Methods for culturing cells with improved culture medium

Abstract

The present disclosure provides methods of preparing immune cells, e.g., T cells and / or NK cells, comprising contacting the cells with programmable cell-signaling scaffolds in a medium, wherein the medium comprises the PCS at an amount of less than about 160 µg per million cells, e.g., less than about 120 µg per million cells. In some aspects, the immune cells are modified, such that the immune cells exhibit reduced expression of a gene and / or protein associated with impaired immune cell function (e.g., NR4A family member), express a ligand-binding protein, and / or exhibit an increased expression of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide). In some aspects, the immune cells are administered to a subject in need thereof.

Description

METHODS FOR CULTURING CELLS WITH IMPROVED CULTURE MEDIUMCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This PCT application claims the priority benefit of U.S. Provisional Application No. 63 / 632,266, filed April 10, 2024, which is herein incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the sequence listing is submitted electronically (Name: 4385_137PC01_SequenceListing_ST26.xml; Size: 252,383 bytes; and Date of Creation: April 9, 2025) with the application and herein incorporated by reference in its entirety.FIELD OF THE DISCLOSURE

[0003] The present disclosure relates to methods of culturing and modifying cells, e.g., pluripotent, multipotent, and / or immune cells (e.g., T cells and / or NK cells), in a medium comprising a programmable cell-signaling scaffold (PCS). In some aspects, the PCS is present in the medium at an amount of less than about 160 pg per million cells (e.g., less than about 120 pg per million cells). In some aspects, the cells are further modified (e.g., edited and / or transduced) to exhibit altered expression of one or more genes and / or proteins as compared to corresponding cells that have not been modified. In some aspects, the one or more genes and / or proteins are associated with impaired immune cell function. For such genes and / or proteins, the cells are modified to decrease the expression. Non-limiting example of a gene and / or protein that is associated with impaired immune cell function comprises a member of the NR4A family. Accordingly, in some aspects, the cells are modified (z.e., edited) to exhibit reduced NR4A expression (e.g., NR4A1, NR4A2, and / or NR4A3), e.g., as compared to corresponding cells that have not been modified. In some aspects, the one or more genes and / or proteins are associated with improved immune cell function. For such genes and / or proteins, the cells are modified to increase the expression. Non-limiting example of a gene and / or protein that is associated with improved immune cell function comprises a c-Jun protein. Accordingly, in some aspects, the cells are further modified (z.e., transduced) to exhibit increased c-Jun expression, e.g., as compared tocorresponding cells that have not been further modified. Cells cultured and modified using the methods disclosed herein can be used for various cell therapies, including but not limited to chimeric antigen receptor (CAR) T cell therapy, TCR T cell therapy including neoantigen directed-T cell therapies, and TIL therapy.BACKGROUND OF THE DISCLOSURE

[0004] Cancer immunotherapy relies on harnessing T cells — the immune system’ s primary killers of infected and diseased cells — to attack and kill tumor cells. However, the ability of immune cells to target and kill tumor cells is dampened by the presence of various inhibitors of the immune response that are present within the tumor microenvironment. Therefore, while CAR T cells have had various successes in treating certain cancers (e.g., KYMRIAH™ (tisagenlecleucel, Novartis) and YESCARTA™ (axicabtagene ciloleucel, Kite / Gilead) have been approved by the FDA), challenges remain. For instance, the success of CAR T cell immunotherapy is often limited by the extent of CAR T expansion in a recipient’s body, which typically requires a large infusion of cells. Additionally, exhaustion and loss of persistence of the transferred CAR T cells have been observed, leading to loss of clinical efficacy and potential relapse.

[0005] Accordingly, there remains a need for manufacturing methods to efficienty, reliably, and consistently expand sufficient cells (e.g., immune cells) with desirable attributes for effective cell therapies. The present disclosure addresses these and related needs.BRIEF SUMMARY OF THE DISCLOSURE

[0006] Provided herein is a method of preparing immune cells for immunotherapy comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than 120 pg per million cells.

[0007] Also provided herein is a method of activating immune cells during ex vivo or in vitro culture comprising contacting the immune with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than 120 pg per million cells.

[0008] Also provided herein is a method of increasing viability of immune cells during ex vivo or in vitro culture comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than 120 pg per million cells. In some aspects, the viability of the immune cells is increased as compared to reference immune cells, wherein the reference immune cells were contacted with the PCS at an amount of 120 pg per million cells or greater. In some aspects, the viability of the immune cells is increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, 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%, or at least about 50%, as compared to the reference immune cells.

[0009] Provided herein is a method of decreasing an amount of background cytokine produced by immune cells during ex vivo or in vitro culture comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than 120 pg per million cells. In some aspects, the amount of background cytokine produced by the immune cells is decreased as compared to reference immune cells, wherein the reference immune cells were contacted with the PCS at an amount of 120 pg per million cells or greater. In some aspects, the amount of background cytokine produced by the immune cells is decreased by 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 80%, at least about 90%, or about 100% , as compared to the reference immune cells.

[0010] Provided herein is a method of decreasing an amount of residual surface cues bound to immune cells after ex vivo or in vitro culture comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than 120 pg per million cells. In some aspects, the amount of residual surface cues bound to the immune cells is decreased as compared to reference immune cells, wherein the reference immune cells were contacted with the PCS at an amount of 120 pg per million cells or greater. In some aspects, the amount of residual surface cues bound to the immune cells is decreased by at least about 5%, 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 80%, at least about 90%, or about 100%, as compared to the reference immune cells.

[0011] In some aspects, any of the methods provided herein (e.g., above) comprises editing the immune cells to exhibit a decrease in an expression level of a gene and / or proteinassociated with impaired immune cell function (e.g., nuclear receptor subfamily 4 A (NR4A) family member) as compared to corresponding immune cells which have not been edited. In some aspects, the editing occurs prior to the contacting. In some aspects, the editing occurs at least about one day, at least about two days, at least about three days, at least about four days, or at least about five days prior to the contacting. In some aspects, the editing occurs about one day prior to the contacting.

[0012] In some aspects, any of the methods provided herein (e.g., above) comprises transducing the immune cells: (a) to express a ligand-binding protein, (b) to exhibit an increase in an expression level of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), or (c) both (a) and (b). In some aspects, the contacting and transducing occur concurrently.

[0013] Provided herein is a method of preparing immune cells for immunotherapy comprising (a) editing the immune cells to exhibit a decrease in an expression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4A (NR4A) family member) as compared to corresponding immune cells which have not been edited, (b) contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, and (c) transducing the immune cells (i) to express a ligandbinding protein (ii) to exhibit an increase in an expression level of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), or (iii) both (i) and (ii); wherein the PCS is present in the medium at an amount of less than 120 pg per million cells.

[0014] Also provided herein is a method of activating immune cells during ex vivo or in vitro culture comprising (a) editing the immune cells to exhibit a decrease in an expression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4A (NR4A) family member) as compared to corresponding immune cells which have not been edited, (b) contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, and (c) transducing the immune cells (i) to express a ligand-binding protein (ii) to exhibit an increase in an expression level of a gene and / or protein associated with impaired immune cell function (e.g., c-Jun polypeptide), or (iii) both (i) and (ii); wherein the PCS is present in the medium at an amount of less than 120 pg per million cells.

[0015] Also provided herein is a method of increasing viability of immune cells during ex vivo or in vitro culture comprising (a) editing the immune cells to exhibit a decrease in anexpression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4A (NR4A) family member) as compared to corresponding immune cells which have not been edited, (b) contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, and (c) transducing the immune cells (i) to express a ligand-binding protein (ii) to exhibit an increase in an expression level of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), or (iii) both (i) and (ii); wherein the PCS is present in the medium at an amount of less than 120 pg per million cells. In some aspects, the viability of the immune cells is increased as compared to reference immune cells, wherein the reference immune cells were contacted with the PCS at an amount of 120 pg per million cells or greater. In some aspects, the viability of the immune cells is increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, 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%, or at least about 50% as compared to the reference immune cells.

[0016] Provided herein is a method of decreasing an amount of background cytokine produced by immune cells during ex vivo or in vitro culture comprising (a) editing the immune cells to exhibit a decrease in an expression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4A (NR4A) family member) as compared to corresponding immune cells which have not been edited, (b) contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, and (c) transducing the immune cells (i) to express a ligand-binding protein (ii) to exhibit an increase in an expression level of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), or (iii) both (i) and (ii); wherein the PCS is present in the medium at an amount of less than 120 pg per million cells. In some aspects, the amount of background cytokine produced by the immune cells is decreased as compared to reference immune cells, wherein the reference immune cells were contacted with the PCS at an amount of 120 pg per million cells or greater. In some aspects, the amount of background cytokine produced by the immune cells is decreased by 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 80%, at least about 90%, or about 100%, as compared to the reference immune cells.

[0017] Provided herein is a method of decreasing an amount of residual antibodies bound to immune cells after ex vivo or in vitro culture comprising (a) editing the immune cells to exhibit a decrease in an expression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4A (NR4A) family member) as compared to corresponding immune cells which have not been edited, (b) contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, and (c) transducing the immune cells (i) to express a ligand-binding protein (ii) to exhibit an increase in an expression level of a gene and / or protein associated with an improved immune cell function (e.g., c-Jun polypeptide), or (iii) both (i) and (ii); wherein the PCS is present in the medium at an amount of less than 120 pg per million cells. In some aspects, the amount of residual antibodies bound to the immune cells is decreased as compared to reference immune cells, wherein the reference immune cells were contacted with the PCS at an amount of 120 pg per million cells or greater. In some aspects, the amount of residual antibodies bound to the immune cells is decreased by at least about 5%, 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 80%, at least about 90%, or about 100%, as compared to the reference immune cells.

[0018] For any of the methods provided herein (e.g., described above), in some aspects, the editing occurs prior to (a) the contacting, (b) the transducing, or (c) both (a) and (b). In some aspects, the editing occurs prior to both the contacting and the transducing. In some aspects, the editing occurs at least about one day, at least about two days, at least about three days, at least about four days, or at least about five days prior to the contacting and / or transducing. In some aspects, the editing occurs about one day prior to the contacting and / or transducing. For any of the methods provided herein (e.g., described above), in some aspects, the contacting and the transducing occur concurrently.

[0019] For any of the methods provided herein (e.g., described above), in some aspects, the amount of the PCS is about 40 pg to less than 120 pg per million cells. In some aspects, the amount of the PCS per million cells is about 45 pg to less than 120 pg, about 50 pg to less than 120 pg, about 55 pg to less than 120 pg, about 60 pg to less than 120 pg, about 65 pg to less than 120 pg, about 70 pg to less than 120 pg, about 75 pg to less than 120 pg, about 80 pg to less than 120 pg, about 85 pg to less than 120 pg, about 90 pg to less than 120 pg, about 95 pg to less than 120 pg, about 100 pg to less than 120 pg, about 105 pg to less than 120 pg, about 110 pg to less than 120 pg, or about 115 pg to less than 120 pg. Insome aspects, the amount of the PCS per million cells is about 40 pg to about 115 pg, about 40 pg to about 110 pg, about 40 pg to about 105 pg, about 40 pg to about 100 pg, about 40 pg to about 95 pg, about 40 pg to about 90 pg, about 40 pg to about 85 pg, about 40 pg to about 80 pg, about 40 pg to about 75 pg, about 40 pg to about 70 pg, about 40 pg to about 65 pg, about 40 pg to about 60 pg, about 40 pg to about 55 pg, about 40 pg to about 50 pg, or about 40 pg to about 45 pg. In some aspects, the amount of the PCS per million cells is about 45 pg to about 115 pg, about 50 pg to about 110 pg, about 55 pg to about 105 pg, about 60 pg to about 100 pg, about 65 pg to about 95 pg, about 70 pg to about 90 pg, or about 75 pg to about 85 pg. In some aspects, the amount of the PCS per million cells is about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg, about 101 pg, about 102 pg, about 103 pg, about 104 pg, about 105 pg, about 106 pg, about 107 pg, about 108 pg, about 109 pg, about 110 pg, about 111 pg, about 112 pg, about 113 pg, about 114 pg, or about 115 pg.

[0020] Provided herein is a method of preparing immune cells for immunotherapy comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of between about 120 pg per million cells and less than about 160 pg per million cells.

[0021] Also provided herein is a method of activating immune cells during ex vivo or in vitro culture comprising contacting the immune with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of between about 120 pg per million cells and less than about 160 pg per million cells.

[0022] Also provided herein is a method of increasing viability of immune cells during ex vivo or in vitro culture comprising contacting the immune cells with a programmable cellsignaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at anamount of between about 120 pg per million cells and less than about 160 pg per million cells.

[0023] Also provided herein is a method of decreasing an amount of background cytokine produced by immune cells during ex vivo or in vitro culture comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of between about 120 pg per million cells and less than about 160 pg per million cells.

[0024] Also provided herein is a method of decreasing an amount of residual antibodies bound to immune cells after ex vivo or in vitro culture comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of between about 120 pg per million cells and less than about 160 pg per million cells.

[0025] For any of the methods provided herein (e.g., described above), in some aspects, the method further comprises editing the immune cells to exhibit a decrease in an expression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4A (NR4A) family member) as compared to corresponding immune cells which have not been edited. In some aspects, the editing occurs prior to the contacting. In some aspects, the editing occurs at least about one day, at least about two days, at least about three days, at least about four days, or at least about five days prior to the contacting. In some aspects, the editing occurs about one day prior to the contacting.

[0026] For any of the methods provided herein (e.g., described above), in some aspects, the method further comprises transducing the immune cells: (a) to express a ligand-binding protein, (b) to exhibit an increase in an expression level of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), or (c) both (a) and (b). In some aspects, the contacting and the transducing occur concurrently.

[0027] For any of the methods provided herein (e.g., described above), in some aspects, the PCS comprises (i) a base layer comprising high surface area mesoporous silica microrods (MSR); (ii) a continuous, fluid-supported lipid bilayer (SLB) layered on the MSRbase layer; and (iii) a plurality of surface cues. In some aspects, the PCS further comprises a plurality of soluble cues. In some aspects, one or more of the plurality of surface cues are loaded onto the SLB layer. In some aspects, one or more of the plurality of soluble cues are loaded onto the MSR base layer. In some aspects, one or more of the plurality of solublecues are released from the PCS in a controlled-release manner. In some aspects, one or more of the plurality of soluble cues are released from the PCS in a sustained manner for at least about 30 days.

[0028] In some aspects, the plurality of soluble cues comprises IL-1, IL-2, IL-4, IL-5, IL- 7, IL- 10, IL- 12, IL- 15, IL- 17, IL-21, transforming growth factor beta (TGF-P), or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof. In some aspects, the plurality of soluble cues comprises (i) IL-2, an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof and (ii) a second soluble cue comprising IL-7, IL-21, IL-15, IL-15 superagonist, or any combination thereof. In some aspects, the plurality of soluble cues comprises (i) IL-2, an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof, (ii) a second soluble cue comprising IL-7, IL- 21, IL- 15, IL- 15 superagonist, or any combination thereof, and (iii) a third soluble cue comprising IL-7, IL-21, IL- 15, IL- 15 superagonist, or any combination thereof. In some aspects, the plurality of soluble cues comprises an N-terminal IL-2 fragment comprising the first 30 amino acids of IL-2 (pl-30), an IL-2 superkine peptide, an IL-2 partial agonist peptide, or a combination thereof.

[0029] In some aspects, the plurality of surface cues comprises a T-cell stimulatory molecule, a T-cell co-stimulatory molecule, or both a T-cell stimulatory molecule and a T cell co-stimulatory molecule. In some aspects, the T-cell stimulatory molecule and / or the T-cell co-stimulatory molecule is loaded onto the SLB. In some aspects, the T-cell stimulatory molecule and / or the T-cell co-stimulatory molecule is loaded via affinity pairing or chemical coupling. In some aspects, the affinity coupling comprises a biotinstreptavidin pair, an antibody-antigen pair, an antibody-hapten pair, an affinity pair, a capture protein pair, an Fc receptor-IgG pair, a metal-chelating lipid pair, or a combination thereof. In some aspects, the chemical coupling comprises azide-alkyne chemical (AAC) reaction, dibenzo-cyclooctyne ligation (DCL), tetrazine-alkene ligation (TAL), or any combination thereof. In some aspects, the T-cell stimulatory molecule and / or the T-cell co- stimulatory molecule is coated onto the SLB. In some aspects, the T-cell stimulatory molecule and / or the T-cell co-stimulatory molecule is partly embedded onto the SLB. In some aspects, the T-cell stimulatory molecule and / or the T-cell co-stimulatory molecule is loaded onto the MSR.

[0030] In some aspects, the T-cell stimulatory molecule and / or the T-cell co-stimulatory molecule comprises antibody molecules or antigen-binding fragments thereof. In some aspects, the T-cell stimulatory molecule comprises an anti-CD3 antibody or an antigenbinding portion thereof, an anti -macrophage scavenger receptor (MSR1) antibody or an antigen-binding portion thereof, an anti-T-cell receptor (TCR) antibody or an antigenbinding portion thereof, an anti-CD2 antibody or an antigen-binding portion thereof, an anti-CD47 antibody or an antigen-binding portion thereof, a major histocompatibility complex (MHC) molecule loaded with an MHC peptide or a multimer thereof, an MHC- immunoglobulin (Ig) conjugate or a multimer thereof, or a combination thereof. In some aspects, the T-cell co-stimulatory molecule comprises an antibody, or an antigen-binding portion thereof, which specifically binds to a co-stimulatory antigen comprising CD28, 4- 1BB (CD137), 0X40 (CD134), CD27 (TNFRSF7), GITR (CD357), CD30 (TNFRSF8), HVEM (CD270), LTfiR (TNFRSF3), DR3 (TNFRSF25), ICOS (CD278), CD226 (DNAM1), CRTAM (CD355), TIM1 (HAVCR1, KIMI), CD2 (LFA2, 0X34), SLAM (CD150, SLAMF1), 2B4 (CD244, SLAMF4), Lyl08 (NTBA, CD352, SLAMF6), CD84 (SLAMF5), Ly9 (CD229, SLAMF3), CRACC (CD319, BLAME), or any combination thereof. In some aspects, the T-cell stimulatory molecule and / or the T-cell co-stimulatory molecule comprises bispecific antibodies or antigen binding portions thereof. In some aspects, the T-cell stimulatory molecule and / or the T-cell co-stimulatory molecule comprises a pair comprising CD3 / CD28, CD3 / ICOS, CD3 / CD27, CD3 / CD137, or a combination thereof.

[0031] In some aspects, the PCS further comprises an immunoglobulin molecule that binds specifically to an Fc-fusion protein. In some aspects, the PCS further comprises a recruitment compound comprising granulocyte macrophage-colony stimulating factor (GM-CSF), chemokine (C-C motif) ligand 21 (CCL-21), chemokine (C-C motif) ligand 19 (CCL-19), Chemokine (C-X-C Motif) ligand 12 (CXCL12), interferon gamma (IFN-y), a FMS-like tyrosine kinase 3 (Flt-3) ligand, or any combination thereof. In some aspects, the recruitment compound comprises granulocyte macrophage colony stimulating factor (GM- CSF).

[0032] In some aspects, the PCS further comprises an antigen. In some aspects, the antigen comprises a tumor antigen. In some aspects, the tumor antigen is adenomatous polyposis coli protein (APC), adenosine deaminase-binding protein (AD Abp), a-fetoprotein, AFP (alpha-fetoprotein), AIM-2, AIM-3, and WT1), ART1, ART4, B7-H3, B7-H6, BAGE,BCMA, B-cyclin, BMI1, Braf, brain glycogen phosphorylase, BRAP, C13orf24, C6orfl53, C9orf 112, CA-125, CA9 (carbonic anhydrase 9), CASP-8, cathepsin B, Cav-1, CCL-1 (C- C motif chemokine ligand 1), CD 123, CD 138, CD 171, CD 19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD352, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD45, CD47, CD5, CD56, CD66e, CD70, CD74, CD74, CD79a, CD79b, CD98, cdc27, CDK-1, CDK4, CEA, CEA (carcinoembryonic antigen), c-erbB-2, Claudin 18.2, Claudin 6, c-MET, Colorectal associated antigen (CRC)- C017-1A / GA733, Connexin 37, COX-2, CT-7, cyclophilin b, CYNL2, Dipeptidyl peptidase IV (DPPIV), DLL3 (delta-like protein 3), DLL4, EBV-encoded nuclear antigen (EBNA)-I, E-cadherin, EGFRvIII, ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase family member 3), EpCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, EPHa2 (ephrine receptor A2), EphA2ZEck, ephrinB2, ERBB dimers, ESO-1, estrogen receptor, ETBR (endothelin B receptor), EZH2, FAP-a (fibroblast activation protein a), FBP (a folate binding protein), FCRL5, fetal AchR (fetal acetylcholine receptor), fodrin, Fra-1 / Fosl 1, FR-a (folate receptor alpha), GAGE-1, GAGE-family of tumor antigens, Ganglioside / GD2, GCC (guanyl cyclase C), GD2, GD2 gangliosides, GD3, GLEA2, GM2, GnT-V, GnT-V„ GOLGA, gplOO (glycoprotein 100), gp75, GPC2 (glypican-2), GPC3, gplOO, GPNMB (glycoprotein NMB), GPRC5D (G Protein Coupled Receptor 5D), GUI, H60, hepatitis B surface antigen, HER2, HER3, HER4, HLA-A complexed with peptides derived from AFP, HLA-A1 (human leukocyte antigen Al), HLA-A2 (human leukocyte antigen A2), HMW-MAA (human high molecular weight-melanoma-associated antigen), HSPH1, Ig kappa, Ig lambda, IGF1R (insulin-like growth factor 1 receptor), Ig-idiotype, IL-13Ra2 (IL- 13 receptor alpha 2), IL13Ralpha, IL- 22Ra (IL-22 receptor alpha), ING4, KDR (kinase insert domain receptor), Ki67, KIAA0376, KRAS, Ku70 / 80, LAGE-I, Lewis Y, LI cell adhesion molecule (LI -CAM), Liv-1, Livin, lmp-1, LRRC8A (leucine rich repeat containing 8 Family member A), MAGE-1, MAGE-2, MAGE-3, MAGE- A, MAGE- A3, MAGE-A6, MART-1 (melan A), MCSP (melanoma-associated chondroitin sulfate proteoglycan), melanoma-associated antigen (MAGE)-Al, mesothelin, MHC / peptide complexes (e.g., MICA, MICB, midkin, MRP-3, MUC16, mucin 1 (MUC1), MUM-1, murine cytomegalovirus (MCMV), NAG, NCAM (neural cell adhesion molecule), Nectin-4, Nestin, NKG2D (natural killer group 2 member D) ligands, NKTR, NSEP1, NY-ESO, NY-ESO-1, OLIG2, oncofetal antigen, P1A, p53, PAP, PD-1, PD-L1, pl20ctn, pl5, Pmell 17, PRAME (preferentially expressed antigen of melanoma), progesterone receptor, PROXI, PSA (prostate specific antigen),PSCA (prostate stem cell antigen ), PSMA (prostate specific membrane antigen), RAE-1 proteins, RAGE, ras, RBPSUH, RCAS1, R0R1, R0R2, RTN4, SART1, SART2, SART3, SCP-I, SIRPa (signal -regulatory protein alpha), SLIT, SLITRK6 (NTRK-like protein 6), Smad family of tumor antigens, SOXIO, SOX11, SOX2, SSX-2 (HOM-MEL-40), SSX-4, SSX-5, SSX-I, SSX-I, STEAP1 (six transmembrane epithelial antigen of the prostate 1), Survivin, survivin, TAG72 (tumor-associated glycoprotein 72), T-cell receptor / CD3-zeta chain, TNKS2, TPBG (trophoblast glycoprotein), TPR, Trop-2, TRP-1, TRP-2, Tyrosinase, U2AF1L, UL16-binding protein-like transcript 1 (Multi), UPAR, VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, WT-1, avP6 or another integrin, P- catenin, pi,6-N, P-catenin, y-catenin, and antigens from HIV, HBV, HCV, HPV, and other pathogens, a patient-specific neoantigen, or an immunogenic peptide thereof, and any combination thereof.

[0033] In some aspects, a weight ratio of the SLB to the MSR is between about 10: 1 and about 1 :20. In some aspects, the SLB comprises a lipid selected from the group consisting of (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), palmitoyl-oleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylethanolamine (DMPE) and dipalmitoylphosphatidylethanolamine (DPPE), l-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (8:0-14:0 PC), or a combination thereof.

[0034] In some aspects, the PCS retains a continuous, fluid architecture for at least 14 days. In some aspects, the dry weight ratio of the MSR to the T-cell stimulatory / co-stimulatory molecules is between 1 : 1 to 50: 1.

[0035] In any of the methods provided herein (e.g., described above), in some aspects, the medium further comprises potassium ion at a concentration higher than 5 mM. In some aspects, the concentration of the potassium ion is higher than about 10 mM, higher than about 15 mM, higher than about 20 mM, higher than about 25 mM, higher than about 30 mM, higher than about 35 mM, higher than about 40 mM, higher than about 45 mM, higher than about 50 mM, higher than about 55 mM, higher than about 60 mM, higher than about 65 mM, higher than about 70 mM, higher than about 75 mM, higher than about 80 mM, higher than about 85 mM, or higher than about 90 mM. In some aspects, the concentration of potassium ion is selected from the group consisting of about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, and about80 mM. In some aspects, the concentration of potassium ion is between about 30 mM and about 80 mM, between about 40 mM and about 80 mM, between about 50 mM and 80 mM, between about 60 mM and about 80 mM, between about 70 mM and about 80 mM, between about 40 mM and about 70 mM, between about 50 mM and about 70 mM, between about 60 mM and about 70 mM, between about 40 mM and about 60 mM, between about 50 mM and about 60 mM, or between about 40 mM and about 50 mM. In some aspects, the concentration of potassium ion is about 50 mM, about 60 mM, or about 70 mM.

[0036] In some aspects, the medium further comprises a sodium ion. In some aspects, the medium further comprises NaCl. In some aspects, the medium comprises less than about 140 mM, less than about 130 mM, less than about 120 mM, less than about 110 mM, less than about 100 mM, less than about 90 mM, less than about 80 mM, less than about 70 mM, less than about 60 mM, less than about 50 mM, or less than about 40 mM NaCl.

[0037] In some aspects, the medium is hypotonic or isotonic. In some aspects, the medium is hypotonic, and wherein the sum of the potassium ion concentration and the sodium ion concentration, multiplied by two is less than 280 mM. In some aspects, the medium is hypotonic, and wherein the sum of the potassium ion concentration and the sodium ion concentration, multiplied by two is more than 240 mM and less than 280 mM. In some aspects, the medium is isotonic, and wherein the sum of the potassium ion concentration and the sodium ion concentration, multiplied by two is more than or equal to 280 mM and less than 300 mM. In some aspects, wherein the concentration of potassium ion is about 60 mM, and the concentration of NaCl is less than about 80 mM, less than about 75 mM, less than about 70 mM, less than about 65 mM, or less than about 60 mM. In some aspects, the concentration of potassium ion is about 55 mM, and the concentration of NaCl is less than about 85 mM, less than about 80 mM, less than about 75 mM, less than about 70 mM, or less than about 65 mM. In some aspects, the concentration of potassium ion is about 50 mM, and the concentration of NaCl is less than about 90 mM, less than about 85 mM, less than about 80 mM, less than about 75 mM, or less than about 70 mM.

[0038] In some aspects, the medium further comprises one or more cytokines. In some aspects, the one or more cytokines comprise Interleukin-2 (IL-2), Interleukin-7 (IL-7), Interleukin-21 (IL-21), Interleukin- 15 (IL-15), or any combination thereof. In some aspects, the one or more cytokines comprise IL-2, IL-7, and IL-15. In some aspects, the medium comprises IL-2 at a concentration from about 50 lU / mL to about 500 lU / mL. In some aspects, the concentration of IL-2 is about 50 lU / mL, about 60 lU / mL, about 70lU / mL, about 80 lU / mL, about 90 lU / mL, about 100 lU / mL, about 125 lU / mL, about 150 lU / mL, about 175 lU / mL, about 200 lU / mL, about 225 lU / mL, about 250 lU / mL, about 275 lU / mL, about 300 lU / mL, about 350 lU / mL, about 400 lU / mL, about 450 lU / mL, or about 500 lU / mL. In some aspects, the concentration of IL-2 is between about 100 lU / mL to about 300 lU / mL. In some aspects, the concentration of IL-2 is about 200 lU / mL. In some aspects, the medium comprises IL-21 at a concentration from about 50 lU / mL to about 500 lU / mL. In some aspects, the concentration of IL-21 is about 50 lU / mL, about 60 lU / mL, about 70 lU / mL, about 80 lU / mL, about 90 lU / mL, about 100 lU / mL, about 125 lU / mL, about 150 lU / mL, about 175 lU / mL, about 200 lU / mL, about 225 lU / mL, about 250 lU / mL, about 275 lU / mL, about 300 lU / mL, about 350 lU / mL, about 400 lU / mL, about 450 lU / mL, or about 500 lU / mL. In some aspects, the concentration of IL-21 is between about 100 lU / mL to about 300 lU / mL. In some aspects, the concentration of IL-21 is about 200 lU / mL. In some aspects, the medium comprises IL-7 at a concentration from about 500 lU / mL to about 1,500 lU / mL. In some aspects, the concentration of IL-7 is about 500 lU / mL, about 550 lU / mL, about 600 lU / mL, about 650 lU / mL, about 700 lU / mL, about 750 lU / mL, about 800 lU / mL, about 850 lU / mL, about 900 lU / mL, about 950 lU / mL, about 1,000 lU / mL, about 1,050 lU / mL, about 1,100 lU / mL, about 1,150 lU / mL, about 1,200 lU / mL, about 1,250 lU / mL, about 1,300 lU / mL, about 1,350 lU / mL, about 1,400 lU / mL, about 1,450 lU / mL, or about 1,500 lU / mL. In some aspects, the concentration of IL-7 is about 1,000 lU / mL to about 1,400 lU / mL. In some aspects, the concentration of IL-7 is about 1,200 lU / mL. In some aspects, the medium comprises IL- 15 at a concentration from about 50 lU / mL to about 500 lU / mL. In some aspects, the concentration of IL- 15 is about 50 lU / mL, about 60 lU / mL, about 70 lU / mL, about 80 lU / mL, about 90 lU / mL, about 100 lU / mL, about 125 lU / mL, about 150 lU / mL, about 175 lU / mL, about 200 lU / mL, about 225 lU / mL, about 250 lU / mL, about 275 lU / mL, about 300 lU / mL, about 350 lU / mL, about 400 lU / mL, about 450 lU / mL, or about 500 lU / mL. In some aspects, the concentration of IL-15 is between about 100 lU / mL to about 300 lU / mL. In some aspects, the concentration of IL- 15 is about 200 lU / mL.

