Methods of culturing immune cells

Abstract

The present disclosure relates to methods of culturing immune cells, e.g., T cells, NK cells, and / or TILs, for an immune cell therapy, wherein the immune cells are contacted with a CD28 agonist, and wherein the immune cells are not contacted with a CD3 agonist. In some aspects, the methods disclosed herein promote the enrichment of immune cells having a less-differentiated phenotype, i.e., expressing markers characteristic of stem cell-like memory T (Tscm) cells. Cells cultured using the methods disclosed herein can be used for various cell therapies, including but not limited to chimeric antigen receptor (CAR) T cell therapy and TCR T cell therapy, including neoantigen directed-T cell therapies.

Description

METHODS OF CULTURING IMMUNE CELLSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the priority benefit of U.S. Provisional Application No. 63 / 714,042, filed on October 30, 2024, which is incorporated by reference herein in its entirety.FIELD

[0002] The present disclosure relates to methods of culturing immune cells, e.g., T cells, NK cells, and / or TILs, for an immune cell therapy. In some aspects, the methods disclosed herein promote the enrichment of immune cells having a less-differentiated phenotype, z.e., expressing markers characteristic of stem cell-like memory T (Tscm) cells. Cells cultured using the methods disclosed herein can be used for various cell therapies, including but not limited to chimeric antigen receptor (CAR) T cell therapy and TCR T cell therapy including neoantigen directed-T cell therapies.BACKGROUND

[0003] Various adoptive T cell therapies, such as chimeric antigen receptor (CAR) and T cell receptor (TCR)-T cell therapies, have been developed to target a range of tumor antigens, aiming to elicit antitumor efficacy. Most current approaches rely on the adoptive transfer of T cells activated and redirected ex vivo using CD3 and CD28 stimulation (see, e.g., Ayala Ceja, el. al., J Exp Med 221 (2024)). This method is essential for the initial activation and expansion of T cells but inevitably generates effector-differentiated T cells. These effector T cells are highly active and capable of immediate cytotoxic responses; however, they are also susceptible to exhaustion and dysfunction due to sustained antigenic stimulation within the tumor microenvironment. This continuous stimulation can lead to a state of chronic activation, characterized by the upregulation of inhibitory receptors and a decrease in proliferative capacity and effector functions, ultimately limiting the efficacy of these therapeutic strategies. Addressing this challenge is crucial for improving the long-term success of adoptive T cell therapies.

[0004] CD8+T cells are an important cytotoxic subset of T cells involved in antitumor immunity. CD3 and CD28 are both essential for CD8+T cell activation, but they initiate distinct intracellular signaling pathways resulting in different cellular outcomes. CD3 signaling, primarily initiated through the TCR complex, activates the MAP kinase pathway, calcium influx, and activation of the transcription factors such as NF AT, AP-1, and NF-KB. This cascade is critical for the initial activation of T cells, promoting proliferation and differentiation into effector cells. Incontrast, CD28 signaling acts as a costimulatory signal that enhances the TCR-mediated activation by further activating the PI3K / Akt and MAP kinase pathways, and upregulating anti-apoptotic proteins. CD28 engagement is essential for sustaining T cell proliferation and survival and enhancing cytokine production. While CD3 stimulation alone can drive T cell activation, it often leads to a more pronounced effector differentiation and a higher propensity for exhaustion due to sustained antigenic stimulation. Moreover, CD3 signaling without adequate CD28 engagement can result in anergy, a state of T cell unresponsiveness characterized by a failure to proliferate and produce cytokines.

[0005] Stem cell-like memory T cells (Tscm) are a rare subset of memory T cells characterized by their enhanced capacity for self-renewal and longevity. These cells are believed to play a significant role in adoptive T cell therapy for cancer due to their ability to induce potent antiviral and antitumor responses. Despite their therapeutic potential, their scarcity in peripheral blood poses a significant challenge for clinical application. Various strategies have been developed to expand Tscm ex vivo, including the addition of cytokines such as IL-7, IL-15, and / or IL-21, the inhibition of MAP kinase or mechanistic target of rapamycin (mTOR), and the induction of Wnt / p- catenin pathway, Notch signaling, or epigenetic modifications. However, these strategies still rely on stimulation of both CD3 and CD28, which leads to undesired differentiation and the acquisition of exhaustion profiles ex vivo prior to infusion. As such, there remains a need in the field of cell immunotherapy for methods of preparing a population of immune cells enriched in Tscm-like cells.BRIEF SUMMARY

[0006] Some aspects of the present disclosure are directed to a method of preparing a population of immune cells for a cell therapy, comprising contacting a population of source immune cells with a CD28 agonist thereby producing a population of stimulated immune cells, wherein neither the population of source immune cells nor the population of stimulated immune cells is contacted with a CD3 agonist.

[0007] In some aspects, the method further comprises introducing into one or more cells of the population of source immune cells a heterologous nucleic acid molecule encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) prior to contacting the source immune cells with the CD28 agonist. In some aspects, the method further comprises introducing into one or more cells of the population of stimulated immune cells a heterologous nucleic acid molecule encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0008] Some aspects of the present disclosure are directed to a method of preparing a population of genetically modified immune cells for an immune cell therapy, comprising: (i) contacting a population of source immune cells with a CD28 agonist thereby generating a population of stimulated immune cells, and (ii) introducing into one or more cells of the population of stimulated immune cells a heterologous nucleic acid molecule encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR); wherein neither the population of source immune cells nor the population of stimulated immune cells is contacted with a CD3 agonist.

[0009] In some aspects, the source immune cells comprise T cells, NK cells, or both. In some aspects, the T cells comprise a[3 T cells, y5 T cells, cytotoxic T cells, helper T cells, regulatory T cells (Treg cells), or any combination thereof. In some aspects, the source immune cells comprise tumor infiltrating lymphocytes (TILs).

[0010] In some aspects, the source immune cells are contacted with the CD28 agonist for about 12 hours to about 7 days. In some aspects, the source immune cells are contacted with the CD28 agonist for about 12 hours to about 6 days, about 12 hours to about 5 days, about 12 hours to about 4 days, about 12 hours to about 3 days, about 12 hours to about 48 hours, about 12 hours to about 36 hours, about 12 hours to about 24 hours, about 24 hours to about 36 hours, about 24 hours to about 48 hours, about 24 hours to about 3 days, about 24 hours to about 4 days, about 24 hours to about 5 days, about 24 hours to about 6 days, about 24 hours to about 7 days, about 36 hours to about 48 hours, about 36 hours to about 3 days, about 36 hours to about 4 days, about 36 hours to about 5 days, about 36 hours to about 6 days, about 36 hours to about 7 days, about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 6 days, about 2 days to about 7 days, about 3 days to about 4 days, about 3 days to about 5 days, about 3 days to about 6 days, about 3 days to about 7 days, about 3 to about 8 days, about 3 to about 9 days, or about 3 to about 10 days.

[0011] In some aspects, the population of source immune cells is further contacted with IL-7, IL-21, an antibody or antigen-binding portion thereof that specifically binds IFN-y, or any combination thereof.

[0012] In some aspects, the population of source immune cells are contacted with the CD28 agonist by culturing the population of source immune cells in a medium comprising the CD28 agonist.

[0013] In some aspects, the medium further comprises IL-7, IL-21, an antibody or antigenbinding portion thereof that specifically binds interferon-gamma (IFN-y), or any combination thereof. In some aspects, the IL-7, IL-21, the antibody or antigen-binding portion thereof thatspecifically binds IFN-y, or any combination thereof is added to the medium on the same day or about 1 day after the source immune cells are contacted with the CD28 agonist. In some aspects, (a) the medium comprises about 1 ng / ml to about 1 pg / ml IL-7; (b) the medium comprises about 1 ng / ml to about 1 pg / ml IL-21; (c) the medium comprises about 0.1 pg / ml to about 100 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y; or (d) any combination of (a) to (c). In some aspects, (a) the medium comprises about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml, about 500 ng / ml, or about 1 pg / ml IL- 7; (b) the medium comprises about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml, about 500 ng / ml, or about 1 pg / ml IL-21; (c) the medium comprises about 0.1 pg / ml, about 0.5 pg / ml, about 1 pg / ml, about 2 pg / ml, about 3 pg / ml, about 4 pg / ml, about 5 pg / ml, about 6 pg / ml, about 7 pg / ml, about 8 pg / ml, about 9 pg / ml, about 10 pg / ml, about 20 pg / ml, about 30 pg / ml, about 40 pg / ml, about 50 pg / ml, or about 100 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y; or (d) any combination of (a) to (c). In some aspects, the medium comprises about 25 ng / ml IL-7, about 25 ng / ml IL-21, and about 2 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y.

[0014] In some aspects, following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD45RA. In some aspects, following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD62L. In some aspects, following the contacting with the CD28 agonist at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CCR7. In some aspects, following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD95.

[0015] In some aspects, following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD45RA and CCR7. In some aspects, following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, and CD95.

[0016] In some aspects, following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, CD95, and CD62L. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof, in the population of stimulated immune cells is increased by at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8- fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, or at least about 20-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of stimulated immune cells is increased by at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 9-fold, at least about 10- fold, at least about 15-fold, or at least about 20-fold, relative to the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of stimulated immune cells is increased by at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, or at least about 20-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of source immune cells.

[0017] In some aspects, following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express TCF 1. In some aspects, following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express KLF2. In some aspects, following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2. In some aspects, following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express TCF1 and / or KLF2 and lack IRF4 expression.

[0018] In some aspects, following the contacting with the CD28 agonist, the expression level of one or more exhaustion makers in the population of stimulated immune cells does not increase relative to the expression level of the one or more exhaustion markers in the population of source immune cells. In some aspects, following the contacting with the CD28 agonist the population of stimulated immune cells has a lower expression level of one or more exhaustion markers, relative to the expression level of the one or more exhaustion markers in a population of immune cells contacted with a CD3 agonist. In some aspects, the one or more exhaustion markers comprise PD-1, TIM-3, LAG-3, TIGIT, or any combination thereof.

[0019] In some aspects, following the contacting with the CD28 agonist the expression level of one or more markers of oxidative stress in the population of stimulated immune cells does not increase relative to the expression level of the one or more markers of oxidative stress in the population of source immune cells. In some aspects, following the contacting with the CD28 agonist the population of stimulated immune cells has a lower expression level of one or more markers of oxidative stress, relative to the expression level of the one or more markers of oxidative stress in a population of immune cells contacted with a CD3 agonist. In some aspects, the one or more markers of oxidative stress comprise NRF2, HIFla or any combination thereof.

[0020] In some aspects, the CD28 agonist comprises an antibody or an antigen-binding portion thereof that specifically binds CD28. In some aspects, the CD28 agonist comprises an anti- CD28 antibody selected from CD28.2, TGN1412, and ANC28.1 / 5D10, or an antigen-binding portion thereof.

[0021] In some aspects, the CD28 agonist is associated with an antigen presenting cell (APC), a bead, an extracellular matrix, or any combination thereof. In some aspects, the CD28 agonist is associated with an APC. In some aspects, the CD28 agonist is bound to the membrane of an APC. In some aspects, the APC is an artificial APC (aAPC). In some aspects, the APC is genetically modified to express the CD28 agonist. In some aspects, the APC does not express B2M, CD32, or both B2M and CD32. In some aspects, the APC is a genetically modified K562 cell. In some aspects, the APC expresses CD83, IL-7, IL-21, or any combination thereof.

[0022] In some aspects, the population of source immune cells comprises peripheral blood mononuclear cells (PBMCs). In some aspects, the population of source immune cells is prepared by isolating naive CD8+ immune cells from PBMCs.

[0023] In some aspects, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the immune cells in the population of source immune cells are naive CD8+ immune cells.

[0024] In some aspects, the population of source immune cells is obtained from a tumor sample.

[0025] In some aspects, the population of source immune cells is expanded prior to the contacting with the CD28 agonist. In some aspects, the population of source immune cells is not expanded prior to the contacting with the CD28 agonist. In some aspects, the population of stimulated immune cells is expanded.

[0026] In some aspects, the CAR or the TCR comprises an antigen-binding domain that specifically binds a tumor antigen. In some aspects, the tumor antigen is CD 19, TRAC, TCR[3, BCMA, CLL-1, CS1, CD38, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1 IRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, S SEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF -I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE- Al, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT- 2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA- 1 / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY- TES1, LCK, AKAP-4, SSX2, RAGE-1, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3s, CD4, CD5, CD7, the extracellular portion of the APRIL protein, or any combinations thereof. In some aspects, the tumor antigen is NY-ESO-1. In some aspects, the one or more cells are transduced with a vector comprising the heterologous nucleic acid molecule encoding the CAR and / or the TCR. In some aspects, the vector is a retroviral vector,a lentiviral vector, an adeno-associated virus (AAV), an adenovirus, an AAV hybrid virus, a baculovirus, or any combination thereof. In some aspects, the vector is a retroviral vector.BRIEF DESCRIPTION OF THE DRAWINGS / FIGURES

[0027] FIGs. 1 A-1L show the effect of CD3 stimulation on T cell effector differentiation, while CD28 superagonist induces proliferation of Tscm-like cells without driving effector differentiation. Three different clones of anti-CD28 antibodies were tested. Soluble anti-CD28 antibodies were added to the culture media and naive CD8+T cells were cultured for 7 days with or without plate-bound CD3 antibody. Proliferation and phenotypes were evaluated by flow cytometry. FIGs. 1 A-1B show CCR7 (x-axes) and CD45RA (y-axes) expression on unstimulated CD8+T cells (FIG. 1A), CD8+T cells stimulated with anti-CD28 antibody (FIG. IB, top), and CD8+T cells stimulated with anti-CD28 antibody and CD3 antibody (FIG. IB, bottom). FIG. 1C shows proliferation calculated as fold expansion from the number of CD8+T cells on day 0. FIGs. 1D-1E show TCF1 and CD95 expression, respectively. Expression of each marker was gated on CD8+cells. FIGs. 1F-1G show representative flow-cytometry analysis of CD8+T cells stimulated with aAPCs, CD8+T cells stimulated with aAPCs+aCD3, and unstimulated CD8+T cells, showing CCR7 and CD45RA expression (FIG. IF) and TCF1 and CD95 expression (FIG. 1G). Naive CD8+T cells were restimulated repeatedly with the same stimulators. FIG. 1H shows representative proliferation as fold expansion. FIGs. II- IL shows expression of CCR7 (FIG. II), CD45RA (FIG. 1J), CD95 (FIG. IK), and TCF1 (FIG. IL) at 35 days after initial stimulation from three independent experiments were shown. Data are mean ± S.D. ** / ?<0.01, *** / ?<0.001 determined using Two-way ANOVA test, n.s., not significant.

[0028] FIGs. 2A-2HH show the effect of aAPC-stimulation on expansion of CD8+T cells maintaining early memory phenotype. CyTOF analysis was performed using naive cells and cells cultured with aAPCs, Beads, or aAPCs+aCD3 for 7 days. FIGs. 2A-2R show CyTOF analysis on T cell differentiation. Combined data from 3 biological replicates are shown. FIG. 2A is a UMAP plot showing the distribution of naive CD8+T cells or cells cultured for 7 days. FIGs. 2B-2R show the expression level of selected proteins on UMAP plot (FIG. 2B) or violin plot (FIGs. 2C-2R). FIGs. 2S-2BB show CyTOF analysis on T cell metabolic status. FIG. 2S is a UMAP plot showing the distribution of cells obtained from three different donors. FIGs. 2T-2BB show the expression level of selected surface markers and transcription factors on UMAP plot (FIG. 2T) or violin plot (FIGs. 2U-2BB). FIGs. 2CC-2FF show electron microscopy imaging of naive CD8+T cells and cells cultured with aAPCs, Beads or aAPCs+aCD3 for 7 days. Scale bar, 200, 500nm. Length, areaof mitochondria and maximum cristae width were measured from 15 images for each stimulation methods using Fiji / ImageJ software. **** / ?<0.0005 determined using One-way ANOVA test. FIGs. 2GG-2HH show the results of a Mito stress test performed with cells stimulated with aAPCs, Beads, or aAPCs+aCD3 on day 7. *** / ?<0.001, **** / ?<0.0001 determined using One-way ANOVA test. Data are mean ± S.D.

[0029] FIGs. 3A-3I show the effect of aAPC-stimulation on the genetic and epigenetic signatures of Tscm. Genetic and epigenetic signatures of Tscm are enriched in aAPC-stimulated cells. FIG. 3A is a PCA plot of bulk RNA-seq performed on naive CD8+T cells, and cells stimulated with aAPCs, Beads, aAPCs+aCD3, or aCD3 for 3 days. Dashed circles indicate stimulation conditions and symbols represent biological replicates. FIG. 3B is a volcano plot showing differentially expressed genes between cells stimulated with aAPCs (right) and Beads (left). Selected genes significantly upregulated in Bead or aAPC-stimulated cells are labelled (log 2-fold change > 1 and adjusted p-value < le'05). FIG. 3C is an expression heatmap for genes upregulated in naive only (first block), naive and aAPC-stimulated cells (second block), aAPC- stimulated cells only (third block), and cells with CD3 stimulation (fourth block). FIG. 3D is a GSEA plot of Tscm and Tpex signatures in aAPC-stimulated cells versus Bead-stimulated cells (left, middle) and Tscm versus naive CD8+T cell signature in aAPC-stimulated cells versus naive cells (right). FIG. 3E is a heatmap showing the expression of genes associated with T cell anergy (left) and apoptosis (right). FIG. 3F is a PCA plot of ATAC-seq in naive and cells stimulated with aAPCs or Beads for 10 days. Dashed circles indicate stimulation conditions and symbols represent biological replicates. FIG. 3G shows chromatin accessibility tracks of TCF7, ENTPD1, and HAVCR2 loci in naive and aAPC or Bead-stimulated cells. Differentially accessible regions (DARs) are marked by boxes. FIG. 3H shows the number of chromatin accessible sites in aAPC- stimulated cells (left of 0 line) and Bead-stimulated cells (right of 0 line). FIG. 31 shows transcription factor binding motifs enriched in aAPC- or Bead-stimulated cells identified using HOMER. Motifs enriched in aAPC-stimulated cells and Bead-stimulated cells are shown, as indicated.

[0030] FIGs. 4A-4P shows the effect of IRF4 KO on effector differentiation and promotion of metabolic switch from glycolysis to the oxidative phosphorylation pathway. IRF4 was knocked out using CRISPR / Cas9 system in naive CD8+T cells. gRNAs targeting safe-harbor site AAVS1 was used as negative control. Cells were stimulated with aAPCs, Beads or aAPCs+aCD3 2 days after electroporation. Phenotypic analysis by flow cytometry and metabolic analysis wereperformed 3 days and 7 days after stimulation, respectively. FIG. 4A-4D show knockout efficiency as analyzed 2 days after electroporation by flow. FIGs. 4E-4I show CD45RO (FIG. 4E), CD25 (FIG. 4F), PD-1 (FIG. 4G), TCF1 (FIG. 4H), and BLIMP 1 (FIG. 41) expression on IRF4 KO or AAVS1 K0 CD8+T cells. *p<0.05, ** / ?<0.01, *** ><0.001, **** / ?<0.0001 determined using Two- way ANOVA test. Data are mean ± S.D., n.s., not significant. In FIGs. 4J-4N, naive CD8+T cells were stimulated with aAPCs for 3 days and electroporation was performed using pmax-IRF4 (IRF4-Q8) and pmax-reverse-IRF4 (control). Phenotype was evaluated by flow cytometry 3 days after electroporation. FIGs. 4J-4K shows representative flow cytometry analysis on IRF4 and CD34 expression. FIGs. 4L-4N show a comparison of CD45RO, PD-1, and CD69 (FIG. 4L), TCF1 (FIG. 4M), and BLIMP 1 (FIG. 4N) on IRF4 overexpressing CD8+T cells and control cells. Expression of all markers were measured after gating on CD34+populations. * / ?<0.05, *** / ?<0.001 determined using multiple-paired t-test. Data are mean ± S.D. FIGs. 4O-4P show the results from ATP rate assays performed using AAVS1 KO aAPC-stimulated cells and IRF4 KO and AAVS1 KO aAPCs+CD3 -stimulated cells. OE: Over Expression.

[0031] FIGs. 5A-5M show the effect of aAPC stimulation on CD8+T cells response and cytotoxicity compared to effector cells upon restimulation. Naive CD8+T cells were stimulated with aAPCs or Beads for 7 days. Cells were restimulated with Beads and cytokine production and degranulation were measured by flow cytometry 5 hours later. For evaluation of cell division, cells were restimulated with Beads for 3 days after labeling with carboxyfluorescein succinimidyl ester (CFSE). FIGs. 5A-5C are representative plots of cells gated on (i) IFN-y and (ii) TNF-a (FIG. 5A), IL-2 (FIG. 5B), or Granzyme B (FIG. 5C), with the inset numbers indicating the percentage of produced cytokines from CD8+T cells. A summary is shown in FIG. 5D. All flow plots were gated on CD8+population. FIGs. 5E-5F show cell division and TCF1 expression 3 days after restimulation by Dynabeads for aAPC-stimulated (FIG. 5E) and Bead-stimulated (FIG. 5F) T cells. Following the stimulation with aAPCs or Beads, cells were transduced with HLA-A2 / NY-ESO-1 TCR (clone 1G4LY) and cytotoxicity assays were performed on day 8. Cytotoxicity assays were performed against A375 (FIG. 5G) and SK-MEL-28 (FIG. 5H). FIGs. 5G-5H show representative data from 3 different donors. ** / ?<0.01, **** / ?<0.0001 determined using Two-way ANOVA-test. Data are mean ± S.D. Statistical analysis was performed between TCR transduced aAPC- and Bead-stimulated cells. FIGs. 5I-5J show the results from xCELLigence Real-Time Cell Analysis, performed with A375 (FIG. 51) and SK-MEL-28 (FIG. 5J) as target cells at 2: 1 E / T ratio. FIG. 5K is a schematic representation of an experimental scheme showing that 5 / I 04of A375 cells weresubcutaneously inoculated into NSG mice (day -13) and I x lO6of TCR or ANGFR alone transduced T cells were intravenously injected. FIGs. 5L-5M show the tumor progression (FIG. 5L) and the Kaplan-Meier curve (FIG. 5M) for event-free survival of mice treated with TCR or ANGFR alone T cells stimulated with either aAPCs or Beads (n = 4 mice per group). *** / ?<0.001, ****p<0.0001 determined using Two-way ANOVA-test. Data are mean ± S.D. Statistical analysis was performed between TCR transduced aAPC- and Bead-stimulated cells.

[0032] FIGs. 6A-6K shows the effect of generating aAPCs expressing membrane-bound anti-CD28 antibody for T cell expansion, in the absence of CD3 stimulation, on limiting effector differentiation. FIG. 6A shows the expression of CCR7, CD45RA, TCF1 and CD95 in naive CD8+T cells stimulated by soluble anti-CD28 antibody (clone. 28.2) with graded concentrations of platebound anti-CD3 antibody for 7 days. FIGs. 6B-6F show the cells’ proliferation and phenotype, evaluated by flow cytometry, after 7 days of stimulation with CD28.2 (FIG. 6C) or TGN1412 (FIG. 6D) microbeads, with or without anti-CD3 antibody. TGN1412 microbeads were added into culture media at 200: 1, 400: 1 and 800: 1 T cell / microbead ratio. FIG. 6B-6D show the representative expression of CCR7 and CD45RA from three different donors. FIGs. 6E-6G show proliferation (FIG. 6E), TCF1 expression (FIG. 6F), and CD95 expression (FIG. 6G) as combined data from three different donors. FIGs. 6H-6K show [EM (FIG. 6H), CD32 (FIG. 61), TGN1412 (FIG. 6J), and CD83 (FIG. 6K) expression on aAPCs measured by flow cytometry.

