Methods for Producing 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, methods are provided herein 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 Patent Application No. 63 / 236,443, filed August 24, 2021, the disclosure of which is incorporated herein by reference in its entirety.

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

[0003] Engineered adoptive cell therapy has been a novel approach for patients with hematological malignancies in recent years, with the first chimeric antigen receptor (CAR)-based therapy approved by the FDA in 2017 (Non-Patent Document 1; Non-Patent Document 2). Since 2017, there has been a surge in the number of clinical trials testing adoptive cell therapy and other cell therapies, such as CAR-T cells, CAR-natural killer (NK) and CAR-NKT cells, T cell receptor (TCR)-T cells, tumor-infiltrating lymphocytes (TIL), and T cells targeting tumor-specific antigens. More recently, the first CAR-macrophage (CAR-M) has entered the clinic for the treatment of solid tumors (Non-Patent Document 3; Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6).

[0004] While cell therapies hold great potential to become curative for patients, many factors limit the widespread development and administration of these drugs: Most cell therapies are currently produced in-house, with variability in the quality of the cell products associated with cytokine release syndrome and other toxicities, extended manufacturing times, high costs, and a limited window during which these therapies can be genetically modified to enhance their efficacy (Non-Patent Document 1).

[0005] Many of the cell therapies currently being tested in the clinic utilize CAR-T or CAR-NK cells, as these subsets of immune cells display potent cytotoxicity. The mature primary human T cells used for these therapies are found in human blood and secondary lymphoid organs, where they protect individuals against 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, THC and regulatory T cells. Many αβ T cell subsets exhibit potent cytotoxic functions that have been exploited for the development of cell therapy.

[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 (Non-Patent Document 7). Like T cells, NK cells exhibit potent cytotoxic functions and are of interest for the development of cell therapy.

[0007] However, primary human immune cells such as T cells and NK cells also have a finite proliferation capacity in vitro and in vivo, limiting their ability to be used to generate broad-spectrum cell therapies. Furthermore, this limited proliferation capacity of mature primary human immune cells impairs their gene editability to mitigate cytokine release syndrome and other potential cell therapy-associated toxicities, overcome tumor microenvironment-associated burdens, and prevent rejection of allogeneic cell therapy products in patients.

[0008] Cell lines of leukemic cells derived from patients have been studied for decades in cell culture, and their transformed state confers long-term proliferative potential, allowing their use in various cellular assays. This, in turn, has facilitated the development of many therapeutics. However, these cells generally lack the potent cytotoxic functions of mature primary human T cells and NK cells, as they are often immature or derived from dysfunctional T cell clones. The transformed nature of these cells can also be mapped onto a constellation of mutations frequently found in patients with T cell acute lymphoblastic leukemia. Furthermore, mature T cells from non-human primates can be transformed by herpes viruses through pathways that converge to some of the same mechanisms involved in the transformation of primary human T cells in patients (Non-Patent Document 8; Non-Patent Document 9; Non-Patent Document 10; Non-Patent Document 11).

[0009] While previous studies suggest that primary human T cells can be immortalized through overexpression of factors such as telomerase reverse transcriptase (TERT) (12; 13; 14), human T-cell leukemia virus type 1 or human T-cell leukemia virus type 2 (HTLV-1 / HTLV-2) transcriptional transactivator protein Tax (15; 16; 17), or by viruses such as Herpesvirus saimiri (8; 9) and HTLV-1 / HTLV-2, these approaches are not very reproducible and may result in reprogramming of modified or infected cells. Furthermore, cells whose proliferative life span has been enhanced through overexpression of TERT still require the use of feeder cells or extensive exogenous stimulation through their T cell receptor to drive proliferation (13; 14). The use of allogeneic feeder cells and extensive repeated stimulation is undesirable when establishing banks of mature primary human T or NK cells, as these methods are difficult to tune and may ultimately drive the cells to a dysfunctional state. Furthermore, because patients are often immunocompromised, the use of infectious agents capable of transforming mature primary human T or NK cells limits the use of these cells in the development of cell therapies.

[0010] In view of these challenges, there is a great need to establish alternative methods to extend the proliferative life span of primary human immune cells to enable large-scale production of allogeneic cytotoxic cells. The present disclosure describes methods and cells that address this unmet need. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Larson & Maus, Nat Rev Cancer 21,145-161(2021) [Non-Patent Document 2] Yu,et al.,Nature Reviews Drug Discovery 19,583-584(2020) [Non-licensed document 3] Mukhopadhyay,Nat Methods 17,561(2020)

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

Non-licensed Document 12

Non-licensed Document 13

[0012] The disclosure herein provides a method of generating a population of replicative senescence-resistant (RRS) primary immune cells, the method comprising: (a) introducing one or more gene edits into primary immune cells; (b) culturing the primary immune cells in medium; which culture induces proliferation of the primary immune cells to result in a population of replicative senescence-resistant (RRS) primary immune cells.

[0013] The disclosure herein also provides a method of generating a population of replicative senescence-resistant (RRS) primary immune cells, comprising: (a) inhibiting expression of one or more endogenous regulators in the primary immune cells, where the endogenous regulators 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 primary immune cells, where the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T cell receptor alpha constant (TRAC); and (c) culturing the primary immune cells in a medium; which culturing induces proliferation of the primary immune cells to give rise to the population of replicative senescence-resistant (RRS) primary immune cells.

[0014] The disclosure herein also provides a method of generating a population of replicative senescence-resistant (RRS) primary immune cells, comprising: (a) inhibiting expression of one or more endogenous regulators in the primary immune cells, where the endogenous regulators 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 primary immune cells, where the endogenous immune-related genes are beta-2 microglobulin (B2M) and / or T cell receptor alpha constant (TRAC); and (c) culturing the primary immune cells in a medium; which culturing induces proliferation of the primary immune cells to give rise to the population of replicative senescence-resistant (RRS) primary immune cells.

[0015] Disclosed herein provides a method of generating a population of replicative senescence-resistant (RRS) primary immune cells, comprising: (a) inhibiting expression of one or more endogenous regulators in primary immune cells, where the endogenous regulators are cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), or S-methyl-5'-thioadenosine phosphorylase (MTAP); (b) culturing the primary immune cells in a medium; which culture induces proliferation of the primary immune cells to generate a population of replicative senescence-resistant (RRS) primary immune cells.

[0016] The disclosure herein also provides modified immune cell populations made according to the methods described. The disclosure herein further provides a pharmaceutical composition comprising the modified immune cell population described above and a pharma- ceutically acceptable carrier. The disclosure herein further provides a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described above.

[0017] The disclosure herein provides modified 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), and / or T cell receptor alpha constant (TRAC).

[0018] The disclosure herein provides modified T cells that express a transgene encoding B-cell large lymphoma (Bcl-XL), where the modified T cells do not express cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), S-methyl-5'-thioadenosine phosphorylase (MTAP), and / or phosphatase and tensin homolog (PTEN). [Brief description of the drawings]