[0039] In some aspects, the medium further comprises a cell expansion agent. In some aspects, the cell expansion agent comprises a GSK3B inhibitor, an ACLY inhibitor, a PI3K inhibitor, an AKT inhibitor, or any combination thereof. In some aspects, the PI3K inhibitor is selected from hydroxyl citrate, LY294002, pictilisib, CAL101, IC87114, and anycombination thereof. In some aspects, the AKT inhibitor is selected from MK2206, A443654, AKTi-VIII, and any combination thereof.

[0040] In some aspects, the medium further comprises calcium ion, glucose, or any combination thereof. In some aspects, the medium further comprises glucose, and wherein the concentration of glucose is more than about 10 mM. In some aspects, the concentration of glucose is from about 10 mM to about 40 mM, from about 10 mM to about 35 mM, from about 10 mM to about 30 mM, from about 10 mM to about 25 mM, from about 10 mM to about 20 mM, from about 15 mM to about 40 mM, from about 15 mM to about 35 mM, from about 15 mM to about 30 mM, from about 15 mM to about 25 mM, from about 15 mM to about 20 mM, from about 15 mM to about 19 mM, from about 15 mM to about 18 mM, from about 15 mM to about 17 mM, from about 15 mM to about 16 mM, from about 16 mM to about 20 mM, from about 16 mM to about 19 mM, from about 16 mM to about 18 mM, from about 16 mM to about 17 mM, from about 17 mM to about 20 mM, from about 17 mM to about 19 mM, or from about 17 mM to about 18 mM. In some aspects, the concentration of glucose is about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, or about 40 mM. In some aspects, the concentration of glucose is about 15.4 mM, about 15.9 mM, about 16.3 mM, about 16.8 mM, about 17.2 mM, or about 17.7 mM.

[0041] In some aspects, the medium further comprises calcium ion, and wherein the concentration of calcium ion is more than about 0.4 mM. In some aspects, the concentration of calcium ion is from about 0.4 mM to about 2.5 mM, from about 0.5 mM to about 2.0 mM, from about 1.0 mM to about 2.0 mM, from about 1.1 mM to about 2.0 mM, from about 1.2 mM to about 2.0 mM, from about 1.3 mM to about 2.0 mM, from about 1.4 mM to about 2.0 mM, from about 1.5 mM to about 2.0 mM, from about 1.6 mM to about 2.0 mM, from about 1.7 mM to about 2.0 mM, from about 1.8 mM to about 2.0 mM, from about 1.2 to about 1.3 mM, from about 1.2 to about 1.4 mM, from about 1.2 to about 1.5 mM, from about 1.2 to about 1.6 mM, from about 1.2 to about 1.7 mM, from about 1.2 to about 1.8 mM, from about 1.3 to about 1.4 mM, from about 1.3 to about 1.5 mM, from about 1.3 to about 1.6 mM, from about 1.3 to about 1.7 mM, from about 1.3 to about 1.8mM, from about 1.4 to about 1.5 mM, from about 1.4 to about 1.6 mM, from about 1.4 to about 1.7 mM, from about 1.4 to about 1.8 mM, from about 1.5 to about 1.6 mM, from about 1.5 to about 1.7 mM, from about 1.5 to about 1.8 mM, from about 1.6 to about 1.7 mM, from about 1.6 to about 1.8 mM, or from about 1.7 to about 1.8 mM. In some aspects, the concentration of calcium ion is about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, or about 2.0 mM.

[0042] In some aspects, the editing comprises introducing a gene editing tool into the immune cells, and wherein the gene editing tool is capable of reducing the expression level of a gene and / or protein associated with impaired immune cell function (e.g., the NR4A family member) in the immune cells. In some aspects, the gene editing tool comprises a guide RNA, shRNA, siRNA, miRNA, antisense oligonucleotides, CRISPR, zinc finger nuclease, TALEN, meganuclease, restriction endonuclease, or any combination thereof. In some aspects, the gene editing tool is CRISPR.

[0043] In some aspects, the transducing comprises introducing into the immune cells a nucleotide sequence encoding a protein that is associated with improved immune cell function (e.g., the c-Jun polypeptide), such that the expression of the protein (e.g., c-Jun polypeptide) is increased after the introducing. In some aspects, the transducing comprises introducing into the immune cells a nucleotide sequence encoding the ligand-binding protein, such that after the introducing, the immune cells express the ligand-binding protein. In some aspects, the transducing comprises introducing into the immune cells a first nucleotide sequence encoding a protein that is associated with improved immune cell function (e.g., the c-Jun polypeptide) and a second nucleotide sequence encoding the ligand-binding protein, such that after the introducing, the expression of the protein that is associated with improved immune cell function (e.g., c-Jun polypeptide) is increased and the immune cells express the ligand-binding protein. In some aspects, the first nucleotide sequence and the second nucleotide sequence are within a single vector. In some aspects, the transducing comprises introducing into the immune cells a transcriptional activator that is capable of increasing the expression level of an endogenous protein that is associated with improved immune cell function (e.g., c-Jun protein) in the immune cells, such that after the introducing, the expression of the endogenous protein that is associated with improved immune cell function (e.g., c-Jun protein) is increased. In some aspects, thetranscriptional activator is attached to a Cas protein, which has been modified to lack endonuclease activity.

[0044] In some aspects, the ligand binding protein is selected from a chimeric antigen receptor (CAR), a T cell receptor (TCR), a chimeric antibody-T cell receptor (caTCR), a chimeric signaling receptor (CSR), T cell receptor mimic (TCR mimic), or combinations thereof. 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 ligand binding protein comprises an antigen-binding domain, a transmembrane domain, a costimulatory domain, an intracellular signaling domain, or combinations thereof.

[0045] In some aspects, the antigen-binding domain specifically binds to an antigen selected from the group consisting of ROR1, ROR2, AFP (alpha-fetoprotein), avP6 or another integrin, BCMA, Braf, B7-H3, B7-H6, CA9 (carbonic anhydrase 9), CCL-1 (C-C motif chemokine ligand 1), CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD45, CD47, CD56, CD66e, CD70, CD74, CD79a, CD79b, CD98, CD 123, CD 138, CD171, CD352, CEA (carcinoembryonic antigen), Claudin 18.2, Claudin 6, c-MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase family member 3), EpCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, ephrinB2, EPHa2 (ephrine receptor A2), ERBB dimers, estrogen receptor, ETBR (endothelin B receptor), FAP-a (fibroblast activation protein a), fetal AchR (fetal acetylcholine receptor), FBP (a folate binding protein), FCRL5, FR-a (folate receptor alpha), GCC (guanyl cyclase C), GD2, GD3, GPC2 (glypican-2), GPC3, gplOO (glycoprotein 100), GPNMB (glycoprotein NMB), GPRC5D (G Protein Coupled Receptor 5D), HER2, HER3, HER4, hepatitis B surface antigen, HLA- A1 (human leukocyte antigen Al), HLA-A2 (human leukocyte antigen A2), HMW-MAA (human high molecular weight-melanoma-associated antigen), IGF1R (insulin-like growth factor 1 receptor), Ig kappa, Ig lambda, IL-22Ra (IL-22 receptor alpha), IL-13Ra2 (IL- 13 receptor alpha 2), KDR (kinase insert domain receptor), LI cell adhesion molecule (LI - CAM), Liv-1, LRRC8A (leucine rich repeat containing 8 Family member A), Lewis Y, melanoma-associated antigen (MAGE)-Al, MAGE-A3, MAGE-A6, MART-1 (melan A), murine cytomegalovirus (MCMV), MCSP (melanoma-associated chondroitin sulfate proteoglycan), mesothelin, mucin 1 (MUC1), MUC16, MHC / peptide complexes (e.g., HLA-A complexed with peptides derived from AFP, KRAS, NY-ESO, MAGE- A, andWT1), NCAM (neural cell adhesion molecule), Nectin-4, NKG2D (natural killer group 2 member D) ligands, NY-ESO, oncofetal antigen, PD-1, PD-L1, PRAME (preferentially expressed antigen of melanoma), progesterone receptor, PSA (prostate specific antigen), PSCA (prostate stem cell antigen ), PSMA (prostate specific membrane antigen), SIRPa (signal-regulatory protein alpha), SLIT, SLITRK6 (NTRK-like protein 6), STEAP1 (six transmembrane epithelial antigen of the prostate 1), survivin, TAG72 (tumor-associated glycoprotein 72), TPBG (trophoblast glycoprotein), Trop-2, VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, and antigens from HIV, HBV, HCV, HPV, and other pathogens, and any combination thereof. In some aspects, the antigen-binding domain specifically binds to R0R1.

[0046] In some aspects, the costimulatory domain 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, costimulatory domain comprises a 4- 1BB / CD137 costimulatory domain.

[0047] In some aspects, the transmembrane domain comprises a transmembrane domain ofKIRDS2, 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, CD l id, ITGAE, CD 103, 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, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKG2D, NKG2C, CD 19, CD8, or any combination thereof. In some aspects, the transmembrane domain comprises a CD28 transmembrane domain.

[0048] In some aspects, the intracellular signaling domain comprises an intracellular signaling domain derived from CD3 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.

[0049] In some aspects, the ligand-binding protein is a TCR, wherein the TCR specifically binds to a tumor antigen / MHC complex. In some aspects, the tumor antigen is derived from ROR1, ROR2, AFP, CD19, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, 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), Kras, Braf, 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, 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, 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, surviving, telomerase, PCTA- 1 / Galectin 8, MelanA / MARTl, Ras mutant (e.g., HRAS, KRAS, NRAS), 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, human telomerase reverse transcriptase, 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, neoantigen, or any combinations thereof.

[0050] In some aspects, the c-Jun polypeptide is linked to the ligand binding protein by a linker.

[0051] In some aspects, the immune cells further express a truncated EGFR (EGFRt). In some aspects, the immune cells have been modified to comprise a nucleotide sequence encoding the EGFRt. In some aspects, the EGFRt comprises an amino acid sequence having 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% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24. In some aspects, the EGFRt comprises the amino acid sequence set forth in SEQ ID NO: 24. In some aspects, the EGFRt is linked to the c-Jun polypeptide and / or the ligand binding protein by a linker.

[0052] Where a linker is involved, in some aspects, the linker comprises a cleavable linker. In some aspects, the linker comprises a P2A linker, a T2A linker, an F2A linker, an E2Alinker, a furin cleavage site, or any combination thereof. In some aspects, the linker comprises an amino acid sequence having 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% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14. In some aspects, the linker comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0053] In some aspects, the immune cells are CD3+, CD45RO-, CCR7+, CD45RA+, CD62L+, CD27+, CD28+, or TCF7+, or any combination thereof, following the culturing. In some aspects, the immune cells comprise T cells, B cells, regulatory T cells (Treg), tumor infiltrating lymphocytes (TIL), natural killer (NK) cells, natural killer T (NKT) cells, or any combination thereof. In some aspects, the immune cells have been engineered in vitro or ex vivo. In some aspects, the immune cells were not previously frozen. In some aspects, the immune cells were previously frozen and subsequently thawed.

[0054] In some aspects, the NR4A family member comprises NR4A1, NR4A2, NR4A3, or combinations thereof. In some aspects, the gene editing tool comprises a guide RNA comprising, consisting of, or consisting essentially of the sequence set forth in any one of SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 194, and SEQ ID NO: 196.

[0055] In some aspects, the c-Jun polypeptide comprises an amino acid sequence having at least about 70%, 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%, at least about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13. In some aspects, the c-Jun polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13. In some aspects, the nucleotide sequence encoding the c-Jun polypeptide comprises: (a) a nucleic acid sequence having at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 1; (b) a nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identityto the nucleic acid sequence as set forth in SEQ ID NO: 2; (c) a nucleic acid sequence having at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 3; (d) a nucleic acid sequence having 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%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 4; (e) a nucleic acid sequence having at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 5; (f) a nucleic acid sequence having at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 6; (g) a nucleic acid sequence having at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 7; (h) a nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 8; (i) a nucleic acid sequence having at least 55%, 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%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 9; (j) a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 10; or (k) a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 11.

[0056] Also provided herein is a population of immune cells prepared by any of the methods provided herein. Some aspects of the present disclosure is related to a pharmaceutical composition comprising any of the population of immune cells described herein, and a pharmaceutically acceptable carrier.

[0057] Also provided herein is a method of treating or preventing a disease or condition in a subject in need thereof comprising administering to the subject the population of immune cells or pharmaceutical composition provided herein. In some aspects, the disease or condition comprises a cancer.

[0058] In some aspects, the method further comprising administering at least one additional therapeutic agent to the subject. In some aspects, the at least one additional therapeutic agent comprises a 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. In some aspects, the immune checkpoint inhibitor comprises an anti-PD-1 antibody, anti-PD-Ll antibody, anti -LAG-3 antibody, anti-CTLA-4 antibody, anti-GITR antibody, anti-TIM3 antibody, or any combination thereof.BRIEF DESCRIPTION OF THE FIGURES

[0059] FIG. 1 is a table of read-out data for characteristics of T cells engineered with anti- ROR1 CAR and manufactured using titrated doses of programmable cell scaffold (PCS). Output metrics for viability, cytokine (IFNY), transduction efficiency and CD3 detection, residual PCS components, and activation / sternness scores are reported for three PCS doses: 160 pg per million cells, 100 pg per million cells, and 45 pg per million cells. Further, at the 160 pg PCS per million cell dose, the culture were diluted at day 2 (“D2”) or day 3 (“D3”) post activation with the PCS.

[0060] FIG. 2 is a table of read-out data for characteristics of T cells engineered with anti- ROR1 CAR and manufactured using additional intermediate doses of PCS. Three PD runs are illustrated. In PD run 1, cells exposed 72 pg PCS per million cells dose were culture in 2 liter or 10 liter RMTX bags to test whether diluted conditions influenced resultant cell product characteristics. PD run 3 included a control T cell product transduced with the same construct encoding the CAR and cJUN, but incorporated the benchmark TransAct activation agent instead of PCS and no NR4A edit.

[0061] FIGs. 3A and 3B show successive anti-RORl lysis of A549-NLR NSCLC (3 A) and Hl 975-NLR NSCLC (3B) tumor cell lines by NR4A3-edited ROR1 CAR T cells with c-Jun overexpression generated using freshly-selected or cryopreserved frozen donor T cells in one independent donor in the sequential stimulation assay at a 1 : 10 effector-to- target ratio. Lysis of A549-NLR and Hl 975-NLR target cells were quantified by measuring total NLR intensity. NLR intensity was normalized relative to the starting intensity after replating for each round of stimulation. NLR - NucLight Red. Each graph shows data from one independent donor.

[0062] FIGs. 4A - 4C show successive anti-RORl lysis of the H1975-NLRNSCLC tumor cell line at an effector-to-target ratio of 1 :25 by NR4A3-edited ROR1 CAR T cells with c- Jun overexpression generated using different amounts of programmable cell-signaling scaffolds (PCS) in three independent donors in the sequential stimulation assay. NR4A3- edited or non-edited ROR1 CAR T cells with c-Jun overexpression generated with a standard reagent were also evaluated. Lysis of H1975-NLR target cells were quantified by measuring total NLR intensity. NLR intensity was normalized relative to the starting intensity after replating for each round of stimulation. NLR - NucLight Red. Each graph shows data from each independent donor.DETAILED DESCRIPTION OF THE DISCLOSURE

[0063] The efficacy of cellular immunotherapy is dependent on a number of factors including the persistence, multipotency, and asymmetric cell division of the cell product that is infused into the patient. The media and methods used in culturing and / or engineering (also referred to herein as "modifying") of the cells used for cell therapy can profoundly affect the metabolic, epigenetic, and phenotypic attributes of these cells thereby affecting their therapeutic potential. In some aspects, the present disclosure is generally directed to the use of certain agents (e.g. a programmable cell-signaling scaffold (PCS) in a medium comprising certain amounts of the PCS) to enhance expansion of cells (e.g., immune cells) with desirable attributes for effective cell therapy applications. In some aspects, the PCS is present in the medium at an amount of less than about 160 pg per million cells. In some aspects, the PCS is present in the medium at an amount of less than about 120 pg per million cells. In some aspects, the medium further comprises one or more additional components (e.g., potassium ion at a concentration higher than 5 mM, sodium ion (e.g., NaCl), saccharide (e.g., glucose), calcium, and / or cytokine). As also described herein, in someaspects, the cells have been modified such that the cells are different (e.g., structurally and / or functionally) as compared to corresponding cells which have not been modified. For instance, in some aspects, the cells have been modified (e.g., edited) to decrease the expression of one or more genes and / or proteins that are associated with impaired immune cell function. In some aspects, the one or more genes and / or proteins associated with impaired immune cell function comprises a nuclear receptor subfamily 4A (NR4A) family member. In some aspects, the cells have been edited to exhibit a reduced expression level of a nuclear receptor subfamily 4A (NR4A) family member as compared to corresponding cells that have not been edited. Unless indicated otherwise, a reduced (or a decrease in) expression level of a NR4A family member can comprise a reduced gene expression and / or reduced protein expression. In some aspects, the cells have been modified e.g. , transduced) to increase the expression of one or more genes and / or proteins that are associated with improved immune cell function. In some aspects, the one or more genes and / or proteins that are associated with improved immune cell function comprises a c-Jun protein. In some aspects, the cells described herein have been transduced to exhibit increased expression of a c-Jun protein, as compared to corresponding cells which have not been further modified. In some aspects, the cells described herein have been transduced to express a ligandbinding protein. Non-limiting examples of the various aspects are provided throughout the present disclosure.

[0064] In some aspects, the present disclosure is generally directed to the use of programmable cell-signaling scaffold at an amount of less than 120 pg per million cells in combination with additional agents (e.g., amedium comprising potassium ion at a concentration higher than 5 mM, gene editing tools that specifically target a NR4A family member, and / or polynucleotides encoding a ligand-binding protein and / or c-Jun polypeptide) to enhance such attributes of cells (e.g., immune cells) that are useful for cell therapy. While the present application provides disclosure related to modifying the expression of a NR4A family member and / or c-Jun protein, a skilled artisan reading the present application would recognize that the medium disclosed herein (e.g., comprising programmable cell-signaling scaffold at an amount of less than 120 pg per million cells) can be used when culturing and / or modifying the expression of other genes and / or proteins in cells. Non-limiting examples of such other genes and / or proteins are provided elsewhere in the present disclosure. As described herein, in some aspects, the gene and / or protein is associated with impaired immune cell function and the medium disclosed herein (e.g.,comprising programmable cell-signaling scaffold at an amount of less than 120 pg per million cells) is used to decrease the expression of the gene and / or protein. In some aspects, the gene and / or protein is associated with improved immune cell function and the medium disclosed herein (e.g., comprising programmable cell-signaling scaffold at an amount of less than 120 pg per million cells) is used to increase the expression of the gene and / or protein.Terms

[0065] 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.

[0066] 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.

[0067] 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).

[0068] 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.

[0069] 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 andMolecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0070] 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.

[0071] Abbreviations used herein are defined throughout the present disclosure. Various aspects of the disclosure are described in further detail in the following subsections.

[0072] 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, i.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%, 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.

[0073] 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).

[0074] 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.

[0075] As used herein, the term "culturing" (including any derivatives thereof) refers to the process in which cells (e.g., T cells and / or NK cells) are maintained, sustained, propagated, and / or modified under a controlled condition. For instance, unless indicated otherwise, culturing can comprise the step of contacting cells with PCS in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, e.g., less than about 120 pg per million cells. In some aspects, culturing can comprise the step of modifying the cells, e.g., to exhibit a reduced expression of a gene and / or protein associated with impaired immune cell function (e.g., aNR4A family member), to exhibit an increased expression of a gene and / or protein associated with improved immune cell function (e.g., c-Jun protein), and / or to express a ligand binding protein (e.g., CAR and / or TCR). In some aspects, culturing can comprise the step of maintaining and / or expanding the cells after the contacting and modifying are performed. As used herein, "culturing" includes the growth of cells, e.g., immune cells, e.g., one or more engineered immune cell disclosed herein, during cell expansion, or cell engineering (e.g, transduction with a construct for expressing a CAR, a TCR, or a TCRm). In some aspects, the cultured 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 / patient. 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.

[0076] The term "control media," “conventional culture media,” or "reference culture media" as used herein refers to any media in comparison to a media (e.g, metabolic reprogramming media (MRM)) disclosed herein, which comprises PCS at an amount of less than about 160 pg per million cells, e.g, less than about 120 pg per million cells. Where the media useful for the present disclosure comprises PCS at an amount of less than about 160 pg per million cells, in some aspects, the control media comprises no PCS or comprises PCS at an amount of 160 pg per million cells or greater. Where the media useful for the present disclosure comprises PCS at an amount of less than about 120 pg per million cells, in some aspects, the control media comprises no PCS or comprises PCS at an amount of 120 pg per million cells or greater. Unless indicated otherwise, the terms "media" and "medium" can be used interchangeably.

[0077] As used herein, the term "edited" (and grammatical variant thereof) refers to the process by which cells (e.g., T cells and / or NK cells) have been modified such that the cells exhibit a decrease in an expression level of one or more genes and / or proteins associated with impaired immune cell function (e.g., a NR4A family member). Unless indicated otherwise, the expression "editing the immune cells to exhibit a reduced expression level" of a gene and / or protein (e.g., NR4A family member) (or equivalent thereof) and the expression "modifying the immune cells to exhibit a reduced expression level" of a gene and / or protein (e.g., NR4A family member) (or equivalent thereof) can be used interchangeably. More specifically, as further described herein, in some aspects, cells (e.g., T cells and / or NK cells) provided herein have been edited such that the cells exhibit reduced expression of a NR4A protein and / or gene as compared to non-edited cells. Accordingly, the term "NR4A-edited" as used herein refers to a reduced expression of one or more members of the NR4A family (e.g., NR4A1, NR4A2, and / or NR4A3). In some aspects, NR4A-edited cells (e.g., NR4Al-edited, NR4A2-edited, and / or NR4 A3 -edited) exhibit no expression of one or more members of the NR4A family. In some aspects, NR4A-edited cells exhibit some expression of a member of the NR4A family but at much reduced level compared to corresponding non-edited cells. In some aspects, compared to corresponding non-edited cells, NR4A expression in a NR4A-edited cells provided herein is reduced 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 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%, or about 100%. Unless indicated otherwise, the terms "edited," "knock out," and "deficient" (including any derivatives thereof) are used interchangeably in the present disclosure. Therefore, in some aspects, "NR4A-edited," "NR4A-knock out," and "NR4A- deficient" refer to the same type of immune cells.

[0078] As used herein, the expression "transducing immune cells to exhibit an increased expression level" of a gene and / or protein (e.g., c-Jun polypeptide) (or equivalent thereof) refers to the process of modifying the immune cells such that the immune cells express higher level of the gene and / or protein (e.g., a c-Jun polypeptide) as compared to immune cells that have not been transduced. Non-limiting examples of transducing immune cells to exhibit increased expression of a gene and / or protein (e.g., a c-Jun) are provided elsewhere in the present disclosure. Unless indicated otherwise, "transducing immune cells to exhibitan increased expression level" of a gene and / or protein (e.g., c-Jun polypeptide) (or equivalent thereof) can be used interchangeably with "modifying immune cells to exhibit an increased expression level" of a gene and / or protein (e.g., c-Jun polypeptide) (or equivalent thereof). Similarly, the expression "transducing immune cells to express a ligand-binding protein" (or equivalent thereof) refers to the process of modifying the immune cells such that the immune cells express a ligand-binding protein. As described herein, in some aspects, the immune cells do not naturally express the ligand-binding protein. Non-limiting examples of transducing immune cells to express a ligand-binding protein is also provided elsewhere in the present disclosure. Unless indicated otherwise, "transducing immune cells to express a ligand-binding protein" (or equivalent thereof) can be used interchangeably with "modifying immune cells to express a ligand-binding protein (or equivalent thereof).

[0079] As further described herein, immune cells described herein differ from corresponding immune cells that naturally exist in nature. For instance, in some aspects, immune cells described herein have been engineered or modified, e.g., (a) contacted with a PCS, (b) edited to exhibit a reduced expression level of a gene and / or protein associated with impaired immune cell function (e.g., NR4A family member), (c) transduced to express a ligand-binding protein, (d) transduced to exhibit an increased expression of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), or (e) any combination of (a) to (d). Based on the disclosures provided in the present application, a skilled artisan will understand that the terms "modified," "edited," "transduced," and "engineered" (or grammatical variants thereof) can be used interchangeably.

[0080] As described herein, in some aspects, an immune cell has been modified to decrease the expression of a gene and / or protein associated with impaired immune cell function. For instance, in some aspects, an immune cell has been modified (e.g., edited) such that the expression of the gene and / or protein associated with impaired immune cell function is decreased as compared to a corresponding immune cell which has not been modified. In some aspects, the expression of the gene and / or protein associated with impaired immune cell function is decreased 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 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%, or at least about 100% as compared to thecorresponding immune cells which has not been modified. As further described elsewhere in the present disclosure, in some aspects, an immune cell has been modified such that the immune cell does not exhibit any expression of the gene and / or protein associated with impaired immune cell function. Not to be bound by any one theory, in some aspects, reducing the expression of one or more genes and / or proteins associated with impaired immune cell function can increase the immune cell function in the cell as compared to a corresponding immune cell which has not been modified. For instance, in some aspects, reducing the expression of one or more genes and / or proteins associated with impaired immune cell function can reduce exhaustion, increase effector function, enhanced proliferative capacity, or combinations thereof. Additional examples of immune cell functions that can be improved are provided elsewhere in the present disclosure. In some aspects, a gene and / or protein that is associated with impaired immune cell function is expressed at a higher level in an exhausted immune cell as compared to a corresponding immune cell that is not exhausted. A non-limiting example of such a gene and / or protein comprises a member of the NR4A family.

[0081] In some aspects, an immune cell has been modified to increase the expression of a gene and / or protein associated with improved immune cell function. In some aspects, an immune cell has been modified (e.g., transduced) such that the expression of the gene and / or protein associated with improved immune cell function such that the expression of the gene and / or protein associated with improved immune cell function is increased as compared to a corresponding immune cell which has not been modified. In some aspects, the expression of the gene and / or protein associated with improved immune cell function 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 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 100%, 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 about8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 45 fold, or at least about 50 fold, as compared to the correspondingimmune cell which has not been modified. Not to be bound by any one theory, in some aspects, increasing the expression of one or more genes and / or proteins associated with improved immune cell function can increase the immune cell function in the cell as compared to a corresponding immune cell which has not been modified. For instance, in some aspects, increasing the expression of one or more genes and / or proteins associated with improved immune cell function can reduce exhaustion, increase effector function, enhanced proliferative capacity, or combinations thereof. Additional examples of immune cell functions that can be improved are provided elsewhere in the present disclosure. Nonlimiting example of such a gene and / or protein comprises c-Jun.

[0082] As used herein, the term "associated with" refers to a close relationship between two or more entities or properties. For instance, where a cell exhibits a decreased expression of a gene and / or protein that is associated with impaired immune cell function, the cell can exhibit improved immune cell function as compared to a corresponding cell that does not exhibit a decreased expression of the gene and / or protein that is associated with impaired immune cell function (e.g., a corresponding cell that has not been modified as described herein). In some aspects, the gene and / or protein causes the impaired immune cell function, such that reducing the expression of the gene and / or protein can improve immune cell function. In some aspects, the gene and / or protein does not necessarily cause the impaired immune cell function but is positively correlated.

[0083] Similarly, where a cell exhibits an increased expression of a gene and / or protein that is associated with improved immune cell function, the cell can exhibit improved immune cell function as compared to a corresponding cell that does not exhibit an increased expression of the gene and / or protein associated with improved immune cell function (e.g., a corresponding cell that has not been modified as described herein). In some aspects, the gene and / or protein causes the improved immune cell function, such that increasing the expression of the gene and / or protein can improve immune cell function. In some aspects, the gene and / or protein does not necessarily cause the improved immune cell function but is positively correlated.

[0084] 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 activatedand 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 cannot change or can decrease.

[0085] 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. Non-limiting example of determining yield is provided in FIG. 2.

