FEEDER CELL REPLACEMENT CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the priority benefit of U.S. Provisional Application Nos. 63/664,692, filed June 26, 2024; 63/709,271, filed October 18, 2024; and 63/725,159, filed November 26, 2024, each of which is incorporated herein by reference in its entirety. FIELD [0002] The present disclosure relates to methods of culturing immune cells, e.g., T cells, NK cells, and/or TILs. BACKGROUND [0003] 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, there is an important stumbling block for immunotherapy: T cells’ ability to kill can fade, a phenomenon often referred to as exhaustion or terminal differentiation of T cells. Immune checkpoint blockade, ex vivo- expanded Tumor-Infiltrating Lymphocytes (TILs) therapy, chimeric antigen receptor (CAR) T cell therapy, and T cell receptor-engineered (TCR) T cell therapy are treatments that make use of functionally active T cells isolated from patients and require highly functional T cells in order to be effective. These T cells are engineered and expanded ex vivo to recognize antigens on target cancer cells. T cell therapies have not been consistently effective at curing solid cancers, in part because the T cells lose their ability to proliferate or kill over time. [0004] TIL expansion traditionally requires coculture with feeder cells, such as irradiated PBMCs. However, the composition and purity of feeder cells can be uncertain. This can lead to potential contamination of the final TIL product. Further, feeder cells are very expensive. As such, there remains a need in the art for methods of expanding TILs while maintaining favorable product quality attributes. BRIEF SUMMARY [0005] Some aspects of the present disclosure are directed to a method of expanding immune cells ex vivo or in vitro comprising culturing the immune cells in a medium comprising a feeder cell replacement (“initial expansion” if an additional expansion is added), wherein the feeder
cell replacement comprises a programmable cell-signaling scaffold (PCS) comprising a surface- exposed CD3 agonist. [0006] In some aspects, the method further comprises culturing the immune cells in an additional medium before or after the culturing (“additional expansion”). In some aspects, the additional expansion is before the initial expansion and does not comprise any feeder cells or feeder cell replacement. In some aspects, the additional expansion is after the initial expansion and comprises culturing the cells in a second medium. [0007] Some aspects of the present disclosure are directed to a method of expanding immune cells ex vivo or in vitro comprising (i) culturing the immune cells in a first medium (“first expansion”) and (ii) culturing the immune cells from (i) in a second medium (“second expansion”), wherein the first medium and/or second medium comprises a feeder cell replacement, wherein the feeder cell replacement comprises a programmable cell-signaling scaffold (PCS) comprising a surface-exposed CD3 agonist. [0008] In some aspects, the feeder cell replacement in the first medium and the feeder cell replacement in the second medium are different. [0009] In some aspects, the method further comprises culturing the immune cells in an additional medium (“additional expansion”) before or after the culturing in the first medium and/or the second medium. In some aspects, the additional expansion is before the first expansion and does not comprise any feeder cells or a feeder cell replacement. [0010] In some aspects, the method further comprises culturing the immune cells in an additional medium (“additional expansion”) between the first expansion and the second expansion. [0011] In some aspects, the medium of the initial expansion does not comprise feeder cells. In some aspects, the second medium does not comprise feeder cells. [0012] In some aspects, one or more of the media comprise potassium ion at a concentration higher than 4 mM. [0013] In some aspects, the medium of the initial expansion further comprises feeder cells. In some aspects, the second medium further comprises feeder cells. [0014] In some aspects, the feeder cells are at an amount less than the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells is less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, or less than about 70% of the amount of reference feeder cells that are required for the
full expansion of the immune cells. In some aspects, the amount of reference feeder cells is between about 1e6 to about 1e10. In some aspects, the amount of reference feeder cells is from about 500e6 to about 8e9. [0015] In some aspects, the amount of the feeder cell replacement is between about 0.01 to about 1 mg feeder cell replacement per 1e6 cells. In some aspects, the amount of the feeder cell replacement is about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, or about 1 mg feeder cell replacement, e.g., PCS, per 1e6 cells. In some aspects, the amount of the feeder cell replacement is about 0.01 to about 1 mg feeder cell replacement per 1e6 cells, and the amount of the feeder cell is less than about 500e6 cells. [0016] In some aspects, 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 loaded onto the scaffold; and wherein the plurality of surface cues comprises (a) the CD3 agonist and (b) one or more of a CD28 agonist, 4-1BBL agonist, an anti-CD2 antibody ("anti-CD2 antibody"), recombinant ICAM1 ("rICAM1"), an anti-SLAM-F6 antibody ("anti-SLAM-F6 antibody"), recombinant OX40L ("OX40L"), recombinant IL-21 ("rIL-21"), an antibody or an antigen-binding portion thereof that binds ICOS ("anti-ICOS antibody"), and a dimer-trimer CD70 polypeptide ("CD70dt"). [0017] In some aspects, the plurality of surface cues is loaded onto the SLB layer. In some aspects, the plurality of surface cues are loaded onto the fluid supported lipid bilayer (SLB). In some aspects, the plurality of surface cues are coated onto the fluid supported lipid bilayer (SLB). In some aspects, the plurality of surface cues are partly embedded onto the fluid supported lipid bilayer (SLB). In some aspects, the plurality of surface cues are loaded onto the mesoporous silica micro-rods (MSR). [0018] In some aspects, the CD3 agonist comprises an anti-CD3 antibody or an antigen- binding portion thereof ("anti-CD3 antibody"). In some aspects, the anti-CD3 antibody comprises OKT3. In some aspects, the CD28 agonist comprises an anti-CD28 antibody or an antigen-binding portion thereof ("anti-CD28 antibody"). In some aspects, the plurality of surface cues comprises (i) an anti-CD3 antibody, (ii) an anti-CD28 antibody, and (iii) a 4-1BB agonist. In some aspects, the plurality of surface cues comprises (i) an anti-CD3 antibody, (ii) an anti-CD28 antibody, (iii) a 4-1BB agonist and (iv) an anti-CD2 antibody or rICAM1. In some aspects, the plurality of surface cues comprises (i) an anti-CD3 antibody, (ii) an anti-CD28 antibody, (iii) a 4-1BB agonist, and
(iv) an anti-CD2 antibody. In some aspects, the plurality of surface cues comprises (i) an anti-CD3 antibody, (ii) an anti-CD28 antibody, (iii) a 4-1BB agonist, (iv) an anti-CD2 antibody, and (v) an anti-SLAM-F6 antibody. In some aspects, the plurality of surface cues comprises (i) an anti-CD3 antibody, (ii) an anti-CD28 antibody, (iii) a 4-1BB agonist, (iv) an anti-CD2 antibody, and (v) CD70dt. In some aspects, the plurality of surface cues comprises (i) an anti-CD3 antibody, (ii) an anti-CD28 antibody, (iii) a 4-1BB agonist, (iv) an anti-CD2 antibody, and (v) rOX40L. In some aspects, the plurality of surface cues comprises (i) an anti-CD3 antibody, (ii) an anti-CD28 antibody, (iii) a 4-1BB agonist, (iv) an anti-CD2 antibody, and (v) rIL21. [0019] In some aspects, the PCS further comprises a plurality of soluble cues. In some aspects, the plurality of surface cues is loaded onto a MSR base layer of the PCS or added directly to the one or more media. [0020] In some aspects, the soluble cue is released from the PCS in a controlled-release manner. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 30 days. [0021] In some aspects, the plurality of soluble cues comprises one or more of the CD3 agonist, a CD28 agonist, a 4-1BB agonist, an anti-CD2 antibody ("anti-CD2 antibody"), recombinant ICAM1 ("rICAM1"), an anti-SLAM-F6 antibody ("anti-SLAM-F6 antibody"), recombinant OX40L ("rOX40L"), recombinant IL-21 ("rIL-21"), and a dimer-trimer CD70 polypeptide ("CD70dt"), IL-1, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12, IL-15, IL-17, IL-18, IL-21, transforming growth factor beta (TGF-β), or an agonist thereof, a mimetic thereof, a variant thereof, and a functional fragment thereof. [0022] 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. [0023] 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 (IFNy), 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). [0024] In some aspects, the PCS comprises about 0.005% to about 1% anti-CD3 antibody, e.g., OKT3. In some aspects, the PCS comprises about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1% anti-CD3 antibody, e.g., OKT3. In some aspects, the PCS comprises about 0.05% anti-CD3 antibody, e.g., OKT3. [0025] In some aspects, the PCS comprises about 0.005% to about 1% anti-CD28 antibody. In some aspects, the PCS comprises about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1% anti-CD28 antibody. In some aspects, the PCS comprises about 0.01% anti-CD28 antibody. [0026] In some aspects, the PCS comprises about 0.005% to about 1% r4-1BBL. In some aspects, the PCS comprises about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1% r4-1BBL. In some aspects, the PCS comprises about 0.15% r4-1BBL. [0027] In some aspects, the PCS comprises about 0.005% to about 1% anti-CD2 antibody. In some aspects, the PCS comprises about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1% anti-CD2 antibody. In some aspects, the PCS comprises about 0.01% anti-CD2 antibody. [0028] In some aspects, the PCS of the initial expansion and the PCS of the additional expansion are the same. In some aspects, the PCS of the initial expansion and the PCS of the additional expansion are different.
[0029] In some aspects, the CD3 agonist comprises an antibody or an antigen-binding portion thereof that specifically binds CD3 ("anti-CD3 antibody"). In some aspects, the anti-CD3 antibody comprises OKT3. [0030] In some aspects, the medium further comprises a CD28 agonist. In some aspects, the CD28 agonist comprises an antibody or an antigen-binding portion thereof that specifically binds CD28 ("anti-CD28 antibody"). [0031] In some aspects, one or more of the media does not comprise a CD3 agonist and/or a CD28 agonist that is not part of the PCS. [0032] In some aspects, the immune cells comprise T cells, TILs, NK cells, TILs, Tregs, or any combination thereof. In some aspects, the TILs are obtained from a human tumor sample. In some aspects, the tumor sample is dissociated prior to the first expansion. In some aspects, the dissociated tumor sample is a single cell suspension. [0033] In some aspects, the immune cells express a chimeric antigen receptor (CAR). In some aspects, the CAR targets CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD70, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL- 13Ra2, mesothelin, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR- 1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-la, MAGE-Al, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT- 2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-1/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, KLK2, STEAP1 (six transmembrane epithelial antigen of the prostate 1), STEAP2, RU1, RU2, intestinal carboxyl esterase, mut hsp70- 2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, CD2, CD3ε, CD4, CD5, CD7, the extracellular portion of the APRIL protein, or any combinations thereof.
[0034] In some aspects, the cells immune cells express an engineered T cell receptor (TCR). In some aspects, the TCR targets AFP, CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL- 13Ra2, mesothelin, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR- 1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-la, MAGE-Al, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT- 2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA- 1/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, 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, CD3ε, CD4, CD5, CD7, the extracellular portion of the APRIL protein, or any combinations thereof. [0035] In some aspects, the initial expansion further comprises contacting the tumor sample with one or more cytokines selected from IL-2, IL-7, IL-15, IL-21, and any combination thereof. In some aspects, the one or more cytokines comprises IL-2 and IL-21. In some aspects, the one or more cytokines comprises IL-2, IL-15, and IL-21. [0036] In some aspects, the PCS further comprises an antigen. In some aspects, the antigen comprises a tumor antigen. [0037] 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), CD123, CD138, CD171, CD19, 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), EphA2/Eck, ephrinB2, ERBB dimers, ESO-1, estrogen receptor, ETBR (endothelin B receptor), EZH2, FAP-α (fibroblast activation protein α), FBP (a folate binding protein), FCRL5, fetal AchR (fetal acetylcholine receptor), fodrin, Fra-l/Fosl 1, FR-α (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, gp100 (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)-A1, 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 l7, PRAME (preferentially expressed antigen of melanoma), progesterone receptor, PROX1, PSA (prostate specific antigen), PSCA (prostate stem cell antigen ), PSMA, PSMA (prostate specific membrane antigen), RAE-1 proteins, RAGE, ras, RBPSUH, RCAS1, ROR1, ROR2, RTN4, SART1, SART2, SART3, SCP-I, SIRPα (signal-regulatory protein alpha), SLIT, SLITRK6 (NTRK-like protein 6), Smad family of tumor antigens, SOX10, 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 (Multl), UPAR, VEGFR1 (vascular endothelial growth factor
receptor 1), VEGFR2, WT-1, αvβ6 or another integrin, β- catenin, β1,6-Ν, β-catenin, γ-catenin, ίίνίηβ, and antigens from HIV, HBV, HCV, HPV, and other pathogens, a patient-specific neoantigen, or an immunogenic peptide thereof, and any combination thereof. In some aspects, the weight ratio of the supported lipid bilayer (SLB) to the mesoporous silica micro-rods (MSR) is between about 10:1 and about 1:20. [0038] In some aspects, the continuous, fluid supported lipid bilayer (SLB) comprises a lipid comprising (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), palmitoyl-oleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylethanolamine (DMPE) and dipalmitoylphosphatidylethanolamine (DPPE), 1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (8:0-14:0 PC), or a combination thereof. [0039] In some aspects, the mesoporous silica microrod-lipid bilayer (MSR-SLB) scaffold retains a continuous, fluid architecture for at least 14 days. In some aspects, the dry weight ratio of the mesoporous silica micro-rods (MSR) to the T-cell activating/co-stimulatory molecules is between 1:1 to 50:1. [0040] In some aspects, the initial expansion lasts about 5-19 days. In some aspects, the TILs are subjected to the initial expansion for less than 11 days. In some aspects, the TILs are subjected to the initial expansion for less than 10 days. In some aspects, the TILs are subjected to the initial expansion for less than 9 days. In some aspects, the TILs are subjected to the initial expansion for about 8 days. In some aspects, the TILs are subjected to the initial expansion for at least about 14 days. [0041] In some aspects, the number of TILs present in the tumor sample is increased by at least about 10-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 following the first expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 20-fold following the first expansion. [0042] In some aspects, the number of TILs following the first expansion is at least about 5 x 107, at least about 1 x 108, at least about 2 x 108, at least about 3 x 108, at least about 4 x 108, at least about 5 x 108, at least about 6 x 108, at least about 7 x 108, at least about 8 x 108, at least about 9 x 108, at least about 1 x 109, at least about 2 x 109, at least about 3 x 109, at least about 4 x 109, at least about 5 x 109, at least about 6 x 109, at least about 7 x 109, at least about 8 x 109, or at least about 9 x 109 TILs.
[0043] In some aspects, the TILs from the initial expansion are subjected to the additional expansion for less than 11 days. In some aspects, the TILs are subjected to the additional expansion for less than 10 days. In some aspects, the TILs are subject to the additional expansion for less than 9 days. In some aspects, the TILs are subjected to the additional expansion for less than 8 days. [0044] In some aspects, the number of TILs present following the initial expansion is increased by at least about 10-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 following the additional expansion. In some aspects, the number of TILs present following the initial expansion is increased by at least about 20-fold following the additional expansion. In some aspects, the number of TILs following the additional expansion is at least about 1 x 109, at least about 2 x 109, at least about 3 x 109, at least about 4 x 109, at least about 5 x 109, at least about 6 x 109, at least about 7 x 109, at least about 8 x 109, at least about 9 x 109, at least about 1 x 1010, at least about 2 x 1010, at least about 3 x 1010, at least about 4 x 1010, at least about 5 x 1010, at least about 6 x 1010, at least about 7 x 1010, at least about 8 x 1010, or at least about 9 x 1010 TILs. [0045] In some aspects, the TILs comprise tumor reactive TILs and non-tumor reactive TILs, wherein the proportion of tumor reactive TILs to non-tumor reactive TILs in the population of expanded TILs is higher than the proportion of tumor reactive TILs to non-tumor reactive TILs in the tumor sample. [0046] In some aspects, the proportion of tumor reactive TILs to non-tumor reactive TILs in the population of expanded TILs is at least about 2-fold, at least about 3-fold, at least about 4- fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, 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 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-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 higher than the proportion of tumor reactive TILs to non-tumor reactive TILs in the tumor sample. In some aspects, the proportion of tumor reactive TILs to non-tumor reactive TILs in the population of expanded TILs is at least about 3-fold higher than the proportion of tumor reactive TILs to non-tumor reactive TILs in the tumor sample. [0047] In some aspects, the TILs comprise CD8+ TILs and CD4+ TILs, wherein the proportion of CD8+ TILs to CD4+ TILs in the population of expanded TILs is higher than the
proportion of CD8+ TILs to CD4+ TILs in the tumor sample. In some aspects, the proportion of CD8+ TILs to CD4+ TILs in the population of expanded TILs is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, 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 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-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 higher than the proportion of CD8+ TILs to CD4+ TILs in the tumor sample. In some aspects, the proportion of CD8+ TILs to CD4+ TILs in the population of expanded TILs is at least about 3-fold higher than the proportion of CD8+ TILs to CD4+ TILs in the tumor sample. [0048] In some aspects, the tumor sample is obtained from a subject that has previously received one or more therapy for treating the tumor. In some aspects, the subject is relapsed or refractory to one or more prior therapy for treating the tumor. In some aspects, the one or more therapy for treating the tumor is selected from a chemotherapy, an immunotherapy, a radiotherapy, a surgery, or any combination thereof. [0049] In some aspects, the tumor is refractory to a checkpoint inhibitor. In some aspects, the checkpoint inhibitor comprises a PD-1 antagonist, a CTLA-4 antagonist, a TIM3 antagonist, a GITR antagonist, a KIR antagonist, a LAG3 antagonist, or any combination thereof. In some aspects, the checkpoint inhibitor comprises an anti-PD1 antibody, an anti-PD-L1 antibody, an anti- CTLA-4 antibody, an anti-TIM3 antibody, an anti-GITR antibody, an anti-KIR antibody, an anti- LAG3 antibody, or any combination thereof. [0050] In some aspects, the tumor is metastatic. In some aspects, the tumor comprises a solid tumor. In some aspects, the solid tumor is derived from a melanoma, a colon cancer, a lung cancer, a cervical cancer, a gastrointestinal cancer, a breast cancer, a prostate cancer, a liver cancer, bone cancer, a pancreatic cancer, a small cell carcinoma of the head and neck, lung squamous cell carcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, testicular germ cell tumors, stomach adenocarcinoma, skin cutaneous melanoma, mesothelioma, kidney renal clear cell carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, esophageal carcinoma, bladder urothelial carcinoma, breast invasive carcinoma, kidney renal papillary cell carcinoma, colon adenocarcinoma, or any combination thereof.
[0051] In some aspects, the concentration of potassium ion in the one or more media is 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, at least about 80 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 concentration of potassium ion in the one or more media is about 55 mM to about 90 mM. In some aspects, the concentration of potassium ion in the one or more media is about 55 mM to about 60 mM, about 60 mM to about 65 mM, about 65 mM to about 70 mM, about 70 mM to about 75 mM, about 80 mM to about 85 mM, or about 85 mM to about 90 mM. In some aspects, the concentration of potassium ion in the one or more media is about 55 mM, about 60 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, or about 90 mM. [0052] In some aspects, one or more of the media further comprise sodium ion, calcium ion, glucose, or any combination thereof. [0053] In some aspects, the one or more media further comprise sodium ion. In some aspects, the concentration of the sodium ion is from about 25 mM to about 100 mM. In some aspects, the concentration of the sodium ion is from 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 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, or about 80 mM. In some aspects, the concentration of the sodium ion is about 55 mM. In some aspects, the concentration of the sodium ion is about 60 mM. In some aspects, the concentration of the sodium ion is about 65 mM. [0054] In some aspects, the one or more media further comprise 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
comprises LY294002, pictilisib, CAL101, IC87114, or any combination thereof. In some aspects, the AKT inhibitor comprises MK2206, A443654, AKTi-VIII, or any combination thereof. [0055] In some aspects, the one or more media further comprise glucose. In some aspects, the concentration of glucose is more than about 10 mM. In some aspects, the concentration of glucose is from about 10 mM to about 25 mM, about 10 mM to about 20 mM, 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 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, or about 25 mM. [0056] In some aspects, the one or more media further comprise calcium ion. In some aspects, 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, 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 1.2 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 about 1.8 mM, or 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. [0057] In some aspects, the one or more media comprises about 60 mM to about 90 mM potassium ion and (i) about 40 mM to about 80 mM sodium ion; (ii) about 10 mM to about 24 mM glucose; (iii) about 0.5 mM to about 2.8 mM calcium ion; or (iv) any combination of (i)-(iii). [0058] Some aspects of the present disclosure are directed to a population of expanded immune cells prepared by a method disclosed herein.
[0059] Some aspects of the present disclosure are directed to a pharmaceutical composition comprising a population of expanded immune cells disclosed herein, and a pharmaceutically acceptable carrier. [0060] Some aspects of the present disclosure are directed to a method of killing target cells, comprising contacting the target cells with a population of expanded immune cells disclosed herein or a pharmaceutical composition disclosed herein under conditions that allow killing of the target cells by the immune cells. [0061] Some aspects of the present disclosure are directed to a method of treating a patient in need thereof, comprising administering a population of expanded TILs disclosed herein or a pharmaceutical composition disclosed herein to the patient. [0062] Some aspects of the present disclosure are directed to a use of a population of human expanded TILs disclosed herein for the manufacture of a medicament for treating a patient in need thereof. [0063] Some aspects of the present disclosure are directed to a culture medium, comprising one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. [0064] In some aspects, the culture medium a modified basal medium, wherein the basal medium further comprises one or more of the CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the basal medium is 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), M199, OpTmizer™ CTS™ T-Cell Expansion Basal Medium (ThermoFisher), OPTMIZER™ Complete, IMMUNOCULT™ XF (STEMCELL™ Technologies), IMMUNOCULT™ XF, AIM V, TEXMACS™ medium, or any combination thereof. In particular aspects, the basal media comprises OPTMIZER™ complete. In some aspects, suitable basal medium includes Click's medium, OpTimizer® (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, RPMI-1640 medium, or any combination thereof. In some aspects, the culture medium is serum free. In some aspects, the culture medium further comprises immune cell serum replacement (ICSR). In some aspects, the medium comprises a MRM. [0065] In some aspects, the culture medium comprises CCL5. In some aspects, the culture medium comprises CXCL10. In some aspects, the culture medium comprises IL-18. In some
aspects, the culture medium comprises IL-8. In some aspects, the culture medium comprises SLAMF-1. [0066] In some aspects, the culture medium comprises two, three, or four of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the culture medium comprises CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the culture medium further comprises one or more of IL-2, IL-17, IL-15, and IL-21, in any combination. BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES [0067] FIG.1A is a combined heat map and table illustrating the MFI values and relative expression of various differentiation, Fc receptors, and costimulatory/adhesion molecules on the APC population obtained from bulk donor feeder populations. Red outlines highlight molecules that were expressed highly on the putative APC population of feeder cells and were used in subsequent PCS formulations. Blue outlines highlight molecules that were not expressed on the screened feeder cells but were identified from other sources as potentially important T cell activation signals and which were tested in subsequent PCS formulations. Data shows the log transform of the mean fluorescent intensity of each marker. Negative mean fluorescent intensity values were omitted from the heat map. [0068] FIGs.1B and 1C are schematics showing illustrative experimental designs used to assess the impact of feeder cell replacement in TIL manufacturing, as well as the metrics used to evaluate the TIL product. The illustrated manufacturing protocols incorporate MRM protocol backbone with multiple rapid expansion protocol (REP) expansion stages. The schematic in FIG. 1B reflects an illustrative design with an 8-day REP1 and an 8-day REP2 (i.e., a 16 day expansion), whereas FIG.1C reflects an additional design with elongated REP ranges, namely 10- or 11-day REP1 with an optional extension up to 18 days, and a 10- or 11-day REP2 (i.e., a 20- to 21-day expansion with optional extension of REP1). [0069] FIG.2 shows the fold expansion of TIL after REP2 Day 8 stimulated in REP2 with either feeder cells or PCS formulations presenting aCD3, aCD28, and r41BBL in different densities and stoichiometries. Formulation PCS-12 demonstrates equivalent or improved TIL expansion compared to feeder cells. The fold expansion was calculated by dividing the total number of viable TIL on REP2 Day 8 by the total number of TIL seeded on REP2 Day 0. Each dot represents a different tumor. Different histologies are aggregated in this graph (comprised of metastatic melanoma, NSCLC, Breast, and CRLM).
[0070] FIG.3 shows the fold expansion of TIL after REP2 Day 8 stimulated in REP2 with either feeder cells or PCS formulations comprised of the PCS-12 base with an additional adhesion/costimulatory signal presented at different densities and stoichiometries (rICAM1 and aCD2). This graph highlights the addition of aCD2 for additional adhesion or costimulation enhances the proliferation of TIL on the PCS-12 base. The fold expansion was calculated by dividing the total number of viable TIL on REP2 Day 8 by the total number of TIL seeded on REP2 Day 0. Each dot represents a different tumor. Different histologies are aggregated in this graph (comprised of metastatic melanoma, NSCLC, Breast, and CRLM). Significance was calculated using a One-Way ANOVA. [0071] FIG.4 shows the fold expansion of TIL after REP2 Day 8 stimulated in REP2 with either feeder cells or PCS formulations comprised of the PCS-1 base with additional signal molecules. This graph shows that presenting additional signal molecules on top of the PCS-1 base (comprising aCD3, aCD28, r41bbL, and aCD2) does not further increase proliferation of TIL stimulated with PCS. The fold expansion was calculated by dividing the total number of viable TIL on REP2 Day 8 by the total number of TIL seeded on REP2 Day 0. Each dot represents a different tumor. Different histologies are aggregated in this graph (comprised of metastatic melanoma, NSCLC, Breast and CRC). Significance was calculated using a One-Way ANOVA. [0072] FIGs.5A and 5B illustrate optimization of stimulatory ligands on PCS formulations based on CD8+ TIL expansion. Different formulations of PCS were used to screen for CD+ T-cell expansion in 5-6 metastatic melanoma (open circle) or NSCLC (open square) tumors. The expansion methodologies used are reflected in the scheme of either FIG.1B or 1C. Lines denote matching products derived from the same tumor sample. Normalized CD8+ TIL expansion was calculated by multiplying the expansion of CD8% of the PCS product and dividing by the equivalent metric of the feeder-expanded TIL. FIG. 5A graphically illustrates preliminary experiments identifying PCS-12, which combined aCD3, aCD28, and r41BBL, as a potent activator of TIL. Upon further evaluation, the addition of aCD2 improved expansion while maintaining high CD8+ skew in the final TIL product (PCS-1). The “*” symbol designates that the formulation of PCS was used multiple times but expansion was not sufficiently high to obtain a CD8+ percentage from the product. FIG. 5B graphically illustrates titration of aCD2 in PCS formulations (PCS-1, PCS2, and PCS-3) to optimize aCD2 intensity. Stronger aCD2 intensities on PCS resulted in lower CD8+ skew in the final product, with respect to matched samples. PCS-1 was identified as an optimal formulation for feeder replacement and was used as the primary formulation for additional analyses.