[0019] [Figure 1] [FIGS. 1A-1B] Figures 1A-1B show that Bcl-xL insertion conferred a selective advantage for 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. [Diagram 2] FIG. 2 shows a method for identifying survival-enhancing transgenes in primary human T cells. [Diagram 3] [FIGS. 3A-3B] FIGS. 3A-3B show that loss of expression of cell cycle regulatory molecules enhanced the proliferative capacity of T cells in long-term culture. [Figure 4] [Figures 4A-4D] Figures 4A-4D show that restimulation of TREX+Bcl-xL cells can promote their proliferation in long-term culture. Figure 4A shows the overall fold increase of TREX+Bcl-xL cells over time. Figures 4B-4D show the overall fold increase of TREX+Bcl-xL cells and PTEN-deleted TREX+Bcl-xL cells restimulated with αCD3 or αCD3 / αCD28 Dynabeads for 3 days after the cells were debeaded. The overall fold increase was followed and graphed for resting and treated cells over time. Arrows indicate the period of restimulation. Black arrows indicate the time points for evaluation of further restimulation modalities. Figures 4A-4D show logarithmic scales. [Diagram 5] [Figures 5A-5B] Figures 5A-5B show that TREX+Bcl-xL cells are dependent on IL-2 for expansion and survival in cell culture. Overall fold expansion (Figure 5A) and cell viability (Figure 5B) were assessed for three TREX+Bcl-xL lines established as in Figure 3 from two different donors grown in the presence of increasing amounts of recombinant human interleukin 2 (IL-2) for 6 days. [Figure 6][Figures 6A-6K] Figures 6A-6K show TREX+Bcl-xL cells phenotypically similar to normal primary human CD8+ T cells. TREX+Bcl-xL cells were stained with fixable viability dyes and a panel of antibodies against CD3, CD4, CD8, CD28, CD45RO, CCR7, PD1 and TIGIT. TREX+Bcl-xL lines showed expression of CD3 (Figure 6A) and contained a high frequency of CD8+ cells (Figure 6B). TREX+Bcl-xL lines displayed donor or cell line specific attributes, as exemplified by expression of markers such as PD1 and TIGIT (Figure 6C), CD28 (Figure 6D) and CCR7 and CD45RO (Figure 6E), regardless of Bcl-xL overexpression (GFP+ and GFP- cells). TREX+Bcl-xL lines show expression of CCR2 (Figure 6F), CCR5 (Figure 6G), and CXCR3 (Figure 6J). Expression of CCR6 (Figure 6H) was heterogeneous, while expression of CCR7 (Figure 6I) and CXCR5 (Figure 6K) was low to absent. [Figure 7] [Figures 7A-7F] Figures 7A-7F show 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 co-cultures 72 hours after addition of effector cells and T cell engagers and analyzed for the presence of interferon gamma (IFN-γ) (Figure 7C), IL-2 (Figure 7D), tumor necrosis factor alpha (TNF-α) (Figure 7E), and granzyme B (Figure 7F). [Figure 8] [Figures 8A-8G] Figures 8A-8G show that TREX+Bcl-xL cells can generate functional CAR-TREX+Bcl-xL cells. Surface CAR expression was assessed 22 days after transduction using flow cytometry (Figure 8A). Percent cell lysis was calculated at 12 hours (Figure 8B) and 24 hours (Figure 8C) after the addition of effector cells. CAR-TREX activity was assessed relative to the activity of CAR-T cells and CAR-CD8+T cells. Supernatants were collected from co-cultures 72 hours after the addition of effector cells and analyzed for the presence of IFN-γ (Figure 8D), IL-2 (Figure 8E), TNF-α (Figure 8F) and granzyme B (Figure 8G). [Figure 9] [Figures 9A-9C] Figures 9A-9C show that TREX cells home to similar locations as primary CD8+ T cells and respond to IL-2 in vivo. [Figure 10] [Figures 10A-10B] Figures 10A-10B show that CAR-TREX cells respond to IL-2 and IL-15 in vivo. [Figure 11] [Figures 11A-11D] Figures 11A-11D show that CAR-TREX cells target solid tumors in vivo. [Figure 12] Figure 12 shows that reproducible REX editing results in enhanced in vitro expansion compared to unmodified same-donor CD8+ T cells. The fold increase of TREX cells or same-donor primary (unedited) CD8+ T cells was followed over time in four additional healthy donors. [Figure 13] [Figures 13A-13B] Figures 13A and 13B show that CAR-TREX cells target BCMA+ tumor cells similarly to unmodified CAR-T cells. [Figure 14] Figure 14 shows that after CAR assembly, anti-BCMA-TREX and anti-HER2-TREX cells produced lower levels of inflammatory cytokines than anti-BCMA-CAR-T cells and anti-HER2-CAR-T cells. [Figure 15] Figure 15 shows that CAR-TREX cells target BCMA+ tumor cells, persist in a serial kill assay, and respond to IL-2. [Figure 16] FIG. 16 shows that the TREX cell phenotype can be generated using different combinations of editing. [Figure 17] FIG. 17 shows that TREX cells are edited at the expected locus. [Figure 18] [Figures 18A-18C] Figures 18A, 18B and 18C show that TREX cells demonstrate enrichment for cell cycle-related gene signatures. [Figure 19]FIG. 19 shows that TREX cells are dependent on IL-2 for survival and proliferation. [Figure 20] [Figures 20A-20B] Figures 20A and 20B show that CAR-TREX cells target HER2hi tumor cells similarly to unmodified CAR-T cells, which produce less cytokines overall. [Figure 21] FIG. 21 shows that REX editing enhances the proliferative potential of CD4+TREX cells. [Figure 22] FIG. 22 shows that γδTREX cells can be generated using REX editing. [Figure 23] FIG. 23 shows that γδTREX cells are active in an in vitro T cell engager (TCE) assay. [Figure 24] FIG. 24 shows that γδ TREX cells can be generated from multiple γδ T cell subsets and diversity is maintained after CAR transduction. [Diagram 25] [Figure 25A-25B] Figures 25A and 25B show that γδ-TREX cells target BCMA+ tumor cells similarly to unmodified CAR-T cells. [Figure 26] FIG. 26 shows that REX editing in NK cells supports the NKREX cell phenotype. [Figure 27] FIG. 27 shows that NKREX cells are dependent on cytokines for proliferation and survival. [Figure 28] FIG. 28 shows that NKREX cells maintain CAR expression over time. [Figure 29] [Figures 29A-29C] Figures 29A-29C show that NKREX cells are cytotoxic in vitro and that CAR expression can further enhance efficacy. [Diagram 30] FIG. 30 shows that TREX cells are sensitive to T cell depleting agents and chemotherapy. [Diagram 31] FIG. 31 shows that B2MKO TREX cells are susceptible to NK cell-mediated depletion, which can be modulated using an anti-CD38 antibody. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present disclosure relates to methods, cells and compositions for preparing cell populations and compositions for adoptive cell therapy. In particular, methods are provided herein for the expansion and propagation of primary immune cells, including T cell populations.

[0021] As utilized in accordance with this disclosure, unless otherwise indicated, all scientific and technical 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.

[0022] As used herein, the terms "comprise" and "include" and variations thereof (e.g., "comprises," "comprising," and "includes" and "including") will be 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. Any of the terms "comprising," "consisting essentially of," and "consisting of" may be substituted with either of the other two terms while retaining their ordinary meaning.

[0023] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0024] The percentages disclosed herein can vary from the disclosed values ​​by ±10, 20, or 30% amounts and remain within the contemplated range of the disclosure.

[0025] Unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values ​​herein expressed as ranges can be considered to be any specific value or subrange within the ranges set forth in various embodiments of the present disclosure, to the tenth of the unit of the lower limit of the range, unless otherwise clearly indicated from the content.

[0026] As used herein, ranges and amounts may be expressed as "about" a particular value or range. The term "about" includes the exact amount. For example, "about 5%" means "about 5%" and also means "5%". The term "about" may 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".

[0027] As used herein, the terms "or" and "and / or" may describe multiple components in combination or mutually exclusive. 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". Replicative senescence resistance (RRS) refers to primary immune cells that are resistant to replicative senescence (RS), which leads to a finite number of population doublings. As a result, the population of primary immune cells described herein advantageously has an increased proliferative capacity.

[0028]

[0004] In one aspect, the disclosure herein provides a method of generating a population of replicative senescence-resistant (RRS) primary immune cells, the method comprising: i) introducing one or more gene edits into primary immune cells; and ii) culturing the primary immune cells in medium; which culture induces proliferation of the primary immune cells to give rise to the population of replicative senescence-resistant (RRS) primary immune cells.

[0029] In one aspect, the disclosure herein provides a method of generating a population of replicative senescence-resistant (RRS) primary immune cells, the method comprising: i) introducing a transgene encoding B-cell lymphoma (Bcl-xL) into the primary immune cells; ii) inhibiting expression of one or more endogenous regulators selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B) and S-methyl-5'-thioadenosine phosphorylase (MTAP) in the primary immune cells; and iii) culturing the primary immune cells in a medium; the culturing induces proliferation of the primary immune cells to generate a population of replicative senescence-resistant (RRS) primary immune cells. In some aspects, the method comprises inhibiting expression of one or more endogenous immune-related genes in the primary immune cells. In certain aspects, the endogenous immune-related genes are beta-2 microglobulin (B2M) or T-cell receptor alpha constant (TRAC). In some embodiments, the methods comprise inhibiting expression of CD38.

[0030] In one aspect, the disclosure herein provides a method of generating a population of replicative senescence-resistant (RRS) primary immune cells, comprising: i) inhibiting expression of one or more endogenous regulators selected from cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B) and S-methyl-5'-thioadenosine phosphorylase (MTAP) in primary immune cells; ii) culturing the primary immune cells in a medium; the culturing induces proliferation of the primary immune cells to generate a population of replicative senescence-resistant (RRS) primary immune cells. In some aspects, the method comprises inhibiting expression of one or more endogenous immune-related genes in the primary immune cells. In certain aspects, the endogenous immune-related genes are beta-2 microglobulin (B2M) or T cell receptor alpha constant (TRAC). In some aspects, the method comprises inhibiting expression of CD38.

[0031] Genetic editing refers to changes to the genetic material of primary immune cells. Genetic editing includes adding, removing, or changing 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 one or more gene edits includes introducing one or more transgenes encoding anti-apoptotic factors or virus-derived factors into the primary immune cells.

[0032] The term "primary immune cells" may refer to any cell involved in the primary immune response, such as T cells, B-cells, and NK cells, neutrophils, and monocytes / macrophages / dendritic cells. In some embodiments, primary immune cells may include total T cells, CD4-positive T cells, CD8-positive 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.

[0033] The term "transgene" refers to any nucleic acid sequence that is introduced into a cell by experimental manipulation. A transgene may be an "endogenous DNA sequence" or a "heterologous DNA sequence." A transgene may be isolated and obtained in suitable quantities using one or more methods well known in the art. These methods, and others 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)).

[0034] Transgenes may be incorporated into "transgene constructs" that contain the gene of interest together with other regulatory DNA sequences required for either temporal or cell-specific or enhanced expression of the transgene of interest.

[0035] The transgene can be introduced into the cell by any suitable method or technique known in the art. In certain embodiments, the transgene is introduced using plasmid-based DNA transposon, lentivirus platform or CRISPR-mediated site-specific integration. The transgene expression in the cell can be constitutive or inducible.

[0036] 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 of cells, particularly cells that have been stressed, have received a signal to undergo apoptosis, or are undergoing abnormal cell proliferation. In certain embodiments, the anti-apoptotic factor is B-cell large lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

[0037] In certain embodiments, the transgene encodes a virus-derived factor. "Virus-derived factor" refers to both native viral peptides, polypeptides, or proteins, as well as peptides, polypeptides, or proteins that exhibit a degree of sequence identity and / or similarity to viral proteins and / or maintain one or more structural, mechanical, or antigenic qualities of the viral proteins. In certain embodiments, the virus-derived factor is derived from Saimiriine gammaherpesvirus 2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified Herpesvirus Ateles-Epstein-Barr virus Tio-LMP1.