[0086] As used herein, the term "metabolic reprogramming media," "metabolic reprogramming medium," or "MRM," refers to a medium, wherein the medium has an increased potassium ion concentration. Accordingly, in some aspects, a medium useful for the present disclosure comprises (a) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and (b) an increased potassium ion concentration. In some aspects, the metabolic reprogramming media comprises potassium ion at a concentration higher than 5 mM. Accordingly, unless indicated otherwise, "medium comprising potassium ion at a concentration higher than 5 mM" and "metabolic reprogramming media" can be used interchangeably in the present disclosure. In some aspects, the metabolic reprogramming media comprises potassium ion at a concentration higher than 40 mM. In some aspects, the metabolic reprogramming media comprises a concentration of potassium ion of at least about 10 mM, at least about 15 mM, at least about20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, or at least about 100 mM. In some aspects, the metabolic reprogramming media comprises about 40 mM to about 80 mM NaCl, about 40 mM to about 90 mM KC1, about 0.5 mM to about 2.8 mM calcium, and about 10 mM to about 24 mM glucose. In some aspects, the metabolic reprogramming media further comprises an osmolality of about 250 to about 300 mOsmol. In some aspects, the metabolic reprogramming medium further comprises programmable cell-signaling scaffolds (PCS) as disclosed herein.

[0087] As used herein, the term "higher than" means greater than but not equal to. For example, "higher than 5 mM" means any amount that is more than 5 mM, but which doesnot include 5 mM. As used herein, the term "less than" means lower than but not equal to. For example, "amount of less than 120 pg per million cells" means any amount greater than 0 pg per million cells but less than 120 pg per million cells, and does not include 120 pg per million cells.

[0088] As used herein, the term "tonicity" refers to the calculated effective osmotic pressure gradient across a cell membrane, represented by the sum of the concentration of potassium ion and the concentration of sodium chloride (NaCl), multiplied by two. Tonicity can be expressed in terms of the osmolality (mOsm / kg) or osmolarity (mOsm / L) of the solution, e.g., the media. Osmolality and osmolarity are measurements of the solute osmotic concentration of a solvent per mass (osmolality) and per volume (osmolarity). As used herein, an isotonic medium has a tonicity of about 280 mOsm / L (e.g., ([K+] + [NaCl]) X 2 = 280).

[0089] As used herein, a hypotonic solution has a tonicity of less than 280 mOsm / L (e.g., ([K+] + [NaCl]) X 2 < 280). In some aspects, a hypotonic medium has a tonicity from at least about 210 mOsm / L to less than about 280 mOsm / L. In some aspects, a hypotonic medium has a tonicity from at least about 220 mOsm / L to less than about 280 mOsm / L. In some aspects, a hypotonic medium has a tonicity from at least about 230 mOsm / L to less than about 280 mOsm / L. In some aspects, a hypotonic medium has a tonicity from at least about 240 mOsm / L to less than about 280 mOsm / L. In some aspects, a hypotonic medium described herein has a tonicity of about 250 mOsm / L.

[0090] As used herein, a hypertonic solution has a tonicity of greater than 300 mOsm / L (e.g., ([K+] + [NaCl]) X 2 > 300). In some aspects, a hypertonic medium described herein has a tonicity of about 320 mOsm / L. In some aspects, the tonicity of the solution, e.g., medium is adjusted by increasing or decreasing the concentration of potassium ions and NaCl. In some aspects, the tonicity of a medium can be maintained by offsetting the increase of one solute with a decrease in a second solute. For example, increasing the concentration of potassium ion in a medium without changing the concentration of sodium ions can increase the tonicity of the medium. However, if the concentration of potassium ions is increased and the concentration of sodium ions is decreased, the tonicity of the original medium can be maintained.

[0091] As used herein, the terms "potassium," "potassium ion," "potassium cation," and "K+" are used interchangeably to refer to elemental potassium. Elemental potassium exists in solution as a positive ion. However, it would be readily apparent to a person of ordinaryskill in the art that standard means of preparing a solution comprising potassium ion include diluting a potassium containing salt (e.g., KC1) into a solution. As such, a solution, e.g., a medium, comprising a molar (M) concentration of potassium ion, can be described as comprising an equal molar (M) concentration of a salt comprising potassium.

[0092] As used herein, the terms "sodium ion" and "sodium cation" are used interchangeably to refer to elemental sodium. Elemental sodium exists in solution as a monovalent cation. However, it would be readily apparent to a person of ordinary skill in the art that standard means of preparing a solution comprising sodium ion include diluting a sodium-containing salt (e.g., NaCl) into a solution. As such, a solution, e.g., a medium, comprising a molar (M) concentration of sodium ion, can be described as comprising an equal molar (M) concentration of a salt comprising sodium.

[0093] As used herein, the terms "calcium ion" and "calcium cation" are used interchangeably to refer to elemental calcium. Elemental calcium exists in solution as a divalent cation. However, it would be readily apparent to a person of ordinary skill in the art that standard means of preparing a solution comprising calcium ion include diluting a calcium-containing salt (e.g., CaCh) into a solution. As such, a solution, e.g., a medium, comprising a molar (M) concentration of calcium ion, can be described as comprising an equal molar (M) concentration of a salt comprising calcium.

[0094] 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 is comprises a heterogeneous mixture of cells, comprising multiple types of cells, e.g., a heterogeneous mixture of immune cells and non- immune cells. In some aspects, the population of cells comprises a plurality of T cells.

[0095] 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 Th 17 cell. In some aspects, the T cell is a Treg cell. In some aspects, the T cell is a tumor-infiltrating cell (TIL).

[0096] 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 stronger 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.

[0097] As used herein, the term "stem cell-like memory T cells," "T memory stem cells," or "TSCM cells" refers to memory T cells that express CD95, CD45RA, CCR7, and CD62L and are endowed with the stem cell-like ability to self-renew and the multipotent capacity to reconstitute the entire spectrum of memory and effector T cell subsets.

[0098] As used herein, the term "central memory T cells" or "TCM cells" refers to memory T cells that express CD45RO, CCR7, and CD62L. Central memory T cells are generally found within the lymph nodes and in peripheral circulation.

[0099] As used herein, the term "effector memory T cells" or "TEM cells" refers to memory T cells that express 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.

[0100] 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.

[0101] As used herein, the term "naive T cells" or "TN cells" refers to T cells that express CD45RA, CCR7, and CD62L, but which do not express CD95. TN cells represent the most undifferentiated 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.

[0102] As used herein, the term "sternness," "stem cell-like," "stem-like," or "less- differentiated" refers to an immune cell (e.g., a T cell, an NK cell, or a TIL), that expresses markers consistent with a more naive phenotype. For example, a less differentiated T cell can express one or more marker characteristic of a TN or a TSCM cell. In some aspects, a "less-differentiated" or "stem-like" T cell expresses CD45RA, CCR7, and CD62L. In someaspects, a "less-differentiated" or "stem-like" T cell expresses CD45RA, CCR7, CD62L, and TCF7. In some aspects, a "less-differentiated" or "stem-like" T cell does not express CD45RO or is CD45ROlow. In some aspects, the methods disclosed herein promote immune cells (e.g, T cells and / or NK cells) having a less-differentiated phenotype. Without being bound by any particular mechanism, in some aspects, the methods disclosed herein block, inhibit, or limit differentiation of less-differentiated immune cells (e.g, T cells and / or NK cells), resulting in an increased number of stem-like cells in culture. For example, it is generally thought that to effectively control tumors, adoptive transfer of less-differentiated immune cells, e.g., T cells and / or NK cells, with a stem cell-like memory or central memory phenotype are preferred. See Gattinoni, L., et al., J. Clin. Invest. 115: 1616-1626 (2005), Gattinoni, L., et al. Nat Med 15(7):808-814 (2009), Lynn, R.C., et al., Nature 576(7786): 293-300 (2019); Gattinoni, L., et al. Nat Rev 12:671-684 (2012), Klebanoff, C., et al., J. Immunother 35(9):651-670 (2012) and Gattinoni, L., et al., Nat Med 17(10): 1290-1297 (2011).

[0103] Sternness is characterized by the capacity to self-renew, the multipotency, and the persistence of proliferative potential. In some aspects, sternness is characterized by a particular gene signature, e.g., a combined pattern of expression across a multitude of genes. In some aspects, the stem-like cells can be identified by a transcriptome analysis, e.g., using sternness gene signatures disclosed herein. In some aspects, the gene signature comprises one or more genes selected from ACINI, DSC1, TSHZ2, MYB, LEF1, TIMD4, MAL, KRT73, SESN3, CDCA7L, LOC283174, TCF7, SLC16A10, LASS6, UBE2E2, IL7R, GCNT4, TAF4B, SULT1B1, SELP, KRT72, STXBP1, TCEA3, FCGBP, CXCR5, GPA33, NELL2, APBA2, SELL, VIPR1, FAM153B, PPFIBP2, FCER1G, GJB6, 0CM2, GCET2, LRRN1, IL6ST, LRRC16A, IGSF9B, EFHA2, LOC129293, APP, PKIA, ZC3H12D, CHMP7, KIAA0748, SLC22A17, FLJ13197, NRCAM, C5orfl3, GIPC3, WNT7A, FAM117B, BEND5, LGMN, FAM63A, FAM153B, ARHGEF11, RBM11, RIC3, LDLRAP1, PELI1, PTK2, KCTD12, LM07, CEP68, SDK2, MCOLN3, ZNF238, EDAR, FAM153C, FAAH2, BCL9, C17orf48, MAP1D, ZSWIM1, SORBS3, IL4R, SERPINF1, C16orf45, SPTBN1, KCNQ1, LDHB, BZW2, NBEA, GAL3ST4, CRTC3, MAP3K1, HLA-DOA, RAB43, SGTB, CNN3, CWH43, KLHL3, PIM2, RGMB, C16orf74, AEBP1, SNORD115-11, SNORD115-11, GRAP, and any combination thereof (see, e.g., Gattinoni et al., Nature Medicine 17(10) : 1290-97 (2011)). In some aspects, the gene signature comprises one or more gene selected from NOG, TIMD4, MYB, UBE2E2,FCER1G, HAVCR1, FCGBP, PPFIBP2, TPST1, ACTN1, IGF1R, KRT72, SLC16A10, GJB6, LRRN1, PRAGMIN, GIPC3, FLNB, ARRB1, SLC7A8, NUCB2, LRRC7, MY015B, MAL, AEBP1, SDK2, BZW2, GAL3ST4, PITPNM2, ZNF496, FAM117B, C16orf74, TDRD6, TSPAN32, C18orf22, C3orf44, LOC129293, ZC3H12D, MLXIP, C7orfl0, STXBP1, KCNQ1, FLJ13197, LDLRAP1, RAB43, RIN3, SLC22A17, AGBL3, TCEA3, NCRNA00185, FAM153B, FAM153C, VIPR1, MMP19, HBS1L, EEF2K, SN0RA5C, UBASH3A, FLJ43390, RP6-213H19.1, INPP5A, PIM2, TNFRSF10D, SNRK, LOC100128288, PIGV, LOC100129858, SPTBN1, PROS1, MMP28, HES1, CACHD1, NSUN5C, LEF1, TTTY14, SNORA54, HSF2, C16orf67, NSUN5B, KIAA1257, NRG2, CAD, TARBP1, STRADB, MT1F, TMEM41B, PDHX, KDM6B, LOC100288322, UXS1, LGMN, NANOS2, PYGB, RASGRP2, C14orf80, XPO6, SLC24A6, FAM113A, MRM1, FBXW8, NDUFS2, KCTD12, and any combination thereof (see, e.g., Gattinoni, L., et al., Nat Med 17(10): 1290-1297 (2011)).

[0104] As used herein, the term "effector-like" or "effector cell-like" refers to tumor cell killing capacity and cytokine polyfunctionality, e.g., ability of a cell to produce inflammatory cytokines and / or cytotoxic molecules. In some aspects, an effector-like cell is characterized by specific markers expressed by the cell. In some aspects, those effectorlike markers comprise one or more of pSTAT5+, STAT5+, pSTAT3+, and STAT3+. In some aspects, the effector-like marker comprises a STAT target selected from the group consisting of AKT1, AKT2, AKT3, BCL2L1, CBL, CBLB, CBLC, CCND1, CCND2, CCND3, CISH, CLCF1, CNTF, CNTFR, CREBBP, CRLF2, CSF2, CSF2RA, CSF2RB, CSF3, CSF3R, CSH1, CTF1, EP300, EPO, EPOR, GH1, GH2, GHR, GRB2, IFNA1, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA2, IFNA21, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNAR1, IFNAR2, IFNB1, IFNE, IFNG, IFNGR1, IFNGR2, IFNK, IFNL1, IFNL2, IFNL3, IFNLR1, IFNW1, IL10, IL10RA, IL10RB, IL11, IL1 IRA, IL12A, IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL19, IL2, IL20, IL20RA, IL20RB, IL21, IL21R, IL22, IL22RA1, IL22RA2, IL23A, IL23R, IL24, IL26, IL2RA, IL2RB, IL2RG, IL3, IL3RA, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7, IL7R, IL9, IL9R, IRF9, JAK1, JAK2, JAK3, LEP, LEPR, LIF, LIFR, MPL, MYC, OSM, OSMR, PIAS1, PIAS2, PIAS3, PIAS4, PIK3CA, PIK3CB, PIK3CD, PIK3CG, PIK3R1, PIK3R2, PIK3R3, PIK3R5, PIM1, PRL, PRLR, PTPN11, PTPN6, SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS7, S0S1, S0S2, SPRED1, SPRED2, SPRY1, SPRY2, SPRY3, SPRY4, STAM, STAM2, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B,STAT6, TPO, TSLP, TYK2, and any combination thereof. In some aspects, the effectorlike cells are characterized by a transcriptome analysis. In some aspects, the effector-like marker comprises a marker disclosed in Kaech et al., Cell 777:837-51 (2002); Tripathi et al., J. Immunology 755:2116-24 (2010); and / or Johnnidis et al., Science Immunology 6:eabe3702 (Jan. 15, 2021), each of which is incorporated by reference herein in its entirety.

[0105] In some aspects, the effector-like cells are characterized using an effector- associated gene set described in Gattinoni, L., et al., Nat Med 17(10): 1290-97 (2011). In some aspects, the gene signature for effector-like cells comprises one or more genes selected from MTCH2, RAB6C, KIAA0195, SETD2, C2orf24, NRD1, GNA13, COP A, SELT, TNIP1, CBFA2T2, LRP10, PRKCI, BRE, ANKS1A, PNPLA6, ARL6IP1, WDFY1, MAPK1, GPR153, SHKBP1, MAP1LC3B2, PIP4K2A, HCN3, GTPBP1, TLN1, C4orf34, KIF3B, TCIRG1, PPP3CA, ATG4D, TYMP, TRAF6, C17orf76, WIPF1, FAM108A1, MYL6, NRM, SPCS2, GGT3P, GALK1, CLIP4, ARL4C, YWHAQ, LPCAT4, ATG2A, IDS, TBC1D5, DMPK, ST6GALNAC6, REEP5, ABHD6, KIAA0247, EMB, TSEN54, SPIRE2, PIWIL4, ZSCAN22, ICAM1, CHD9, LPIN2, SETD8, ZC3H12A, ULBP3, IL15RA, HLA-DQB2, LCP1, CHP, RUNX3, TMEM43, REEP4, MEF2D, ABL1, TMEM39A, PCBP4, PLCD1, CHST12, RASGRP1, Clorf58, Cl lorf63, C6orfl29, FHOD1, DKFZp434F142, PIK3CG, ITPR3, BTG3, C4orf50, CNNM3, IFI16, AK1, CDK2AP1, REL, BCL2L1, MVD, TTC39C, PLEKHA2, FKBP11, EML4, FANCA, CDCA4, FUCA2, MFSD10, TBCD, CAPN2, IQGAP1, CHST11, PIK3R1, MY05A, KIR2DL3, DLG3, MXD4, RALGDS, S1PR5, WSB2, CCR3, TIPARP, SP140, CD151, SOX13, KRTAP5-2, NF1, PEA15, PARP8, RNF166, UEVLD, LIMK1, CACNB1, TMX4, SLC6A6, LBA1, SV2A, LLGL2, IRF1, PPP2R5C, CD99, RAPGEF1, PPP4R1, OSBPL7, FOXP4, SLA2, TBC1D2B, ST7, JAZF1, GGA2, PI4K2A, CD68, LPGAT1, STX11, ZAK, FAM160B1, RORA, C8orf80, APOBEC3F, TGFBI, DNAJC1, GPR114, LRP8, CD69, CMI, NAT13, TGFBI, FLJ00049, ANTXR2, NR4A3, IL12RB1, NTNG2, RDX, MLLT4, GPRIN3,, ADCY9, CD300A, SCD5, ABB, PTPN22, LGALS1, SYTL3, BMPR1A, TBK1, PMAIP1, RASGEF1A,, GCNT1, GABARAPL1, STOM, CALHM2, ABCA2, PPP1R16B, SYNE2, PAM, C12orf75, CLCF1, MXRA7, APOBEC3C, CLSTN3, ACOT9, HIP1, LAG3, TNFAIP3, DCBLD1, KLF6, CACNB3, RNF19A, RAB27A, FADS3, DLG5, APOBEC3D, TNFRSF1B, ACTN4, TBKBP1, ATXN1, ARAP2, ARHGEF12, FAM53B, MAN1A1, FAM38A, PLXNC1, GRLF1, SRGN, HLA-DRB5, B4GALT5, WIPI1, PTPRJ, SLFN11, DUSP2, ANXA5, AHNAK, NEO1, CLIC1, EIF2C4, MAP3K5,IL2RB, PLEKHG1, MY06, GTDC1, EDARADD, GALM, TARP, ADAM8, MSC, HNRPLL, SYT11, ATP2B4, NHSL2, MATK, ARHGAP18, SLFN12L, SPATS2L, RAB27B, PIK3R3, TP53INP1, MB0AT1, GYG1, KATNAL1, FAM46C, ZC3HAV1L, ANXA2P2, CTNNA1, NPC1, C3AR1, CRIM1, SH2D2A, ERN1, YPEL1, TBX21, SLC1A4, FASLG, PHACTR2, GALNT3, ADRB2, PIK3AP1, TLR3, PLEKHA5, DUSP10, GNAO1, PTGDR, FRMD4B, ANXA2, EOMES, CADM1, MAF, TPRG1, NBEAL2, PPP2R2B, PELO, SLC4A4, KLRF1, FOSL2, RGS2, TGFBR3, PRF1, MYO1F, GAB3, C17orf66, MICAL2, CYTH3, TOX, HLA-DRA, SYNE1, WEE1, PYHIN1, F2R, PLD1, THBS1, CD58, FAS, NETO2, CXCR6, ST6GALNAC2, DUSP4, AUTS2, Clorf21, KLRG1, TNIP3, GZMA, PRR5L, PRDM1, ST8SIA6, PLXND1, PTPRM, GFPT2, MYBL1, SLAMF7, FLJ16686,, GNLY, ZEB2, CST7, IL18RAP, CCL5, KLRD1, KLRB1, and any combination thereof (see, e.g., Gattinoni, L., et al., Nat Med 17(10): 1290-97 (2011).

[0106] In the presence of prolonged antigen exposure, such as in many cancers, more differentiated immune cells, e.g., effector and effector memory T cells, often become exhausted and lose their anti -tumor function. Biomarkers, e.g., T cell markers, can be measured using any methods. In some aspects, T cells are identified using antibody-staining following by gated flow cytometry.

[0107] As used herein, the term "basal" media refers to any starting media that is supplemented with one or more of the additional elements disclosed herein, e.g. , potassium, sodium, calcium, glucose, IL-2, IL-7, IL- 15, IL-21, programmable cell-signaling scaffold (PCS), or any combination thereof. The basal media can be any media for culturing immune cells, e.g., T cells and / or NK cells. In some aspects, the basal media comprises a balanced salt solution (e.g., PBS, DPBS, HBSS, EBSS), Dulbecco's Modified Eagle's Medium (DMEM), Click’s medium, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), F-10, F-12, RPMI 1640, Glasgow Minimal Essential Medium (GMEM), alpha Minimal Essential Medium (alpha MEM), Iscove's Modified Dulbecco's Medium (IMDM), Ml 99, OPTMIZER™ Pro, OPTMIZER™ CTS™ T-Cell Expansion Basal Medium (ThermoFisher), OPTMIZER™, OPTMIZER™ Complete, IMMUNOCULT™ XF (STEMCELL™ Technologies), AIM V™, TEXMACS™ medium, PRIME-XV® T cell CDM, X-VIVO™ 15 (Lonza), TRANSACT™ TIL expansion medium, or any combination thereof. In some aspects, the basal medium is serum free. In some aspects, the basal media comprises PRIME-XV® T cell CDM. In some aspects, the basal media comprisesOPTMIZER™. In some aspects, the basal media comprises OPTMIZER™ Pro. In some aspects, the basal medium further comprises immune cell serum replacement (ICSR). For example, in some aspects, the basal medium comprises OPTMIZER™ Complete supplemented with ICSR, AIM V™ supplemented with ICSR, IMMUNOCULT™ XF supplemented with ICSR, RPMI supplemented with ICSR, TEXMACS™ supplemented with ICSR, or any combination thereof. In some aspects, suitable basal media include Click's medium, OPTMIZER™ (CTS™) medium, STEMLINE® T cell expansion medium (Sigma-Aldrich), AIM V™ medium (CTS™), TEXMACS™ medium (Miltenyi Biotech), IMMUNOCULT™ medium (Stem Cell Technologies), PRIME-XV® T-Cell Expansion XSFM (Irvine Scientific), Iscoves medium, and / or RPMI- 1640 medium. In some aspects, the basal media comprises NaCl free CTS™ OPTMIZER™. In some aspects, the basal media comprises one or more sodium salt in addition to the NaCl.

[0108] As used herein, the term "cytokine" refers to small, secreted proteins released by cells that have a specific effect on the interactions and communications between cells. Nonlimiting examples of cytokines include interleukins (e.g., interleukin (IL)-l, IL-2, IL-4, IL- 7, IL-9, IL-13, IL-15, IL-3, IL-5, IL-6, IL-11, IL-10, IL-20, IL-14, IL-16, IL-17, IL-21 and IL-23), interferons (IFN; e.g., IFN-a, IFN-P, and IFN-y), tumor necrosis factor (TNF) family members, and transforming growth factor (TGF) family members. Some aspects of the present disclosure are directed to methods of culturing and / or expanding immune cells, e.g., T cells and / or NK cells or one or more engineered immune cell disclosed herein, in a medium comprising a cytokine. In some aspects, the cytokine is an interleukin. In some aspects, the cytokine comprises IL-2, IL-7, IL- 15, IL-21 or any combination thereof. IL-2 (UniProtKB - P60568) is produced by T cells in response to antigenic or mitogenic stimulation. IL-2 is known to stimulate T cell proliferation and other activities crucial to regulation of the immune response. IL-7 (UniProtKB - Pl 3232) is a hematopoietic growth factor capable of stimulating the proliferation of lymphoid progenitors. IL-7 is believed to play a role in proliferation during certain stages of B-cell maturation. IL-15 (UniProtKB - P40933), like IL-2, is a cytokine that stimulates the proliferation of T-lymphocytes. IL-21 (UniProtKB - Q9HBE4) is a cytokine with immunoregulatory activity. IL-21 is thought to promote the transition between innate and adaptive immunity and to induce the production of IgGl and IgG3 in B-cells. IL-21 can also play a role in proliferation and maturation of natural killer (NK) cells in synergy with IL- 15, and IL-21 can regulate proliferation of mature B- and T-cells in response to activating stimuli. In synergy with IL-15 and IL-18,IL-15 also stimulates interferon gamma production in T-cells and NK cells, and IL-21 can also inhibit dendritic cell activation and maturation during a T-cell-mediated immune response.

[0109] As used herein, the term “endogenous expression” or “endogenous expression levels” or “endogenous levels” (or grammatical variants thereof) refers to gene and / or protein expression (e.g., amount, kinetics, etc.) that is naturally occurring (e.g., the gene and / or protein is not directly manipulated by non-naturally-occurring engineering). For example, in some aspects, a modified cell disclosed herein does not express endogenous levels of a NR4A3 gene and / or protein (e.g., expresses no NR4A3 or expresses reduced level of NR4A3 as compared to corresponding non-modified cell), but because the two NR4A1 and NR4A2 genes have not been knocked down (e.g., by CRISPR, e.g., a non- naturally occurring engineering) the modified cells exhibit endogenous expression of NR4A1 and NR4A2 gene and / or NR4A1 and NR4A2 protein. Similarly, in some aspects, a modified cell disclosed herein has been edited such that the modified cell does not express endogenous level of NR4A2 (e.g., expresses no NR4A2 or expresses reduced level of NR4A2 as compared to corresponding non-modified cell), but because the NR4A1 and NR4A3 have not been knocked down, the modified cell exhibits endogenous expression of the NR4A1 and NR4A2 (gene and / or protein). In some aspects, a modified cell disclosed herein has been edited such that modified cell does not express endogenous level of NR4A1 (e.g., expresses no NR4A1 or expresses reduced level of NR4A1 as compared to corresponding non-modified cell), but because the NR4A2 and NR4A3 have not been knocked down, the modified cell exhibits endogenous expression of the NR4A2 and NR4A3 (gene and / or protein). In some aspects, a modified cell disclosed herein has been edited such that the modified cell does not express endogenous level of NR4A1 and NR4A2 (e.g., expresses no NR4A1 and NR4A2 or expresses reduced level of NR4A1 and NR4A2 as compared to corresponding non-modified cell), but because the NR4A3 has not been knocked down, the modified cell exhibits endogenous expression of the NR4A3 (gene and / or protein). In some aspects, a modified cell disclosed herein has been edited such that the modified cell does not express endogenous level of NR4A1 and NR4A3 (e.g., expresses no NR4A1 and NR4A3 or expresses reduced level of NR4A1 and NR4A3 as compared to corresponding non-modified cell), but because the NR4A2 has not been knocked down, the modified cell exhibits endogenous expression of the NR4A2 (gene and / or protein). In some aspects, a modified cell disclosed herein has been edited such that the modified cell doesnot express endogenous level of NR4A1 and NR4A2 (e.g., expresses no NR4A1 and NR4A2 or expresses reduced level of NR4A1 and NR4A2 as compared to corresponding non-modified cell), but because the NR4A3 has not been knocked down, the modified cell exhibits endogenous expression of the NR4A3 (gene and / or protein). In some aspects, a modified cell disclosed herein has been edited such that the modified cell does not express endogenous level of NR4A2 and NR4A3 (e.g., expresses no NR4A2 and NR4A3 or expresses reduced level of NR4A2 and NR4A3 as compared to corresponding nonmodified cell), but because the NR4A1 has not been knocked down, the modified cell exhibits endogenous expression of the NR4A1 (gene and / or protein).

[0110] As used herein the term "reduced levels," "lower levels," "reduced expression levels," "lower levels," "decreased levels," or "decreased in levels" (or variants thereof) refers both to reduction in physical levels (e.g., less gene sequence due to edition from the genome, or less protein due a decrease in protein expression) and to reduction in function. For example, a reduction in level of NR4A3 gene can refer to a decrease in gene function, e.g., due to the introduction of a mutation introducing a stop codon or a frame shift, to an epigenetic modification that would alter transcription, or to a mutation or other change on a promoter gene or another gene that regulates NR4A3 expression. In some aspects, a reduction in level of NR4A3 gene in a modified cell refers to a decrease in the amount (e.g., concentration) of genomic DNA, pre-mRNA, and / or mRNA that is capable of encoding a functional NR4A3 protein, e.g., wild type NR4A3 protein, compared to a reference cell. Similarly, a reduction in NR4A3 protein can refer to changes resulting in the expression of a functional NR4A3 protein, e.g., wild type NR4A3 protein, including but not limited to changes (e.g., mutations or post-translational modifications) that cause a loss of function (partial or complete), or to the activity of molecules that bind to functional sites of NR4A3 altering, e.g., its interaction with other cell signaling partners.[OHl] 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 are described herein (e.g., an immune cell or a population of immune cells modified to express a reduced level of one or more members of the NR4A family, and cultured as described herein). Exemplary routes of administration include intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrastema, oral, rectal, topical, epidermal, mucosal, intranasal, vaginal, rectal, sublingual administration, and combinations thereof. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0112] The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intratumoral, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, intratracheal, pulmonary, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraventricular, intravitreal, epidural, and intrastemal injection and infusion, as well as in vivo electroporation.

[0113] Alternatively, a therapeutic agent described herein (e.g., an immune cell contacted with PCS in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and further modified and cultured as described herein) can be administered via a non-parenteral route, such as a topical, epidermal, or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0114] 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 binding protein) introduction of one or more endogenous genes that improve T cell function, introduction of one or more synthetic genes that improve immune cell, e.g., T cell, function, or any combination thereof. As further described elsewhere in the present disclosure, in some aspects, a cell can be engineered or modified with a transcription activator (e.g., CRISPR / Cas system-based transcription activator), wherein the transcription activator is capable of inducing and / or increasing the endogenous expression of a protein of interest. In some aspects, a cell (e.g., T cells and / or NK cells) can be engineered or modified with a gene editing tool (e.g., gRNAs provided herein) to reduce the expression of one or more of genes and / or proteins associated with impaired immune cell function (e.g., the NR4A family (e.g., NR4A1, NR4A2, and / or NR4 A3)).

[0115] 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.

[0116] 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.

[0117] 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 are also referred to as "liquid tumors." Liquid tumor cancers include, but are not limited to, leukemias, myelomas, and lymphomas, as well as other hematological malignancies.

[0118] A "solid tumor," as used herein, refers to an abnormal mass of tissue. Solid tumors can be benign or malignant. Non-limiting 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 can provide a supporting microenvironment.

[0119] 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, nonsmall 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, myxoid / round cell liposarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, 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, or telangiectaltic sarcoma. In some aspects, the cancer is selected from acra- lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma, juvenile 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, bronchiolarcarcinoma, 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, mucoepidernoid 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 someaspects, the cancer is selected from metastatic melanoma, non-small cell lung cancer, myeloma, esophageal cancer, synovial sarcoma, gastric cancer, breast cancer, hepatocellular cancer, head and neck cancer, ovarian cancer, prostate cancer, bladder cancer, or any combination thereof.