[0073] FIGs. 6A-6C illustrate TIL expansion using various PCS formulations in REP2 compared to expansion using feeder cells. FIG.6A shows the CD8+ frequency of TIL products that were stimulated in REP2 with PCS compared to feeder cells. Titrating aCD2 on the PCS-12 base impacts the CD8+ frequency in the TIL product, as indicated in FIG. 5B, above. FIG. 6B shows a comparison of fold expansion between metastatic melanoma-derived TIL products activated with PCS and matched samples activated with feeder cells in REP2 according to the expansion scheme illustrated in FIG.1C. Using a paired t-test with n=14, no statistical difference was observed. FIG. 6C compares metastatic melanoma-derived TIL CD8% between products activated with PCS and those activated with feeder cells in REP2, according to the expansion scheme illustrated in FIG. 1C. Using a paired t-test with n=14, no statistical difference was observed. [0074] FIGs. 7A-7L show the stem-like characteristics of the CD8+ T cells in the TIL product after expansion with PCS-1 or feeder cells in the REP2 process. FIGs. 7A-7D show the percentage of CD8+ T cells that are CD39-CD69- for the following cancers: melanoma (FIG.7A), NSCLC (FIG. 7B), CRLM (FIG. 7C), or breast (FIG. 7D). FIGs. 7E-7H show the percentage of CD8 T cells that are CD27+ for the following cancers: melanoma (FIG. 7E), NSCLC (FIG. 7F), CRLM (FIG. 7G), or breast (FIG. 7H). FIGs. 7I-7L show the percentage of CD8+ T cells that are CD27+CD62L+ for the following cancers: melanoma (FIG. 7I), NSCLC (FIG. 7J), CRLM (FIG. 7K), or breast (FIG. 7L). Each symbol represents TIL expanded from a unique tumor of a given histology. Lines connecting symbols represent the PCS expanded compared to feeder expanded TIL of the same tumor produced in the same production run. The circle symbol represents TIL that received costimulatory signals aCD3 and 4-1BBL with feeder cells in the REP1 process. The square symbol represents TIL that received costimulatory signals aCD3, 4-1BBL, and others (see FIG. 13 and 13B) along with feeder cells in the REP1 process. [0075] FIGs. 8A-8D are graphical representations of data derived from an additional analysis showing that melanoma-derived The TIL for this analysis were produced according to the expansion scheme illustrated in FIG. 1C. FIG. 8A compares a stemlike phenotype (CD39-CD69-) in CD8+ T cells between products activated with PCS-1 and those activated with feeder cells in REP2. Using a paired t-test with n=14, no statistical difference was observed. FIG. 8B compares a terminally differentiated phenotype (CD39+CD69+) on CD8+ T cells between products activated with PCS-1 and those activated with feeder cells in REP2. Using a paired t-test with n=14, no statistical difference was observed. FIG. 8C compares a stemlike phenotype (CD62L+CD27+) on CD8+ T cells between
products activated with PCS-1 and those activated with feeder cells in REP2. Using a paired t-test with n=14, no statistical difference was observed. FIG.8D compares a stemlike marker (CD27+) on CD8+ T cells between products activated with PCS-1 and those activated with feeder cells in REP2. Using a paired t-test with n=14, no statistical difference was observed. [0076] FIGs. 9A-9F show the flow cytometry gating strategy for a representative melanoma sample in the data presented in FIG.6A. [0077] FIGs.10A-10E show the Simpson clonality of TIL products expanded with PCS-1 in REP2 compared to TIL products expanded with feeder cells in REP2. Lower Simpson clonality represents a more diverse TCR repertoire in the TIL product. Individual symbols represent different tumors from respective histologies [melanoma (FIG.10A), NSCLC (FIG.10B), CRLM (FIG.10C), or breast (FIG.10D)], lines between symbols represent matched Simpson clonalities between the PCS and feeder-expanded TIL of the same tumor. FIG. 10E shows an additional analysis of Simpson clonality of melanoma-derived TIL products that were activated either with PCS-1 or feeder cells in REP2, according to the expansion scheme illustrated in FIG. 1C. The comparably low Simpson clonality suggests PCS is expanding a polyclonal product (n=10). Lines showed paired data sets derived from the same tumor sample. [0078] FIGs.11A-11E show the abundance of TCRs in the TIL product compared to the top 200 clones in the original tumor normalized to 30,000 template reads. In this graph, the top 200 clones are stratified into segments of 50 clones and their subsequent distribution is shown in the TIL product of the four different histologies tested: melanoma (FIG. 11A), CRLM (FIG. 11B), breast (FIG. 11C), or NSCLC (FIG. 11D). The circle symbol represents the top 1-50 clones in terms of abundance from the original tumor. The square symbol represents clones 51-100, the triangle represents clones 101-150, and the diamond represents clone 151-200. FIG.11E shows an additional analysis of the estimated number of PTRCs in melanoma-derived TIL products activated with either PCS1 or feeder cells in REP2, according to the expansion scheme illustrated in FIG. 1C. A paired t-test was run and no statistical difference was observed (n=10). Estimated PTRCs was calculated by extrapolating the number of PTRCs detected to a clinical scale production. Lines connect paired data sets derived from the same tumor sample. [0079] FIGs. 12A-12D summarize the sum productive frequency of the top 200 clones from the tumor sample and the number of top 200 clones from the tumor sample retained in the TIL products expanded with either PCS or feeder cells in REP2. The sum productive frequency represents the total percentage of TCRs in the TIL product that are from the top 200 in the original tumor [melanoma (FIG.12A), CRLM (FIG.12B), breast (FIG.12C), or NSCLC (FIG.12D)]. The
total clones retained metric describes the number of the top 200 clones from the original tumor in the TIL product at any frequency. [0080] FIGs. 13A-13B summarize the metrics used to evaluate TIL products produced using PCS-1 in REP2. FIG.13A shows TIL samples expanded with aCD3 and r4-1BBL in REP1 (along with feeder cells). FIG. 13B shows TIL samples expanded with additional costimulatory ligands in REP1 (along with feeder cells). Regardless of the costimulatory ligands used in REP1, PCS-1 in REP2 maintains or increases the proliferation, CD8+ percentage of CD3+ cells, and stem- like quality of the TIL product compared to feeder cells in REP2. In "stemness phenotype” and “clone retention” columns, green cells signify PCS TIL product was similar to feeder cell product and yellow cells signify there were modest differences in some compartments. [0081] FIGs.14A-14C show the expansion of TIL utilizing PCS-1 in a feeder-free process in the MRM two-REP process after REP1 (FIG.14A), REP2 (FIG.14B), and overall (FIG.14C) final expansion. A standard feeder cell arm using the MRM two-REP process was used as a control. The total fold expansion was calculated by multiplying the fold expansion in REP1 by the fold expansion in REP2. [0082] FIGs.15A-15B show the CD8+ frequency of TIL products that were stimulated with PCS-1 in both REP1 (FIG. 15A) and REP2 (FIG. 15B) (feeder-free process) compared to TIL products stimulated using feeders in both REP1 (FIG. 15A) and REP2 (FIG. 15B). PCS-1- expanded TIL showed similar CD8+ frequency as feeder-expanded TIL at the end of REP1 (FIG. 15A) and REP2 (FIG.15B) (TIL product). Each symbol represents TIL expanded from a unique melanoma tumor. Lines connecting symbols represent the PCS expanded compared to feeder expanded TIL of the same tumor. [0083] FIGs. 16A-16C show the stem-like characteristics of the CD8+ T cells in the TIL product after expansion in a feeder free (PCS-1) or feeder MRM two-REP process. FIG.16A shows the percentage of CD8+ T cells that are CD39-CD69-. This population represents a regenerative source of non-terminally differentiated cells that are capable of persisting in the tumor. FIG.16B shows the percentage of CD8+ T cells that are CD27+. These cells are capable of being activated via the CD27 receptor for survival and persistence. FIG.16C shows the percentage of CD8+ T cells that are CD27+CD62L+, which represent a population of memory-like T cells. Each symbol represents TIL expanded from a unique melanoma tumor. Lines connecting symbols represent the PCS expanded compared to feeder expanded TIL of the same tumor produced in the same production run.
[0084] FIG. 17 shows the fold expansion of TIL after REP2 stimulated in REP2 with a PCS formulation presenting aCD3, aCD28, aCD2 and r41BBL (PCS-1) or a PCS formulation presenting aCD3 and aCD28 (PCS-18). The fold expansion was calculated by dividing the total number of viable TIL on REP2 at the end of production by the total number of TIL seeded on REP2 Day 0. Each dot represents a different tumor. Round dots represent tumors that were stimulated at a 1:10 (TIL:feeder) in REP1. Square dots represent tumors that were stimulated at a 1:25 (TIL:feeder) in REP1. [0085] FIG.18 is a schematic showing the PCS technology to replace feeder cell use in a two-REP TIL expansion process. [0086] FIG.19 is a box plot showing the TIL product yield in an MRM two-REP process is comparable when using either PCS-1 or feeder cells for TIL stimulation in “expansion 2”.2.5 x 105 TIL were stimulated with PCS-1 and 5 x 106 TIL were stimulated in the feeder cell condition. Total fold expansion, the product of expansions in the “expansion 1” and “expansion 2” steps, is shown in a box and whisker plot with median expansion listed for each condition. Statistical significance was determined via paired t-test. ns, not significant. [0087] FIGs.20A-20C compare expansion of TIL in a control feeder-based process (i.e., a MRM-based two-REP process as illustrated in FIGs. 1B and 1C) to the use of PCS-1 feeder replacement in the REP2 stage at either a scaled-up 6M expansion format or a research-scale 24W TC expansion format. Expansions were compared for TIL derived from NSCLC (FIG. 20A), melanoma (FIG. 20B), and CRC/CRLM (FIG.20C). The box in FIG. 20A highlights a subset of NSCLC tumors in the 6M format that demonstrated lower expansion with PCS-1 feeder replacement formulation. [0088] FIGs.21A-21D show the clonality and estimated representation of putative tumor reactive cells in TIL produced at large scale using PCS feeder replacement (i.e., the PCS-1 formulation) in REP2 in comparison to a feeder-based control process. FIGs. 21A and 21B specifically show the fraction of the TIL products that are comprised of the top 20% of T cell clones from the original NSCLC (FIG.21A) and melanoma tumors (FIG.21B). Mean fraction is shown above each data set. FIGs.21C and 21D show the number of putative tumor reactive cells in a clinical scale production as estimated by multiplying the cells that go into REP2 (1.5e9) by the expansion in REP2 previously observed at research scale by the fraction of the top 20% of the product observed at research scale. [0089] FIGs. 22A-22C show the reactivity of a melanoma (FIG. 22A) and two NSCLC (FIGs.22B-22C) TIL products in co-culture assays. Reactivity was inferred by presence of CD3+
cells with upregulated 4-1BB (CD137) expression upon co-culture of the TIL product with CD45- depleted gMACs samples from the same tumor from which the respective TIL were derived (TIL + gMACS). The co-culture was also run in the presence of a TCR block as an additional control (TIL + gMACS + block). [0090] FIGs. 23A-23D are graphs demonstrating that supplementing PCS-1 feeder replacement expansion culture with feeder-secreted proteins (FSPs) at the start of REP2 increases the expansion of TIL from both melanoma (circles) and NSCLC (square) compared to a control of PCS-1 without FSP supplement, while maintaining similar CD8+ representation in the final product. FIGs. 23A and 23B show the TIL expansion after REP2 and the percent of CD8 in the final expanded product, respectively, in fresh samples. FIGs.23C and 23D show the TIL expansion after REP2 and the percent of CD8 in the final expanded product, respectively, in previously frozen samples. [0091] FIGs.24A-24D show TIL expansion characteristics using two different PCS feeder replacement formulations, PCS-1 and PCS-21 (PCS-1 plus anti-ICOS antibody), and a feeder- based control in either fresh (FIGs. 24A and 24B) or previously frozen (FIGs. 24C and 24D) samples. FIGs.24A and 24C illustrate expansion of TIL after the REP2 stage using the PCS feeder replacement formulations or control, and FIGs. 24B and 24D illustrate the percentage of CD8+ cells in the final expanded products. Increased TIL expansion was observed when utilizing PCS- 21 compared to both PCS-1 feeder replacement and feeder-based MRM control in the REP2 expansion protocol. Both feeder replacement formulations were implemented in the assay with the supplementation of the FSPs into the REP2 media. No loss in CD8+ skew in the final TIL product was observed with the addition of the anti-ICOS signal in the PCS-21 group. The TIL were expanded from either melanoma or NSCLC tumors. Individual data points are represented by diamonds representing highly activated tumors or circles representing lowly activated tumors, based on CD39+PD-1+ status of the cells at day zero of the process. [0092] FIGs.25A-25D illustrate the stemlike qualities of the TIL product produced in the 6M format using FSPs and feeder replacement formulations PCS-1 and PCS-21, or in the feeder- based MRM control, which did not include FSP supplement. No changes in stemlike phenotype was observed with the addition of the anti-ICOS antibody into the feeder replacement formulation or the inclusion of FSPs, indicating an equivalent product phenotype profile using the feeder replacement formulations. [0093] FIGs. 26A and 26B show that overall putative tumor reactivity is retained in the TIL product when replacing feeder cells with PCS-21 and supplementing the media with the FSP
supplements. The dataset represents a mix of both melanoma and NSCLC tumors. FIG. 26A illustrates the fraction of the top 20% of clones from the original tumor in the TIL products expanded with feeder cells in the REP2 stage (feeder control) compared to TIL expanded with PCS-21 + FSPs in the REP2 stage (PCS-21). FIG. 26B shows the estimated number of putative tumor reactive cells in a therapeutic dose calculated by multiplying the REP2 fold expansion by the 1.5 billion cells (i.e., number of cells seeded into REP2) by the fraction of the top 20% of the product measured via TCR sequencing. [0094] FIGs.27A and 27B graphically represent expansion characteristics of TIL after the REP2 stage using the PCS feeder replacement formulation PCS-22 in REP2. FIG.27A illustrates higher REP2 stage expansion over cultures of TIL using the PCS-21 formulation of feeder replacement or feeder control. FIG.27B indicates maintenance of a high proportion of CD8+ cells after the REP2 expansion, although the PCS-22 formulation resulted in slightly lower proportion of CD8+ cells compared to TIL expanded with PCS-21 or feeder control. DETAILED DESCRIPTION [0095] Some aspects of the present disclosure are directed to a method of expanding immune cells ex vivo or in vitro comprising culturing the immune cells in a medium comprising a feeder cell replacement (“initial expansion” if an additional expansion is added), wherein the feeder cell replacement comprises a programmable cell-signaling scaffold (PCS) comprising a surface- exposed CD3 agonist. In some aspects, the method further comprises culturing the immune cells in an additional medium before or after the culturing (“additional expansion”). In some aspects, the additional expansion is before the initial expansion and does not comprise any feeder cells or feeder cell replacement. In some aspects, the additional expansion is after the initial expansion and comprises culturing the cells in a second medium. [0096] Some aspects of the present disclosure are directed to a method of expanding immune cells ex vivo or in vitro comprising (i) culturing the immune cells in a first medium (“first expansion”) and (ii) culturing the immune cells from (i) in a second medium (“second expansion”), wherein the first medium and/or second medium comprises a feeder cell replacement, wherein the feeder cell replacement comprises a programmable cell-signaling scaffold (PCS) comprising a surface-exposed CD3 agonist. In some aspects, the feeder cell replacement in the first medium and the feeder cell replacement in the second medium are different. In some aspects, the method further
comprises culturing the immune cells in an additional medium (“additional expansion”) before or after the culturing in the first medium and/or the second medium. [0097] The methods disclosed herein can be used in the culture of any immune cells that heretofore typically included the use of feeder cells. Immune cells that can be cultured according to the methods disclosed herein include, but are not limited to, T cells, natural killer (NK) cells, and tumor infiltrating lymphocytes. In some aspects, the immune cells comprise helper T-cells (CD4+ T cells), cytotoxic T-cells (CD8+ T cells), memory T-cells (CD45RO+ T cells), suppressor T-cells (Ts cells), regulatory T-cells (Tregs), natural killer T-cells (NKT cells), mucosal associated invariant (MAITs), gamma delta T cells, (γδ T cells), or any combination thereof. [0098] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to the particular compositions or process steps described, as such can, of course, vary. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. [0099] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. I. Terms [0100] 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. [0101] 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.
[0102] 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). [0103] 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. [0104] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure. [0105] Units, prefixes, and symbols are denoted in their Système International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, unless otherwise explicitly stated. [0106] Abbreviations used herein are defined throughout the present disclosure. Various aspects of the disclosure are described in further detail in the following subsections. [0107] 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%, 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, as used herein, when "about" is used to modify a concentration (e.g., by weight or percent) or an amount (e.g., by weight or percent) of an agent, "about" refers to plus or minus 10% of the value. For example "about 55 mM," as used herein, includes 49.5 mM 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. [0108] 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). [0109] 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. [0110] The term "control media" as used herein refers to any media in comparison to the metabolic reprogramming media ("MRM") disclosed herein. Control media can comprise the same components as the metabolic reprogramming media except certain ion concentrations, e.g, potassium ion. In some aspects, metabolic reprogramming media described herein are prepared from control media by adjusting one or more ion concentrations, e.g., potassium ion concentration, as described herein. In some aspects, control media comprise basal media, e.g., CTS™ OPTIMIZER™. In some aspects, control media comprise AIM V, RPMI, or a mixture comprising AIM V and RPMI. In some aspects, control media comprise (i) 50% AIM V, (ii) 50% RPMI1640, (iii) 5% or 10% human serum, and (iv) IL-2. In some aspects, control media thus comprises one or more additional components, including, but not limited to, amino acids, glucose, glutamine, T cell stimulators, antibodies, substituents, etc. that are also being added in the metabolic reprogramming media, but control media have certain ion concentrations different from the metabolic reprogramming media. Unless indicated otherwise, the terms "media" and "medium" can be used interchangeably. [0111] As used herein, the term "immune cell" refers to a cell of the immune system. In some aspects, the immune cell comprises a T lymphocyte ("T cell"), B lymphocyte ("B cell"), natural killer (NK) cell, regulatory T cell ("Treg"), macrophage, eosinophil, mast cell, dendritic cell or neutrophil, or any combination thereof. In some aspects, the immune cell is a tumor-
infiltrating cell (TIL). As used herein, a "TIL" refers to T cell that has at least once entered into a tumor or is capable of entering a tumor, e.g., within the parenchyma of a tumor. In some aspects, the tumor is a solid tumor. In some aspects, the tumor is a liquid tumor, e.g., a hematopoietic cancer. TILs prepared by the present methods can have one or more properties that are the same as the naturally occurring TILs. In some aspects, TILs prepared by the present methods have one or more properties that are not present in the naturally occurring TILs. TILs can be obtained using any methods. In some aspects, the TILs are obtained from a tumor sample from a subject. In some aspects, the tumor sample, or a portion thereof, is cultured under conditions that promote evasion of the TILs from the tumor tissue, proliferation of the TILs, and/or expansion of the TILs. In some aspects, the medium used to promote evasion, proliferation, and/or expansion of the TILs is any metabolic reprogramming medium, e.g., hyperkalemic medium, disclosed herein. [0112] In some aspects, the term "immune cells" refers to helper T-cells (CD4+ T cells). In some aspects, the term "immune cells" refers to cytotoxic T-cells (CD8+ T cells). In some aspects, the term "immune cells" refers to memory T-cells (CD45RO+ T cells). In some aspects, the term "immune cells" refers to suppressor T-cells (Ts cells). In some aspects, the term "immune cells" refers to regulatory T-cells (Tregs). In some aspects, the term "immune cells" refers to natural killer T-cells (NKT cells). In some aspects, the term "immune cells" refers to mucosal associated invariant (MAITs). In some aspects, the term "immune cells" refers to gamma delta T cells (γδ T cells). [0113] 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., T cell, NK cell, and/or TIL. In some aspects, the population of cells comprises more than one TILs, e.g., a plurality of TILs. 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, e.g., TILs and cells other than TILs. In some aspects, the population of cells comprises a population comprising one or more cells that have been selected based on a particular cell characteristic. In some aspects, the population of cells comprises a population comprising one or more cells that do not include cells that have been selected based on a particular cell characteristic. [0114] TILs include, but are not limited to, CD8+ T cells (i.e. cytotoxic T cells), CD4+ T cells, B cells, and natural killer cells. TILs include both primary (e.g., obtained from a patient tissue sample) and secondary TILs (e.g., TIL cell populations that have been cultured, expanded or proliferated from primary TILs. In some aspects the TILs are genetically modified. In some
aspects, the TIL is a CD8+ T cell. CD8+ TILs are generally considered to be the subpopulation of TILs responsible for destroying cancer cells. Conversely, CD4+ TILs are generally considered to act as suppressors of the immune response, which can limit the immune response against the tumor. [0115] In some aspects, TILs can be defined biochemically using cell surface markers. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR αβ, CD27, CD28, CD56, CCR7, CD45RA, CD95, PD-1, and CD25. In some aspects, TILs can be defined functionally by their ability to infiltrate tumors and selectively kill the cancer cells. [0116] 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 (such as CD8+ T cell) and Th cells (such as CD4+ T cells). In some aspects, the immune cell is a Th1 cell. In some aspects, the immune cell is a Th2 cell. In some aspects, the immune cell is a Tc17 cell. In some aspects, the immune cell is a Th17 cell. In some aspects, the immune cell is a Treg cell. In some aspects, the immune cell is a gamma delta T cell (γδ T cell). [0117] The term "culturing" as used herein refers to the controlled growth of cells ex vivo and/or in vitro. As used herein, "culturing" includes the growth of cells, e.g., immune cells, e.g., T cells, TILs, NK cells, TILs, Tregs, or any combination thereof, 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, e.g., T cells, TILs, NK cells, TILs, Tregs, or any combination thereof, obtained from a human subject. In some aspects, the cultured cells comprise one or more engineered immune cell disclosed herein. In some aspects, the cultured cells comprise T cells or NK cells obtained from a human subject/patient. In some aspects, the T cells and/or NK cells are purified prior to the culture. In some aspects, the T cells and/or NK cells are tumor- infiltrating T cells and/or NK cells. In some aspects, the cultured cells comprise one or more engineered immune cell disclosed herein. [0118] In some aspects, the culturing comprises placing a tumor sample or tumor fragment into a medium disclosed herein, wherein the medium promotes TIL evasion from the tumor sample and TIL expansion. In some aspects, the tumor sample or tumor fragment is a dissociated tumor sample (e.g., single cell suspension). In some aspects, the dissociated tumor sample (e.g., single cell suspension) is dissociated in a medium disclosed herein. In some aspects, the culturing comprises placing a dissociated tumor sample or tumor fragment into a medium disclosed herein. In some aspects, the TILs are isolated or purified prior to the culture. In some aspects, the cell
culturing is intended to expand the number of cultured cells, e.g., to increase proliferation of the cells. [0119] The term "expand," "expansion," or "expanding" as used herein in reference to immune cell culture refers to the process of stimulating or activating the cells and culturing the cells. The expansion process can lead to an increase in the proportion or the total number of desired cells, e.g., an increase in the proportion or total number of less differentiated immune cells, in a population of cultured cells, after the cells are stimulated or activated and cultured. Expansion does not require that all cell types in a population of cultured cells are increased in number. Rather, in some aspects, only a subset of cells in a population of cultured cells are increased in number during expansion, while the number of other cell types may not change or may decrease. [0120] As used here, the term "D0" refers to the starting point of a given process. For example, in some aspects of the methods disclosed herein, "D0" refers to the beginning of the entire culture process, such as the day on which a single cell suspension disclosed herein is first contacted with IL-2. If no particular process is defined, then "D0" refers to the initiation of the entire culture process. As such, if the dissociated tumor sample, i.e., the single cell suspension is contacted with IL-2 at 9:00 am on a Monday, D0 runs from 9:00 am to 11:59 pm on that Monday. "D1" then refers to the next full day, e.g., from 12:00 am until 11:59 pm on Tuesday; "D2" then refers to the next full day, e.g., from 12:00 am until 11:59 pm on Wednesday; and so forth. The term "D#" can be used interchangeably with the term "day #", wherein the "#" is any whole number. Unless otherwise specified, any action taken on a particular day can occur at any time between 12:00 am and 11:59 pm on that day. [0121] 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 and/or a TCR), 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 (e.g., a polynucleotide encoding a c-Jun polypeptide, such that the immune cell exhibits increased c-Jun expression), genetic knockout or knockdown of functional expression of a gene to improve T cell function, or any combination thereof. [0122] As used herein, the term "rapid expansion protocol" or "REP" refers to a process of expanding a population of immune cells, e.g., TILs, by culturing the immune cells, e.g., TILs, in the presence of one or more agents that stimulate the immune cells. In some aspects, the one or
more agents that stimulate the immune cells comprise (i) a CD3 agonist, (ii) antigen presenting cells (APCs), or (iii) both a CD3 agonist and APCs. See, e.g., Dudley, et al., Science 298:850-54 (2002); Dudley, et al., J. Clin. Oncol.23:2346-57 (2005); Dudley, et al., J. Clin. Oncol.26:5233- 39 (2008); Riddell, et al., Science 257:238-41 (1992); and Dudley, et al., J. Immunother.26:332- 42 (2003), each of which is incorporated by reference herein in its entirety. Any CD3 agonist can be used in a REP step disclosed herein. In some aspects, the CD3 agonist comprises OKT3. The term "APCs," as used herein, refers to natural cells and to synthetic equivalents (i.e., artificial antigen presenting cells, or aAPCs), such as vesicles and/or microparticles. Artificial antigen presenting cells (aAPCs) such as genetically engineered human K562 aAPCs can be used for rapid expansion of TILs. In some aspects, the aAPC is generated by transducing K562 cells with a polycistronic lentiviral vector comprising genes encoding CD70, CD80, CD86, 41BB ligand, and OX40 ligand. K562 cells do not express HLA-A, HLA-B, or HLA-DR molecules, which makes them a powerful tool for T cell expansion when transduced with the above mentioned co- stimulatory ligands (See, e.g., Suhoski et al., Molecular therapy, 2007). In some aspects, K562 cells, e.g., genetically engineered human K562 aAPCs, are used during a first REP step disclosed herein. [0123] The term "anti-CD3 antibody" as used herein refers to an antibody or variant thereof, e.g., a monoclonal antibody and including human, humanized, chimeric or murine antibodies which are directed against the CD3 complex in T cells. In some aspects, an anti-CD3 antibody comprises OKT-3, also known as muromonab, and UCHT-1. Other anti-CD3 antibodies include, for example, visilizumab otelixizumab, and teplizumab. [0124] The term "OKT-3" or "OKT3" refers to a monoclonal antibody or biosimilar or variant thereof, including human, humanized, chimeric, or murine antibodies, directed against the CD3 receptor in the T cell antigen receptor of mature T cells, and includes commercially-available forms such as OKT-3 (30 ng/mL, MACS GMP CD3 pure, Miltenyi Biotech, Inc., San Diego, Calif., USA) and muromonab or variants, conservative amino acid substitutions, glycoforms, or biosimilars thereof. A hybridoma capable of producing OKT-3 is deposited with European Collection of Authenticated Cell Cultures (ECACC) and assigned Catalogue No. 86022706. A hybridoma capable of producing OKT-3 is also deposited with the American Type Culture Collection and assigned the ATCC accession number CRL 8001. [0125] 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 TILs in a population of TILs. The yield can be determined using any methods, including, but not limited to, estimating the yield based on a representative sample. [0126] As used herein, the term "stem cell-like," "stem-like," or "less-differentiated" refers to a cell, e.g., an immune cell (e.g., a T cell, NK cell, or TIL), that expresses markers consistent with a more naïve phenotype. For example, a less differentiated T cell or TIL can express one or more markers characteristic of a TN or a TSCM cell. In some aspects, a "less-differentiated" or "stem- like" T cell or TIL expresses CD45RA, CCR7, and CD62L. In some aspects, a "less-differentiated" or "stem-like" T cell or TIL expresses CD45RA, CCR7, and CD62L, and is CD45ROlow. In some aspects, a "less-differentiated" or "stem-like" immune cell (e.g., T cell or TIL) expresses CD45RA, CCR7, and CD62L, and does not express CD45RO. In some aspects, a "less-differentiated" or "stem-like" T cell expresses CD45RA, CCR7, CD62L, and TCF7. In some aspects, the methods disclosed herein promote the growth and/or proliferation of cells, e.g., immune cells, e.g., T cells, NK cells, or TILs, 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 cells, e.g., immune cells, e.g., T cells, NK cells, or TILs, 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, NK cells, or TILs, with a stem cell-like memory or central memory phenotype are preferred. See, e.g., 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., J. Clin. Invest.115:1616–1626 (2005); Gattinoni, L., et al. Nat Med 15(7):808-814 (2009); and Gattinoni, L., et al., Nat Med 17(10): 1290-1297 (2011). [0127] Stemness is characterized by the capacity to self-renew, the multipotency, and the persistence of proliferative potential. In some aspects, stemness is characterized by a particular gene signature, e.g., a combined pattern of expression across a multitude of genes. In some aspects, the gene signature comprises one or more genes selected from ACTN1, 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, OCM2, GCET2, LRRN1, IL6ST, LRRC16A, IGSF9B, EFHA2, LOC129293, APP, PKIA, ZC3H12D, CHMP7, KIAA0748, SLC22A17, FLJ13197, NRCAM, C5orf13, GIPC3, WNT7A, FAM117B, BEND5, LGMN, FAM63A, FAM153B, ARHGEF11, RBM11, RIC3, LDLRAP1, PELI1, PTK2, KCTD12, LMO7, 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 (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, MYO15B, MAL, AEBP1, SDK2, BZW2, GAL3ST4, PITPNM2, ZNF496, FAM117B, C16orf74, TDRD6, TSPAN32, C18orf22, C3orf44, LOC129293, ZC3H12D, MLXIP, C7orf10, STXBP1, KCNQ1, FLJ13197, LDLRAP1, RAB43, RIN3, SLC22A17, AGBL3, TCEA3, NCRNA00185, FAM153B, FAM153C, VIPR1, MMP19, HBS1L, EEF2K, SNORA5C, 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) or Galletti et al. Nat Immunol 21, 1552-1562 (2020)). In some aspects, the gene signature comprises one or more gene selected from SELL, CCR7, S1PR1, KLF3, TCF7, GPR183, SC5D, FAAH2, LTB, SESN3, MAL, TSHZ2, LEF1, AP3M2, SLC2A3, ICAM2, PLAC8, SCML1, IL7R, ABLIM1, RASGRP2, TRABD2A, SATB1, ALG13, ARID5A, BACH2, PABPC1, GPCPD1, NELL2, TAF4B, FCMR, ARRDC2, C1orf162, FAM177A1, ANKRD12, TXK, SORL1, AQP3, ADTRP, FXYD7, CD28, P2RY8, CRYBG1, TNFSF8, BEX2, PGAP1, PTGER4, MAML2, BEX3, PCSK1N, INPP4B, AC119396.1, CXCR5, LINC00402, CCR4, IL6R, ZBTB10, ITGA6, ARMH1, RILPL2, FOXP1, TESPA1, YPEL5, LPAR6, CMSS1, RIPOR2, ZNF331, EMP3, GIMAP7, WDR74, RIC3, CYSLTR1, ITGB1, CD5, SAMHD1, SERINC5, and any combination thereof (see e.g., Caushi et al., Nature 596: 126-132 (2021)). [0128] 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. [0129] As used herein, the term "fragmenting," "fragment," and "fragmented" describe processes for disrupting a tumor, including mechanical fragmentation methods such as crushing,