[0038] In other embodiments, the transgene encodes a protein that is associated with an activation signal in a cell.

[0039] In some aspects, the method of the present disclosure further comprises inhibiting the expression of one or more endogenous regulators in primary immune cells such that the activity of the endogenous regulator 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. An endogenous regulator 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, TALENs, zinc finger nucleases, meganucleases, neutralizing antibodies, small molecule inhibitors, chemical inhibitors that block downstream signaling pathways, and the like. Inhibition of an endogenous regulator can be complete inhibition, partial inhibition, downregulation of gene expression or reduction of the activity of a 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 certain 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).

[0040] The term "endogenous" refers to originating within or having a molecular structure in a cell, tissue, or organism, or part of a cell, tissue, or organism.

[0041] In some aspects, the methods of the present disclosure further comprise inhibiting expression of one or more endogenous immune-related genes in primary immune cells such that the activity of the immune-related genes is eliminated or reduced. As used herein, the term "immune-related genes" refers to genes that encode proteins involved in eliciting an immune response. In certain aspects, immune-related genes encode proteins that are involved in host-versus-graft (HvG) and graft-versus-host (GvH) alloimmune responses. Immune-related genes can be downregulated or inhibited 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, and the like. Inhibition of endogenous immune-related genes can be complete inhibition, partial inhibition, downregulation of gene expression, or reduction of the activity of a factor. In some embodiments, endogenous immune-related gene 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%). Immune-related genes include genes encoding proteins involved in eliciting an immune response. Immune-related genes may 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 (TRAC).In certain aspects, the one or more endogenous immune-related genes are 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), TIM3, or TIGIT genes.

[0042] In a further aspect, the methods disclosed herein include inhibiting cluster of differentiation 38 (CD38) expression in primary immune cells such that the activity of CD38 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 the 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 aspects, 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%).

[0043] In a further aspect, the method disclosed herein comprises inhibiting the expression of phosphatase and tensin homolog (PTEN) in primary immune cells such that the activity of PTEN is eliminated or reduced. PTEN 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 PTEN 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 aspects, PTEN activity or gene expression is decreased by between 1% and 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%).

[0044] "T REX The term "renewably expandable T cells" refers to T cells that can be regeneratively expanded, for example, using the techniques and genetic modifications provided herein. More specifically, T REX The cell refers to a cell 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.

[0045] In some embodiments, the inhibition of expression of one or more endogenous regulators occurs after the introduction of 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 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 the inhibition of one or more endogenous regulators occurs. In further embodiments, the inhibition of expression of PTEN occurs after the introduction of one or more transgenes into the cells. In some embodiments, the method comprises the successive 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, at least 20 days; ii) inhibiting one or more endogenous regulators 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, at least 20 days; and iii) inhibiting PTEN expression.

[0046] The primary immune cells are cultured under appropriate conditions to promote proliferation and expansion. In vitro expansion using a culture step activates and induces proliferation of the primary immune cells to obtain an expanded population containing primary immune cells in sufficient numbers for therapeutic use.

[0047] The methods disclosed herein are carried out ex vivo, which means that the methods are carried out outside of a living organism. Treating immune cells ex vivo means exposing the cells to certain biological molecules in vitro, preferably under sterile conditions. In some cases, the ex vivo method further comprises culturing the immune cells that have been isolated from a human before administering them back to the same or a different human subject.

[0048] The primary immune cells, including the expanded populations and / or modified T cells of this disclosure, may include total T cells, CD4-positive T cells, CD8-positive 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 divided into cells that express CD4 on their surface (also referred to as CD4-positive cells) and cells that express CD8 on their surface (also referred to as CD8-positive cells). T cells suitable for use with the methods provided herein are mononuclear lymphocytes from the bone marrow (BM), peripheral blood (PB), or umbilical cord blood (CB) of a human donor. These cells may be harvested directly from the BM, PB, or CB, or following 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 an allogeneic or autologous donor. Those skilled in the art will recognize that there are many established protocols for isolating peripheral blood mononuclear cells (PBMCs) from peripheral blood. Isolation of PBMCs may be facilitated by density gradient separation protocols, usually using Ficoll®-Hypaque or Histopaque® density gradient centrifugation techniques for separation of lymphocytes from other elements in the blood. Preferably, PBMC isolation is performed under sterile conditions. Isolation of PBMCs may also use negative selection kits. Alternatively, cell elutriation methods may be used to separate the mononuclear cell population. In some embodiments, the primary immune cells are human.

[0049] In some cases, the method of the disclosure further comprises introducing a genetically modified antigen receptor or chimeric antigen receptor into the activated T cells, thereby generating an expanded population comprising T cells expressing the genetically modified antigen receptor 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 onto immune effector cells. In general, 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 cells. In one embodiment, a polynucleotide encoding the chimeric antigen receptor is introduced into the primary cells. In one embodiment, a nucleic acid vector encoding the chimeric antigen receptor or the genetically modified receptor is introduced into the 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)), B-cell maturation antigen (BCMA). In certain embodiments, the CAR can bind to any target for use in immunotherapy.

[0050] Design of the CAR construct: The CAR construct of the present disclosure may have several components, many of which may be selected based on the desired or sophisticated function of the resulting CAR construct. In addition to the antigen binding domain, the CAR construct may have a spacer domain, a hinge domain, a signal peptide domain, a transmembrane domain, and one or more costimulatory domains. The selection of one component over another (i.e., the selection of a particular costimulatory domain from one receptor versus a costimulatory domain from a different receptor) may affect clinical efficacy and safety profile.

[0051] Antigen-binding domain: The antigen-binding domain 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 directly linked together or linked together via a flexible linker (e.g., G4S repeats with 1, 2, 3 or more repeats).

[0052] Spacer domain: CAR constructs may have a spacer domain to provide conformational freedom to facilitate binding to target antigens on target cells. The optimal length of the spacer domain may 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 epitopes. In addition to facilitating binding of the CAR to the target antigen, achieving optimal distance between the CAR cell and the cancer cell may also help sterically block large inhibitory molecules from the immunological synapse formed between the CAR cell and the target cancer cell. CARs may have long, intermediate or shorter spacers. Long spacers may include the CH2CH3 domains (about 220 amino acids) of immunoglobulin G1 (IgG1) or IgG4 (either native or with modifications common to therapeutic antibodies such as the S228P mutation), while the CH3 region may be used to construct the intermediate spacer (about 120 amino acids) by itself. 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 from any IgG isotype and may or may not contain mutations common to therapeutic antibodies, such as the S228P mutation described above.

[0053] 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 may be used alone or together with a spacer sequence. The terms "hinge" and "spacer" are often used interchangeably, for example, an IgG4 sequence may be considered both a "hinge" and a "spacer" sequence (i.e., a hinge / spacer sequence).

[0054] Signal peptide: The CAR construct may further comprise a sequence containing a signal peptide. The signal peptide serves to prompt the cell to translocate the CAR to the cell membrane. Examples include IgG1 heavy chain signal polypeptide, Ig kappa or lambda light chain signal peptide, granulocyte macrophage colony stimulating factor receptor 2 (GM-CSFR2 or CSFR2) signal peptide, CD8a signal polypeptide, or CD33 signal peptide.

[0055] Transmembrane domain: The CAR construct may further comprise a sequence that comprises 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 studied as extensively as other aspects of the CAR construct, but may potentially affect CAR expression and binding to endogenous membrane proteins. The transmembrane domain may be derived, for example, from CD4, CD8α, or CD28.

[0056] 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 sequences from one or more of, for example, 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 cells. For example, costimulation of CD28 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 modified with CARs having a 4-1BB costimulatory domain tend to proliferate and persist longer in vivo, have increased oxidative metabolism, are less susceptible to exhaustion, and have an increased capacity to generate central memory T cells.

[0057] In certain embodiments, the methods disclosed herein impair the initial stimulation of primary immune cells to ensure that the cells are in cycle before introducing one or more gene edits into the cells. In other embodiments, the methods disclosed herein impair the later stimulation (also called "restimulation") of primary immune cells. Once the primary immune cells have exited the cell cycle, the cells are restimulated to allow the cells to re-enter the cell cycle (i.e., proliferate).

[0058] In certain embodiments, the methods disclosed herein further comprise stimulating the primary immune cells prior to the introduction of one or more gene edits into the primary immune cells. In certain embodiments, the primary immune cells are 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 prior to the introduction of one or more gene edits into the primary immune cells. Thus, in certain embodiments, a method of generating a population of replicative senescence-resistant (RRS) primary immune cells is provided herein, the method comprising: i) stimulating the primary immune cells; ii) introducing one or more gene edits into the primary immune cells; iii) culturing the primary immune cells in a medium; the culturing induces proliferation of the primary immune cells to produce a population of replicative senescence-resistant (RRS) primary immune cells.

[0059] In certain embodiments, the methods disclosed herein further comprise stimulating the primary immune cells after the introduction of one or more gene edits into the primary immune cells. In certain embodiments, the primary immune cells are 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 primary immune cells. Thus, in certain embodiments, a method of generating a population of replicative senescence-resistant (RRS) primary immune cells is provided herein, the method comprising: i) introducing one or more gene edits into the primary immune cells; ii) culturing the primary immune cells in a medium; iii) stimulating the primary immune cells; iv) culturing the primary immune cells in a medium; the culturing induces proliferation of the primary immune cells to generate a population of replicative senescence-resistant (RRS) primary immune cells. In further embodiments, 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. Thus, in certain aspects, provided herein is a method of generating a population of replicative senescence-resistant (RRS) primary immune cells, the method comprising: i) introducing one or more gene edits into a primary immune cell; ii) culturing the primary immune cell in medium; iii) stimulating the primary immune cell; iv) culturing the primary immune cell in medium; v) restimulating the primary immune cell; and vi) culturing the primary immune cell in medium, which induces proliferation of the primary immune cell to generate a population of replicative senescence-resistant (RRS) primary immune cells. Any suitable stimulus known in the art may be used to stimulate the immune cells.