[0033] FIGs. 7A-7J show the effect of PI3K-mTOR inhibition on the proliferation of aAPC-stimulated cells. FIGs. 7A-7D show the phosphorylation of S6 (FIGs. 7A and 7C) and Akt (FIGs. 7B and 7D) upon aAPC- or aAPCs+aCD3- stimulation measured by flow cytometry. FIGs. 7A-7B show representative flow cytometry data, and FIGs. 7C-7D show combined data from three different donors. **p<0.01 determined using One-way ANOVA test. Data are mean ± S.D., n.s., not significant. FIGs. 7E-7G show the effect of inhibiting PI3K on CTV labelled naive CD8+T cells stimulated with aAPCs or aAPCs+aCD3. LY294002 (PI3K inhibitor) was added to culture media at three different concentrations: 1, 2.5, and 5 pM. Cell division was evaluated as dye dilution by flow cytometry on day 5. FIG. 7E shows representative data from three different donors. FIGs. 7F show the cell number, counted by flow cytometry using counting beads, on day 7. FIG. 7G shows divided cells upon each stimulation gated as CTV population under various concentrations of LY294002. Combined data from 3 biological replicates shown. FIGs. 7H-7J show the effect of adding Rapamycin (mTORCl / 2 inhibitor) to culture media on expansion (FIG. 71) and cell division upon each stimulation (FIG. 7J) at four different concentrations: 0.1, 0.25, 0.5and 1 pg / ml. Cell division and proliferation upon each stimulation were evaluated as indicated above. Combined data from 3 biological replicates is shown. Flow plots and bar graphs were gated on CD8+populations. *p<0.05, ** / ?<0.01, *** / ?<0.001, **** / ?<0.0001 determined using One-way ANOVA test. Data are mean ± S.D., n.s., not significant.

[0034] FIGs. 8A-8H show the effect of aAPC-stimulation on expanding CD8+T cells maintaining early memory phenotype. FIG. 8A is a UMAP plot for T cell phenotypic analysis, showing the distribution of naive CD8+T cells or cells cultured for 7 days for three different donors separated per donor. FIG. 8B is a heatmap, showing the expression level of all markers used for the UMAP analysis. FIG. 8C is a UMAP plot for metabolomic analysis, showing the three different donors separately. FIG. 8D is a heatmap showing the expression level of all markers used for the UMAP analysis. FIGs. 8E-8F show glucose uptake (FIG. 8E) and fatty acid uptake (FIG. 8F), which were evaluated by flow cytometry. FIG. 8G is a bar graph showing combined data from four different donors. * / ?<0.05 determined using one-way ANOVA. Data are mean ± S.D., n.s., not significant. FIG. 8H shows ATP rate assays were performed with cells stimulated with aAPCs, Beads or aAPCs+aCD3 on day 7. ATP level in cultured cells was measured using bioluminescence. Combined data from three different donors shown. * / ?<0.05 determined using One-way ANOVA test. Data are mean ± S.D.

[0035] FIGs. 9A-9V show the effect of aAPC-stimulation on the genetic and epigenetic signatures of Tscm. FIG. 9A shows CyTOF analysis for naive cells and cells stimulated with aAPC, Beads, or aAPCs+aCD3 for 3 days. FIGs. 9B-9R show the expression level of selected proteins shown on UMAP plot (FIG. 9B) or violin plot (FIGs. 9C-9R). FIG. 9S is a volcano plot showing the differentially expressed genes between cells stimulated aAPCs and aCD3. Data points representing upregulated genes in aCD3 -stimulated cells and data points representing upregulated genes in aAPC-stimulated cells are labelled. X-axis represents Log2 fold changes (cut off value; 1) and Y-axis represents -LoglO adjusted / J- values (cut off value; le-0.5). FIG. 9T shows GSEA plots of Tscm and Tpex signatures in aAPC-stimulated cells versus aCD3 -stimulated cells. FIGs. 9U-9V show the result of GSEA performed using MSigDB canonical pathway gene sets with aAPC-stimulated cells compared to aCD3 -stimulated cells. FIG. 9U shows gene sets enriched in aAPC-stimulated cells. FIG. 9V shows GSEA plots for WNT pathway or hematopoietic stem cell differentiation gene set in aAPC versus aCD3 -stimulated cells.

[0036] FIGs. 10A-10N show the effect of aAPC stimulation on CD8+T cells cytotoxicity compared to effector cells. FIGs. 10A-10B show degranulation capacity of aAPC-stimulated (FIG.10A) or Bead-stimulated (FIG. 10B) cells when restimulated with Beads. FIGs. 10C-10D show TCR expression before (FIG. IOC) and after (FIG. 10D) magnetic sorting for transduced cells. FIGs. 10E-10G show degranulation assessed against A375 (FIG. 10E) and SK-MEL-28 (FIG. 10F) by CD 107a staining on cells after 5-hour coculture with target cells at 1 : 1 E / T ratio, with FIG. 10G showing control no-target cells. Representative data from 3 different donors. FIG. 10H shows images captured during xCELLigence Real-Time Cell Analysis at three different time points, and representative images are shown. FIGs. 10I-10J show representative phenotypes of CD8+T cells after 3-day coculture with A375 analyzed by flow cytometry based on expression of PD-1 and Tim-3 (FIG. 101) or CCR7 ad CD25 (FIG. 10J). FIGs. 10K-10N are bar graphs showing expression of CCR7 (FIG. 10K), CD25 (FIG. 10L), PD-1 (FIG. 10M), and Tim-3 (FIG. ION), wherein data was combined from three different donors. * / ?<0.05 determined using paired t-test. Data are mean ± S.D., n.s., not significant.

[0037] FIGs. 11A-11C provide data showing aAPCs elicit a fundamentally distinct activation program compared with weak CD3 engagement. FIG. 11A is a bar graph illustrating fold expansion of naive CD8+T cells following stimulation with graded doses of ocCD3 or aAPCs for seven days. Combined data from three technical triplicates are shown as mean ± S.D., and representative data from three donors are shown (*p < 0.05, **p < 0.01 determined using one-way ANOVA. n.s., not significant) (FIG. 11 A). FIG. 11B illustrates expression of early activation markers CD69 and CD25 (top) or PD-1 (bottom) assessed two days after stimulation with ocCD3 or aAPCs. FIG. 11C shows expression of TCF1 (top) and CD95 (bottom) assessed seven days after stimulation with ocCD3 or aAPCs. MFI is shown for the histograms.

[0038] FIGs. 12A-12D show that engagement of adhesion molecules is required for aAPC- induced T cell proliferation. FIG. 12A provides graphical representations of surface expression of adhesion and co-stimulatory molecules on aAPCs, including CD15, CD58, CD59, ICAM1, and ICAM2, as assessed by flow cytometry. Corresponding isotype controls are shown in the bottom panels (FIG. 12A). FIGs. 12B-12D are bar graphs showing the results of naive CD8+T cells stimulated with aAPCs in the presence of blocking antibodies against CD 18, CD2, or both, and phenotypes evaluated after seven days. Cell proliferation (FIG. 12B), TCF1 expression (FIG. 12C), and CD95 expression (FIG. 12D) are shown. Isotype-treated and unstimulated controls are also included. Dotted lines indicate unstimulated levels. *p < 0.05, **p < 0.01, *** / ? < 0.001 determined using one-way ANOVA. n.s., not significant. Data are from three donors and shown as mean ± S.D.

[0039] FIGs. 13A-13H show aAPC-stimulated CD8+T cells exhibit enhanced responses and higher cytotoxicity compared to effector cells. Naive CD8+T cells were expanded with aAPCs or beads for 7 days. FIGs. 13A-13B are graphical representations of cytotoxicity of mesothelin (MSLN) CAR-T cells against parental MSLN+(FIG. 13 A) and MSLN KO (FIG. 13B) AsPC-1 cells. Data representative of three independent donors are shown (FIGs. 13A-13B). *p < 0.05 determined using two-way ANOVA. Data are shown as mean ± S.D. FIGs. 13C-13E show data for an in vivo TCR-T cell therapy model, wherein 5 * 104A375 cells were subcutaneously injected into NSG mice on day -13 and 1 x 106TCR- or ANGFR-transduced CD8+T cells were intravenously injected on day 0 (FIG. 13C). Tumor growth (FIG. 13D) and survival (FIG. 13E) are shown (n=12-13 / group). FIGs. 13F-13H show data for an in vivo CAR-T cell therapy model, wherein 5 * 105AsPC-1 cells were subcutaneously injected into NSG mice on day -7, followed by intravenous injection of 1 x 106MSLN CAR-transduced CD8+T cells on day 0 (FIG. 13F). Tumor growth (FIG. 13G) and survival (FIG. 13H) are shown (n=l 1-12 / group). Data are pooled from three independent experiments. (FIGs. 13D, 13E, 13G, and 13H). Tumor growth: **p < 0.01 determined using one-way ANOVA (FIGs. 13D and 13G). Data are shown as mean ± S.D. Survival: **p < 0.01, ***p < 0.001 determined using log-rank test.

[0040] FIGs. 14A-14E provide data showing aAPC-stimulated cells are enriched in epigenetic signatures associated with memory T cells. ATAC-seq analysis of chromatin accessibility in naive and day 10 aAPC- or bead-stimulated cells. FIGs. 14A-14C are genome browser tracks of chromatin accessibility at representative loci: LEF1 (FIG. 14A), CCR7 (FIG. 14B), and IRF4 (FIG. 14C). FIGs. 14D-14E are graphical representations of transcription factor binding motifs enriched in aAPC-stimulated cells (FIG. 14D) and bead-stimulated cells (FIG. 14E), identified by HOMER. Histograms represent motif frequencies within differentially accessible regions associated with aAPC or bead stimulated cells (FIGs. 14D and 14E).

[0041] FIGs. 15A-15D provide data showing aAPC stimulation does not induce CD3 signaling and promotes proliferation independently of CD3. Naive CD8+T cells were stimulated with aAPCs or ocCD3 (FIG. 15 A). FIG. 15A shows graphical representations of ZAP70 phosphorylation (pY319) assessed at multiple time points ranging from 1 to 60 minutes poststimulation. Data representative of three independent donors are shown (FIG. 15 A). Naive CD8+T cells were electroporated with CRISPR-Cas9 ribonucleoproteins targeting CD3Z or the safeharbor locus AAVS1 (negative control), followed by stimulation with aAPCs 10 days after electroporation (FIGs. 15B-15D). CD3 expression was assessed 10 days after electroporation forCD3Z knock out (FIG. 15B) or AAVS1 knock out (FIG. 15C). Proliferation was evaluated 7 days after aAPC-stimulation (FIG. 15D). Unstimulated cells cultured for 7 days were used as a negative control. Expansion of cell populations segregated by gene knockout and CD3 expression is shown as follows: CD3" (1) and CD3+(2) populations in CD3Z KO cells, and CD3+population (3) in AAVS1 KO cells (FIG. 15D). *p < 0.05, **p < 0.01, ***p < 0.001 determined using two-way ANOVA. n.s., not significant. Data are from two donors and shown as mean ± S.D.DETAILED DESCRIPTION

[0042] Adoptive T cell therapies generally use effector T cells ex vivo expanded using CD3 / CD28 stimulation, which inevitably makes them prone to exhaustion and dysfunction, thereby limiting therapeutic efficacy. Tscm cells, with their enhanced self-renewal and longevity, present an improved alternative for adoptive T cell therapy. However, the scarcity of Tscm in peripheral blood and the reliance of existing expansion methods on CD3 / CD28 stimulation pose significant challenges. Some aspects of the present disclosure are directed to a novel strategy to generate Tscm-like cells using CD28 signaling, without CD3 stimulation. In some aspects, the method comprises preparing a population of immune cells for a cell therapy, comprising contacting a population of source immune cells, e.g., T cells, NK cells, or TILs, with a CD28 agonist thereby producing a population of stimulated immune cells, wherein neither the population of source immune cells nor the population of stimulated immune cells is contacted with a CD3 agonist.

[0043] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to the particular compositions or process steps described, as such can, of course, vary. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0044] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.I. Terms

[0045] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0046] Throughout the disclosure, the term "a" or "an" entity refers to one or more of that entity; for example, "a chimeric polypeptide," is understood to represent one or more chimeric polypeptides. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. In addition, "or" is used to mean an open list of the components in the list. For example, “wherein X comprises A or B” means X comprises A, X comprises B, X comprises A and B, or X comprises A or B and any other components.

[0047] Furthermore, "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0048] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of' and / or "consisting essentially of are also provided.

[0049] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0050] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, unless otherwise explicitly stated.

[0051] Abbreviations used herein are defined throughout the present disclosure. Various aspects of the disclosure are described in further detail in the following subsections.

[0052] The terms “about” or “comprising essentially of’ refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, z.e., the limitations of the measurement system. For example, “about” or “comprising essentially of’ can mean within 1 or more than 1 standard deviation per the practice in the art. Alternatively, “about” or “comprising essentially of’ can mean a range of up to 10% (e.g., a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value)). For example, “about 55 mM,” as used herein, includes 49.5 mM to 60.5 mM. Furthermore, particularly with respect to biological systems or processes, the terms can mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of "about" or "comprising essentially of should be assumed to be within an acceptable error range for that particular value or composition.

[0053] As used herein, the term "approximately," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some aspects, the term "approximately," like the term, “about,” refers to a range of values that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0054] As described herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0055] As used herein, the term "immune cell" refers to a cell of the immune system. In some aspects, the immune cell is selected from a T lymphocyte ("T cell"), B lymphocyte ("B cell"), natural killer (NK) cell, natural killer T lymphocytes (NKT cells), macrophage, eosinophil, mast cell, dendritic cell or neutrophil. As used herein, a "population" of cells refers to a collection of more than one cell, e.g., a plurality of cells. In some aspects, the population of cells comprises more than one immune cell, e.g., a plurality of immune cells. In some aspects, the population of cells comprises a heterogeneous mixture of cells, comprising multiple types of cells, e.g., a heterogeneous mixture of immune cells and non-immune cells or a heterogeneous mixture ofimmune cells at different stages of differentiation. In some aspects, the population of cells comprises a plurality of T cells.

[0056] As used herein, the terms "T cell" and "T lymphocyte" are interchangeable and refer to any lymphocytes produced or processed by the thymus gland. Non-limiting classes of T cells include effector T cells and T helper (Th) cells (such as CD4+or CD8+T cells). In some aspects, the T cell is a Th 1 cell. In some aspects, the T cell is a Th2 cell. In some aspects, the T cell is a Tcl7 cell. In some aspects, the T cell is a Thl7 cell. In some aspects, the T cell is a Treg cell. In some aspects, the T cell is a tumor-infiltrating cell (TIL).

[0057] As used herein, the term "memory" T cells refers to T cells that have previously encountered and responded to their cognate antigen (e.g., in vivo, in vitro, or ex vivo) or which have been stimulated, e.g., with an anti-CD3 antibody (e.g., in vitro or ex vivo). Immune cells having a "memory-like" phenotype upon secondary exposure, such memory T cells can reproduce to mount a faster and strong immune response than during the primary exposure. In some aspects, memory T cells comprise central memory T cells (TCM cells), effector memory T cells (TEM cells), tissue resident memory T cells (TRM cells), stem cell-like memory T cells (TSCM cells), or any combination thereof.

[0058] As used herein, the term "stem cell-like memory T cells," "T memory stem cells," "Tscm" or "Tscm cells" refers to memory T cells that express at least CD95, CD45RA, CCR7, and CD62L and are endowed with the stem cell-like ability to self-renew and the multipotent capacity to reconstitute the entire spectrum of memory and effector T cell subsets.

[0059] As used herein, the term "central memory T cells" or "TCM cells" refers to memory T cells that express at least CD45RO, CCR7, and CD62L. Central memory T cells are generally found within the lymph nodes and in peripheral circulation.

[0060] As used herein, the term "effector memory T cells" or "TEM cells" refers to memory T cells that express at least CD45RO but lack expression of CCR7 and CD62L. Because effector memory T cells lack lymph node-homing receptors (e.g., CCR7 and CD62L), these cells are typically found in peripheral circulation and in non-lymphoid tissues.

[0061] As used herein, the term "tissue resident memory T cells" or "TRM cells" refers to memory T cells that do not circulate and remain resident in peripheral tissues, such as skin, lung, and gastrointestinal tract. In some aspects, tissue resident memory T cells are also effector memory T cells.

[0062] As used herein, the term "naive T cells" or "TN cells" refers to T cells that express at least CD45RA, CCR7, and CD62L, but which do not express CD95. TN cells represent the mostundifferentiated cell in the T cell lineage. The interaction between a TN cell and an antigen presenting cell (APC) induces differentiation of the TN cell towards an activated TEFF cell and an immune response.

[0063] As used herein, the term "Tscm-like" or "less differentiated" refers to an immune cell (e.g., a T cell or an NK cell) that expresses markers consistent with a more naive phenotype. For example, a Tscm-like cell can express one or more marker characteristic of a Tscm cell. In some aspects, a "Tscm-like" cell or a "less differentiated" cell expresses CD45RA, CCR7, and CD62L. In some aspects, the methods disclosed herein promote immune cells (e.g., T cells and / or NK cells) having a less-differentiated phenotype.

[0064] As used herein, the term "source immune cells" refers to a population of cells that is used at the start of a culture. A population of source immune cells can comprise any population of immune cells. In some aspects, the population of source immune cells comprises PBMCs. In some aspects, the population of source immune cells comprises CD8+ T cells. In some aspects, the population of source immune cells comprises CD4+ T cells. In some aspects, the population of source immune cells comprises CD8+ and CD4+ T cells. In some aspects, the population of source immune cells comprises tumor infiltrating lymphocytes. In some aspects, the population of source immune cells comprises a selected population enriched for CD8+ T cells. In some aspects, the population of source immune cells comprises a selected population enriched naive T cells (Tn), a selected population enriched for Tn and Tscm cells. In some aspects, the population of source immune cells comprises genetically engineered T cells (e.g., CAR T cells and / or TCR T cells).

[0065] As used herein the term "stimulated immune cell" refers to an immune cell that has been contacted with an antigen, a CD3 agonist, a CD28 agonist, or any other agent that activates an immune response in a cell.

[0066] As used herein the term "exhaustion" or "exhausted" refers to a state of immune cells following prolonged antigen exposure, wherein the immune cells lose their anti-tumor function.

[0067] The term "culturing" as used herein refers to the controlled growth of cells ex vivo and / or in vitro. As used herein, "culturing" includes the growth of cells, e.g., immune cells (e.g., T cells, NK cells, TILs), including engineered immune cells, during cell expansion and / or cell engineering (e.g., transduction with a construct for expressing a CAR or a TCR). In some aspects, the cultured immune cells are obtained from a subject, e.g., a human subject / patient. In some aspects, the cultured cells comprise immune cells obtained from a human subject. In some aspects, the cultured cells comprise one or more engineered immune cell disclosed herein. In some aspects,the cultured cells comprise T cells or NK cells obtained from a human subject / patient. In some aspects, the T cells and / or NK cells are purified prior to the culture. In some aspects, the T cells and / or NK cells are tumor-infiltrating T cells and / or NK cells. In some aspects, the cultured cells comprise one or more engineered immune cell disclosed herein.

[0068] The term "expand" or "expansion," as used herein in reference to immune cell culture refers to the process of stimulating or activating the cells and culturing the cells. The expansion process can lead to an increase in the proportion or the total number of desired cells, e.g., an increase in the proportion or total number of less differentiated immune cells, in a population of cultured cells, after the cells are stimulated or activated and cultured. Expansion does not require that all cell types in a population of cultured cells are increased in number. Rather, in some aspects, only a subset of cells in a population of cultured cells are increased in number during expansion, while the number of other cell types may not change or may decrease.

[0069] As used herein, the term "yield" refers to the total number of cells following a culture method or a portion thereof. In some aspects, the term "yield" refers to a particular population of cells, e.g., stem-like T cells in a population of T cells. The yield can be determined using any methods, including, but not limited to, estimating the yield based on a representative sample.

[0070] As used herein, "administering" refers to the physical introduction of a therapeutic agent or a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems. The different routes of administration for a therapeutic agent described herein (e.g., an immune cell cultured according to the methods disclosed herein) include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion. Administering can be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0071] "Treatment" or "therapy" (including any grammatical derivatives thereof) of a subj ect refers to any type of intervention or process performed on, or the administration of an active agent to, a subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical indicia associated with a disease. In some aspects, the term refers to inducing an immune response in a subject against an antigen.

[0072] The terms "prevent," "preventing," and variants thereof as used herein, refer partially or completely delaying onset of an disease, disorder and / or condition; partially or completely delaying onset of one or more symptoms, features, or clinical manifestations of aparticular disease, disorder, and / or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular disease, disorder, and / or condition; partially or completely delaying progression from a particular disease, disorder and / or condition; and / or decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some aspects, preventing an outcome is achieved through prophylactic treatment.

[0073] As used herein, "cell engineering" or "cell modification" (including derivatives thereof) refers to the targeted modification of a cell, e.g., an immune cell disclosed herein. In some aspects, the cell engineering comprises viral genetic engineering, non-viral genetic engineering, introduction of receptors to allow for tumor specific targeting (e.g., a chimeric antigen receptor (CAR) and / or a TCR) introduction of one or more endogenous genes that improve immune cell (e.g., T cell) function, introduction of one or more synthetic genes that improve immune cell (e.g., T cell) function, or any combination thereof.

[0074] As used herein, the term "antigen" refers to any natural or synthetic immunogenic substance, such as a protein, peptide, or hapten. As used herein, the term "cognate antigen" refers to an antigen which an immune cell (e.g., T cell) recognizes and thereby, induces the activation of the immune cell (e.g., triggering intracellular signals that induce effector functions, such as cytokine production, and / or for proliferation of the cell). In some aspects, the antigen comprises a tumor antigen. In some aspects, the antigen comprises a neoantigen.

[0075] A "cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. "Cancer" as used herein comprises primary, metastatic and recurrent cancers. Unless indicated otherwise, the terms "cancer" and "tumor" can be used interchangeably.

[0076] The term "hematological malignancy" or "hematological cancer" refers to mammalian cancers and tumors of the hematopoietic and lymphoid tissues. Non-limiting examples of hematological malignancies include those affecting tissues of the blood, bone marrow, lymph nodes, and lymphatic system, including acute lymphoblastic leukemia (ALL), chronic lymphocytic lymphoma (CLL), small lymphocytic lymphoma (SLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CIVIL), acute monocytic leukemia (AMoL), Hodgkin's lymphoma, and non-Hodgkin's lymphomas. Hematological malignancies can also be referred to as "liquid tumors." Liquid tumor cancers include, but are not limited to, leukemias, myelomas, and lymphomas, as well as other hematological malignancies.

[0077] A "solid tumor," as used herein, refers to an abnormal mass of tissue. Solid tumors may be benign or malignant. Nonlimiting examples of solid tumors include sarcomas, carcinomas, and lymphomas, such as cancers of the lung, breast, prostate, colon, rectum, and bladder. The tissue structure of a solid tumor includes interdependent tissue compartments including the parenchyma (cancer cells) and the supporting stromal cells in which the cancer cells are dispersed, and which may provide a supporting microenvironment.

[0078] In some aspects, the cancer is selected from adrenal cortical cancer, advanced cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, brain tumors, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon / rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft tissue, basal and squamous cell skin cancer, melanoma, small intestine cancer, stomach cancer, testicular cancer, throat cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor and secondary cancers caused by cancer treatment. In some aspects, the cancer is selected from chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, myxoid / round cell liposarcoma, or telangiectaltic sarcoma. In someaspects, the cancer is selected from acra-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanomajuvenile melanoma, lentigo maligna melanoma, malignant melanoma, metastatic melanoma, nodular melanoma, subungal melanoma, or superficial spreading melanoma. In some aspects, the cancer is selected from acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidemoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, small-cell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, or carcinoma viflosum. In some aspects, the cancer is selected from Leukemia, Hodgkin's Disease, Non- Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer,malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, papillary thyroid cancer, neuroblastoma, neuroendocrine cancer, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, prostate cancer, Mullerian cancer, ovarian cancer, peritoneal cancer, fallopian tube cancer, or uterine papillary serous carcinoma. In some aspects, the cancer is selected from metastatic melanoma, non-small cell lung cancer, myeloma, esophageal cancer, synovial sarcoma, myxoid / round cell liposarcoma, gastric cancer, breast cancer, hepatocellular cancer, head and neck cancer, ovarian cancer, prostate cancer, bladder cancer, or any combination thereof.

