Use of engineered T cells for the manufacture of a medicament for treating cancer in a patient, wherein the engineered T cells do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and the engineered T cells comprise a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants.

166. The use of claim 165, wherein the one or more STAT5A mutants may be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants may be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

167. 167. The use of claim 165 or 166, wherein the engineered T cells further comprise a polynucleotide encoding a chimeric antigen receptor (CAR).

168. 168. The use of any one of claims 165 to 167, wherein the engineered T cells further comprise a transgene encoding either diffuse large B-cell lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

169. 169. The use of any one of claims 165 to 168, wherein the engineered T cells further comprise a transgene encoding TERT.

170. 170. The use of any one of claims 165 to 169, wherein the engineered T cells do not express one or more endogenous immune-related genes.

171. The use of claim 166, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC).

172. 172. The use of any one of claims 165 to 171, wherein the engineered T cells do not express cluster of differentiation 38 (CD38), phosphatase tensin homolog (PTEN), and / or p53.

173. 173. The use according to any one of claims 165 to 172, wherein the engineered T cells comprise a transgene encoding MYC and / or a transgene encoding KRAS.

174. 1. Use of an engineered T cell for the manufacture of a medicament for treating cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP).

175. 175. The use of claim 174, wherein the engineered T cells comprise a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC.

176. 176. The use of claim 174 or 175, wherein the engineered T cells do not express p53.

177. 177. The use of any one of claims 174 to 176, wherein the engineered T cells comprise a transgene encoding KRAS.

178. 178. The use of claim 177, wherein the KRAS comprises the KRAS A146V mutation.

179. 179. The use of any one of claims 174 to 178, wherein the engineered T cells do not express phosphatase tensin homolog (PTEN).

180. The use of claim 179, wherein PTEN expression is inhibited by the CRISPR / Cas system.

181. 1. Use of engineered T cells for the manufacture of a medicament for treating cancer in a patient, wherein the engineered T cells do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and the engineered T cells comprise a transgene encoding TERT.

182. 182. The use of claim 181, wherein the engineered T cells comprise a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC.

183. 183. The use of claim 181 or 182, wherein the engineered T cells comprise a transgene encoding KRAS.

184. 184. The use of claim 183, wherein the KRAS comprises the KRAS A146V mutation.

185. 185. The use of any one of claims 181 to 184, wherein the engineered T cells further comprise a polynucleotide encoding a chimeric antigen receptor (CAR).

186. 186. The use of any one of claims 174 to 185, wherein said engineered T cells do not express one or more endogenous immune-related genes in said primary immune cells in said population of primary immune cells.

187. The use of claim 186, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant region (TRAC).

188. 188. The use of any one of claims 174 to 187, wherein the engineered T cells do not express cluster of differentiation 38 (CD38).

189. The engineered T cells are CD8 ＋ T cells, CD4 ＋ 189. The use of any one of claims 165 to 188, wherein the T cells are gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells, natural killer T (NKT) cells, or combinations thereof.

190. The engineered T cells are CD8 ＋ The use according to any one of claims 165 to 188, which is a T cell.

191. The engineered T cells are CD4 ＋ The use according to any one of claims 165 to 188, which is a T cell.

192. 192. The use of any one of claims 165 to 191, wherein the engineered T cells are human.

193. 1. An engineered T cell for the treatment of cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and the engineered T cell comprises a transgene encoding one or more STAT5A mutants and / or one or more STAT5B mutants.

194. 194. The engineered T cell of claim 193, wherein the one or more STAT5A mutants can be H299R, N642H, Y665F, S711F, and combinations thereof, and / or the one or more STAT5B mutants can be H298R, R430C, E433K, N642H, Y665F, P702A, V712E, S715F, and combinations thereof.

195. 195. The engineered T cell of claim 193 or 194, wherein the engineered T cell further comprises a transgene encoding either diffuse large B-cell lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

196. 196. The engineered T cell of any one of claims 193-195, wherein the engineered T cell further comprises a transgene encoding TERT.

197. 197. The engineered T cell of any one of claims 193-196, wherein the engineered T cell does not express one or more endogenous immune-related genes.

198. 200. The engineered T cell of claim 197, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

199. 200. The engineered T cell of any one of claims 193-198, wherein the engineered T cell does not express cluster of differentiation 38 (CD38), PTEN, and / or p53.

200. 200. The engineered T cell of any one of claims 193 to 199, wherein the engineered T cell comprises a transgene encoding MYC and / or a transgene encoding KRAS.

201. 201. The engineered T cell of any one of claims 193-200, wherein the engineered T cell further comprises a polynucleotide encoding a chimeric antigen receptor (CAR).

202. 1. An engineered T cell for the treatment of cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP).

203. 203. The engineered T cell of claim 202, further comprising a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC.

204. 204. The engineered T cell of claim 202 or 203, wherein the engineered T cell does not express p53.

205. 205. The engineered T cell of any one of claims 202-204, further comprising a transgene encoding KRAS.

206. 206. The engineered T cell of claim 205, wherein KRAS comprises a KRAS A146V mutation.

207. 207. The engineered T cell of any one of claims 202-206, wherein the engineered T cell does not express phosphatase tensin homolog (PTEN).

208. The engineered T cell of claim 207, wherein said PTEN expression is inhibited by a CRISPR / Cas system.

209. 1. An engineered T cell for the treatment of cancer in a patient, wherein the engineered T cell does not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), and / or S-methyl-5'-thioadenosine phosphorylase (MTAP), and comprises a transgene encoding TERT.

210. 210. The engineered T cell of claim 209, wherein said engineered T cell comprises a transgene encoding diffuse large B-cell lymphoma (Bcl-xL) and a transgene encoding MYC.

211. 211. The engineered T cell of claim 209 or 210, further comprising a transgene encoding KRAS.

212. 212. The engineered T cell of claim 211, wherein KRAS comprises a KRAS A146V mutation.

213. 213. The engineered T cell of any one of claims 202-212, wherein said engineered T cell does not express one or more endogenous immune-related genes in said primary immune cells in said population of primary immune cells.

214. 214. The engineered T cell of claim 213, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant region (TRAC).

215. 215. The engineered T cell of any one of claims 202-214, wherein the engineered T cell does not express cluster of differentiation 38 (CD38).

216. 216. The engineered T cell of any one of claims 202-215, wherein the engineered T cell further comprises a polynucleotide encoding a chimeric antigen receptor (CAR).

217. The engineered T cells are CD8 ＋ T cells, CD4 ＋ 217. The engineered T cell of any one of claims 193-216, which is a T cell, a gamma-delta T cell, a mucosal-associated invariant T (MAIT) T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, or a combination thereof.

218. The engineered T cells are CD8 ＋ 217. The engineered T cell of any one of claims 193 to 216, which is a T cell.

219. The engineered T cells are CD4 ＋ 217. The engineered T cell of any one of claims 193 to 216, which is a T cell.

220. 220. The engineered T cell of any one of claims 193-219, wherein the engineered T cell is human.

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