[0060] In certain embodiments, primary immune cells exhibit at least about a 50-fold increase, at least about a 500-fold increase, at least about a 5000-fold increase, at least about a 250,000-fold increase, at least about a 500,000-fold increase, at least about a 10 6 A fold increase of at least about 10 7A fold increase of at least about 10 8 A fold increase of at least about 10 9 A fold increase or at least about 10 10 A fold increase in the number of primary immune cells is achieved. In certain embodiments, the expanded population of primary immune cells is replicative senescence resistant. Furthermore, these cells are not functionally exhausted after long-term expansion and can be directed to carry out cytotoxic functions through engagement of their TCRs by T cell engager antibodies or through engagement of chimeric antigen receptors (CARs) (or through natural or transgenic TCRs).

[0061] In certain embodiments, primary immune cells are cultured in media that includes supportive cytokines but does not include 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 in the absence of extensive T cell restimulation or feeder cells advantageously obviates the challenges of scaling up the methods and generating dysfunctional populations of immune cells.

[0062] The methods disclosed herein advantageously provide human CD8 T cells that have the capacity to expand for substantial periods of time in the absence of restimulation through their T cell receptor (TCR), increasing millions of fold in long-term culture. + T cells, human CD4 + Expanded populations of primary immune cells are provided, including T cells, human regulatory T cells, human gamma-delta T cells, or human natural killer T cells. In certain embodiments, the populations of primary immune cells are 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.

[0063] In a further aspect, provided herein is a modified T cell that expresses a transgene encoding cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B) and / or large B-cell lymphoma that does not express S-methyl-5'-thioadenosine phosphorylase (MTAP) (Bcl-XL).

[0064] In a further aspect, provided herein are modified 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).

[0065] In a further aspect, provided herein is an engineered T cell that expresses a transgene encoding cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), S-methyl-5'-thioadenosine phosphorylase (MTAP), and / or large B-cell lymphoma not expressing phosphatase and tensin homolog (PTEN) (Bcl-XL).

[0066] In certain aspects, modified T cells as disclosed herein do not express one or more endogenous immune-related genes in primary immune cells, hi some aspects, the endogenous immune-related genes are beta-2 microglobulin (B2M) or T cell receptor alpha constant (TRAC).

[0067] In certain aspects, modified T cells as disclosed herein do not express cluster of differentiation 38 (CD38).

[0068] In a further aspect, the disclosure herein provides modified 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 (TRAC), cluster of differentiation 38 (CD38), and / or phosphatase and tensin homolog (PTEN).

[0069] In certain embodiments, the modified T cells as disclosed 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)).

[0070] In certain aspects, the modified T cells as 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.

[0071] In some embodiments, the modified T cells are replicative senescence resistant (RRS). In some embodiments, the modified T cells are CD8 + In some embodiments, the modified T cells are CD4 + T cells. In some embodiments, the modified T cells are human.

[0072] The expanded T cell populations disclosed herein are useful for cellular immunotherapy, including but not limited to T cell therapy, adoptive cellular therapy (ACT), and CAR T cell therapy.

[0073] The expanded populations of T cells 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.

[0074] As used herein, the term "treatment" or "treat" refers to both therapeutic treatment and prophylactic or preventative measures. Those 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, those in need of treatment include subjects with tumors as well as subjects prone to have tumors or subjects in which tumors are to be prevented. In certain aspects, the methods, compositions, and combinations disclosed herein can be used for the treatment of tumors. In other aspects, the treatment of tumors includes inhibiting tumor growth, promoting tumor reduction, or both inhibiting tumor growth and promoting tumor reduction.

[0075] In some cases, the T cells obtained by the methods provided herein can be administered as a pharmaceutical composition comprising a therapeutically effective amount of the T cells as a therapeutic agent (i.e., for therapeutic applications).

[0076] The term "pharmaceutical composition" or "therapeutic composition," as used herein, refers to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a subject. In some aspects, the present disclosure provides a pharmaceutical composition comprising a pharma- ceutical acceptable carrier and a therapeutically effective amount of at least one immune cell of the present disclosure.

[0077] The term "pharmacologically acceptable carrier" or "physiologically acceptable carrier" as used herein refers to one or more formulation materials suitable for achieving or enhancing delivery of one or more immune cells of the present disclosure.

[0078] The term "subject" is intended to include human and non-human animals, particularly mammals. In certain embodiments, the subject is a human patient.

[0079] 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, by inhalation, and implantation.

[0080] Without limiting the disclosure, several aspects of the disclosure are described herein for purposes of illustration. EXAMPLES

[0081] The following examples illustrate specific embodiments of the present disclosure and various uses thereof, they are set forth for illustrative purposes only and should not be construed as limiting the scope of the disclosure in any way.

[0082] Example 1: Overexpression of anti-apoptotic or virus-derived factors can provide a selective survival advantage to gene-transferred T cells in long-term culture. Transposon frequency was assessed in T cell subsets over a period of 66-137 days using flow cytometry. Total primary human T cells were isolated from the blood of healthy donors, activated with Dynabeads (human T-activators CD3 / CD28) for 72 hours, and then transfected with plasmids containing transposons encoding fluorescent reporters plus anti-apoptotic factors, virus-derived factors, mutant cytokine receptors, mutant signaling molecules, and / or mutant cell cycle regulatory molecules. Cells were simultaneously transfected with mRNA encoding the transposase to allow chromosomal integration of the transposase. In total, 52 different transposon constructs were processed across the 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 promote increased T cell survival include CD8 + and CD4 + As demonstrated in T cells, these are expected to be enriched in their starting frequency within the total T cell pool (Figure 1, Figure 2). From these screens, we found that the anti-apoptotic factor, large B cell lymphoma (Bcl-xL), was a key regulator of CD4 T cells that were driven through multiple rounds of proliferation over an extended period of in vitro culture. + and CD8 + We found that 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).

[0083] While 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) demonstrated enhanced viability in long-term culture with repeated stimulation through their TCR compared with non-transfected control cells in the same wells, these cells did not show a large and sustained boost in their proliferative potential. These data suggest that T REX It is suggested that further editing may be required to confer the desired phenotype of the cell.

[0084] Example 2: Loss of expression of CDKN2A, CDKN2B and MTAP substantially increases the proliferative potential of primary human T cells in long-term culture. Patient-derived leukemia cell lines have been used for many years in laboratories to perform various cellular assays. The transformed nature of these cells can be mapped in a group of mutations that are also frequently found in T-cell acute lymphoblastic leukemia (T-ALL) patients (Table 1). Mutations in T-ALL patients can be divided into several large 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 modification of pleiotropic factors such as transcription factors, epigenetic regulators, and other cellular mechanisms.

[0085] [Table 1]

[0086] As mentioned above, in addition to providing survival factors such as Bcl-XL, it may be necessary to modify T cell expression of the panel of genes mentioned above to generate the desired phenotype (Table 1). Thus, total CD8 + T cells were isolated from the blood of healthy donors and activated using αCD3 / αCD28 Dynabeads, and after 72 hours, purified CD8 +These cells were then expanded for 17 days before reactivation with αCD3 / αCD28 Dynabeads and subsequent loss of expression of factors identified from leukemic T cell lines and T-ALL patients. REX One of the main features we wanted to engineer into the cells was therefore the effect of truncating the expression of molecules from the "cell cycle arrest" bin: cyclin-dependent kinase inhibitor 2A (CDKN2A) and CDKN2B, which were tested in these cells (Figure 3A). S-methyl-5'-thioadenosine phosphorylase (MTAP) is chromosomally adjacent to CDKN2A and CDKN2B and is also frequently defective in patients lacking CDKN2A and CDKN2B. 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 are henceforth referred to as "T REX +Bcl-xL” cells (Table 2), and CDKN2A / CDKN2B / MTAP-edited (without Bcl-XL) cells were called “T REX " cells.

[0087] [Table 2]

[0088] Bcl-xL-editing, Bcl-xL and CDKN2A / CDKN2B-editing and T REX +Bcl-xL cells were maintained in culture for nearly 100 days without further stimulation through their TCR, and the overall fold increase of each population was assessed (Figure 3A). Approximately 31 days after introduction of these edits, T REX The proliferation of +Bcl-xL cells was distinct from other populations and was associated with T cell proliferation in the absence of further TCR stimulation. REX +Bcl-xL cell expansion was sustained, achieving a >400,000-fold increase during this period. Meanwhile, Bcl-xL-edited and Bcl-xL / CDKN2A / CDKN2B-edited CD8 +T cells achieved lower levels of expansion at this time point, between 73 and 286 fold (Figure 3A, Table 3). Furthermore, even when unedited or Bcl-xL-edited cells were repeatedly restimulated through their TCR with αCD3 / αCD28 Dynabeads to drive proliferation, they did not increase the T REX +Bcl-xL cells achieved only a low level of overall fold increase, far below that of +Bcl-xL cells (Fig. 1A, Table 3).