[0079] As used herein, the term "immune response" refers to a biological response within a vertebrate against foreign agents, which response protects the organism against these agents and diseases caused by them. An immune response is mediated by the action of a cell of the immune system (e.g., a T lymphocyte, B lymphocyte, natural killer (NK) cell, NKT cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and / or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell or a Th cell, such as a CD4+or CD8+T cell, or the inhibition of a Treg cell.

[0080] As used herein, the term "anti-tumor immune response" refers to an immune response against a tumor antigen.

[0081] A "subject" includes any human or nonhuman animal. The term "nonhuman animal" includes, but is not limited to, vertebrates such as nonhuman primates, sheep, dogs, and rodents such as mice, rats and guinea pigs. In some aspects, the subject is a human. The terms "subject," "patient," "individual," and "host" are used interchangeably herein. As used herein, the phrase "subject in need thereof includes subjects, such as mammalian subjects, that would benefit, e.g., from administration of immune cells, e.g., modified to express a CAR or TCR, and cultured using the methods provided herein, to control tumor growth.

[0082] The term "therapeutically effective amount" or "therapeutically effective dosage" refers to an amount of an agent (e.g., an immune cell modified to express a c-Jun polypeptide and a chimeric binding protein, and cultured as described herein) that provides the desired biological, therapeutic, and / or prophylactic result. That result can be reduction, amelioration, palliation,lessening, delaying, and / or alleviation of one or more of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In reference to solid tumors, an effective amount comprises an amount sufficient to cause a tumor to shrink and / or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some aspects, an effective amount is an amount sufficient to delay tumor development. In some aspects, an effective amount is an amount sufficient to prevent or delay tumor recurrence. An effective amount can be administered in one or more administrations.

[0083] The effective amount of the composition (e.g., immune cultured as described herein) can, for example, (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, delay, slow to some extent and can stop cancer cell infiltration into peripheral organs; (iv) inhibit (z.e., slow to some extent and can stop tumor metastasis); (v) inhibit tumor growth; (vi) prevent or delay occurrence and / or recurrence of tumor; and / or (vii) relieve to some extent one or more of the symptoms associated with the cancer.

[0084] In some aspects, a "therapeutically effective amount" is the amount of a composition disclosed herein (e.g., an immune cell cultured as described herein), which is clinically proven to effect a significant decrease in cancer or slowing of progression (regression) of cancer, such as an advanced solid tumor. The ability of a therapeutic agent of the present disclosure (e.g., an immune cell modified and cultured as described herein) to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0085] The terms "effective" and "effectiveness" with regard to a treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a composition disclosed herein (e.g., immune cells cultured as described herein) to promote cancer regression in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ, and / or organism level (adverse effects) resulting from administration of a composition disclosed herein (e.g., immune cells cultured as described herein).

[0086] The terms "chimeric antigen receptor" and "CAR," as used herein, refer to a set of polypeptides, typically two in the simplest form, which when in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and with intracellular signal generation. In some aspects, a CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "anintracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and / or costimulatory molecule as defined below. In some aspects, the set of polypeptides are in the same polypeptide chain, e.g., comprise a chimeric fusion protein. In some aspects, the set of polypeptides are not contiguous with each other, e.g., are in different polypeptide chains. In some aspects, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen-binding domain to an intracellular signaling domain. In some aspects, the stimulatory molecule of the CAR is the zeta chain associated with the T cell receptor complex (e.g., CD3 zeta). In some aspects, the cytoplasmic signaling domain comprises a primary signaling domain (e.g., a primary signaling domain of CD3-zeta). In some aspects, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In some aspects, the costimulatory molecule is chosen from the costimulatory molecules described herein, e.g., CD-28, 4-1BB (i.e., CD137), and / or CD27.

[0087] In some aspects, the CAR comprises a chimeric fusion protein comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule, wherein the antigen-binding domain and the transmembrane domain are linked by a CAR spacer. In some aspects, the CAR comprises a chimeric fusion protein comprising an antigen-binding domain linked to a transmembrane domain via a CAR spacer and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some aspects, the CAR comprises a chimeric fusion protein comprising an antigen-binding domain linked to a transmembrane domain via a CAR spacer and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some aspects, the CAR comprises a chimeric fusion protein comprising an antigen-binding domain linked to a transmembrane domain via a CAR spacer and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some aspects, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR. In some aspects, the CAR further comprises a leader sequence at the N-terminus of the antigen-binding domain, wherein the leader sequence is optionally cleaved from the antigen-binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane.

[0088] The antigen-specific extracellular domain of a chimeric antigen receptor recognizes and specifically binds an antigen, typically a surface-expressed antigen of a malignancy. An antigen-specific extracellular domain specifically binds an antigen when, for example, it binds the antigen with an affinity constant or affinity of interaction (KD) between about 0.1 pM to about 10 pM, for example, about 0.1 pM to about 1 pM or about 0.1 pM to about 100 nM. Methods for determining the affinity of interaction are known in the art. An antigen-specific extracellular domain suitable for use in a CAR of the present disclosure can be any antigen-binding polypeptide, a wide variety of which are known in the art. In some aspects, the antigen-binding domain is a single chain Fv (scFv). Other antibody-based recognition domains such as cAb VHH (camelid antibody variable domains) and humanized versions thereof, IgNAR VH (shark antibody variable domains) and humanized versions thereof, sdAb VH (single domain antibody variable domains), and "camelized" antibody variable domains are also suitable for use in a CAR of the present disclosure. In some aspects, T cell receptor (TCR) based recognition domains, such as single chain TCR (scTv, i.e., single chain two-domain TCR containing VaVP) are also suitable for use in the chimeric binding proteins of the present disclosure.

[0089] As used herein, the term "T cell receptor" or "TCR" refers to a heterodimer composed of 2 different transmembrane polypeptide chains: an a chain and a P chain, each consisting of a constant region, which anchors the chain inside the T-cell surface membrane, and a variable region, which recognizes and binds to the antigen presented by MHCs. The TCR complex is associated with 6 polypeptides forming 2 heterodimers, CD3ys and CD35s, and 1 homodimer CD3which together forms the CD3 complex. T-cell receptor-engineered T-cell therapy utilizes the modification of T cells that retain these complexes to specifically target the antigens expressed by particular tumor cells. As used herein, the term "TCR" includes naturally occurring TCRs and engineered TCRs.

[0090] A "TCR mimic" or a "TCRm" refers to a type of antibody that recognize epitopes comprising both the peptide and the MHC-I molecule, similar to the recognition of such complexes by the TCR on T cells.

[0091] The terms "nucleic acids," "nucleic acid molecules, "nucleotides," "nucleotide(s) sequence," and "polynucleotide" can be used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Single stranded nucleic acid sequencesrefer to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double stranded DNA- DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes doublestranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes. In discussing the structure of particular doublestranded DNA molecules, sequences can be described herein according to the normal convention of giving only the sequence in the 5’ to 3’ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi -synthetic DNA. A "nucleic acid composition" of the disclosure comprises one or more nucleic acids as described herein. As described herein, in some aspects, a polynucleotide of the present disclosure can comprise a single nucleotide sequence encoding a single protein (e.g., codon-optimized c-Jun nucleotide sequence) ("monocistronic"). In some aspects, a polynucleotide of the present disclosure is polycistronic (i.e., comprises two or more cistrons). In some aspects, each of the cistrons of a polycistronic polynucleotide can encode for a protein disclosed herein (e.g., c-Jun protein, chimeric binding protein, or EGFRt). In some aspects, each of the cistrons can be translated independently of one another.

[0092] As used herein, the term “polypeptide” encompasses both peptides and proteins, unless indicated otherwise. Polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a single polypeptide or can be a multi- molecular complex such as a dimer, trimer or tetramer. They can also comprise single chain or multichain polypeptides. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid. In some aspects, a "peptide" can be less than or equal to 50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0093] As used herein, the term "fragment" or "portion" of a polypeptide (e.g., an antigenbinding fragment or portion of an antibody) refers to an amino acid sequence of a polypeptide that is shorter than the naturally-occurring sequence, N- and / or C-terminally deleted or any part of the polypeptide deleted in comparison to the naturally occurring polypeptide. Thus, a fragment doesnot necessary need to have only N- and / or C- terminal amino acids deleted. A polypeptide in which internal amino acids have been deleted with respect to the naturally occurring sequence is also considered a fragment.

[0094] A "recombinant" polypeptide or protein refers to a polypeptide or protein produced via recombinant DNA technology. Recombinantly produced polypeptides and proteins expressed in engineered host cells are considered isolated for the purpose of the disclosure, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0095] The term "expression" as used herein refers to a process by which a polynucleotide produces a gene product. It includes, without limitation, transcription of the polynucleotide into messenger RNA (mRNA) and the translation of an mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g. , methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.

[0096] As used herein, the terms "isolated," "purified," "extracted," and grammatical variants thereof are used interchangeably and refer to the state of a preparation of desired composition of the present disclosure that has undergone one or more processes of purification. In some aspects, isolating or purifying as used herein is the process of removing, partially removing (e.g, a fraction) of a composition of the present disclosure.

[0097] As used herein, the terms "ug" and "uM" are used interchangeably with "pg" and "pM," respectively.

[0098] Various aspects of the disclosure are described in further detail in the following subsections.II. Methods of the Disclosure

[0099] Some aspects of the present disclosure are directed to methods of preparing a population of immune cells for a cell therapy, comprising contacting a population of source immune cells with a CD28 agonist thereby producing a population of stimulated immune cells. In some aspects, in the method, neither the population of source immune cells nor the population of stimulated immune cells is contacted with a CD3 agonist.

[0100] In some aspects, the method further comprises introducing into one or more cells of the population of source immune cells a heterologous nucleic acid molecule encoding a CAR prior to contacting the source immune cells with the CD28 agonist. In some aspects, the method further comprises introducing into one or more cells of the population of source immune cells a heterologous nucleic acid molecule encoding a TCR prior to contacting the source immune cells with the CD28 agonist.

[0101] In some aspects, the present methods are directed to a method of stimulating a population of immune cells in preparation of an immune cell therapy, comprising: (i) culturing a population of source immune cells in a medium comprising a CD28 agonist, but not comprising a CD3 agonist, thereby generating a population of stimulated immune cells.

[0102] In some aspects, the method further comprises introducing into one or more cells of the population of stimulated immune cells a heterologous nucleic acid molecule encoding a CAR. In some aspects, the method further comprises introducing into one or more cells of the population of stimulated immune cells a heterologous nucleic acid molecule encoding a TCR.

[0103] Some aspects of the present disclosure are directed to a method of preparing a population of genetically modified immune cells for an immune cell therapy, comprising: (i) contacting a population of source immune cells with a CD28 agonist thereby generating a population of stimulated immune cells, and (ii) introducing into one or more cells of the population of stimulated immune cells a heterologous nucleic acid molecule encoding a CAR; wherein neither the population of source immune cells nor the population of stimulated immune cells is contacted with a CD3 agonist.

[0104] Some aspects of the present disclosure are directed to a method of preparing a population of genetically modified immune cells for an immune cell therapy, comprising: (i) contacting a population of source immune cells with a CD28 agonist thereby generating a population of stimulated immune cells, and (ii) introducing into one or more cells of the population of stimulated immune cells a heterologous nucleic acid molecule encoding a TCR; wherein neither the population of source immune cells nor the population of stimulated immune cells is contacted with a CD3 agonist.

[0105] In some aspects, the present methods are directed to a method of stimulating a population of genetically modified immune cells for an immune cell therapy, comprising: (i) culturing a population of source immune cells in medium comprising a CD28 agonist, but not comprising a CD3 agonist, thereby generating a population of stimulated immune cells, and (ii)introducing into the medium comprising one or more cells of the population of stimulated immune cells a heterologous nucleic acid molecule encoding a CAR or a TCR.ILA. Immune Cells

[0106] Any immune cells can be used as the population of source immune cells. In some aspects, the population of source immune cells comprises T cells. In some aspects, the population of source immune cells comprises NK cells. In some aspects, the population of source immune cells comprises T cells and NK cells. In some aspects, the population of source immune cells comprises a[3 T cells, y5 T cells, cytotoxic T cells, helper T cells, regulatory T cells (Treg cells), or any combination thereof. In some aspects, the population of source immune cells comprises a[3 T cells. In some aspects, the population of source immune cells comprises y5 T cells. In some aspects, the population of source immune cells comprises cytotoxic T cells. In some aspects, the population of source immune cells comprises helper T cells. In some aspects, the population of source immune cells comprises regulatory T cells (Treg cells).

[0107] In some aspects, the population of source immune cells comprises tumor infiltrating lymphocytes (TILs). In some aspects, the population of source immune cells comprises TILs obtained from a tumor obtained from a human subject. In some aspects, the population of source immune cells comprises a dissociated tumor sample.

[0108] In some aspects, the population of source immune cells comprises CD8+ T cells. In some aspects, the population of source immune cells comprises CD4+ T cells. In some aspects, the population of source immune cells comprises CD8+ T cells and CD4+ T cells. In some aspects, the population of source immune cells comprises CD8+ T cells and CD4+ T cells at a ratio of about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8. 1 :9, or l : 10 CD8+ T cells:CD4+ T cells. In some aspects, the population of source immune cells comprises an about 1 : 1 ratio of CD8+ T cells to CD4+ T cells.

[0109] In some aspects, the population of source immune cells comprises a mix of a selected population of CD8+ T cells and a selected population of CD4+ T cells, wherein the two selected populations are combined to arrive at a mixed population of CD8+ T cells and CD4+ T cells at a desired ration, e.g. , 1 : 1.

[0110] In some aspects, the population of source immune cells comprises immune cells that have been enriched for naive T or Tscm cells by selecting for immune cells expressing one or more marker expressed by naive T or Tscm cells. In some aspects, the population of source immune cells is prepared by isolating naive CD8+ immune cells (e.g., naive CD8+ T cells) from PBMCs.In some aspects, the population of source immune cells comprises T cells that have been selected for positive expression of CD62L, CCR7, CD95, and / or CD45RA. In some aspects, the population of source immune cells comprises T cells that have been selected for positive expression of CD62L. In some aspects, the population of source immune cells comprises T cells that have been selected for positive expression of CCR7. In some aspects, the population of source immune cells comprises T cells that have been selected for positive expression of CD95. In some aspects, the population of source immune cells comprises T cells that have been selected for positive expression of CD45RA. In some aspects, the population of source immune cells comprises T cells that have been selected for positive expression of CD45RA and CCR7. In some aspects, the population of source immune cells comprises T cells that have been selected for positive expression of CD45RA, CCR7, and CD95. In some aspects, the population of source immune cells comprises T cells that have been selected for positive expression of CD45RA, CCR7, CD95, and CD62L.

[0111] In some aspects, the population of source immune cells is prepared by isolating naive CD8+ immune cells (e.g., naive CD8+ T cells) from PBMCs. In some aspects, the population of source immune cells comprises T cells that have been selected for positive expression of (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, the population of source immune cells comprises T cells that have been selected for positive expression of (i) CD8 and (ii) CD62L. In some aspects, the population of source immune cells comprises T cells that have been selected for positive expression of (i) CD8 and (ii) CCR7. In some aspects, the population of source immune cells comprises T cells that have been selected for positive expression of (i) CD8 and (ii) CD95. In some aspects, the population of source immune cells comprises T cells that have been selected for positive expression of (i) CD8 and (ii) CD45RA. In some aspects, the population of source immune cells comprises T cells that have been selected for positive expression of (i) CD8, (ii) CD45RA, and (iii) CCR7. In some aspects, the population of source immune cells comprises T cells that have been selected for positive expression of (i) CD8, (ii) CD45RA, (iii) CCR7, and (iv) CD95. In some aspects, the population of source immune cells comprises T cells that have been selected for positive expression of (i) CD8, (ii) CD45RA, (ii) CCR7, (iii) CD95, and (iv) CD62L.

[0112] In some aspects, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 10% of the immune cells in the population of source immune cells(e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 15% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 20% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 25% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 30% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 35% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 40% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 45% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 50% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 60% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 70% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 75% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 80% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 90% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 95% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 96% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 97% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 98% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells). In some aspects, at least about 99% of the immune cells in the population of source immune cells (e.g., T cells) are naive CD8+ immune cells (e.g., CD8+ naive T cells).

[0113] In some aspects, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 10% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 15% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 20% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 25% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 30% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 35% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 40% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 45% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 50% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 60% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 70% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 75% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 80% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 90% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 95% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 96% of the immune cellsin the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 97% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 98% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA. In some aspects, at least about 99% of the immune cells in the population of source immune cells (e.g., T cells) express (i) CD8 and (ii) CD62L, CCR7, CD95, and / or CD45RA.

[0114] In some aspects, the population of source immune cells do not comprise one or more cells that were selected based on the expression of one or more marker. In some aspects, the population of source immune cells comprises PBMCs.

[0115] In some aspects, the population of source immune cells comprises immune cells that have been previously expanded ex vivo, wherein the prior expansion did not comprise contacting the immune cells with a CD3 agonist. In some aspects, the population of source immune cells is expanded prior to the contacting with the CD28 agonist, wherein the prior expansion does not comprise contacting the immune cells with a CD3 agonist.

[0116] In some aspects, the population of source immune cells comprises immune cells that have not been previously expended ex vivo. In some aspects, the population of source immune cells does not comprise immune cells that have been previous expended ex vivo.

[0117] In some aspects, the resulting immune cells, e.g., the population of stimulated immune cells, e.g., T cells, cultured using the methods disclosed herein, exhibit an increased number of Tscm-like cells relative to a population of immune cells cultured using conventional methods, e.g., immune cells stimulated with a CD3 agonist alone or in combination with a CD28 agonist. In some aspects, the resulting immune cells, e.g., the population of stimulated immune cells, e.g., T cells, cultured using the methods disclosed herein, exhibit an increased proportion of Tscm-like cells relative to the proportion of Tscm-like cells in the population of source immune cells. In some aspects, the population of stimulated immune cells exhibit increased expression of markers characteristic of stem-like cells relative to the population of source immune cells (i.e., prior to the culturing.

[0118] In some aspects, the number of Tscm-like cells in the culture is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, relative to the number of Tscm-like cells in the population of source immune cells. In some aspects,the number of Tscm-like cells in the culture is increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, or at least about 20-fold, relative to the number of Tscm-like cells in the population of source immune cells.

[0119] In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, Tscm-like T cells constitute at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the total number of T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm-like T cells constitute at least about 50% of the total number of T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm-like T cells constitute at least about 60% of the total number of T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm-like T cells constitute at least about 70% of the total number of T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm- like T cells constitute at least about 75% of the total number of T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm-like T cells constitute at least about 80% of the total number of T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm-like T cells constitute at least about 85% of the total number of T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm-like T cells constitute at least about 90% of the total number of T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm-like T cells constitute at least about 95% of the total number of T cells in the population of stimulated immune cells.

[0120] In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, Tscm-like T cells constitute at least about 50%, at least about 55%, at least about 60%, at least about 65%, at leastabout 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the total number of CD8+T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm- like T cells constitute at least about 50% of the total number of CD8+ T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm-like T cells constitute at least about 60% of the total number of CD8+ T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm- like T cells constitute at least about 70% of the total number of CD8+ T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm-like T cells constitute at least about 75% of the total number of CD8+ T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm- like T cells constitute at least about 80% of the total number of CD8+ T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm-like T cells constitute at least about 85% of the total number of CD8+ T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm- like T cells constitute at least about 90% of the total number of CD8+ T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm-like T cells constitute at least about 95% of the total number of CD8+ T cells in population of stimulated immune cells.

[0121] In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, Tscm-like T cells constitute at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the total number of CD4+T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm- like T cells constitute at least about 50% of the total number of CD4+ T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm-like T cells constitute at least about 60% of thetotal number of CD4+ T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm- like T cells constitute at least about 70% of the total number of CD4+ T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm-like T cells constitute at least about 75% of the total number of CD4+ T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm- like T cells constitute at least about 80% of the total number of CD4+ T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm-like T cells constitute at least about 85% of the total number of CD4+ T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm- like T cells constitute at least about 90% of the total number of CD4+ T cells in the population of stimulated immune cells. In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, Tscm-like T cells constitute at least about 95% of the total number of CD4+ T cells in population of stimulated immune cells.

[0122] In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, z.e., following the contacting with the CD28 agonist, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD45RA. In some aspects, following the contacting with the CD28 agonist, at least about 50% of the immune cells in the population of stimulated immune cells express CD45RA. In some aspects, following the contacting with the CD28 agonist, at least about 55% of the immune cells in the population of stimulated immune cells express CD45RA. In some aspects, following the contacting with the CD28 agonist, at least about 60% of the immune cells in the population of stimulated immune cells express CD45RA. In some aspects, following the contacting with the CD28 agonist, at least about 65% of the immune cells in the population of stimulated immune cells express CD45RA. In some aspects, following the contacting with the CD28 agonist, at least about 70% of the immune cells in the population of stimulated immune cells express CD45RA. In some aspects, following the contacting with the CD28 agonist, at least about 75% of the immune cells in the population of stimulated immune cells express CD45RA. In some aspects, following the contacting with the CD28 agonist, at least about 80% of the immune cellsin the population of stimulated immune cells express CD45RA. In some aspects, following the contacting with the CD28 agonist, at least about 85% of the immune cells in the population of stimulated immune cells express CD45RA. In some aspects, following the contacting with the CD28 agonist, at least about 90% of the immune cells in the population of stimulated immune cells express CD45RA. In some aspects, following the contacting with the CD28 agonist, at least about 95% of the immune cells in the population of stimulated immune cells express CD45RA.

[0123] In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, z.e., following the contacting with the CD28 agonist, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 50% of the immune cells in the population of stimulated immune cells express CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 55% of the immune cells in the population of stimulated immune cells express CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 60% of the immune cells in the population of stimulated immune cells express CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 65% of the immune cells in the population of stimulated immune cells express CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 70% of the immune cells in the population of stimulated immune cells express CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 75% of the immune cells in the population of stimulated immune cells express CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 80% of the immune cells in the population of stimulated immune cells express CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 85% of the immune cells in the population of stimulated immune cells express CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 90% of the immune cells in the population of stimulated immune cells express CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 95% of the immune cells in the population of stimulated immune cells express CD62L.

[0124] In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, z.e., following the contacting with the CD28 agonist, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 50% of the immune cells in the population of stimulated immune cells express CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 55% of the immune cells in the population of stimulated immune cells express CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 60% of the immune cells in the population of stimulated immune cells express CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 65% of the immune cells in the population of stimulated immune cells express CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 70% of the immune cells in the population of stimulated immune cells express CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 75% of the immune cells in the population of stimulated immune cells express CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 80% of the immune cells in the population of stimulated immune cells express CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 85% of the immune cells in the population of stimulated immune cells express CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 90% of the immune cells in the population of stimulated immune cells express CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 95% of the immune cells in the population of stimulated immune cells express CCR7.

[0125] In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, z.e., following the contacting with the CD28 agonist, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD95. In some aspects, following the contacting with the CD28 agonist, at least about 50% of the immune cells in the population of stimulated immune cells express CD95. In some aspects, following the contacting with the CD28 agonist, at least about 55% of the immune cells in the population of stimulated immune cells express CD95. In some aspects, following the contacting with the CD28 agonist, at least about 60% of the immune cells in the population of stimulated immune cells express CD95. In some aspects, following the contacting with the CD28 agonist, at least about 65% of the immune cells in the population of stimulated immune cells express CD95. In some aspects, following the contacting with the CD28 agonist, at least about 70% of the immune cells in the population of stimulated immune cellsexpress CD95. In some aspects, following the contacting with the CD28 agonist, at least about 75% of the immune cells in the population of stimulated immune cells express CD95. In some aspects, following the contacting with the CD28 agonist, at least about 80% of the immune cells in the population of stimulated immune cells express CD95. In some aspects, following the contacting with the CD28 agonist, at least about 85% of the immune cells in the population of stimulated immune cells express CD95. In some aspects, following the contacting with the CD28 agonist, at least about 90% of the immune cells in the population of stimulated immune cells express CD95. In some aspects, following the contacting with the CD28 agonist, at least about 95% of the immune cells in the population of stimulated immune cells express CD95.