slicing, dividing, and morcellating tumor tissue as well as any other methods for disrupting the physical structure of tumor tissue. [0130] The term "clonotype," as used herein, refers to a population of T cells with unique DNA sequences that result from TCRα or TCRß rearrangements. A unique variable α chain (VA) sequence may pair up with more than one variable ß chain (VB) sequence. Conversely, a unique VB sequence may pair up with more than one VA sequence. [0131] As used herein, the term "tonicity" refers to the measure of the effective osmotic pressure gradient across a cell membrane. Tonicity can be measured or calculated based on the level of potassium ion and sodium chloride (NaCl) in a solution. Herein, tonicity is calculated as the sum of the concentration of potassium ion (K+) and the concentration of sodium chloride (NaCl), multiplied by two. Tonicity can be expressed in terms of the osmolality of the solution, e.g., the media. As used herein, a solution, e.g., medium, is considered "isotonic" when the concentration of solutes in the media is equivalent to the concentration of solutes inside the cell. As used herein, an isotonic medium has an osmolality of about 280 mOsm/L (e.g., ([K+] + [NaCl]) X 2 = 280). [0132] As used herein, a solution, e.g., a medium, is considered "hypotonic" if the concentration of solutes in the solution is lower than the concentration of solutes in the cell. 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 described herein has an osmolality of about 240 mOsm/L or about 250 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 (e.g., ([K+] + [NaCl]) X 2 = 250). [0133] As used herein, a solution, e.g., a medium, is considered "hypertonic" if the concentration of solutes in the solution is higher than the concentration of solutes in the cell. As used herein, a hypertonic solution has an osmolality of greater than 300 mOsm/L (e.g., ([K+] + [NaCl]) X 2 > 280). In some aspects, a hypertonic medium described herein has an osmolality of about 320 mOsm/L. In certain aspects, the tonicity of the solution, e.g., medium is adjusted by increasing or decreasing the concentration of one or more solute selected from potassium ions, sodium ions, glucose, and any combination thereof. 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. As used herein, the tonicity of a medium is defined by the sum of the potassium concentration and the NaCl concentration, multiplied by two. [0134] 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 ordinary skill in the art that standard means of preparing a solution comprising potassium ion include diluting a potassium containing salt (e.g., KCl) 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. [0135] 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. [0136] 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., CaCl2) 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. [0137] As used herein, the term "hyperkalemic," e.g., "hyperkalemic medium," refers to a medium that has an increased potassium concentration. In some aspects, the hyperkalemic medium comprises potassium ion at a concentration of greater than 5 mM. In some aspects, the hyperkalemic medium comprises potassium ion at a concentration higher than 40 mM. In some aspects, the hyperkalemic medium a concentration of potassium ion of 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, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM. The term "metabolic reprogramming media," "metabolic reprogramming medium," or "MRM," as used herein, refers to a hyperkalemic medium of the present disclosure. In some aspects, the expansion medium (e.g., MRM) comprises potassium ion at a concentration of about 50 mM. In some aspects, the expansion medium (e.g., MRM) comprises potassium ion at a concentration of about 55 mM. In some aspects, the expansion medium (e.g., MRM) comprises potassium ion at a concentration of about 60 mM. In some aspects, the expansion medium (e.g., MRM) comprises potassium ion at a concentration of about 65 mM. In some aspects, the expansion medium (e.g., MRM) comprises potassium ion at a concentration of about 70 mM. In certain aspects, the expansion medium (e.g., MRM) comprises about 40 mM to about 80 mM NaCl, about 40 mM to about 90 mM KCl, about 0.5 mM to about 2.8 mM calcium, and about 10 mM to about 24 mM glucose. In some aspects, the expansion medium (e.g., MRM) further comprises an osmolality of about 250 to about 340 mOsmol. [0138] 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, or any combination thereof. The basal media can be any media for culturing immune cells, e.g., TILs. In some aspects, the basal media is selected from 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), M199, OPTMIZER™ CTS™ T-Cell Expansion Basal Medium (ThermoFisher), OPTMIZER™ Complete, IMMUNOCULT™ XF (STEMCELL™ Technologies), IMMUNOCULT™ XF, AIM V, TEXMACS™ medium, TRANSACT™ TIL expansion medium, TIL rapid expansion protocol medium, and 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 comprises OPTMIZERTM. In some aspects, the basal media comprises OPTMIZERTM Pro. In some aspects, the basal media comprises X-VIVOTM 15 (LONZA). In some aspects, the basal media comprises IMMUNOCULTTM. In some aspects, the basal media comprises Click's medium. In some aspects, the basal media comprises TRANSACTTM TIL expansion medium. In some aspects, the basal media comprises TIL rapid expansion medium. 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, OpTimizer® (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™ OPTIMIZER™. In some aspects, suitable basal media include Click's medium, OpTimizer® (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™ OpTimizer™. In some aspects, the basal media comprises one or more sodium salt in addition to the NaCl that is added to control the tonicity, e.g., NaCl added in combination with potassium ion. [0139] 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. Non-limiting examples of cytokines include interleukins (e.g., interleukin (IL)-1, IL-2, IL-4, IL-7, IL-9, IL-13, IL-15, IL-3, IL-5, IL-6, IL-11, IL-10, IL-20, IL-14, IL-16, IL-17, IL-18, IL-21, IL-23, and IL-29), interferons (IFN; e.g., IFN-α, IFN-β, and IFN-γ), tumor necrosis factor (TNF) family members, and transforming growth factor (TGF) family members. Some aspects of the present disclosure are directed to methods of culturing cells, e.g., T cells and/or NK cells, in a medium comprising a cytokine. Some aspects of the present disclosure are directed to methods of culturing TILs in a medium comprising a cytokine. Some aspects of the present disclosure are directed to methods of expanding TILs in a medium comprising a cytokine. In some aspects, the cytokine is an interleukin. In some aspects, the cytokine is selected from IL-2, IL-7, IL-15, IL-21, and a 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 – P13232) 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 IgG1 and IgG3 in B-cells. IL-21 may also play a role in proliferation and maturation of natural killer (NK) cells in synergy with IL-15, and IL-21 may 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 may also inhibit dendritic cell activation and maturation during a T-cell-mediated immune response. [0140] As used herein, the term "higher than" means greater than but not equal to. For example, "higher than 4 mM" means any amount that is more than 4 mM, but which does not include 4 mM. As used herein, the term "less than" means less than but not equal to. For example, "less than 4 mM" means any amount that is less than 4 mM, but which does not include 4 mM. [0141] 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. [0142] 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 necessary need to have only N- and/or C- terminal amino acids deleted. A polypeptide in which internal amino acids have been deleted with respect to the naturally occurring sequence is also considered a fragment. [0143] As used herein, the term "functional fragment" or "functional portion" 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 the antigen-binding domain (e.g., an scFv) can effectively interact with the target epitope (e.g., a tumor antigen). Similarly, in some aspects, a c-Jun functional fragment is a fragment that when expressed
in an immune cell (e.g., CAR T cell), results in an immune cell with, e.g., 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 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 99%, or about 100% of the activity of a reference immune cell expressing a corresponding full length c-Jun. Non-limiting examples of such activity are further described elsewhere in the present disclosure. [0144] 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 or modification with at least one polynucleotide or vector encoding the polypeptides described here. [0145] 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. [0146] The term "express" or "expression" as used herein refers to a process by which a polynucleotide produces a gene product, for example, a CAR or TCR. 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 that 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. [0147] 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. [0148] In some aspects, the CAR comprises a chimeric fusion protein comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule, wherein the antigen-binding domain and the transmembrane domain are linked by a CAR spacer. In some aspects, the CAR comprises a chimeric fusion protein comprising an antigen-binding domain linked to a transmembrane domain via a CAR spacer and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some aspects, the CAR comprises a chimeric fusion protein comprising an antigen-binding domain linked to a transmembrane domain via a CAR spacer and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some aspects, the CAR comprises a chimeric fusion protein comprising an antigen-binding domain linked to a transmembrane domain via a CAR spacer and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some aspects, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR. In some aspects, the CAR further comprises a leader
sequence at the N-terminus of the antigen-binding domain, wherein the leader sequence is optionally cleaved from the antigen-binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane. [0149] 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 µM, for example, about 0.1 pM to about 1 µM 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, lgNAR 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 VαVβ) are also suitable for use in the chimeric binding proteins of the present disclosure. [0150] As used herein, the term "T cell receptor" or "TCR" refers to a heterodimer composed of 2 different transmembrane polypeptide chains: (i) an α chain and a β chain or (ii) a γ chain and a δ 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, CD3γε and CD3δε, 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. [0151] A "TCR mimic" or a "TCRm" refers to a type of antibody that recognize epitopes comprising both the peptide and the MHC-I molecule, similar to the recognition of such complexes by the TCR on T cells. [0152] 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 single-stranded RNA (ssRNA). Double stranded DNA- DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes 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 DNA molecules, sequences can be described herein according to the normal convention of giving only the sequence in the 5’ to 3’ direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic DNA. A "nucleic acid composition" of the disclosure comprises one or more nucleic acids as described herein. As described herein, in some aspects, a polynucleotide of the present disclosure can comprise a single nucleotide sequence encoding a single protein (e.g., codon-optimized c-Jun nucleotide sequence) ("monocistronic"). In some aspects, a polynucleotide of the present disclosure is polycistronic (i.e., comprises two or more cistrons). In some aspects, each of the cistrons of a polycistronic polynucleotide can encode for a protein disclosed herein (e.g., c-Jun protein, chimeric binding protein, or EGFRt). In some aspects, each of the cistrons can be translated independently of one another. [0153] 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. [0154] 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., miR-485 nucleic acid sequence). 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. [0155] 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. [0156] 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. [0157] A "solid tumor," as used herein, refers to an abnormal mass of tissue. Solid tumors may be benign or malignant. Nonlimiting examples of solid tumors include sarcomas, carcinomas, and lymphomas, such as cancers of the lung, breast, prostate, colon, rectum, and bladder. The tissue structure of a solid tumor includes interdependent tissue compartments including the parenchyma
(cancer cells) and the supporting stromal cells in which the cancer cells are dispersed, and which may provide a supporting microenvironment. [0158] In some aspects, the cancer comprises adrenal cortical cancer, advanced cancer, anal cancer, aplastic anemia, bileduct cancer, bladder cancer, bone cancer, bone metastasis, brain tumors, brain cancer, breast cancer, childhood cancer, cancer of unknown primary origin, Castleman disease, cervical cancer, colon/rectal cancer, endometrial cancer, esophagus cancer, Ewing family of tumors, eye cancer, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, Hodgkin disease, Kaposi sarcoma, renal cell carcinoma, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myelomonocytic leukemia, liver cancer, non-small cell lung cancer, small cell lung cancer, lung carcinoid tumor, lymphoma of the skin, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma in adult soft tissue, basal and squamous cell skin cancer, melanoma, small intestine cancer, stomach cancer, testicular cancer, throat cancer, thymus cancer, thyroid cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, Wilms tumor, secondary cancers caused by cancer treatment, or any combination thereof. In some aspects, the cancer comprises chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy’s sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms’ tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing’s sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin’s sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphoma, immunoblastic sarcoma of T-cells, Jensen’s sarcoma, Kaposi’s sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, myxoid/round cell liposarcoma, or telangiectaltic sarcoma. In some aspects, the cancer comprises 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
comprises acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiermoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher’s carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, 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 comprises 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, Müllerian cancer, ovarian cancer,
peritoneal cancer, fallopian tube cancer, or uterine papillary serous carcinoma. In some aspects, the cancer comprises metastatic melanoma, non-small cell lung cancer, myeloma, esophageal cancer, synovial sarcoma, myxoid/round cell liposarcoma, gastric cancer, breast cancer, hepatocellular cancer, head and neck cancer, ovarian cancer, prostate cancer, bladder cancer, or any combination thereof. [0159] 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 (e.g., a TIL), B lymphocyte, natural killer (NK) 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+ TIL, 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 TIL is a CD8+ TIL. In some aspects, a TIL is a CD4+ TIL. [0160] As used herein, the term "anti-tumor immune response" refers to an immune response against a tumor antigen. [0161] 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. [0162] "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 term refers to inducing an immune response in a subject against an antigen. In some aspects, the therapy
comprises chemotherapy, an immunotherapy, a radiotherapy, a surgery, or any combination thereof. [0163] "Administering" (and grammatical variants thereof) refers to the physical introduction of a therapeutic agent (e.g., an engineered cell described herein) to a subject, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration include intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasterna, 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. [0164] "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 term refers to inducing an immune response in a subject against an antigen. [0165] 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/or condition; partially or completely delaying progression from a particular disease, disorder and/or condition; and/or decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. In some aspects, preventing an outcome is achieved through prophylactic treatment. [0166] The term "therapeutically effective amount" or "therapeutically effective dosage" refers to an amount of an agent (e.g., an immune cell, e.g., a T cell, an NK cell, or a TIL, 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. [0167] The effective amount of the composition (e.g., cells 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. [0168] In some aspects, a "therapeutically effective amount" is the amount of a composition disclosed herein (e.g., T cells 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., T cells 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. [0169] 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., cells cultured as described herein) to promote cancer regression in the patient. Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ, and/or organism level (adverse effects) resulting from administration of a composition disclosed herein (e.g., cells cultured as described herein). [0170] As used herein, the term "tumor reactive" refers to the ability of an immune cell, e.g., an engineered T cell, an engineered NK cell, or a TIL, to target and kill a tumor cell. As used herein, the term "tumor specific" refers to a tumor reactive immune cell, e.g., engineered T cell, an engineered NK cell, or TIL, that specifically targets a tumor cell. [0171] As used herein, the term "putative tumor reactive" refers to immune cells or clones that are potentially tumor reactive based on functional or phenotype characteristics (e.g., expression of genes such as CXCL13, 4-1BB, PD-1, and TIGIT). [0172] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μΜ," respectively.
[0173] Various aspects of the disclosure are described in further detail in the following subsections. II. Methods of the Disclosure [0174] Some aspects of the present disclosure are directed to methods of expanding immune cells ex vivo or in vitro comprising culturing the immune cells in a medium comprising a feeder cell replacement (“initial expansion” if an additional expansion is added), wherein the feeder cell replacement comprises a programmable cell-signaling scaffold (PCS) comprising a surface- exposed CD3 agonist. [0175] In some aspects, the method further comprises culturing the immune cells in an additional medium before or after the culturing (“additional expansion”). In some aspects, the additional expansion is before the initial expansion. In some aspect, the additional expansion before the initial expansion does not comprise any feeder cells or feeder cell replacement. In some aspects, the additional expansion is after the initial expansion. In some aspect, the additional expansion after the initial expansion comprises culturing the cells in a second medium. [0176] Some aspects of the present disclosure are directed to a method of expanding immune cells ex vivo or in vitro comprising (i) culturing the immune cells in a first medium (“first expansion”) and (ii) culturing the immune cells from (i) in a second medium (“second expansion”), wherein the first medium and/or second medium comprises a feeder cell replacement, wherein the feeder cell replacement comprises a programmable cell-signaling scaffold (PCS) comprising a surface-exposed CD3 agonist. In some aspects, the feeder cell replacement in the first medium and the feeder cell replacement in the second medium are different. In some aspects, the method further comprises culturing the immune cells in an additional medium (“additional expansion”) before or after the culturing in the first medium and/or the second medium. [0177] The methods disclosed herein can be used in the culture of any immune cells that heretofore typically included the use of feeder cells. Immune cells that can be cultured according to the methods disclosed herein include, but are not limited to, T cells, natural killer (NK) cells, and tumor infiltrating lymphocytes. In some aspects, the immune cells comprise helper T-cells (CD4+ T cells), cytotoxic T-cells (CD8+ T cells), memory T-cells (CD45RO+ T cells), suppressor T-cells (Ts cells), regulatory T-cells (Tregs), natural killer T-cells (NKT cells), mucosal associated invariant (MAITs), gamma delta T cells, (γδ T cells), or any combination thereof. [0178] In some aspects, the additional expansion is before the first expansion and does not comprise any feeder cells or a feeder cell replacement. In some aspects, the method further
comprising culturing the immune cells in an additional medium (“additional expansion”) between the first expansion and the second expansion. [0179] In some aspects, the medium of the initial expansion does not comprise feeder cells. In some aspects, the medium of the initial expansion comprises feeder cells. In some aspects, the medium of the first expansion does not comprise feeder cells. In some aspects, the medium of the first expansion comprises feeder cells. [0180] In some aspects, the second medium does not comprise feeder cells. In some aspects, the second medium comprises feeder cells. [0181] In some aspects, the medium of the initial expansion comprises feeder cells, and the second medium does not comprise feeder cells. In some aspects, the medium of the initial expansion does not comprise feeder cells, and the second medium comprises feeder cells. In some aspects, the medium of the initial expansion comprises feeder cells, and the second medium comprises feeder cells. [0182] In some aspects, the medium of the first expansion comprises feeder cells, and the second medium does not comprise feeder cells. In some aspects, the medium of the first expansion does not comprise feeder cells, and the second medium comprises feeder cells. In some aspects, the medium of the first expansion comprises feeder cells, and the second medium comprises feeder cells. [0183] In some aspects, the feeder cells are present in a medium disclosed herein at an amount less that the amount of reference feeder cells that are required for the full expansion of the immune cells, using conventional methods that do not utilize a feeder cell replacement, e.g., a PCS. In some aspects, the amount of reference feeder cells is between about 1 X 106 to about 1 X 1010. In some aspects, the amount of reference feeder cells is from about 500 X 106 to about 8 X 109. In some aspects, the amount of feeder cells in the medium is less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, less than about 55%, less than about 60%, less than about 65%, or less than about 70% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 5% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 10% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 15% of the amount of reference feeder cells that are required for the full expansion
of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 20% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 25% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 30% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 35% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 40% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 45% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 50% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 55% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 60% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 65% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 70% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 75% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 80% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 85% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 90% of the amount of reference feeder cells that are required for the full expansion of the immune cells. In some aspects, the amount of feeder cells in the medium is less than about 95% of the amount of reference feeder cells that are required for the full expansion of the immune cells. [0184] In some aspects, the methods of expanding immune cells ex vivo or in vitro, and media use therein, do not comprise feeder cells.
[0185] In some aspects, a medium disclosed herein comprises about 0.01-1 mg feeder cell replacement, e.g., PCS, per 1 X 106 cells. In some aspects, the medium comprises about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, or about 1 mg feeder cell replacement, e.g., PCS, per 1 X 106 cells. II.A. Programmable Cell-signaling Scaffolds (PCS) [0186] Some aspects of the present disclosure are directed to methods of expanding immune cells ex vivo or in vitro comprising culturing the immune cells in a medium comprising a feeder cell replacement (“initial expansion” if an additional expansion is added), wherein the feeder cell replacement comprises a programmable cell-signaling scaffold (PCS) comprising a surface- exposed CD3 agonist. Non-limiting examples of programmable cell-signaling scaffolds (PCS) are described in WO2018/013797 and Chung et al. (Nature Biotechnology 36(2): 160-169 (2018), the contents of which are incorporated by reference. In some aspects, the PCS of the disclosure comprise a first layer comprising high surface area mesoporous silica micro rods (MSRs); a second layer comprising lipids coating said first layer; and a plurality of functional molecules loaded onto the scaffold. In some aspects, the scaffolds are biodegradable. [0187] The scaffolds described herein are capable of mimicking and/or replacing functions commonly associated with antigen-presenting cells (APCs), which allows the scaffolds to elicit various functions on target cells, e.g., eliciting effector functions of T-cells. As contemplated herein, the scaffolds mediate these effects via either direct or indirect interactions between the cell surface molecules residing in target cells (e.g., T cells, NK cells, and/or TILs) and the various functional molecules presented by the scaffolds. In some aspects, the scaffold modulates survival of target cells (e.g., T cells, NK cells, and/or TILs), growth of targeted cells (e.g., T cells, NK cells, and/or TILs), and/or function of target cells (e.g., T cells, NK cells, and/or TILs) through the physical or chemical characteristics of a scaffold itself. [0188] In some aspects, the scaffold composition is modified to comprise one or more surface cues and/or soluble cues (e.g., cell signaling molecules). In some aspects, the surface cues and/or soluble cues act to mediate various effector functions. Non-limiting examples of effector functions that can be affected by the surface cues and/or soluble cues include activation, division, promoting differentiation, growth, expansion, survival, increase yield, reprogramming, anergy, quiescence, senescence, apoptosis, death of target cells, or any combination thereof. In some
aspects, the one or more surface cues and/or soluble cues act to increase “stemness.” In some aspects, cells, e.g., immune cells, contacted with the PCS described herein in the media described herein exhibit superior growth and function compared to cells, e.g., immune cells, contacted with other substrate platforms, such as magnetic beads, e.g., DYNABEADS™, or commercial particles, e.g., TRANSACT™ (Miltenyi Biotech). [0189] In some aspects, the methods described herein comprise contacting human immune cells with PCS comprising a surface-exposed CD3 agonist, and further contacting the immune cells with one or more additional stimulatory molecules, cytokines, and/or other co-factors. In some aspects, the one or more additional stimulatory molecules, cytokines, and/or other co-factors are present in the medium. In some aspects, the one or more stimulatory molecules, cytokines, and/or other co-factors are present in the scaffold. In some aspects, non-targeted cells (e.g., cells other than T cells), which have otherwise infiltrated a scaffold, are rejected or removed using negative selection agents, cues, or through passive non-stimulation. [0190] In some aspects, the specific components of a scaffold are modulated. The permeability of a scaffold composition can be regulated, for example, by selecting or engineering a material for greater or smaller pore size, density, polymer cross-linking, stiffness, toughness, ductility, or elasticity. A scaffold composition can contain physical channels or paths through which targeted cells interact with a scaffold and/or move into a specific compartment or region of a scaffold. As needed, to facilitate compartmentalization, a scaffold composition can be optionally organized into compartments or layers, each with a different permeability, so that cells can be sorted or filtered to allow access to only a certain sub-population of cells. Sequestration of target cell populations in the scaffold can also be regulated by the degradation, dehydration, re-hydration, oxygenation, chemical alteration, pH alteration, ongoing self-assembly of the scaffold composition, or any combination thereof. Further, the functional molecules of a scaffold can vary in type and relative abundance to elicit specific interactions with desired cells. [0191] In some aspects, 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; (iii) a plurality of surface cues loaded onto the scaffold; and/or (iv) a plurality of soluble cues loaded onto the scaffold. II.A.1. Mesoporous silica [0192] In some aspects, the scaffold comprises mesoporous silica. Mesoporous silica is a porous body with hexagonal close-packed, cylinder-shaped, uniform pores. In some aspects, the
mesoporous silica is synthesized by using a rod-like micelle of a surfactant as a template, which is formed in water by dissolving and hydrolyzing a silica source such as alkoxysilane, sodium silicate solution, kanemite, silica fine particle in water or alcohol in the presence of acid or basic catalyst. See, US Pub. No.2015-0072009 and Hoffmann et al., Angewandte Chemie International Edition, 45, 3216-3251, 2006, each of which is incorporated by reference herein in its entirety. Many kinds of surfactants can be used in the synthesis of the mesoporous silica, including, but not limited to, cationic, anionic, and nonionic surfactants. In some aspects, the surfactant is an alkyl trimethylammonium salt of cationic surfactant. An alkyl trimethylammonium salt of cationic surfactant can yield a mesoporous silica having the increased specific surface area and pore volume. See U.S. Publication No. 2013/0052117 and Katiyar et al. (Journal of Chromatography 1122 (1-2): 13-20), each of which is incorporated by reference herein in its entirety. The terms "mesoscale," "mesopore," "mesoporous" and the like, as used herein, refer to structures having feature sizes in the range of about 1 nm to about 60 nm. In some aspects, the mesoporous material includes pores having a diameter in the range of about 1 nm to about 50 nm. In some aspects, the mesoporous material includes pores having a diameter in the range of about 5 nm to about 60 nm. In some aspects, the mesoporous material includes pores having a diameter in the range of about 2 nm to about 50 nm. In some aspects, the pores are orderly distributed. In some aspects, the pores are randomly distributed. [0193] The mesoporous silica used in scaffolds of the disclosure can be provided in various forms. In some aspects, the scaffolds are provided in a form selected from microspheres, irregular particles, rectangular rods, round nanorods, and any combination thereof. In some aspects, the scaffolds are provided as structured rod-shaped forms (MSR). The particles can have any pre- determined shape. In some aspects, the particles have a spheroid shape. In some aspects, the particles have an ellipsoid shape. In some aspects, the particles have a rod-like shape. In some aspects, the particles have a curved cylindrical shape. Non-limiting examples of methods of assembling mesoporous silica to generate microrods can be found, e.g., in Wang et al, Journal of Nanoparticle Research, 15:1501, 2013, which is incorporated by reference herein in its entirety. In some aspects, mesoporous silica nanoparticles are synthesized by reacting tetraethyl orthosilicate with a template made of micellar rods. The template can then be removed by washing with a solvent adjusted to the proper pH. In this example, after removal of surfactant templates, hydrophilic silica nanoparticles characterized by a uniform, ordered, and connected mesoporosity are prepared with a specific surface area of, for example, about 600 m2/g to about 1200 m2/g, particularly about 800 m2/g to about 1000 m2/g and especially about 850 m2/g to about 950 m2/g.