[0089] [Table 3]

[0090] T REX +Bcl-xL cells were compared with control CD8 + While these cells showed substantially enhanced proliferative capacity compared to T cells, we aim to determine whether these edits could confer a similar phenotype in other donors by ablating the expression of signal suppressors that are frequently mutated in T-ALL patients. REX Further experiments were performed to test whether it was possible to further promote the +Bcl-xL phenotype (Table 1). The phosphatase and tensin homolog (PTEN) locus shows a high frequency of loss-of-function mutations in patient-derived leukemia cell lines and T-ALL patients and is known to negatively regulate cell cycle progression (Table 1). T REX +Bcl-xL cells were generated, and approximately 2 weeks after "triplex" editing, these T REX PTEN expression was ablated in one of the +Bcl-xL cell lines (40B32) (Figure 3B). REX Both sets of cells (CDKN2A / CDKN2B / MTAP deficient) exhibited substantial proliferative capacity in the absence of further TCR stimulation, achieving overall fold increases of >3.7e8 and >1.8e7 by day 118 of culture. Moreover, consistent with its established role in negatively regulating cell cycle progression through control of AKT signaling, T REX Loss of PTEN expression in +Bcl-xL cells was observed in donor B-2TREX It further enhanced the proliferative potential of +Bcl-xL cells, allowing these cells to achieve an overall fold increase of >2.0e8 by day 118 of culture (Figure 3B). The additional effect of loss of PTEN expression took 49 days to emerge, reflecting either a low initial editing efficiency or a later competitive advantage of this editing after cells had expanded >3e6-fold.

[0091] T with intact PTEN expression REX +Bcl-xL cells dramatically increased in the absence of further TCR stimulation, whereas their proliferation was ultimately suppressed by T REX +Bcl-xL cells (Fig. 3B). REX Even +Bcl-xL cells eventually showed a reduced proliferation rate (Figure 3B). REX To determine whether restimulation of +Bcl-xL cells could be a viable alternative or complementary approach to the loss of PTEN expression, we tested whether αCD3 or αCD3 / αCD28 Dynabeads could reinstate cells into the cell cycle (Figure 4). REX + Bcl-xL or PTEN deletion T REX +Bcl-xL cells were left untreated or restimulated with Dynabeads as above, the beads were removed (debeaded), and the overall fold increase of each population was followed (Figure 4). REX +Bcl-xL and PTEN deletion T REX +Bcl-xL line was enhanced in proliferation ability.

[0092] Example 3: REX +Bcl-xL cells resemble primary human T cells with respect to cytokine dependency and cell phenotype. Primary T cells depend on cytokines such as IL-2 for survival and proliferation in vitro and in vivo, but some leukemia cell lines proliferate independently of IL-2. REX +Bcl-xL cells and PTEN deletion T REXWe generated +Bcl-xL+ cells from mice. We investigated whether these cells remained similar to normal primary human T cells in terms of cytokine dependency by tracking cell proliferation and survival over a range of IL-2 concentrations for 6 days in culture (Figure 5). Consistent with normal T cells, T REX +Bcl-xL cells and PTEN deletion T REX +Bcl-xL cells were highly dependent on IL-2 for both proliferation and survival.

[0093] T REX +Bcl-xL cells and PTEN deletion T REX We investigated whether +Bcl-xL cells maintain a phenotype similar to normal T cells after modification and expansion in in vitro culture, or whether these conditions are related to T REX We also tested whether +Bcl-xL+ cells could be driven to 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 displayed a differentiation phenotype defined by surface expression of CD45RO and CCR7 that was donor-dependent (Figure 6E). These data suggest that despite substantial expansion and increased duration of in vitro culture, T REX + Bcl-xL cells resemble normal T cells and do not display a surface phenotype associated with a dysfunctional state.

[0094] Chemokine receptors are important for the trafficking of immune cells to sites of inflammation. Therefore, flow cytometry was used to detect T 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 (Figures 6F-K).REX +Bcl-xL strain and PTEN deletion 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, while expression of CCR7 (Fig. 6I) and CXCR5 (Fig. 6K) was low or absent. Thus, T REX +Bcl-xL cells and PTEN deletion T REX +Bcl-xL cells maintain expression of important chemokine receptors that enable them to be trafficked to sites of inflammation.

[0095] Example 4: T REX +Bcl-xL cells are cytotoxic. T REX +Bcl-xL lines were confirmed to be similar to normal primary human T cells, REX We determined whether +Bcl-xL cells maintained potent cytotoxic function after long-term culture and expansion. REX To quantify the cytotoxic function of +Bcl-xL cells, a T cell engager was used in the presence of target tumor cells and the impedance-based xCELLigence platform (Figure 7). REX +Bcl-xL lines inhibited the expression of unmodified primary total T cells and unmodified primary CD8 + These co-cultures demonstrated a tumor cell targeting ability comparable to that of 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 lower levels of these cytokines compared to unmodified primary T cells, despite a similar ability to lyse target cells in an antigen-dependent manner. These data indicate that T REX It is shown that +Bcl-xL cells are not functionally exhausted and maintain their cytotoxic capacity.

[0096] Example 5: T REX +Bcl-xL cells are functional CAR-T REX The cells may be generated. T REX In order for +Bcl-xL cells to be developed into a promising cell therapy, these cells must be capable of expressing targeting molecules, such as chimeric antigen receptors (CARs), to direct their cytotoxic function. REX A cell line of +Bcl-xL cells was 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 line is a normal primary total T cell and normal primary CD8 + We found that they expressed the GPC3 CAR well, at levels similar to T cells (Figure 8A).

[0097] By performing the impedance-based xCELLigence assay with target tumor cells of various antigen expression levels, REX The cytotoxic function of +Bcl-xL cells against CAR was evaluated: OE21 (antigen negative), HuH-7 (antigen-medium) and Hep3B (antigen-high) (Figures 8B and 8C). REX +Bcl-xL cells were compared with normal CAR-T cells and normal CAR-CD8 + CAR-T cells 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). Seventy-two hours after addition of T cells, supernatants were collected from these co-cultures and subsequently analyzed for secretion of IFN-γ, IL-2, TNF-α, and granzyme B (Figures 8D-8G). Consistent with their potent cytotoxic function, CAR-T cells 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). Seventy-two hours after addition of T cells, supernatants were collected from these co-cultures and subsequently analyzed for secretion of IFN-γ, IL-2, TNF-α, and granzyme B (Figures 8D-8G). REX +Bcl-xL cells were compared with normal CAR-T cells and normal CAR-CD8 + The effector cytokine secretion capacity of CAR-T cells was comparable to that of T cells (Figures 8D-8G). However, in general, IFN-γ and TNF-α levels were significantly lower in CAR-T cells than in T cells (Figures 8C-8D). REX+Bcl-xL cells.

[0098] These data suggest that even after significant in vitro expansion, T REX +Bcl-xL and PTEN deletion T REX +Bcl-xL cells are confirmed to be capable of expressing CAR and carrying out cytotoxic function against the CAR in an antigen-dependent manner.

[0099] Example 6: T REX Cells are primary CD8 + They home to a location similar to T cells and are responsive to IL-2 in vivo. Because 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), T REX The cells were assessed for their ability to respond to different human cytokines in vivo. Briefly, primary human CD8 + T cells or 278-day-old T REX Cells were labeled with a luciferase reporter and 3E6 luciferase-expressing cells were injected into NSG mice with or without supplementation of 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, images at 216 hours revealed primary human CD8+ T cells and T REX Furthermore, mice supplemented with recombinant human IL-2 fusion protein showed similar localization of T REX It was clear that cell proliferation was promoted (Figure 9A, right). The ventral radiance was plotted over time (Figure 9B), and the T REXThe ability of the cells to respond to exogenously supplemented IL-2 in vivo was demonstrated. Ten days after adoptive cell transfer, mice were sacrificed and primary CD8+ T cells or T REX Their blood, spleen and bone marrow were assessed for the presence of T cells (Figure 9C). REX While CD8+ T cells were found in similar organs as primary CD8+ T cells (Figure 9C), they showed slower decay kinetics, and administration of recombinant human IL-2 fusion protein resulted in a large increase in T REX These data suggest that T REX It is suggested that the cells are directed to similar sites as primary CD8+ T cells and remain responsive to exogenous cytokine cues.

[0100] Example 7: CAR-T REX The cells respond to IL-2 and IL-15 in vivo. CAR-T targeting GPC3 REX +Bcl-xL cells were assessed for their ability to respond to various human cytokines in vivo. NSG, hIL-2 NOG, or hIL-15 NOG mice were inoculated with Hep3B tumor cells expressing GPC3. Once tumors were established, mice were either left untreated or treated with 10E6 GPC3-targeting CAR-T cells. REX +Bcl-xL cells. REX +Bcl-xL cells were 121 days old at the time of infusion. REX Mice were sacrificed 8 days after +Bcl-xL cell injection and weighed (FIG. 10A), with no discernible differences observed, indicating no toxicity. Tumors, blood, and spleens were harvested and CAR-T REX +Bcl-xL cells were analyzed (Figure 10B). REX +Bcl-xL cell numbers are enhanced, and CAR-T REXThis demonstrates that +Bcl-xL cells are responsive to exogenous cytokine cues in vivo. The augmentation profile was specific to the particular cytokine support provided (FIG. 10B).