[0126] In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, z.e., following the contacting with the CD28 agonist, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD45RA and CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 50% of the immune cells in the population of stimulated immune cells express CD45RA and CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 55% of the immune cells in the population of stimulated immune cells express CD45RA and CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 60% of the immune cells in the population of stimulated immune cells express CD45RA and CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 65% of the immune cells in the population of stimulated immune cells express CD45RA and CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 70% of the immune cells in the population of stimulated immune cells express CD45RA and CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 75% of the immune cells in the population of stimulated immune cells express CD45RA and CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 80% of the immune cells in the population of stimulated immune cells express CD45RA and CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 85% of the immune cells in the population of stimulated immune cells express CD45RA and CCR7. In some aspects, following the contacting with the CD28 agonist, at least about 90% of the immune cells in the population of stimulated immune cells express CD45RA and CCR7. In some aspects, following the contacting with the CD28 agonist, atleast about 95% of the immune cells in the population of stimulated immune cells express CD45RA and CCR7.

[0127] In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, z.e., following the contacting with the CD28 agonist, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, and CD95. In some aspects, following the contacting with the CD28 agonist, at least about 50% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, and CD95. In some aspects, following the contacting with the CD28 agonist, at least about 55% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, and CD95. In some aspects, following the contacting with the CD28 agonist, at least about 60% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, and CD95. In some aspects, following the contacting with the CD28 agonist, at least about 65% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, and CD95. In some aspects, following the contacting with the CD28 agonist, at least about 70% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, and CD95. In some aspects, following the contacting with the CD28 agonist, at least about 75% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, and CD95. In some aspects, following the contacting with the CD28 agonist, at least about 80% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, and CD95. In some aspects, following the contacting with the CD28 agonist, at least about 85% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, and CD95. In some aspects, following the contacting with the CD28 agonist, at least about 90% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, and CD95. In some aspects, following the contacting with the CD28 agonist, at least about 95% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, and CD95.

[0128] In some aspects, following culture of immune cells (e.g., T cells) according to the methods disclosed herein, z.e., following the contacting with the CD28 agonist, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the populationof stimulated immune cells express CD45RA, CCR7, CD95, and CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 50% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, CD95, and CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 55% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, CD95, and CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 60% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, CD95, and CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 65% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, CD95, and CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 70% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, CD95, and CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 75% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, CD95, and CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 80% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, CD95, and CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 85% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, CD95, and CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 90% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, CD95, and CD62L. In some aspects, following the contacting with the CD28 agonist, at least about 95% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, CD95, and CD62L.

[0129] In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof, in the population of stimulated immune cells is increased by at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5- fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, or at least about 20-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof, in the population of stimulated immune cells is increased by at least about 1- fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof in the population of source immune cells. In some aspects, the percent ofimmune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof, in the population of stimulated immune cells is increased by at least about 1.5-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof, in the population of stimulated immune cells is increased by at least about 2-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof, in the population of stimulated immune cells is increased by at least about 3-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof, in the population of stimulated immune cells is increased by at least about 4-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof, in the population of stimulated immune cells is increased by at least about 5-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof, in the population of stimulated immune cells is increased by at least about 6-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof, in the population of stimulated immune cells is increased by at least about 7-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof, in the population of stimulated immune cells is increased by at least about 8-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof, in the population of stimulated immune cells is increased by at least about 9-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof in the population of source immune cells. In some aspects, thepercent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof, in the population of stimulated immune cells is increased by at least about 10-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof, in the population of stimulated immune cells is increased by at least about 15-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof, in the population of stimulated immune cells is increased by at least about 20-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof in the population of source immune cells.

[0130] In some aspects, the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of stimulated immune cells is increased by at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5- fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, or at least about 20-fold, relative to the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of stimulated immune cells is increased by at least about 1-fold, relative to the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of stimulated immune cells is increased by at least about 1.5-fold, relative to the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of stimulated immune cells is increased by at least about 2-fold, relative to the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of stimulated immune cells is increased by at least about 3-fold, relative to the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of stimulated immune cells is increased by at least about 4-fold, relative to the percent of immune cells that express CD45RA, CCR7, and CD95 in the populationof source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of stimulated immune cells is increased by at least about 5-fold, relative to the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of stimulated immune cells is increased by at least about 6-fold, relative to the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of stimulated immune cells is increased by at least about 7-fold, relative to the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of stimulated immune cells is increased by at least about 8-fold, relative to the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of stimulated immune cells is increased by at least about 9-fold, relative to the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of stimulated immune cells is increased by at least about 10-fold, relative to the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of stimulated immune cells is increased by at least about 15-fold, relative to the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of stimulated immune cells is increased by at least about 20-fold, relative to the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of source immune cells.

[0131] In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L, in the population of stimulated immune cells is increased by at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, or at least about 20-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of source immune cells. In some aspects, the percent of immune cells that expressCD45RA, CCR7, CD95, and CD62L, in the population of stimulated immune cells is increased by at least about 1-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L, in the population of stimulated immune cells is increased by at least about 1.5-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L, in the population of stimulated immune cells is increased by at least about 2-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L, in the population of stimulated immune cells is increased by at least about 3-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L, in the population of stimulated immune cells is increased by at least about 4-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L, in the population of stimulated immune cells is increased by at least about 5-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L, in the population of stimulated immune cells is increased by at least about 6-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L, in the population of stimulated immune cells is increased by at least about 7-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L, in the population of stimulated immune cells is increased by at least about 8-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L, in the population of stimulated immune cells is increased by at least about 9-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L, in the population of stimulated immune cells is increased byat least about 10-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L, in the population of stimulated immune cells is increased by at least about 15-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of source immune cells. In some aspects, the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L, in the population of stimulated immune cells is increased by at least about 20-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of source immune cells.

[0132] In some aspects, following culturing the immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express TCF1. In some aspects, following the contacting with the CD28 agonist, at least about 50% of the immune cells in the population of stimulated immune cells express TCF1. In some aspects, following the contacting with the CD28 agonist, at least about 55% of the immune cells in the population of stimulated immune cells express TCF1. In some aspects, following the contacting with the CD28 agonist, at least about 60% of the immune cells in the population of stimulated immune cells express TCF1. In some aspects, following the contacting with the CD28 agonist, at least about 65% of the immune cells in the population of stimulated immune cells express TCF1. In some aspects, following the contacting with the CD28 agonist, at least about 70% of the immune cells in the population of stimulated immune cells express TCF 1. In some aspects, following the contacting with the CD28 agonist, at least about 75% of the immune cells in the population of stimulated immune cells express TCF1. In some aspects, following the contacting with the CD28 agonist, at least about 80% of the immune cells in the population of stimulated immune cells express TCF1. In some aspects, following the contacting with the CD28 agonist, at least about 85% of the immune cells in the population of stimulated immune cells express TCF1. In some aspects, following the contacting with the CD28 agonist, at least about 90% of the immune cells in the population of stimulated immune cells express TCF1. In some aspects, following the contacting with the CD28 agonist, at least about 95% of the immune cells in the population of stimulated immune cells express TCF1.

[0133] In some aspects, following culturing the immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, at least about50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 50% of the immune cells in the population of stimulated immune cells express KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 55% of the immune cells in the population of stimulated immune cells express KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 60% of the immune cells in the population of stimulated immune cells express KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 65% of the immune cells in the population of stimulated immune cells express KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 70% of the immune cells in the population of stimulated immune cells express KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 75% of the immune cells in the population of stimulated immune cells express KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 80% of the immune cells in the population of stimulated immune cells express KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 85% of the immune cells in the population of stimulated immune cells express KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 90% of the immune cells in the population of stimulated immune cells express KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 95% of the immune cells in the population of stimulated immune cells express KLF2.

[0134] In some aspects, following culturing the immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 50% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 55% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 60% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 65% ofthe immune cells in the population of stimulated immune cells express TCF1 and KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 70% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 75% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 80% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 85% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 90% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2. In some aspects, following the contacting with the CD28 agonist, at least about 95% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2.

[0135] In some aspects, following culturing the immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 50% of the immune cells in the population of stimulated immune cells lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 55% of the immune cells in the population of stimulated immune cells lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 60% of the immune cells in the population of stimulated immune cells lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 65% of the immune cells in the population of stimulated immune cells lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 70% of the immune cells in the population of stimulated immune cells lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 75% of the immune cells in the population of stimulated immune cells lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 80% of the immune cells in the population of stimulated immune cells lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 85% of the immune cells in the population of stimulated immune cells lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 90% of the immune cells in the population ofstimulated immune cells lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 95% of the immune cells in the population of stimulated immune cells lack IRF4 expression.

[0136] In some aspects, following culturing the immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2 and lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 50% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2 and lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 55% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2 and lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 60% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2 and lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 65% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2 and lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 70% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2 and lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 75% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2 and lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 80% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2 and lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 85% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2 and lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 90% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2 and lack IRF4 expression. In some aspects, following the contacting with the CD28 agonist, at least about 95% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2 and lack IRF4 expression.

[0137] In some aspects, following culturing the immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, the expression level of one or more exhaustion makers in the population of stimulated immune cells does notincrease, relative to the expression level of the one or more exhaustion markers in the population of source immune cells.

[0138] In some aspects, following culturing the immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, the population of stimulated immune cells has a lower expression level of one or more exhaustion markers, relative to the expression level of the one or more exhaustion markers in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more exhaustion markers is less than about 90%, less than about 85%, less than about 80%, less than about 70%, less than about 75%, less than about 60%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% of the expression level of the one or more exhaustion markers in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more exhaustion markers is less than about 90% of the expression level of the one or more exhaustion markers in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more exhaustion markers is less than about 80% of the expression level of the one or more exhaustion markers in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more exhaustion markers is less than about 75% of the expression level of the one or more exhaustion markers in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more exhaustion markers is less than about 70% of the expression level of the one or more exhaustion markers in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more exhaustion markers is less than about 60% of the expression level of the one or more exhaustion markers in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more exhaustion markers is less than about 50% of the expression level of the one or more exhaustion markers in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more exhaustion markers is less than about 40% of the expression level of the one or more exhaustion markers in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more exhaustion markers is less than about 30% of the expression level of the one or more exhaustion markers in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more exhaustion markers is less than about 20% of the expression level of the one or more exhaustion markers in a population of immune cells contacted with a CD3 agonist.

[0139] In some aspects, following culturing the immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, the population of stimulated immune cells has a lower expression level of PD-1 relative to the expression level of PD-1 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of PD-1 is less than about 90%, less than about 85%, less than about 80%, less than about 70%, less than about 75%, less than about 60%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% of the expression level of PD-1 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of PD-1 is less than about 90% of the expression level of PD-1 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of PD-1 is less than about 80% of the expression level of PD-1 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of PD-1 is less than about 75% of the expression level of PD-1 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of PD-1 is less than about 70% of the expression level of PD-1 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of PD-1 is less than about 60% of the expression level of PD-1 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of PD-1 is less than about 50% of the expression level of PD-1 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of PD-1 is less than about 40% of the expression level of PD-1 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of PD- 1 is less than about 30% of the expression level of PD-1 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of PD-1 is less than about 20% of the expression level of PD-1 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist.

[0140] In some aspects, following culturing the immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, the population of stimulated immune cells has a lower expression level of TIM-3 relative to the expression level of TIM-3 in (i) the population of source immune cells or (ii) a population of immune cells contactedwith a CD3 agonist. In some aspects, the expression of TIM-3 is less than about 90%, less than about 85%, less than about 80%, less than about 70%, less than about 75%, less than about 60%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% of the expression level of TIM-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIM-3 is less than about 90% of the expression level of TIM-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIM-3 is less than about 80% of the expression level of TIM-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIM-3 is less than about 75% of the expression level of TIM-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIM-3 is less than about 70% of the expression level of TIM-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIM-3 is less than about 60% of the expression level of TIM-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIM- 3 is less than about 50% of the expression level of TIM-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIM-3 is less than about 40% of the expression level of TIM-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIM-3 is less than about 30% of the expression level of TIM-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIM-3 is less than about 20% of the expression level of TIM-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist.

[0141] In some aspects, following culturing the immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, the population of stimulated immune cells has a lower expression level of LAG-3 relative to the expression level of LAG-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of LAG-3 is less than about 90%, less than about 85%, less than about 80%, less than about 70%, less than about 75%, less than about 60%, less than about 65%, less than about 60%, less than about 55%, less than about 50%,less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% of the expression level of LAG-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of LAG-3 is less than about 90% of the expression level of LAG-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of LAG-3 is less than about 80% of the expression level of LAG-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of LAG-3 is less than about 75% of the expression level of LAG-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of LAG-3 is less than about 70% of the expression level of LAG-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of LAG- 3 is less than about 60% of the expression level of LAG-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of LAG-3 is less than about 50% of the expression level of LAG-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of LAG-3 is less than about 40% of the expression level of LAG-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of LAG-3 is less than about 30% of the expression level of LAG-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of LAG-3 is less than about 20% of the expression level of LAG-3 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist.

[0142] In some aspects, following culturing the immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, the population of stimulated immune cells has a lower expression level of TIGIT relative to the expression level of TIGIT in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIGIT is less than about 90%, less than about 85%, less than about 80%, less than about 70%, less than about 75%, less than about 60%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% of the expression level of TIGIT in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expressionof TIGIT is less than about 90% of the expression level of TIGIT in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIGIT is less than about 80% of the expression level of TIGIT in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIGIT is less than about 75% of the expression level of TIGIT in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIGIT is less than about 70% of the expression level of TIGIT in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIGIT is less than about 60% of the expression level of TIGIT in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIGIT is less than about 50% of the expression level of TIGIT in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIGIT is less than about 40% of the expression level of TIGIT in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIGIT is less than about 30% of the expression level of TIGIT in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of TIGIT is less than about 20% of the expression level of TIGIT in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist.

[0143] In some aspects, following culturing the immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, the expression level of one or more makers of oxidative stress in the population of stimulated immune cells does not increase relative to the expression level of the one or more markers of oxidative stress in the population of source immune cells.

[0144] In some aspects, following culturing the immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, the population of stimulated immune cells has a lower expression level of one or more markers of oxidative stress, relative to the expression level of the one or more markers of oxidative stress in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more markers of oxidative stress is less than about 90%, less than about 85%, less than about 80%, less than about 70%, less than about 75%, less than about 60%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 40%, less than about 30%, lessthan about 20%, less than about 10%, less than about 5%, or less than about 1% of the expression level of the one or more markers of oxidative stress in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more markers of oxidative stress is less than about 90% of the expression level of the one or more markers of oxidative stress in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more markers of oxidative stress is less than about 80% of the expression level of the one or more markers of oxidative stress in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more markers of oxidative stress is less than about 75% of the expression level of the one or more markers of oxidative stress in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more markers of oxidative stress is less than about 70% of the expression level of the one or more markers of oxidative stress in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more markers of oxidative stress is less than about 60% of the expression level of the one or more markers of oxidative stress in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more markers of oxidative stress is less than about 50% of the expression level of the one or more markers of oxidative stress in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more markers of oxidative stress is less than about 40% of the expression level of the one or more markers of oxidative stress in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more markers of oxidative stress is less than about 30% of the expression level of the one or more markers of oxidative stress in a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of the one or more markers of oxidative stress is less than about 20% of the expression level of the one or more markers of oxidative stress in a population of immune cells contacted with a CD3 agonist.

[0145] In some aspects, following culturing the immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, the population of stimulated immune cells has a lower expression level of NRF2 relative to the expression level of NRF2 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of NRF2 is less than about 90%, less than about 85%, less than about 80%, less than about 70%, less than about 75%, less than about 60%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, orless than about 1% of the expression level of NRF2 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of NRF2 is less than about 90% of the expression level of NRF2 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of NRF2 is less than about 80% of the expression level of NRF2 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of NRF2 is less than about 75% of the expression level of NRF2 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of NRF2 is less than about 70% of the expression level of NRF2 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of NRF2 is less than about 60% of the expression level of NRF2 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of NRF2 is less than about 50% of the expression level of NRF2 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression ofNRF2 is less than about 40% of the expression level of NRF2 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of NRF2 is less than about 30% of the expression level of NRF2 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of NRF2 is less than about 20% of the expression level of NRF2 in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist.

[0146] In some aspects, following culturing the immune cells (e.g., T cells) according to the methods disclosed herein, i.e., following the contacting with the CD28 agonist, the population of stimulated immune cells has a lower expression level of HIFla relative to the expression level of HIFla in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of HIFla is less than about 90%, less than about 85%, less than about 80%, less than about 70%, less than about 75%, less than about 60%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% of the expression level of HIFla in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of HIFla is less than about 90% of the expression level of HIFla in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects,the expression of HIFla is less than about 80% of the expression level of HIFla in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of HIFla is less than about 75% of the expression level of HIFla in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of HIFla is less than about 70% of the expression level of HIFla in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of HIFla is less than about 60% of the expression level of HIFla in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of HIFla is less than about 50% of the expression level of HIFla in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of HIFla is less than about 40% of the expression level of HIFla in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of HIFla is less than about 30% of the expression level of HIFla in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist. In some aspects, the expression of HIFla is less than about 20% of the expression level of HIFla in (i) the population of source immune cells or (ii) a population of immune cells contacted with a CD3 agonist.

[0147] In some aspects, following the contacting with the CD28 agonist, the population of stimulated immune cells (e.g., T cells) is expanded, wherein the expansion does not comprise contacting the stimulated immune cells e.g., T cells) with a CD3 agonist.II.B. Stimulation

[0148] Some aspects of the present disclosure are directed to methods of preparing a population of immune cells for a cell therapy, comprising contacting a population of source immune cells with a CD28 agonist thereby producing a population of stimulated immune cells, wherein neither the population of source immune cells nor the population of stimulated immune cells is contacted with a CD3 agonist. Some aspects of the present disclosure are directed to methods of preparing a population of genetically modified immune cells for an immune cell therapy, comprising: (i) contacting a population of source immune cells with a CD28 agonist thereby generating a population of stimulated immune cells, and (ii) introducing into one or more cells of the population of stimulated immune cells a heterologous nucleic acid molecule encodinga CAR or a TCR; wherein neither the population of source immune cells nor the population of stimulated immune cells is contacted with a CD3 agonist.

[0149] Any CD28 agonist can be used in the methods disclosed herein. In some aspects, the CD28 agonist comprises an antibody or an antigen-binding portion thereof that specifically binds CD28. In some aspects, the CD28 agonist comprises an anti-CD28 antibody comprising CD28.2 or an antigen-binding portion thereof. In some aspects, the CD28 agonist comprises an anti-CD28 antibody comprising TGN1412 or an antigen-binding portion thereof. In some aspects, the CD28 agonist comprises an anti-CD28 antibody comprising ANC28.1 / 5D10 or an antigenbinding portion thereof. In some aspects, the CD28 agonist comprises an antibody or an antigenbinding portion thereof that cross competes for binding to human CD28 with CD28.2, TGN1412, or ANC28.1 / 5D10.

[0150] In some aspects, the CD28 agonist comprises a small molecule. In some aspects, the CD28 agonist comprises a ligand. In some aspects, the CD28 agonist comprises a CD80 polypeptide or a portion thereof that is capable of interacting with CD28. In some aspects, the CD28 agonist comprises a CD86 polypeptide or a portion thereof that is capable of interacting with CD28.

[0151] In some aspects, the immune cells (e.g., T cells) are contacted with a soluble CD28 agonist. In some aspects, the CD28 agonist is associated with an antigen presenting cell (APC), a bead, an extracellular matrix, or any combination thereof.

[0152] In some aspects, the CD28 agonist is associated with an APC. In some aspects, the CD28 agonist is bound to the membrane of an APC. Any APC can be used in the methods disclosed herein. In some aspects, the APC is a K562 cell. In some aspects, the APC is a genetically modified K562 cell. In some aspects, the APC is an artificial APC ("aAPC"). In some aspects, the APC is genetically modified to express the CD28 agonist. In some aspects, the APC is genetically modified to express an antigen-binding protein that specifically binds human CD28. In some aspects, the APC is genetically modified to express on the cell surface of the APC an antigen-binding protein that specifically binds human CD28. In some aspects, the APC is genetically modified to express CD28.2 or an antigen-binding portion of CD28.2 that specifically binds human CD28. In some aspects, the APC is genetically modified to express on the cell surface of the APC CD28.2 or an antigen-binding portion of CD28.2 that specifically binds human CD28. In some aspects, the APC is genetically modified to express TGN1412 or an antigen-binding portion of TGN1412 that specifically binds human CD28. In some aspects, the APC is genetically modified to express on the cell surface of the APC TGN1412 or an antigen-binding portion of TGN1412 that specificallybinds human CD28. In some aspects, the APC is genetically modified to express ANC28.1 / 5D10 or an antigen-binding portion of ANC28.1 / 5D10 that specifically binds human CD28. In some aspects, the APC is genetically modified to express on the cell surface of the APC ANC28.1 / 5D10 or an antigen-binding portion of ANC28.1 / 5D10 that specifically binds human CD28. In some aspects, the population of source immune cells (e.g., T cells) is contacted with a genetically modified APC that has surface expression of TGN1412 or an antigen-binding portion of TGN1412.

[0153] In some aspects, the APC has decreased or no expression of B2M. In some aspects, the APC has decreased or no expression of CD32. In some aspects, the APC has decreased or no expression of B2M, and the APC has decreased or no expression of CD32. In some aspects, the APC has decreased or no expression of CD 32 A. In some aspects, the APC has decreased or no expression of B2M, and the APC has decreased or no expression of CD 32 A. In some aspects, the APC does not express B2M. In some aspects, the APC does not express CD32. In some aspects, the APC does not express B2M, and the APC does not express CD 32. In some aspects, the APC does not express CD 32 A. In some aspects, the APC does not express B2M, and the APC does not express CD32A.

[0154] In some aspects, the APC expresses CD83, IL-7, IL-21, or any combination thereof. In some aspects, the APC expresses CD83. In some aspects, the APC expresses IL-7. In some aspects, the APC expresses IL-21. In some aspects, the APC expresses CD83 and IL-7. In some aspects, the APC expresses CD83 and IL-21. In some aspects, the APC expresses IL-7 and IL-21. In some aspects, the APC expresses CD83, IL-7, and IL-21.

[0155] In some aspects, the APC expresses (i) TGN1412 or an antigen-binding portion of TGN1412 that specifically binds human CD28 and (ii) CD83; and the APC does not express CD32A. In some aspects, the APC expresses (i) TGN1412 or an antigen-binding portion of TGN1412 that specifically binds human CD28 and (ii) CD83; the APC does not express CD32A,' and the APC does not express B2M.

[0156] In some aspects, the APC expresses (i) an antibody or an antigen-binding portion thereof that cross competes for binding to human CD28 with TGN1412 and (ii) CD83; wherein the APC has decreased or no expression of CD 32 A. In some aspects, the APC expresses (i) an antibody or an antigen-binding portion thereof that cross competes for binding to human CD28 with TGN1412 and (ii) CD83; wherein the APC has decreased or no expression of CD32A and wherein the APC has decreased or no expression of B2M.

[0157] In some aspects, the APC expresses (i) an antibody or an antigen-binding portion thereof that cross competes for binding to human CD28 with TGN1412 and (ii) CD83; whereinthe APC does not express CD 32 A. In some aspects, the APC expresses (i) an antibody or an antigen-binding portion thereof that cross competes for binding to human CD28 with TGN1412 and (ii) CD83; wherein the APC does not express CD32A and wherein the APC does not express B2M.

[0158] In some aspects, the source immune cells are contacted with the CD28 agonist for about 12 hours to about 7 days. In some aspects, the source immune cells are contacted with the CD28 agonist for about 12 hours to about 8 days. In some aspects, the source immune cells are contacted with the CD28 agonist for about 12 hours to about 9 days. In some aspects, the source immune cells are contacted with the CD28 agonist for about 12 hours to about 10 days. In some aspects, the source immune cells are contacted with the CD28 agonist for about 12 hours to about 6 days, about 12 hours to about 5 days, about 12 hours to about 4 days, about 12 hours to about 3 days, about 12 hours to about 48 hours, about 12 hours to about 36 hours, about 12 hours to about 24 hours, about 24 hours to about 36 hours, about 24 hours to about 48 hours, about 24 hours to about 3 days, about 24 hours to about 4 days, about 24 hours to about 5 days, about 24 hours to about 6 days, about 24 hours to about 7 days, about 36 hours to about 48 hours, about 36 hours to about 3 days, about 36 hours to about 4 days, about 36 hours to about 5 days, about 36 hours to about 6 days, about 36 hours to about 7 days, about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 6 days, about 2 days to about 7 days, about 3 days to about 4 days, about 3 days to about 5 days, about 3 days to about 6 days, about 3 days to about 7 days, about 3 to about 8 days, about 3 to about 9 days, or about 3 to about 10 days.