In some aspects, the mesoporous particle is synthesized using a simple sol-gel method or a spray drying method. Tetraethyl orthosilicate can also be used with an additional polymer monomer (e.g., as a template). In some aspects, one or more tetraalkoxy-silanes and one or more (3- cyanopropyl)trialkoxy-silanes are co-condensed to provide the mesoporous silicate particles as rods. See US Publication Nos.2013-0145488, 2012-0264599 and 2012-0256336, each of which is hereby incorporated by reference in its entirety. [0194] The MSR can comprise pores of between 1-60 nm in diameter, e.g., pores of between 2-5 nm, 10-20 nm, 10-30 nm, 10-40 nm, 20-30 nm, 30-50 nm, 30-40 nm, 40-50 nm, 50- 60 nm. In some aspects, the microrods comprise pores of approximately 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, or more in diameter. The pore size can be altered depending on the type of application. [0195] In some aspects, the length of the MSR is in the micrometer range, ranging from about 5 µm to about 500 µm. In some aspects, the microrods comprise a length of about 5-50 µm, e.g., about 10-20 µm, about 10-30 µm, about 10-40 µm, about 20-30 µm, about 30-50 µm, about 30-40 µm, or about 40-50 µm. In some aspects, the MSR comprise a length of about 50 µm to about 250 µm, e.g., about 60 µm, about 70 µm, about 80 µm, about 90 µm, about 100 µm, about 120 µm, about 150 µm, about 180 µm, about 200 µm, about 225 µm, or more. For recruitment of cells, MSR compositions having a higher aspect ratio can be employed, e.g., with rods comprising a length of 50 µm to 200 µm, particularly a length of 80 µm to 120 µm, especially a length of about 100 µm or more. [0196] In some aspects, the width of the MSR is in the micrometer range, ranging from about 0.1 µm to about 100 µm. In some aspects, the microrods comprise a width of about 0.1-75 µm, e.g., about 1-55 µm, about 1-50 µm, about 2-50 µm, about 1-40 µm. In some aspects, the MSR comprise a width of about 1.0 µm, about 2 µm, about 5 µm, about 10 µm, about 15 µm, about 20 µm, about 25 µm, about 30 µm, about 35 µm, about 40 µm, about 45 µm, about 50 µm, about 55 µm or more. [0197] In some aspects, the MSR provides a high surface area for attachment and/or binding to target cells, e.g., T cells, NK cells, and/or TILs. Non-limiting methods of obtaining high surface area mesoporous silicates can be found, for example, in US patent No.8,883,308 and US Publication No. 2011-0253643, each of which is incorporated by reference herein in its entirety. In some aspects, the high surface area is due to the fibrous morphology of the nanoparticles, which makes it possible to obtain a high concentration of highly dispersed and easily accessible moieties on the surface. In some aspects, the MSR has a surface area of at least about 100 m2/g, at least 150
m2/g, at least about 200 m2/g, at least about 250 m2/g or at least 300 m2/g. In some aspects, the MSR has a surface area from about 100 m2/g to about 1500 m2/g, including all values or sub-ranges in between, e.g., 50 m2/g, 100 m2/g, 200 m2/g, 300 m2/g, 400 m2/g, 500 m2/g, 600 m2/g, 700 m2/g, 800 m2/g, 100-500 m2/g, 100-300 m2/g, 500-800 m2/g, 100-700 m2/g, 200-600 m2/g, 500-1000 m2/g or 500-1500 m2/g. [0198] In some aspects, the MSR is sufficiently porous such that scaffolds sustain antigen presentation and attract and manipulate immune cells. In some aspects, scaffolds contain porous matrices, wherein the pores have a diameter of at least 10 nm. In some aspects, the pores have a diameter of at least 500 µm. In some aspects, the pores have a diameter from 10 nm to 500 µm. In some aspects, the pores have a diameter from 100 nm to 100 µm. In these aspects, the scaffolds comprise mesoporous scaffolds. In some aspects, scaffolds contain porous matrices, wherein the pores are as large or larger than the cell population infiltrating the scaffold. Non-limiting examples of methods of making polymer matrices having desired pore sizes and pore alignments are described, e.g., in US pub. No. 2011/0020216 and US patent No. 6,511,650, each of which is incorporated herein by reference in its entirety. II.A.2. Lipids [0199] The scaffolds of the disclosure comprise a second layer comprising lipids coating said first layer. The term "lipid" generally denotes a heterogeneous group of substances associated with living systems which have the common property of being insoluble in water, can be extracted from cells by organic solvents of low polarity such as chloroform and ether. In some aspects, "lipid" refers to any substance that comprises long, fatty-acid chains, preferably containing 10-30 carbon units, particularly containing 14-23 carbon units, especially containing 16-18 carbon units. [0200] In some aspects, the layer comprising lipids is provided as a monolayer. In some aspects, the layer comprising lipids is provided as a bilayer. Preferably, the lipid bilayer is fluid, wherein individual lipid molecules are able to diffuse within the bilayer. The membrane lipid molecules are preferably amphipathic. [0201] In some aspects, the layer comprising lipids comprises one or more continuous bilayers, e.g., resembling those found in natural biological membranes such as cellular plasma membranes. In some aspects, the layer comprising lipids is provided in the form of a supported bilayer. In some aspects, the layer comprising lipids is a continuous, fluid supported liposome. In some aspects, the layer comprising lipids is a continuous, fluid supported lipid bilayer. As used herein, a supported bilayer is a planar structure sitting on a solid support. In such an arrangement,
the upper face of the supported bilayer is exposed, while the inner face of the supported bilayer is in contact with the support. The scaffolds of the disclosure generally are stable and remain largely intact even when subject to high flow rates or vibration. The layer comprising lipids of scaffolds of the disclosure are also amenable to modification, derivatization, and/or chemical conjugation with any chemical and/or biological moiety. [0202] In some aspects, the layer comprising lipids of scaffolds of the disclosure is immobilized on the MSR layer. The lipid layer can be immobilized on the MSR using any method, including, but not limited to, covalent and non-covalent interactions. In some aspects, the layer comprising lipids is adsorbed on the MSR layer. In some aspects, the layer comprising lipids is attached or tethered to the MSR via one or more covalent interactions. Non-limiting examples of methods for attaching lipids to silicates include surface absorption and physical immobilization, e.g., using a phase change to entrap the substance in the scaffold material. In some aspects, the layer comprising lipids is layered onto the MSR layer. For example, a lipid film (containing for example, a solution of DPPC/cholesterol/DSPE-PEG at a molar ratio of 77.5:20:2.5 in chloroform) can be spotted onto the MSR layer and the solvent is evaporated using a rotary evaporator. See Meng et al, ACS Nano, 9 (4), 3540-3557, 2015. In some aspects, the lipid bilayer is prepared by extrusion of hydrated lipid films through a filter with pore size of, e.g., about 100 nm. The filtered lipid films can then be fused with the porous particle cores, for example, by a pipette mixing. In some aspects, lipid nanoparticles or “liposomes” are mixed with the particles resulting in the formation of the lipid layer on the MSRs. In some aspects, the liposomes are prepared using homogenization. [0203] In some aspects, covalent coupling via alkylating or acylating agents are used to provide a stable, structured, and long-term retention of the layer comprising lipids on the MSR layer. In some aspects, the lipid bilayers are reversibly or irreversibly immobilized onto the MSR layer. For example, the MSR layer can be hydrophilic and can be further treated to provide a more hydrophilic surface, e.g., with ammonium hydroxide and hydrogen peroxide. The lipid bilayer can be fused, e.g., using any coupling technique, onto the porous MSR layer to form scaffolds of the disclosure. [0204] In some aspects, the layer comprising lipids comprises a phospholipid. Representative examples of such lipids include, but are not limited to, amphoteric liposomes described in U.S. Patent Nos.9,066,867 and 8,3676,28, each of which is incorporated by reference herein in its entirety. In some aspects, the layer comprising lipids comprising dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC),
distearoylphosphatidylcholine (DSPC), palmitoyl-oleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE), dimyristoyl- phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), 1-stearoyl-2- myristoyl-sn-glycero-3-phosphocholine (8:0-14:0 PC), or any combination thereof. In some aspects, the layer comprising lipids comprises palmitoyl-oleoylphosphatidylcholine (POPC). In some aspects, the layer comprising lipids comprises a lipid composition that mimics the lipid composition of a mammalian cell membrane (e.g., a human cell plasma membrane). The lipid compositions of many mammalian cell membranes have been characterized and are readily ascertainable by one of skill in the art (see, e.g., Essaid et al. Biochim. Biophys. Acta 1858(11): 2725- 36 (2016), the entire contents of which are incorporated herein by reference). The composition of the layer comprising lipids can be altered to modify the charge or fluidity of the lipid bilayer. In some aspects, the layer comprising lipids comprises cholesterol. In some aspects, the layer comprising lipids comprises a sphingolipid. In some aspects, the layer comprising lipids comprises a phospholipid. In some aspects, the lipid is a phosphatidylethanolamine, a phosphatidylcholine, a phosphatidylserine, a phosphoinositide a phosphosphingolipid with saturated or unsaturated tails comprising 6-20 carbons, or a combination thereof. In some aspects, the lipid is a DIYNE PC lipid. In some aspects, the layer comprising lipids comprises a lipid composition that favors the spontaneous partitioning of lipid species into liquid-ordered domains (see, e.g., Wang T-Y et al. Biochemistry 40(43): 1303 1-40 (2001), which is incorporated by reference herein in its entirety). [0205] In some aspects, the layer comprising lipids is stabilized by compounds such as ionic or non-ionic surfactants. Non-limiting examples of surfactants useful in the compositions disclosed herein include: synthetic phospholipids, their hydrogenated derivatives and mixtures thereof; sphingolipids and glycosphingolipids; saturated or unsaturated fatty acids; fatty alcohols; polyoxyethylene-polyoxypropylene copolymers; ethoxylated fatty acids as well as esters or ethers thereof; dimyristoyl phosphatidyl choline; dimyristoyl phosphatidyl glycerol; or a combination thereof. In some aspects, the surfactant comprises dimyristoyl phosphatidyl glycerol. [0206] In some aspects, once in contact with a cell, a scaffold of the disclosure retains a continuous, fluid architecture for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 25 days, at least 30 days, at least 35 days, at least 40 days, at least 50 days, or more.
[0207] In some aspects, the weight ratio of the supported lipid bilayer (SLB) to the mesoporous silica micro-rods (MSR) is between about 10:1 and about 1:20. In some aspects, the continuous, fluid supported lipid bilayer (SLB) comprising (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), palmitoyl- oleoylphosphatidylcholine (POPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphatidylethanolamine (DMPE) and dipalmitoylphosphatidylethanolamine (DPPE), 1-stearoyl-2-myristoyl-sn-glycero-3- phosphocholine (8:0-14:0 PC), or a combination thereof. In some aspects, the mesoporous silica microrod-lipid bilayer (MSR-SLB) scaffold retains a continuous, fluid architecture for at least 14 days. In some aspects, the dry weight ratio of the mesoporous silica micro-rods (MSR) to the T- cell activating/co-stimulatory molecules is between 1 :1 to 50:1. II.A.3. Biodegradable scaffolds [0208] In some aspects, the scaffolds of the disclosure are biodegradable. In some aspects, the scaffold structure substantially degrades when exposed to a biological milieu. In some aspects, the biological milieu comprises a tissue culture condition, e.g., tissue culture media that has been optionally adapted to culture lymphocytes such as T cells. In some aspects, the biological milieu comprises a biological fluid, e.g., blood, lymph, CSF, peritoneal fluid, or the like. In some aspects, the biological milieu is the tissue environment at the site of implant, e.g., blood vessels, lymphatic system, adipose tissue, or the like. [0209] In some aspects, the biodegradable scaffolds are substantially degraded following contact with a biological milieu in vivo over 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 30 days, 45 days, 60 days, 90 days, or more. In some aspects, the biodegradable scaffolds are substantially degraded following contact with a biological milieu in vivo in less than 1 week. In some aspects, the biodegradable scaffolds are substantially degraded following contact with a biological milieu in vitro over 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7, days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 30 days, 45 days, 60 days, 90 days, or more. In some aspects, the biodegradable scaffolds are substantially degraded following contact with a biological milieu in vitro in less than 1 week. As used herein, "substantial degradation" means that at least 30%, at least 50%, at least 60%, at least 70%, at least 90%, at least 95%, or more of a scaffold composition is degraded when a scaffold composition is contacted with the biological milieu.
[0210] Accordingly, in some aspects, it is advantageous to tailor the degradation kinetics of a scaffold composition by modifying the properties of mesoporous silica rods, such as size, geometry, and/or porosity. Alternately, the degradation kinetics of a scaffold compositions can be modified by changing the culture conditions (e.g., by adjusting the pH of the media). [0211] In accordance with the aforementioned aspects, a scaffold of the disclosure can comprise a plurality of functional molecules which are optionally biodegradable. In some aspects, the scaffolds of the instant disclosure are encapsulated into other biodegradable scaffolds. Non- limiting examples of reagents and techniques useful in making such composite biodegradable scaffold compositions are described in Liao et al, J. Biomed. Mater. Res. B. Appl. Biomater., 102(2):293-302, 2014, which is incorporated by reference herein its entirety. In some aspects, the scaffolds are made up of physiologically-compatible and optionally biodegradable polymers. Non- limiting examples of polymers that are employable in the scaffolds are described in U.S. Patent No. 6,642,363, U.S. Publication No. 2011/0020216, Martinsen et al., Biotech. & Bioeng., 33 (1989) 79-89), (Matthew et al. Biomateriah, 16 (1995) 265-274), Atala et al., J Urology, 152 (1994) 641-643), and Smidsrod, TIBTECH 8 (1990) 71-78), the entire contents of which are incorporated herein by reference. [0212] Aspects described herein further relate to programmable cell signaling scaffolds with one or more functional molecules, e.g., surface cues and soluble cues, optionally together with one or more additional agents. In some aspects, the disclosure provides compositions comprising a scaffold and T cells clustered therein. In some aspects, the compositions and/or scaffolds are provided with one or more reagents for selecting, culturing, expanding, sustaining, and/or transplanting the cells of interest. II.A.4. Functional molecules [0213] In some aspects, the scaffolds comprise one or more functional molecules. In some aspects, the functional molecule interacts with cells, e.g., T cells, NK cells, and/or TILs, to elicit interaction and/or provoke or inhibit a response. In some aspects, the functional molecule is a surface cue, such as a surface-exposed CD3 agonist. In some aspects, the functional molecule is a soluble cue. In some aspects, a scaffold comprises at least one surface cue. In some aspects, a scaffold comprises at least one soluble cue. In some aspects, a scaffold comprises at least one surface cue and at least one soluble cue. [0214] Non-limiting examples of such functional molecules include polypeptides, antigens, antibodies, DNA, RNA, carbohydrates, haptens, other small molecules, and any
combination thereof. In some aspects, the functional molecules of the disclosure comprise a polypeptide (used interchangeably herein with protein and peptide). II.A.5. Surface cues [0215] In some aspects, the scaffolds comprise one or more surface cues. As used herein “surface cue” refers to molecules capable of binding to a cell surface receptor. In some aspects, the surface cue is in contact with, or coupled to, the layer comprising lipids of the scaffold structure. In some aspects, the surface cue mediates direct, indirect, or semi-direct modulation of one or more biological activities of a target population of cells, e.g., T cells, NK cells, and/or TILs. In some aspects, the surface cue mediates direct activation of T cells, NK cells, and/or TILs. In some aspects, the surface cue directly activates T cells, NK cells, and/or TILs, e.g., via binding to cell surface receptors on target cells. In some aspects, the surface cue comprises a stimulatory molecule that is an activation signal to T cells. As used herein, a T cell "stimulatory molecule" refers to any agent that increases one or more T cell activity, increases the expression of one or more cytokine by the T cell, increases the cytotoxicity of the T cell, increases T cell proliferation, reduces T cell death, or any combination thereof. In some aspects, the surface cue comprises a co-stimulatory molecule. [0216] In some aspects, the surface cue of a scaffold of the disclosure is an antibody or an antigen-binding portion thereof. The term "antibody," as used herein, broadly refers to any immunoglobulin (Ig) molecule comprising one or more polypeptide chains. In some aspects, the antibody comprises two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. As used herein, "antibody fragments" refer to a portion of an antibody, which is capable of binding an epitope on an antigen. The term “antigen-binding portion” of an antibody, as used herein, refers one or more part of an antibody that facilitates recognition of and/or binding to an antigen. [0217] Non-limiting examples of antigen-binding portions within the scope of the present disclosure include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab’) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment; and (vi) an isolated complementarity determining region (CDR). In some aspects, the
antibody comprises a VHH antibody, a vNAR antibody, an IgNAR antibody, a camelid antibody, a diabody, a monobody, or any combination thereof. [0218] In some aspects of the surface cues of the disclosure, the antibody is monospecific, bispecific, dual specific, or multi-specific formats; specifically binding to one, or two or more different, antigens. [0219] In some aspects, the surface cues include, but are not limited to, a stimulatory molecule that activates T cells (T cell activating molecules). In some aspects, a stimulatory molecule activates T cells by engaging and/or clustering components of the T cell receptor complex. In some aspects, the stimulatory molecule comprises an anti-CD3 antibody or antigen- binding portion thereof. In some aspects, the stimulatory molecule comprises an anti-CD2 antibody or an antigen-binding portion thereof. In some aspects, the stimulatory molecule comprises an anti- CD47 antibody or an antigen-binding portion thereof. In some aspects, the stimulatory molecule comprises an anti-CD81 antibody or antigen-binding portion thereof. In some aspects, the stimulatory molecule comprises an anti-macrophage scavenger receptor (MSR1) antibody or an antigen-binding portion thereof. In some aspects, the stimulatory molecule comprises an anti-T- cell receptor (TCR) antibody or an antigen-binding portion thereof. In some aspects, the surface cue comprises a major histocompatibility complex (MHC) molecule or a multimer thereof. In some aspects, the major histocompatibility complex (MHC) molecule or a multimer thereof is loaded with an MHC peptide. In some aspects, the surface cue comprises a conjugate containing MHC and immunoglobulin (Ig) or a multimer thereof. [0220] T cells can be activated in a CD3-dependent or independent manner, for example, via binding and/or ligation of CD3 or one or more cell-surface receptors other than CD3. Representative examples of such CD3-independent cell-surface molecules include, e.g., CD2, CD47, CD81, MSR1, etc. The process of T cell activation is characterized, for example, in Ryan et al, Nature Reviews Immunology 10, 7, 2010, which is incorporated by reference in its entirety. [0221] In some aspects, the surface cue used in a scaffold of the disclosure is an anti-CD3 antibody or antigen-binding portion thereof. Representative examples of anti-CD3 antibodies include, but are not limited to, muromonab (OKT3), otelixizumab (TRX4), teplizumab (hOKT3yl(Ala- Ala)), visilizumab, an antibody recognizing 17-19 kD C-chain of CD3 within the CD3 antigen/T cell antigen receptor (TCR) complex (HIT3a), and an antibody recognizing a 20 kDa subunit of the TCR complex within CD3e (UCHT1), or an antigen-binding portion thereof. Additional non-limiting examples of anti-CD3 antibodies and antigen-binding portions thereof are
described in US patent pub. No.2014-0088295, which is incorporated herein by reference herein in its entirety. [0222] In some aspects, the surface cue comprises a surface-exposed CD3 agonist. In some aspects, the CD3 agonist comprises an antibody or an antigen-binding portion thereof that specifically binds CD3 ("anti-CD3 antibody"). In some aspects, the anti-CD3 agonist comprises OKT3. In some aspects, the anti-CD3 agonist comprises UCHT1. In some aspects, the anti-CD3 agonist comprises SP34. [0223] In some aspects, the scaffold (e.g., PCS) comprises about 0.005% to about 1% anti- CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.01% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.02% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.03% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.04% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.05% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.06% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.07% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.08% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.09% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.1% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.15% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.2% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.25% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.5% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.6% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.7% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.8% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.9% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 1.0% anti-CD3 antibody, e.g., OKT3.
[0224] In some aspects, a medium of the present disclosure does not comprise a CD3 agonist that is not associated with the PCS, e.g., any CD3 agonist that is present is associated with the PCS. [0225] In some aspects, the surface cue comprises a CD2 agonist. In some aspects, the surface cue comprises an anti-CD2 antibody or antigen-binding portion thereof. Representative examples of anti-CD2 antibodies include, but are not limited to, siplizumab (MEDI-507) and LO- CD2b, or an antigen-binding portion thereof. See, e.g., ATCC accession No. PTA-802; deposited June 22, 1999. In some aspects, the CD2 agonist comprises an antibody or antigen-binding portion thereof that specifically binds CD2 ("anti-CD2 antibody"). In some aspects, the scaffold (e.g., PCS) comprises about 0.005% to about 1% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.01% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.005% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.006% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.007% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.008% anti- CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.009% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.01% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.02% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.03% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.04% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.05% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.06% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.07% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.08% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.09% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.1% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.2% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.3% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.4% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.5% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.6% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.7% anti-CD2
antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.8% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.9% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 1% anti-CD2 antibody. [0226] In some aspects, the surface cue comprises a CD28 agonist. In some aspects, the CD28 agonist comprises an antibody or antigen-binding portion thereof that specifically binds CD28 ("anti-CD28 antibody"). In some aspects, the scaffold (e.g., PCS) comprises about 0.005% to about 1% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.01% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.005% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.006% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.007% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.008% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.009% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.01% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.02% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.03% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.04% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.05% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.06% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.07% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.08% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.09% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.1% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.2% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.3% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.4% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.5% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.6% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.7% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.8% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.9% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 1% anti-CD28 antibody.
[0227] In some aspects, a medium of the present disclosure does not comprise a CD28 agonist that is not associated with the PCS, e.g., any CD28 agonist that is present is associated with the PCS. [0228] In some aspects, the surface cue comprises a 4-1BB agonist. In some aspects, the 4-1BB agonist comprises an antibody or an antigen-binding portion thereof that binds 4-1BB. In some aspects, the 4-1BB agonist comprises urelumab. In some aspects, the 4-1BB agonist comprises utolimumab. In some aspects, the surface cue comprises 4-1BB ligand ("4-1BBL"). In some aspects, the scaffold (e.g., PCS) comprises about 0.005% to about 1% r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.01% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.005% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.006%4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.007% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.008% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.009% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.01% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.02% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.03% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.04% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.05% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.06% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.07% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.08% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.09% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.1% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.15% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.2% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.25% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.3% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold
(e.g., PCS) comprises about 0.35% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.4% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.45% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.5% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.6% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.7% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.8% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.9% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 1% 4-1BB agonist, e.g., r4-1BBL. [0229] In some aspects, the surface cue comprises recombinant ICAM1 ("rICAM1"). In some aspects, the surface cue comprises an antibody or antigen-binding portion thereof that specifically binds SLAM-F6 ("anti-SLAM-F6 antibody"). In some aspects, the surface cue comprises an antibody or antigen-binding portion thereof that specifically binds ICOS ("anti-ICOS antibody"). In some aspects, the surface cue comprises a dimer-trimer CD70 polypeptide ("CD70dt"). In some aspects, the surface cue comprises recombinant IL-21 ("rIL-21"). In some aspects, the surface cue comprises recombinant OX40L ("rOX40L"). In some aspects, the surface cue comprises CCL5 (e.g., recombinant CCL5). In some aspects, the surface cue comprises CXCL10 (e.g., recombinant CXCL10). In some aspects, the surface cue comprises IL-18 (e.g., recombinant IL-18). In some aspects, the surface cue comprises IL-8 (e.g., recombinant IL-8). In some aspects, the surface cue comprises SLAMF-1 (e.g., recombinant SLAMF-1). [0230] In some aspects, the surface cue comprises CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the surface cue comprises CCL5 and CXCL10. In some aspects, the surface cue comprises CCL5, CXCL10, and IL-18. In some aspects, the surface cue comprises CCL5, CXCL10, IL-18, and IL-8. In some aspects, the surface cue comprises CCL5, CXCL10, IL- 18, IL-8, and SLAMF-1. In some aspects, the surface cue comprises CCL5, and IL-18. In some aspects, the surface cue comprises CCL5, IL-18, and IL-8. In some aspects, the surface cue comprises CCL5, IL-18, IL-8, and SLAMF-1. In some aspects, the surface cue comprises CCL5, CXCL10, and IL-8. In some aspects, the surface cue comprises CCL5, CXCL10, IL-8, and SLAMF-1. In some aspects, the surface cue comprises CCL5 and IL-8. In some aspects, the surface cue comprises CCL5, IL-8, and SLAMF-1. In some aspects, the surface cue comprises CCL5 and SLAMF-1. In some aspects, the surface cue comprises CXCL10, and IL-18. In some aspects, the surface cue comprises CXCL10, IL-18, and IL-8. In some aspects, the surface cue comprises CXCL10, IL-18, IL-8, and SLAM-1. In some aspects, the surface cue comprises CXCL10, IL-18,
and SLAMF-1. In some aspects, the surface cue comprises CXCL10, IL-8, and SLAMF-1. In some aspects, the surface cue comprises CXCL10 and SLAMF-1. In some aspects, the surface cue comprises IL-18, and IL-8. In some aspects, the surface cue comprises IL-18 and SLAMF-1. In some aspects, the surface cue comprises IL-8 and SLAMF-1. [0231] In some aspects, the surface cue comprises an anti-ICOS antibody and CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the surface cue comprises an anti-ICOS antibody and CCL5 and CXCL10. In some aspects, the surface cue comprises an anti-ICOS antibody and CCL5, CXCL10, and IL-18. In some aspects, the surface cue comprises an anti-ICOS antibody and CCL5, CXCL10, IL-18, and IL-8. In some aspects, the surface cue comprises an anti- ICOS antibody and CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the surface cue comprises an anti-ICOS antibody and CCL5, and IL-18. In some aspects, the surface cue comprises an anti-ICOS antibody and CCL5, IL-18, and IL-8. In some aspects, the surface cue comprises an anti-ICOS antibody and CCL5, IL-18, IL-8, and SLAMF-1. In some aspects, the surface cue comprises an anti-ICOS antibody and CCL5, CXCL10, and IL-8. In some aspects, the surface cue comprises an anti-ICOS antibody and CCL5, CXCL10, IL-8, and SLAMF-1. In some aspects, the surface cue comprises an anti-ICOS antibody and CCL5 and IL-8. In some aspects, the surface cue comprises an anti-ICOS antibody and CCL5, IL-8, and SLAMF-1. In some aspects, the surface cue comprises an anti-ICOS antibody and CCL5 and SLAMF-1. In some aspects, the surface cue comprises an anti-ICOS antibody and CXCL10, and IL-18. In some aspects, the surface cue comprises an anti-ICOS antibody and CXCL10, IL-18, and IL-8. In some aspects, the surface cue comprises an anti-ICOS antibody and CXCL10, IL-18, IL-8, and SLAM-1. In some aspects, the surface cue comprises an anti-ICOS antibody and CXCL10, IL-18, and SLAMF-1. In some aspects, the surface cue comprises an anti-ICOS antibody and CXCL10, IL-8, and SLAMF-1. In some aspects, the surface cue comprises an anti-ICOS antibody and CXCL10 and SLAMF-1. In some aspects, the surface cue comprises an anti-ICOS antibody and IL-18, and IL-8. In some aspects, the surface cue comprises an anti-ICOS antibody and IL-18 and SLAMF-1. In some aspects, the surface cue comprises an anti-ICOS antibody and IL-8 and SLAMF-1. [0232] In some aspects, the surface cue used in a scaffold of the disclosure comprises an anti-CD47 antibody or antigen-binding portion thereof. Representative examples of anti-CD47 antibodies include, but are not limited to, monoclonal antibody Hu5F9-G4, monoclonal antibody MABL-1, and monoclonal antibody MABL-2 (FERM Deposit Nos. BP-6100 and BP-6101), or an antigen-binding portion thereof. See, e.g., WO1999/12973, the disclosure in which is incorporated by reference herein.