[0101] Example 8: CAR-T REX The cells are targeted to solid tumors in vivo. CAR-T targeting GPC3 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 The cells (Figure 11A) were injected into mice. REX CAR-TREX cells or 2E6 CAR-T cells were injected into the mice, and tumor volumes were then measured and graphed over time (Figure 11B, left). CAR-TREX cells showed tumor growth inhibition and control of Hep3B tumors. Eighteen days after CAR-TREX cell transfer, mice were sacrificed and tumors, blood, and spleens were collected for further analysis (Figures 11B, 11C, and 11D). Intratumoral CAR-T REX We examined the cell phenotype (Figure 11B) and found that CAR-T REX The cell number was determined (Figure 11C). REX T cells were found in greatest numbers in mouse tumors, and these cells displayed an activated phenotype, actively secreting effector cytokines and degranulating (Figure 11D). REX The cells further exhibit enhanced proliferative capacity in vitro and can be multiplied millions of fold and maintained in culture for over 100 days without further stimulation through their TCR (Figure 12). + When the above target genes were simultaneously edited in T cells, these edits reproducibly conferred the REX phenotype across different donors (Figure 12).

[0102] Example 9: Further gene editing for clinical profiling REX Editing is T REX This results in enhanced proliferation of cell products while inhibiting T cell proliferation by the patient's immune system. REXFurther edits were made at the B2M and CD38 loci to delay rejection of the cell product. 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 also associates with non-classical MHC I-like molecules such as CD1, MR1, neonatal Fc receptor, and Qa-1. Despite being located outside the MHC locus, B2M is required for successful expression of classical and non-classical MHC I molecules on the surface of nucleated cells. CRISPR / Cas9 was used to engineer T REX By eliminating B2M expression in T cells, these cells are shielded from patient CD8+ T cells. Furthermore, the loss of B2M expression and the resulting T REX MHC-I expression by a cell product renders it susceptible to rejection by the patient's NK cells. REX The knockout of B2M was performed simultaneously with targeted knock-in of CAR (e.g. GPC3, HER2, BCMA) in cell products.

[0103] NK cells express high levels of CD38, and NK cells are depleted in patients with certain cancers, e.g., multiple myeloma patients, who are treated with anti-CD38 monoclonal antibodies such as daratumumab and isatuximab. To extend the persistence of this homogeneous cell population in patients, we used CRISPR / Cas9 to generate NK cells. REX CD38 was knocked out in T cells, and daratumumab or isatuximab was administered to T cells. REX It may be co-administered with the cells (see Figures 30 and 31).

[0104] homogeneous CD8 + Like T cell populations, T REX It is expected that T cells can target healthy cells of HLA-mismatched patients through their TCR, resulting in GvHD. To prevent the progression of this pathology, T cells are required to express T cells at the T cell receptor alpha constant (TRAC) locus, which encodes the TCR α chain. REX CRISPR / Cas9 editing of TRAC led to loss of expression of the TCR α chain and subsequently to T REXPrevents surface expression of TCR by cells.

[0105] [Table 4]

[0106] Example 10: Anti-BCMA-T REX Allogeneic cell therapy T REX We expressed a CAR targeting BCMA in cells (i.e., cells lacking CDKN2A / CDKN2B / MTAP) (Figure 13A). REX The genome of the cells was further edited to remove the expression of human leukocyte antigen (HLA) class I and αβ T cell receptor (TCR) by inactivation of the B2M and TRAC genes, respectively, to minimize host-versus-graft (HvG) and graft-versus-host (GvH) alloresponses, respectively. Additionally, we used CRISPR / Cas9 to confer resistance to anti-CD38-depleting monoclonal antibodies to anti-BCMA-T REX The CD38 gene was inactivated in IL-1 cells. Inactivation of these three genes reduced peripheral blood CD8 + The total in vitro expansion capacity of T cells was enhanced, and downstream cell populations were increased by 100% in unedited peripheral blood CD8 + These cells retain the characteristic proliferation properties of primary T cells (dependence on both anti-CD3 stimulation and IL-2 prior to TRAC inactivation for expansion / survival) but have a higher potential for expansion. REX The cells maintain their cytotoxic function but are not associated with mixed CD4 + and CD8 + It shows reduced cytokine release compared to conventional CAR-T cell preparations composed of T cell populations.

[0107] anti-BCMA-T REXEvaluation of the cells indicates that these cells appear to control BCMA-expressing tumors similar to primary anti-BCMA-CAR-T cells, while exhibiting a potentially improved safety profile in the form of reduced cytokine release and potentially reduced risk of CRS (Figure 13B). REX Cells and anti-BCMA-CAR-T cells were cultured. Tumor cell lysates were measured at different time points after the initiation of co-culture at various effector:target cell ratios (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).

[0108] Anti-BCMA-T with BCMA-expressing tumor cells REX Cells (82 days in culture) or anti-BCMA-CAR-T cells were cultured. Supernatants were collected 72 hours after the start of co-culture and assessed for levels of IFN-γ using the MSD kit (Figure 14, left). The data show that, despite similar control of tumor cells, anti-BCMA-T cells were more potent than co-cultured with anti-BCMA-CAR-T cells. REX These data show that IFN-γ levels are 90% lower in co-culture with CAR-T cells (83 days in culture). REX The cells are found to exhibit a cytokine secretion profile that may confer a lower risk of CRS than CAR-T cells.

[0109] anti-BCMA-T REX Cells (112 days in culture) were assessed for their persistence in a serial kill assay with or without IL-2 support. Briefly, anti-BCMA-T cells were cultured with JJN3 cells expressing BCMA at a 1:1 effector:target cell ratio. REX Anti-BCMA-CAR-T cells were serially cultured. Tumor cell control (% cytolysis), effector cell numbers and effector cytokine secretion were measured after each round of co-culture and graphed (Figure 15). REXThe cells sustained a comparable number of rounds in this serial kill assay as anti-BCMA-CAR-T cells, and the inclusion of IL-2 in the cell culture medium significantly outperformed the anti-BCMA-CAR-T cells. REX This further increased the number of rounds that anti-BCMA-CAR-T cells could control tumor cell proliferation. 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 duration in co-cultures with IL-2 in the medium (Figure 15, top row vs. bottom row). These data suggest that anti-BCMA-T REX It is noted that the cells demonstrate similar cytotoxicity to anti-BCMA-CAR-T cells in vitro and also show similar responsiveness 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.

[0110] Example 11: Anti-HER2-T REX Allogeneic cell therapy CAR-T REX To generate CAR-T cells, T REX HER2-targeting CAR was expressed in primary T cells (Figure 20A). REX Anti-HER2-CAR-T cells and anti-HER2-CAR-T cells were evaluated for their ability to target HER2-overexpressing OE21 cells at various effector:target cell ratios (Figure 20B, left). REX The cells revealed 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 start of co-culture and subsequently examined for the presence of effector cytokines (Figure 20B, right). As previously observed, despite comparable or improved tumor cell control, anti-HER2-T REXThe cells secreted lower levels of cytokines (IFN-γ, TNF-α, and IL-2) than anti-HER2-CAR-T cells, which suggests that CAR-T REX These results suggest that anti-BCMA-T cells may be less prone to causing CRS in patients. REX As shown above for HER2-expressing tumor cells and anti-HER2-T cells, the results were comparable to those of the supernatants from co-culture with anti-HER2-CAR-T cells. REX We also observed a decrease in IFN-γ secretion in the supernatants obtained from co-culture of the cells (Figure 14, right). These data again suggest that CAR-T REX The cells are found to exhibit a cytokine secretion profile that may confer a lower risk of CRS than CAR-T cells.

[0111] Example 12: T REX Cell phenotypes can be generated using different combinations of edits. Isolated CD8 from two donors (shown as G and H). + We assessed the requirement of overexpression of Bcl-xL and various REX target genes to confer the REX phenotype in T cells. Briefly, CD8+ T cells were negatively selected and then activated with αCD3 / αCD28 Dynabeads for 3 days. Some cells were transfected with Bcl-xL while others were cultured and various combinations of REX target genes were knocked out using CRISPR / Cas9 (Figure 16). Cell expansion was monitored and graphed over time. (CDKN2A and CDKN2A' reflect single vs multiple isoform targeting). While the three REX target genes yielded consistent phenotypes across donors, Bcl-xL was found to be dispensable for the REX phenotype (Figure 16, right).

[0112] Example 13: T REX The cells are edited at the targeted locus. Western blot analysis revealed that loss of expression of REX target genes was observed in T REX Cells and γδT REXThe cells were examined (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 unedited control cells. Furthermore, Sanger sequencing data indicates a high incidence of indels at these three loci in edited TREX cells (Figure 17, right).

[0113] Example 14: T REX The cells show enrichment for cell cycle-related gene signatures. T cells overexpressing Bcl-xL REX Cells and unedited control CD8 from the same donor + T cells were cultured over time. RNAseq analysis was performed on cell pellets generated at various points to identify gene signatures of Bcl-xL T REX As expected, TREX cells showed enrichment for gene signatures associated with the cell cycle, including E2F target genes and G2M checkpoint target genes (Figure 18A). REX The cells also showed higher levels of expression of MYC target genes, consistent with the observed proliferation rate of these cells (Figure 18B). REX The cells showed upregulated expression of multiple cell cycle-related genes (Figure 22C). These data confirm that the REX phenotype is associated with cell cycle progression and enhanced proliferation.

[0114] Example 15: T REX The cells are dependent on IL-2 for survival and proliferation. T with various doses of IL-2 for 12–14 days REX The cells were cultured. Cell proliferation was followed and graphed over time (Figure 19). Bcl-xL T REX As shown above for cells (see, e.g., FIG. 5), T REX T cells are highly dependent on IL-2 for proliferation and survival in vitro. REXT cells showed dose-dependent proliferation in response to IL-2; in the absence of IL-2, REX Cells showed a rapid decline in survival, with over 60% T REX Cells were eliminated within the first 4 days.