[0120] 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. As used herein, the terms "T cell" and "T lymphocytes" are interchangeable and refer to any lymphocytes produced or processed by the thymus gland. In some aspects, a T cell is a CD4+T cell. In some aspects, a T cell is a CD8+T cell. In some aspects, a T cell is a NKT cell.

[0121] As used herein, the term "anti -turn or immune response" refers to an immune response against a tumor antigen.

[0122] 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., contacted with PCS in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and further modified and cultured using the methods provided herein, as described herein to control tumor growth.

[0123] The term "therapeutically effective amount" or "therapeutically effective dosage" refers to an amount of an agent (e.g., an immune cell contacted with PCS in a medium,wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and further modified 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.

[0124] The effective amount of the composition (e.g., immune cells as described herein, e.g., contacted with PCS in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and further modified and 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 (i.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.

[0125] In some aspects, a "therapeutically effective amount" is the amount of a composition disclosed herein (e.g., an immune cell contacted with PCS in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and further modified and 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.

[0126] 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 modified and 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 modified and cultured as described herein).

[0127] 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 "an intracellular 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., 4-1BB (i.e., CD137), CD27, and / or CD28.

[0128] 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-bindingdomain 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 aminoterminus (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.

[0129] 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.

[0130] 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 CD3 which 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.

[0131] As used herein, an "engineered TCR" or "engineered T-cell receptor" refers to a T- cell receptor (TCR) engineered to specifically bind with a desired affinity to a major histocompatibility complex (MHC) / peptide target antigen that is selected, cloned, and / or subsequently introduced into a population of immune cells, e.g., T cells and / or NK cells.

[0132] A "TCR mimic" or a "TCRm" refers to a type of engineered chimeric TCR comprising an antigen binding domain (e.g., derived from an 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. The TCR mimic further comprises a T cell receptor module (TCRM) capable of recruiting at least one TCR-associated signaling molecule. Exemplary TCR mimics are described for example in U.S. Patent No. 10,822,413, which is incorporated herein by reference in its entirety.

[0133] 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 sequences refer to single-stranded DNA (ssDNA) or singlestranded 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 double-stranded 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 double-stranded DNAmolecules, 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 (z.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 semisynthetic 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 ("monocistronic"). In some aspects, a polynucleotide of the present disclosure is polycistronic (z.e., comprises two or more cistrons). In some aspects, each of the cistrons of a polycistronic polynucleotide can encode for a protein disclosed herein. In some aspects, each of the cistrons can be translated independently of one another.

[0134] 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.

[0135] As used herein, the term "fragment" of a polypeptide 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 does not necessarily 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.

[0136] As used herein, the term "functional fragment" refers to a polypeptide fragment that retains polypeptide function. Accordingly, in some aspects, a functional fragment of an Ig hinge, retains the ability to position an antigen-binding domain (e.g., an scFv) in a chimeric binding protein at a distance from a target epitope (e.g., a tumor antigen) such that theantigen-binding domain e.g., an scFv) can effectively interact with the target epitope e.g., a tumor antigen). Non-limiting examples of such activity are further described elsewhere in the present disclosure.

[0137] 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. The polypeptides encoded by the polynucleotides disclosed herein can be recombinantly produced using methods known in the art. In some aspects, the polypeptides encoded by the polynucleotides of the present disclosure are produced by cells, e.g., T cells, following transfection with at least one polynucleotide or vector encoding the polypeptides described here.

[0138] As used herein, a "coding region," "coding sequence," or "translatable sequence" is a portion of polynucleotide which consists of codons translatable into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5' terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3' terminus, encoding the carboxyl terminus of the resulting polypeptide.

[0139] The terms "complementary" and "complementarity" refer to two or more oligomers (i.e., each comprising a nucleobase sequence), or between an oligomer and a target gene, that are related with one another by Watson-Crick base-pairing rules. For example, the nucleobase sequence "T-G-A (5' to 3')," is complementary to the nucleobase sequence "A- C-T (3' to 5')." Complementarity can be "partial," in which less than all of the nucleobases of a given nucleobase sequence are matched to the other nucleobase sequence according to base pairing rules. For example, in some aspects, complementarity between a given nucleobase sequence and the other nucleobase sequence can be about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. Accordingly, in some aspects, the term "complementary" refers to at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%match or complementarity to a target nucleic acid sequence e.g., a gene associated with impaired immune cell function, e.g., NR4A1, NR4A2, and / or NR4A3). Or, there can be "complete" or "perfect" (100%) complementarity between a given nucleobase sequence and the other nucleobase sequence to continue the example. In some aspects, the degree of complementarity between nucleobase sequences has significant effects on the efficiency and strength of hybridization between the sequences.

[0140] The term "expression" as used herein refers to a process by which a polynucleotide produces a gene product, for example, a ligand-binding protein. 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.

[0141] As used herein, the term "identity" refers to the overall monomer conservation between polymeric molecules, e.g., between polynucleotide molecules. The term "identical" without any additional qualifiers, e.g., polynucleotide A is identical to polynucleotide B, implies the polynucleotide sequences are 100% identical (100% sequence identity). Describing two sequences as, e.g., "70% identical," is equivalent to describing them as having, e.g., "70% sequence identity." A "reference nucleotide sequence," when used herein as a comparison to a nucleotide sequence of the disclosure, refers to a polynucleotide sequence essentially identical to the nucleotide sequence of the disclosure except that sequence is not optimized. For example, in some aspects, the reference nucleotide sequence comprises the wild-type JUN nucleic acid sequence set forth in SEQ ID NO: 11.

[0142] Calculation of the percent identity of two polypeptide or polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second polypeptide or polynucleotide sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In some aspects, the length of a sequence aligned for comparison purposes is at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or about 100% of the length of the reference sequence. The amino acids at corresponding amino acid positions, or bases in the case of polynucleotides, are then compared.

[0143] When a position in the first sequence is occupied by the same amino acid or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.

[0144] Suitable software programs that can be used to align different sequences (e.g., polynucleotide sequences) are available from various sources. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of programs available from the U.S. government's National Center for Biotechnology Information BLAST web site (blast.ncbi.nlm.nih.gov). B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, e.g., Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at worldwideweb.ebi.ac.uk / Tools / psa.

[0145] Sequence alignments can be conducted using methods known in the art such as MAFFT, Clustal (ClustalW, Clustal X or Clustal Omega), MUSCLE, etc.

[0146] Different regions within a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.

[0147] In some aspects, the percentage identity (%ID) or of a first amino acid sequence (or nucleic acid sequence) to a second amino acid sequence (or nucleic acid sequence) is calculated as %ID = 100 x (Y / Z), where Y is the number of amino acid residues (or nucleobases) scored as identical matches in the alignment of the first and second sequences(as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0148] One skilled in the art will appreciate that the generation of a sequence alignment for the calculation of a percent sequence identity is not limited to binary sequence-sequence comparisons exclusively driven by primary sequence data. It will also be appreciated that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g., crystallographic protein structures), functional data (e.g., location of mutations), or phylogenetic data. A suitable program that integrates heterogeneous data to generate a multiple sequence alignment is T-Coffee, available at worldwidewebtcoffee.org, and alternatively available, e.g., from the EBI. It will also be appreciated that the final alignment used to calculate percent sequence identity can be curated either automatically or manually.

[0149] 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, including partially removing (e.g., a fraction), a composition of the present disclosure (e.g., an immune cell contacted with a PCS in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells and further modified to express a reduced level of a gene and / or protein associated with impaired immune cell function (e.g., NR4A1, NR4A2, and / or NR4A3) from a sample containing contaminants.

[0150] In some aspects, an isolated composition has no detectable undesired activity or, alternatively, the level or amount of the undesired activity is at or below an acceptable level or amount. In some aspects, an isolated composition has an amount and / or concentration of desired composition of the present disclosure, at or above an acceptable amount and / or concentration and / or activity. In some aspects, the isolated composition is enriched as compared to the starting material from which the composition is obtained. This enrichment can be by 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 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, at least about 99.9%, at least about 99.99%, at least about 99.999%, at least about 99.9999%, or greater than 99.9999% as compared to the starting material.

[0151] In some aspects, isolated preparations are substantially free of residual biological products. In some aspects, the isolated preparations are 100% free, at least about 99% free, at least about 98% free, at least about 97% free, at least about 96% free, at least about 95% free, at least about 94% free, at least about 93% free, at least about 92% free, at least about 91% free, or at least about 90% free of any contaminating biological matter. Residual biological products can include abiotic materials (including chemicals) or unwanted nucleic acids, proteins, lipids, or metabolites.

[0152] The term "linked" as used herein refers to a first amino acid sequence or polynucleotide sequence covalently or non-covalently joined to a second amino acid sequence or polynucleotide sequence, respectively. The first amino acid or polynucleotide sequence can be directly joined or juxtaposed to the second amino acid or polynucleotide sequence or alternatively an intervening sequence can covalently join the first sequence to the second sequence. The term "linked" means not only a fusion of a first polynucleotide sequence to a second polynucleotide sequence at the 5'-end or the 3'-end, but also includes insertion of the whole first polynucleotide sequence (or the second polynucleotide sequence) into any two nucleotides in the second polynucleotide sequence (or the first polynucleotide sequence, respectively). The first polynucleotide sequence can be linked to a second polynucleotide sequence by a phosphodiester bond or a linker. The linker can be, e.g., a polynucleotide.

[0153] Treatment" or "therapy" (including any grammatical derivatives thereof) of a subject 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 terms refers to inducing an immune response in a subject against an antigen.

[0154] 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 a particular disease, disorder, and / or condition; partially or completely delaying onset of one or more symptoms, features, or manifestations of a particular disease, disorder, and / orcondition; 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.

[0155] As used herein the term "therapeutically effective amount" is the amount of reagent or pharmaceutical compound comprising a composition disclosed herein (e.g., modified immune cell described herein) that is sufficient to a produce a desired therapeutic effect, pharmacologic and / or physiologic effect on a subject in need thereof.

[0156] A therapeutically effective amount can be a "prophylactically effective amount" as prophylaxis can be considered therapy. As used herein, "prophylactic" refers to a therapeutic or course of action used to prevent the onset of a disease or condition, or to prevent or delay a symptom associated with a disease or condition. As used herein, a "prophylaxis" refers to a measure taken to maintain health and prevent the onset of a disease or condition, or to prevent or delay a symptom associated with a disease or condition.

[0157] As used herein, the term "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters." Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as "cell-specific promoters" or "tissue-specific promoters." Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as "developmentally-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as "inducible promoters" or "regulatable promoters." It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.

[0158] As used herein, the terms "ug" and "uM" are used interchangeably with "pg" and "pM," respectively.

[0159] Various aspects of the disclosure are described in further detail in the following subsections.Methods of Modifying and Culturing

[0160] Some aspects of the present disclosure provide a method of preparing immune cells (or a population of immune cells (e.g., human immune cells)) for immunotherapy comprising contacting immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells. Some aspects of the present disclosure provide a method of activating immune cells during ex vivo or in vitro culture comprising contacting immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells.

[0161] As further described and demonstrated herein, contacting immune cells in a medium with a specific amount of PCS described herein (e.g., less than about 160 pg per million cells, e.g., less than 120 pg per million cells) can enhance certain properties of the immune cells as compared to reference immune cells. In some aspects, where the immune cells are contacted with PCS at an amount less than about 160 pg per million cells, the reference immune cells comprise corresponding immune cells that were contacted with the PCS at an amount of 160 pg per million cells or greater. In some aspects, where the immune cells are contacted with PCS at an amount less than 120 pg per million cells, the reference immune cells comprise corresponding immune cells that were contacted with the PCS at an amount of 120 pg per million cells or greater. In some aspects, the reference immune cells are corresponding immune cells that are not contacted with the PCS. Non-limiting examples of improved properties of immune cells include: (a) increased viability of the immune cells during ex vivo or in vitro culture, (b) decreased production of background cytokines during ex vivo or in vitro culture, or (c) both (a) and (b). Additional examples of improved properties are provided elsewhere in the present disclosure.

[0162] Some aspects of the present disclosure relate to methods of increasing the viability of immune cells during ex vivo or in vitro culture comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS ispresent in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells. In some aspects, after the contacting, the viability of the immune cells is increased by at least about 1%, at least about 2 %, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50%, as compared to the reference immune cells (e.g., corresponding immune cells that were contacted with the PCS at an amount of 120 pg per million cells or greater).

[0163] Many immune cell-based therapies are associated with certain toxicities. For instance, there have been increased incidences of the occurrence of excessive cytokine production (e.g., cytokine release syndrome) with T-cell engaging therapies (e.g., bispecific T-cell engaging (BiTE) single-chain antibodies) and chimeric antigen receptors. See Shimabukuro-Vornhagen el al...J Immunolher Cancer 6:56 (2018). Accordingly, the ability to produce immune cell-based therapies that are associated with reduced background cytokine production could be useful. As used herein, the term "background cytokine" refers to a cytokine that is produced by a cell in an antigen-independent manner. Non-limiting examples of such cytokines include IFN-y, TNF-a, IL-2, or combinations thereof. As will be apparent to those skilled in the arts, cytokines can have many positive effects on an immune response (e.g., help in the induction of an immune response). However, excessive or inappropriate cytokine production (particularly in the absence of any specific antigen) could lead to excessive inflammation, which can result in the destruction of healthy tissues.

[0164] Accordingly, some aspects of the present disclosure relate to a method of decreasing an amount of background cytokine produced by immune cells during ex vivo or in vitro culture comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells. In some aspects, after the contacting, the amount of background cytokine produced by the immune cells is decreased 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 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 about90%, at least about 95%, or about 100%, as compared to the reference immune cells e.g., corresponding immune cells that were contacted with the PCS at an amount of 120 pg per million cells or greater).

[0165] Contacting immune cells with PCS during ex vivo or in vitro can aid in the activation of the immune cells, such that they are more suitable for immune cell-based therapies (e.g., CAR-based therapies). See, e.g., WO2018 / 013797, which is incorporated herein by reference in its entirety. As further described elsewhere in the present disclosure, in some aspects, a PCS comprises one or more surface cues, which is capable of binding to a receptor expressed on immune cells, and thereby, aid in the activation of the immune cells. Non-limiting example of such a surface cue is a stimulatory molecule, such as an anti-CD3 antibody which binds to CD3 molecules expressed on immune cells. To reduce excessive activation of the immune cells, it would be useful to wash away or remove any bound surface cues after the immune cells have been sufficiently activated with the PCS. As demonstrated herein, contacting immune cells with PCS at the specific amounts described herein allows effective activation of the cultured immune cells while decreasing the amount of residual surface cues (e.g., stimulatory antibodies) bound to the immune cells after ex vivo or in vitro culture to avoid potentially deleterious levels of background cytokine production. As used herein, the term "residual surface cues" refers to surface cues that remain bound to the immune cells have after the PCS has been removed from culture.

[0166] Accordingly, some aspects of the present disclosure relate to a method of decreasing an amount of residual surface cues bound to immune cells after ex vivo or in vitro culture comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells. In some aspects, after the contacting, the amount of residual surface cues bound to the immune cells is decreased 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 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%, or about 100%, as compared to the reference immune cells (e.g., corresponding immune cells that were contacted with the PCS at an amount of 120 pg per million cells or greater).

[0167] As further described herein, in some aspects, the immune cells useful for the present disclosure (e.g., contacted with a PCS in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) are edited to exhibit a reduced expression level of one or more genes and / or proteins that are associated with impaired immune cell function. For instance, in some aspects, the immune cells useful for the present disclosure (e.g. , contacted with a PCS in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) are edited to exhibit a reduced expression level of a nuclear receptor subfamily 4A (NR4A) family member as compared to corresponding immune cells which have not been edited.

[0168] Some aspects of the present disclosure are related to a method of preparing immune cells (or a population of immune cells (e.g., human immune cells)) for immunotherapy comprising: (a) contacting immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and (b) editing the immune cells to exhibit a reduced expression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4A (NR4A) family member) as compared to corresponding immune cells which have not been edited. Some aspects of the present disclosure provide a method of activating immune cells during ex vivo or in vitro culture comprising: (a) contacting immune cells with a programmable cellsignaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and (b) editing the immune cells to exhibit a reduced expression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4 A (NR4A) family member) as compared to corresponding immune cells which have not been edited. Some aspects of the present disclosure provide a method of increasing the viability of immune cells during ex vivo or in vitro culture comprising: (a) contacting immune cells with programmable cell-signaling scaffolds (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and (b) editing the immune cells to exhibit a reduced expression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4A (NR4A) family member) as compared to corresponding immune cells which have not been edited. Some aspects of the present disclosure relate to a methodof decreasing an amount of background cytokine produced by immune cells during ex vivo or in vitro culture comprising: (a) contacting immune cells with a programmable cellsignaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and (b) editing the immune cells to exhibit a reduced expression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4 A (NR4A) family member) as compared to corresponding immune cells which have not been edited. Some aspects of the present disclosure relate to a method of decreasing an amount of residual surface cues bound to immune cells after ex vivo or in vitro culture comprising: (a) contacting immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and (b) editing the immune cells to exhibit a reduced expression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4A (NR4A) family member) as compared to corresponding immune cells which have not been edited.

[0169] In some aspects, the contacting of the immune cells with PCS and the editing the immune cells to exhibit a reduced expression level of the gene and / or protein associated with impaired immune cell function (e.g., NR4A family member) can occur non- concurrently. In some aspects, the editing occurs prior to the contacting. Where the editing occurs prior to the contacting, the immune cells express a reduced expression level of the gene and / or protein associated with impaired immune cell function (e.g., NR4A family member) when contacted with the PCS. In some aspects, the editing occurs at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about seven days, at least about eight days, at least about nine days, or at least about 10 days prior to the contacting. In some aspects, the editing occurs about one day prior to the contacting. In some aspects, the editing occurs after the contacting. For example, in some aspects, the editing occurs at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about seven days, at least about eight days, at least about nine days, or at least about 10 days after the contacting. In some aspects, the editing occurs about two days after the contacting of the immune cells with the PCS. In some aspects, the contacting can occur both before the editing and after the editing. In some aspects, the editing and the contacting occur concurrently.

[0170] In some aspects, immune cells described herein (e.g., contacted with a PCS in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) are transduced (a) to express a ligand-binding protein, (b) to exhibit an increase in an expression level of a gene and / or protein associated with improved immune cell function (e.g. , c-Jun polypeptide), or (c) both (a) and (b).

[0171] Accordingly, some aspects of the present disclosure are related to a method of preparing immune cells (or a population of immune cells (e.g., human immune cells)) for immunotherapy comprising: (a) contacting immune cells with a programmable cellsignaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and (b) transducing the immune cells (i) to express a ligand-binding protein, (ii) to exhibit an increase in an expression level of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), or (iii) both (i) and (ii). Some aspects of the present disclosure provide a method of activating immune cells during ex vivo or in vitro culture comprising: (a) contacting immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and (b) transducing the immune cells (i) to express a ligand-binding protein, (ii) to exhibit an increase in an expression level of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), or (iii) both (i) and (ii). Some aspects of the present disclosure provide a method of increasing the viability of immune cells during ex vivo or in vitro culture comprising: (a) contacting immune cells with programmable cell-signaling scaffolds (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and (b) transducing the immune cells (i) to express a ligand-binding protein, (ii) to exhibit an increase in an expression level of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), or (iii) both (i) and (ii). Some aspects of the present disclosure relate to a method of decreasing an amount of background cytokine produced by immune cells during ex vivo or in vitro culture comprising: (a) contacting immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and (b) transducing the immune cells (i) to express a ligand-binding protein, (ii) to exhibit an increase in an expression level of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), or (iii) both (i) and (ii). Some aspects of the present disclosure relate to a method of decreasing an amount of residual surface cues bound to immune cells after ex vivo or in vitro culture comprising: (a) contacting immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and (b) transducing the immune cells (i) to express a ligand-binding protein, (ii) to exhibit an increase in an expression level of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), or (iii) both (i) and (ii).

[0172] In some aspects, the contacting of the immune cells with PCS and the transducing the immune cells can occur non-concurrently. In some aspects, the contacting and the transducing occur concurrently (e.g., within a single day).

[0173] As is apparent from the present disclosure, in some aspects, immune cells (e.g., T cells and / or NK cells) are: (i) contacted with PCS in a medium which comprises the PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells), (ii) transduced with a ligand-binding construct (such that the immune cells express the encoded ligand-binding protein), and (iii) edited to exhibit reduced expression of a gene and / or protein associated with impaired immune cell function (e.g., NR4A family member). In some aspects, immune cells (e.g, T cells and / or NK cells) are: (i) contacted with PCS in a medium which comprises the PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells), (ii) transduced to exhibit an increased expression of a gene and / or protein associated with improved immune cell function (e.g, c-Jun polypeptide), and (iii) edited to exhibit reduced expression of a NR4A family member. In some aspects, immune cells (e.g., T cells and / or NK cells) are: (i) contacted with PCS in a medium which comprises the PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells), (ii) transduced to both express a ligand-binding protein and exhibit an increased expression of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), and (iii) edited to exhibit reduced expression of a gene and / or protein associated with impaired immune cell function (e.g., NR4A family member). As further described herein, for such aspects, the contacting, editing, and transducing can occur concurrently (e.g., within a single day). Insome aspects, the contacting, editing, and / or transducing occur sequentially (e.g., over a course of two or more days).

[0174] In some aspects, the contacting with PCS, transducing to express a ligand-binding protein, and editing to exhibit reduced expression of a gene and / or protein associated with impaired immune cell function (e.g., NR4A family member) all occur concurrently (e.g., within a single day). As further described herein, in some aspects, immune cells can be further transduced to exhibit an increased expression level of a gene and / or protein that is associated with improved immune cell function (e.g., c-Jun polypeptide). For example, in some aspects, the ligand-binding construct can further comprise a nucleotide sequence encoding the protein associated with improved immune cell function (e.g., c-Jun polypeptide). In some aspects, each of the contacting with PCS, transducing with a ligandbinding construct, editing to reduce expression of a gene and / or protein associated with impaired immune cell function (e.g., NR4A family member), and transducing the immune cells to increase expression of a gene and / or protein associated with improved immune cell function (e.g., c-Jun) can all occur concurrently.

[0175] In some aspects, two of the above steps (i.e., contacting, transducing, and editing) occur concurrently (e.g., within a single day), while the third step occurs later on a separate day. For instance, in some aspects, the contacting with PCS and transducing (i.e., to express a ligand-binding protein and / or to increase the expression of a gene and / or protein associated with improved immune cell function, e.g., c-Jun) occur concurrently, and the editing occurs after the activating and the transducing. In some aspects, the editing occurs at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about seven days, at least about eight days, at least about nine days, or at least about 10 days after the activating and the transduce. In some aspects, the editing occurs about two days later.

[0176] In some aspects, the editing (i.e., to reduce the expression of one or more genes associated with impaired immune cell function, e.g., NR4A) occurs prior to the cells being contacted with PCS. In some aspects, the editing occurs prior to the cells being transduced (e.g., to express a ligand-binding protein and / or to increase the expression of a gene and / or protein associated with improved immune cell function, e.g., c-Jun). In some aspects, the editing occurs prior to both the contacting and the transducing. For example, in some aspects, the editing occurs at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at leastabout seven days, at least about eight days, at least about nine days, or at least about 10 days prior to the cells being contacted with PCS. In some aspects, the editing occurs at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about seven days, at least about eight days, at least about nine days, or at least about 10 days prior to the immune cells being transduced (e.g., to express a ligand-binding protein and / or to exhibit increased expression of a gene and / or protein that is associated with improved immune cell function, e.g., c-Jun). In some aspects, the editing occurs at least about one day, at least about two days, at least about three days, at least about four days, at least about five days, at least about six days, at least about seven days, at least about eight days, at least about nine days, or at least about 10 days prior to both the contacting and the transducing. In some aspects, the editing occurs about one day prior to the contacting and the transducing.

[0177] As demonstrated herein, in some aspects, the contacting and the editing can occur concurrently. As used herein, the term "concurrently" refers to two or more events (e.g., contacting with PCS and editing to exhibit reduced expression of the gene and / or protein associated with impaired immune cell function, e.g., NR4A family member) occurring at the same time or within a single day (i.e., 24-hour period). For instance, as is apparent from the present disclosure, any of the individual steps described herein (e.g., contacting with PCS, transducing (e.g., with a ligand-binding construct, construct comprising a nucleic acid encoding a protein associated with improved immune cell function (e.g., c-Jun), or both), and / or editing with a gene editing tool targeting a gene associated with impaired immune cell function, e.g., NR4A family member) can occur sequentially (e.g., cells are first contacted with PCS and then subsequently edited to exhibit reduced expression of a gene and / or protein associated with impaired immune cell function, e.g., NR4A family member) and yet still be considered to occur concurrently, where the individual steps all occur within a single day. Accordingly, in some aspects, methods can be performed sequentially and yet be considered to occur concurrently. As used herein, the term "non-concurrently" refers to when two or more events (e.g., contacting with PCS and editing to exhibit reduced expression of the gene and / or protein associated with impaired immune cell function, e.g., NR4A family member) do not occur within a single day (e.g., cells are first contactedwith PCS and then two days later, the cells are edited to exhibit reduced expression of a gene and / or protein associated with impaired immune cell function, e.g., NR4A family member).

[0178] Where the individual steps of the methods provided herein (e.g., contacting with PCS, transducing with a ligand-binding and / or construct comprising a nucleic acid encoding a protein associated with improved immune cellf unction (e.g., c-Jun), and / or editing with a gene editing tool targeting a gene associated with impaired immune cell function (e.g., NR4A family member)) occur sequentially, in some aspects, the individual steps occur continuously, such that there can be some overlap in the individual steps. For example, in some aspects, after the immune cells are contacted with PCS in a medium, wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, the immune cells are immediately transduced such that the transducing occurs in the same medium as the contacting. In some aspects, where the individual steps occur continuously (e.g., contacting and transducing), one or more additional components can be added to the medium during the later steps, such that the medium used during the earlier steps and the medium used during the later steps differ. For example, in some aspects, immune cells are contacted with PCS in a medium that lacks any cytokine (and wherein the PCS is present in the medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells), and then immediately transduced with a ligand-binding and / or construct comprising a nucleic acid encoding a protein associated with improved immune cell function (e.g., c-Jun) and / or edited with a gene editing tool targeting a gene associated with impaired immune cell function (e.g., NR4A family member) in the same medium, except that exogenous cytokine (e.g., IL-2, IL-7, and / or IL- 15) is added to the medium during the editing and / or transducing.

[0179] In some aspects, where the individual steps of the methods occur sequentially, two or more of the steps are performed non-continuously (i.e., two or more of the steps are separated by an intervening step). For example, in some aspects, after the immune cells are contacted with PCS in a medium described herein, the immune cells are not immediately edited. Instead, the immune cells are cultured in a separate medium (i.e., different from the medium used to contact the cells with PCS) and then subsequently edited, such that the contacting and the transducing are performed in different media.

[0180] To further illustrate, in some aspects, immune cells (e.g., T cells and / or NK cells) are cultured in a medium comprising a programmable cell-signaling scaffold (PCS) (e.g., at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) and a gene editing tool that specifically targets a gene associated with impairedimmune cell function (e.g., a NR4A family member). Where immune cells are cultured in such a medium, in some aspects, the immune cells are contacted with both the PCS and the gene editing tool. In some aspects, the immune cells can be first contacted with PCS and then edited to exhibit reduced expression level of the gene and / or protein associated with impaired immune cell function (e.g., NR4A family member) with the proviso that both the contacting and the editing occur within a single day.

[0181] As further described herein, in some aspects, the medium can additionally comprises an agent that is capable of increasing the expression of a gene and / or protein that is associated with improved immune cell function (e.g., c-Jun protein) in the immune cells (e.g., nucleotide sequence encoding a c-Jun protein and / or a transcriptional activator that is capable of increasing the expression level of an endogenous c-Jun protein in the immune cells). Accordingly, in some aspects, immune cells (e.g., T cells and / or NK cells) are cultured in a medium comprising: (a) a programmable cell-signaling scaffold (PCS) at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (b) a gene editing tool that specifically targets a gene associated with impaired immune cell function (e.g., NR4A family member), and (c) an agent that is capable of increasing the expression of a gene and / or protein associated with improved immune cell function (e.g., c-Jun protein) in the immune cells. Where immune cells are cultured in such a medium, in some aspects, the immune cells are contacted with the PCS, the gene editing tool, and the agent. In some aspects, the immune cells are first contacted with PCS and then subsequently edited to exhibit reduced expression level of a gene and / or protein associated with impaired immune cell function (e.g., NR4A family member) and transduced to exhibit an increased expression of a gene and / or protein associated with improved immune cell function (e.g., c-Jun protein), with the proviso that the contacting and the editing all occur within a single day.

[0182] As described and demonstrated herein, the culturing and modifying (e.g., editing and / or transducing) methods provided herein can be useful in enhancing some aspects of immune cells (e.g., T cells and / or NK cells). In some aspects, the one or more properties of immune cells that can be enhanced using the methods provided herein include: (a) an ability to produce a cytokine in response to an antigen, (b) an ability to down-regulate an exhaustion marker, (c) an ability to proliferate, (d) an ability to kill tumor cells, (e) increased viability during ex vivo or in vitro culture, and (f) any combination thereof. In some aspects, the cytokine comprises an IFN-y, TNF-a, IL-2, or combinations thereof.