[0233] In some aspects, the surface cue used in a scaffold of the disclosure comprises an anti-CD81 antibody or antigen-binding portion thereof. Representative examples of anti-CD81 antibodies include, but are not limited to, monoclonal antibody 5A6, or an antigen-binding portion thereof. See, e.g., Maecker et al., BMC Immunol., 4:1, 2003, the disclosure in which is incorporated by reference herein. [0234] In some aspects, the surface cue used in a scaffold of the disclosure comprises an anti-MSRI antibody or antigen-binding portion thereof. Representative examples of anti-MSRI antibodies include, but are not limited to, rat anti-human CD204 antibody (Thermo Catalog No. MA5-16494) and goat anti-human CD204/MSR1 antibody (Biorad Catalog No. AHP563), or an antigen-binding portion thereof. [0235] In some aspects, the surface cue used in a scaffold of the disclosure comprises an anti-TCR antibody or antigen-binding portion thereof. Representative examples of anti-TCR antibodies include, but are not limited to, mouse anti-human TCR monoclonal antibody IMMU510 (Immunotech, Beckman Coulter, Fullerton, CA) (described in Zhou et a , Cell Mol Immunol., 9(1): 34-44, 2012) and monoclonal antibody defining alpha/beta TCR WT31 (described in Gupta et al, Cell Immunol, 132(l):26-44, 1991), or an antigen-binding portion thereof. [0236] In some aspects, the surface cue comprises a bispecific antibody. In some aspects, a bispecific antibody is used to bring a cell of interest, e.g., a cancer cell or a pathogen, in close proximity with a target effector cell of the disclosure, e.g., a cytotoxic T-cell, such that the effector function of the target effector cell is mediated specifically upon the cell of interest. In some aspects, the surface cue comprises a bispecific antibody, wherein one arm of the antibody is specific to a T cell antigen and the other arm of the antibody is specific to a tumor-associated antigen or a pathogen-specific antigen or mutants thereof. [0237] In some aspects, a bispecific antibody functions in an activation and co-stimulatory capacity. In some aspects, the bispecific antibody specifically binds CD3 and CD28. Such surface cues can be referred to herein as, e.g., “anti-CD3/anti-CD28,” or “anti-CD3×CD28” or “CD3×CD28” bispecific molecules, or other similar terminology. The human CD28 protein has the amino acid sequence shown in GENBANK accession Nos. NP_001230006.1, NP_001230007.1, or NP_006130.1. The mouse CD28 protein has the amino acid sequence shown in GENBANK accession No. NP_031668.3. The various polypeptide sequences encompassed by the aforementioned accession numbers, include, the corresponding mRNA and gene sequences, and are incorporated by reference herein in their entirety. Additional examples of bispecific antibodies envisaged within the scope of the instant disclosure include, but are not limited to,
solitomab (CD3xEpCAM), blinatumomab (CD3xCD19), MAB MT-111 (CD3xCEA), and BAY- 2010112 (CD3xPSMA). [0238] In some aspects, the surface cue used in a scaffold of the disclosure comprises a major histocompatibility complex (MHC) molecule which binds to CD3. Representative examples include, but are not limited to, MHC type I, which binds to TCR and CD8, and MHC type II, which binds to TCR and CD4. In some aspects, MHC molecules include HLA-A, HLA-B, HLA-C, DP, DQ, and DR, or a combination thereof. In some aspects, the surface cues comprise two or more MHC molecules attached to a linker. In some aspects, the MHC molecule is monovalent. In some aspects, the MHC molecule is bivalent. [0239] In some aspects, the MHC molecules are loaded with a specific peptide (e.g., a peptide derived from a viral antigen, a bacterial antigen, allergen antigen, or tumor-associated antigen). [0240] In some aspects, the surface cue comprises a fusion protein. In some aspects, the fusion protein has T cell stimulatory properties. T cell stimulatory properties can be constructed by using a linker which allows for delivery of a second signal to the T cell in addition to the signal delivered via the TCR. This can be accomplished by using a linker that has binding affinity for a cell surface structure on another cell, that cell being capable of delivering a second signal to the T cell. Thus, the linker serves to bridge the T cell and the other cell. By bringing the other cell into close proximity to the T cell, the other cell can deliver a second signal to the T cell. [0241] In some aspects, the surface cue of the disclosure comprises one or more co- stimulatory molecules. As used herein “co-stimulatory molecule” refers to a polypeptide that binds to and provides a secondary or co-stimulatory signal to a cell, such as an immune cell (e.g., a T cell). Some co-stimulatory molecules include immune cell surface receptor/ligands, which engage between T cells and antigen presenting cells and generate a stimulatory signal in T cells, which combines with the stimulatory signal (i.e., "co-stimulation") in T cells that results from T cell receptor ("TCR") recognition of antigen on antigen presenting cells. As used herein, a soluble form of a co-stimulatory molecule "derived from an APC" refers to a co-stimulatory molecule normally expressed by B cells, macrophages, monocytes, dendritic cells and other APCs. See, Huppa et al., Nature Reviews Immunology.3, 973- 983 (2003). A "co-stimulator of T cell activation" refers to the ability of a co-stimulatory ligand to bind and to activate T cells which have been activated via any of the aforementioned mechanisms or pathways, e.g., via CD3-dependent or CD3-independent T-cell activation. Co-stimulatory activation can be measured for T cells by the production of cytokines and by proliferation assays that are well known (e.g., CFSE staining).
[0242] Such co-stimulatory molecules can mediate direct, indirect, or semi-direct stimulation of a target population of cells. In some aspects, the co-stimulatory molecules mediate activation of T-cells in the presence of one or more surface cues. [0243] In some aspects, the co-stimulatory molecule comprises molecules that specifically bind to a co-stimulatory receptor (e.g., recombinant ligands, purified natural ligands, or derivatives thereof). In some aspects, the co-stimulatory molecule comprises an antibody or antigen-binding portion thereof, which binds specifically to one or more co-stimulatory antigens. Representative examples of co-stimulatory molecules include, but are not limited to, molecules that specifically bind to CD28, 4-1BB (CD137), OX40 (CD134), CD27 (TNFRSF7), GITR (CD357), CD30 (TNFRSF8), HVEM (CD270), LT R (TNFRSF3), DR3 (TNFRSF25), ICOS (CD278), CD226 (DNAM1), CRTAM (CD355), TIM1 (HAVCR1, KIM1), CD2 (LFA2, 0X34), SLAM (CD150, SLAMF1), 2B4 (CD244, SLAMF4), Lyl08 (NTBA, CD352, SLAMF6), CD84 (SLAMF5), Ly9 (CD229, SLAMF3), CD279 (PD-1) and/or CRACC (CD319, BLAME). [0244] In this context, CD28 is the prototypic T cell co-stimulatory receptor and binds to molecules of the B7 family expressed on APCs such as dendritic cells and activated B cells. The ligands for CD28 include CD80 (B7-1) and CD86 (B7-2), which are immunoglobulin superfamily monomeric transmembrane glycoproteins. [0245] In some aspects, the co-stimulatory molecule comprises an anti-CD28 antibody or antigen-binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti- ICOS (CD278) antibody or antigen-binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti-CD152 (CTLA4) antibody or antigen-binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti-CD81 antibody or antigen-binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti-CD137 antibody or antigen-binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti- OX40 (CD134) antibody or antigen-binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti-CD27 (TNFRSF7) antibody or antigen-binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti-GITR (CD357) antibody or antigen- binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti-CD30 (TNFRSF8) antibody or antigen-binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti-HVEM (CD270) antibody or antigen-binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti-LTpR (TNFRSF3) antibody or antigen- binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti-DR3 (TNFRSF25) antibody or antigen-binding portion thereof. In some aspects, the co-stimulatory
molecule comprises an anti-CD226 (DNAM1) antibody or antigen-binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti-CRTAM (CD355) antibody or antigen-binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti- TIM1 (HAVCR1, KIM1) antibody or antigen-binding portion thereof. In some aspects, the co- stimulatory molecule comprises an anti-SLAM (CD 150, SLAMF1) antibody or antigen-binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti-2B4 (CD244, SLAMF4) antibody or antigen-binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti-Lyl08 (NTBA, CD352, SLAMF6). In some aspects, the co-stimulatory molecule comprises an anti-CD84 (SLAMF5) antibody or antigen-binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti-CD229 (Ly9, SLAMF3) antibody or antigen-binding portion thereof. In some aspects, the co-stimulatory molecule comprises an anti- PD-1 (CD279). In some aspects, the co-stimulatory molecule comprises an anti-CRACC (CD319, BLAME) antibody or antigen-binding portion thereof. Representative examples of co-stimulatory molecules include, but are not limited to, those referenced in e.g., U.S. Patent No.8.785,604; Int’l Publication No. WO 2010/078526; Maecker et al., BMC Immunol., 4:1, (2003); Ramakrishna et al., Journal for ImmunoTherapy of Cancer, 3:37, (2015); Cheung et al, J. Immunol, 185:1949, (2010); Hobo et al, J. Immunol. 189:39, (2012); Reddy et al , J. Virol , 86 (19) 10606- 10620, (2012); Wolf et al., Transplantation, 27;94(6):569-74, (2012); Flaig et al., J. Immunol.172:6524- 6527, (2004); and Stark et al., J. Immunol. Methods 296: 149-158, (2005), each of which is incorporated by reference herein in its entirety. In some aspects, the co-stimulatory molecule comprises a recombinant or purified natural ligand or derivative thereof. [0246] In some aspects, the scaffolds comprise a pair of surface cues. In some aspects, a pair of surface cues provide a primary stimulatory signal and co-stimulatory signal to a target cell, such as a T cell. Representative examples of such pairs include, but are not limited to, antibodies capable of binding to CD3/CD28, CD3/ICOS, CD3/CD27, and CD3/CD137, or a combination thereof. [0247] In some aspects, the scaffolds comprise a binding pair comprising an antibody binding to CD3 and at least one co-stimulatory molecule. In some aspects, the at least one co- stimulatory molecule comprises an anti-CD28 antibody. In some aspects, the at least one co- stimulatory molecule comprises an anti-CD28 antibody and a second co-stimulatory molecule. In some aspects, the second costimulatory molecule comprises an antibody that specifically binds ICOS, CD27, or CD137. In some aspects, the scaffold comprises a combination of functional molecules selected from the following combinations: (a) antibodies that specifically bind CD3,
CD28, and ICOS, (b) antibodies that specifically bind to CD3, CD28, and CD27, (c) antibodies that specifically bind to CD3, CD28, and CD137, (d) antibodies that specifically bind to CD3, CD28, ICOS and CD27. [0248] In some aspects, the scaffolds comprise a binding pair comprising at least two monospecific antibodies, wherein a first antibody binds to a first member of the pair, e.g., CD3, and a second antibody binds to a second member of the pair, e.g., CD28. In some aspects, the binding pair comprises a bispecific antibody comprising an antigen-binding domain that specifically binds CD3 and an antigen-binding domain that specifically binds CD28. [0249] Alternately, in some aspects, the binding pair comprises at least two monospecific antibodies, wherein a first antibody binds to CD3 and a second antibody binds to ICOS. In some aspects, the binding pair comprises an antibody or antigen-binding portion thereof the specifically binds to ICOS. In some aspects, the antibody is an antagonistic antibody or antigen-binding portion that neutralizes ICOS. [0250] In some aspects, the binding pair comprises at least two monospecific antibodies, wherein a first antibody binds to CD3 and a second antibody binds to CD27. In some aspects, both antibodies are stimulatory antibodies. In some aspects, both antibodies are agonist antibodies. In some aspects, the binding scaffold comprises a bispecific antibody comprising an agonist anti-CD3 binding domain and an agonist CD27 binding domain. [0251] In some aspects, the binding pair comprises at least two monospecific antibodies, wherein a first antibody binds to CD3 and a second antibody binds to CD137. In some aspects, both antibodies are stimulatory antibodies. In some aspects, both antibodies are agonist antibodies. In some aspects, the binding scaffold comprises a bispecific antibody comprising an agonist anti- CD3 binding domain and an agonist anti-CD137 binding domain. [0252] In some aspects, the scaffold comprises a plurality of surface cues. In one aspect, the scaffold comprises multiple antibodies where each antibody preferentially binds to a different receptor on the surface of a target cell. [0253] The amount of different surface cue molecules present on the scaffolds, such as surface cue 1/surface cue 2, for example, can be understood as functional molecule density, calculated as either the theoretical number of molecules per surface area or scaffold or calculated based on the mol percent of coating lipid used for functional molecule presentation or stoichiometry of said functional molecules. The surface cue density can be determined by the percentage of the lipids in the layer comprising lipids being used for function molecule affinity pairing, wherein the surface cues are affixed to the layer comprising lipids. The ratio or
stoichiometry of said functional molecules can be expressed as the relative proportion of the various functional molecules being affixed. The density of functional molecule presentation can also be determined by the dry weight ratio of the MSR to the dry weight of the combined surface cues. [0254] The term "affinity pair" as used herein includes antigen-antibody, receptor- hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, nucleic acid (RNA or DNA) hybridizing sequences, Fc receptor or mouse IgG-protein A, avidin-biotin, streptavidin-biotin, biotin/biotin binding agent, Ni2+ or Cu2+ chelator (e.g., NTA or other chelator/metal pair)/HisTag (6x histidine or other polyhistidine tag) and virus-receptor interactions. Various other specific binding pairs are contemplated for use in practicing the methods of this disclosure. [0255] As used herein, "biotin-binding agent" encompasses avidin, streptavidin and other avidin analogs such as streptavidin or avidin conjugates, highly purified and fractionated species of avidin or streptavidin, and non or partial amino acid variants, recombinant or chemically synthesized avidin analogs with amino acid or chemical substitutions, which still accommodate biotin binding. [0256] In some aspects, each biotin-binding agent molecule binds at least two biotin moieties. In some aspects, each biotin-binding agent molecule binds at least four biotin moieties. As used herein, "biotin" encompasses biotin in addition to biocytin and other biotin analogs such as biotin amido caproate N-hydroxysuccinimide ester, biotin 4- amidobenzoic acid, biotinamide caproyl hydrazide and other biotin derivatives and conjugates. Other derivatives include biotin- dextran, biotin-disulfide-N-hydroxysuccinimide ester, biotin-6 amido quinoline, biotin hydrazide, d-biotin-N hydroxysuccinimide ester, biotin maleimide, d-biotin p- nitrophenyl ester, biotinylated nucleotides and biotinylated amino acids such as Nε-biotinyl-l -lysine. [0257] The ligands that can be functionalized via affinity pairing include, but are not limited to, receptors, monoclonal or polyclonal antibodies, viruses, chemotherapeutic agents, receptor agonists and antagonists, antibody fragments, lectin, albumin, peptides, proteins, hormones, amino sugars, lipids, fatty acids, nucleic acids, and cells prepared or isolated from natural or synthetic sources. Any site-specific ligand for any molecular epitope or receptor to be detected through the practice of the disclosure can be utilized. In some aspects, the ligand is a membrane-anchored protein. [0258] The functional molecules, as noted hereinabove, can be any protein or peptide. In some aspects, the proteins are involved in ligand-receptor interactions. For example, an important event of T cell activation is a result of membrane-membrane contact between T cells and APCs,
wherein a variety of ligand-receptor interactions take place between the two opposing membranes, including, MHC- peptide and TCR, LFA-1 and ICAM-1, CD2 and CD48, as well as B7 or CTLA- 4 and CD28. [0259] In some aspects, the PCS comprises a plurality of surface cues loaded onto the scaffold. In some aspects, 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 loaded onto the scaffold. In some aspects, the plurality of surface cues comprises (a) the CD3 agonist and (b) one or more of a CD28 agonist, a 4-1BB agonist (e.g., r4-1BBL), an anti-CD2 antibody, rICAM1, an anti-SLAM-F6 antibody, an anti-ICOS antibody, rOX40L, rIL-21, and a CD70dt. In some aspects, the plurality of surface cues comprises (i) an anti-CD3 antibody, (ii) an anti-CD28 antibody, and (iii) 4-1BB agonist, e.g., r4-1BBL. In some aspects, the plurality of surface cues comprises (i) an anti-CD3 antibody, (ii) an anti-CD28 antibody, (iii) 4-1BB agonist, e.g., r4-1BBL and (iv) an anti-CD2 antibody) or rICAM1. In some aspects, the plurality of surface cues comprises (i) an anti-CD3 antibody, (ii) an anti-CD28 antibody, (iii) 4-1BB agonist, e.g., r4-1BBL, and (iv) an anti-CD2 antibody. In some aspects, the plurality of surface cues comprises (i) an anti-CD3 antibody, (ii) an anti-CD28 antibody, (iii) 4-1BB agonist, e.g., r4-1BBL, (iv) an anti-CD2 antibody, and (v) an anti- SLAM-F6 antibody. In some aspects, the plurality of surface cues comprises (i) an anti-CD3 antibody, (ii) an anti-CD28 antibody, (iii) 4-1BB agonist, e.g., r4-1BBL, (iv) an anti-CD2 antibody, and (v) CD70dt. In some aspects, the plurality of surface cues comprises (i) an anti-CD3 antibody, (ii) an anti-CD28 antibody, (iii) 4-1BB agonist, e.g., r4-1BBL, (iv) an anti-CD2 antibody, and (v) rOX40L. In some aspects, the plurality of surface cues comprises (i) an anti-CD3 antibody, (ii) an anti-CD28 antibody, (iii) 4-1BB agonist, e.g., r4-1BBL, (iv) an anti-CD2 antibody, and (v) rIL21. [0260] In some aspects, the plurality of surface cues comprises (a) the CD3 agonist; (b) one or more of a CD28 agonist, a 4-1BB agonist (e.g., r4-1BBL), an anti-CD2 antibody, rICAM1, an anti-SLAM-F6 antibody, an anti-ICOS antibody, rOX40L, rIL-21, and a CD70dt; and (c) one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the plurality of surface cues comprises (a) the CD3 agonist; (b) one or more of a CD28 agonist, a 4-1BB agonist (e.g., r4- 1BBL), an anti-CD2 antibody, rICAM1, an anti-SLAM-F6 antibody, an anti-ICOS antibody, rOX40L, rIL-21, and a CD70dt; and (c) CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. [0261] In some aspects, the plurality of surface cues comprises (a) the CD3 agonist; (b) one or more of a CD28 agonist, a 4-1BB agonist (e.g., r4-1BBL), an anti-CD2 antibody, rICAM1, an anti-SLAM-F6 antibody, rOX40L, rIL-21, and a CD70dt; (c) an anti-ICOS antibody, and (d)
one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the plurality of surface cues comprises (a) the CD3 agonist; (b) one or more of a CD28 agonist, a 4-1BB agonist (e.g., r4-1BBL), an anti-CD2 antibody, rICAM1, an anti-SLAM-F6 antibody, rOX40L, rIL-21, and a CD70dt; (c) an anti-ICOS antibody; and (d) CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. [0262] In some aspects, the plurality of surface cues is loaded onto the SLB layer. In some aspects, the plurality of surface cues are loaded onto the fluid supported lipid bilayer (SLB). In some aspects, the plurality of surface cues are coated onto the fluid supported lipid bilayer (SLB). In some aspects, the plurality of surface cues are partly embedded onto the fluid supported lipid bilayer (SLB). [0263] In some aspects, the plurality of surface cues are loaded onto the mesoporous silica micro-rods (MSR). [0264] Incorporation of predefined amounts of a biotinylated phospholipid into liposome formulations enables the precise surface attachment of biotinylated surface cues via streptavidin- biotin interactions, mimicking the cell surface presentation of cues by natural APCs to T cells. [0265] In some aspects, the density of surface cues or the combinations of surface cues is determined by percentage of affinity paired (e.g., biotinylated) lipid used in the scaffold. In some aspects, the percentage of biotinylated lipid is between about 0.01% to about 1.1%. In some aspects, the percentage of biotinylated lipid is between about 0.1% to about 0.9%. In some aspects, the percentage of biotinylated lipid is between about 0.1% to about 2.5%. In some aspects, the percentage of biotinylated lipid is about 0.01%. In some aspects, the percentage of biotinylated lipid is about 0.05%. In some aspects, the percentage of biotinylated lipid is about 0.1%. In some aspects, the percentage of biotinylated lipid is about 0.2%. In some aspects, the percentage of biotinylated lipid is about 0.25%. In some aspects, the percentage of biotinylated lipid is about 0.3%. In some aspects, the percentage of biotinylated lipid is about 0.4%. In some aspects, the percentage of biotinylated lipid is about 0.5%. In some aspects, the percentage of biotinylated lipid is about 0.6%. In some aspects, the percentage of biotinylated lipid is about 0.7%. In some aspects, the percentage of biotinylated lipid is about 0.8%. In some aspects, the percentage of biotinylated lipid is about 0.9%. In some aspects, the percentage of biotinylated lipid is about 1.0%. In some aspects, the percentage of biotinylated lipid is about 1.1%. In some aspects, the percentage of biotinylated lipid is about 1.5%. In some aspects, the percentage of biotinylated lipid is about 2.0%. In some aspects, the percentage of biotinylated lipid is about 2.5%. [0266] In some aspects, the density of surface cues or the combination of surface cues is determined by the mass of each affinity paired surface cue added during scaffold loading, provided
there is an excess of affinity paired lipid in the scaffold, e.g., when using a metal-chelating lipid and his-tagged surface cue. [0267] In some aspects, the dry weight ratio of the MSR to the surface cues (stoichiometry) is from about 1:1 to about 100:1. In some aspects, the dry weight ratio of the MSR to the surface cues (stoichiometry) is from about 10:1 to about 50:1. In some aspects, the dry weight ratio of the MSR to the surface cues (stoichiometry) is from about 20:1 to about 50:1. In some aspects, the dry weight ratio of the MSR to the surface cue of the scaffolds is from about 10,000:1 to about 1:1. In some aspects, the dry weight ratio of the MSR to the surface cues of the scaffolds is from about 5,000:1 to about 1:1, from about 1,000:1 to about 1:1, from about 500:1 to about 1:1, or from about 100:1 to about 1:1. In some aspects, the dry weight ratio of the MSR to the surface cues of the scaffolds is about 10,000:1, about 5,000:1, about 2,500:1, about 1,000:1, about 750:1, about 500:1, about 250:1, about 100:1, about 75:1, about 50:1, about 40:1, about 30:1, about 25:1, about 20:1, about 10:1, or about 1:1. II.A.6. Soluble cues [0268] In some aspects, the scaffolds (e.g., PCS) comprise a plurality of soluble cues. As used herein “soluble cues” refers to cell-signaling molecules in contact with the scaffold structure. In some aspects, the plurality of surface cues is loaded onto a MSR base layer of the scaffold (e.g., PCS) or added directly to the media. In some aspects, the soluble cues are in contact with, or coupled to, the layer comprising MSR of the scaffold structure. In some aspects, the scaffolds of the instant disclosure contain a plurality of soluble comprising CD3 agonist, a CD28 agonist, a 4- 1BB agonist, an anti-CD2 antibody ("anti-CD2 antibody"), recombinant ICAM1 ("rICAM1"), an anti-SLAM-F6 antibody ("anti-SLAM-F6 antibody"), an anti-ICOS antibody, CCL5, CXCL10, recombinant OX40L ("rOX40L"), recombinant IL-21 ("rIL-21"), and a dimer-trimer CD70 polypeptide ("CD70dt"), IL-1, IL-2, IL-4, IL-5, IL-7, IL-8, IL-10, IL-12, IL-15, IL-17, IL-18, IL- 21, SLAMF-1, Wnt proteins, or transforming growth factor beta (TGF- β), or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or in any combination. [0269] In some aspects, the plurality of soluble cues comprises a CD3 agonist disclosed herein. In some aspects, the scaffold (e.g., PCS) comprises about 0.005% to about 1% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about
0.9%, about 1% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.01% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.02% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.03% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.04% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.05% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.06% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.07% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.08% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.09% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.1% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.15% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.2% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.25% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.5% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.6% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.7% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.8% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 0.9% anti-CD3 antibody, e.g., OKT3. In some aspects, the scaffold (e.g., PCS) comprises about 1.0% anti-CD3 antibody, e.g., OKT3. [0270] In some aspects the plurality of soluble cues comprises a CD28 agonist disclosed herein. In some aspects, the scaffold (e.g., PCS) comprises about 0.005% to about 1% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.01% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.005% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.006% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.007% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.008% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.009% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.01% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises
about 0.02% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.03% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.04% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.05% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.06% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.07% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.08% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.09% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.1% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.2% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.3% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.4% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.5% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.6% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.7% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.8% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.9% anti-CD28 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 1% anti-CD28 antibody. [0271] In some aspects the plurality of soluble cues comprises a 4-1BB agonist, e.g., r4- 1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.005% to about 1% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.01% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.005% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.006% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.007% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.008% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.009% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.01% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.02% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.03% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.04% 4-1BB agonist, e.g., r4-1BBL. In
some aspects, the scaffold (e.g., PCS) comprises about 0.05% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.06% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.07% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.08% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.09% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.1% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.15% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.2% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.25% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.3% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.35% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.4% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.45% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.5% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.6% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.7% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.8% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 0.9% 4-1BB agonist, e.g., r4-1BBL. In some aspects, the scaffold (e.g., PCS) comprises about 1% 4-1BB agonist, e.g., r4-1BBL. [0272] In some aspects the plurality of soluble cues comprises an anti-CD2 antibody disclosed herein. In some aspects, the scaffold (e.g., PCS) comprises about 0.005% to about 1% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.01% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.005% anti- CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.006% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.007% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.008% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.009% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.01% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.02% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS)
comprises about 0.03% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.04% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.05% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.06% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.07% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.08% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.09% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.1% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.2% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.3% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.4% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.5% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.6% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.7% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.8% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 0.9% anti-CD2 antibody. In some aspects, the scaffold (e.g., PCS) comprises about 1% anti-CD2 antibody. [0273] In some aspects the plurality of soluble cues comprises rICAM1. In some aspects the plurality of soluble cues comprises an anti-SLAM-F6 antibody disclosed herein. In some aspects the plurality of soluble cues comprises an anti-ICOS antibody disclosed herein. In some aspects the plurality of soluble cues comprises rOX40L. In some aspects the plurality of soluble cues comprises rIL-21. In some aspects the plurality of soluble cues comprises a CD70dt. In some aspects the plurality of soluble cues comprises IL-1. In some aspects the plurality of soluble cues comprises IL-2. In some aspects the plurality of soluble cues comprises IL-4. In some aspects the plurality of soluble cues comprises IL-5. In some aspects the plurality of soluble cues comprises IL-7. In some aspects the plurality of soluble cues comprises IL-10. In some aspects the plurality of soluble cues comprises IL-12. In some aspects the plurality of soluble cues comprises IL-15. In some aspects the plurality of soluble cues comprises IL-17. In some aspects the plurality of soluble cues comprises IL-21. In some aspects the plurality of soluble cues comprises Wnt proteins. In some aspects the plurality of soluble cues comprises TGF- β. [0274] In some aspects, the plurality of soluble cues comprises CCL5, CXCL10, IL-18, IL-8, and/or SLAMF-1, in any combination. In some aspects, the plurality of soluble cues comprises CCL5 and CXCL10. In some aspects, the plurality of soluble cues comprises CCL5, CXCL10, and IL-18. In some aspects, the plurality of soluble cues comprises CCL5, CXCL10, IL- 18, and IL-8. In some aspects, the plurality of soluble cues comprises CCL5, CXCL10, IL-18, IL-
8, and SLAMF-1. In some aspects, the plurality of soluble cues comprises CCL5, and IL-18. In some aspects, the plurality of soluble cues comprises CCL5, IL-18, and IL-8. In some aspects, the plurality of soluble cues comprises CCL5, IL-18, IL-8, and SLAMF-1. In some aspects, the plurality of soluble cues comprises CCL5, CXCL10, and IL-8. In some aspects, the plurality of soluble cues comprises CCL5, CXCL10, IL-8, and SLAMF-1. In some aspects, the plurality of soluble cues comprises CCL5 and IL-8. In some aspects, the plurality of soluble cues comprises CCL5, IL-8, and SLAMF-1. In some aspects, the plurality of soluble cues comprises CCL5 and SLAMF-1. In some aspects, the plurality of soluble cues comprises CXCL10, and IL-18. In some aspects, the plurality of soluble cues comprises CXCL10, IL-18, and IL-8. In some aspects, the plurality of soluble cues comprises CXCL10, IL-18, IL-8, and SLAM-1. In some aspects, the plurality of soluble cues comprises CXCL10, IL-18, and SLAMF-1. In some aspects, the plurality of soluble cues comprises CXCL10, IL-8, and SLAMF-1. In some aspects, the plurality of soluble cues comprises CXCL10 and SLAMF-1. In some aspects, the plurality of soluble cues comprises IL-18, and IL-8. In some aspects, the plurality of soluble cues comprises IL-18 and SLAMF-1. In some aspects, the plurality of soluble cues comprises IL-8 and SLAMF-1. [0275] Representative soluble cues, include, but are not limited to, the following NCBI accession numbers of human and/or mouse homologs thereof: IL-1, NP_000566.3 (human); IL- 1α, NP_034684.2 (mouse); IL-1, NP_000567.1 (human); IL-1β, NP_032387.1 (mouse); IL-2, NP_000577.2 (human) and NP_032392.1 (mouse); IL-4, NP_000580.1, NP_758858.1 (human) and NP_067258.1 (mouse); IL-5, NP_000870.1 (human) and NP_034688.1 (mouse); IL-7, NP_000871.1, NP_001186815.1, NP_001186816.1, NP_001186817.1 (human) and NP_032397.1 (mouse); IL-10, NP_000563 (human) and NP_034678.1 (mouse); IL-12A, NP_000873.2 (human) and NP_001152896.1, NP_032377.1 (mouse); IL-12B, NP_002178.2 (human) and NP_001290173.1 (mouse); IL-15, NP_000576.1, NP_751915.1 (human) and NP_001241676.1, NP_032383.1 (mouse); IL-17(a), NP_002181.1, NP_034682.1 (human); NP_002181.1; NP_034682.1 (mouse); TGF-beta 1, NP_000651.3 (human) and NP_035707.1 (mouse); TGF-beta 2, NP_001129071.1, NP_003229.1 (human) and NP_033393.2 (mouse); and TGF-beta, NP_003230.1 (human). Non-limiting examples of fragments and variants of the aforementioned soluble cues are presented, for example, in the database UNIPROT. [0276] In some aspects, the soluble cue comprises interleukin-2 (IL-2) or an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof with one or more additional soluble cues listed above. Non-limiting examples of IL-2 agonists, mimetics thereof, variants thereof, and functional fragments thereof include those provided in U.S.