[0115] Example 17: REX editing is a novel method for the treatment of CD4+ T REX Supports cell proliferation. REX editing reproducibly results in CD8 + Conferring enhanced replicative senescence resistance in T cells. We determined the ability of abolishing the expression of REX target genes (CDKN2A, CDKN2B, and MTAP) to promote replicative senescence resistance in CD4+ T cells. + T cells were isolated and stimulated using αCD3 / αCD28 Dynabeads and then edited at these loci. + T REX Cells and unedited CD4 from the same donor + The proliferation of T cell controls was tracked over time and graphed (Figure 21). CD8 + As previously shown with T cells, CD4 + Targeting the REX gene in T cells reproducibly supported the proliferative potential of these cells and conferred on them resistance to replicative senescence.

[0116] Example 18: Using REX editing, γδT REX The cells may be produced. γδ T cells are another cytotoxic subset of T cells. Using γδ T cells from eight different donors, we demonstrated that REX editing in γδ T cells is a potent T cell cytotoxic agent. REX The ability of γδ T cells to confer a cellular phenotype was examined (Figure 22). γδ T cells were isolated and stimulated with αCD3 / αCD28 Dynabeads or αCD3 antibody, and then the REX locus was edited using CRISPR / Cas9. γδ T cells and γδ T REX Cell proliferation was monitored and graphed over time. REX editing reproducibly promoted replicative senescence resistance in γδT cells, and REX This led to the generation of a cellular phenotype.

[0117] γδT REX Having established that the cell lines exhibit enhanced replicative senescence resistance, γδT REX We determined whether the cells maintained their potent cytotoxic function after long-term culture and expansion. REX To quantify the cytotoxic function of the cells (Figure 23), T cell engagers were used in the presence of target tumor cells and the impedance-based xCELLigence platform. On days 79 and 88, unmodified primary CD8 + In the presence of T cell engagers as T cells, γδT REX The cell lines demonstrated comparable target tumor cell lysis capacity (Figure 23, top). Supernatants from these co-cultures were collected 72 hours after addition of effector cells and active T cell engager or control T cell engager molecules and analyzed for the presence of IFN-γ, IL-2 and TNF-α (Figure 23, bottom). REX Although the cell lines were similar in their ability to lyse target cells in an antigen-dependent manner, the levels of these cytokines produced were significantly higher than those of unmodified primary CD8 + These data suggest that even after 79 days in culture and subsequent expansion, γδT REX The cells are shown not to be functionally exhausted and to maintain their cytotoxic capacity.

[0118] γδ T cells are generally 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.

[0119] γδT REX cells, γδCAR-T REX Cell- and donor-identical unedited γδ T cells were stained and analyzed for Vδ1 and Vδ2 expression (FIG. 24). FACS analysis revealed that γδ T REX The cells are composed of multiple γδ T cell subtypes (Vδ1, Vδ2 and Vδ1 - Vδ2- ), indicating that REX editing can promote replicative senescence resistance in multiple γδ T cell subtypes. REX The diversity of γδ T cell subtypes was maintained in the cells ( FIG. 28 , bottom).

[0120] Next, γδT REX The cells were examined for their ability to be commanded by tumor-targeting moieties such as BCMA-targeting CARs (Figure 25). REX Cells were transduced to express a CAR targeting BCMA (Figure 25A) and 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 start of co-culture. γδT REX The cells exhibited similar ability to suppress BCMA-expressing tumor cells as primary CAR-T cell controls, but they generally secreted lower levels of effector cytokines, including IFN-γ, TNF-α, and IL-2 (Figure 25B, bottom). These data suggest that γδ CAR-T REX It shows that the cells can receive instructions from the CAR to target tumors and may be less likely to cause CRS than primary CAR-T cells.

[0121] Example 19: REX editing in NK cells enhances NK REX Supports cell phenotype. REX editing enhances replicative senescence resistance of T cells, but they also inhibit NK REX It was unclear whether NK cells 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. CRISPR / Cas9 was then used to edit NK cells at the REX locus to enhance NK cell phenotype. REXThe proliferation of REX-edited NK cells and unedited NK cells from the same donor was monitored over time (Figure 26). Across all donors and cytokine conditions, REX editing reproducibly increased the proliferation of NK cells. REX The results showed that the NK cells were able to promote resistance to replicative senescence (Figure 26). Unedited NK cells were unable to proliferate and died within 80 days, whereas the NK cells REX The cells can be cultured for up to 90 days and >10 6 ~>10 10 It increases by twofold.

[0122] Considering the enhanced resistance to replicative senescence, NK REX It was important to determine whether the NK cells maintained their dependence on cytokine support. REX Cells were grown in medium containing IL-2 or a combination of IL-2 and IL-15. In experiments where cytokines were removed from the growth medium, NK cell proliferation and proliferation in these cytokines was significantly improved. REX Cell dependency was determined and NK cells were cultured for 37 days. REX Cell counts were monitored. NK REX The cells were unable to proliferate after cytokine withdrawal and these cells showed a rapid decline in cell viability and viable cell diameter, enhancing their dependence on cytokine support despite REX gene editing (Figure 27).

[0123] To determine whether these cells were able to stably express tumor-targeting CARs, NK REX Cells were transduced to express a CAR targeting BCMA (Figure 28). REX CAR expression was maintained in the cells, with expression levels (mean fluorescence intensity, MFI) similar to those in purified CAR-T cells. These data support the conclusion that CAR-NK REX We show that the cells can stably express CAR and that the expression levels are comparable to standard CAR-T cells.

[0124] N.K. REXWhile the cells could be expanded in culture for extended periods of time, it was unclear: 1) whether their cytotoxic potential was maintained after sustained expansion; and 2) whether they could be directed by CARs to target tumors. REX The cells were cultured in two different NK REX These CAR-NK REX One of the strains had a CAR of >95% + CAR-NK REX NK cells from donors 50-1 and 47-1 were purified based on CAR expression to generate NK cell lines (Figure 29A, bottom right). NK cells from donors 50-1 and 47-1 were screened for their ability to lyse BCMA-expressing tumor cells in the xCELLigence assay. REX and CAR-NK REX After 78 and 86 days in culture, NK REX and CAR-NK REX The cells were potently cytotoxic. These lines rapidly lysed BCMA-expressing target cells and inhibited CAR-T REX At lower effector:target cell ratios, NK cells achieved higher levels of regulation more rapidly than NK cells (Figure 29B). REX and CAR-NK REX Due to the engagement of activating receptors on NK cells, REX While NK cells were able to lyse tumor cells independent of CAR expression, a contribution of cytotoxicity to the CAR could be observed for both donors 50-1 and 47-1. After 48 hours of co-culture, supernatants were collected and levels of IFN-γ, IL-2 and TNF-α were determined using MSD (Figure 29C). REX The cells are CAR-NK REX These cytokines were secreted at lower levels than CAR-T cells. REX The cells secreted the maximal levels of these factors (Figure 29C). REX We show that the cells can stably express and receive instructions from tumor-targeting CARs, and furthermore, these cells rapidly lyse tumor cells and accumulate lower levels of IFN-γ, IL-2, and TNF-α in co-culture supernatants.

[0125] Example 20: T REX The cells are sensitive to T cell depleting agents and chemotherapeutic agents. T REX The cells were modified to improve their replicative senescence resistance, but they displayed normal T cell characteristics. REX To understand in more detail their ability to regulate cells, we determined their sensitivity to standard T cell depletion agents and chemotherapeutic agents compared to unedited freshly activated whole T cells or T 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 survival was assessed using the Cell Titer Glo assay. Unedited freshly activated whole T cells or T REX Cells were also 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 were as sensitive to these drugs as unedited, freshly activated total T cells.

[0126] 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 The cells are modified to enhance 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 CD38KO B2M KO T REX The cells were co-cultured. REX The cells were CD38 KO B2M KO T REX While the cells were susceptible to NK cell-mediated lysis as expected, they did not show a decrease in numbers when co-cultured with PBMCs (Figure 31, top). NK cells express high levels of CD38 and CD38 KO B2M KO Total T cells or CD38 KO B2M KO T REX Preincubation of NK cells with the CD38-targeting antibody daratumamab (Dara) prior to coculture with cells reduced cytolysis by >50%. These data suggest that CD38 KO B2M KO T REX It is shown 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.

Claims

1. 1. A method for generating a population of replicative senescence-resistant (RRS) primary immune cells, comprising: (a) introducing one or more gene edits into primary immune cells; (b) culturing the primary immune cells in a culture medium; Including, the culturing induces proliferation of the primary immune cells to generate a population of replicative senescence-resistant (RRS) primary immune cells. method.

2. 10. The method of claim 1, further comprising stimulating the primary immune cells prior to introducing the one or more gene edits into the primary immune cells.

3. 10. The method of claim 1, further comprising stimulating the primary immune cells after introducing the one or more gene edits into the primary immune cells.

4. 10. The method of claim 1, further comprising inhibiting expression of one or more endogenous regulatory factors in the primary immune cells.

5. The method of claim 4, wherein the endogenous regulator is cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), or S-methyl-5'-thioadenosine phosphorylase (MTAP).

6. 10. The method of claim 1, further comprising inhibiting expression of one or more endogenous immune-related genes in the 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 (TRAC).

8. 10. The method of claim 1, wherein introducing one or more gene edits comprises introducing one or more transgenes encoding anti-apoptotic factors or virus-derived factors into the primary immune cells.

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

10. 9. The method of claim 8, wherein the virus-derived factor is any one of Saimiriine gammaherpesvirus 2 StpA A11, Herpesvirus saimiri StpC, Herpesvirus saimiri Tip, or modified herpesvirus Ateles-Epstein-Barr virus Tio-LMP1.