[0183] In some aspects, immune cells useful for the present disclosure are CD3+, CD45RO' , CCR7+, CD45RA+, CD62L+, CD27+, CD28+, or TCF7+, or any combination thereof, following the culturing and the modifying. In some aspects, the immune cells comprise T cells, B cells, regulatory T cells (Treg), tumor infiltrating lymphocytes (TIL), natural killer (NK) cells, natural killer T (NKT) cells, or any combination thereof. In some aspects, the immune cells have been engineered in vitro or ex vivo.

[0184] Additional disclosure relating to the PCS, culture medium, gene editing tools targeting a gene associated with impaired immune cell function (e.g., NR4A family member), and agents that are capable of increasing the expression of a gene and / or protein associated with improved immune cell function (e.g., c-Jun protein) that can be used with the present disclosure are provided below.Culture Media

[0185] Some aspects of the present disclosure are directed to methods of contacting immune cells (e.g., T cells and / or NK cells) with PCS in a culture condition (e.g., media), wherein the PCS is present in the culture medium at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells.

[0186] In some aspects, the PCS is present in the medium at an amount of less than about 160 pg per millions cells. In some aspects, the PCS is present in the medium at an amount of less than about 160 pg per million cells and about 120 pg per million cells. In some aspects, the PCS is present in the medium at an amount of between about 150 pg per million cells and about 120 pg per million cells. In some aspects, the PCS is present in the medium at an amount of between about 140 pg per million cells and about 120 pg per million cells. In some aspects, the PCS is present in the medium at an amount of between about 130 pg per million cells and about 120 pg per million cells. In some aspects, the PCS is present in the medium at an amount of less than about 150 pg per million cells and about 130 pg per million cells. In some aspects, the PCS is present in the medium at an amount of less than about 140 pg per million cells and about 130 pg per million cells. In some aspects, the PCS is present in the medium at an amount of about 150 pg per million cells, about 140 pg per million cells, about 130 pg per million cells, or about 120 pg per million cells.

[0187] In some aspects, the PCS is present in the medium at an amount of less than 120 pg per millions cells. In some aspects, the PCS is present in the medium at an amount of between about 40 pg per million cells and less than 120 pg per million cells. In someaspects, the PCS is present in the medium at an amount of about 45 pg per million cells to less than 120 pg per million cells, about 50 pg per million cells to less than 120 pg per million cells, about 55 pg per million cells to less than 120 pg per million cells, about 60 pg per million cells to less than 120 pg per million cells, about 65 pg per million cells to less than 120 pg per million cells, about 70 pg per million cells to less than 120 pg per million cells, about 75 pg per million cells to less than 120 pg per million cells, about 80 pg per million cells to less than 120 pg per million cells, about 85 pg per million cells to less than 120 pg per million cells, about 90 pg per million cells to less than 120 pg per million cells, about 95 pg per million cells to less than 120 pg per million cells, about 100 pg per million cells to less than 120 pg per million cells, about 105 pg per million cells to less than 120 pg per million cells, about 110 pg per million cells to less than 120 pg per million cells, or about 115 pg per million cells to less than 120 pg per million cells. In some aspects, the PCS is present in the medium at an amount of about 40 pg to about 115 pg, about 40 pg per million cells to about 110 pg per million cells, about 40 pg per million cells to about 105 pg per million cells, about 40 pg per million cells to about 100 pg per million cells, about 40 pg per million cells to about 95 pg per million cells, about 40 pg per million cells to about 90 pg per million cells, about 40 pg per million cells to about 85 pg per million cells, about 40 pg per million cells to about 80 pg per million cells, about 40 pg per million cells to about 75 pg per million cells, about 40 pg per million cells to about 70 pg per million cells, about 40 pg per million cells to about 65 pg per million cells, about 40 pg per million cells to about 60 pg per million cells, about 40 pg per million cells to about 55 pg per million cells, about 40 pg per million cells to about 50 pg per million cells, or about 40 pg per million cells to about 45 pg per million cells. In some aspects, the amount of PCS present in the medium is about 45 pg per million cells to about 115 pg per million cells, about 50 pg per million cells to about 110 pg per million cells, about 55 pg per million cells to about 105 pg per million cells, about 60 pg per million cells to about 100 pg per million cells, about 65 pg per million cells to about 95 pg per million cells, about 70 pg per million cells to about 90 pg per million cells, or about 75 pg per million cells to about 85 pg per million cells. In some aspects, the amount of PCS present in the medium is about 40 pg per million cells, about 41 pg per million cells, about 42 pg per million cells, about 43 pg per million cells, about 44 pg per million cells, about 45 pg per million cells, about 46 pg per million cells, about 47 pg per million cells, about 48 pg per million cells, about 49 pg per million cells, about 50 pg per million cells, about 51 pg permillion cells, about 52 pg per million cells, about 53 pg per million cells, about 54 pg per million cells, about 55 pg per million cells, about 56 pg per million cells, about 57 pg per million cells, about 58 pg per million cells, about 59 pg per million cells, about 60 pg per million cells, about 61 pg per million cells, about 62 pg per million cells, about 63 pg per million cells, about 64 pg per million cells, about 65 pg per million cells, about 66 pg per million cells, about 67 pg per million cells, about 68 pg per million cells, about 69 pg per million cells, about 70 pg per million cells, about 71 pg per million cells, about 72 pg per million cells, about 73 pg per million cells, about 74 pg per million cells, about 75 pg per million cells, about 76 pg per million cells, about 77 pg per million cells, about 78 pg per million cells, about 79 pg per million cells, about 80 pg per million cells, about 81 pg per million cells, about 82 pg per million cells, about 83 pg per million cells, about 84 pg per million cells, about 85 pg per million cells, about 86 pg per million cells, about 87 pg per million cells, about 88 pg per million cells, about 89 pg per million cells, about 90 pg per million cells, about 91 pg per million cells, about 92 pg per million cells, about 93 pg per million cells, about 94 pg per million cells, about 95 pg per million cells, about 96 pg per million cells, about 97 pg per million cells, about 98 pg per million cells, about 99 pg per million cells, about 100 pg per million cells, about 101 pg per million cells, about 102 pg per million cells, about 103 pg per million cells, about 104 pg per million cells, about 105 pg per million cells, about 106 pg per million cells, about 107 pg per million cells, about 108 pg per million cells, about 109 pg per million cells, about 110 pg per million cells, about H l pg per million cells, about 112 pg per million cells, about 113 pg per million cells, about 114 pg per million cells, or about 115 pg per million cells.

[0188] In some aspects, a medium useful for the present disclosure (e.g., used in contacting immune cells with PCS) comprises one or more additional components that can further aid in regulating the differentiation of the immune cells, thereby affecting or improving their use in cell therapy, e.g., adoptive cell therapy. Non-limiting examples of such additional components are described further below.Potassium

[0189] Some aspects of the disclosure are directed to methods of culturing in a medium, wherein the medium comprises PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells. In some aspects, the medium further comprises potassium ion. In some aspects, the potassium ion is present in the medium at aconcentration higher than 5 mM. Such medium is also referred to herein as a "metabolic reprogramming medium." Accordingly, in some aspects, a medium useful for the present disclosure comprises (a) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and (b) potassium ion at a concentration higher than 5 mM. In some aspects, the cultured cells are also edited to exhibit reduced expression of a gene and / or protein associated with impaired immune cell function, e.g., NR4A family member) and / or transduced to express a ligand-binding protein and / or to exhibit increased expression of a gene and / or protein associated with improved immune cell function, e.g., c-Jun polypeptide) immune cells (e.g., T cells and / or NK cells), as described elsewhere herein.

[0190] In some aspects, the metabolic reprogramming medium comprises at least about 5 mM to at least about 100 mM of potassium ion, at least about 5 mM to at least about 90 mM of potassium ion, at least about 5 mM to at least about 80 mM of potassium ion, at least about 5 mM to at least about 75 mM of potassium ion, at least about 5 mM to at least about 70 mM of potassium ion, at least about 5 mM to at least about 65 mM of potassium ion, at least about 5 mM to at least about 60 mM of potassium ion, at least about 5 mM to at least about 55 mM of potassium ion, at least about 5 mM to at least about 50 mM of potassium ion, at least about 5 mM to at least about 45 mM of potassium ion, at least about 5 mM to at least about 40 mM of potassium ion, at least about 10 mM to at least about 80 mM of potassium ion, at least about 10 mM to at least about 75 mM of potassium ion, at least about 10 mM to at least about 70 mM of potassium ion, at least about 10 mM to at least about 65 mM of potassium ion, at least about 10 mM to at least about 60 mM of potassium ion, at least about 10 mM to at least about 55 mM of potassium ion, at least about 10 mM to at least about 50 mM of potassium ion, at least about 10 mM to at least about 45 mM of potassium ion, at least about 10 mM to at least about 40 mM of potassium ion, at least about 20 mM to at least about 80 mM of potassium ion, at least about 20 mM to at least about 75 mM of potassium ion, at least about 20 mM to at least about 70 mM of potassium ion, at least about 20 mM to at least about 65 mM of potassium ion, at least about 20 mM to at least about 60 mM of potassium ion, at least about 20 mM to at least about 55 mM of potassium ion, at least about 20 mM to at least about 50 mM of potassium ion, at least about 20 mM to at least about 45 mM of potassium ion, at least about 20 mM to at least about 40 mM of potassium ion, at least about 30 mM to at least about 80 mM of potassium ion, at least about 30 mM to at least about 75 mM of potassium ion, at least about30 mM to at least about 70 mM of potassium ion, at least about 30 mM to at least about 65 mM of potassium ion, at least about 30 mM to at least about 60 mM of potassium ion, at least about 30 mM to at least about 55 mM of potassium ion, at least about 30 mM to at least about 50 mM of potassium ion, at least about 30 mM to at least about 45 mM of potassium ion, at least about 30 mM to at least about 40 mM of potassium ion, at least about 40 mM to at least about 80 mM of potassium ion, at least about 40 mM to at least about 75 mM of potassium ion, at least about 40 mM to at least about 70 mM of potassium ion, at least about 40 mM to at least about 65 mM of potassium ion, at least about 40 mM to at least about 60 mM of potassium ion, at least about 40 mM to at least about 55 mM of potassium ion, at least about 40 mM to at least about 50 mM of potassium ion, at least about 40 mM to at least about 45 mM of potassium ion, at least about 45 mM to at least about 80 mM of potassium ion, at least about 45 mM to at least about 75 mM of potassium ion, at least about 45 mM to at least about 70 mM of potassium ion, at least about 45 mM to at least about 65 mM of potassium ion, at least about 45 mM to at least about 60 mM of potassium ion, at least about 45 mM to at least about 55 mM of potassium ion, at least about 45 mM to at least about 50 mM of potassium ion, at least about 50 mM to at least about 80 mM of potassium ion, at least about 50 mM to at least about 75 mM of potassium ion, at least about 50 mM to at least about 70 mM of potassium ion, at least about 50 mM to at least about 65 mM of potassium ion, at least about 50 mM to at least about 60 mM of potassium ion, or at least about 50 mM to at least about 55 mM of potassium ion.

[0191] In some aspects, the metabolic reprogramming medium comprises at least about 5 mM, at least about 10 mM, at least about 15 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, or at least about 80 mM of potassium ion. In some aspects, the metabolic reprogramming medium comprises at least about 5 mM of potassium ion. In some aspects, the metabolic reprogramming medium comprises at least about 10 mM of potassium ion. In some aspects, the metabolic reprogramming medium comprises at least about 15 mM of potassium ion. In some aspects, the metabolic reprogramming medium comprises at least about 20 mM of potassium ion. In some aspects, the metabolic reprogramming medium comprises at least about 25 mM of potassium ion. In some aspects, the metabolic reprogramming medium comprises at least about 30 mM of potassium ion. In some aspects, the metabolic reprogramming medium comprises at leastabout 35 mM of potassium ion. In some aspects, the metabolic reprogramming medium comprises at least about 40 mM of potassium ion. In some aspects, the metabolic reprogramming medium comprises at least about 45 mM of potassium ion. In some aspects, the metabolic reprogramming medium comprises at least about 50 mM of potassium ion. In some aspects, the metabolic reprogramming medium comprises at least about 55 mM of potassium ion. In some aspects, the metabolic reprogramming medium comprises at least about 60 mM of potassium ion. In some aspects, the metabolic reprogramming medium comprises at least about 65 mM of potassium ion. In some aspects, the metabolic reprogramming medium comprises at least about 70 mM of potassium ion. In some aspects, the metabolic reprogramming medium comprises at least about 75 mM of potassium ion. In some aspects, the metabolic reprogramming medium comprises at least about 80 mM of potassium ion.

[0192] In some aspects, the metabolic reprogramming medium comprises an increased concentration of potassium ion, e.g., at least about 5 mM of potassium ion, and the medium is hypotonic. As further described elsewhere in the present disclosure, in some aspects, the metabolic reprogramming medium comprises potassium ion and NaCl, wherein the potassium ion is at a concentration between about 40 mM and about 80 mM and NaCl is at a concentration between about 30 mM and about 100 mM, and wherein the total concentration of potassium ion and NaCl is between about 110 and about 140 mM.

[0193] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 5 mM to about 100 mM. In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 5 mM to about 100 mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 5 mM to about 90 mM, about 5 mM to about 80 mM, about 5 mM to about 70 mM, about 5 mM to about 60 mM, or about 5 mM to about 50 mM. In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 5 mM to about 90 mM, about 5 mM to about 80 mM, about 5 mM to about 70 mM, about 5 mM to about 60 mM, or about 5 mM to about 50 mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 25 mM to about 100 mM. In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 25 mM to about 100 mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 25 mM to about 90 mM,about 25 mM to about 80 mM, about 25 mM to about 70 mM, about 25 mM to about 60 mM, or about 25 mM to about 50 mM. In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 25 mM to about 90 mM, about 25 mM to about 80 mM, about 25 mM to about 70 mM, about 25 mM to about 60 mM, or about 25 mM to about 50 mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 40 mM to about 100 mM. In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 40 mM to about 100 mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ion is about 40 mM to about 90 mM, about 40 mM to about 85 mM, about 40 mM to about 80 mM, about 40 mM to about 75 mM, about 40 mM to about 70 mM, about 40 mM to about 65 mM, about 40 mM to about 60 mM, about 40 mM to about 55 mM, or about 40 mM to about 50 mM. In some aspects, the concentration of potassium ion is about 40 mM to about 90 mM, about 40 mM to about 85 mM, about 40 mM to about 80 mM, about 40 mM to about 75 mM, about 40 mM to about 70 mM, about 40 mM to about 65 mM, about 40 mM to about 60 mM, about 40 mM to about 55 mM, or about 40 mM to about 50 mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ion is about 50 mM to about 90 mM, about 50 mM to about 85 mM, about 50 mM to about 80 mM, about 50 mM to about 75 mM, about 50 mM to about 70 mM, about 50 mM to about 65 mM, about 50 mM to about 60 mM, or about 50 mM to about 55 mM. In some aspects, the concentration of potassium ion is about 50 mM to about 90 mM, about 50 mM to about 85 mM, about 50 mM to about 80 mM, about 50 mM to about 75 mM, about 50 mM to about 70 mM, about 50 mM to about 65 mM, about 50 mM to about 60 mM, or about 50 mM to about 55 mM, and wherein the medium is hypotonic. In some aspects, the metabolic reprogramming medium comprises at least about 50 mM potassium ion and less than about 90 mM NaCl. In some aspects, the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0194] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 50 mM to about 120 mM. In some aspects, the concentration of potassium ion is about 50 mM to about 115 mM, about 50 mM to about 110 mM, about 50 mM to about 105 mM, about 50 mM to about 100 mM, about 50 mM to about 95 mM, about 50 mM to about 90 mM, about 50 mM to about 85 mM, about 50 mM to about 80 mM, about 50 mM to about 75 mM, about 50 mM to about 70 mM, about 50 mM to about 65 mM, about 50 mM to about 60 mM, or about 50 mM to about 55 mM. In some aspects, themedium is hypotonic. In some aspects, the medium comprises at least about 50 mM to about 120 mM potassium ion and less than about 90 mM to about 20 mM NaCl. In some aspects, the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0195] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 55 mM to about 120 mM. In some aspects, the concentration of potassium ion is about 55 mM to about 115 mM, about 55 mM to about 110 mM, about 55 mM to about 105 mM, about 55 mM to about 100 mM, about 55 mM to about 95 mM, about 55 mM to about 90 mM, about 55 mM to about 85 mM, about 55 mM to about 80 mM, about 55 mM to about 75 mM, about 55 mM to about 70 mM, about 55 mM to about 65 mM, or about 55 mM to about 60 mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming medium comprises at least about 55 mM to about 120 mM potassium ion and less than about 85 mM to about 20 mM NaCl. In some aspects, the total concentration of potassium ion and NaCl in a metabolic reprogramming medium of the present disclosure is between 110 mM and 140 mM.

[0196] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 60 mM to about 120 mM. In some aspects, the concentration of potassium ion is about 60 mM to about 115 mM, about 60 mM to about 110 mM, about 60 mM to about 105 mM, about 60 mM to about 100 mM, about 60 mM to about 95 mM, about 60 mM to about 90 mM, about 60 mM to about 85 mM, about 60 mM to about 80 mM, about 60 mM to about 75 mM, about 60 mM to about 70 mM, or about 60 mM to about 65 mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming medium comprises at least about 60 mM to about 120 mM potassium ion and less than about 80 mM to about 20 mM NaCl. In some aspects, the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0197] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 65 mM to about 120 mM. In some aspects, the concentration of potassium ion is about 65 mM to about 115 mM, about 65 mM to about 110 mM, about 65 mM to about 105 mM, about 65 mM to about 100 mM, about 65 mM to about 95 mM, about 65 mM to about 90 mM, about 65 mM to about 85 mM, about 65 mM to about 80 mM, about 65 mM to about 75 mM, or about 65 mM to about 70 mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming medium comprises at least about 65 mM to about 120 mM potassium ion and less than about 75 mM to about 20 mM NaCl.In some aspects, the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0198] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 70 mM to about 120 mM. In some aspects, the concentration of potassium ion is about 70 mM to about 115 mM, about 70 mM to about 110 mM, about 70 mM to about 105 mM, about 70 mM to about 100 mM, about 70 mM to about 95 mM, about 70 mM to about 90 mM, about 70 mM to about 85 mM, about 70 mM to about 80 mM, or about 70 mM to about 75 mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming medium comprises at least about 70 mM to about 120 mM potassium ion and less than about 70 mM to about 20 mM NaCl. In some aspects, the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0199] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 75 mM to about 120 mM. In some aspects, the concentration of potassium ion is about 75 mM to about 115 mM, about 75 mM to about 110 mM, about 75 mM to about 105 mM, about 75 mM to about 100 mM, about 75 mM to about 95 mM, about 75 mM to about 90 mM, about 75 mM to about 85 mM, or about 75 mM to about 80 mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming medium comprises at least about 75 mM to about 120 mM potassium ion and less than about 65 mM to about 20 mM NaCl. In some aspects, the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0200] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 80 mM to about 120 mM. In some aspects, the concentration of potassium ion is about 80 mM to about 115 mM, about 80 mM to about 110 mM, about 80 mM to about 105 mM, about 80 mM to about 100 mM, about 80 mM to about 95 mM, about 80 mM to about 90 mM, or about 80 mM to about 85 mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming medium comprises at least about 80 mM to about 120 mM potassium ion and less than about 60 mM to about 20 mM NaCl. In some aspects, the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0201] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 85 mM to about 120 mM. In some aspects, the concentration of potassium ion is about 85 mM to about 115 mM, about 85 mM to about 110 mM, about 85 mM to about 105 mM, about 85 mM to about 100 mM, about 85 mM to about 95 mM, or about85 mM to about 90 mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming medium comprises at least about 85 mM to about 120 mM potassium ion and less than about 65 mM to about 20 mM NaCl. In some aspects, the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0202] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 90 mM to about 120 mM. In some aspects, the concentration of potassium ion is about 90 mM to about 115 mM, about 90 mM to about 110 mM, about 90 mM to about 105 mM, about 90 mM to about 100 mM, or about 90 mM to about 95 mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming medium comprises at least about 90 mM to about 120 mM potassium ion and less than about 50 mM to about 20 mM NaCl. In some aspects, the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0203] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 95 mM to about 120 mM. In some aspects, the concentration of potassium ion is about 95 mM to about 115 mM, about 95 mM to about 110 mM, about 95 mM to about 105 mM, or about 95 mM to about 100 mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming medium comprises at least about 95 mM to about 120 mM potassium ion and less than about 55 mM to about 20 mM NaCl. In some aspects, the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0204] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 100 mM to about 120 mM. In some aspects, the concentration of potassium ion is about 100 mM to about 115 mM, about 100 mM to about 110 mM, or about 100 mM to about 105 mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming medium comprises at least about 100 mM to about 120 mM potassium ion and less than about 50 mM to about 20 mM NaCl. In some aspects, the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0205] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 105 mM to about 120 mM. In some aspects, the concentration of potassium ion is about 105 mM to about 115 mM, or about 105 mM to about 110 mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming medium comprises at least about 105 mM to about 120 mM potassium ion and less thanabout 35 mM to about 20 mM NaCl. In some aspects, the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0206] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 110 mM to about 120 mM. In some aspects, the concentration of potassium ion is about 110 mM to about 115 mM. In some aspects, the medium is hypotonic. In some aspects, the metabolic reprogramming medium comprises at least about 110 mM to about 120 mM potassium ion and less than about 30 mM to about 20 mM NaCl. In some aspects, the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0207] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 50 mM to about 90 mM. In some aspects, the concentration of potassium ion is about 50 mM to about 80 mM. In some aspects, the concentration of potassium ion is about 60 mM to about 90 mM. In some aspects, the concentration of potassium ion is about 60 mM to about 80 mM. In some aspects, the concentration of potassium ion is about 70 mM to about 90 mM. In some aspects, the concentration of potassium ion is about 70 mM to about 80 mM. In some aspects, the concentration of potassium ion is about 80 mM to about 90 mM.

[0208] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 50 mM to about 90 mM, and the concentration of NaCl is less than about 90 mM to about 50 mM. In some aspects, the concentration of potassium ion is about 50 mM to about 80 mM, and the concentration of NaCl is less than about 90 mM to about 60 mM. In some aspects, the concentration of potassium ion is about 60 mM to about 90 mM, and the concentration of NaCl is less than about 90 mM to about 60 mM. In some aspects, the concentration of potassium ion is about 60 mM to about 80 mM, and the concentration of NaCl is less than about 80 mM to about 60 mM. In some aspects, the concentration of potassium ion is about 70 mM to about 90 mM, and the concentration of NaCl is less than about 70 mM to about 50 mM. In some aspects, the concentration of potassium ion is about 70 mM to about 80 mM, and the concentration of NaCl is less than about 70 mM to about 60 mM. In some aspects, the concentration of potassium ion is about 80 mM to about 90 mM, and the concentration of NaCl is less than about 60 mM to about 50 mM. In some aspects, the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0209] In some aspects, the concentration of potassium ion in a metabolic reprogramming medium is about 50 mM to about 55 mM. In some aspects, the concentration of potassium ion is about 50 mM to about 55 mM, and the concentration of NaCl is less than about 90 to about 85. In some aspects, the concentration of potassium ion is about 55 mM to about 60 mM. In some aspects, the concentration of potassium ion is about 55 mM to about 60 mM, and the concentration of NaCl is less than about 85 to about 80. In some aspects, the concentration of potassium ion is about 60 mM to about 65 mM. In some aspects, the concentration of potassium ion is about 60 mM to about 65 mM, and the concentration of NaCl is less than about 80 mM to about 75 mM. In some aspects, the concentration of potassium ion is about 65 mM to about 70 mM. In some aspects, the concentration of potassium ion is about 65 mM to about 70 mM, and the concentration of NaCl is less than about 75 mM to about 70 mM. In some aspects, the concentration of potassium ion is about 70 mM to about 75 mM. In some aspects, the concentration of potassium ion is about 70 mM to about 75 mM, and the concentration of NaCl is less than about 70 mM to about 65 mM. In some aspects, the concentration of potassium ion is about 75 mM to about 80 mM. In some aspects, the concentration of potassium ion is about 75 mM to about 80 mM, and the concentration of NaCl is less than about 65 mM to about 60 mM. In some aspects, the concentration of potassium ion is about 80 mM to about 85 mM. In some aspects, the concentration of potassium ion is about 80 mM to about 85 mM, and the concentration of NaCl is less than about 60 mM to about 55 mM. In some aspects, the concentration of potassium ion is about 85 mM to about 90 mM. In some aspects, the concentration of potassium ion is about 85 mM to about 90 mM, and the concentration of NaCl is less than about 55 mM to about 50 mM. In some aspects, the concentration of potassium ion is about 90 mM to about 95 mM. In some aspects, the concentration of potassium ion is about 90 mM to about 95 mM, and the concentration of NaCl is less than about 50 to about 45. In some aspects, the concentration of potassium ion is about 95 mM to about 100 mM. In some aspects, the concentration of potassium ion is about 95 mM to about 100 mM, and the concentration of NaCl is less than about 45 mM to about 40 mM. In some aspects, the concentration of potassium ion is about 100 mM to about 105 mM. In some aspects, the concentration of potassium ion is about 100 mM to about 105 mM, and the concentration of NaCl is less than about 40 mM to about 35 mM. In some aspects, the concentration of potassium ion is about 105 mM to about 110 mM. In some aspects, the concentration of potassium ion is about 105 mM to about 110 mM, and the concentration of NaCl is lessthan about 35 to about 30. In some aspects, the concentration of potassium ion is about 110 mM to about 115 mM. In some aspects, the concentration of potassium ion is about 110 mM to about 115 mM, and the concentration of NaCl is less than about 30 mM to about 25 mM. In some aspects, the concentration of potassium ion is about 115 mM to about 120 mM. In some aspects, the concentration of potassium ion is about 115 mM to about 120 mM, and the concentration of NaCl is less than about 25 mM to about 20 mM. In some aspects, the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0210] In some aspects, the concentration of potassium ion is about 40 mM to about 90 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 40 mM to about 80 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 40 mM to about 70 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 50 mM to about 90 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 50 mM to about 80 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 50 mM to about 70 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 55 mM to about 90 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 55 mM to about 80 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 55 mM to about 70 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 60 mM to about 90 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 60 mM to about 80 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 60 mM to about 70 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 65 mM to about 90 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 65 mM to about 80 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 65 mM to about 70 mM, wherein the medium is hypotonic or isotonic.

[0211] In some aspects, the concentration of potassium ion is higher than about 4 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration ofpotassium ion is about 4 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 5 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 5 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 6 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 6 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 7 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 7 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 8 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 8 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 9 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 9 mM, wherein the medium is hypotonic or isotonic.

[0212] In some aspects, the concentration of potassium ion is higher than about 10 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 10 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 11 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 11 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 12 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 12 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 13 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 13 mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ion is higher than about 14 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 14 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 15 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 15 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 16 mM, wherein the medium ishypotonic or isotonic. In some aspects, the concentration of potassium ion is about 16 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 17 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 17 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 18 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 18 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 19 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 19 mM, wherein the medium is hypotonic or isotonic.

[0213] In some aspects, the concentration of potassium ion is higher than about 20 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 20 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 21 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 21 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 22 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 22 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 23 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 23 mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ion is higher than about 24 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 24 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 25 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 25 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 26 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 26 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 27 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 27 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher thanabout 28 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 28 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 29 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 29 mM, wherein the medium is hypotonic or isotonic.

[0214] In some aspects, the concentration of potassium ion is higher than about 30 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 30 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 31 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 31 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 32 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 32 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 33 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 33 mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ion is higher than about 34 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 34 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 35 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 35 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 36 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 36 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 37 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 37 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 38 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 38 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 39 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 39 mM, wherein the medium is hypotonic or isotonic.

[0215] In some aspects, the concentration of potassium ion is higher than about 40 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 40 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 41 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 41 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 42 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 42 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 43 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 43 mM, wherein the medium is hypotonic. In some aspects, the concentration of potassium ion is higher than about 44 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 44 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 45 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 45 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 46 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 46 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 47 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 47 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 48 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 48 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is higher than about 49 mM, wherein the medium is hypotonic or isotonic. In some aspects, the concentration of potassium ion is about 49 mM, wherein the medium is hypotonic or isotonic.

[0216] In some aspects, the metabolic reprogramming medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and a high concentration of potassium ion) is prepared by adding a sufficient amount of a potassium salt in a medium. In some aspects, non-limiting examples of potassium salt include potassium aminetrichloroplatinate,potassium aquapentachlororuthenate, potassium bis(oxalato)platinate(II) dihydrate, potassium bisulfate, potassium borohydride, potassium bromide, potassium carbonate, potassium chloride, potassium chromate, potassium dichromate, potassium dicyanoargentate, potassium dicyanoaurate, potassium fluoride, potassium fluorosulfate, potassium hexachloroiridate, potassium hexachloroosmate, potassium hexachloropalladate, potassium hexachloroplatinate, potassium hexachlororhenate, potassium hexacyanochromate, potassium hexacyanoferrate, potassium hexacyanoruthenate(II) hydrate, potassium hexafluoroantimonate, potassium hexafluoronickel ate, potassium hexafluorophosphate, potassium hexafluorotitanate, potassium hexafluorozirconate, potassium hexahydroxoantimonate, potassium hexaiodoplatinate, potassium hexaiodorhenate, potassium hydroxide, potassium iodate, potassium iodide, potassium manganate, potassium metavanadate, potassium molybdate, potassium nitrate, potassium nitrosodi sulfonate, potassium osmate(VI) dihydrate, potassium pentachloronitrosylruthenate, potassium perchlorate, potassium perrhenate, potassium perruthenate, potassium persulfate, potassium phosphate dibasic, potassium phosphate monobasic, potassium pyrophosphate, potassium selenocyanate, potassium selenocyanate, potassium stannate trihydrate, potassium sulfate, potassium tellurate hydrate, potassium tellurite, potassium tetraborate tetrahydrate, potassium tetrabromoaurate, potassium tetrabromopalladate, potassium tetrachloropalladate, potassium tetrachloroplatinate, potassium tetracyanopalladate, potassium tetracyanoplatinate, potassium tetrafluoroborate, potassium tetranitroplatinate, potassium tetrathionate, potassium p-toluenethiosulfonate, potassium hydroxycitrate tribasic monohydrate, or any combination thereof. In some aspects, the potassium salt comprises potassium chloride (KC1). In some aspects, the potassium salt comprises potassium gluconate. In some aspects, the potassium salt comprises potassium citrate. In some aspects, the potassium salt comprises potassium hydroxy citrate.Sodium

[0217] As is apparent from at least the above disclosure, in some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) further comprises a sodium ion. Accordingly, in some aspects, a medium described herein comprises: (a) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, and(b) a sodium ion. In some aspects, the medium comprises: (a) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (b) a sodium ion, and (c) a potassium ion at a concentration higher than 5 mM.