[0159] In some aspects, the source immune cells are contacted with the CD28 agonist for about 12 hours. In some aspects, the source immune cells are contacted with the CD28 agonist for about 18 hours. In some aspects, the source immune cells are contacted with the CD28 agonist for about 48 hours. In some aspects, the source immune cells are contacted with the CD28 agonist for about 60 hours. In some aspects, the source immune cells are contacted with the CD28 agonist for about 72 hours. In some aspects, the source immune cells are contacted with the CD28 agonist for about 84 hours. In some aspects, the source immune cells are contacted with the CD28 agonist for about 96 hours. In some aspects, the source immune cells are contacted with the CD28 agonist for about 12 hours.

[0160] In some aspects, the source immune cells are contacted with the CD28 agonist for about 1 day. In some aspects, the source immune cells are contacted with the CD28 agonist for about 2 days. In some aspects, the source immune cells are contacted with the CD28 agonist for about 3 days. In some aspects, the source immune cells are contacted with the CD28 agonist forabout 4 days. In some aspects, the source immune cells are contacted with the CD28 agonist for about 5 days. In some aspects, the source immune cells are contacted with the CD28 agonist for about 6 days. In some aspects, the source immune cells are contacted with the CD28 agonist for about 7 days. In some aspects, the source immune cells are contacted with the CD28 agonist for about 8 days. In some aspects, the source immune cells are contacted with the CD28 agonist for about 9 days. In some aspects, the source immune cells are contacted with the CD28 agonist for about 10 days.

[0161] In some aspects, the population of stimulated immune cells is washed to remove the CD28 agonist after the contacting. In some aspects, the CD28 agonist is removed about 24 hours after initiation of the contacting. In some aspects, the CD28 agonist is removed about 36 hours after initiation of the contacting. In some aspects, the CD28 agonist is removed about 48 hours after initiation of the contacting. In some aspects, the CD28 agonist is removed about 60 hours after initiation of the contacting. In some aspects, the CD28 agonist is removed about 72 hours after initiation of the contacting. In some aspects, the CD28 agonist is removed about 84 hours after initiation of the contacting. In some aspects, the CD28 agonist is removed about 96 hours after initiation of the contacting.

[0162] In some aspects, the population of source immune cells is further contacted with IL-7, IL-21, an antibody or antigen-binding portion thereof that specifically binds IFN-y, or any combination thereof concurrently with the CD28 agonist. In some aspects, the IL-7, IL-21, the antibody or antigen-binding portion thereof that specifically binds IFN-y, or any combination thereof is added to the culture medium comprising the source immune cells on the same day or about 1 day after the source immune cells are contacted with the CD28 agonist.

[0163] As used herein, a "CD3 agonist" refers to any agent capable of binding to and activating (i.e., stimulating) CD3. The methods disclosed herein exclude contacting the population of source immune cells and / or the population of stimulated immune cells with any CD3 agonist. A CD3 agonist is any molecule that is capable of binding to CD3 complex and activating CD3. In some aspects, a CD3 agonist is a small molecule. In some aspects, a CD3 agonist is a protein. In some aspects, a CD3 agonist is an anti-CD3 antibody. The term "anti-CD3 antibody" as used herein refers to an antibody or variant thereof, e.g., a monoclonal antibody and including human, humanized, chimeric or murine antibodies, which are directed against the CD3 complex in T cells. In some aspects, an anti-CD3 antibody comprises OKT-3, also known as muromonab. In some aspects, an anti-CD3 antibody comprises UCHT-1. Other anti-CD3 antibodies include, for example, visilizumab, otelixizumab, and teplizumab.

[0164] The term "OKT-3" or "OKT3" refers to a monoclonal antibody or biosimilar or variant thereof, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially-available forms such as OKT-3 (30 ng / mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, Calif., USA) and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. A hybridoma capable of producing OKT-3 is deposited with European Collection of Authenticated Cell Cultures (ECACC) and assigned Catalogue No. 86022706. A hybridoma capable of producing OKT-3 is also deposited with the American Type Culture Collection and assigned the ATCC accession number CRL 8001.

[0165] Some aspects of the present disclosure are directed to methods of preparing a population of immune cells for a cell therapy, comprising contacting a population of source immune cells with a CD28 agonist thereby producing a population of stimulated immune cells, wherein neither the population of source immune cells nor the population of stimulated immune cells is contacted with OKT-3.

[0166] Some aspects of the present disclosure are directed to a method of preparing a population of genetically modified immune cells for an immune cell therapy, comprising: (i) contacting a population of source immune cells with a CD28 agonist thereby generating a population of stimulated immune cells, and (ii) introducing into one or more cells of the population of stimulated immune cells a heterologous nucleic acid molecule encoding a CAR; wherein neither the population of source immune cells nor the population of stimulated immune cells is contacted with OKT-3.

[0167] Some aspects of the present disclosure are directed to a method of preparing a population of genetically modified immune cells for an immune cell therapy, comprising: (i) contacting a population of source immune cells with a CD28 agonist thereby generating a population of stimulated immune cells, and (ii) introducing into one or more cells of the population of stimulated immune cells a heterologous nucleic acid molecule encoding a CAR; wherein neither the population of source immune cells nor the population of stimulated immune cells is contacted with OKT-3.

[0168] Some aspects of the present disclosure are directed to a method of preparing a population of genetically modified immune cells for an immune cell therapy, comprising: (i) contacting a population of source immune cells with a CD28 agonist thereby generating a population of stimulated immune cells, and (ii) introducing into one or more cells of the population of stimulated immune cells a heterologous nucleic acid molecule encoding a TCR; wherein neitherthe population of source immune cells nor the population of stimulated immune cells is contacted with OKT-3.II.C. Genetic Engineering

[0169] In some aspects, the population of source immune cells, e.g., T cells, is transduced before, during, or after culturing the immune cells according to the methods disclosed herein. In some aspects, the population of source immune cells, e.g., T cells, is transduced before contacting the immune cells with the CD28 agonist. In some aspects, the population of source immune cells, e.g., T cells, is transduced after contacting the immune cells with the CD28 agonist.

[0170] In some aspects, the immune cells are transduced about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 50 hours, about 62 hours, about 74 hours, about 86 hours, about 98 hours, or about 120 hours after contacting the population of source immune cells (e.g., T cells) with the CD28 agonist. In some aspects, the immune cells are transduced about 6 hours after contacting the population of source immune cells (e.g., T cells) with the CD28 agonist. In some aspects, the immune cells are transduced about 12 hours after contacting the population of source immune cells (e.g., T cells) with the CD28 agonist. In some aspects, the immune cells are transduced about 24 hours after contacting the population of source immune cells (e.g., T cells) with the CD28 agonist. In some aspects, the immune cells are transduced about 36 hours after contacting the population of source immune cells (e.g., T cells) with the CD28 agonist. In some aspects, the immune cells are transduced about 48 hours after contacting the population of source immune cells (e.g., T cells) with the CD28 agonist. In some aspects, the immune cells are transduced about 60 hours after contacting the population of source immune cells (e.g., T cells) with the CD28 agonist. In some aspects, the immune cells are transduced about 72 hours after contacting the population of source immune cells (e.g., T cells) with the CD28 agonist. In some aspects, the immune cells are transduced about 84 hours after contacting the population of source immune cells (e.g., T cells) with the CD28 agonist. In some aspects, the immune cells are transduced about 96 hours after contacting the population of source immune cells (e.g., T cells) with the CD28 agonist. In some aspects, the immune cells are transduced about 120 hours after contacting the population of source immune cells (e.g., T cells) with the CD28 agonist.

[0171] In certain aspects, the immune cells are transduced using a viral vector comprising a heterologous nucleic acid molecule encoding the CAR and / or the TCR. In some aspects, the vector comprises a retroviral vector, a lentiviral vector, an adeno-associated virus (AAV), anadenovirus, an AAV hybrid virus, a baculovirus, or any combination thereof. In some aspects, the vector comprises a vaccinia vector, herpes simplex viral vector, or Epstein-Barr viral vector. In some aspects, the viral vector comprises a retroviral vector (e.g., a retrovirus). In some aspects, the viral vector comprises a lentivirus. In some aspects, the viral vector comprises an AAV.

[0172] In some aspects, the immune cells are transduced using a non-viral method. In some aspects, the non-viral method includes the use of a transposon. In some aspects, use of a non-viral method of delivery permits reprogramming of immune cells, e.g., T cells and / or NK cells, and direct infusion of the cells into the subject. In some aspects, the polynucleotide can be inserted into the genome of a target cell (e.g., a T cell) or a host cell (e.g., a cell for recombinant expression of the encoded proteins) by using CRISPR / Cas systems and genome edition alternatives such as zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases (MNs).

[0173] In some aspects, the immune cell is transduced with a vector comprising a heterologous nucleic acid encoding a CAR. In some aspects, a CAR-expressing cell disclosed herein is a CAR T cell, e.g., a mono CAR T cell, a genome-edited CAR T cell, a dual CAR T cell, or a tandem CAR T cell.

[0174] In some aspects, the CAR is designed as a standard CAR, a split CAR, an off-switch CAR, an on-switch CAR, a first-generation CAR, a second-generation CAR, a third-generation CAR, or a fourth-generation CAR. In some aspects, the CAR comprises an antigen-binding domain, a transmembrane domain, a costimulatory domain, an intracellular signaling domain, or combinations thereof.

[0175] In some aspects, the CAR specifically binds (i.e., targets) one or more antigens expressed on a tumor cell, such as a malignant B cell, a malignant T cell, or a malignant plasma cell. In some aspects, the CAR specifically binds to (i.e., targets) an antigen selected from CD19, TRAC, TCRP, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1 IRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE- Al, legumain, HPV E6,E7, MAGE Al,ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT- 2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA- 1 / Galectin 8, MelanA / MARTl, Ras mutant, human telomerase reverse transcriptase (hTERT), sarcoma translocation breakpoints, ML- IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3s, CD4, CD5, CD7, the extracellular portion of the APRIL protein, or any combinations thereof. In some aspects, the TCR targets AFP, CD19, TRAC, TCRP, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, R0R1, R0R2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CX0RF61, CD97, CD 179a, ALK, Poly sialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE- Al, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT- 2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA- 1 / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3s, CD4, CD5, CD7, the extracellular portion of the APRIL protein, or any combinations thereof In some aspects, the CAR specifically binds NY-ESO-1.

[0176] In some aspects, the CAR comprises a costimulatory domain of an interleukin-2 receptor (IL-2R), interleukin- 12 receptor (IL-12R), IL-7, IL-21, IL-23, IL-15, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, 4-1BB / CD137, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, 0X40, DAP10, or any combination thereof. In some aspects, the costimulatory domain comprises a CD28 costimulatory domain. In some aspects, the costimulatory domain comprises a 4-1BB / CD137 costimulatory domain.

[0177] In some aspects, the CAR comprises a transmembrane domain of KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 160, CD 19, IL2R beta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKG2D, NKG2C, CD 19, or any combination thereof. In some aspects, the transmembrane domain comprises a CD28 transmembrane domain.

[0178] In some aspects, the CAR comprises an intracellular signaling domain derived fromCD3 zeta, FcR gamma, common FcR gamma (FCER1G), Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, CD278 (“ICOS”), FcsRI, CD66d, CD32, DAP10, DAP12, or any combination thereof. In some aspects, the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain.

[0179] In some aspects, the immune cell is transduced with a vector comprising a heterologous nucleic acid encoding a TCR. In some aspects, the TCR specifically binds (i.e., targets) one or more antigens expressed on a tumor cell, such as a malignant B cell, a malignant T cell, or a malignant plasma cell. In some aspects, the CAR specifically binds to (i.e., targets) an antigen selected from CD19, TRAC, TCRP, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPC AM, B7H3, KIT, IL-13Ra2, mesothelin, IL- HRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD 179a, ALK, Poly sialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT- 2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA- 1 / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC,TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3s, CD4, CD5, CD7, the extracellular portion of the APRIL protein, or any combinations thereof. In some aspects, the TCR targets AFP, CD19, TRAC, TCRp, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, R0R1, R0R2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1 IRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o- acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CX0RF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT- 2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA- 1 / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, , RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3s, CD4, CD5, CD7, the extracellular portion of the APRIL protein, or any combinations thereof In some aspects, the TCR specifically binds NY-ESO-1.

[0180] In some aspects, the TCR comprises an intracellular gamma / delta domain. In some aspects, the TCR is an antibody-T-cell receptor (AbTCR) (see, e.g., Xu et al., Cell Discovery 4:62 (2018), which is incorporated by reference herein in its entirety.II.D. Culture Conditions

[0181] In some aspects, the population of source immune cells are contacted with the CD28 agonist by culturing the population of source immune cells in a medium. Various cell culture media are commercially available, and any culture medium suitable for immune cell culture can be used in the methods disclosed herein. In some aspects, the medium comprises soluble CD28 agonist. In some aspects, the medium is supplemented with soluble CD28 agonist. In some aspects, themedium comprises CD28 agonist associated with a bead, e.g., a CD28 agonist linked to a bead. In some aspects, the medium is supplemented with APC that express the CD28 agonist.

[0182] In some aspects, the medium comprises IL-7, IL-21, an antibody or antigen-binding portion thereof that specifically binds interferon-gamma (IFN-y), or any combination thereof. In some aspects, the medium comprises IL-7. In some aspects, the medium comprises about 1 ng / ml to about 1 pg / ml IL-7. In some aspects, the medium comprises about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml, about 500 ng / ml, or about 1 pg / ml IL-7. In some aspects, the medium comprises about 5 ng / ml IL-7. In some aspects, the medium comprises about 10 ng / ml IL-7. In some aspects, the medium comprises about 15 ng / ml IL-7. In some aspects, the medium comprises about 20 ng / ml IL-7. In some aspects, the medium comprises about 25 ng / ml IL-7. In some aspects, the medium comprises about 30 ng / ml IL-7. In some aspects, the medium comprises about 35 ng / ml IL-7. In some aspects, the medium comprises about 40 ng / ml IL-7. In some aspects, the medium comprises about 45 ng / ml IL-7. In some aspects, the medium comprises about 50 ng / ml IL-7. In some aspects, the medium comprises about 100 ng / ml IL-7.

[0183] In some aspects, the medium comprises about 1 ng / ml to about 1 pg / ml IL-21. In some aspects, the medium comprises about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml, about 500 ng / ml, or about 1 pg / ml IL-21. In some aspects, the medium comprises about 5 ng / ml IL-21. In some aspects, the medium comprises about 10 ng / ml IL-21. In some aspects, the medium comprises about 15 ng / ml IL-21. In some aspects, the medium comprises about 20 ng / ml IL-21. In some aspects, the medium comprises about 25 ng / ml IL-21. In some aspects, the medium comprises about 30 ng / ml IL-21. In some aspects, the medium comprises about 35 ng / ml IL-21. In some aspects, the medium comprises about 40 ng / ml IL-21. In some aspects, the medium comprises about 45 ng / ml IL-21. In some aspects, the medium comprises about 50 ng / ml IL-21. In some aspects, the medium comprises about 100 ng / ml IL-21.

[0184] In some aspects, the medium comprises about 0.1 pg / ml to about 100 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. the medium comprises about 0.1 pg / ml, about 0.5 pg / ml, about 1 pg / ml, about 2 pg / ml, about 3 pg / ml, about 4 pg / ml, about 5 pg / ml, about 6 pg / ml, about 7 pg / ml, about 8 pg / ml, about 9 pg / ml, about 10 pg / ml, about 20 pg / ml, about 30 pg / ml, about 40 pg / ml, about 50 pg / ml, or about 100 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 0.1 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 0.5 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 1 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 2 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 3 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 4 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 5 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 6 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 7 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 8 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 9 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 10 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 20 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 30 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 40 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 50 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 100 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y.

[0185] In some aspects, the medium comprises about 20 to about 30 ng / ml IL-7 and about 20 to about 30 ng / ml IL-21. In some aspects, the medium comprises about 20 to about 30 ng / ml IL-7 and about 1 to about 5 pg / ml of an antibody or antigen-binding portion thereof thatspecifically binds IFN-y. In some aspects, the medium comprises about 20 to about 30 ng / ml IL- 21 and about 1 to about 5 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 20 to about 30 ng / ml IL-7, about 20 to about 30 ng / ml IL-21, and about 1 to about 5 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y.

[0186] In some aspects, the medium comprises about 25 ng / ml IL-7 and about 25 ng / ml IL-21. In some aspects, the medium comprises about 25 ng / ml IL-7 and about 2 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 25 ng / ml IL-21 and about 2 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y. In some aspects, the medium comprises about 25 ng / ml IL-7, about 25 ng / ml IL-21, and about 2 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y.

[0187] In some aspects, the IL-7, IL-21, the antibody or antigen-binding portion thereof that specifically binds IFN-y, or any combination thereof is added to the medium before the source immune cells are contacted with the CD28 agonist. In some aspects, the IL-7, IL-21, the antibody or antigen-binding portion thereof that specifically binds IFN-y, or any combination thereof is added to the medium on the same day the source immune cells are contacted with the CD28 agonist. In some aspects, the IL-7, IL-21, the antibody or antigen-binding portion thereof that specifically binds IFN-y, or any combination thereof is added to the medium about 1 day after the source immune cells are contacted with the CD28 agonist. In some aspects, the IL-7, IL-21, the antibody or antigen-binding portion thereof that specifically binds IFN-y, or any combination thereof is added to the medium about 12 hours after the source immune cells are contacted with the CD28 agonist. In some aspects, the IL-7, IL-21, the antibody or antigen-binding portion thereof that specifically binds IFN-y, or any combination thereof is added to the medium about 18 hours after the source immune cells are contacted with the CD28 agonist. In some aspects, the IL-7, IL-21, the antibody or antigen-binding portion thereof that specifically binds IFN-y, or any combination thereof is added to the medium about 24 hours after the source immune cells are contacted with the CD28 agonist. In some aspects, the IL-7, IL-21, the antibody or antigen-binding portion thereof that specifically binds IFN-y, or any combination thereof is added to the medium about 36 hours after the source immune cells are contacted with the CD28 agonist. In some aspects, the IL-7, IL- 21, the antibody or antigen-binding portion thereof that specifically binds IFN-y, or any combination thereof is added to the medium about 48 hours after the source immune cells are contacted with the CD28 agonist. In some aspects, the IL-7, IL-21, the antibody or antigen-bindingportion thereof that specifically binds IFN-y, or any combination thereof is added to the medium about 60 hours after the source immune cells are contacted with the CD28 agonist. In some aspects, the IL-7, IL-21, the antibody or antigen-binding portion thereof that specifically binds IFN-y, or any combination thereof is added to the medium about 72 hours after the source immune cells are contacted with the CD28 agonist. In some aspects, the IL-7, IL-21, the antibody or antigen-binding portion thereof that specifically binds IFN-y, or any combination thereof is added to the medium about 84 hours after the source immune cells are contacted with the CD28 agonist. In some aspects, the IL-7, IL-21, the antibody or antigen-binding portion thereof that specifically binds IFN-y, or any combination thereof is added to the medium about 96 hours after the source immune cells are contacted with the CD28 agonist.III. Compositions of the Disclosure

[0188] Some aspects of the present disclosure are directed to a cell composition comprising a population of stimulated immune cells (e.g., T cells and / or NK cells) cultured according to the methods disclosed herein. Cell populations cultured according to the methods disclosed herein have an increased number of Tscm-like cells as compared to comparable cells cultured according to conventional methods, e.g., wherein the cells are contacted with a CD3 agonist. In some aspects, the cells cultured according to the methods disclosed herein exhibit increased expression of one or more marker typical of Tscm cell. In some aspects, the cells cultured according to the methods disclosed herein exhibit greater proliferative potential compared to cells cultured according to conventional methods, e.g., wherein the cells are contacted with a CD3 agonist. In some aspects, the cells cultured according to the methods disclosed herein exhibit increased transduction efficiency. In some aspects, the cells cultured according to the methods disclosed herein exhibit increased in vivo viability upon transplantation in a subject. In some aspects, the cells cultured according to the methods disclosed herein exhibit increased in vivo persistence upon transplantation in a subject. In some aspects, the cells cultured according to the methods disclosed herein exhibit increased cell potency. In some aspects, the cells cultured according to the methods disclosed herein exhibit decreased cell exhaustion. In some aspects, the cells cultured according to the methods disclosed herein exhibit increased in vivo cytotoxicity upon transplantation in a subject. In some aspects, the cells cultured according to the methods disclosed herein exhibit increased in vitro cytotoxicity compared to cells cultured according to conventional methods, e.g., wherein the cells are contacted with a CD3 agonist. In some aspects, the cells cultured accordingto the methods disclosed herein exhibit a more durable in vivo response upon transplantation in a subject. In some aspects, the subject is a human.

[0189] In some aspects, at least about 25% of the cells in the cell composition have a Tscm- like phenotype. In some aspects, at least about 30% of the cells in the cell composition have a Tscm-like phenotype. In some aspects, at least about 35% of the cells in the cell composition have a Tscm-like phenotype. In some aspects, at least about 40% of the cells in the cell composition have a Tscm-like phenotype. In some aspects, at least about 45% of the cells in the cell composition have a Tscm-like phenotype. In some aspects, at least about 50% of the cells in the cell composition have a Tscm-like phenotype. In some aspects, at least about 55% of the cells in the cell composition have a Tscm-like phenotype. In some aspects, at least about 60% of the cells in the cell composition have a Tscm-like phenotype. In some aspects, at least about 65% of the cells in the cell composition have a Tscm-like phenotype. In some aspects, at least about 70% of the cells in the cell composition have a Tscm-like phenotype. In some aspects, at least about 75% of the cells in the cell composition have a Tscm-like phenotype. In some aspects, at least about 80% of the cells in the cell composition have a Tscm-like phenotype. In some aspects, at least about 85% of the cells in the cell composition have a Tscm-like phenotype. In some aspects, at least about 90% of the cells in the cell composition have a Tscm-like phenotype. In some aspects, at least about 95% of the cells in the cell composition have a Tscm-like phenotype.

[0190] In some aspects, following culture of T cells according to the methods disclosed herein, Tscm-like cells constitute at least about 10% to at least about 90% of the total number of T cells in the culture. In some aspects, following culture of T cells according to the methods disclosed herein, Tscm-like cells constitute at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the total number of CD8+T cells in the culture. In some aspects, following culture of T cells according to the methods disclosed herein, Tscm-like cells constitute at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the total number of CD4+T cells in the culture.

[0191] In some aspects, at least about 25% of the cells in the cell composition express CD45RA, CD62L, CCR7, CD95, or any combination thereof. In some aspects, at least about 30% of the cells in the cell composition express CD45RA, CD62L, CCR7, CD95, or any combination thereof. In some aspects, at least about 35% of the cells in the cell composition express CD45RA,CD62L, CCR7, CD95, or any combination thereof. In some aspects, at least about 40% of the cells in the cell composition express CD45RA, CD62L, CCR7, CD95, or any combination thereof. In some aspects, at least about 45% of the cells in the cell composition express CD45RA, CD62L,CCR7, CD95, or any combination thereof. In some aspects, at least about 50% of the cells in the cell composition express CD45RA, CD62L, CCR7, CD95, or any combination thereof. In some aspects, at least about 55% of the cells in the cell composition express CD45RA, CD62L, CCR7, CD95, or any combination thereof. In some aspects, at least about 60% of the cells in the cell composition express CD45RA, CD62L, CCR7, CD95, or any combination thereof. In some aspects, at least about 65% of the cells in the cell composition express CD45RA, CD62L, CCR7, CD95, or any combination thereof. In some aspects, at least about 70% of the cells in the cell composition express CD45RA, CD62L, CCR7, CD95, or any combination thereof. In some aspects, at least about 75% of the cells in the cell composition express CD45RA, CD62L, CCR7,CD95, or any combination thereof. In some aspects, at least about 80% of the cells in the cell composition express CD45RA, CD62L, CCR7, CD95, or any combination thereof. In some aspects, at least about 85% of the cells in the cell composition express CD45RA, CD62L, CCR7, CD95, or any combination thereof. In some aspects, at least about 90% of the cells in the cell composition express CD45RA, CD62L, CCR7, CD95, or any combination thereof. In some aspects, at least about 95% of the cells in the cell composition express CD45RA, CD62L, CCR7, CD95, or any combination thereof.