Patent No.5,496,924; U.S. Patent No.6,955,807; Margolin et al, Clin Cancer Res.1;13(11):3312- 9 (2007); Eckenberg et al, J Immunol 165:4312-4318 (2000); Levin et al, Nature 484, 529-533, (2012); and Zurawski et al., EMBO Journal, 9(12): 3899-3905 (1990), each of which is incorporated herein by reference in its entirety. [0277] In some aspects, the scaffolds comprise a plurality of soluble cues. In some aspects, the scaffold comprises a first soluble cue comprising IL-2 and a second soluble cue comprising IL- 7, IL-21, IL-15, or IL-15 superagonist. IL-15 superagonist (IL-15 SA) is a combination of IL-15 with soluble IL-15 receptor-a, which possesses greater biological activity than IL-15 alone. In some aspects, the scaffold comprises a first soluble cue comprising IL-2, a second soluble cue comprising IL-7, and a third soluble cue comprising IL-15. In some aspects, the scaffold comprises a first soluble cue comprising IL-2 and a second and third soluble cue comprising IL-7, IL-21, IL- 15, or IL-15 superagonist. [0278] 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. [0279] In some aspects, the soluble cue is released from the PCS in a controlled-release manner. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 5 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 6 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 7 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 8 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 9 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 10 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 11 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 12 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 13 days. In some aspects, the soluble cue is released from the PCS in a sustained manner
for at least 14 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 15 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 20 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 25 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 30 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 35 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 40 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 45 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 50 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 55 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 60 days. In some aspects, the soluble cue is released from the PCS in a sustained manner for at least 65 days. [0280] In some aspects, the total soluble cue input to MSR mass ratio (µg total soluble cue input to µg MSR) is about 0.001 to about 0.005. In some aspects, the total soluble cue input to MSR mass ratio is about 0.001. In some aspects, the total soluble cue input to mass ratio is about 0.002. In some aspects, the total soluble cue input to MSR mass ratio is about 0.003. In some aspects, the total soluble cue input to MSR mass ratio is about 0.004. In some aspects, the total soluble cue input to MSR mass ratio is about 0.005. In some aspects, wherein a scaffold comprises more than one soluble cue, the cues are present in equal amounts. In some aspects, the scaffold comprises more than one soluble cue, wherein the cues are present in unequal amounts. II.A.7. Further aspects of scaffolds [0281] In some aspects the scaffolds comprise a plurality of surface cues and soluble cues. A typical scaffold can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or more of each of the aforementioned functional molecules. [0282] In some aspects, the functional molecules are recombinant. In some aspects, the functional molecules are humanized derivatives of mammalian counterparts. Exemplary mammalian species from which the functional molecules are derived include, but are not limited to, mouse, rat, hamster, guinea pig, ferret, cat, dog, monkey, or primate. In some aspects, the functional molecules are human or humanized version of the aforementioned functional molecules. [0283] The functional molecules can be modified to increase protein stability in vivo. Alternatively, the functional molecules can be engineered to be more or less immunogenic. For
instance, insofar as the structures of the various functional molecules are known, the sequences can be modified at one or more of amino acid residues, e.g., glycosylation sites, to generate immunogenic variants. [0284] Any functional molecule (e.g., any antigen, antibody, protein, enzyme, fragment thereof, recombinant or purified natural ligands or derivatives thereof, or any combination thereof) can be directly or indirectly immobilized onto the layer comprising MSR and/or the layer comprising lipids using routine techniques. In some aspects, the functional molecules are provided in an organelle (e.g., golgi membrane or plasma membrane), a cell, a cell cluster, a tissue, a microorganism, an animal, a plant, or an extract thereof, which in turn is immobilized onto the layer comprising MSR or the layer comprising lipids. In some aspects, the functional molecule is synthesized by genetic engineering or chemical reactions at the desired situs, e.g., outer face of the layer comprising lipids. [0285] Each of the aforementioned functional molecules, e.g., surface cues and soluble cues can, independently from one another, be loaded, adsorbed or integrated into/onto the layer comprising MSR or the layer comprising lipids. Therefore, in some aspects, the surface cues are loaded, adsorbed or integrated into/onto the layer of the scaffold comprising MSR. In one aspect, the surface cues are loaded, adsorbed or integrated into/onto the layer comprising lipids. In some aspects, the surface cues are loaded, adsorbed or integrated into/onto both the layer comprising MSR as well as the layer comprising lipids. In some aspects, the surface cue comprises a co- stimulatory molecule loaded, adsorbed or integrated into/onto the layer comprising MSR. In some aspects, the co-stimulatory molecule is loaded, adsorbed or integrated into/onto the layer comprising lipids. In some aspects, the surface cue comprises a co-stimulatory molecule, which is loaded, adsorbed or integrated into/onto both the layer comprising MSR as well as the layer comprising lipids. In some aspects, the soluble cues are loaded, adsorbed or integrated into/onto the layer comprising MSR. In some aspects, the soluble cues are loaded, adsorbed or integrated into/onto the layer comprising lipids. In some aspects, the soluble cues are loaded, adsorbed or integrated into/onto both the layer comprising MSR as well as the layer comprising lipids. [0286] In general, the functional molecules and the layer comprising MSR and/or the layer comprising lipids, can be linked together through the use of reactive groups, which are typically transformed by the linking process into a new organic functional group or unreactive species. The reactive functional group(s) can be located in any of the aforementioned components. Reactive groups and classes of reactions useful in practicing the present disclosure are generally those that are well known in the art of bioconjugate chemistry. Currently favored classes of reactions
available with reactive chelates are those that proceed under relatively mild conditions. These include, but are not limited to, nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions) and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reaction, Diels-Alder addition). In some aspects, chemical coupling comprises click chemistry, discussed in, for example, clickchemistrytools.com. In some aspects, chemical coupling comprises a click chemistry reagent (e.g., DBCO or azide). These and other useful reactions are discussed in, for example, March, Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, Bioconjugate Techniques, Academic Press, San Diego, 1996; and Feeney et al, Modification of Proteins; vol.198, American Chemical Society, Washington, D.C., 1982. [0287] In some aspects, the scaffolds comprise one or more additional molecules. In some aspects, the one or more additional molecules are naturally-occurring, synthetically produced, or recombinant compounds. In some aspects, the one or more additional molecules comprise peptides, polypeptides, nucleic acids, small molecules, haptens, carbohydrates, or agents, including fragments thereof or combinations thereof. [0288] In some aspects, an anchor is used to connect a functional molecule to a pore wall. However, the anchor is not an essential component. In some aspects, each pore of the mesoporous silica accommodates at least one functional molecule. The pore size depends on the size of the functional molecule to be immobilized. In some aspects, the functional molecule is immobilized in a pore. In some aspects, the functional molecule is loaded or adsorbed on an inner surface of the pore by electrostatic bonding. In some aspects, the functional molecule is loaded or adsorbed on an inner surface of the pore by a noncovalent bond. [0289] In some aspects, the anchor reduces a large structural change of the functional molecule to hold it stably. In some aspects, the anchor comprises substantially the same component as the mesoporous material. In some aspects, the anchor comprises one or more functional groups to permit binding to a desired functional molecule: a hydroxyl group, an amide group, an amino group, a pyridine group, a urea group, a urethane group, a carboxyl group, a phenol group, an azo group, a hydroxyl group, a maleimide group, a silane derivative, an aminoalkylene group, or a combination thereof. [0290] In some aspects, a scaffold comprises an antigen. In some aspects, the antigen comprises a polypeptide. In some aspects, the antigen is purified. In some aspects, the antigen is a self-antigen. In some aspects, the antigen is a non-self antigen. Self-antigens are specifically associated with a human disease or a disorder including, but not limited to, autoimmune disorders
and cancer. Non-self antigens are specifically associated with pathogens including, but not limited to, a virus, a bacteria, a protozoan, a parasite, or a fungus. In some aspects, the antigens are loaded onto MHC molecules, e.g., HLA-A, HLA-B, HLA-C, DP, DQ, and DR, which are then incorporated into/onto the scaffolds. [0291] 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), CD123, CD138, CD171, CD19, 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), EphA2/Eck, ephrinB2, ERBB dimers, ESO-1, estrogen receptor, ETBR (endothelin B receptor), EZH2, FAP-α (fibroblast activation protein α), FBP (a folate binding protein), FCRL5, fetal AchR (fetal acetylcholine receptor), fodrin, Fra-l/Fosl 1, FR-α (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, gp100 (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)-A1, 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 l7, PRAME (preferentially expressed antigen of melanoma), progesterone receptor, PROX1, PSA (prostate specific antigen), PSCA (prostate stem cell antigen ), PSMA, PSMA (prostate specific membrane antigen), RAE-1 proteins, RAGE, ras, RBPSUH, RCAS1, ROR1, ROR2, RTN4, SART1, SART2, SART3, SCP-I, SIRPα (signal-regulatory protein alpha), SLIT, SLITRK6 (NTRK-like protein 6), Smad family of tumor antigens, SOX10, 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 (Multl), UPAR, VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, WT-1, αvβ6 or another integrin, β- catenin, β1,6-Ν, β-catenin, γ-catenin, ίίνίηβ, and antigens from HIV, HBV, HCV, HPV, and other pathogens, a patient-specific neoantigen, or an immunogenic peptide thereof, and any combination thereof. [0292] In some aspects, the antigen is formulated to interact with the immune cell via direct binding or indirect binding. Types of direct binding include, for example, engagement or coupling of the antigen with the cognate receptor, e.g., T-cell receptor. Indirect binding can occur through the intermediacy of one or more secondary agents or cell-types. For example, the antigen can first bind to a B-cell or an antigen-presenting cell (APC), get processed (e.g., degraded) and presented on cell-surface major-histocompatibility complexes (MHC), to which the target cell population, e.g., T-cell, binds. Alternately, the antigen can recruit other intermediary cells that secrete various cytokines, growth factors, chemokines, etc., which in turn attract the target immune cell population. In some aspects, the antigen is CD19, CD22, or a fragment thereof. [0293] In some aspects, the scaffold comprises a membrane-associated protein, which is anchored directly or indirectly to the layer comprising lipids. In some aspects, the membrane- associated protein comprises a selective or non-selective membrane transport protein, ion channel, pore forming protein, membrane-resident receptors, or any combination thereof. [0294] In some aspects, the scaffold comprises a growth factor, a cytokine (e.g., IL-2, IL- 7, IL-15, and/or IL-21), a chemokine, an interleukin, an adhesion signaling molecule, an integrin signaling molecule, a fragment thereof, or any combination thereof. In some aspects, these
molecules can be used as soluble cues and/or surface cues and can be loaded to either the layer comprising the MSR or the layer comprising the lipids. [0295] In some aspects, the scaffold comprises adhesion molecules. In some aspects, the adhesion molecules further serve as signaling agents. Representative examples of adhesion signaling molecules include, but are not limited to, fibronectin, laminin, collagen, thrombospondin 1, vitronectin, elastin, tenascin, aggrecan, agrin, bone sialoprotein, cartilage matrix protein, fibronogen, fibrin, fibulin, mucins, entactin, osteopontin, plasminogen, restrictin, serglycin, SPARC/osteonectin, versican, von Willebrand Factor, polysaccharide heparin sulfate, connexins, collagen, RGD (Arg-Gly-Asp) and YIGSR (Tyr-Ile-Gly- Ser-Arg) peptides and cyclic peptides, glycosaminoglycans (GAGs), hyaluronic acid (HA), condroitin-6-sulfate, integrin ligands, selectins, cadherins, and members of the immunoglobulin superfamily. [0296] In some aspects, the functional molecules are conjugated to membrane- associated proteins, which associate with and/or insert into the layer comprising lipids, e.g. gramicidin; a- helix bundles, e.g. bacteriorhodopsin or K+ channels; β-barrels, e.g., a-hemolysin, leukocidin or E. coli porins; or combinations thereof. [0297] In some aspects, the scaffold further comprises one or more recruiting agents. In some aspects, the recruiting agent comprises a T-cell recruiting agent, a B-cell recruiting agent, a dendritic cell recruiting agent, or a macrophage recruiting agent. Examples of such recruiting agents include, but are not limited to chemokines, chemokine ligands, or fragments, variants, homologs, and combinations thereof. In some aspects, the recruitment compound comprises 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. In some aspects, the recruitment compound comprises granulocyte macrophage-colony stimulating factor (GM-CSF). In some aspects, the recruitment compound comprises chemokine (C-C motif) ligand 21 (CCL-21). In some aspects, the recruitment compound comprises chemokine (C-C motif) ligand 19 (CCL-19). In some aspects, the recruitment compound comprises chemokine (C-X-C Motif) ligand 12 (CXCL12). In some aspects, the recruitment compound comprises interferon gamma (IFNy). In some aspects, the recruitment compound comprises a FMS-like tyrosine kinase 3 (Flt-3) ligand. Preferential recruitment is characterized by an accumulation of at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 2-fold, at least 5-fold, at least 8-fold, at least 10-fold, or greater increase in one or more of a particular type of immune cells compared to other types of immune cells.
[0298] Depending on need, the scaffolds can be specifically formulated to comprise a subset of recruitment agents and adhesion molecules so as to manipulate a particular subset of immune cells, e.g., pan-T cells or a particular sub-population of T-cells. In some aspects, the scaffolds are formulated/fabricated using agents that specifically bind to cell-surface markers that are expressed in the target cells. For example, in the context of T-cells, the scaffolds can be adapted for the preferential recruitment of helper T-cells (CD4+ T cells), cytotoxic T-cells (CD8+ T cells), memory T-cells (CD45RO+ T cells), suppressor T-cells (Ts which cells), regulatory T-cells (Tregs; further characterized as FOXP3+ Treg cells and FOXP3- Treg), natural killer T-cells (NK cells; differentially express CDld+), mucosal associated invariant (MAITs; differentially express MR1), gamma delta T cells, (γδ T cells; comprise TCRs containing one γ -chain and one δ-chain). Such agents which bind to cell-surface markers can include, for example, haptens, peptides, ligands, antibodies, or the like, or any combination thereof. Other routine techniques for enriching the isolates with one or more cell subtype can be used in situ or ex situ. II.A.8. Methods of making [0299] The scaffolds of the disclosure can be generated in a variety of ways and used for various applications, including, but not limited to, modulating the type and abundance of functional molecules or additional agents in accordance with a scaffold, for use in the manipulation of target effector cells, e.g., T-cells, isolation of a specific population of effector cells, e.g., a sub-population of CD8+ T-cells, therapy of diseases, and the production of compositions and kits. Examples of methods of making and using such scaffolds is described in PCT Publication No. WO 2018/013797 A1 and Chung et al. (Nature Biotechnology 36(2): 160-169 (2018)), the entire contents of which are incorporated by reference herein. [0300] For isolation and/or expansion of a desired population of cells, the concentration of cells and scaffold surface can be varied. In some aspects, the volume in which the scaffolds and cells are mixed is decreased (i.e., increasing the concentration of cells), to ensure maximum contact of cells and scaffolds. In some aspects, a concentration of 2 billion cells/ml is used. In some aspects, a concentration of 1 billion cells/ml is used. In a further aspect, greater than 100 million cells/ml is used. In a further aspect, a concentration of cells of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells/ml is used. In yet another aspect, a concentration of cells from 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells/ml is used. In further aspects, a concentration of 125 million or 150 million cells/ml is used. Using high concentrations can result in increased cell yield, cell activation, and cell
expansion. Further, use of high cell concentrations can allow more efficient capture of cells that can weakly express target antigens of interest, such as CD28− negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc.). Such populations of cells can have a therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression. [0301] In other aspects, it is desirable to use lower concentrations of cells. This can be achieved by lowering the scaffold:cell ratio, such that interactions between the scaffolds and cells are minimized. This method selects for cells that express high amounts of desired antigens to be bound to the scaffolds. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In some aspects, the concentration of cells used is 5x106/ml. In other aspects, the concentration used can be from about 1×104/ml to 1×109/ml, and any integer value in between, e.g., 1×105/ml to 1×108/ml, 1×106/ml to 1×107/ml, 1×107/ml to 1×109/ml. II.B. Cell Isolation, Expansion, and Harvest [0302] Some aspects of the present disclosure are directed to methods of expanding immune cells ex vivo or in vitro comprising culturing the immune cells in a medium comprising a feeder cell replacement (“preliminary culture” if an additional expansion is added), wherein the feeder cell replacement comprises a PCS comprising a surface-exposed CD3 agonist, and wherein the immune cells comprise TILs. As such, some aspects of the present disclosure are directed to methods of expanding TILs ex vivo or in vitro comprising culturing the TILs in a medium comprising a feeder cell replacement (“preliminary culture” if an additional expansion is added), wherein the feeder cell replacement comprises a PCS comprising a surface-exposed CD3 agonist. [0303] In some aspects, the method further comprises obtaining a population of TILs from a tumor. In some aspects, the tumor is dissociated to generate a population of TILs. In some aspects, the dissociated tumor sample is a single cell suspension. Any means of generating a single cell suspension can be used in the methods disclosed herein (e.g., enzymatic and/or mechanical dissociation). Solely as an example, in some aspects, a single cell suspension is prepared using a gentleMACS system. In some aspects, the tumor is cultured in a medium comprising IL-2 to generate the population of TILs. In some aspects, the population of TILs is cultured in a medium comprising IL-2 ("preliminary culture"). In some aspects, the method further comprises culturing the selected population of TILs in a medium comprising IL-2.
[0304] In some aspects, the method further comprises culturing the population of TILs in a first expansion medium comprising a cell-support matrix, a cytokine, and a T cell activator (a "initial expansion"). In some aspects, the method further comprises culturing the population of selected TILs after the initial expansion in a second expansion medium comprising a cell-support matrix, cytokine, and a T cell activator (a "additional expansion"). In some aspects, the cell support matrix of the initial expansion comprises a feeder cell replacement disclosed herein, e.g., a PCS comprising a CD3 agonist, e.g., OKT3. In some aspects, the cell support matrix of the additional expansion comprises a feeder cell replacement disclosed herein, e.g., a PCS comprising a CD3 agonist, e.g., OKT3. In some aspects, the cell support matrix of the initial expansion and the additional expansion comprises a feeder cell replacement disclosed herein, e.g., a PCS comprising a CD3 agonist, e.g., OKT3. [0305] In some aspects, the method comprises culturing the population of TILs in an expansion medium comprising a cytokine and a feeder cell replacement disclosed herein, e.g., a PCS comprising a CD3 agonist, e.g., OKT3 (a "single expansion step"). [0306] Some aspects of the present disclosure are directed to methods of culturing TILs ex vivo or in vitro for a therapy to a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a population of TILs; (ii) culturing the population of TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix (a "initial expansion"); and (iii) culturing the population of TILs from the initial expansion in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix (a "additional expansion"); wherein the cell support matrix of the initial expansion, the additional expansion, or both comprises a feeder cell replacement disclosed herein comprising the T cell activator, e.g., a PCS comprising a CD3 agonist, e.g., OKT3. In some aspects, the method further comprises culturing the population of TILs in a medium comprising IL-2 before the initial expansion. [0307] Some aspects of the present disclosure are directed to methods of culturing TILs ex vivo or in vitro for a therapy to a subject in need thereof comprising: (i) fragmenting a tumor sample obtained from the subject comprising TILs; (ii) culturing the tumor fragments in a medium comprising IL-2 to produce a population of TILs; (iii) culturing the population of TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix (a "initial expansion"); and (iv) culturing the population of TILs from the initial expansion in a second expansion medium, wherein the second expansion medium comprises a T
cell activator and a cell support matrix (a "additional expansion"); wherein the cell support matrix of the initial expansion, the additional expansion, or both comprises a feeder cell replacement disclosed herein comprising the T cell activator, e.g., a PCS comprising a CD3 agonist, e.g., OKT3. In some aspects, the cell support matrix of the initial expansion, the additional expansion, or both comprises one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the first expansion medium, the second expansion medium, or both comprises one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. [0308] Some aspects of the present disclosure are directed to methods of culturing TILs ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a population of TILs; (ii) culturing the population of TILs in a preliminary culture medium, wherein the preliminary culture medium comprises IL-2; and (ii) culturing the population of TILs from the preliminary culture medium in a rapid expansion medium, wherein the rapid expansion medium comprises a feeder cell replacement disclosed herein, e.g., a PCS comprising a CD3 agonist, e.g., OKT3 (a "single expansion step"). In some aspects, the cell support matrix of the feeder cell replacement, e.g., the PCS comprising a CD3 agonist, e.g., OKT3, further comprises one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the preliminary culture medium, the rapid expansion medium, or both comprises one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. [0309] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a population of TILs; (ii) culturing the population of TILs in a preliminary culture medium, wherein the preliminary culture medium comprises IL-2; (iii) culturing the population of TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix (a "initial expansion"); and (iv) culturing the TILs from the initial expansion in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix (a "additional expansion"); wherein the cell support matrix of the initial expansion, the additional expansion, or both comprises a feeder cell replacement disclosed herein comprising the T cell activator, e.g., a PCS comprising a CD3 agonist, e.g., OKT3. In some aspects, the cell support matrix of the initial expansion, the additional expansion, or both comprises one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the preliminary culture medium, the first expansion medium, the second expansion
medium, or any combination thereof comprises one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. [0310] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a population of TILs; (ii) culturing the population of TILs in a preliminary culture medium, wherein the preliminary culture medium comprises IL-2; (iii) culturing the population of TILs in a first expansion medium, wherein the first expansion medium comprises a feeder cell replacement disclosed herein comprising a T cell activator, e.g., a PCS comprising a CD3 agonist, e.g., OKT3 (a "initial expansion"); and (iv) culturing the TILs from the initial expansion in a second expansion medium, wherein the second expansion medium comprises a T cell activator and a cell support matrix (a "additional expansion"). In some aspects, the feeder cell replacement comprising a T cell activator, e.g., the PCS comprising a CD3 agonist, e.g., OKT3, of the first expansion medium comprises one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the preliminary culture medium, the first expansion medium, the second expansion medium, or any combination thereof comprises one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. [0311] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a population of TILs; (ii) culturing the population of TILs in a preliminary culture medium, wherein the preliminary culture medium comprises IL-2; (iii) culturing the population of TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix (a "initial expansion"); and (iv) culturing the TILs from the initial expansion in a second expansion medium, wherein the second expansion medium comprises a feeder cell replacement disclosed herein comprising a T cell activator, e.g., a PCS comprising a CD3 agonist, e.g., OKT3 (a "additional expansion"). In some aspects, the cell support matrix; the feeder cell replacement comprising a T cell activator, e.g., the PCS comprising a CD3 agonist, e.g., OKT3; or both comprises one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the preliminary culture medium, the first expansion medium, the second expansion medium, or any combination thereof comprises one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1.