[0218] In some aspects, the medium is hypotonic or isotonic. In some aspects, the target concentration of sodium ion is reached by starting with a basal medium comprising a higher concentration of sodium ion, and diluting the solution to reach the target concentration of sodium ion. In some aspects, the target concentration of sodium ion is reached by adding one or more sodium salts. Non-limiting examples of sodium salts include sodium (meta)periodate, sodium arsenyl tartrate hydrate, sodium azide, sodium benzyloxide, sodium bromide, sodium carbonate, sodium chloride, sodium chromate, sodium cyclohexanebutyrate, sodium ethanethiolate, sodium fluoride, sodium fluorophosphate, sodium formate, sodium hexachloroiridate(III) hydrate, sodium hexachloroiridate(IV) hexahydrate, sodium hexachloroplatinate(IV) hexahydrate, sodium hexachlororhodate(III), sodium hexafluoroaluminate, sodium hexafluoroantimonate(V), sodium hexafluoroarsenate(V), sodium hexafluoroferrate(III), sodium hexafluorophosphate, sodium hexafluorosilicate, sodium hexahydroxyplatinate(IV), sodium hexametaphosphate, sodium hydrogen difluoride, sodium hydrogen sulfate, sodium hydrogencyanamide, sodium hydroxide, sodium iodide, sodium metaborate tetrahydrate, sodium metasilicate nonahydrate, sodium metavanadate, sodium molybdate, sodium nitrate, sodium nitrite, sodium oxalate, sodium perborate monohydrate, sodium percarbonate, sodium perchlorate, sodium periodate, sodium permanganate, sodium perrhenate, sodium phosphate, sodium pyrophosphate, sodium selenate, sodium selenite, sodium stannate, sodium sulfate, sodium tellurite, sodium tetraborate, sodium tetrachloroaluminate, sodium tetrachloroaurate(III), sodium tetrachloropalladate(II), sodium tetrachloroplatinate(II), sodium thiophosphate tribasic, sodium thiosulfate, sodium thiosulfate pentahydrate, sodium yttrium oxyfluoride, Trisodium trimetaphosphate, or any combination thereof. In some aspects, the sodium salt comprises sodium chloride (NaCl). In some aspects, the sodium salt comprises sodium gluconate. In some aspects, the sodium salt comprises sodium bicarbonate. In some aspects, the sodium salt comprises sodium hydroxycitrate. In some aspects, the sodium salt comprises sodium phosphate.

[0219] In some aspects, the concentration of the sodium ion (e.g., NaCl) in a medium of the present disclosure is less than that of the basal medium. In some aspects, where the medium further comprises a potassium ion (e.g., metabolic reprogramming medium), theconcentration of the sodium ion (e.g., NaCl) is reduced as the concentration of potassium ion is increased. In some aspects, the concentration of the sodium ion (e.g., NaCl) is from about 25 mM to about 115 mM. In some aspects, the concentration of the sodium (e.g., NaCl) ion is from about 25 mM to about 100 mM, about 30 mM to about 40 mM, about 30 mM to about 50 mM, about 30 mM to about 60 mM, about 30 mM to about 70 mM, about 30 mM to about 80 mM, about 40 mM to about 50 mM, about 40 mM to about 60 mM, about 40 mM to about 70 mM, about 40 mM to about 80 mM, about 50 mM to about 55 mM, about 50 mM to about 60 mM, about 50 mM to about 65 mM, about 50 mM to about 70 mM, about 50 mM to about 75 mM, about 50 mM to about 80 mM, about 55 mM to about 60 mM, about 55 mM to about 65 mM, about 55 mM to about 70 mM, about 55 mM to about 75 mM, about 55 mM to about 80 mM, about 60 mM to about 65 mM, about 60 mM to about 70 mM, about 60 mM to about 75 mM, about 60 mM to about 80 mM, about 70 mM to about 75 mM, about 70 mM to about 80 mM, or about 75 mM to about 80 mM. In some aspects, the concentration of the sodium ion (e.g., NaCl) is from about 40 mM to about 80 mM. In some aspects, the concentration of the sodium ion (e.g., NaCl) is from about 50 mM to about 85 mM. In some aspects, the concentration of the sodium ion (e.g., NaCl) is from about 55 mM to about 80 mM. In some aspects, the concentration of the sodium ion (e.g., NaCl) is from about 30 mM to about 35 mM. In some aspects, the concentration of the sodium ion (e.g., NaCl) is from about 35 mM to about 40 mM. In some aspects, the concentration of the sodium ion (e.g., NaCl) is from about 40 mM to about 45 mM. In some aspects, the concentration of the sodium ion (e.g., NaCl) is from about 45 mM to about 50 mM. In some aspects, the concentration of the sodium ion (e.g., NaCl) is from about 50 mM to about 55 mM. In some aspects, the concentration of the sodium ion (e.g., NaCl) is from about 55 mM to about 60 mM. In some aspects, the concentration of the sodium ion (e.g., NaCl) is from about 60 mM to about 65 mM. In some aspects, the concentration of the sodium ion (e.g., NaCl) is from about 65 mM to about 70 mM. In some aspects, the concentration of the sodium ion (e.g., NaCl) is from about 70 mM to about 75 mM. In some aspects, the concentration of the sodium ion (e.g., NaCl) is from about 75 mM to about 80 mM. In some aspects, the concentration of the sodium ion (e.g., NaCl) is from about 80 mM to about 85 mM.

[0220] In some aspects, the concentration of the sodium ion (e.g., NaCl) is about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, or about 90 mM.In some aspects, the concentration of sodium ion (e.g., NaCl) is about 40 mM. In some aspects, the concentration of sodium ion (e.g, NaCl) is about 45 mM. In some aspects, the concentration of sodium ion (e.g, NaCl) is about 50 mM. In some aspects, the concentration of sodium ion (e.g, NaCl) is about 55 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 55.6 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 59.3 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 60 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 63.9 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 65 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 67.6 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 70 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 72.2 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 75 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 76 mM. In some aspects, the concentration of sodium ion (e.g., NaCl) is about 80 mM. In some aspects, the concentration of sodium ion (e.g, NaCl) is about 80.5 mM. In some aspects, the metabolic reprogramming medium comprises about 40 mM to about 90 mM potassium ion and about 40 mM to about 80 mM sodium ion (e.g., NaCl).

[0221] In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 50 mM to about 75 mM of potassium ion and about 80 mM to about 90 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 55 mM to about 75 mM of potassium ion and about 80 mM to about 90 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 60 mM to about 75 mM of potassium ion and about 80 mM to about 90 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 65 mM to about 75 mM of potassium ion and about 80 mM to about 85 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160pg per million cells, such as less than 120 pg per million cells) comprises about 65 mM of potassium ion and about 80 mM to about 85 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 66 mM of potassium ion and about 80 mM to about 85 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 67 mM of potassium ion and about 80 mM to about 85 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 68 mM of potassium ion and about 80 mM to about 85 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 69 mM of potassium ion and about 80 mM to about 85 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 70 mM of potassium ion and about 80 mM to about 85 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 71 mM of potassium ion and about 80 mM to about 85 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 72 mM of potassium ion and about 80 mM to about 85 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 73 mM of potassium ion and about 80 mM to about 85 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 74 mM of potassium ion and about 80 mM to about 85 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 75 mM ofpotassium ion and about 80 mM to about 85 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 65 mM of potassium ion and about 80 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 65 mM of potassium ion and about 85 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 65 mM of potassium ion and about 90 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 70 mM of potassium ion and about 80 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 70 mM of potassium ion and about 85 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 70 mM of potassium ion and about 90 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 75 mM of potassium ion and about 80 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 75 mM of potassium ion and about 85 mM of sodium ion (e.g., NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 75 mM of potassium ion and about 90 mM of sodium ion (e.g., NaCl).

[0222] In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 40 mM to about 90 mM of potassium ion and about 30 mM to about 109 mM of NaCl, wherein the concentration of NaCl (mM) is equal to or lower than (135- potassium ion concentration, meaning 135 minus the concentration of potassium ion). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 40 mM of potassium ion and less than or equal to about 95 mM of NaCl (e.g., about 95 mM, about 94 mM, about 93 mM, about 92 mM, about 91 mM, about 90 mM, about 85 mM, about 80 mM, about 75 mM, about 70 mM, about 65 mM, about 60 mM, about 55 mM, or about 50 mM of NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 45 mM of potassium ion and less than or equal to about 90 mM of NaCl (e.g., about 90 mM, about 89 mM, about 88 mM, about 87 mM, about 86 mM, about 85 mM, about 80 mM, about 75 mM, about 70 mM, about 65 mM, about 60 mM, about 55 mM, or about 50 mM of NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 50 mM of potassium ion and less than or equal to about 85 mM of NaCl (e.g., about 85 mM, about 84 mM, about 83 mM, about 82 mM, about 81 mM, about 80 mM, about 75 mM, about 70 mM, about 65 mM, about 60 mM, about 55 mM, or about 50 mM of NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 55 mM of potassium ion and less than or equal to about 80 mM of NaCl (e.g., about 80 mM, about 79 mM, about 78 mM, about 77 mM, about 76 mM, about 75 mM, about 70 mM, about 65 mM, about 60 mM, about 55 mM, or about 50 mM of NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 60 mM of potassium ion and less than or equal to about 75 mM of NaCl (e.g., about 75 mM, about 74 mM, about 73 mM, about 72 mM, about 71 mM, about 70 mM, about 69 mM, about 68 mM, about 67 mM, about 66 mM, about 65 mM, about 64 mM, about 63 mM, about 62 mM, about 61 mM, about 60 mM, about 55 mM, or about 50 mM of NaCl). In some aspects, a medium useful for the present disclosure (e.g. , comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 65 mM of potassium ion and less than or equal to about 70 mM of NaCl (e.g., about 70 mM, about 69 mM, about 68 mM, about 67 mM, about 66 mM, about 65 mM, about 64 mM, about 63 mM, about 62 mM, about 61 mM, about 60 mM,about 55 mM, or about 50 mM of NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 70 mM of potassium ion and less than or equal to about 70 mM of NaCl (e.g., about 65 mM, about 64 mM, about 63 mM, about 62 mM, about 61 mM, about 60 mM, about 55 mM, or about 50 mM of NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 75 mM of potassium ion and less than or equal to about 60 mM of NaCl (e.g., about 60 mM, about 59 mM, about 58 mM, about 57 mM, about 56 mM, about 55 mM, about 50 mM, about 45 mM, or about 40 mM of NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 80 mM of potassium ion and less than or equal to about 55 mM of NaCl (e.g, about 55 mM, about 54 mM, about 53 mM, about 52 mM, about 51 mM, about 50 mM, about 45 mM, about 40 mM, or about 35 mM of NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 85 mM of potassium ion and less than or equal to about 50 mM of NaCl (e.g., about 50 mM, about 49 mM, about 48 mM, about 47 mM, about 46 mM, about 45 mM, about 40 mM, about 35 mM, or about 30 mM of NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 90 mM of potassium ion and less than or equal to about 45 mM of NaCl (e.g., about 45 mM, about 44 mM, about 43 mM, about 42 mM, about 41 mM, about 40 mM, about 35 mM, about 30 mM, or about 25 mM of NaCl). In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 70 mM of potassium ion and about 60 mM of NaCl. In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 70 mM of potassium ion and about 61 mM of NaCl. In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 70 mM of potassium ion and about 62 mM of NaCl.

[0223] In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 50 mM of potassium ion and about 75 mM of NaCl. In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 55 mM of potassium ion and about 70 mM of NaCl. In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 60 mM of potassium ion and about 65 mM of NaCl. In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises about 65 mM of potassium ion and about 60 mM of NaCl. In some aspects, the medium is hypotonic. In some aspects, the medium is isotonic.

[0224] Some aspects of the present disclosure are directed to methods of culturing and / or modifying immune cells (e.g., T cells and / or NK cells) in a medium comprising (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 5 mM, and (iii) NaCl at a concentration of less than about 135 mM. Some aspects of the present disclosure are directed to methods of culturing and / or modifying immune cells, e.g., T cells and / or NK cells, in a medium comprising (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 40 mM, and (iii) NaCl at a concentration of less than about 100 mM. Some aspects of the present disclosure are directed to methods of culturing and / or modifying immune cells, e.g., T cells and / or NK cells, in a medium comprising (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 50 mM, and (iii) NaCl at a concentration of less than about 90 mM. Some aspects of the present disclosure are directed to methods of culturing and / or modifying immune cells, e.g., T cells and / or NK cells, in a medium comprising (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 55 mM, and (iii) NaCl at a concentration of less than about 70 mM. Some aspects of the present disclosure are directed to methods of culturing and / or modifying immune cells, e.g., T cells and / or NK cells, in a medium comprising (i) PCS at an amount of less than about 160 pg per million cells, suchas less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 60 mM, and (iii) NaCl at a concentration of less than about 70 mM.Tonicity

[0225] In some aspects, the tonicity of a medium described herein (e.g., (concentration of potassium ion and concentration of NaCl) X 2) is adjusted based on the concentration of potassium ion and / or NaCl. In some aspects, the tonicity of the medium is lower than that of the basal medium. In some aspects, the tonicity of the medium is higher than that of the basal medium. In some aspect, the tonicity of the medium is the same as that of the basal medium. The tonicity of the medium can be affected by modifying the concentration of potassium ion and / or NaCl in the media. In some aspects, increased potassium ion concentration is paired with an increase or a decrease in the concentration of NaCl. In some aspects, this pairing affects the tonicity of the medium. In some aspects, the concentration of potassium ion is increased while the concentration of NaCl is decreased.

[0226] In some aspects, the medium useful for the present disclosure (e.g. , comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) is prepared based on the function of potassium ion and tonicity. For example, in some aspects, if the medium useful for the present disclosure is hypotonic (e.g., less than 280 mOsm) and comprises at least about 50 mM of potassium ion, a concentration of NaCl that is sufficient to maintain the medium as hypotonic can be determined based on the following formula: NaCl concentration = (desired tonicity (280) / 2) - potassium ion concentration, (i.e., the concentration of NaCl (mM) is equal to or lower than (140 - potassium ion concentration)). In some aspects, a hypotonic medium disclosed herein comprises a total concentration of potassium ion and NaCl between 110 mM and 140 mM. Therefore, for hypotonic medium, the concentration of potassium ion can be set at a concentration between 50 mM and 90 mM, and the NaCl concentration can be between 90 mM and 50 mM, or lower, so long as the total concentration of potassium ion and NaCl is between 110 mM and 140 mM. In some aspects, a hypotonic medium disclosed herein comprises a total concentration of potassium ion and NaCl between 115 mM and 140 mM. In some aspects, the hypotonic medium disclosed herein comprises a total concentration of potassium ion and NaCl between 120 mM and 140 mM.

[0227] In some aspects, the medium is isotonic (between 280 mOsm and 300 mOsm) and comprises a concentration of potassium ion between about 50 mM and 70 mM. Thecorresponding concentration of NaCl can be again calculated based on the formula: NaCl concentration = (desired tonicity / 2) - potassium ion concentration. For example, if the concentration of potassium is 50 mM and the desired tonicity is 300 mOsm, the NaCl concentration can be 100 mM.

[0228] In some aspects, the medium is isotonic. In some aspects, the medium has a tonicity of about 280 mOsm / L. In some aspects, the medium has a tonicity of 280 mOsm / L. In some aspects, the medium has a tonicity of 280 mOsm / L ± 1 mOsm / L. In some aspects, the medium has a tonicity of 280 mOsm / L ± 2 mOsm / L. In some aspects, the medium has a tonicity of 280 mOsm / L ± 3 mOsm / L. In some aspects, the medium has a tonicity of 280 mOsm / L ± 4 mOsm / L. In some aspects, the medium has a tonicity of 280 mOsm / L ± 5 mOsm / L. In some aspects, the medium has a tonicity of 280 mOsm / L ± 6 mOsm / L. In some aspects, the medium has a tonicity of 280 mOsm / L ± 7 mOsm / L. In some aspects, the medium has a tonicity of 280 mOsm / L ± 8 mOsm / L. In some aspects, the medium has a tonicity of 280 mOsm / L ± 9 mOsm / L. In some aspects, the medium has a tonicity of 280 mOsm / L ± 10 mOsm / L. In some aspects, the medium has a tonicity of about 280 mOsm / L to about 285 mOsm / L, about 280 mOsm / L to about 290 mOsm / L, about 280 mOsm / L to about 295 mOsm / L, about 280 mOsm / L to about 300 mOsm / L, about 280 mOsm / L to about 305 mOsm / L, about 280 mOsm / L to about 310 mOsm / L, about 280 mOsm / L to about 315 mOsm / L, or about 280 mOsm / L to less than 320 mOsm / L. In some aspects, the medium has a tonicity of about 285 mOsm / L, about 290 mOsm / L, about 295 mOsm / L, about 300 mOsm / L, about 305 mOsm / L, about 310 mOsm / L, or about 315 mOsm / L.

[0229] In some aspects, the medium is hypotonic. In some aspects, the medium has a tonicity lower than about 280 mOsm / L. In some aspects, the medium has a tonicity lower than about 280 mOsm / L; as measured by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than 280 mOsm / L. In some aspects, the medium has a tonicity lower than 280 mOsm / L; as measured by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than 275 mOsm / L. In some aspects, the medium has a tonicity lower than 275 mOsm / L; as measured by adding the potassium ion concentration and the NaCl concentration, and multiplying by two; as measured by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than 270 mOsm / L. In some aspects, the medium has a tonicity lower than 270 mOsm / L; asmeasured by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than 265 mOsm / L. In some aspects, the medium has a tonicity lower than 265 mOsm / L; as measured by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than 260 mOsm / L. In some aspects, the medium has a tonicity lower than 260 mOsm / L; as measured by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than 265 mOsm / L. In some aspects, the medium has a tonicity lower than 265 mOsm / L; as measured by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than 260 mOsm / L. In some aspects, the medium has a tonicity lower than 260 mOsm / L; as measured by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than 255 mOsm / L. In some aspects, the medium has a tonicity lower than 255 mOsm / L; as measured by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than about 250 mOsm / L. In some aspects, the medium has a tonicity lower than about 250 mOsm / L; as measured by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than about 245 mOsm / L. In some aspects, the medium has a tonicity lower than about 245 mOsm / L; as measured by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than about 240 mOsm / L. In some aspects, the medium has a tonicity lower than about 240 mOsm / L; as measured by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than about 235 mOsm / L. In some aspects, the medium has a tonicity lower than about 235 mOsm / L; as measured by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the metabolic reprogramming medium has a tonicity lower than about 230 mOsm / L. In some aspects, the medium has a tonicity lower than about 230 mOsm / L; as measured by adding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity lower than about 225 mOsm / L. In some aspects, the medium has a tonicity lower than about 225 mOsm / L. In some aspects, the tonicity is higher than about 220 mOsm / L; as measured byadding the potassium ion concentration and the NaCl concentration, and multiplying by two. In some aspects, the medium has a tonicity from about 230 mOsm / L to about 280 mOsm / L. In some aspects, the medium has a tonicity from about 240 mOsm / L to about 280 mOsm / L.

[0230] In some aspects, the medium has an osmolality lower than about 220 mOsm / L. In some aspects, the medium has an osmolality lower than about 215 mOsm / L. In some aspects, the medium has an osmolality lower than about 210 mOsm / L. In some aspects, the medium has an osmolality lower than about 205 mOsm / L. In some aspects, the medium has an osmolality lower than about 200 mOsm / L.

[0231] In some aspects, the medium has a tonicity from about 100 mOsm / L to about 280 mOsm / L, about 125 mOsm / L to about 280 mOsm / L, about 150 mOsm / L to about 280 mOsm / L, about 175 mOsm / L to about 280 mOsm / L, about 200 mOsm / L to about 280 mOsm / L, about 210 mOsm / L to about 280 mOsm / L, about 220 mOsm / L to about 280 mOsm / L, about 225 mOsm / L to about 280 mOsm / L, about 230 mOsm / L to about 280 mOsm / L, about 235 mOsm / L to about 280 mOsm / L, about 240 mOsm / L to about 280 mOsm / L, about 245 mOsm / L to about 280 mOsm / L, about 250 mOsm / L to about 280 mOsm / L, about 255 mOsm / L to about 280 mOsm / L, about 260 mOsm / L to about 280 mOsm / L, about 265 mOsm / L to about 280 mOsm / L, about 270 mOsm / L to about 280 mOsm / L, or about 275 mOsm / L to about 280 mOsm / L. In some aspects, the medium has a tonicity from about 250 mOsm / L to about 270 mOsm / L. In some aspects, the medium has a tonicity from about 250 mOsm / L to about 255 mOsm / L, about 250 mOsm / L to about 260 mOsm / L, about 250 mOsm / L to about 265 mOsm / L, about 255 mOsm / L to about 260 mOsm / L, about 255 mOsm / L to about 265 mOsm / L, about 255 mOsm / L to about 265 mOsm / L, about 260 mOsm / L to about 265 mOsm / L, or about 254 mOsm / L to about 263 mOsm / L. In some aspects, the medium has a tonicity from about 254 mOsm / L to about 255 mOsm / L. In some aspects, the medium has a tonicity from about 255 mOsm / L to about 256 mOsm / L. In some aspects, the medium has a tonicity from about 256 mOsm / L to about 257 mOsm / L. In some aspects, the medium has a tonicity from about 257 mOsm / L to about 258 mOsm / L. In some aspects, the medium has a tonicity from about 258 mOsm / L to about 259 mOsm / L. In some aspects, the medium has a tonicity from about 260 mOsm / L to about 261 mOsm / L. In some aspects, the medium has a tonicity from about 261 mOsm / L to about 262 mOsm / L. In some aspects, the medium has a tonicity from about 262 mOsm / L to about 263 mOsm / L. In some aspects, the medium has a tonicity from about 263 mOsm / L to about 264mOsm / L. In some aspects, the medium has a tonicity from about 264 mOsm / L to about 265 mOsm / L. In some aspects, the medium has a tonicity from about 220 mOsm / L to about 280 mOsm / L.

[0232] In some aspects, the medium has a tonicity of about 100 mOsm / L, about 125 mOsm / L, about 150 mOsm / L, about 175 mOsm / L, about 200 mOsm / L, about 210 mOsm / L, about 220 mOsm / L, about 225 mOsm / L, about 230 mOsm / L, about 235 mOsm / L, about 240 mOsm / L, about 245 mOsm / L, about 250 mOsm / L, about 255 mOsm / L, about 260 mOsm / L, about 265 mOsm / L, about 270 mOsm / L, or about 275 mOsm / L.

[0233] In some aspects, the medium has a tonicity of about 250 mOsm / L. In some aspects, the medium has a tonicity of about 262.26 mOsm / L. In some aspects, the medium has a tonicity of about 260 mOsm / L. In some aspects, the medium has a tonicity of about 259.7 mOsm / L. In some aspects, the medium has a tonicity of about 257.5 mOsm / L. In some aspects, the medium has a tonicity of about 257.2 mOsm / L. In some aspects, the medium has a tonicity of about 255.2 mOsm / L. In some aspects, the medium has a tonicity of about 254.7. In some aspects, the medium has a tonicity of about 255 mOsm / L. In some aspects, the medium has a tonicity of about 260 mOsm / L.

[0234] In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises: (i) about 50 mM of potassium ion; (ii) about 80.5 mM of NaCl; (iii) about17.7 mM of glucose; and (iv) about 1.8 mM of calcium ion.

[0235] In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises: (i) about 55 mM of potassium ion; (ii) about 76 mM of NaCl; (iii) about17.2 mM of glucose; and (iv) about 1.7 mM of calcium ion.

[0236] In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises: (i) about 60 mM of potassium ion; (ii) about 72.2 mM of NaCl; (iii) about16.8 mM of glucose; and (iv) about 1.6 mM of calcium ion.

[0237] In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises: (i) about 65 mM of potassium ion; (ii) about 67.6 mM of NaCl; (iii) about16.3 mM of glucose; and (iv) about 1.5 mM of calcium ion.

[0238] In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises: (i) about 70 mM of potassium ion; (ii) about 63.9 mM of NaCl; (iii) about 15.9 mM of glucose; and (iv) about 1.4 mM of calcium ion.

[0239] In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises: (i) about 75 mM of potassium ion; (ii) about 59.3 mM of NaCl; (iii) about 15.4 mM of glucose; and (iv) about 1.3 mM of calcium ion.

[0240] In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) comprises: (i) about 80 mM of potassium ion; (ii) about 55.6 mM of NaCl; (iii) about 15 mM of glucose; and (iv) about 1.2 mM of calcium ion.

[0241] The tonicity of the medium can be adjusted, e.g., to an isotonic or hypotonic state disclosed herein, at any point. In some aspects, the tonicity of the medium can be adjusted, e.g., to an isotonic or hypotonic state disclosed herein, before the cells are added to the medium. In some aspects, the cells are cultured in the hypotonic or isotonic medium prior to cell engineering, e.g., prior to transduction with a construct expressing a CAR, TCR or TCR mimic. In some aspects, the cells are cultured in the hypotonic or isotonic medium during cell engineering, e.g., during transduction with a construct expressing a CAR, TCR or TCR mimic. In some aspects the cells are cultured in the hypotonic or isotonic medium after cell engineering, e.g., after transduction with a construct expressing a CAR, TCR or TCR mimic. In some aspects, the cells are cultured in the hypotonic or isotonic medium throughout cell expansion.Saccharides

[0242] In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells,) further comprises a saccharide. Accordingly, some aspects of the present disclosure are directed to methods of culturing immune cells in a medium comprising (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 5 mM, and (iii) a saccharide. In some aspects, the medium is hypotonic or isotonic. In some aspects, a medium that can be used with the present disclosure comprises (i) PCS at an amount of less than about 160 pg permillion cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 5 mM, (iii) a saccharide, and (iv) a sodium ion (e.g., NaCl). In some aspects, the T cells and / or NK cells contacted with PCS are edited to exhibit a reduced expression of a member of the gene and / or protein associated with impaired immune cell function, e.g., NR4A family and / or transduced to express a ligand-binding protein and / or to exhibit increased expression of a gene and / or protein associated with improved immune cell function, e.g., c-Jun polypeptide, as described elsewhere herein.

[0243] In some aspects, the target concentration of the saccharide is reached by starting with a basal medium comprising a higher concentration of the saccharide, and diluting the solution to reach the target concentration of the saccharide. In some aspects, the target concentration of the saccharide is reached by raising the concentration of the saccharide by adding the saccharide until the desired concentration is reached. In some aspects, the saccharide is a monosaccharide, a disaccharide, or a polysaccharide. In some aspects, the saccharide is selected from glucose, fructose, galactose, mannose, maltose, sucrose, lactose, trehalose, or any combination thereof. In some aspects, the saccharide is glucose.

[0244] In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration of at least about 5 mM, and (iii) glucose. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration of at least about 5 mM, (iii) a sodium ion, and (iv) glucose. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 40 mM, and (iii) glucose. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 40 mM, (iii) a sodium ion, and (iv) glucose.

[0245] In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration of at least about 5 mM, and (iii) mannose. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration of at least about 5 mM, (iii) a sodium ion, and (iv) glucose. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per millioncells, (ii) potassium ion at a concentration of at least about 50 mM, and (iii) mannose. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration of at least about 50 mM, (iii) a sodium ion, and (iv) mannose.

[0246] In some aspects, the medium is hypotonic. In some aspects, the medium is isotonic. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 40 mM, (iii) NaCl, and (iv) glucose; wherein the total concentration of potassium ion and NaCl is between 110 mM and 140 mM. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 50 mM, (iii) NaCl, and (iv) glucose; wherein the total concentration of potassium ion and NaCl is between 110 mM and 140 mM. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration of at least about 40 mM, (iii) NaCl, and (iv) mannose; wherein the total concentration of potassium ion and NaCl is between 110 mM and 140 mM. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration of at least about 50 mM, (iii) NaCl, and (iv) mannose; wherein the total concentration of potassium ion and NaCl is between 110 mM and 140 mM.