[0192] In some aspects, at least about 25% of the cells in the cell composition express CD45RA. In some aspects, at least about 30% of the cells in the cell composition express CD45RA. In some aspects, at least about 35% of the cells in the cell composition express CD45RA. In some aspects, at least about 40% of the cells in the cell composition express CD45RA. In some aspects, at least about 45% of the cells in the cell composition express CD45RA. In some aspects, at least about 50% of the cells in the cell composition express CD45RA. In some aspects, at least about 55% of the cells in the cell composition express CD45RA. In some aspects, at least about 60% of the cells in the cell composition express CD45RA. In some aspects, at least about 65% of the cells in the cell composition express CD45RA. In some aspects, at least about 70% of the cells in the cell composition express CD45RA. In some aspects, at least about 75% of the cells in the cell composition express CD45RA. In some aspects, at least about 80% of the cells in the cell composition express CD45RA. In some aspects, at least about 85% of the cells in the cell composition express CD45RA. In some aspects, at least about 90% of the cells in the cellcomposition express CD45RA. In some aspects, at least about 95% of the cells in the cell composition express CD45RA.

[0193] In some aspects, at least about 25% of the cells in the cell composition expressCD62L. In some aspects, at least about 30% of the cells in the cell composition express CD62L. In some aspects, at least about 35% of the cells in the cell composition express CD62L. In some aspects, at least about 40% of the cells in the cell composition express CD62L. In some aspects, at least about 45% of the cells in the cell composition express CD62L. In some aspects, at least about 50% of the cells in the cell composition express CD62L. In some aspects, at least about 55% of the cells in the cell composition express CD62L. In some aspects, at least about 60% of the cells in the cell composition express CD62L. In some aspects, at least about 65% of the cells in the cell composition express CD62L. In some aspects, at least about 70% of the cells in the cell composition express CD62L. In some aspects, at least about 75% of the cells in the cell composition express CD62L. In some aspects, at least about 80% of the cells in the cell composition express CD62L. In some aspects, at least about 85% of the cells in the cell composition express CD62L. In some aspects, at least about 90% of the cells in the cell composition express CD62L. In some aspects, at least about 95% of the cells in the cell composition express CD62L.

[0194] In some aspects, at least about 25% of the cells in the cell composition express CCR7. In some aspects, at least about 30% of the cells in the cell composition express CCR7. In some aspects, at least about 35% of the cells in the cell composition express CCR7. In some aspects, at least about 40% of the cells in the cell composition express CCR7. In some aspects, at least about 45% of the cells in the cell composition express CCR7. In some aspects, at least about 50% of the cells in the cell composition express CCR7. In some aspects, at least about 55% of the cells in the cell composition express CCR7. In some aspects, at least about 60% of the cells in the cell composition express CCR7. In some aspects, at least about 65% of the cells in the cell composition express CCR7. In some aspects, at least about 70% of the cells in the cell composition express CCR7. In some aspects, at least about 75% of the cells in the cell composition express CCR7. In some aspects, at least about 80% of the cells in the cell composition express CCR7. In some aspects, at least about 85% of the cells in the cell composition express CCR7. In some aspects, at least about 90% of the cells in the cell composition express CCR7. In some aspects, at least about 95% of the cells in the cell composition express CCR7.

[0195] In some aspects, at least about 25% of the cells in the cell composition express CD95. In some aspects, at least about 30% of the cells in the cell composition express CD95. Insome aspects, at least about 35% of the cells in the cell composition express CD95. In some aspects, at least about 40% of the cells in the cell composition express CD95. In some aspects, at least about 45% of the cells in the cell composition express CD95. In some aspects, at least about 50% of the cells in the cell composition express CD95. In some aspects, at least about 55% of the cells in the cell composition express CD95. In some aspects, at least about 60% of the cells in the cell composition express CD95. In some aspects, at least about 65% of the cells in the cell composition express CD95. In some aspects, at least about 70% of the cells in the cell composition express CD95. In some aspects, at least about 75% of the cells in the cell composition express CD95. In some aspects, at least about 80% of the cells in the cell composition express CD95. In some aspects, at least about 85% of the cells in the cell composition express CD95. In some aspects, at least about 90% of the cells in the cell composition express CD95. In some aspects, at least about 95% of the cells in the cell composition express CD95.

[0196] In some aspects, at least about 25% of the cells in the cell composition express CD45RA and CCR7. In some aspects, at least about 30% of the cells in the cell composition express CD45RA and CCR7. In some aspects, at least about 35% of the cells in the cell composition express CD45RA and CCR7. In some aspects, at least about 40% of the cells in the cell composition express CD45RA and CCR7. In some aspects, at least about 45% of the cells in the cell composition express CD45RA and CCR7. In some aspects, at least about 50% of the cells in the cell composition express CD45RA and CCR7. In some aspects, at least about 55% of the cells in the cell composition express CD45RA and CCR7. In some aspects, at least about 60% of the cells in the cell composition express CD45RA and CCR7. In some aspects, at least about 65% of the cells in the cell composition express CD45RA and CCR7. In some aspects, at least about 70% of the cells in the cell composition express CD45RA and CCR7. In some aspects, at least about 75% of the cells in the cell composition express CD45RA and CCR7. In some aspects, at least about 80% of the cells in the cell composition express CD45RA and CCR7. In some aspects, at least about 85% of the cells in the cell composition express CD45RA and CCR7. In some aspects, at least about 90% of the cells in the cell composition express CD45RA and CCR7. In some aspects, at least about 95% of the cells in the cell composition express CD45RA and CCR7.

[0197] In some aspects, at least about 25% of the cells in the cell composition express CD45RA, CCR7, and CD95. In some aspects, at least about 30% of the cells in the cell composition express CD45RA, CCR7, and CD95. In some aspects, at least about 35% of the cells in the cell composition express CD45RA, CCR7, and CD95. In some aspects, at least about 40% of the cells in the cell composition express CD45RA, CCR7, and CD95. In some aspects, at least about 45%of the cells in the cell composition express CD45RA, CCR7, and CD95. In some aspects, at least about 50% of the cells in the cell composition express CD45RA, CCR7, and CD95. In some aspects, at least about 55% of the cells in the cell composition express CD45RA, CCR7, and CD95. In some aspects, at least about 60% of the cells in the cell composition express CD45RA, CCR7, and CD95. In some aspects, at least about 65% of the cells in the cell composition express CD45RA, CCR7, and CD95. In some aspects, at least about 70% of the cells in the cell composition express CD45RA, CCR7, and CD95. In some aspects, at least about 75% of the cells in the cell composition express CD45RA, CCR7, and CD95. In some aspects, at least about 80% of the cells in the cell composition express CD45RA, CCR7, and CD95. In some aspects, at least about 85% of the cells in the cell composition express CD45RA, CCR7, and CD95. In some aspects, at least about 90% of the cells in the cell composition express CD45RA, CCR7, and CD95. In some aspects, at least about 95% of the cells in the cell composition express CD45RA, CCR7, and CD95.

[0198] In some aspects, at least about 25% of the cells in the cell composition express CD45RA, CD62L, CCR7, and CD95. In some aspects, at least about 30% of the cells in the cell composition express CD45RA, CD62L, CCR7, and CD95. In some aspects, at least about 35% of the cells in the cell composition express CD45RA, CD62L, CCR7, and CD95. In some aspects, at least about 40% of the cells in the cell composition express CD45RA, CD62L, CCR7, and CD95. In some aspects, at least about 45% of the cells in the cell composition express CD45RA, CD62L, CCR7, and CD95. In some aspects, at least about 50% of the cells in the cell composition express CD45RA, CD62L, CCR7, and CD95. In some aspects, at least about 55% of the cells in the cell composition express CD45RA, CD62L, CCR7, and CD95. In some aspects, at least about 60% of the cells in the cell composition express CD45RA, CD62L, CCR7, and CD95. In some aspects, at least about 65% of the cells in the cell composition express CD45RA, CD62L, CCR7, and CD95. In some aspects, at least about 70% of the cells in the cell composition express CD45RA, CD62L, CCR7, and CD95. In some aspects, at least about 75% of the cells in the cell composition express CD45RA, CD62L, CCR7, and CD95. In some aspects, at least about 80% of the cells in the cell composition express CD45RA, CD62L, CCR7, and CD95. In some aspects, at least about 85% of the cells in the cell composition express CD45RA, CD62L, CCR7, and CD95. In some aspects, at least about 90% of the cells in the cell composition express CD45RA, CD62L, CCR7, and CD95. In some aspects, at least about 95% of the cells in the cell composition express CD45RA, CD62L, CCR7, and CD95.IV. Methods of Treatment

[0199] Some aspects of the present disclosure are directed to methods of treating a subject in need thereof comprising administering to the subject a population of immune cells, e.g, T cells and / or NK cells, cultured according to the methods disclosed herein (e.g., by contacting a source population of immune cells, e.g, T cells, with a CD28 agonist, wherein the cells are not contacted with a CD3 agonist).

[0200] In some aspects, the population of immune cells administered (e.g., the population of stimulated immune cells) comprises T cells. In some aspects, the T cells are autologous T cells. In some aspects the T cells are allogeneic T cells. In some aspects, the T cells comprise a CAR, i.e., CAR-T cells, as described herein. In some aspects, the T cells comprise a heterologous TCR, as described herein.

[0201] In some aspects, the subj ect is afflicted with a cancer, e.g. , a tumor. In some aspects, administering the population of immune cells reduces a tumor volume in the subject compared to a reference tumor volume. In some aspects, the reference tumor volume is the tumor volume in the subject prior to the administration of the engineered cell. In further aspects, the reference tumor volume is the tumor volume in a corresponding subject that did not receive the administration. In some aspects, the tumor volume in the subject is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after the administration compared to the reference tumor volume.

[0202] In some aspects, treating a tumor comprises reducing a tumor weight in the subject. In certain aspects, administering the population of immune cells reduces the tumor weight in a subject when administered to the subject. In some aspects, the tumor weight is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after the administration compared to a reference tumor weight. In some aspects, the reference tumor weight is the tumor weight in the subject prior to the administration of the population of immune cells of the disclosure. In further aspects, the reference tumor weight is the tumor weight in a corresponding subject that did not receive the administration.

[0203] In some aspects, administering the population of immune cells to a subject, e.g., suffering from a tumor, increases the number and / or percentage of TILs (e.g., CD4+or CD8+) in a tumor and / or a tumor microenvironment (TME) of the subject. In certain aspects, the numberand / or percentage of TILs in a tumor and / or TME is increased by at least about 5%, at least about10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, or at least about 300% or more compared to a reference (e.g., corresponding value in a subject that did not receive the cell composition of the present disclosure or the same subject prior to the administration of the cell composition of the present disclosure).

[0204] In some aspects, administering the population of immune cells to a subject, e.g., suffering from a tumor, can increase the duration of an immune response in a subject relative to the duration of an immune response in a subject administered a similar cell therapy comprising cells prepared according to conventional methods, e.g., cell stimulated with a CD3 agonist alone or in combination with a CD28 agonist. In certain aspects, the duration of the immune response is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 1000% or more. In certain aspects, the duration of the immune response is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold or more.

[0205] As described herein, the population of immune cells, e.g., the population of stimulated immune cells, can be used to treat variety of cancer types, e.g., a tumor derived from a cancer comprising a breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, renal cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (gastric) cancer, gastrointestinal cancer, ovarian cancer, carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof.

[0206] In some aspects, the population of immune cells can be used in combination with other therapeutic agents (e.g., anti-cancer agents and / or immunomodulating agents). Accordingly,in certain aspects, a method of treating a tumor disclosed herein comprises administering the population of immune cells in combination with one or more additional therapeutic agents.

[0207] In some aspects, the subject is a nonhuman animal such as a rat or a mouse. In some aspects, the subject is a human.

[0208] In some aspects, the population of immune cells is used in combination with other therapeutic agents (e.g., anti-cancer agents and / or immunomodulating agents). Accordingly, in certain aspects, a method of treating a tumor disclosed herein comprises administering a population of immune cells in combination with one or more additional therapeutic agents to a subject. Such agents can include, for example, chemotherapeutic drug, targeted anti-cancer therapy, oncolytic drug, cytotoxic agent, immune-based therapy, cytokine, surgical procedure, radiation procedure, activator of a costimulatory molecule, immune checkpoint inhibitor, a vaccine, a cellular immunotherapy, or any combination thereof.

[0209] In some aspects, the population of immune cells cultured is used in combination with a standard of care treatment (e.g., surgery, radiation, and chemotherapy). Methods described herein can also be used as a maintenance therapy, e.g., a therapy that is intended to prevent the occurrence or recurrence of tumors.

[0210] In some aspects, the population of immune cells is used in combination with one or more anti-cancer agents, such that multiple elements of the immune pathway can be targeted. Nonlimiting of such combinations include: a therapy that enhances tumor antigen presentation (e.g., dendritic cell vaccine, GM-CSF secreting cellular vaccines, CpG oligonucleotides, imiquimod); a therapy that inhibits negative immune regulation e.g., by inhibiting CTLA-4 and / or PD-l / PD- L1 / PD-L2 pathway and / or depleting or blocking Tregs or other immune suppressing cells (e.g., myeloid-derived suppressor cells); a therapy that stimulates positive immune regulation, e.g., with agonists that stimulate the CD-137, OX-40, and / or CD40 or GITR pathway and / or stimulate T cell effector function; a therapy that increases systemically the frequency of anti-tumor T cells; a therapy that depletes or inhibits Tregs, such as Tregs in the tumor, e.g., using an antagonist of CD25 (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion; a therapy that impacts the function of suppressor myeloid cells in the tumor; a therapy that enhances immunogenicity of tumor cells (e.g., anthracyclines); adoptive T cell or NK cell transfer including genetically engineered cells, e.g., cells engineered to express a chimeric antigen receptor (CAR-T therapy); a therapy that inhibits a metabolic enzyme such as indoleamine dioxigenase (IDO), dioxigenase, arginase, or nitric oxide synthetase; a therapy that reverses / prevents T cell anergy or exhaustion; a therapy that triggers an innate immune activation and / or inflammation at a tumor site;administration of immune stimulatory cytokines; blocking of immuno repressive cytokines; or any combination thereof.

[0211] In some aspects, an anti-cancer agent comprises an immune checkpoint inhibitor (z.e., blocks signaling through the particular immune checkpoint pathway). Non-limiting examples of immune checkpoint inhibitors that can be used in the present methods comprise a CTLA-4 antagonist (e.g., anti-CTLA-4 antibody), PD-1 antagonist (e.g., anti-PD-1 antibody, anti-PD-Ll antibody), TIM-3 antagonist (e.g., anti-TIM-3 antibody), or combinations thereof. Non-limiting examples of such immune checkpoint inhibitors include the following: anti-PDl antibody e.g., nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®; MK-3475), pidilizumab (CT-011), PDR001, MEDI0680 (AMP-514), TSR-042, REGN2810, JS001, AMP-224 (GSK-2661380), PF- 06801591, BGB-A317, BI 754091, SHR-1210, and combinations thereof); anti-PD-Ll antibody (e.g., atezolizumab (TECENTRIQ®; RG7446; MPDL3280A; RO5541267), durvalumab (MEDI4736, IMFINZI®), BMS-936559, avelumab (BAVENCIO®), LY3300054, CX-072 (Proclaim-CX-072), FAZ053, KN035, MDX-1105, and combinations thereof); and anti-CTLA-4 antibody (e.g., ipilimumab (YERVOY®), tremelimumab (ticilimumab; CP-675,206), AGEN-1884, ATOR-1015, and combinations thereof).

[0212] In some aspects, an anti-cancer agent comprises an immune checkpoint activator (i.e., promotes signaling through the particular immune checkpoint pathway). In certain aspects, immune checkpoint activator comprises 0X40 agonist (e.g., anti-OX40 antibody), LAG-3 agonist (e.g. anti-LAG-3 antibody), 4-1BB (CD137) agonist (e.g., anti-CD137 antibody), GITR agonist (e.g., anti-GITR antibody), TIM3 agonist (e.g., anti-TIM3 antibody), or combinations thereof.

[0213] In some aspects, the population of immune cells is administered to the subject prior to or after the administration of the additional therapeutic agent. In other aspects, the population of immune cells disclosed herein is administered to the subject concurrently with the additional therapeutic agent. In certain aspects, the population of immune cells disclosed herein and the additional therapeutic agent can be administered concurrently as a single composition in a pharmaceutically acceptable carrier. In other aspects, the population of immune cells disclosed herein and the additional therapeutic agent are administered concurrently as separate compositions. In some aspects, the additional therapeutic agent and the population of immune cells disclosed herein are administered sequentially.

[0214] Certain aspects of the present disclosure are directed to methods of treating an autoimmune disease, comprising administering a population of immune cells, e.g., comprising a Treg cell, cultured according to any of the methods disclosed herein. Other aspects of the presentdisclosure are directed to methods of treating an inflammatory pathology, comprising administering a population of immune cells, e.g., comprising a Treg cell, cultured according to any of the methods disclosed herein. In some aspects, the inflammatory pathology comprises cytokine release syndrome. In some aspects, the inflammatory pathology comprises sepsis. In some aspects, the inflammatory pathology comprises graft-versus host disease. In some aspects, the immune cell, e.g., the Treg cell, is engineered.EXAMPLESExample 1.

[0215] Materials and Methods

[0216] In vitro Culture of Primary Human T Cells

[0217] Peripheral blood mononuclear cells (PBMC) were obtained from healthy donors, and processed by Ficoll-Paque PLUS gradient centrifugation (GE healthcare). Naive CD8+T cells were isolated using EasySep Human Naive CD8+T cell Isolation kit II (STEMCELL Technologies). K562-based artificial antigen-presenting cells (aAPC) were modified to express the membrane-bound form of superagonistic anti-CD28 antibody clone TGN1412 (aAPCs). Isolated naive CD8+T cells were stimulated with aAPCs, Dynabeads Human T-Activator CD3 / 28 (Thermo Fisher Scientific) (Beads), or aAPCs with plate-bound anti-CD3 antibody (aAPCs+aCD3) (clone SK7, BioLegend). For aAPCs and Beads stimulation, naive CD8+T cells were stimulated at a T cell to aAPC ratio of 10: 1 or a T cell to Beads ratio of 1 :2. For aAPCs+aCD3, 2 pg / mL of anti- CD3 antibody was coated on a flat-bottom culture plate and incubated at 4C° overnight. On the following day, naive CD8+T cells and aAPCs were seeded at a ratio of 10: 1. Human IL-7 (25 ng / mL), human IL-21 (25 ng / mL) (PeproTech) and anti -human IFN-g (2 pg / ml) (BioXcell, clone B133.5) were added to the aAPC and aAPCs+aCD3 -stimulation conditions. 100 lU / ml of human IL-2 (Novartis) was added to the Bead-stimulation condition. Culture media was replenished every 2 or 3 days. aAPCs were irradiated at 200 Gy using a cesium-137 source.

[0218] For PI3K / mT0R pathway inhibition assays, naive CD8+T cells were labelled with CellTrace Violet (CTV) (Thermo Fisher Scientific) and stimulated with aAPCs or aAPCs+aCD3 in the presence of LY294002 (PI3K inhibitor) (Selleck Chemicals) or Rapamycin (mTORCl and 2 inhibitor) (Selleck Chemicals). Cell division was evaluated as dye dilution by flow cytometry on day 5, and the cell number was counted by flow cytometry using counting beads on day 7. Allprimary T cells were cultured in RPMI 1640 with 25mM HEPES (Gibco) supplemented with 10% human AB serum (Gemini Bio-Products), 50 pg / mL gentamicin (Bioshop).

[0219] Cytokine and CD107a Degranulation Assays

[0220] T cells were prepared as described above. For cytokine assays, IxlO5cultured T cells were restimulated with Beads and incubated in culture media. After 1 hour of incubation, brefeldin A solution (IX) (BioLegend) was added and cells were incubated for 4 hours. Cytokine production was evaluated by intracellular staining using Cyto-Fast Fix / Perm Buffer Set (BioLegend) and analyzed by flow cytometry. For CD107a degranulation assays, IxlO5cultured T cells were restimulated with Beads or cocultured with target cells at an E:T ratio of 1 : 1 and cultured for 5 hours in the presence of APC conjugated CD 107a antibody (BioLegend, clone H4A3). Monensin (BioLegend) was added after 1 hour of incubation. Degranulation capacity was measured by flow cytometry.

[0221] Cytotoxicity Assays

[0222] T cells were transduced using retrovirus on three consecutive days after 3 days of aAPC or Bead-stimulation. Transduced cells expressing a truncated nerve growth factor receptor (DNGFR) were magnetically sorted using human CD271 Microbeads kit (Miltenyi Biotec) on day 7. Magnetically sorted T cells were co-cultured with CTV labelled target cells at E:T ratios of 4: 1, 2: 1, and 1 : 1. After 24-hour culture, cytotoxicity was evaluated by measuring TO-PRO-3 (Thermo Fisher Scientific) labeled dead cells using flow cytometry.

[0223] For xCELLigence Real-Time Cell Analysis, T cells were prepared as described above. IxlO4target cells were seeded on E-plates the day before and cultured CD8+T cells were seeded at an E:T ratio of 2: 1. Cytotoxicity was measured using xCELLigence eSight (Agilent Technologies) following the manufacturer’s instructions.

[0224] Flow Cytometry

[0225] The following antibodies were used for flow cytometry analysis: PE-Cy7-anti-CD8 (SKI, BioLegend), Pacific-blue-anti-CD8 (RPA-T8, BioLegend), PE-anti-CD8 (RPA-T8, BioLegend), AF700-anti-CD8 (RPA-T8, BioLegend), PE-anti-CD34 (QBEnd / 10, R&D systems), FITC-anti-CD45RO (UCHL1, BioLegend), APC-anti-CD45RA (HI100, BioLegend), APC-Cy7- anti-CD45RA (HI100, BioLegend), Pacific-blue-anti-CCR7 (G043H7, BioLegend), FITC-anti- CD95 (DX2, BioLegend), PE-Cy7-anti-CD25 (BC96, BioLegend), FITC-anti-CD271 (ME20.4, BioLegend), APC-anti-PD-l(EH12.2H7, BioLegend), APC-anti-TIM-3 (F38-2E2, BioLegend), PE-Cy7-anti-LAG-3 (11C3C65, BioLegend), Pacific blue-anti-CD69 (FN50, BioLegend), PE- TCR-Vbl3.1 (IMMU222, Beckman Coulter), PE-anti-TCFl (7F11A10, BioLegend), Alexa Fluor647-TCF1 (7F11A10, BioLegend), Alexa Fluor 488-anti-IRF4 (IRF4.3E4, BioLegend), Alexa- Flour647-anti-IRF4 (IRF4.3E4, BioLegend), Alexa-Flour647-anti-BLIMPl (646702, R&D systems), PE-Cy7-anti-IFNgannma (B27, BioLegend), PE-anti-TNFalfa (Mabl l, BioLegend), APC-anti-IL2 (MQ1-17H12, BioLegend), APC-anti-CD107a (H4A3, BioLegend), FITC-anti- Granzyme B (GB11, BioLegend), Alexa Fluor-phosphoS6 Ribosomal Protein (Ser235 / 236) (D57.2.2E, Cell Signaling Technology), Alexa-Fluor 647-phospho-Akt (Ser473) (D9E, Cell Signaling Technology), PE-phospho-Akt (Thr308) (D25E6, Cell Signaling Technology). Dead cells were discriminated with 7-AAD Viability Staining Solution (BioLegend) or LIVE / DEAD Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific). Stained cells were analyzed with a CytoFLEX S Flow Cytometer (Beckman Coulter). Data analysis was performed using FlowJo software (BD Life Sciences).