[0312] Some aspects of the present disclosure are directed to methods of culturing tumor infiltrating lymphocytes (TILs) ex vivo or in vitro for a therapy for a subject in need thereof comprising: (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a population of TILs; (ii) culturing the population of TILs in a preliminary culture medium, wherein the preliminary culture medium comprises IL-2; (iii) culturing the population of TILs in a first expansion medium, wherein the first expansion medium comprises a feeder cell replacement disclosed herein comprising a T cell activator, e.g., a PCS comprising a CD3 agonist, e.g., OKT3 (a "initial expansion"); and (iv) culturing the TILs from the initial expansion in a second expansion medium, wherein the second expansion medium comprises a feeder cell replacement disclosed herein comprising a T cell activator, e.g., a PCS comprising a CD3 agonist, e.g., OKT3 (a "additional expansion"). In some aspects, the cell support matrix of; the feeder cell replacement comprising a T cell activator, e.g., the PCS comprising a CD3 agonist, e.g., OKT3; or both comprises one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the preliminary culture medium, the first expansion medium, the second expansion medium, or any combination thereof comprises one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. [0313] Any method of TIL isolation, culture, and/or expansion can be modified according to the methods disclosed herein, e.g., by culturing and/or expanding the TILs in a culture medium described herein. II.B.1. Tumor Sample [0314] In general, TILs are obtained from a tumor sample obtained from a human subject. Any methods for obtaining a tumor biopsy from a subject can be used in the methods disclosed herein, so long as the tumor sample contains a mixture of tumor and TILs. In some aspects, the tumor sample is isolated through a tumor resection. In some aspects, the tumor sample is isolated by a needle biopsy (see, e.g., US Publication No. US 2020/0277573, which is incorporated by reference herein in its entirety). In some aspects, the tumor sample comprises a solid tumor, including a primary tumor, invasive tumor or metastatic tumor. In other aspects, the tumor sample comprises a liquid tumor, such as a tumor obtained from a hematological malignancy. The tumor may be of any cancer type, including, but not limited to, breast, pancreatic, prostate, colorectal, cervical, lung, brain, renal, stomach, liver (including but not limited to hepatocellular carcinoma) and skin (including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma). In some aspects, the tumor comprises a melanoma. In some aspects, the tumor
comprises a colorectal cancer. In some aspects, the tumor comprises a pancreatic cancer. In some aspects, the tumor comprises a head and neck cancer. In some aspects, the tumor comprises a cervical cancer. In some aspects, the tumor comprises an ovarian cancer. In some aspects, the tumor comprises a non-small cell lung cancer. In some aspects, the tumor comprises a breast cancer. [0315] In some aspects, the tumor sample is cryopreserved after the tumor sample is dissociated. In some aspects, the tumor sample is dissociated. In some aspects, the tumor sample is dissociated in MRM prior to cryopreservation. In some aspects, the tumor sample is cryopreserved prior to TIL isolation/expansion. In some aspects, the tumor sample is dissociated in MRM prior to a first expansion. In some aspects, the dissociated tumor sample is a single cell suspension. In some aspects, the tumor sample is cryopreserved after initial TIL expansion. In some aspects the tumor sample is fresh, e.g., not cryopreserved. In some aspects, the tumor sample is placed directly into MRM media. [0316] In some aspects, the donor patient (e.g., the subject from which the tumor is obtained) is treatment naïve (i.e., the patient has not received a prior therapy for the treatment of the tumor). In some aspects, the donor patient has received one or more prior therapy for the treatment of the tumor. In some aspects, the subject has received at least one prior therapy, at least two prior therapies, at least three prior therapies, or at least four prior therapies. In some aspects, the subject is relapsed or refractory to one or more prior therapy. In some aspects, the one or more prior therapy comprises a BRAF inhibitor. In some aspects, the one or more prior therapy comprises an MEK inhibitor. [0317] In some aspects, the subject has received one or more prior anticancer therapy. In some aspects, the prior anticancer therapy comprises a standard of care therapy. In some aspects, the prior anticancer therapy comprises an immunotherapy. In some aspects, the prior therapy comprises an immunotherapy comprising a checkpoint inhibitor. In some aspects, the prior therapy comprises an immunotherapy comprising an anti-PD-1 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, or any combination thereof. [0318] In some aspects, the subject is administered one or more therapy that enhances the isolation and/or expansion of TILs prior to resection of the tumor sample. In some aspects, the subject is administered a kinase inhibitor or an ITK inhibitor. Examples of kinase inhibitors and/or ITK inhibitors can be found, for example, in Int'l Publication No. WO2019217753, which is incorporated by reference herein in its entirety. In some aspects, the kinase inhibitor and/or the ITK inhibitor is added to the culture medium during the preliminary culture and/or the second expansion. In some aspects, the ITK inhibitor comprises aminothiazole-based ITK inhibitors,
benzimidazole-based ITK inhibitors, aminopyrimidine-based ITK inhibitors, 3-aminopyride-2- ones-based ITK inhibitors, indolylndazole-based ITK inhibitors, pyrazolyl-indole-based inhibitors, thienopyrazole inhibitors, or ITK inhibitors targeting cysteine-442 in the ATP pocket. In some aspects, the ITK inhibitor comprises ibrutinib, dasatinib, bosutinib, nilotinib, erlotinib, BMS509744, CTA056, GSK2250665A, PF06465469, or any combination thereof. [0319] In some aspects, the tumor sample is cut into smaller fragments. In some aspects, the one or more of the smaller fragments is at least about 1 mm2, at least about 1.5 mm2, at least about 2 mm2, at least about 2.5 mm2, at least about 3 mm2, at least about 3.5 mm2, at least about 4 mm2, at least about 4.5 mm2, at least about 5 mm2, at least about 5.5 mm2, at least about 6 mm2, or at least about 6.5 mm2. In some aspects, the one or more of the smaller fragments is at least about 1 mm3, at least about 1.5 mm3, at least about 2 mm3, at least about 2.5 mm3, at least about 3 mm3, at least about 3.5 mm3, at least about 4 mm3, at least about 4.5 mm3, at least about 5 mm3, at least about 5.5 mm3, at least about 6 mm3, at least about 6.5 mm3, at least about 7 mm3, at least about 7.5 mm3, at least about 8 mm3, at least about 8.5 mm3, at least about 9 mm3, at least about 9.5 mm3, or at least about 10 mm3. In some aspects, the tumor samples are subjected to an enzymatic digest, by culturing the tumor samples in an enzymatic media (e.g., RPMI 1640 buffer or MRM supplemented with glutamate (e.g., about 2 mM), gentamicine (e.g., about 10 mcg/mL), DNase (e.g., about 30 units/mL), and collagenase (e.g., about 1.0 mg/mL)). In some aspects, the tumor digests are produced by placing the tumor in the enzymatic media and/or mechanically dissociating (i.e., disaggregating) the tumor (e.g., for about 1 minute), followed by incubation at 37° C. in 5% CO2 (e.g., for 30 minutes), followed by repeated cycles of mechanical dissociation and incubation under the foregoing conditions until only small tissue pieces are present. At the end of this process, if the cell suspension contains a large number of red blood cells or dead cells, a density gradient separation using FICOLL branched hydrophilic polysaccharide can be performed to remove these cells. The mechanical and/or enzymatic dissociation can be performed in any medium. In some aspects, the mechanical and/or enzymatic dissociation is performed in an MRM medium disclosed herein. [0320] In some aspects, the mechanical dissociation comprises applying a physical pressure to the resected tumor. In some aspects, the mechanical dissociation comprises repeated physical pressure. In some aspects, the repeated physical pressure is applied at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times, at least about 110 times, at least about 120 times, at least about 130 times, at least about 140 times, at least about 150 times, at least about 160 times, at least about 170 times,
at least about 180 times, at least about 190 times, at least about 200 times, at least about 210 times, at least about 220 times, at least about 230 times, at least about 240 times, at least about 250 times, at least about 260 times, at least about 270 times, at least about 280 times, at least about 290 times, at least about 300 times, at least about 310 times, at least about 320 times, at least about 330 times, at least about 340 times, at least about 350 times, or at least about 360 times per minute. In some aspects, the repeated physical pressure is applied at least about 120 to 260 times per minute. In some aspects, the repeated physical pressure is applied up to about 6 N/cm2, up to about 5.5 N/cm2, up to about 5.0 N/cm2, up to about 4.5 N/cm2, up to about 4.0 N/cm2, up to about 3.5 N/cm2, up to about 3.0 N/cm2. In some aspects, the mechanical dissociation proceeds for about 90 minutes or less, about 85 minutes or less, about 80 minutes or less, about 75 minutes or less, about 70 minutes or less, about 65 minutes or less, about 60 minutes or less, about 55 minutes or less, or about 50 minutes or less. In some aspects, the mechanical dissociation is applied at room temperature. In some aspects, the mechanical dissociation is applied at less than room temperature. In some aspects, the mechanical dissociation is applied according to the methods disclosed in and/or using a device disclosed in Int'l Publication No. WO 2021/123832, which is incorporated by reference herein in its entirety. [0321] In some aspects, the tumor sample is dissociated to prepare a single cell suspension comprising a population of TILs. Any methods of dissociation can be used in the methods disclosed herein. In some aspects, the tumor sample is cut into small fragments. In some aspects, the small fragments are then collected in one or more gentleMACS tube and subjected to dissociation according to the gentleMACS system. [0322] In some aspects, the single cell suspension is taken directly into a preliminary culture step, e.g., wherein the population of TILs is cultured in a preliminary culture medium comprising IL-2. [0323] In some aspects, the single cell suspension is taken directly into a first expansion step, e.g., an initial expansion disclosed herein. In some aspects, the singe cell suspension is cryopreserved prior to advancing to a first expansion step, e.g., an initial expansion. II.B.2. Preliminary Culture [0324] In some aspects, the tumor sample (e.g., the dissociated tumor sample, e.g., the single cell suspension) is placed into a preliminary culture medium comprising IL-2. In some aspects, the preliminary culture medium comprises from about 1000 IU/ml to at least about 5000 IU/ml IL-2. In some aspects, the preliminary culture medium comprises at least about 1000 IU/ml,
at least about 1100 IU/ml, at least about 1200 IU/ml, at least about 1300 IU/ml, at least about 1400 IU/ml, at least about 1500 IU/ml, at least about 1600 IU/ml, at least about 1700 IU/ml, at least about 1800 IU/ml, at least about 1900 IU/ml, at least about 2000 IU/ml, at least about 2100 IU/ml, at least about 2200 IU/ml, at least about 2300 IU/ml, at least about 2400 IU/ml, at least about 2500 IU/ml, at least about 2600 IU/ml, at least about 2700 IU/ml, at least about 2800 IU/ml, at least about 2900 IU/ml, at least about 3000 IU/ml, at least about 3100 IU/ml, at least about 3200 IU/ml, at least about 3300 IU/ml, at least about 3400 IU/ml, at least about 3500 IU/ml, at least about 3600 IU/ml, at least about 3700 IU/ml, at least about 3800 IU/ml, at least about 3900 IU/ml, at least about 4000 IU/ml, at least about 4100 IU/ml, at least about 4200 IU/ml, at least about 4300 IU/ml, at least about 4400 IU/ml, at least about 4500 IU/ml, at least about 4600 IU/ml, at least about 4700 IU/ml, at least about 4800 IU/ml, at least about 4900 IU/ml, at least about 5000 IU/ml, at least about 5100 IU/ml, at least about 5200 IU/ml, at least about 5300 IU/ml, at least about 5400 IU/ml, at least about 5500 IU/ml, at least about 5600 IU/ml, at least about 5700 IU/ml, at least about 5800 IU/ml, at least about 5900 IU/ml, at least about 6000 IU/ml, at least about 6100 IU/ml, at least about 6200 IU/ml, at least about 6300 IU/ml, at least about 6400 IU/ml, at least about 6500 IU/ml, at least about 6600 IU/ml, at least about 6700 IU/ml, at least about 6800 IU/ml, at least about 6900 IU/ml, at least about 7000 IU/ml, at least about 7500 IU/ml, at least about 8000 IU/ml, at least about 9000 IU/ml, at least about 10,000 IU/ml IL-2. In some aspects, the preliminary culture medium comprises about 6000 IU/ml IL-2. [0325] In some aspects, the preliminary culture medium comprises IL-21. In some aspects, the preliminary culture medium comprises from about 1 ng/ml to at least about 100 ng/ml IL-21. In some aspects, the preliminary culture medium comprises at least about 1 ng/ml, at least about 2 ng/ml, at least about 3 ng/ml, at least about 4 ng/ml, at least about 5 ng/ml, at least about 6 ng/ml, at least about 7 ng/ml, at least about 8 ng/ml, at least about 9 ng/ml, at least about 10 ng/ml, at least about 11 ng/ml, at least about 12 ng/ml, at least about 13 ng/ml, at least about 14 ng/ml, at least about 15 ng/ml, at least about 20 ng/ml, at least about 25 ng/ml, at least about 30 ng/ml, at least about 35 ng/ml, at least about 40 ng/ml, at least about 45 ng/ml, at least about 50 ng/ml, at least about 60 ng/ml, at least about 70 ng/ml, at least about 80 ng/ml, at least about 90 ng/ml, or at least about 100 ng/ml IL-21. In some aspects, the preliminary culture medium comprises about 10 ng/ml IL-21. [0326] In some aspects, the preliminary culture does not comprise a T cell activator (e.g., a CD3 agonist). In some aspects, the preliminary culture does not comprise a cell-support matrix
(e.g., a population of APCs). In some aspects, the preliminary culture does not comprise a feeder cell replacement. [0327] In some aspects, the TILs are cultured in the preliminary culture step for less than about 7 days. In some aspects, the TILs are cultured in the preliminary culture step for less than about 6 days. In some aspects, the TILs are cultured in the preliminary culture step for less than about 5 days. In some aspects, the TILs are cultured in the preliminary culture step for less than about 4 days. In some aspects, the TILs are cultured in the preliminary culture step for less than about 3 days. In some aspects, the TILs are cultured in the preliminary culture step for less than about 2 days. In some aspects, the TILs are cultured in the preliminary culture step for about 4 days. II.B.3. Initial expansion [0328] In some aspects, the tumor sample (e.g., the dissociated tumor sample, e.g., the single cell suspension comprising the population of TILs) is placed into a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix (a "initial expansion"). In some aspects, the cell support matrix comprises a feeder cell replacement disclosed herein, e.g., a PCS, comprising the T cell activator, e.g., a CD3 agonist, e.g., OKT3. In some aspects, the method comprises placing the tumor sample, (e.g., the dissociated tumor sample, e.g., the single cell suspension comprising the population of TILs) into a first expansion medium, wherein the first expansion medium comprises a PCS comprising a CD3 agonist, e.g., OKT3. [0329] In some aspects, the method comprises (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a population of TILs; and (ii) culturing the population of TILs in a first expansion medium, wherein the first expansion medium comprises a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "initial expansion"). In some aspects, the method comprises (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a population of TILs; and (ii) culturing the population of TILs in a first expansion medium, wherein the first expansion medium comprises a PCS comprising a CD3 agonist, e.g., OKT3 (a "initial expansion"). [0330] In some aspects, the method comprises (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a population of TILs; (ii) culturing of TILs in a preliminary culture medium disclosed herein; and (iii) culturing the population of TILs in a first expansion medium, wherein the first expansion medium comprises
a T cell activator and a cell support matrix at a first ratio of cell support matrix to TIL (a "initial expansion"). In some aspects, the method comprises (i) dissociating a tumor sample obtained from the subject comprising TILs to generate a single cell suspension comprising a population of TILs; (ii) culturing of TILs in a preliminary culture medium disclosed herein; and (iii) culturing the population of TILs in a first expansion medium, wherein the first expansion medium comprises a PCS comprising a CD3 agonist, e.g., OKT3 (a "initial expansion"). [0331] In some aspects, the initial expansion lasts for about 5-19 days. In some aspects, the TILs are cultured in the initial expansion for about 19 days. In some aspects, the TILs are cultured in the initial expansion for less than about 19 days. In some aspects, the TILs are cultured in the initial expansion for less than about 18 days. In some aspects, the TILs are cultured in the initial expansion for less than about 17 days. In some aspects, the TILs are cultured in the initial expansion for less than about 16 days. In some aspects, the TILs are cultured in the initial expansion for less than about 15 days. In some aspects, the TILs are cultured in the initial expansion for less than about 14 days. In some aspects, the TILs are cultured in the initial expansion for less than about 13 days. In some aspects, the TILs are cultured in the initial expansion for less than about 12 days. In some aspects, the TILs are cultured in the initial expansion for less than about 11 days. In some aspects, the TILs are cultured in the initial expansion for less than about 10 days. In some aspects, the TILs are cultured in the initial expansion for less than about 9 days. In some aspects, the TILs are cultured in the initial expansion for less than about 8 days. In some aspects, the TILs are cultured in the initial expansion for less than about 7 days. In some aspects, the TILs are cultured in the initial expansion for less than about 6 days. In some aspects, the TILs are cultured in the initial expansion for less than about 5 days. [0332] In some aspects, the initial expansion lasts for about 5-19 days. In some aspects, the TILs are cultured in the initial expansion for about 19 days. In some aspects, the TILs are cultured in the initial expansion for about 18 days. In some aspects, the TILs are cultured in the initial expansion for about 17 days. In some aspects, the TILs are cultured in the initial expansion for about 16 days. In some aspects, the TILs are cultured in the initial expansion for about 15 days. In some aspects, the TILs are cultured in the initial expansion for about 14 days. In some aspects, the TILs are cultured in the initial expansion about 13 days. In some aspects, the TILs are cultured in the initial expansion for about 12 days. In some aspects, the TILs are cultured in the initial expansion for about 11 days. In some aspects, the TILs are cultured in the initial expansion for about 10 days. In some aspects, the TILs are cultured in the initial expansion for about 9 days. In some aspects, the TILs are cultured in the initial expansion for about 8 days. In some aspects, the
TILs are cultured in the initial expansion for about 7 days. In some aspects, the TILs are cultured in the initial expansion for about 6 days. In some aspects, the TILs are cultured in the initial expansion for about 5 days. [0333] In some aspects, the TILs are cultured in the initial expansion for a period of time until a certain number of cells is reached. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 10-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 following culture in the initial expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 10-fold following culture in the initial expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 15-fold following culture in the initial expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 20-fold following culture in the initial expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 25-fold following culture in the initial expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 30-fold following culture in the initial expansion. In some aspects, the number of TILs preset in the dissociated tumor sample is increased by at least about 35-fold following culture in the initial expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 40-fold following culture in the initial expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 45-fold following culture in the initial expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 50-fold following culture in the initial expansion. [0334] In some aspects, the number of TILs following culture in the initial expansion is at least about 5 x 107, at least about 1 x 108, at least about 2 x 108, at least about 3 x 108, at least about 4 x 108, at least about 5 x 108, at least about 6 x 108, at least about 7 x 108, at least about 8 x 108, at least about 9 x 108, at least about 1 x 109, at least about 2 x 109, at least about 3 x 109, at least about 4 x 109, at least about 5 x 109, at least about 6 x 109, at least about 7 x 109, at least about 8 x 109, at least about 9 x 109, at least about 1 x 1010, or at least about 2 x 1010 TILs. In some aspects, the number of TILs following culture in the initial expansion is at least about 1 x 109. In some aspects, the number of TILs following culture in the initial expansion is at least about 2 x 109. In some aspects, the number of TILs following culture in the initial expansion is at least about 3 x 109. In some aspects, the number of TILs following culture in the initial expansion is at least about
4 x 109. In some aspects, the number of TILs following culture in the initial expansion is at least about 5 x 109. In some aspects, the number of TILs following culture in the initial expansion is at least about 6 x 109. In some aspects, the number of TILs following culture in the initial expansion is at least about 7 x 109. In some aspects, the number of TILs following culture in the initial expansion is at least about 8 x 109. In some aspects, the number of TILs following culture in the initial expansion is at least about 9 x 109. In some aspects, the number of TILs following culture in the initial expansion is at least about 10 x 109. [0335] In some aspects, the initial expansion continues until at least about 1 x 109 TILs are obtained. In some aspects, the initial expansion continues until at least about 2 x 109 TILs are obtained. In some aspects, the initial expansion continues until at least about 3 x 109 TILs are obtained. In some aspects, the initial expansion continues until at least about 4 x 109 TILs are obtained. In some aspects, the initial expansion continues until at least about 5 x 109 TILs are obtained. In some aspects, the initial expansion continues until at least about 6 x 109 TILs are obtained. In some aspects, the initial expansion continues until at least about 7 x 109 TILs are obtained. In some aspects, the initial expansion continues until at least about 8 x 109 TILs are obtained. In some aspects, the initial expansion continues until at least about 9 x 109 TILs are obtained. In some aspects, the initial expansion continues until at least about 10 x 109 TILs are obtained. [0336] In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 1 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 2 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 3 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 4 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 5 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 6 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 7 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 8 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 9 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 10 x 109 TILs are obtained. [0337] In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 1 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 2 x 109 TILs are obtained. In some aspects, the initial expansion lasts for
less than 9 days, wherein at least about 3 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 4 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 5 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 6 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 7 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 8 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 9 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 10 x 109 TILs are obtained. [0338] In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 1 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 2 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 3 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 4 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 5 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 6 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 7 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 8 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 9 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 10 x 109 TILs are obtained. [0339] In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 1 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 2 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 3 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 4 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 5 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 6 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 7 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 8 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 9 x 109 TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 10 x 109 TILs are obtained.
[0340] In some aspects, the feeder cell replacement, e.g., PCS, comprises a CD3 agonist. In some aspects, the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the CD3 agonist comprises OKT3. In some aspects, the first expansion medium comprises from about 1 to about 100 ng/mL OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the concentration of OKT3 is at least about 1 ng/ml, at least about 5 ng/ml, at least about 10 ng/ml, at least about 15 ng/ml, at least about 20 ng/ml, at least about 25 ng/ml, at least about 30 ng/ml, at least about 35 ng/ml, at least about 40 ng/ml, at least about 45 ng/ml, at least about 50 ng/ml, at least about 60 ng/ml, at least about 70 ng/ml, at least about 80 ng/ml, at least about 90 ng/ml, or at least about 100 ng/ml. In some aspects, the first expansion medium comprises about 10 ng/ml OKT3. In some aspects, the first expansion medium comprises about 15 ng/ml OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises about 20 ng/ml OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises about 25 ng/ml OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises about 30 ng/ml OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises about 35 ng/ml OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises about 40 ng/ml OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises about 45 ng/ml OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises about 50 ng/ml OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. [0341] In some aspects, the cells are passed through a strainer following the dissociation step. In some aspects, the cells are passed through a strainer following the preliminary culture step. In some aspects, the cells are passed through a strainer following the initial expansion. In some aspects, the cells are passed through an at least about 10 µm, an at least about 15 µm, an at least
about 20 µm, an at least about 25 µm, an at least about 30 µm, an at least about 35 µm, an at least about 40 µm, an at least about 45 µm, an at least about 50 µm strainer following the initial expansion. In some aspects, the cells are passed through an about 40 µm strainer following the initial expansion. [0342] In some aspects, the initial expansion is carried out in one or more gas permeable flasks (e.g., G-Rex® flasks). In some aspects, the initial expansion is carried out in static G-Rex®. In some aspects, the initial expansion is carried out in a stirred tank. In some aspects, the initial expansion is carried out in a bioreactor. In some aspects, the initial expansion is carried out in a closed system (e.g., using a G-Rex® closed system). [0343] In some aspects, the initial expansion further comprises contacting the TILs with a 4-1BB agonist, e.g., 4-1BB ligand. In some aspects, the 4-1BB agonist, e.g., 4-1BBL (4-1BB ligand, CD137L), is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the initial expansion comprises contacting the TILs with 4-1BB ligand on about day 1, about day 2, about day 3, about day 4, about day 5, about day 6, or about day 7 of the initial expansion; wherein initial expansion is performed for less than about 14 days. In some aspects, the initial expansion comprises contacting the TILs with 4-1BB ligand on about day 3, about day 4, about day 5, about day 6, or about day 7 of the initial expansion; wherein initial expansion is performed less than about 13 days. In some aspects, the initial expansion comprises contacting the TILs with 4-1BB ligand on about day 1, about day 2, about day 3, about day 4, about day 5, about day 6, or about day 7 of the initial expansion; wherein initial expansion is performed less than about 12 days. In some aspects, the initial expansion comprises contacting the TILs with 4-1BB ligand on about day 1, about day 2, about day 3, about day 4, about day 5, about day 6, or about day 7 of the initial expansion; wherein initial expansion is performed less than about 11 days. In some aspects, the initial expansion comprises contacting the TILs with 4-1BB ligand on about day 1, about day 2, about day 3, about day 4, about day 5, about day 6, or about day 7 of the initial expansion; wherein initial expansion is performed less than about 10 days. In some aspects, the initial expansion comprises contacting the TILs with 4-1BB ligand on about day 1, about day 2, about day 3, about day 4, about day 5, about day 6, or about day 7 of the initial expansion; wherein initial expansion is performed less than about 9 days. In some aspects, the initial expansion comprises contacting the TILs with 4-1BB ligand on about day 1, about day 2, about day 3, about day 4, about day 5, about day 6, or about day 7 of the initial expansion; wherein initial expansion is performed less than about 8 days.