[0247] In some aspects, the concentration of the saccharide, e.g., glucose, is about 10 mM to about 25 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is less than about 4.29 g / L. In some aspects, the concentration of the saccharide, e.g., glucose, is less than about 25 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is more than about 5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 5 mM to about 50 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 10 mM to about 20 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 10 mM to about 50 mM, about 10 mM to about 45 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 15 mM, about 15 mM to about 50 mM, about 15 mM to about 45 mM, about 15 mM to about 40 mM, about 15 mM to about 35 mM, about 15 mM to about 30 mM, about15 mM to about 25 mM, about 15 mM to about 20 mM, about 20 mM to about 50 mM, about 20 mM to about 45 mM, about 20 mM to about 40 mM, about 20 mM to about 35 mM, about 20 mM to about 30 mM, about 20 mM to about 25 mM, about 25 mM to about 50 mM, about 25 mM to about 45 mM, about 25 mM to about 40 mM, about 25 mM to about 35 mM, about 25 mM to about 30 mM, about 30 mM to about 50 mM, about 30 mM to about 45 mM, about 30 mM to about 40 mM, about 30 mM to about 35 mM, about 35 mM to about 50 mM, about 35 mM to about 45 mM, about 35 mM to about 40 mM, about 40 mM to about 50 mM, about 40 mM to about 45 mM, or about 45 mM to about 50 mM.

[0248] In some aspects, the concentration of the saccharide, e.g., glucose, is from about 15 mM to about 25 mM, about 15 mM to about 20 mM, about 15 mM to about 19 mM, about 15 mM to about 18 mM, about 15 mM to about 17 mM, about 15 mM to about 16 mM, about 16 mM to about 20 mM, about 16 mM to about 19 mM, about 16 mM to about 18 mM, about 16 mM to about 17 mM, about 17 mM to about 20 mM, about 17 mM to about 19 mM, or about 17 mM to about 18 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 5 mM to about 20 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 10 mM to about 20 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 10 mM to about 15 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 14 mM to about 14.5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 14.5 mM to about 15 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 15 mM to about 15.5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 15.5 mM to about 16 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 16 mM to about 16.5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 16.5 mM to about 17 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 17 mM to about 17.5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is from about 17.5 mM to about 18 mM.

[0249] In some aspects, the concentration of the saccharide, e.g., glucose, is about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, is about 10.5 mM, about 11 mM, about 11.5 mM, about 12 mM, about 12.5 mM, about 13 mM, about 13.5 mM, about 14 mM, about 14.5 mM, about 15 mM, about 15.5 mM, about 16 mM, about 16.5 mM, about 17 mM, about 17.5 mM, about 18 mM, about 18.5 mM, about 19 mM, about 19.5 mM, about 20 mM, about 20.5 mM, about 21 mM, about 22 mM, about 23 mM,about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, or about 50 mM.Calcium

[0250] In some aspects, a medium useful for the present disclosure (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells) further comprises a calcium ion. Accordingly, some aspects of the present disclosure are directed to methods of culturing immune cells in a medium comprising (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 5 mM, and (iii) calcium ion. In some aspects, the medium is hypotonic or isotonic. In some aspects, a medium that can be used with the present disclosure comprises (a) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (b) potassium ion at a concentration higher than 5 mM, (c) a sodium ion (e.g., NaCl), and (d) a calcium ion. In some aspects, a medium useful for the present disclosure comprises (a) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (b) potassium ion at a concentration higher than 5 mM, (c) a saccharide, and (d) a calcium ion. In some aspects, a medium useful for the present disclosure comprises (a) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (b) potassium ion at a concentration higher than 5 mM, (c) a sodium ion (e.g., NaCl), (d) a saccharide, and (e) a calcium ion. In some aspects, the T cells and / or NK cells are contacted with PCS and edited to exhibit a reduced expression of a gene and / or protein associated with impaired immune cell function (e.g., NR4A family member) and / or transduced to express a ligandbinding protein and / or to exhibit increased expression of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), as described elsewhere herein.

[0251] In some aspects, the target concentration of calcium is reached by starting with a basal medium comprising a higher concentration of calcium ion, and diluting the solution to reach the target concentration of calcium ion. In some aspects, the target concentration of calcium is reached by raising the concentration of calcium ion by adding one or morecalcium salts. Non-limiting examples of calcium salts include calcium bromide, calcium carbonate, calcium chloride, calcium cyanamide, calcium fluoride, calcium hydride, calcium hydroxide, calcium iodate, calcium iodide, calcium nitrate, calcium nitrite, calcium oxalate, calcium perchlorate tetrahydrate, calcium phosphate monobasic, calcium phosphate tribasic, calcium sulfate, calcium thiocyanate tetrahydrate, hydroxyapatite, or any combination thereof. In some aspects, the calcium salt comprises calcium chloride (CaCh). In some aspects, the calcium salt comprises calcium gluconate.

[0252] In some aspects, the concentration of the calcium ion is less than that of the basal medium. In some aspects, the concentration of the calcium ion is greater than that of the basal medium. In some aspects, the concentration of calcium ion is more than about 0.4 mM. In some aspects, the concentration of calcium ion is less than about 2.8 mM. In some aspects, the concentration of calcium ion is less than about 2.5 mM. In some aspects, the concentration of calcium ion is less than about 2.0 mM. In some aspects, the concentration of calcium ion is less than about 1.9 mM. In some aspects, the concentration of calcium ion is less than about 1.8 mM. In some aspects, the concentration of calcium ion is less than about 1.7 mM. In some aspects, the concentration of calcium ion is less than about 1.6 mM. In some aspects, the concentration of calcium ion is less than about 1.5 mM. In some aspects, the concentration of calcium ion is less than about 1.4 mM. In some aspects, the concentration of calcium ion is less than about 1.3 mM. In some aspects, the concentration of calcium ion is less than about 1.2 mM. In some aspects, the concentration of calcium ion is less than about 1.1 mM. In some aspects, the concentration of calcium ion is less than about 1.0 mM.

[0253] In some aspects, the concentration of calcium ion is from about 0.4 mM to about 2.8 mM, about 0.4 mM to about 2.7 mM, about 0.4 mM to about 2.5 mM, about 0.5 mM to about 2.0 mM, about 1.0 mM to about 2.0 mM, about 1.1 mM to about 2.0 mM, about 1.2 mM to about 2.0 mM, about 1.3 mM to about 2.0 mM, about 1.4 mM to about 2.0 mM, about 1.5 mM to about 2.0 mM, about 1.6 mM to about 2.0 mM, about 1.7 mM to about 2.0 mM, about 1.8 mM to about 2.0 mM, about 0.8 to about 0.9 mM, about 0.8 to about 1.0 mM, about 0.8 to about 1.1 mM, about 0.8 to about 1.2 mM, about 0.8 to about 1.3 mM, about 0.8 to about 1.4 mM, about 0.8 to about 1.5 mM, about 0.8 to about 1.6 mM, about 0.8 to about 1.7 mM, about 0.8 to about 1.8 mM, about 0.9 to about 1.0 mM, about 0.9 to about 1.1 mM, about 0.9 to about 1.2 mM, about 0.9 to about 1.3 mM, about 0.9 to about 1.4 mM, about 0.9 to about 1.5 mM, about 0.9 to about 1.6 mM, about 0.9 to about 1.7 mM,about 0.9 to about 1.8 mM, about 1.0 to about 1.1 mM, about 1.0 to about 1.2 mM, about 1.0 to about 1.3 mM, about 1.0 to about 1.4 mM, about 1.0 to about 1.5 mM, about 1.0 to about 1.6 mM, about 1.0 to about 1.7 mM, about 1.0 to about 1.8 mM, about 1.1 to about1.2 mM, about 1.1 to about 1.3 mM, about 1.1 to about 1.4 mM, about 1.1 to about 1.5 mM, about 1.1 to about 1.6 mM, about 1.1 to about 1.7 mM, about 1.1 to about 1.8 mM, about1.2 to about 1.3 mM, about 1.2 to about 1.4 mM, about 1.2 to about 1.5 mM, about 1.2 to about 1.6 mM, about 1.2 to about 1.7 mM, about 1.2 to about 1.8 mM, about 1.3 to about 1.4 mM, about 1.3 to about 1.5 mM, about 1.3 to about 1.6 mM, about 1.3 to about 1.7 mM, about 1.3 to about 1.8 mM, about 1.4 to about 1.5 mM, about 1.4 to about 1.6 mM, about 1.4 to about 1.7 mM, about 1.4 to about 1.8 mM, about 1.5 to about 1.6 mM, about 1.5 to about 1.7 mM, about 1.5 to about 1.8 mM, about 1.6 to about 1.7 mM, about 1.6 to about1.8 mM, or about 1.7 to about 1.8 mM.

[0254] In some aspects, the concentration of calcium ion is from about 0.8 mM to about1.8 mM. In some aspects, the concentration of calcium ion is from about 0.9 mM to about1.8 mM. In some aspects, the concentration of calcium ion is from about 1.0 mM to about1.8 mM. In some aspects, the concentration of calcium ion is from about 1.1 mM to about1.8 mM. In some aspects, the concentration of calcium ion is from about 1.2 mM to about1.8 mM. In some aspects, the concentration of calcium ion is from about 0.8 mM to about1.8 mM. In some aspects, the concentration of calcium ion is from about 0.8 mM to about0.9 mM. In some aspects, the concentration of calcium ion is from about 0.9 mM to about1.0 mM. In some aspects, the concentration of calcium ion is from about 1.0 mM to about1.1 mM. In some aspects, the concentration of calcium ion is from about 1.1 mM to about1.2 mM. In some aspects, the concentration of calcium ion is from about 1.2 mM to about1.3 mM. In some aspects, the concentration of calcium ion is from about 1.3 mM to about1.4 mM. In some aspects, the concentration of calcium ion is from about 1.4 mM to about1.5 mM. In some aspects, the concentration of calcium ion is from about 1.5 mM to about1.6 mM. In some aspects, the concentration of calcium ion is from about 1.7 mM to about1.8 mM.

[0255] In some aspects, the concentration of calcium ion is about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, or about 2.0 mM. In some aspects, the concentration of calcium ion is about 0.6 mM. In some aspects, the concentration of calcium ion is about 0.7 mM. In some aspects, theconcentration of calcium ion is about 0.8 mM. In some aspects, the concentration of calcium ion is about 0.9 mM. In some aspects, the concentration of calcium ion is about 1.0 mM. In some aspects, the concentration of calcium ion is about 1.1 mM. In some aspects, the concentration of calcium ion is about 1.2 mM. In some aspects, the concentration of calcium ion is about 1.3 mM. In some aspects, the concentration of calcium ion is about 1.4 mM. In some aspects, the concentration of calcium ion is about 1.5 mM. In some aspects, the concentration of calcium ion is about 1.6 mM. In some aspects, the concentration of calcium ion is about 1.7 mM. In some aspects, the concentration of calcium ion is about 1.8 mM.Cytokines

[0256] In some aspects, the medium useful for the present disclosure (e.g. , comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells,) comprises a cytokine. Accordingly, some aspects of the present disclosure are related to methods of culturing immune cells in a medium comprising (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 5 mM, and (iii) a cytokine. In some aspects, the medium is hypotonic. In some aspects, the medium is isotonic. In some aspects, the medium is hypertonic. In some aspects, a medium useful for the present disclosure comprises (a) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (b) potassium ion at a concentration higher than 5 mM, (c) sodium ion (e.g., NaCl), and (d) a cytokine. In some aspects, a medium that can be used with the present disclosure comprises (a) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (b) potassium ion at a concentration higher than 5 mM, (c) a saccharide, and (c) a cytokine. In some aspects, a medium useful for the present disclosure comprises (a) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (b) potassium ion at a concentration higher than 5 mM, (c) a calcium ion, and (d) a cytokine. In some aspects, a medium useful for the present disclosure comprises (a) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (b) potassium ion at a concentration higher than 5 mM, (c) sodium ion (e.g., NaCl), (d) a saccharide, (e) a calcium ion, and (f) a cytokine. In some aspects, the T cells and / or NK cells are contacted with PCS and edited to exhibit a reduced expression of a gene and / or protein associated with impaired immune cell function (e.g.,NR4A family member) and / or transduced to express a ligand-binding protein and / or to exhibit increased expression of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), as described elsewhere herein.

[0257] In some aspects, the cytokine is selected from IL-2, IL-7, IL-15, IL-21, or any combination thereof. In some aspects, the medium provided herein (e.g., comprising PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells,) does not comprise IL-2. In some aspects, the medium comprises IL-2 and IL-21. In some aspects, the medium comprises IL-2, IL-21, and IL-15. The cytokine can be added to the medium at any point. In some aspects, the cytokine is added to the medium before the immune cells, e.g., T cells and / or NK cells, are added to the medium.

[0258] In some aspects, the medium useful for the present disclosure comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 5 mM, and (iii) IL-2. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) more than about 40 mM of potassium ion, and (iii) IL-2. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) at least about 50 mM of potassium ion, and (iii) IL-2. In some aspects, the medium of the present disclosure comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 5 mM, and (iii) IL-7. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) more than about 40 mM of potassium ion, and (iii) IL-7. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) at least about 50 mM of potassium ion, and (iii) IL-7. In some aspects, the medium that can be used with the present disclosure comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 5 mM, and (iii) IL-15. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) more than about 40 mM of potassium ion, and (iii) IL-15. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) at least about 50 mM of potassium ion, and (iii) IL-15. In some aspects, the medium useful for the present disclosurecomprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 5 mM, and (iii) IL-21. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) more than about 40 mM of potassium ion, and (iii) IL-21. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) at least about 50 mM of potassium ion, and (iii) IL-21.

[0259] In some aspects, the medium provided herein comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 5 mM, and (iii) IL-2, wherein the medium does not comprise IL-7. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) more than about 40 mM of potassium ion, and (iii) IL-2, wherein the medium does not comprise IL-7. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) at least about 50 mM of potassium ion, and (iii) IL-2, wherein the medium does not comprise IL-7. In some aspects, the medium useful for the present disclosure comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) potassium ion at a concentration higher than 5 mM, and (iii) IL-2, wherein the medium does not comprise IL-15. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) more than about 40 mM of potassium ion, and (iii) IL-2, wherein the medium does not comprise IL-15. In some aspects, the medium comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per million cells, (ii) at least about 50 mM of potassium ion, and (iii) IL-2, wherein the medium does not comprise IL-15. In some aspects, the medium provided herein comprises (i) PCS at an amount of less than about 160 pg per million cells, such as less than 120 pg per mi...

Claims

What is Claimed is:

1. A method of preparing immune cells for immunotherapy comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than 120 pg per million cells.

2. A method of activating immune cells during ex vivo or in vitro culture comprising contacting the immune with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than 120 pg per million cells.

3. A method of increasing viability of immune cells during ex vivo or in vitro culture comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than 120 pg per million cells.

4. The method of claim 3, wherein the viability of the immune cells is increased as compared to reference immune cells, wherein the reference immune cells were contacted with the PCS at an amount of 120 pg per million cells or greater.

5. The method of claim 4, wherein the viability of the immune cells is increased by at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, 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%, or at least about 50%, as compared to the reference immune cells.

6. A method of decreasing an amount of background cytokine produced by immune cells during ex vivo or in vitro culture comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than 120 pg per million cells.

7. The method of claim 6, wherein the amount of background cytokine produced by the immune cells is decreased as compared to reference immune cells, wherein the reference immune cells were contacted with the PCS at an amount of 120 pg per million cells or greater.

8. The method of claim 7, wherein the amount of background cytokine produced by the immune cells is decreased by at least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100%, as compared to the reference immune cells.

9. A method of decreasing an amount of residual surface cues bound to immune cells after ex vivo or in vitro culture comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of less than 120 pg per million cells.

10. The method of claim 9, wherein the amount of residual surface cues bound to the immune cells is decreased as compared to reference immune cells, wherein the reference immune cells were contacted with the PCS at an amount of 120 pg per million cells or greater.

11. The method of claim 10, wherein the amount of residual surface cues bound to the immune cells is decreased by at least about 5%, 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 80%, at least about 90%, or about 100%, as compared to the reference immune cells.

12. The method of any one of claims 1 to 11, further comprising editing the immune cells to exhibit a decrease in an expression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4A (NR4A) family member) as compared to corresponding immune cells which have not been edited.

13. The method of claim 12, wherein the editing occurs prior to the contacting.

14. The method of claim 13, wherein the editing occurs at least about one day, at least about two days, at least about three days, at least about four days, or at least about five days prior to the contacting.

15. The method of claim 14, wherein the editing occurs about one day prior to the contacting.

16. The method of any one of claims 1 to 15, further comprising transducing the immune cells: (a) to express a ligand-binding protein, (b) to exhibit an increase in an expression level of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), or (c) both (a) and (b).

17. The method of claim 16, wherein the contacting and transducing occur concurrently.

18. A method of preparing immune cells for immunotherapy comprising (a) editing the immune cells to exhibit a decrease in an expression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4 A (NR4A) family member) as compared to corresponding immune cells which have not been edited, (b) contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, and (c) transducing the immune cells (i) to express a ligand-binding protein (ii) to exhibit an increase in an expression level of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), or (iii) both (i) and (ii); wherein the PCS is present in the medium at an amount of less than 120 pg per million cells.

19. A method of activating immune cells during ex vivo or in vitro culture comprising (a) editing the immune cells to exhibit a decrease in an expression level of a gene and / or protein associated with impaired immune cell function (e.g. , nuclear receptor subfamily 4A (NR4A) family member) as compared to corresponding immune cells which have not been edited, (b) contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, and (c) transducing the immune cells (i) to express a ligand-binding protein (ii) to exhibit an increase in an expression level of a gene and / or protein associated with impaired immune cell function (e.g., c-Jun polypeptide), or (iii) both (i) and (ii); wherein the PCS is present in the medium at an amount of less than 120 pg per million cells.

20. A method of increasing viability of immune cells during ex vivo or in vitro culture comprising (a) editing the immune cells to exhibit a decrease in an expression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4 A (NR4A) family member) as compared to corresponding immune cells which have not been edited, (b) contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, and (c) transducing the immune cells (i) to express a ligand-binding protein (ii) to exhibit an increase in an expression level of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), or (iii) both (i) and (ii); wherein the PCS is present in the medium at an amount of less than 120 pg per million cells.

21. The method of claim 20, wherein the viability of the immune cells is increased as compared to reference immune cells, wherein the reference immune cells were contacted with the PCS at an amount of 120 pg per million cells or greater.

22. The method of claim 21, wherein the viability of the immune cells is increased by at leat about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, 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%, or at least about 50% as compared to the reference immune cells.

23. A method of decreasing an amount of background cytokine produced by immune cells during ex vivo or in vitro culture comprising (a) editing the immune cells to exhibit a decrease in an expression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4A (NR4A) family member) as compared to corresponding immune cells which have not been edited, (b) contacting the immune cells with a programmable cellsignaling scaffold (PCS) in a medium, and (c) transducing the immune cells (i) to express a ligandbinding protein (ii) to exhibit an increase in an expression level of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), or (iii) both (i) and (ii); wherein the PCS is present in the medium at an amount of less than 120 pg per million cells.

24. The method of claim 23, wherein the amount of background cytokine produced by the immune cells is decreased as compared to reference immune cells, wherein the reference immune cells were contacted with the PCS at an amount of 120 pg per million cells or greater.

25. The method of claim 24, wherein the amount of background cytokine produced by the immune cells is decreased by 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 80%, at least about 90%, or about 100%, as compared to the reference immune cells.

26. A method of decreasing an amount of residual antibodies bound to immune cells after ex vivo or in vitro culture comprising (a) editing the immune cells to exhibit a decrease in an expression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4A (NR4A) family member) as compared to corresponding immune cells which have not been edited, (b) contacting the immune cells with a programmable cellsignaling scaffold (PCS) in a medium, and (c) transducing the immune cells (i) to express a ligandbinding protein (ii) to exhibit an increase in an expression level of a gene and / or protein associated with an improved immune cell function (e.g., c-Jun polypeptide), or (iii) both (i) and (ii); wherein the PCS is present in the medium at an amount of less than 120 pg per million cells.

27. The method of claim 26, wherein the amount of residual antibodies bound to the immune cells is decreased as compared to reference immune cells, wherein the reference immune cells were contacted with the PCS at an amount of 120 pg per million cellsor greater.

28. The method of claim 27, wherein the amount of residual antibodies bound to the immune cells is decreased by at least about 5%, 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 80%, at least about 90%, or about 100%, as compared to the reference immune cells.

29. The method of any one of claims 18 to 28, wherein the editing occurs prior to (a) the contacting, (b) the transducing, or (c) both (a) and (b).

30. The method of claim 29, wherein the editing occurs prior to both the contacting and the transducing.

31. The method of claim 29 or 30, wherein the editing occurs at least about one day, at least about two days, at least about three days, at least about four days, or at least about five days prior to the contacting and / or transducing.

32. The method of claim 31, wherein the editing occurs about one day prior to the contacting and / or transducing.

33. The method of any one of claims 18 to 32, wherein the contacting and the transducing occur concurrently.

34. The method of any one of claims 1 to 33, wherein the amount of the PCS is about 40 pg to less than 120 pg per million cells.

35. The method of claim 34, wherein the amount of the PCS per million cells is about 45 pg to less than 120 pg, about 50 pg to less than 120 pg, about 55 pg to less than 120 pg, about 60 pg to less than 120 pg, about 65 pg to less than 120 pg, about 70 pg to less than 120 pg, about 75 pg to less than 120 pg, about 80 pg to less than 120 pg, about 85 pg to less than 120 pg, about 90 pg to less than 120 pg, about 95 pg to less than 120 pg, about 100 pg to less than 120 pg, about 105 pg to less than 120 pg, about 110 pg to less than 120 pg, or about 115 pg to less than 120 pg.

36. The method of claim 34, wherein the amount of the PCS per million cells is about 40 pg to about 115 pg, about 40 pg to about 110 pg, about 40 pg to about 105 pg, about 40 pg to about 100 pg, about 40 pg to about 95 pg, about 40 pg to about 90 pg, about 40 pg to about 85 pg, about 40 pg to about 80 pg, about 40 pg to about 75 pg, about 40 pg to about 70 pg, about 40 pg to about 65 pg, about 40 pg to about 60 pg, about 40 pg to about 55 pg, about 40 pg to about 50 pg, or about 40 pg to about 45 pg.

37. The method of claim 34, wherein the amount of the PCS per million cells is about 45 pg to about 115 pg, about 50 pg to about 110 pg, about 55 pg to about 105 pg, about 60 pg to about 100 pg, about 65 pg to about 95 pg, about 70 pg to about 90 pg, or about 75 pg to about 85 pg.

38. The method of claim 34, wherein the amount of the PCS per million cells is about 40 pg, about 41 pg, about 42 pg, about 43 pg, about 44 pg, about 45 pg, about 46 pg, about 47 pg, about 48 pg, about 49 pg, about 50 pg, about 51 pg, about 52 pg, about 53 pg, about 54 pg, about 55 pg, about 56 pg, about 57 pg, about 58 pg, about 59 pg, about 60 pg, about 61 pg, about 62 pg, about 63 pg, about 64 pg, about 65 pg, about 66 pg, about 67 pg, about 68 pg, about 69 pg, about 70 pg, about 71 pg, about 72 pg, about 73 pg, about 74 pg, about 75 pg, about 76 pg, about 77 pg, about 78 pg, about 79 pg, about 80 pg, about 81 pg, about 82 pg, about 83 pg, about 84 pg, about 85 pg, about 86 pg, about 87 pg, about 88 pg, about 89 pg, about 90 pg, about 91 pg, about 92 pg, about 93 pg, about 94 pg, about 95 pg, about 96 pg, about 97 pg, about 98 pg, about 99 pg, about 100 pg, about 101 pg, about 102 pg, about 103 pg, about 104 pg, about 105 pg, about 106 pg, about 107 pg, about 108 pg, about 109 pg, about 110 pg, about 111 pg, about 112 pg, about 113 pg, about 114 pg, or about 115 pg.

39. A method of preparing immune cells for immunotherapy comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of between about 120 pg per million cells and less than about 160 pg per million cells.

40. A method of activating immune cells during ex vivo or in vitro culture comprising contacting the immune with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of between about 120 pg per million cells and less than about 160 pg per million cells.

41. A method of increasing viability of immune cells during ex vivo or in vitro culture comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of between about 120 pg per million cells and less than about 160 pg per million cells.

42. A method of decreasing an amount of background cytokine produced by immune cells during ex vivo or in vitro culture comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of between about 120 pg per million cells and less than about 160 pg per million cells.

43. A method of decreasing an amount of residual antibodies bound to immune cells after ex vivo or in vitro culture comprising contacting the immune cells with a programmable cell-signaling scaffold (PCS) in a medium, wherein the PCS is present in the medium at an amount of between about 120 pg per million cells and less than about 160 pg per million cells.

44. The method of any one of claims 39 to 43, further comprising editing the immune cells to exhibit a decrease in an expression level of a gene and / or protein associated with impaired immune cell function (e.g., nuclear receptor subfamily 4A (NR4A) family member) as compared to corresponding immune cells which have not been edited.

45. The method of claim 44, wherein the editing occurs prior to the contacting.

46. The method of claim 45, wherein the editing occurs at least about one day, at least about two days, at least about three days, at least about four days, or at least about five days prior to the contacting.

47. The method of claim 46, wherein the editing occurs about one day prior to the contacting.

48. The method of any one of claims 39 to 47, further comprising transducing the immune cells: (a) to express a ligand-binding protein, (b) to exhibit an increase in an expression level of a gene and / or protein associated with improved immune cell function (e.g., c-Jun polypeptide), or (c) both (a) and (b).

49. The method of claim 48, wherein the contacting and the transducing occur concurrently.

50. The method of any one of claims 1 to 49, wherein the PCS comprises (i) a base layer comprising high surface area mesoporous silica micro-rods (MSR); (ii) a continuous, fluid- supported lipid bilayer (SLB) layered on the MSR base layer; and (iii) a plurality of surface cues.

51. The method of claim 50, wherein the PCS further comprises a plurality of soluble cues.

52. The method of claim 50or 51, wherein one or more of the plurality of surface cues are loaded onto the SLB layer.

53. The method of claim 51 or 52, wherein one or more of the plurality of soluble cues are loaded onto the MSR base layer.

54. The method of any one of claims 51 to 53, wherein one or more of the plurality of soluble cues are released from the PCS in a controlled-release manner.

55. The method of any one of claims 51 to 54, wherein one or more of the plurality of soluble cues are released from the PCS in a sustained manner for at least about 30 days.

56. The method of any one of claims 51 to 55, wherein the plurality of soluble cues comprises IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-21, transforming growth factor beta (TGF-P), or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof.

57. The method of any one of claims 51 to 56, wherein the plurality of soluble cues comprises (i) IL-2, an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof and (ii) a second soluble cue comprising IL-7, IL-21, IL- 15, IL- 15 superagonist, or any combination thereof.

58. The method of any one of claims 51 to 57, wherein the plurality of soluble cues comprises (i) IL-2, an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof, (ii) a second soluble cue comprising IL-7, IL-21, IL- 15, IL- 15 superagonist, or any combination thereof, and (iii) a third soluble cue comprising IL-7, IL-21, IL-15, IL-15 superagonist, or any combination thereof.

59. The method of any one of claims 51 to 58, wherein the plurality of soluble cues comprises an N-terminal IL-2 fragment comprising the first 30 amino acids of IL-2 (pl-30), an IL-2 superkine peptide, an IL-2 partial agonist peptide, or a combination thereof.

60. The method of any one of claims 51 to 59, wherein the plurality of surface cues comprises a T-cell stimulatory molecule, a T-cell co-stimulatory molecule, or both a T-cell stimulatory molecule and a T cell co-stimulatory molecule.

61. The method of claim 60, wherein the T-cell stimulatory molecule and / or the T-cell co- stimulatory molecule is loaded onto the SLB.

62. The method of claim 60 or 61, wherein the T-cell stimulatory molecule and / or the T-cell co-stimulatory molecule is loaded via affinity pairing or chemical coupling.

63. The method of claim 62, wherein the affinity coupling comprises a biotin-streptavidin pair, an antibody-antigen pair, an antibody-hapten pair, an affinity pair, a capture protein pair, an Fc receptor-IgG pair, a metal-chelating lipid pair, or a combination thereof.

64. The method of claim 62 or 63, wherein the chemical coupling comprises azide-alkyne chemical (AAC) reaction, dibenzo- cyclooctyne ligation (DCL), tetrazine-alkene ligation (TAL), or any combination thereof.

65. The method of any one of claims 60 to 64, wherein the T-cell stimulatory molecule and / or the T-cell co-stimulatory molecule is coated onto the SLB.

66. The method of any one of claims 60 to 65, wherein the T-cell stimulatory molecule and / or the T-cell co-stimulatory molecule is partly embedded onto the SLB.

67. The method of any one of claims 60 to 66, wherein the T-cell stimulatory molecule and / or the T-cell co-stimulatory molecule is loaded onto the MSR.

68. The method of any one of claims 60 to 67, wherein the T-cell stimulatory molecule and / or the T-cell co-stimulatory molecule comprises antibody molecules or antigen-binding fragments thereof.

69. The method of any one of claims 60 to 68, wherein the T-cell stimulatory molecule comprises an anti-CD3 antibody or an antigen-binding portion thereof, an anti -macrophagescavenger receptor (MSR1) antibody or an antigen-binding portion thereof, an anti-T-cell receptor (TCR) antibody or an antigen-binding portion thereof, an anti-CD2 antibody or an antigen-binding portion thereof, an anti-CD47 antibody or an antigen-binding portion thereof, a major histocompatibility complex (MHC) molecule loaded with an MHC peptide or a multimer thereof, an MHC -immunoglobulin (Ig) conjugate or a multimer thereof, or a combination thereof.

70. The method of any one of claims 60 to 69, wherein the T-cell co-stimulatory molecule comprises an antibody, or an antigen-binding portion thereof, which specifically binds to a costimulatory antigen comprising CD28, 4-1BB (CD137), 0X40 (CD134), CD27 (TNFRSF7), GITR (CD357), CD30 (TNFRSF8), HVEM (CD270), LTfiR (TNFRSF3), DR3 (TNFRSF25), ICOS (CD278), CD226 (DNAM1), CRTAM (CD355),TIM1 (HAVCR1, KIMI), CD2 (LFA2, 0X34), SLAM (CD150, SLAMF1), 2B4 (CD244, SLAMF4), Lyl08 (NTBA, CD352, SLAMF6), CD84 (SLAMF5), Ly9 (CD229, SLAMF3), CRACC (CD319, BLAME), or any combination thereof.