[0226] Generation of TGN1412 Antibody and TGN1412 Microbeads

[0227] TGN1412 protein was produced using the Expi293 Expression System Kit (ThermoFisher Scientific following manufacturer’s instructions with the following exceptions. Cotransfections of TGN1412 light chain (LC) and TGN1412 heavy chain (HC) containing a C- terminal AviTag sequence were performed at an LC:HC ratio of 7:3 in pcDNA3.1 expression vectors. Protein was isolated using Protein A agarose (Thermo Fisher Scientific) following manufacturer’s instructions with lx PBS pH 7.4 binding buffer, 0.1 M glycine pH 3 elution buffer, and 1.5M Tris-HCl pH 8.8 neutralization buffer. Eluates were assessed for total protein using Pierce BCA Protein Assay Kits (Thermo Fisher Scientific) and combined and washed 3x in PBS using Amicon Ultra 0.5 mL centrifugal filters, 10K MWCO (Millipore Sigma), and resuspended in PBS. Protein purity was assessed by SDS-PAGE followed by Coomassie Brilliant Blue R-250 staining (Bio-Rad). Biotin was added to the TGN1412 protein using the BirA500 Biotin-Protein Ligase Standard Reaction Kit (Avidity Biosciences) following manufacturer’s instructions. Biotinylation was confirmed by SA-agarose (Thermo Fisher Scientific) pulldown and twin SDS- PAGE Western blot and Coomassie stain. Biotin was detected using a Biotin-HRP antibody (Jackson ImmunoResearch, 1 : 1,000) and imaged using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 1 : 100,000). TGN1412 microbeads were generated using the abovementioned biotinylated protein and 25 pm SA-coated polystyrene microbeads (Spherotech). 0.5 pg of biotinylated TGN1412 protein was added for every 200,000 beads. TGN1412-bound beads were resuspended in PBS containing 0.1% Pluronic F-68 surfactant (Thermo Fisher Scientific).

[0228] Generation of K562-Based Artificial Antigen-Presenting Cells Expressing Membrane-Bound TGN1412

[0229] Retroviral vectors encoding TGN1412 and CD83 coding sequence were transduced into K562 cells using 293GPG cell-based retrovirus system (see Ory, D.S., Neugeboren, B.A. & Mulligan, R.C. A stable human-derived packaging cell line for production of high titer retrovirus / vesicular stomatitis virus G pseudotypes. Proc Natl Acad Sci U S A 93, 11400-11406 (1996)). CRISPR / Cas9 vectors targeting CD32A (ORIGEN) were transfected into TGN1412 and CD83 -expressing K562 cells using Lipofectamine LTX reagent (Thermo Fisher Scientific) following the manufacturer’s protocol. B2M knockout in TGN1412 and CD83 -expressing CD 32 A knockout K562 cells were performed as previously described (see Kagoya, Y., et al. Genetic Ablation of HLA Class I, Class II, and the T-cell Receptor Enables Allogeneic T Cells to Be Used for Adoptive T-cell Therapy. Cancer Immunol Res 8, 926-936 (2020)).

[0230] Cell Lines

[0231] The human erythroleukemic cell line K562, the human melanoma cell line, A375 and SK-MEL-28, and the PG13 retroviral packaging cell line were obtained from the American Type Culture Collection (ATCC). All cell lines were routinely assessed for the presence of mycoplasma contamination using a PCR-based technology.

[0232] Retroviral Transduction of T Cells

[0233] pMX retroviral vector encoding 1G4LY TCR and ANGFR coding sequences linked by 2A sequences was transduced into PG13 packaging cells. T cell transduction was performed using PG13 retroviral supernatant on day 3, 4 and 5 following the stimulation with aAPCs or Beads.

[0234] Metabolic Analysis

[0235] Seahorse cell plates were pre-coated with poly-D-lysine (50 pg / mL). 4xl05cultured T cells were resuspended in Seahorse XF assay media (RPMI, pH 7.4, 10 mmol / L glucose, 2 mmol / L L-glutamine, and 1 mmol / L sodium pyruvate), then were seed on Seahorse cell plates. Cells were incubated in a CCh-free incubator at 37°C for 60 minutes before Oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were measured using XF Real-Time ATP Rate Assay kit or Seahorse XF Cell Mito Stress Test Kit (Agilent Technologies) with XFe96 extracellular analyzer (Agilent Technologies), following the manufacturer’s protocols. Seahorse data was analyzed using Wave software with the appropriate assay templates (Agilent Technologies). Glucose uptake was measured using 2-NBDG (Invitrogen), and fatty acid uptake using BODIPY (Molecular Probes). Briefly, after 4-hour glucose starvation, IxlO5cells were stained with 25 pM of 2-NBDG at 37 °C for 30 minutes. For fatty acid uptake assays, IxlO5cellswere stained with 2 pM of BODIPY at 37 °C for 15 minutes. Cells were then analyzed by flow cytometry.

[0236] CRISPR / Cas9-mediated gene knockout in human primary T cells

[0237] Two IRF4 and AAVS1 -targeting CRISPR RNAs (crRNA) were designed and purchased from IDT. Electroporation was performed using P3 Primary Cell 4D-Nucleofector X Kit (Lonza), following the manufacturer’s protocols. Briefly, 160 pM crRNA and 160 pM tracrRNA (IDT) were annealed using a thermal cycler to generate crRNA: tracrRNA duplex. Subsequently, 180 pmol crRNA: tracrRNA duplex was mixed with 60 pmol TrueCut Cas9 protein v2 (Thermo Fisher Scientific) to form CRISPR ribonucleoprotein (crRNP). 5 mL of crRNP was mixed with 20 pL of 5-10 x 105T cells resuspended in P3 buffer, and nucleofection was performed using 4D-Nucleofector (Lonza). Immediately after nucleofection, cells were transferred to culture plates containing warmed complete media. Genomic DNA was extracted using DNeasy Blood and Tissue Kit (QIAGEN) and targeted sites were PCR amplified and sent for Sanger sequencing (The Centre for Applied Genomics). Knockout efficiency was determined by analyzing the sequencing results using ICE Analysis Tool (Synthego).

[0238] Overexpression of IRF4

[0239] IRF4 overexpression construct was generated by cloning human IRF4 cDNA into pmax expression vector downstream of CMV promoter. As a negative control, pmax vector containing IRF4 cDNA in the inverse orientation was used. Naive CD8+T cells were stimulated with aAPCs 3 days prior to electroporation and pmax-IRF4 was electroporated using 4D- Nucleofector (Lonza).

[0240] Mass Cytometry Using CyTOF

[0241] Custom conjugation of antibodies to heavy-metal ions was performed at the SickKids Centre for Advanced Single Cell Analysis (CASCA) using commercially available Maxpar Antibody Labeling Kits (Stanard Biotools) according to the Maxpar Antibody Labeling protocol PRD002 Rev 17(Standard Biotools). Palladium barcoding was employed to reduce variability associated with staining samples separately. Briefly, samples were washed twice with PBS, then incubated with 1 mM Cell-ID Intercal at or-Rh viability stain in RPMI for 15minutes in a 37°C incubator. Following a PBS wash, samples were fixed and permeabilized in the eBioscience Foxp3 / Trancription Factor Staining Buffer Set for 15 minutes at RT, then washed with lx Maxpar Barcode Perm Buffer. Each barcode was resuspended with 100 pL of lx Maxpar Barcode Perm Buffer, added to the designated cell sample, and incubated for 30 minutes at RT. Barcoded samples were washed with Maxpar Cell Staining Buffer twice, then pooled into a composite sample forstaining. Cells were incubated in 1 : 10 Fc block in lx eBioscience Permeabilization Buffer for 10 minutes. Antibody master mix in lx eBioscience Permeabilization Buffer was filtered through a 0.1 gm Ultra-free MC Centrifugal Filter to remove antibody aggregates, then added to the cell sample. After overnight incubation for 19 hours at 4°C, cells were washed with Cell Staining Media (CSM; PBS + 2% FBS), then stained with 125 pM Cell-ID Intercalator Ir in Maxpar Fix and Perm Buffer to distinguish single cells from doublets. Finally, cells were stored in PBC + 1.6% formaldehyde at 4°C until acquisition. Before acquisition, cells were washed once in PBS and filtered through a cell strainer then resuspended at 1 x 106cells per mL of Cell Acquisition Solution (CAS) supplemented with 10% EQ Four Element Calibration Beads. Acquisition was completed on a Helios mass cytometer (Standard Biotools).

[0242] Transmission Electron Microscopy

[0243] Samples for TEM were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer for 2 hours. Rinsed in buffer, post-fixed in 1% osmium tetroxide in buffer for 90 minutes, dehydrated in a graded ethanol series (50%, 70%, 90%, and 100%, 20 minutes each step) followed by two propylene oxide changes for 30 minutes, and embedded in Quetol-Spurr resin. Blocks were cured overnight in the oven at 60C. Sections 70nm thick were cut on a Leica EM UC7 ultramicrotome, stained with uranyl acetate and lead citrate. Length and area of mitochondria were measured from 15 images for each stimulation methods. Maximum cristae width was measured from 15 images using Fiji / ImageJ software. The investigators were blinded to allocation during measurement.

[0244] Bulk ATAC-Seq Library Preparation and Sequencing

[0245] Samples were prepared as described. Briefly 60,000 viable cells per sample were pelleted at 500 RCF at 4°C for 5 minutes. Supernatant was removed and cells were re-suspended in 50 gL of cold resuspension buffer (RSB) containing 0.1% NP40, 0.1% Tween-20 and 0.01% digitonin. Cell suspension was incubated on ice for 3 minutes before being washed out with 1 ml of cold RSB containing 0.1% Tween-20. Cells were mixed by inversion prior to pelleting at 500 RCF at 4°C for 5 minutes. Supernatant was removed, cells were re-suspended in 50 gL of transposition mix and incubated at 37°C for 30 minutes in a thermomixer (Eppendorf) set to 1,000 RPM. Transposition mix was purified using the Qiagen MinElute Reaction Cleanup Kit (Qiagen) and eluted in 20 gL of water. 1 gL of sample was taken into a qPCR reaction to determine the optimal number of PCR cycles required for amplification without reaching saturation and based off the measured cycle number, the remaining 19 gL of each sample was amplified. Libraries werepurified using AMPure XP beads (Beckman Coulter) using a double-sided bead cleanup protocol set to 0.7x-1.0x. This clean-up was performed twice to remove the large molecular fragments.

[0246] Purified libraries were evaluated for enrichment by qPCR using primers designed against open regions (KA T6B and GAPDH) compared against closed regions (QML93 and SLC22A3). Samples that had a fold enrichment greater than 10 were sequenced.

[0247] The libraries were quantified by qPCR then normalized and pooled to 1.25 nM. Each 1.25 nM pool was denatured using 4 pL of 0.2N NaOH (Sigma) for 8 minutes at room temperature before being neutralized with 5 pL of 400mM Tris-HCl (Sigma). The neutralized pool was loaded immediately onto a NovaSeq 6000 SP flow cell. Samples were sequenced with the following run parameters: read 1 - 50 cycles, read 2 - 50 cycles, index 1 - 8 cycles, index 2 - 0 cycles to achieve ~60 million reads per sample.

[0248] Bulk RNAseq Library Preparation and Sequencing

[0249] Total RNA samples were quantified by qubit RNA kit (Life Technologies) and quality assessed by Agilent Bioananlyzer. All samples have RIN >9.2. Libraries were prepared with 100 ng of total RNA using Stranded Total RNA prep with Ribo-Zero Plus kit (Illumina) with 13 cycles of amplification used. Final cDNA libraries were size validated using Agilent Bioanalyzer or Tapestation and concentration validated by qPCR (Kapa Biosy stems / Roche). All libraries were normalized and pooled together, denatured with 0.2N NaOH and diluted to a final concentration of 250 pM. Pooled libraries were loaded onto an Illumina Novaseq VI.5 cartridge for cluster generation and sequencing on an Illumina Novaseq 6000 instrument (Illumina). Samples were sequenced using the following parameters: read 1-101 cycles, read 2-101 cycles, index 1-8 cycles, index 2-8 cycles to achieve ~40 million reads per sample.

[0250] Mouse Studies

[0251] In the mouse experiments, male NSG mice that were bred at the Princess Margaret Cancer Centre animal facility were used. Mice were subcutaneously injected with 5* 104A375 cells. T cells stimulated with aAPCs or Beads were retrovirally transduced with 1G4LY TCR- encoding gene and cultured for 11 days. l * 106TCR-T cells were intravenously injected into the mice 13 days after the transplantation of A375. The volume of the inoculated tumors was monitored every 2-3 days until they reached >15 mm or an ulcer had formed in the tumor. Mice were monitored at least once daily and euthanized by CO2 inhalation after they reached the humane endpoints described above. The mice were assigned to treatment groups based on the tumor size assessed the day before T cell injection in each experiment. No statistical methods were used topredetermine sample size. The investigators were not blinded to allocation during experiments and outcome assessment.Example 2.

[0252] CD28 stimulation alone induces Tscm-like phenotype without driving effector differentiation

[0253] CD3 stimulation is well-documented for driving T cell effector differentiation. Even a weak CD3 stimulation of naive T cells resulted in a significant reduction in the expression of molecules marking naive and early memory T cells such as CCR7, CD45RA, and TCF1 (FIG. 6A). To deliver CD28 stimulation without CD3 engagement, three anti-CD28 antibody clones were used: 28.2, TGN1412, and ANC28.1 / 5D10. Both CD28.2 and TGN1412 increased TCF1 expression without inducing cell proliferation, whereas ANC28.1 / 5D10 enhanced proliferation and CD95 expression but reduced the CCR7+and CD45RA+population (FIGs. 1A-1E). In all conditions, the addition of CD3 stimulation skewed CD8+T cells towards effector differentiation. Cells stimulated using TGN1412-coated microbeads (TGN1412 microbeads) rapidly expanded while retaining the CCR7+TCF1+population, which was abrogated by CD3 costimulation. However, TGN1412 microbeads induced partial effector differentiation (FIGs. 6C-6G).

[0254] To stimulate naive CD8+T cells through CD28 more effectively, K562-based artificial antigen-presenting cells expressing membrane-bound TGN1412 (aAPCs) were generated. K562 is a human leukemic cell line that has been used as a backbone cell line to expand T cells and NK cells in research and clinical settings. B2M and CD32 were knocked out to remove potential alloreactivity and Fc-mediated antibody binding, respectively (FIGs. 6H-6K). Cellbased stimulation using aAPCs facilitated moderate proliferation of T cells while maintaining a Tscm-like phenotype without driving effector differentiation (FIGs. 1F-1G). Importantly, an early memory-like phenotype was maintained even after five repeated stimulations with aAPCs, in contrast to cells stimulated through CD3, which resulted in the downregulation of CCR7, CD45RA, and TCF1, demonstrating effector differentiation. While the proliferation of cells stimulated through CD3 peaked at the third or fourth restimulation before undergoing contraction, cells stimulated with aAPCs exhibited moderate but persistent proliferation even after repeated rounds of stimulations (FIGs. 1H-1L).Example 3.

[0255] Proliferation of aAPC-Stimulated Cells is Sensitive to PI3K-mTOR Inhibition

[0256] The PI3K pathway plays a pivotal role in T cell proliferation and effector / memory formation. To investigate whether cell proliferation induced by CD28 stimulation, in the absence of CD3 coengagement, depends on the PI3K / mTOR pathway, the levels of phosphorylated S6 and AKT, which are downstream of PI3K, were measured. S6 and AKT were both phosphorylated when cells were stimulated with aAPCs alone, although the extent of phosphorylation was enhanced by CD3 costimulation (FIGs. 7A-7D). To examine whether PI3K / mT0R was necessary for aAPC-mediate T cell proliferation, we added LY294002, a PI3Kalfa / delta / beta inhibitor and rapamycin, an mTORCl / 2 inhibitor. Expansion and cell division of aAPC-stimulated cells was significantly suppressed by LY294002 and rapamycin. On the other hand, cells stimulated with CD3 were mostly unaffected (FIGs. 7E-7 J). These results indicate that the proliferation of aAPC- stimulated cells is dependent PI3K / mT0R pathway.Example 4.

[0257] aAPC-stimulation expands CD8+T cells displaying early memory-like phenotype

[0258] Cytometry by Time-of-Flight (CyTOF) analysis was performed to compare T cells stimulated with aAPCs, aAPCs plus CD3 coengagement (aAPCs+aCD3), CD3 / CD28 Dynabeads (Beads), or unstimulated naive T cells. Uniform Manifold Approximation and Projection (UMAP) analysis revealed that aAPC-stimulated cells formed a cluster distinct from naive cells and those stimulated with CD3 and CD28 (Beads or aAPCs+aCD3) (FIGs. 2A, 2S, 8A, and 8C). As expected, CD3 and CD28-stimulated cells presented expression signatures of differentiated effector T cells, including elevated expression of effector differentiation markers (CD45RO and KLRG1), activation markers (CD25 and CD69), and exhaustion markers (PD-1, LAG-3, TIM-3, and TIGIT). In contrast, aAPC-stimulated cells showed higher expression of CD45RA, CD62L, CCR7, and CD95, which are markers defining Tscm. Transcription factor expression also differed between clusters, with TCF1 enriched in aAPC-stimulated cells, while IRF-4, BLIMP-1, and T- bet were enriched in cells with CD3 stimulation (Bead and aAPCs+aCD3) (FIGs. 2B-2R and FIG. 8B)

[0259] Metabolic characterization using CyTOF revealed that aAPC-stimulated cells exhibit a metabolic phenotype relying more on fatty acid oxidation than glycolysis, indicated by higher expression of CPTla and lower expression of GLUT1 and G6PD (FIGs. 2T-2BB and FIG. 8D) These cells also displayed reduced oxidative stress as shown by the lower expression of NRF2 and HIFla. In contrast, cells stimulated with CD3 and CD28 showed elevated expression ofexhaustion markers, CD39 and TOX (FIGs. 2T-2BB and FIG. 8D). Flow cytometric analysis using 2-NBDG and BODIPY substrates confirmed the differing use of metabolic programs by aAPC-stimulated cells, concordant with the CyTOF data (FIGs. 8E-8F).

[0260] Electron microscopy (EM) analysis found that aAPC-stimulated cells had significantly more elongated and enlarged mitochondria compared to naive T cells and those stimulated through CD3. These cells also had more tightly packed mitochondria (FIGs. 2CC-2FF). Seahorse Mito Stress tests showed that aAPC-stimulated cells had higher oxygen consumption rate (OCR) and spare respiratory capacity (SRC) (FIGs. 2GG-2HH). This increased mitochondrial function was linked to a higher proportion of adenosine triphosphate (ATP) produced from oxidative phosphorylation than glycolysis. In contrast, Bead or aAPCs+aCD3 -stimulated cells primarily produced ATP through glycolysis (FIG. 8G). Furthermore, aAPC-stimulated cells stored more ATP compared to CD3 -stimulated cells (FIG. 8H). These results suggest that aAPC- stimulated cells are in a dormant state typical of early memory T cells or Tscm, allowing them to mount a rapid and potent response upon antigen restimulation.Example 5.

[0261] Genetic Signatures of Tscm Are Enriched in aAPC-Stimulated Cells

[0262] Bulk RNA-seq was performed on naive CD8+T cells that were unstimulated or stimulated with aAPCs, Beads, aCD3 alone or aAPCs+aCD3. Principal component analysis (PC A) showed that aAPC-stimulated cells had a distinct transcriptomic profile compared to naive cells and those receiving CD3 stimulation (FIG. 3A). Bead-stimulated cells had elevated expression of genes associated with T cell effector differentiation (TBX21, ZEB2, PRF1, GZMB, PRDM1, and IRF4) and genes associated with T cell exhaustion (HA VCR2. PDCD1, TIGIT, and TOX). Conversely, aAPC-stimulated cells expressed higher level of TCF7 and KLF2, crucial for Tscm formation, and genes associated with early differentiated memory T cells, such as CCR7 (FIGs. 3B-3C) The transcript-level findings from RNA-seq were further corroborated by proteomic analysis using CyTOF (FIGs. 9A-9R).

[0263] Gene set enrichment analysis (GSEA) indicated that Tscm signatures were enriched in aAPC-stimulated cells, whereas Bead-stimulated cells were characterized by an enrichment of precursor exhausted T (Tpex) signatures (FIG. 3D). This was more pronounced when comparing aAPC-stimulated cells to those stimulated with CD3 alone (FIGs. 9T). Additionally, the WNT pathway as well as hematopoietic stem cell differentiation signatures were enriched in aAPC- stimulated cells (FIGs. 9U-9V).

[0264] Continuous CD3 stimulation or CD3 stimulation without necessary costimulatory signals can induce apoptosis or anergy. However, aAPC-stimulated cells did not upregulate genes driving these processes. Instead, anti-apoptotic genes were expressed at higher levels in aAPC- stimulated cells compared to CD3 stimulated cells (FIG. 3E). These findings suggest that while CD28 stimulation alone maintains TCF1 expression and a Tscm-like phenotype, CD3 stimulation drives effector differentiation.Example 6.

[0265] aAPC Stimulation Generates T cells Bearing Epigenetic Profile Similar to Tscm

[0266] To explore the epigenetic landscape of Tscm-like cells generated using the aAPCs, an assay was performed for transposase-accessible chromatin using sequencing (ATAC-seq) after aAPC or Bead-stimulation. PCA showed that aAPC-stimulated cells had a distinct chromatin accessibility profile from naive and Bead-stimulated cells (FIG. 3F). Genes associated with T cell memory (TCF7, LEF, SLAMF6, and CCR7) were more accessible in aAPC-stimulated cells, while those associated with T cell effector differentiation (TBX21, PRDM1, and IRF4) and exhaustion (ENTPD1, HAVCR2, TIGIT. and NFKBP) were more accessible in Bead-stimulated cells (FIGs. 3G-3H) Motif enrichment analysis using HOMER in differentially accessible regions (DARs) revealed that aAPC-stimulated cells were enriched in binding motifs of transcription factors associated with naive and early memory cells (RUNX and FOXPF) and an ETS family transcription factor, GABBA, involved in homeostasis and formation of memory T cells. TCF7L2 and E2A, known to enhance TCF1 expression and memory T cell formation, were also enriched in aAPC- stimulated cells. Conversely, transcription factors associated with effector differentiation (IRF4, BA TE, PRDM1, and T-bef) and activation (NFAT, Jun, and AP-P) were enriched in Bead- stimulated cells. NFAT induces T cell exhaustion, especially in the absence of AP-1, by elevating TOX. The NFAT motif was enriched in DARs associated with Bead but not aAPC-stimulated cells, suggesting that CD28 stimulation alone does not drive T cell exhaustion like CD3 (FIG. 31). Collectively, sole stimulation through CD28 generates T cells characterized by a distinct epigenetic profile coupled with transcriptomic and phenotypic differences, resembling the known characteristics of Tscm.Example 7.

[0267] IRF4 Deletion Limits Effector Differentiation and Promotes Metabolic Switch from Glycolysis to Oxidative Phosphorylation Pathway

[0268] IRF4 is rapidly upregulated in T cells upon antigen stimulation and its expression is positively correlated with the strength of TCR stimulation. Upregulated IRF4 interacts with other TCR-induced transcription factors, such as NF AT and BATF, and plays a crucial role in T cell effector differentiation and formation of exhaustion phenotype. IRF4 was among the most differentially expressed genes between aAPC and Bead-stimulated cells in our RNA-seq result, with minimal expression in Tscm-like cells generated by aAPC stimulation (FIG. 3B). To explore the role of IRF4 induced by CD3 but not by CD28, we used the CRISPR / Cas9 system to knock out IRF4 in naive CD8+T cells before stimulating with aAPCs, Beads or aAPCs+aCD3 (FIGs. 4A- 4D) IRF4 deletion reduced the expression of effector differentiation and activation markers, including BLIMP1, and restored TCF1 expression in Bead or aAPCs+aCD3 -stimulated cells, suggesting that CD3-induced IRF4 promotes BLIMP-1 upregulation and TCF1 downregulation (FIGs. 4E-4I)

[0269] In a reciprocal experiment, aAPC-stimulated cells overexpressing IRF4 exhibited increased CD45RO, PD-1, CD69 and BLIMP 1 expression, and decreased TCF1 expression (FIGs. 4L^4N). Consistent with surface marker profiles, OXHOS was restored upon IRF4 deletion in CD3 -stimulated cells, resembling the metabolic phenotype of aAPC-stimulated cells (FIGs. 40- 4P) These data reveal that IRF4 is crucial in T cell differentiation by controlling TCF1 and BLIMP 1 expression, as well as the metabolic switch from OXHOS to glycolysis. The absence of IRF4 expression in aAPC-stimulated cells critically prevents effector differentiation in our system.Example 8.