11. 10. The method of claim 1, further comprising inhibiting expression of cluster of differentiation 38 (CD38).

12. 10. The method of claim 1, further comprising inhibiting expression of phosphatase and tensin homolog (PTEN).

13. The method of claim 1 , wherein the primary immune cells comprise whole T cells.

14. The primary immune cells are CD8 ＋ The method of claim 1 , comprising T cells.

15. The primary immune cells are CD4 ＋ The method of claim 1 , comprising T cells.

16. 2. The method of claim 1, wherein the primary immune cells comprise gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells and / or natural killer T (NKT) cells.

17. The method of claim 1 , wherein the primary immune cells are human.

18. 10. The method of claim 1, further comprising introducing a polynucleotide encoding a chimeric antigen receptor (CAR).

19. 10. The method of claim 1, wherein the population of primary immune cells can be cultured with or without TCR stimulation for at least 100 days.

20. The primary immune cells are cultured in a medium containing at least about 10 ６ The method of claim 1, wherein the amount of the ion exchange material is increased by a factor of 1.

21. 10. The method of claim 1, wherein the primary immune cells are cultured in a medium that does not contain a primary immune cell stimulus.

22. 3. The method of claim 2, further comprising (c) restimulating the primary immune cells.

23. The primary immune cells are cultured for at least about 10 ８ 23. The method of claim 22, wherein the number of times ...

24. 10. The method of claim 1, wherein the transgene is introduced using a plasmid-based DNA transposon.

25. The method of claim 1 , wherein the transgene is introduced using a lentiviral platform.

26. 2. The method of claim 1, wherein the transgene is introduced using CRISPR-mediated site-specific integration.

27. 1. A method for generating a population of replicative senescence-resistant (RRS) primary immune cells, comprising: (a) inhibiting the expression of one or more endogenous regulatory factors in said primary immune cells; wherein the endogenous regulator is cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B), or S-methyl-5'-thioadenosine phosphorylase (MTAP); (b) inhibiting the expression of one or more endogenous immune-related genes in said primary immune cells. wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant (TRAC); (c) culturing the primary immune cells in a culture medium. Including, The method, wherein said culturing induces proliferation of said primary immune cells to generate a population of primary immune cells that are replicative senescence resistant (RRS).

28. 28. The method of claim 27, further comprising introducing into the primary immune cells a transgene encoding either B-cell large lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

29. 28. The method of Claim 27, further comprising stimulating the primary immune cells before the one or more gene edits are introduced into the primary immune cells.

30. 28. The method of Claim 27, further comprising stimulating the primary immune cells after the one or more gene edits are introduced into the primary immune cells.

31. 28. The method of claim 27, further comprising inhibiting expression of cluster of differentiation 38 (CD38).

32. 28. The method of claim 27, further comprising inhibiting expression of phosphatase and tensin homolog (PTEN).

33. 28. The method of claim 27, wherein the primary immune cells comprise whole T cells.

34. The primary immune cells are CD8 ＋ 28. The method of claim 27, comprising T cells.

35. The primary immune cells are CD4 ＋ 28. The method of claim 27, comprising T cells.

36. 28. The method of claim 27, wherein the primary immune cells comprise gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells and / or natural killer T (NKT) cells.

37. 28. The method of claim 27, wherein the primary immune cells are human.

38. 28. The method of claim 27, further comprising introducing a polynucleotide encoding a chimeric antigen receptor (CAR).

39. 28. The method of claim 27, wherein said population of primary immune cells can be cultured for at least 100 days.

40. The primary immune cells are cultured in a medium containing at least about 10 ６ 28. The method of claim 27, wherein the number of cells is increased by a factor of 2.

41. 28. The method of claim 27, wherein the primary immune cells are cultured in a medium that does not contain a primary immune cell stimulus.

42. 28. The method of claim 27, further comprising (d) restimulating the primary immune cells.

43. The primary immune cells are cultured in a medium containing at least about 10 ８ 43. The method of claim 42, wherein the number of times ...

44. 28. The method of claim 27, wherein the transgene is introduced using a plasmid-based DNA transposon.

45. 28. The method of claim 27, wherein the transgene is introduced using a lentiviral platform.

46. 28. The method of claim 27, wherein the transgene is introduced using CRISPR-mediated site-specific integration.

47. 1. A method for generating a population of replicative senescence-resistant (RRS) primary immune cells, comprising: (a) inhibiting the expression of one or more endogenous regulatory factors in said primary immune cells; (The endogenous regulator is cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase inhibitor 2B (CDKN2B) or S-methyl-5'-thioadenosine phosphorylase (MTAP)); (b) culturing the primary immune cells in a culture medium. Including, The method, wherein said culturing induces proliferation of said primary immune cells to generate a population of primary immune cells that are replicative senescence resistant (RRS).

48. 48. The method of Claim 47, further comprising stimulating the primary immune cells before the one or more gene edits are introduced into the primary immune cells.

49. 48. The method of Claim 47, further comprising stimulating the primary immune cells after the one or more gene edits are introduced into the primary immune cells.

50. 48. The method of claim 47, further comprising inhibiting expression of one or more endogenous immune-related genes in said primary immune cells.

51. 51. The method of claim 50, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T-cell receptor alpha constant (TRAC).

52. 48. The method of claim 47, further comprising inhibiting expression of cluster of differentiation 38 (CD38).

53. 48. The method of claim 47, further comprising inhibiting expression of phosphatase and tensin homolog (PTEN).

54. 48. The method of claim 47, wherein the primary immune cells comprise whole T cells.

55. The primary immune cells are CD8 ＋ 48. The method of claim 47, comprising T cells.

56. The primary immune cells are CD4 ＋ 48. The method of claim 47, comprising T cells.

57. 48. The method of claim 47, wherein the primary immune cells comprise gamma-delta T cells, mucosal-associated invariant T (MAIT) T cells, natural killer (NK) cells and / or natural killer T (NKT) cells.

58. 48. The method of claim 47, wherein the primary immune cells are human.

59. 48. The method of claim 47, further comprising introducing a polynucleotide encoding a chimeric antigen receptor (CAR).

60. 48. The method of claim 47, wherein said population of primary immune cells can be cultured for at least 100 days.

61. The primary immune cells are cultured in a medium containing at least about 10 ６ 48. The method of claim 47, wherein the number of times ...

62. 48. The method of claim 47, wherein the primary immune cells are cultured in medium that does not contain a primary immune cell stimulus.

63. 48. The method of claim 47, further comprising (c) stimulating the primary immune cells.

64. The primary immune cells are cultured for at least about 10 ８ 64. The method of claim 63, wherein the number of times ...

65. 48. The method of claim 47, wherein the transgene is introduced using a plasmid-based DNA transposon.

66. 48. The method of claim 47, wherein the transgene is introduced using a lentiviral platform.

67. 48. The method of claim 47, wherein the transgene is introduced using CRISPR-mediated site-specific integration.

68. 68. A modified immune cell population produced according to the method of any one of claims 1 to 67.

69. 69. A pharmaceutical composition comprising the modified immune cell population of claim 68 and a pharmaceutically acceptable carrier.

70. 70. 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 69.

71. Modified 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).

72. 72. The modified T cell of claim 71, further comprising a transgene encoding either B-cell large lymphoma (Bcl-xL) or B-cell lymphoma 2 (Bcl-2).

73. 73. The modified T cell of claim 71 or 72, which does not express one or more endogenous immune-related genes in the primary immune cell.

74. 74. The modified T cell of claim 73, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) and / or T cell receptor alpha constant (TRAC).

75. 72. The modified T cell of claim 71, which does not express cluster of differentiation 38 (CD38).

76. 72. The modified T cell of claim 71, further comprising a polynucleotide encoding a chimeric antigen receptor (CAR).

77. CD8 ＋ T cells, CD4 ＋ 72. The modified T cell of claim 71, 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.

78. CD8 ＋ 72. The modified T cell of claim 71, which is a T cell.

79. CD4 ＋ 72. The modified T cell of claim 71, which is a T cell.

80. 72. The modified T cell of claim 71, which is human.

81. Modified 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), and / or T cell receptor alpha constant (TRAC).

82. 82. The modified T cell of claim 81, which does not express cluster of differentiation 38 (CD38).

83. 83. The modified T cell of claim 81 or claim 82, further comprising a polynucleotide encoding a chimeric antigen receptor (CAR).

84. 82. The modified T cell of claim 81, which 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.

85. CD8 ＋ 82. The modified T cell of claim 81, which is a T cell.

86. CD4 ＋ 82. The modified T cell of claim 81, which is a T cell.

87. 82. The modified T cell of claim 81, which is human.

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

89. 89. The modified T cell of claim 88, which does not express one or more endogenous immune-related genes in primary immune cells.

90. 90. The modified T cell of claim 89, wherein the endogenous immune-related gene is beta-2 microglobulin (B2M) or T cell receptor alpha constant (TRAC).

91. 91. The modified T cell of any one of claims 88 to 90, which does not express cluster of differentiation 38 (CD38).

92. 89. The modified T cell of claim 88, further comprising a polynucleotide encoding a chimeric antigen receptor (CAR).

93. CD8 ＋ T cells, CD4 ＋ 89. The modified T cell of claim 88, 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.

94. CD8 ＋ 89. The modified T cell of claim 88, which is a T cell.

95. CD4 ＋ 89. The modified T cell of claim 88, which is a T cell.

96. 89. The modified T cell of claim 88, which is human.

Images