71. The method of any one of claims 60 to 70, wherein the T-cell stimulatory molecule and / or the T-cell co-stimulatory molecule comprises bispecific antibodies or antigen binding portions thereof.

72. The method of any one of claims 60 to 71, wherein the T-cell stimulatory molecule and / or the T-cell co-stimulatory molecule comprises a pair comprising CD3 / CD28, CD3 / ICOS, CD3 / CD27, CD3 / CD137, or a combination thereof.

73. The method of any one of claims 50 to 72, wherein the PCS further comprises an immunoglobulin molecule that binds specifically to an Fc-fusion protein.

74. The method of any one of claims 50 to 73, wherein the PCS further comprises a recruitment compound comprising granulocyte macrophage-colony stimulating factor (GM-CSF), chemokine (C-C motif) ligand 21 (CCL-21), chemokine (C-C motif) ligand 19 (CCL-19), Chemokine (C-X- C Motif) ligand 12 (CXCL12), interferon gamma (IFNy), a FMS-like tyrosine kinase 3 (Flt-3) ligand, or any combination thereof.

75. The method of claim 74, wherein the recruitment compound comprises granulocyte macrophage colony stimulating factor (GM-CSF).

76. The method of any one of claims 50 to 75, wherein the PCS further comprises an antigen.

77. The method of claim 76, wherein the antigen comprises a tumor antigen.

78. The method of claim 77, wherein the tumor antigen is adenomatous polyposis coli protein (APC), adenosine deaminase-binding protein (AD Abp), a-fetoprotein, AFP (alpha-fetoprotein), AIM-2, AIM-3, and WT1), ART1, ART4, B7-H3, B7-H6, BAGE, BCMA, B-cyclin, BMI1, Braf, brain glycogen phosphorylase, BRAP, C13orf24, C6orfl53, C9orf 112, CA-125, CA9 (carbonic anhydrase 9), CASP-8, cathepsin B, Cav-1, CCL-1 (C-C motif chemokine ligand 1), CD123, CD 138, CD171, CD 19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD352, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD45, CD47, CD5, CD56, CD66e, CD70, CD74, CD74, CD79a, CD79b, CD98, cdc27, CDK-1, CDK4, CEA, CEA (carcinoembryonic antigen), c-erbB-2, Claudin 18.2, Claudin 6, c-MET, Colorectal associated antigen (CRC)- C017-1A / GA733, Connexin 37, COX-2, CT-7, cyclophilin b, CYNL2, Dipeptidyl peptidase IV (DPPIV), DLL3 (delta-like protein 3), DLL4, EBV-encoded nuclear antigen (EBNA)-I, E-cadherin, EGFRvIII, ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase family member 3), EpCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, EPHa2 (ephrine receptor A2), EphA2ZEck, ephrinB2, ERBB dimers, ESO-1, estrogen receptor, ETBR (endothelin B receptor), EZH2, FAP-a (fibroblast activation protein a), FBP (a folate binding protein), FCRL5, fetal AchR (fetal acetylcholine receptor), fodrin, Fra-1 / Fosl 1, FR-a (folate receptor alpha), GAGE-1, GAGE-family of tumor antigens, Ganglioside / GD2, GCC (guanyl cyclase C), GD2, GD2 gangliosides, GD3, GLEA2, GM2, GnT-V, GnT-V„ GOLGA, gplOO (glycoprotein 100), gp75, GPC2 (glypican-2), GPC3, gplOO, GPNMB (glycoprotein NMB), GPRC5D (G Protein Coupled Receptor 5D), GUI, H60, hepatitis B surface antigen, HER2, HER3, HER4, HLA-A complexed with peptides derived from AFP, HLA-A1 (human leukocyte antigen Al), HLA-A2 (human leukocyte antigen A2), HMW- MAA (human high molecular weight-melanoma-associated antigen), HSPH1, Ig kappa, Ig lambda, IGF1R (insulin-like growth factor 1 receptor), Ig-idiotype, IL-13Ra2 (IL-13 receptor alpha 2), IL13Ralpha, IL-22Ra (IL-22 receptor alpha), ING4, KDR (kinase insert domain receptor), Ki67, KIAA0376, KRAS, Ku70 / 80, LAGE-I, Lewis Y, LI cell adhesion molecule (LI -CAM), Liv-1, Livin, lmp-1, LRRC8A (leucine rich repeat containing 8 Family member A), MAGE-1, MAGE-2, MAGE-3, MAGE-A, MAGE-A3, MAGE-A6, MART-1 (melan A), MCSP (melanoma-associated chondroitin sulfate proteoglycan), melanoma-associated antigen (MAGE)-Al, mesothelin, MHC / peptide complexes (e.g., MICA, MICB, midkin, MRP-3, MUC16, mucin 1 (MUC1), MUM- 1, murine cytomegalovirus (MCMV), NAG, NCAM (neural cell adhesion molecule), Nectin-4, Nestin, NKG2D (natural killer group 2 member D) ligands, NKTR, NSEP1, NY-ESO, NY-ESO-1, 0LIG2, oncofetal antigen, P1A, p53, PAP, PD-1, PD-L1, pl20ctn, pl5, Pmell 17, PRAME (preferentially expressed antigen of melanoma), progesterone receptor, PR0X1, PSA (prostate specific antigen), PSCA (prostate stem cell antigen ), PSMA (prostate specific membrane antigen), RAE-1 proteins, RAGE, ras, RBPSUH, RCAS1, R0R1, R0R2, RTN4, SART1, SART2, SART3, SCP-I, SIRPa (signal-regulatory protein alpha), SLIT, SLITRK6 (NTRK-like protein 6), Smad family of tumor antigens, SOXIO, SOX11, SOX2, SSX-2 (HOM-MEL-40), SSX-4, SSX-5, SSX- I, SSX-I, STEAP1 (six transmembrane epithelial antigen of the prostate 1), Survivin, survivin, TAG72 (tumor-associated glycoprotein 72), T-cell receptor / CD3-zeta chain, TNKS2, TPBG (trophoblast glycoprotein), TPR, Trop-2, TRP-1, TRP-2, Tyrosinase, U2AF1L, UL16-binding protein-like transcript 1 (Multi), UP AR, VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, WT-1, avP6 or another integrin, P- catenin, pi,6-N, P-catenin, y-catenin, and antigens from HIV, HBV, HCV, HPV, and other pathogens, a patient-specific neoantigen, or an immunogenic peptide thereof, and any combination thereof.

79. The method of any one of claims 50 to 78, wherein a weight ratio of the SLB to the MSR is between about 10: 1 and about 1 :20.

80. The method of any one of claims 50 to 79, wherein the SLB comprises a lipid selected from the group consisting of (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), palmitoyl-oleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylethanolamine (DMPE) and dipalmitoylphosphatidylethanolamine (DPPE), l-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (8:0-14:0 PC), or a combination thereof.

81. The method of any one of claims 50 to 80, wherein the PCS retains a continuous, fluid architecture for at least 14 days.

82. The method of any one of claims 50 to 81, wherein the dry weight ratio of the MSR to the T-cell stimulatory / co-stimulatory molecules is between 1 : 1 to 50:1.

83. The method of any one of claims 1 to 82, wherein the medium further comprises potassium ion at a concentration higher than 5 mM.

84. The method of claim 83, wherein the concentration of the potassium ion is higher than about 10 mM, higher than about 15 mM, higher than about 20 mM, higher than about 25 mM, higher than about 30 mM, higher than about 35 mM, higher than about 40 mM, higher than about 45 mM, higher than about 50 mM, higher than about 55 mM, higher than about 60 mM, higher than about 65 mM, higher than about 70 mM, higher than about 75 mM, higher than about 80 mM, higher than about 85 mM, or higher than about 90 mM.

85. The method of claim 83, wherein the concentration of potassium ion is selected from the group consisting of about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, and about 80 mM.

86. The method of claim 83, wherein the concentration of potassium ion is between about 30 mM and about 80 mM, between about 40 mM and about 80 mM, between about 50 mM and 80 mM, between about 60 mM and about 80 mM, between about 70 mM and about 80 mM, between about 40 mM and about 70 mM, between about 50 mM and about 70 mM, between about 60 mM and about 70 mM, between about 40 mM and about 60 mM, between about 50 mM and about 60 mM, or between about 40 mM and about 50 mM.

87. The method of claim 83, wherein the concentration of potassium ion is about 50 mM, about 60 mM, or about 70 mM.

88. The method of any one of claims 83 to 87, wherein the medium further comprises sodium ion.

89. The method of claim 88, wherein the medium further comprises NaCl.

90. The method of claim 89, wherein the medium comprises less than about 140 mM, less than about 130 mM, less than about 120 mM, less than about 110 mM, less than about 100 mM, less than about 90 mM, less than about 80 mM, less than about 70 mM, less than about 60 mM, less than about 50 mM, or less than about 40 mM NaCl.

91. The method of any one of claims 88 to 90, wherein the medium is hypotonic or isotonic.

92. The method of claim 91, wherein the medium is hypotonic, and wherein the sum of the potassium ion concentration and the sodium ion concentration, multiplied by two is less than 280 mM.

93. The method of claim 91, wherein the medium is hypotonic, and wherein the sum of the potassium ion concentration and the sodium ion concentration, multiplied by two is more than 240 mM and less than 280 mM.

94. The method of claim 91, wherein the medium is isotonic, and wherein the sum of the potassium ion concentration and the sodium ion concentration, multiplied by two is more than or equal to 280 mM and less than 300 mM.

95. The method of any one of claims 88 to 94, wherein the concentration of potassium ion is about 60 mM, and the concentration of NaCl is less than about 80 mM, less than about 75 mM, less than about 70 mM, less than about 65 mM, or less than about 60 mM.

96. The method of any one of claims 88 to 94, wherein the concentration of potassium ion is about 55 mM, and the concentration of NaCl is less than about 85 mM, less than about 80 mM, less than about 75 mM, less than about 70 mM, or less than about 65 mM.

97. The method of any one of claims 88 to 94, wherein the concentration of potassium ion is about 50 mM, and the concentration of NaCl is less than about 90 mM, less than about 85 mM, less than about 80 mM, less than about 75 mM, or less than about 70 mM.

98. The method of any one of claims 83 to 97, wherein the medium further comprises one or more cytokines.

99. The method of claim 98, wherein the one or more cytokines comprise Interleukin-2 (IL- 2), Interleukin-7 (IL-7), Interleukin-21 (IL-21), Interleukin- 15 (IL- 15), or any combination thereof.

100. The method of claim 99, wherein the one or more cytokines comprise IL-2, IL-7, and IL- 15.

101. The method of claim 99 or 100, wherein the medium comprises IL-2 at a concentration from about 50 HJ / mL to about 500 lU / mL.

102. The method of claim 101, wherein the concentration of IL-2 is about 50 lU / mL, about 60 lU / mL, about 70 lU / mL, about 80 lU / mL, about 90 lU / mL, about 100 lU / mL, about 125 lU / mL, about 150 lU / mL, about 175 lU / mL, about 200 lU / mL, about 225 lU / mL, about 250 lU / mL,about 275 lU / mL, about 300 lU / mL, about 350 lU / mL, about 400 lU / mL, about 450 lU / mL, or about 500 lU / mL.

103. The method of claim 101, wherein the concentration of IL-2 is between about 100 lU / mL to about 300 lU / mL.

104. The method of claim 101, wherein the concentration of IL-2 is about 200 lU / mL.

105. The method of any one of clams 99 to 104, wherein the medium comprises IL-21 at a concentration from about 50 lU / mL to about 500 lU / mL.

106. The method of claim 105, wherein the concentration of IL-21 is about 50 lU / mL, about 60 lU / mL, about 70 lU / mL, about 80 lU / mL, about 90 lU / mL, about 100 lU / mL, about 125 lU / mL, about 150 lU / mL, about 175 lU / mL, about 200 lU / mL, about 225 lU / mL, about 250 lU / mL, about 275 lU / mL, about 300 lU / mL, about 350 lU / mL, about 400 lU / mL, about 450 lU / mL, or about 500 lU / mL.

107. The method of claim 105, wherein the concentration of IL-21 is between about 100 lU / mL to about 300 lU / mL.

108. The method of claim 105, wherein the concentration of IL-21 is about 200 lU / mL.

109. The method of any one of claims 99 to 108, wherein the medium comprises IL-7 at a concentration from about 500 lU / mL to about 1,500 lU / mL.

110. The method of claim 109, wherein the concentration of IL-7 is about 500 lU / mL, about 550 lU / mL, about 600 lU / mL, about 650 lU / mL, about 700 lU / mL, about 750 lU / mL, about 800 lU / mL, about 850 lU / mL, about 900 lU / mL, about 950 lU / mL, about 1,000 lU / mL, about 1,050 lU / mL, about 1,100 lU / mL, about 1,150 lU / mL, about 1,200 lU / mL, about 1,250 lU / mL, about 1,300 lU / mL, about 1,350 lU / mL, about 1,400 lU / mL, about 1,450 lU / mL, or about 1,500 lU / mL.

111. The method of claim 109, wherein the concentration of IL-7 is about 1,000 lU / mL to about 1,400 lU / mL.

112. The method of claim 109, wherein the concentration of IL-7 is about 1,200 lU / mL.

113. The method of any one of claims 99 to 112, wherein the medium comprises IL-15 at a concentration from about 50 lU / mL to about 500 lU / mL.

114. The method of claim 113, wherein the concentration of IL-15 is about 50 lU / mL, about 60 lU / mL, about 70 lU / mL, about 80 lU / mL, about 90 lU / mL, about 100 lU / mL, about 125 lU / mL, about 150 lU / mL, about 175 lU / mL, about 200 lU / mL, about 225 lU / mL, about 250 lU / mL, about 275 lU / mL, about 300 lU / mL, about 350 lU / mL, about 400 lU / mL, about 450 lU / mL, or about 500 lU / mL.

115. The method of claim 113, wherein the concentration of IL-15 is between about 100 lU / mL to about 300 lU / mL.

116. The method of claim 113, wherein the concentration of IL-15 is about 200 lU / mL.

117. The method of any one of claims 83 to 116, wherein the medium further comprises a cell expansion agent.

118. The method of claim 117, wherein the cell expansion agent comprises a GSK3B inhibitor, an ACLY inhibitor, a PI3K inhibitor, an AKT inhibitor, or any combination thereof.

119. The method of claim 118, wherein the PI3K inhibitor is selected from hydroxyl citrate, LY294002, pictilisib, CAL101, IC87114, and any combination thereof.

120. The method of claim 118 or 119, wherein the AKT inhibitor is selected from MK2206, A443654, AKTi-VIII, and any combination thereof.

121. The method of any one of claims 83 to 120, wherein the medium further comprises calcium ion, glucose, or any combination thereof.

122. The method of claim 121, wherein the medium further comprises glucose, and wherein the concentration of glucose is more than about 10 mM.

123. The method of claim 122, wherein the concentration of glucose is from about 10 mM to about 40 mM, from about 10 mM to about 35 mM, from about 10 mM to about 30 mM, from about 10 mM to about 25 mM, from about 10 mM to about 20 mM, from about 15 mM to about 40 mM, from about 15 mM to about 35 mM, from about 15 mM to about 30 mM, from about 15 mM to about 25 mM, from about 15 mM to about 20 mM, from about 15 mM to about 19 mM,from about 15 mM to about 18 mM, from about 15 mM to about 17 mM, from about 15 mM to about 16 mM, from about 16 mM to about 20 mM, from about 16 mM to about 19 mM, from about 16 mM to about 18 mM, from about 16 mM to about 17 mM, from about 17 mM to about 20 mM, from about 17 mM to about 19 mM, or from about 17 mM to about 18 mM.

124. The method of claim 122, wherein the concentration of glucose is about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, or about 40 mM.

125. The method of claim 122, wherein the concentration of glucose is about 15.4 mM, about 15.9 mM, about 16.3 mM, about 16.8 mM, about 17.2 mM, or about 17.7 mM.

126. The method of any one of claims 121 to 125, wherein the medium further comprises calcium ion, and wherein the concentration of calcium ion is more than about 0.4 mM.

127. The method of claim 126, wherein the concentration of calcium ion is from about 0.4 mM to about 2.5 mM, from about 0.5 mM to about 2.0 mM, from about 1.0 mM to about 2.0 mM, from about 1.1 mM to about 2.0 mM, from about 1.2 mM to about 2.0 mM, from about 1.3 mM to about 2.0 mM, from about 1.4 mM to about 2.0 mM, from about 1.5 mM to about 2.0 mM, from about 1.6 mM to about 2.0 mM, from about 1.7 mM to about 2.0 mM, from about 1.8 mM to about 2.0 mM, from about 1.2 to about 1.3 mM, from about 1.2 to about 1.4 mM, from about 1.2 to about 1.5 mM, from about 1.2 to about 1.6 mM, from about 1.2 to about 1.7 mM, from about 1.2 to about 1.8 mM, from about 1.3 to about 1.4 mM, from about 1.3 to about 1.5 mM, from about 1.3 to about 1.6 mM, from about 1.3 to about 1.7 mM, from about 1.3 to about 1.8 mM, from about 1.4 to about 1.5 mM, from about 1.4 to about 1.6 mM, from about 1.4 to about 1.7 mM, from about 1.4 to about 1.8 mM, from about 1.5 to about 1.6 mM, from about 1.5 to about 1.7 mM, from about 1.5 to about 1.8 mM, from about 1.6 to about 1.7 mM, from about 1.6 to about 1.8 mM, or from about 1.7 to about 1.8 mM.

128. The method of claim 126, wherein the concentration of calcium ion is about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, or about 2.0 mM.

129. The method of any one of claims 12 to 38 and 44 to 128, wherein the editing comprises introducing a gene editing tool into the immune cells, and wherein the gene editing tool is capable of reducing the expression level of a gene and / or protein associated with impaired immune cell function (e.g., the NR4A family member) in the immune cells.

130. The method of any one of claims 16 to 38 and 48 to 129, wherein the transducing comprises introducing into the immune cells a nucleotide sequence encoding a protein that is associated with improved immune cell function (e.g., the c-Jun polypeptide), such that the expression of the protein (e.g., c-Jun polypeptide) is increased after the introducing.

131. The method of any one of claims 16 to 38 and 48 to 129, wherein the transducing comprises introducing into the immune cells a nucleotide sequence encoding the ligand-binding protein, such that after the introducing, the immune cells express the ligand-binding protein.

132. The method of any one of claims 16 to 38 and 48 to 129, wherein the transducing comprises introducing into the immune cells a first nucleotide sequence encoding a protein that is associated with improved immune cell function (e.g., the c-Jun polypeptide) and a second nucleotide sequence encoding the ligand-binding protein, such that after the introducing, the expression of the protein that is associated with improved immune cell function (e.g., the c-Jun polypeptide) is increased and the immune cells express the ligand-binding protein.

133. The method of claim 132, wherein the first nucleotide sequence and the second nucleotide sequence are within a single vector.

134. The method of any one of claims 16 to 38 and 48 to 129, wherein the transducing comprises introducing into the immune cells a transcriptional activator that is capable of increasing the expression level of an endogenous protein that is associated with improved immune cell function (e.g, c-Jun protein) in the immune cells, such that after the introducing, the expression of the endogenous protein that is associated with improved immune cell function (e.g., c-Jun protein) is increased.

135. The method of claim 134, wherein the transcriptional activator is attached to a Cas protein, which has been modified to lack endonuclease activity.

136. The method of any one of claims 16 to 38 and 48 to 135, wherein the ligand binding protein is selected from a chimeric antigen receptor (CAR), a T cell receptor (TCR), a chimeric antibody- T cell receptor (caTCR), a chimeric signaling receptor (CSR), T cell receptor mimic (TCR mimic), or combinations thereof.

137. The method of claim 136, wherein 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.

138. The method of claim 136 or 137, wherein the ligand binding protein comprises an antigenbinding domain, a transmembrane domain, a costimulatory domain, an intracellular signaling domain, or combinations thereof.

139. The method of claim 138, wherein the antigen-binding domain specifically binds to an antigen selected from the group consisting of R0R1, R0R2, AFP (alpha-fetoprotein), avP6 or another integrin, BCMA, Braf, B7-H3, B7-H6, CA9 (carbonic anhydrase 9), CCL-1 (C-C motif chemokine ligand 1), CD5, CD19, CD20, CD21, CD22, CD23, CD24, CD30, CD33, CD38, CD40, CD44, CD44v6, CD44v7 / 8, CD45, CD47, CD56, CD66e, CD70, CD74, CD79a, CD79b, CD98, CD 123, CD 138, CD171, CD352, CEA (carcinoembryonic antigen), Claudin 18.2, Claudin 6, c- MET, DLL3 (delta-like protein 3), DLL4, ENPP3 (ectonucleotide pyrophosphatase / phosphodiesterase family member 3), EpCAM, EPG-2 (epithelial glycoprotein 2), EPG-40, ephrinB2, EPHa2 (ephrine receptor A2), ERBB dimers, estrogen receptor, ETBR (endothelin B receptor), FAP-a (fibroblast activation protein a), fetal AchR (fetal acetylcholine receptor), FBP (a folate binding protein), FCRL5, FR-a (folate receptor alpha), GCC (guanyl cyclase C), GD2, GD3, GPC2 (glypican-2), GPC3, gplOO (glycoprotein 100), GPNMB (glycoprotein NMB), GPRC5D (G Protein Coupled Receptor 5D), HER2, HER3, HER4, hepatitis B surface antigen, HLA-A1 (human leukocyte antigen Al), HLA-A2 (human leukocyte antigen A2), HMW-MAA (human high molecular weight-melanoma-associated antigen), IGF1R (insulinlike growth factor 1 receptor), Ig kappa, Ig lambda, IL-22Ra (IL-22 receptor alpha), IL-13Ra2 (IL- 13 receptor alpha 2), KDR (kinase insert domain receptor), LI cell adhesion molecule (LI -CAM), Liv-1, LRRC8A (leucine rich repeat containing 8 Family member A), Lewis Y, melanoma- associated antigen (MAGE)-Al, MAGE-A3, MAGE-A6, MART-1 (melan A), murine cytomegalovirus (MCMV), MCSP (melanoma-associated chondroitin sulfate proteoglycan), mesothelin, mucin 1 (MUC1), MUC16, MHC / peptide complexes (e.g., HLA-A complexed withpeptides derived from AFP, KRAS, NY-ESO, MAGE-A, and WT1), NCAM (neural cell adhesion molecule), Nectin-4, NKG2D (natural killer group 2 member D) ligands, NY-ESO, oncofetal antigen, PD-1, PD-L1, PRAME (preferentially expressed antigen of melanoma), progesterone receptor, PSA (prostate specific antigen), PSCA (prostate stem cell antigen ), PSMA (prostate specific membrane antigen), SIRPa (signal-regulatory protein alpha), SLIT, SLITRK6 (NTRK- like protein 6), STEAP1 (six transmembrane epithelial antigen of the prostate 1), survivin, TAG72 (tumor-associated glycoprotein 72), TPBG (trophoblast glycoprotein), Trop-2, VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, and antigens from HIV, HBV, HCV, HPV, and other pathogens, and any combination thereof.

140. The method of claim 139, wherein the antigen-binding domain specifically binds to R0R1.

141. The method of any one of claims 138 to 140, wherein the costimulatory domain 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.

142. The method of claim 141, wherein the costimulatory domain comprises a 4-1BB / CD137 costimulatory domain.

143. The method of any one of claims 138 to 142, wherein the transmembrane domain comprises a transmembrane domain ofKIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rbeta, IL2R gamma, IL7R a, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 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), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKG2D, NKG2C, CD 19, CD8, or any combination thereof.

144. The method of claim 143, wherein the transmembrane domain comprises a CD28 transmembrane domain.

145. The method of any one of claims 138 to 144, wherein the intracellular signaling domain comprises an intracellular signaling domain derived from CD3 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.

146. The method of claim 145, wherein the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain.

147. The method of claim 136, wherein the TCR specifically binds to a tumor antigen / MHC complex.

148. The method of claim 147, wherein the tumor antigen is derived from R0R1, R0R2, AFP, CD19, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPC AM, B7H3, KIT, IL- 13Ra2, mesothelin, IL-1 IRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), Kras, Braf, 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, 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, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT- 2, Fos-related antigen1, p53, p53 mutant, prostein, surviving, telomerase, PCTA- 1 / Galectin 8, MelanA / MARTl, Ras mutant (e.g., HRAS, KRAS, NRAS), 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, human telomerase reverse transcriptase, 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, neoantigen, or any combinations thereof.

149. The method of any one of claims 16 to 38 and 48 to 148, wherein the c-Jun polypeptide is linked to the ligand binding protein by a linker.

150. The method of any one of claims 1 to 149, wherein the immune cells further express a truncated EGFR (EGFRt).

151. The method of claim 150, wherein the immune cells have been modified to comprise a nucleotide sequence encoding the EGFRt.

152. The method of claim 150 or 151, wherein the EGFRt comprises an amino acid sequence having 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% sequence identity to the amino acid sequence set forth in SEQ ID NO: 24.

153. The method of claim 152, wherein the EGFRt comprises the amino acid sequence set forth in SEQ ID NO: 24.

154. The method of any one of claims 150 to 153, wherein the EGFRt is linked to the c-Jun polypeptide and / or the ligand binding protein by a linker.

155. The method of any one of claims 149 to 154, wherein the linker comprises a cleavable linker.

156. The method of claim 155, wherein the linker comprises a P2A linker, a T2A linker, an F2A linker, an E2A linker, a furin cleavage site, or any combination thereof.

157. The method of any one of claims 149 to 156, wherein the linker comprises an amino acid sequence having 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% sequence identity to the amino acid sequence set forth in SEQ ID NO: 14.

158. The method of claim 157, wherein the linker comprises the amino acid sequence set forth in SEQ ID NO: 14.

159. The method of any one of claims 1 to 158, wherein the immune cells are CD3+, CD45RO' , CCR7+, CD45RA+, CD62L+, CD27+, CD28+, or TCF7+, or any combination thereof, following the culturing.

160. The method of any one of claims 1 to 159, wherein the immune cells comprise T cells, B cells, regulatory T cells (Treg), tumor infiltrating lymphocytes (TIL), natural killer (NK) cells, natural killer T (NKT) cells, or any combination thereof.

161. The method of any one of claims 1 to 160, wherein the immune cells have been engineered in vitro or ex vivo.

162. The method of any one of claims 1 to 161, wherein the immune cells were not previously frozen.

163. The method of any one of claims 1 to 161 , wherein the immune cells were previously frozen and subsequently thawed.

164. The method of any one of claims 129 to 163, wherein the gene editing tool comprises a guide RNA, shRNA, siRNA, miRNA, antisense oligonucleotides, CRISPR, zinc finger nuclease, TALEN, meganuclease, restriction endonuclease, or any combination thereof.

165. The method of claim 164, wherein the gene editing tool is CRISPR.

166. The method of any one of claims 12 to 38 and 44 to 165, wherein the NR4A family member comprises NR4A1, NR4A2, NR4A3, or combinations thereof.

167. The method of any one of claims 164 to 166, wherein the gene editing tool comprises a guide RNA comprising, consisting of, or consisting essentially of the sequence set forth in any one of SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 161, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 186, SEQ ID NO: 194, and SEQ ID NO: 196.

168. The method of any one of claims 16 to 38 and 48 to 167, wherein the c-Jun polypeptide comprises an amino acid sequence having at least about 70%, 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%, at least about 99%, or about 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 13.

169. The method of claim 168, wherein the c-Jun polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 13.

170. The method of any one of claims 130 to 169, wherein the nucleotide sequence encoding the c-Jun polypeptide comprises:(a) a nucleic acid sequence having at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 1;(b) a nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 2;(c) a nucleic acid sequence having at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence set forth in SEQ ID NO: 3;(d) a nucleic acid sequence having 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%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 4;(e) a nucleic acid sequence having at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 5;(f) a nucleic acid sequence having at least 88%, at least 89%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 6;(g) a nucleic acid sequence having at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 7;(h) a nucleic acid sequence having at least 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 8;(i) a nucleic acid sequence having at least 55%, 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%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 9;(j) a nucleic acid sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 10; or(k) a nucleic acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% sequence identity to the nucleic acid sequence as set forth in SEQ ID NO: 11.

171. A population of immune cells prepared by the method of any one of claims 1 to 170.

172. A pharmaceutical composition comprising the population of immune cells of claim 171, and a pharmaceutically acceptable carrier.

173. A method of treating or preventing a disease or condition in a subject in need thereof comprising administering to the subject the population of immune cells of claim 171 or the pharmaceutical composition of claim 172.

174. The method of claim 173, wherein the disease or condition comprises a cancer.

175. The method of claim 173 or 174, further comprising administering at least one additional therapeutic agent to the subject.

176. The method of claim 175, wherein the at least one additional therapeutic agent comprises a chemotherapeutic drug, targeted anti-cancer therapy, oncolytic drug, cytotoxic agent, immunebased therapy, cytokine, surgical procedure, radiation procedure, activator of a costimulatory molecule, immune checkpoint inhibitor, a vaccine, a cellular immunotherapy, or any combination thereof.

177. The method of claim 176, wherein the immune checkpoint inhibitor comprises an anti-PD- 1 antibody, anti-PD-Ll antibody, anti -LAG-3 antibody, anti-CTLA-4 antibody, anti-GITR antibody, anti-TIM3 antibody, or any combination thereof.

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