[0270] aAPC-Stimulated Cells Demonstrate Higher Anti-Tumor Function Compared to Conventional Effector Cells

[0271] Tscm exhibit strong responses upon encountering the cognate antigen similar to other memory T cell subsets. Cells stimulated with aAPCs demonstrated enhanced cytokine production and cytokine polyfunctionality after restimulation with Beads (FIGs. 5A-5D) as well as higher degranulation than Bead-stimulated cells (FIGs. 10A-10B). To assess the capacity for self-renewal and maintenance of the Tscm-like phenotype, cells stimulated with aAPC or Beads were restimulated with Beads, and their phenotype after cell division was monitored. In aAPC-stimulated cells, the CFSE low fraction retained the TCF1+population, whereas Bead-stimulated cells largely lost TCF1 expression in divided cells, indicating that aAPC-stimulated cells possess self-renewal capacity (FIGs. 5E-5F).

[0272] The antigen-specific functionality of Tscm-like cells generated through aAPC- mediated CD28 stimulation compared to conventional Bead-stimulated effector cells was evaluated. CD8+T cells were engineered to express an HLA-A2 / NY-ESO-1 TCR. Both aAPC and Bead-stimulated cells had similar TCR expression levels (FIGs. 10C-10D). However, aAPC- stimulated cells demonstrated higher cytotoxicity against A2+NY-ESO-1+A375 cells across all the E:T ratios tested (FIGs. 5G-5H). Degranulation measured by the surface expression of CD 107a was higher in aAPC-stimulated cells than Bead-stimulated cells (FIGs. 10E-10G). In long-term cytotoxicity assays, aAPC-stimulated cells showed potent cytotoxicity quickly after being added to the target cells and continued to exert anti-tumor control throughout the assays (FIGs. 5I-5J and FIG. 10H) Moreover, aAPC-stimulated TCR-T cells maintained a less differentiated CD25lowCCR7highpopulation after 3 days of co-culture with target cells, unlike Bead-stimulated TCR-T cells. Furthermore, aAPC-stimulated cells showed significantly lower expression of exhaustion markers PD-1 and TIM-3 (FIGs. 10I-10N). In immunodeficient mice bearing subcutaneous A375 tumors, aAPC-stimulated TCR-T cells exerted improved tumor control compared to Beads stimulated TCR-T cells (FIGs. 5K-5M). Overall, TCR-T cells generated using CD28-stimulating aAPCs demonstrated superior cytotoxicity and resistance to tumor-driven exhaustion.Example 9.

[0273] Materials and methods

[0274] Cell lines

[0275] The human melanoma cell lines HLA-A2+NY-ESO-1+A375 and HLA-A2" NY- ESO-1" SK-MEL-28, the human pancreatic cancer line Mesothelin (MSLN)+AsPC-1 were obtained from the American Type Culture Collection (ATCC). All cell lines were routinely assessed for the presence of mycoplasma contamination using a PCR-based technology.

[0276] Flow cytometry analysis

[0277] The following antibodies were used for flow cytometric analysis: APC-anti-CD15 (W6D3, BioLegend), APC-anti-CD58 (TS2 / 9, BioLegend), PE-anti-CD59 (p282(H19), BioLegend), PE-anti-ICAMl (HCD54, BioLegend), FITC-anti-ICAM2 (CBR-IC2 / 2, BioLegend).Stained cells were acquired on a CytoFLEX S Flow Cytometer (Beckman Coulter) and data were analyzed using Flow Jo software version 10 (BD Life Sciences).

[0278] Adhesion molecule blocking assay

[0279] Naive CD8+T cells were treated with 10 pg / ml of ocCD18 (clone TS1 / 18, BioLegend), ocCD2 (clone RAP -2.10, BioLegend), a combination of ocCD18 and ocCD2, or isotype control antibody (clone MOPC-21, BioLegend) for 1 hour at 37 °C prior to aAPC-stimulation. Cells were cultured as described above. After seven days, proliferation and phenotype were evaluated by flow cytometry.

[0280] CRISPR-Cas9-mediated gene knockout

[0281] CRISPR RNAs (crRNA) targeting CD3Z, MSLN, and the safe harbor locus A A VS1 were designed and synthesized (Integrated DNA Technologies (IDT)). Electroporation was performed using the P3 Primary Cell 4D-Nucleofector X Kit (Lonza) according to the manufacturer’s instructions. Briefly, 160 pM crRNA and 160 pM tracrRNA (IDT) were annealed using a thermal cycler to form crRNA:tracrRNA duplexes. A total of 180 pmol of the duplex was then mixed with 60 pmol TrueCut Cas9 protein v2 (Thermo Fisher Scientific) to form CRISPR ribonucleoproteins (crRNPs). For each reaction, 5 pL of crRNP was mixed with 20 pL of 5-10 x 105T cells resuspended in P3 buffer, and nucleofection was performed using the 4D-Nucleofector System (Lonza). Immediately after nucleofection, cells were transferred into pre-warmed complete culture medium.

[0282] Mouse studies

[0283] NSG mice bred at the Princess Margaret Cancer Centre Animal Resource Centre (ARC) were used for all in vivo experiments. For the melanoma model, male mice were subcutaneously (s.c.) injected with 5 x io4A375 cells. Thirteen days later, mice received intravenous (i.v.) injections of 1 x io6TCR-engineered T cells, which had been generated by retroviral transduction of T cells with A2 / NY-ESO-1 TCR-encoding genes following stimulation with either aAPCs or beads and cultured for a total of 11 days. T cells transduced with ANGFR alone were used as a control. For the pancreatic tumor model, 5 x io5AsPC-1 cells were s.c. injected to female mice, and 1 x 106anti-MSLN CAR-T cells were administered i.v. on day 8 post- engraftment. Tumor volumes were measured every 2-3 days and monitored until they exceeded 10 mm in diameter or developed ulceration, at which point mice were euthanized by CO2 inhalation in accordance with humane endpoint guidelines. Mice were monitored at least once daily throughout the experiments. Treatment groups were assigned based on tumor size measured theday prior to T cell infusion. No statistical methods were used to predetermine sample size, and investigators were not blinded to group allocation or outcome assessment.

[0284] Cytotoxicity assays

[0285] Naive CD8+T cells were expanded for three days using either aAPCs or beads and subsequently engineered to express either the HLA-A2 / NY-ESO-1 -specific TCR (clone 1G4LY) or the MSLN-targeting 4-1BB CAR (clone SSI), each linked to ANGFR via a furin-2A selfcleaving peptide sequence. T cells transduced with ANGFR alone served as controls. Retroviral transduction was performed using PG13 -derived supernatant three days after the initial stimulation with aAPCs or beads, generating gene-modified T cells. On day 7, ANGFR-positive cells were isolated using the human CD271 Microbeads Kit (Miltenyi Biotec) and co-cultured with CTV- labeled target cells at E:T ratios of 4: 1, 1 : 1, and 1 :4. After 24 hours, cytotoxicity was assessed based on TO-PRO-3 (Thermo Fisher Scientific) uptake in dead target cells.

[0286] Results

[0287] aAPC stimulation is not functionally equivalent to weak CD3 engagement

[0288] To delineate the differences between weak CD3 engagement and aAPC stimulation, we titrated CD3 stimulation to achieve equivalent expansion. Despite equal proliferation, weak CD3 engagement still resulted in robust upregulation of CD25 and CD69, increased expression of the activation / exhaustion marker PD-1, and a marked reduction in TCF1 levels (FIGs. 11 A-l 1C). These changes were not observed following aAPC stimulation, highlighting that aAPCs elicit a fundamentally distinct activation program from that induced by low-level CD3 signaling (FIGs. 11A-11C).

[0289] Adhesion molecules promote effective aAPC-mediated T cell proliferation.

[0290] The observation that aAPCs induced changes to the phenotype of T cells in the absence of direct CD3 -engagement led us to explore their unique properties. In addition to TCR and co-stimulatory engagement, optimal T cell activation depends on the coordinated activity of adhesion molecules to stabilize the interaction between T cells and aAPCs (see, e.g., Wulfing C, et al. PNAS USA. 1998;95(l l):6302-7; Zuckerman LA, et al. J Immunol. 1998;160(7):3259-68). We confirmed the surface expression of adhesion molecules, including CD15, CD58, CD59, ICAM1 and ICAM2, on aAPCs (FIG. 12A). To assess the functional significance of these molecules, we performed blocking experiments targeting CD 18 and CD2, the respective ligands for ICAMs and CD58 on T cells. Antibody-mediated blockade of these interactions markedly reduced T cell proliferation (FIG. 12B) and abolished the upregulation of CD95 (FIG. 12D),although TCF1 expression remained unchanged (FIG. 12C), suggesting that stable engagement of CD28, facilitated by adhesion molecule interactions, is critical for aAPCs-induced T cell proliferation and the induction of the Tscm-associated marker CD95.

[0291] aAPC-stimulated cells demonstrate higher antitumor function upon their first CD3 engagement compared to conventional effector cells undergoing restimulation.

[0292] Functionally, T scm demonstrates the robust responses upon encountering a “target” antigen. To assess these features, we compared antigen-specific cytotoxic capacity of TCR- and CAR-engineered T cells generated with aAPCs or beads. Mesothelin (MSLN) CAR-T cells generated via aAPC stimulation demonstrated significantly enhanced cytotoxicity against MSLN+AsPC-1 cells but not against MSLN-knockout controls across all E:T ratios (FIGs. 13A-13B). In vivo experiments using NSG mice bearing A375 or AsPC-1 tumors revealed that CD8+ TCR- or CAR-T cells generated with aAPCs-without CD4+T cell help or exogenous cytokine support- achieved significantly superior tumor control compared to those expanded with beads (FIGs. 13C- 13H).

[0293] aAPCs generate T cells with an epigenomic program resembling Tscm.

[0294] To investigate whether the transcriptomic differences were associated with epigenetic alterations, we performed an assay for transposase-accessible chromatin using sequencing (ATAC-seq) on naive T cells and T cells stimulated with aAPCs or beads. Notably, genomic regions of T cell memory genes (LEF1, FIG. 14 A; and CCR7, FIG. 14B) exhibited increased accessibility in aAPC-stimulated cells. Conversely, genes associated with T cell effector differentiation, such as IRF4, showed decreased accessibility (FIG. 14C). Motif enrichment and variability analyses revealed that chromosomal regions accessible in aAPC-stimulated cells were enriched in transcription factor binding motifs associated with naive and early memory cells such as RUNX and FOXP1 (see, e.g., Galletti G, et al. Nat Immunol. 2020;21(12): 1552-62), as well as GABPA, an ETS family transcription factor involved in homeostasis and formation of memory T cells (FIG. 14D). Additionally, motifs for TCF7L2 and E2A, known enhancers of TCF1 expression and memory T cell development (see, e.g., Schauder DM, et al. PNAS USA. 2021; 118(16)), were enriched in aAPC-stimulated cells. In contrast, binding motifs for transcription factors associated with effector differentiation (IRF4, BATF, BLIMP1 and T-bet) and activation (NF AT, Jun and AP-1) were enriched in bead-stimulated cells (FIG. 14E). Notably, increased accessibility of NF AT binding sites has been associated with the induction of T cell exhaustion through elevation of TOX (see, e.g., Martinez GJ, et al. Immunity. 2015;42(2):265-78; Chen C, et al. Cancer Gene Ther.2023;30(l): 1-10). Collectively, transcriptomic and epigenomic analyses indicate that CD28 stimulation using aAPCs generates T cells with molecular characteristics resembling Tscm.

[0295] aAPCs promote expansion independently of CD3-mediated signaling.

[0296] To determine whether aAPCs drive proliferation without engaging canonical CD3 signaling, we compared early ZAP70 phosphorylation between aAPC and ocCD3 stimulation. Whereas ocCD3 induced robust phosphorylation of ZAP70 (pY319), aAPC stimulation did not elicit detectable CD3-proximal signaling, indicating minimal direct CD3 engagement (FIG. 15 A). We then knocked out CD3Z or, as a negative control, the safe-harbor locus AAVS1 in naive CD8+ T cells. Loss of CD3 surface expression was confirmed in CD3Z knockout cells (FIGs. 15B-15C), and these cells were stimulated with aAPCs. CD3-deficient cells expanded comparably to their CD3-intact cells following aAPC stimulation (FIG. 15D). Together, these data demonstrate that aAPC stimulation drives T cell proliferation independently of CD3 signaling.

[0297] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary embodiments of the present disclosure as contemplated by the inventor(s), and thus, are not intended to limit the present disclosure and the appended claims in any way.

[0298] The present disclosure has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

[0299] The foregoing description of the specific embodiments will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0300] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

[0301] The contents of all cited references (including literature references, U.S. or foreign patents or patent applications, and websites) that are cited throughout this application are hereby expressly incorporated by reference as if written herein in their entireties for any purpose, as are the references cited therein. Where any inconsistencies arise, material literally disclosed herein controls.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing a population of immune cells for a cell therapy, comprising contacting a population of source immune cells with a CD28 agonist thereby producing a population of stimulated immune cells, wherein neither the population of source immune cells nor the population of stimulated immune cells is contacted with a CD3 agonist.

2. The method of claim 1, further comprising introducing into one or more cells of the population of source immune cells a heterologous nucleic acid molecule encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR) prior to contacting the source immune cells with the CD28 agonist.

3. The method of claim 1, further comprising introducing into one or more cells of the population of stimulated immune cells a heterologous nucleic acid molecule encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

4. A method of preparing a population of genetically modified immune cells for an immune cell therapy, comprising:(i) contacting a population of source immune cells with a CD28 agonist thereby generating a population of stimulated immune cells, and(ii) introducing into one or more cells of the population of stimulated immune cells a heterologous nucleic acid molecule encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR); wherein neither the population of source immune cells nor the population of stimulated immune cells is contacted with a CD3 agonist.

5. The method of any one of claims 1 to 4, wherein the source immune cells comprise T cells, NK cells, or both.

6. The method of claim 5, wherein the T cells comprise a[3 T cells, y5 T cells, cytotoxic T cells, helper T cells, regulatory T cells (Treg cells), or any combination thereof.

7. The method of any one of claims 1 to 6, wherein the source immune cells comprise tumor infiltrating lymphocytes (TILs).

8. The method of any one of claims 1 to 7, wherein the source immune cells are contacted with the CD28 agonist for about 12 hours to about 7 days.

9. The method of any one of claims 1 to 8, wherein the source immune cells are contacted with the CD28 agonist for about 12 hours to about 6 days, about 12 hours to about 5 days, about 12 hours to about 4 days, about 12 hours to about 3 days, about 12 hours to about 48 hours, about 12 hours to about 36 hours, about 12 hours to about 24 hours, about 24 hours to about 36 hours, about 24 hours to about 48 hours, about 24 hours to about 3 days, about 24 hours to about 4 days, about 24 hours to about 5 days, about 24 hours to about 6 days, about 24 hours to about 7 days, about 36 hours to about 48 hours, about 36 hours to about 3 days, about 36 hours to about 4 days, about 36 hours to about 5 days, about 36 hours to about 6 days, about 36 hours to about 7 days, about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 6 days, about 2 days to about 7 days, about 3 days to about 4 days, about 3 days to about 5 days, about 3 days to about 6 days, about 3 days to about 7 days, about 3 to about 8 days, about 3 to about 9 days, or about 3 to about 10 days.

10. The method of any one of claims 1 to 9, wherein the population of source immune cells is further contacted with IL-7, IL-21, an antibody or antigen-binding portion thereof that specifically binds IFN-y, or any combination thereof.

11. The method of any one of claims 1 to 10, wherein the population of source immune cells are contacted with the CD28 agonist by culturing the population of source immune cells in a medium comprising the CD28 agonist.

12. The method of claim 11, wherein the medium further comprises IL-7, IL-21, an antibody or antigen-binding portion thereof that specifically binds interferon-gamma (IFN-y), or any combination thereof.

13. The method of claim 11 or 12, wherein the IL-7, IL-21, the antibody or antigen-binding portion thereof that specifically binds IFN-y, or any combination thereof is added to the medium on the same day or about 1 day after the source immune cells are contacted with the CD28 agonist.

14. The method of any one of claims 11 to 13, wherein:(a) the medium comprises about 1 ng / ml to about 1 pg / ml IL-7;(b) the medium comprises about 1 ng / ml to about 1 pg / ml IL-21;(c) the medium comprises about 0.1 pg / ml to about 100 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y; or(d) any combination of (a) to (c).

15. The method of any one of claims 11 to 14, wherein:(a) the medium comprises about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml, about 500 ng / ml, or about 1 pg / ml IL-7;(b) the medium comprises about 1 ng / ml, about 2 ng / ml, about 3 ng / ml, about 4 ng / ml, about 5 ng / ml, about 6 ng / ml, about 7 ng / ml, about 8 ng / ml, about 9 ng / ml, about 10 ng / ml, about 11 ng / ml, about 12 ng / ml, about 13 ng / ml, about 14 ng / ml, about 15 ng / ml, about 20 ng / ml, about 25 ng / ml, about 30 ng / ml, about 35 ng / ml, about 40 ng / ml, about 45 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, about 100 ng / ml, about 200 ng / ml, about 300 ng / ml, about 400 ng / ml, about 500 ng / ml, or about 1 pg / ml IL-21;(c) the medium comprises about 0.1 pg / ml, about 0.5 pg / ml, about 1 pg / ml, about 2 pg / ml, about 3 pg / ml, about 4 pg / ml, about 5 pg / ml, about 6 pg / ml, about 7 pg / ml, about 8 pg / ml, about 9 pg / ml, about 10 pg / ml, about 20 pg / ml, about 30 pg / ml, about 40 pg / ml, about 50 pg / ml, or about 100 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y; or(d) any combination of (a) to (c).

16. The method of any one of claims 11 to 15, wherein the medium comprises about 25 ng / ml IL-7, about 25 ng / ml IL-21, and about 2 pg / ml of an antibody or antigen-binding portion thereof that specifically binds IFN-y.

17. The method of any one of claims 1 to 16, wherein following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD45RA.

18. The method of any one of claims 1 to 17, wherein following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD62L.

19. The method of any one of claims 1 to 18, wherein following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CCR7.

20. The method of any one of claims 1 to 19, wherein following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD95.

21. The method of any one of claims 1 to 20, wherein following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD45RA and CCR7.

22. The method of any one of claims 1 to 21, wherein following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, and CD95.

23. The method of any one of claims 1 to 22, wherein following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express CD45RA, CCR7, CD95, and CD62L.

24. The method of any one of claims 1 to 23, wherein the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof, in the population of stimulated immune cells is increased by at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, or at least about 20-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, CD62L, or any combination thereof in the population of source immune cells.

25. The method of any one of claims 1 to 24, wherein the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of stimulated immune cells is increased by at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, or at least about 20-fold,relative to the percent of immune cells that express CD45RA, CCR7, and CD95 in the population of source immune cells.

26. The method of any one of claims 1 to 25, wherein the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of stimulated immune cells is increased by at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, or at least about 20-fold, relative to the percent of immune cells that express CD45RA, CCR7, CD95, and CD62L in the population of source immune cells.

27. The method of any one of claims 1 to 26, wherein following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express TCF1.

28. The method of any one of claims 1 to 27, wherein following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express KLF2.

29. The method of any one of claims 1 to 28, wherein following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express TCF1 and KLF2.

30. The method of any one of claims 1 to 29, wherein following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells lack IRF4 expression.

31. The method of any one of claims 1 to 30, wherein following the contacting with the CD28 agonist at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the immune cells in the population of stimulated immune cells express TCF1 and / or KLF2 and lack IRF4 expression.

32. The method of any one of claims 1 to 31, wherein following the contacting with the CD28 agonist, the expression level of one or more exhaustion makers in the population of stimulated immune cells does not increase relative to the expression level of the one or more exhaustion markers in the population of source immune cells.

33. The method of any one of claims 1 to 32, wherein following the contacting with the CD28 agonist the population of stimulated immune cells has a lower expression level of one or more exhaustion markers, relative to the expression level of the one or more exhaustion markers in a population of immune cells contacted with a CD3 agonist.

34. The method of claim 32 or 33, wherein the one or more exhaustion markers comprise PD- 1, TIM-3, LAG-3, TIGIT, or any combination thereof.

35. The method of any one of claims 1 to 34, wherein following the contacting with the CD28 agonist the expression level of one or more markers of oxidative stress in the population of stimulated immune cells does not increase relative to the expression level of the one or more markers of oxidative stress in the population of source immune cells.

36. The method of any one of claims 1 to 35, wherein following the contacting with the CD28 agonist the population of stimulated immune cells has a lower expression level of one or more markers of oxidative stress, relative to the expression level of the one or more markers of oxidative stress in a population of immune cells contacted with a CD3 agonist.

37. The method of claim 35 or 36, wherein the one or more markers of oxidative stress comprise NRF2, HIFla or any combination thereof.

38. The method of any one of claims 1 to 37, wherein the CD28 agonist comprises an antibody or an antigen-binding portion thereof that specifically binds CD28.

39. The method of any one of claims 1 to 38, wherein the CD28 agonist comprises an anti- CD28 antibody selected from CD28.2, TGN1412, and ANC28.1 / 5D10, or an antigenbinding portion thereof.

40. The method of any one of claims 1 to 39, wherein the CD28 agonist is associated with an antigen presenting cell (APC), a bead, an extracellular matrix, or any combination thereof.

41. The method of any one of claims 1 to 40, wherein the CD28 agonist is associated with an APC.

42. The method of any one of claims 1 to 41, wherein the CD28 agonist is bound to the membrane of an APC.

43. The method of any one of claims 40 to 42, wherein the APC is an artificial APC (aAPC).

44. The method of any one of claims 40 to 43, wherein the APC is genetically modified to express the CD28 agonist.

45. The method of any one of claims 40 to 44, wherein the APC does not express B2M, CD32, or both B2M and CD32.

46. The method of any one of claims 40 to 45, wherein the APC is a genetically modified K562 cell.

47. The method of any one of claims 40 to 46, wherein the APC expresses CD83, IL-7, IL- 21, or any combination thereof.

48. The method of any one of claims 1 to 47, wherein the population of source immune cells comprises peripheral blood mononuclear cells (PBMCs).

49. The method of any one of claims 1 to 48, wherein the population of source immune cells is prepared by isolating naive CD8+ immune cells from PBMCs.

50. The method of any one of claims 1 to 49, wherein at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% of the immune cells in the population of source immune cells are naive CD8+ immune cells.

51. The method of any one of claims 1 to 47, wherein the population of source immune cells is obtained from a tumor sample.

52. The method of any one of claims 1 to 51, wherein the population of source immune cells is expanded prior to the contacting with the CD28 agonist.

53. The method of any one of claims 1 to 51, wherein the population of source immune cells is not expanded prior to the contacting with the CD28 agonist.

54. The method of any one of claims 1 to 53, wherein the population of stimulated immune cells is expanded.

55. The method of any one of claims 2 to 54, wherein the CAR or the TCR comprises an antigen-binding domain that specifically binds a tumor antigen.

56. The method of claim 55, wherein the tumor antigen is CD19, TRAC, TCRP, BCMA, CLL-1, CS1, CD38, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, R0R1, R0R2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD 179a, ALK, Poly sialic acid, PLAC1, GloboH, NY-BR- 1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO- 1, LAGE-la, MAGE- Al, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT- 2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA- 1 / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene),- I l l -NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3s, CD4, CD5, CD7, the extracellular portion of the APRIL protein, or any combinations thereof.

57. The method of claim 55 or 56, wherein the tumor antigen is NY-ESO-1.

58. The method of any one of claims 2 to 57, wherein the one or more cells are transduced with a vector comprising the heterologous nucleic acid molecule encoding the CAR and / or the TCR.

59. The method of claim 58, wherein the vector is a retroviral vector, a lentiviral vector, an adeno-associated virus (AAV), an adenovirus, an AAV hybrid virus, a baculovirus, or any combination thereof.

60. The method of claim 58 or 59, wherein the vector is a retroviral vector

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