[0344] In some aspects, the cells are split during the additional expansion once the cells reach high confluence. In some aspects, the cells are split without opening the closed system. [0345] In some aspects, the expanded TILs are cryopreserved following the initial expansion. In some aspects, the expanded TILs from the initial expansion are administered to a subject. In some aspects, the TILs from the initial expansion are inoculated directly into an additional expansion, e.g., as described herein, following the initial expansion. II.B.3.i. Cytokines [0346] In some aspects, the initial expansion further comprises contacting the tumor sample with one or more cytokines selected from IL-2, IL-7, IL-15, IL-21, and any combination thereof. In some aspects, the initial expansion medium comprises IL-2. In some aspects, the IL-2 is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises at least about 50 ng/mL, at least about 60 ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, at least about 90 ng/mL, at least about 100 ng/mL, at least about 110 ng/mL, at least about 120 ng/mL, at least about 130 ng/mL, at least about 140 ng/mL, at least about 150 ng/mL, at least about 160 ng/mL, at least about 170 ng/mL, at least about 180 ng/mL, at least about 190 ng/mL, at least about 200 ng/mL, at least about 210 ng/mL, at least about 220 ng/mL, at least about 230 ng/mL, at least about 240 ng/mL, at least about 250 ng/mL, at least about 260 ng/mL, at least about 270 ng/mL, at least about 280 ng/mL, at least about 290 ng/mL, at least about 300 ng/mL, at least about 310 ng/mL, at least about 320 ng/mL, at least about 330 ng/mL, at least about 340 ng/mL, at least about 350 ng/mL, at least about 360 ng/mL, at least about 370 ng/mL, at least about 380 ng/mL, at least about 390 ng/mL, at least about 400 ng/mL, at least about 410 ng/mL, at least about 420 ng/mL, at least about 430 ng/mL, at least about 440 ng/mL, at least about 450 ng/mL, at least about 460 ng/mL, at least about 470 ng/mL, at least about 480 ng/mL, at least about 490 ng/mL, at least about 500 ng/mL, at least about 510 ng/mL, at least about 520 ng/mL, at least about 530 ng/mL, at least about 540 ng/mL, at least about 550 ng/mL, at least about 560 ng/mL, at least about 570 ng/mL, at least about 580 ng/mL, at least about 590 ng/mL, or at least about 600 ng/mL IL-2. In some aspects, the first expansion medium comprises at least about 50 ng/mL IL-2. In some aspects, the first expansion medium comprises at least about 60 ng/mL IL-2. In some aspects, the first expansion medium comprises at least about 70 ng/mL IL-2. In some aspects, the first expansion medium comprises at least about 73.6 ng/mL IL-2. In some aspects, the first expansion medium comprises at least about 75 ng/mL IL-2. In some aspects, the first expansion medium comprises at least about 80 ng/mL
IL-2. In some aspects, the first expansion medium comprises at least about 90 ng/mL IL-2. In some aspects, the first expansion medium comprises at least about 100 ng/mL IL-2. In some aspects, the first expansion medium comprises at least about 200 ng/mL IL-2. In some aspects, the first expansion medium comprises at least about 300 ng/mL IL-2. In some aspects, the first expansion medium comprises at least about 400 ng/mL IL-2. In some aspects, the first expansion medium comprises at least about 500 ng/mL IL-2. In some aspects, the first expansion medium comprises at least about 600 ng/mL IL-2. [0347] In some aspects, the first expansion medium comprises at least about 1500 IU/mL IL-2. In some aspects, the first expansion medium comprises from about 1500 IU/mL to about 12,000 IU/mL IL-2. In some aspects, the first expansion medium comprises at least about 1500 IU/mL, at least about 1600 IU/mL, at least about 1700 IU/mL, at least about 1800 IU/mL, at least about 1900 IU/mL, at least about 2000 IU/mL, at least about 2100 IU/mL, at least about 2200 IU/mL, at least about 2300 IU/mL, at least about 2400 IU/mL, at least about 2500 IU/mL, at least about 2600 IU/mL, at least about 2700 IU/mL, at least about 2800 IU/mL, at least about 2900 IU/mL, at least about 3000 IU/mL, at least about 3100 IU/mL, at least about 3200 IU/mL, at least about 3300 IU/mL, at least about 3400 IU/mL, at least about 3500 IU/mL, at least about 3600 IU/mL, at least about 3700 IU/mL, at least about 3800 IU/mL, at least about 3900 IU/mL, at least about 4000 IU/mL, at least about 4100 IU/mL, at least about 4200 IU/mL, at least about 4300 IU/mL, at least about 4400 IU/mL, at least about 4500 IU/mL, at least about 4600 IU/mL, at least about 4700 IU/mL, at least about 4800 IU/mL, at least about 4900 IU/mL, at least about 5000 IU/mL, at least about 5100 IU/mL, at least about 5200 IU/mL, at least about 5300 IU/mL, at least about 5400 IU/mL, at least about 5500 IU/mL, at least about 5600 IU/mL, at least about 5700 IU/mL, at least about 5800 IU/mL, at least about 5900 IU/mL, at least about 6000 IU/mL, at least about 6100 IU/mL, at least about 6200 IU/mL, at least about 6300 IU/mL, at least about 6400 IU/mL, at least about 6500 IU/mL, at least about 6600 IU/mL, at least about 6700 IU/mL, at least about 6800 IU/mL, at least about 6900 IU/mL, at least about 7000 IU/mL IL-2, at least about 7100 IU/mL, at least about 7200 IU/mL, at least about 7300 IU/mL, at least about 7400 IU/mL, at least about 7500 IU/mL, at least about 7600 IU/mL, at least about 7700 IU/mL, at least about 7800 IU/mL, at least about 7900 IU/mL, or at least about 8000 IU/mL IL-2. In some aspects, the first expansion medium comprises at least about 1500 IU/mL IL-2. In some aspects, the first expansion medium comprises at least about 3000 IU/mL IL-2. In some aspects, the first expansion medium comprises at least about 3100 IU/mL IL-2. In some aspects, the first expansion medium comprises at least about 3200 IU/mL IL-2. In some aspects, the first expansion medium comprises at least
about 3300 IU/mL IL-2. In some aspects, the first expansion medium comprises at least about 3400 IU/mL IL-2. In some aspects, the first expansion medium comprises at least about 3500 IU/mL IL- 2. In some aspects, the first expansion medium comprises at least about 6000 IU/mL IL-2. [0348] In some aspects, the initial expansion medium comprises IL-21. In some aspects, the IL-21 is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises at least about 0.1 ng/mL IL-21. In some aspects, the first expansion medium comprises from about 0.1 ng/mL to about 50 ng/mL, about 1 ng/mL to about 50 ng/mL, about 1 ng/mL to about 40 ng/mL, about 1 ng/mL to about 35 ng/mL, about 10 ng/mL to about 40 ng/mL, about 10 ng/mL to about 35 ng/mL, about 10 ng/mL to about 30 ng/mL, about 15 ng/mL to about 40 ng/mL, about 15 ng/mL to about 35 ng/mL, about 15 ng/mL to about 30 ng/mL, about 20 ng/mL to about 40 ng/mL, about 20 ng/mL to about 35 ng/mL, about 20 ng/mL to about 30 ng/mL, about 25 ng/mL to about 35 ng/mL, about 25 ng/mL to about 30 ng/mL, or about 30 ng/mL to about 35 ng/mL IL-21. [0349] In some aspects, the first expansion medium comprises at least about 0.1 ng/mL, at least about 0.5 ng/mL, at least about 1 ng/mL, at least about 2 ng/mL, at least about 3 ng/mL, at least about 4 ng/mL, at least about 5 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about 8 ng/mL, at least about 9 ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least about 12 ng/mL, at least about 13 ng/mL, at least about 14 ng/mL, at least about 15 ng/mL, at least about 16 ng/mL, at least about 17 ng/mL, at least about 18 ng/mL, at least about 19 ng/mL, at least about 20 ng/mL, at least about 25 ng/mL, at least about 30 ng/mL, at least about 35 ng/mL, or at least about 40 ng/mL IL-21. In some aspects, the first expansion medium comprises at least about 1.0 ng/mL IL-21. In some aspects, the first expansion medium comprises at least about 2.0 ng/mL IL-21. In some aspects, the first expansion medium comprises at least about 3.0 ng/mL IL-21. In some aspects, the first expansion medium comprises at least about 4.0 ng/mL IL-21. In some aspects, the first expansion medium comprises at least about 5.0 ng/mL IL-21. In some aspects, the first expansion medium comprises at least about 6.0 ng/mL IL-21. In some aspects, the first expansion medium comprises at least about 7.0 ng/mL IL-21. In some aspects, the first expansion medium comprises at least about 8.0 ng/mL IL-21. In some aspects, the first expansion medium comprises at least about 9.0 ng/mL IL-21. In some aspects, the first expansion medium comprises at least about 10 ng/mL IL-21. In some aspects, the first expansion medium comprises at least about 15 ng/mL IL-21. In some aspects, the first expansion medium comprises at least about 20 ng/mL IL-21. In some aspects, the first expansion medium comprises at least about 25 ng/mL IL-
21. In some aspects, the first expansion medium comprises at least about 30 ng/mL IL-21. In some aspects, the first expansion medium comprises at least about 35 ng/mL IL-21. [0350] In some aspects, the first expansion medium comprises between about 50 IU/mL to about 500 IU/mL of IL-21. In some aspects, the first expansion medium comprises about 50 IU/mL, about 60 IU/mL, about 70 IU/mL, about 80 IU/mL, about 90 IU/mL, about 100 IU/mL, about 125 IU/mL, about 150 IU/mL, about 175 IU/mL, about 200 IU/mL, about 225 IU/mL, about 250 IU/mL, about 275 IU/mL, about 300 IU/mL, about 350 IU/mL, about 400 IU/mL, about 450 IU/mL, or about 500 IU/mL of IL-21. [0351] In some aspects, the first expansion medium does not comprise IL-7. In some aspects, the first expansion medium comprises IL-7. In some aspects, the IL-7 is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises at least about 0.1 ng/mL IL-7. In some aspects, the first expansion medium comprises from about 0.1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about 1 ng/mL to about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 2 ng/mL, about 5 ng/mL to about 15 ng/mL, about 5 ng/mL to about 10 ng/mL, about 10 ng/mL to about 20 ng/mL, about 10 ng/mL to about 15 ng/mL, or about 15 ng/mL to about 20 ng/mL IL-7. [0352] In some aspects, the first expansion medium comprises at least about 0.1 ng/mL, at least about 0.5 ng/mL, at least about 1 ng/mL, at least about 1.3 ng/mL, at least about 1.5 ng/mL, at least about 1.7 ng/mL, at least about 2 ng/mL, at least about 2.3 ng/mL, at least about 2.5 ng/mL, at least about 2.7 ng/mL, at least about 3 ng/mL, at least about 3.3 ng/mL, at least about 3.5 ng/mL, at least about 3.7 ng/mL, at least about 4 ng/mL, at least about 4.3 ng/mL, at least about 4.5 ng/mL, at least about 4.7 ng/mL, at least about 5 ng/mL, at least about 5.3 ng/mL, at least about 5.5 ng/mL, at least about 5.7 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about 8 ng/mL, at least about 9 ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least about 12 ng/mL, at least about 13 ng/mL, at least about 14 ng/mL, at least about 15 ng/mL, at least about 16 ng/mL, at least about 17 ng/mL, at least about 18 ng/mL, at least about 19 ng/mL, or at least about 20 ng/mL IL-7. In some aspects, the medium comprises at least about 1.0 ng/mL IL-7. In some aspects, the first expansion medium comprises at least about 2.0 ng/mL IL-7. In some aspects, the
first expansion medium comprises at least about 2.3 ng/mL IL-7. In some aspects, the first expansion medium comprises at least about 2.5 ng/mL IL-7. In some aspects, the first expansion medium comprises at least about 2.7 ng/mL IL-7. In some aspects, the first expansion medium comprises at least about 3.0 ng/mL IL-7. In some aspects, the first expansion medium comprises at least about 3.3 ng/mL IL-7. In some aspects, the first expansion medium comprises at least about 3.5 ng/mL IL-7. In some aspects, the first expansion medium comprises at least about 3.7 ng/mL IL-7. [0353] In some aspects, the first expansion medium comprises between about 500 IU/mL to about 1,500 IU/mL of IL-7. In some aspects, the first expansion medium comprises about 500 IU/mL, about 550 IU/mL, about 600 IU/mL, about 650 IU/mL, about 700 IU/mL, about 750 IU/mL, about 800 IU/mL, about 850 IU/mL, about 900 IU/mL, about 950 IU/mL, about 1,000 IU/mL, about 1,050 IU/mL, about 1,100 IU/mL, about 1,150 IU/mL, about 1,200 IU/mL, about 1,250 IU/mL, about 1,300 IU/mL, about 1,350 IU/mL, about 1,400 IU/mL, about 1,450 IU/mL, or about 1,500 IU/mL of IL-7. [0354] In some aspects, the first expansion medium does not comprise IL-15. In some aspects, the first expansion medium comprises IL-15. In some aspects, the IL-15 is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises at least about 0.1 ng/mL IL-15. In some aspects, the first expansion medium comprises from about 0.1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about 1 ng/mL to about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 2 ng/mL, about 5 ng/mL to about 15 ng/mL, about 5 ng/mL to about 10 ng/mL, about 10 ng/mL to about 20 ng/mL, about 10 ng/mL to about 15 ng/mL, or about 15 ng/mL to about 20 ng/mL IL-15. [0355] In some aspects, the first expansion medium comprises at least about 0.1 ng/mL, at least about 0.2 ng/mL, at least about 0.3 ng/mL, at least about 0.4 ng/mL, at least about 0.5 ng/mL, at least about 0.6 ng/mL, at least about 0.7 ng/mL, at least about 0.8 ng/mL, at least about 0.9 ng/mL, at least about 1 ng/mL, at least about 2 ng/mL, at least about 3 ng/mL, at least about 4 ng/mL, at least about 5 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about 8 ng/mL, at least about 9 ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least about 12
ng/mL, at least about 13 ng/mL, at least about 14 ng/mL, at least about 15 ng/mL, at least about 16 ng/mL, at least about 17 ng/mL, at least about 18 ng/mL, at least about 19 ng/mL, or at least about 20 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 0.1 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 0.2 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 0.3 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 0.4 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 0.5 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 0.6 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 0.7 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 0.8 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 0.9 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 1.0 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 2.0 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 3.0 ng/mL IL- 15. In some aspects, the first expansion medium comprises at least about 4.0 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 5.0 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 6.0 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 7.0 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 8.0 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 9.0 ng/mL IL-15. In some aspects, the first expansion medium comprises at least about 10 ng/mL IL-15. [0356] In some aspects, the first expansion medium comprises between about 50 IU/mL to about 500 IU/mL of IL-15. In some aspects, the first expansion medium comprises about 50 IU/mL, about 60 IU/mL, about 70 IU/mL, about 80 IU/mL, about 90 IU/mL, about 100 IU/mL, about 125 IU/mL, about 150 IU/mL, about 175 IU/mL, about 200 IU/mL, about 225 IU/mL, about 250 IU/mL, about 275 IU/mL, about 300 IU/mL, about 350 IU/mL, about 400 IU/mL, about 450 IU/mL, or about 500 IU/mL of IL-15. [0357] In some aspects, the first expansion medium comprises about 3000 IU/mL IL-2 and about 30 ng/M IL-21, wherein the IL-2, the IL-21, or both are associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises about 3000 IU/mL IL-2 and about 30 ng/M IL-21; wherein the IL-2, the IL-21, or both are associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein; and wherein the medium does not comprise IL-7, IL-15, or both IL-7 and IL-15.
II.B.4. Additional Expansion [0358] In some aspects, the TILs are subjected to an additional expansion, wherein the additional expansion comprises culturing the TILs in a second expansion medium comprising a T cell activator and a cell support matrix (an "additional expansion"). In some aspects, the cell support matrix of the second expansion medium comprises a feeder cell replacement, e.g., PCS, comprising the T cell activator, e.g., a CD3 agonist, as disclosed herein. In some aspects, the feeder cell replacement, e.g., PCS, of the initial expansion medium and the feeder cell replacement, e.g., PCS, of the additional expansion medium are the same. In some aspects, the feeder cell replacement, e.g., PCS, of the initial expansion medium and the feeder cell replacement, e.g., PCS, of the additional expansion medium are different. [0359] In some aspects, the additional expansion is carried out in one or more gas permeable flasks (e.g., G-Rex® flasks). In some aspects, the TILs are transitioned to the additional expansion without opening the closed system. In some aspects, the additional expansion is carried out in a bioreactor. In some aspects, the TILs from the initial expansion are screened for tumor- specific cytolytic activity prior to advancing the TILs to the additional expansion. In some aspects, the TILs are screened for expression of one or more biomarkers prior to advancing to the additional expansion. In some aspects, the biomarker comprises expression of one or more gene typically expressed by more naïve TILs, e.g., CD8+, CD27+, CD3+, CD95+, CD45RA+, CCR7+, CD62L+, TCF7+, or any combination thereof. In some aspects, the TILs are screened for expression of PD- 1 prior to advancing to the additional expansion. [0360] In some aspects, the TILs from the initial expansion are not screened prior to advancing the TILs to the additional expansion. In some aspects, all TILs obtained in the initial expansion are subjected to the additional expansion. In some aspects, the TILs from the initial expansion are pooled prior to advancement to the additional expansion. [0361] In some aspects, the TILs are cultured in the additional expansion for less than about 15 days. In some aspects, the TILs are cultured in the additional expansion for less than about 14 days. In some aspects, the TILs are cultured in the additional expansion for less than about 13 days. In some aspects, the TILs are cultured in the additional expansion for less than about 12 days. In some aspects, the TILs are cultured in the additional expansion for less than about 11 days. In some aspects, the TILs are cultured in the additional expansion for less than about 10 days. In some aspects, the TILs are cultured in the additional expansion for less than about 9 days. In some aspects, the TILs are cultured in the additional expansion for less than about 8 days.
[0362] In some aspects, the TILs are cultured in the additional expansion for about 14 days. In some aspects, the TILs are cultured in the additional expansion for about 13 days. In some aspects, the TILs are cultured in the additional expansion for about 12 days. In some aspects, the TILs are cultured in the additional expansion for about 11 days. In some aspects, the TILs are cultured in the additional expansion for about 10 days. In some aspects, the TILs are cultured in the additional expansion for about 9 days. In some aspects, the TILs are cultured in the additional expansion for about 8 days. In some aspects, the TILs are cultured in the additional expansion for about 7 days. In some aspects, the TILs are cultured in the additional expansion for about 6 days. In some aspects, the TILs are cultured in the additional expansion for about 5 days. In some aspects, the TILs are cultured in the additional expansion for about 4 days. In some aspects, the TILs are cultured in the additional expansion for about 3 days. [0363] In some aspects, the TILs are cultured in the additional expansion for a period of time until a certain number of cells is reached. In some aspects, the number of TILs present following the initial expansion is increased by at least about 10-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 following culture in the additional expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 10-fold following culture in the additional expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 15-fold following culture in the additional expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 20-fold following culture in the additional expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 25-fold following culture in the additional expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 30-fold following culture in the additional expansion. In some aspects, the number of TILs preset in the dissociated tumor sample is increased by at least about 35-fold following culture in the additional expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 40-fold following culture in the additional expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 45-fold following culture in the additional expansion. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 50-fold following culture in the additional expansion.
[0364] In some aspects, the number of TILs following culture in the additional expansion is at least about 1 x 109, at least about 2 x 109, at least about 3 x 109, at least about 4 x 109, at least about 5 x 109, at least about 6 x 109, at least about 7 x 109, at least about 8 x 109, at least about 9 x 109, at least about 1 x 1010, at least about 2 x 1010, at least about 3 x 1010, at least about 4 x 1010, at least about 5 x 1010, at least about 6 x 1010, at least about 7 x 1010, at least about 8 x 1010, or at least about 9 x 1010 TILs. In some aspects, the number of TILs following culture in the additional expansion is at least about 5 x 109. In some aspects, the number of TILs following culture in the additional expansion is at least about 1 x 1010. In some aspects, the number of TILs following culture in the additional expansion is at least about 2 x 1010. In some aspects, the number of TILs following culture in the additional expansion is at least about 3 x 1010. In some aspects, the number of TILs following culture in the additional expansion is at least about 4 x 1010. In some aspects, the number of TILs following culture in the additional expansion is at least about 5 x 1010. In some aspects, the number of TILs following culture in the additional expansion is at least about 6 x 1010. In some aspects, the number of TILs following culture in the additional expansion is at least about 7 x 1010. In some aspects, the number of TILs following culture in the additional expansion is at least about 8 x 1010. In some aspects, the number of TILs following culture in the additional expansion is at least about 9 x 1010. In some aspects, the number of TILs following culture in the additional expansion is at least about 10 x 1010. [0365] In some aspects, the additional expansion continues until at least about 5 x 109 TILs are obtained. In some aspects, the additional expansion continues until at least about 1 x 1010 TILs are obtained. In some aspects, the additional expansion continues until at least about 2 x 1010 TILs are obtained. In some aspects, the additional expansion continues until at least about 3 x 1010 TILs are obtained. In some aspects, the additional expansion continues until at least about 4 x 1010 TILs are obtained. In some aspects, the additional expansion continues until at least about 5 x 1010 TILs are obtained. In some aspects, the additional expansion continues until at least about 6 x 1010 TILs are obtained. In some aspects, the additional expansion continues until at least about 7 x 1010 TILs are obtained. In some aspects, the additional expansion continues until at least about 8 x 1010 TILs are obtained. In some aspects, the additional expansion continues until at least about 9 x 1010 TILs are obtained. In some aspects, the additional expansion continues until at least about 10 x 1010 TILs are obtained. [0366] In some aspects, the additional expansion lasts for less than 8 days, wherein at least about 1 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 8 days, wherein at least about 2 x 1010 TILs are obtained. In some aspects, the additional expansion
lasts for less than 108 days, wherein at least about 3 x 100 TILs are obtained. In some aspects, the additional expansion lasts for less than 8 days, wherein at least about 4 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 8 days, wherein at least about 5 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 8 days, wherein at least about 6 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 8 days, wherein at least about 7 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 8 days, wherein at least about 8 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 8 days, wherein at least about 9 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 8 days, wherein at least about 10 x 1010 TILs are obtained. [0367] In some aspects, the additional expansion lasts for less than 9 days, wherein at least about 1 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 9 days, wherein at least about 2 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 9 days, wherein at least about 3 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 9 days, wherein at least about 4 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 9 days, wherein at least about 5 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 9 days, wherein at least about 6 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 9 days, wherein at least about 7 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 9 days, wherein at least about 8 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 9 days, wherein at least about 9 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 9 days, wherein at least about 10 x 1010 TILs are obtained. [0368] In some aspects, the additional expansion lasts for less than 10 days, wherein at least about 1 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 10 days, wherein at least about 2 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 10 days, wherein at least about 3 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 10 days, wherein at least about 4 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 10 days, wherein at least about 5 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 10 days, wherein at least about 6 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 10 days, wherein at least about 7 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 10 days, wherein at least about 8 x 1010 TILs are obtained.
In some aspects, the additional expansion lasts for less than 10 days, wherein at least about 9 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 10 days, wherein at least about 10 x 1010 TILs are obtained. [0369] In some aspects, the additional expansion lasts for less than 11 days, wherein at least about 1 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 11 days, wherein at least about 2 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 11 days, wherein at least about 3 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 11 days, wherein at least about 4 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 11 days, wherein at least about 5 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 11 days, wherein at least about 6 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 11 days, wherein at least about 7 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 11 days, wherein at least about 8 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 11 days, wherein at least about 9 x 1010 TILs are obtained. In some aspects, the additional expansion lasts for less than 11 days, wherein at least about 10 x 1010 TILs are obtained. [0370] In some aspects, the TILs are cultured in the initial expansion for 7 to 10 days, and the TILs are cultured in the additional expansion for 7 to 10 days, wherein the total number of TILs following culture in the additional expansion is at least about 1 x 1010 TILs. In some aspects, the TILs are cultured in the initial expansion for 7 days, and the TILs are cultured in the additional expansion for 7 days, wherein the total number of TILs following culture in the additional expansion is at least about 1 x 1010 TILs. In some aspects, the TILs are cultured in the initial expansion for 10 days or less, and the TILs are cultured in the additional expansion for 10 days or less, wherein the total number of TILs following culture in the additional expansion is at least about 1 x 1010 TILs. [0371] In some aspects, the feeder cell replacement, e.g., PCS, comprises a CD3 agonist. In some aspects, the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the CD3 agonist comprises OKT3. In some aspects, the first expansion medium comprises from about 1 to about 100 ng/mL OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the concentration of OKT3 is at least about 1 ng/ml, at least about 5 ng/ml, at least about 10 ng/ml, at least about 15 ng/ml, at least about 20 ng/ml, at least about 25 ng/ml, at least about 30 ng/ml, at least about 35 ng/ml, at least about 40
ng/ml, at least about 45 ng/ml, at least about 50 ng/ml, at least about 60 ng/ml, at least about 70 ng/ml, at least about 80 ng/ml, at least about 90 ng/ml, or at least about 100 ng/ml. In some aspects, the first expansion medium comprises about 10 ng/ml OKT3. In some aspects, the first expansion medium comprises about 15 ng/ml OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises about 20 ng/ml OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises about 25 ng/ml OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises about 30 ng/ml OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises about 35 ng/ml OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises about 40 ng/ml OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises about 45 ng/ml OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the first expansion medium comprises about 50 ng/ml OKT3, wherein the CD3 agonist is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. [0372] In some aspects, the cells are passed through a strainer following the second expansion. In some aspects, the cells are passed through an at least about 10 µm, an at least about 15 µm, an at least about 20 µm, an at least about 25 µm, an at least about 30 µm, an at least about 35 µm, an at least about 40 µm, an at least about 45 µm, an at least about 50 µm strainer following the additional expansion. In some aspects, the cells are passed through an about 40 µm strainer following the additional expansion. [0373] In some aspects, the additional expansion is carried out in one or more gas permeable flasks (e.g., G-Rex® flasks). In some aspects, the additional expansion is carried out in static G-Rex®. In some aspects, the additional expansion is carried out in a stirred tank. In some aspects, the additional expansion is carried out in a bioreactor. In some aspects, the additional expansion is carried out in a closed system (e.g., using a G-Rex® closed system).
[0374] In some aspects, the TILs are cryopreserved following a REP step. In some aspects, the TILs from a REP step are administered directly to a subject. In some aspects, the TILs are cryopreserved following the additional expansion. In some aspects, the TILs from the additional expansion are administered directly to a subject. II.B.4.i. Cytokines [0375] In some aspects, the second expansion medium comprises IL-2. In some aspects, the IL-2 is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the second expansion medium comprises at least about 50 ng/mL, at least about 60 ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, at least about 90 ng/mL, at least about 100 ng/mL, at least about 110 ng/mL, at least about 120 ng/mL, at least about 130 ng/mL, at least about 140 ng/mL, at least about 150 ng/mL, at least about 160 ng/mL, at least about 170 ng/mL, at least about 180 ng/mL, at least about 190 ng/mL, at least about 200 ng/mL, at least about 210 ng/mL, at least about 220 ng/mL, at least about 230 ng/mL, at least about 240 ng/mL, at least about 250 ng/mL, at least about 260 ng/mL, at least about 270 ng/mL, at least about 280 ng/mL, at least about 290 ng/mL, at least about 300 ng/mL, at least about 310 ng/mL, at least about 320 ng/mL, at least about 330 ng/mL, at least about 340 ng/mL, at least about 350 ng/mL, at least about 360 ng/mL, at least about 370 ng/mL, at least about 380 ng/mL, at least about 390 ng/mL, at least about 400 ng/mL, at least about 410 ng/mL, at least about 420 ng/mL, at least about 430 ng/mL, at least about 440 ng/mL, at least about 450 ng/mL, at least about 460 ng/mL, at least about 470 ng/mL, at least about 480 ng/mL, at least about 490 ng/mL, at least about 500 ng/mL, at least about 510 ng/mL, at least about 520 ng/mL, at least about 530 ng/mL, at least about 540 ng/mL, at least about 550 ng/mL, at least about 560 ng/mL, at least about 570 ng/mL, at least about 580 ng/mL, at least about 590 ng/mL, or at least about 600 ng/mL IL-2. In some aspects, the second expansion medium comprises at least about 50 ng/mL IL-2. In some aspects, the second expansion medium comprises at least about 60 ng/mL IL-2. In some aspects, the second expansion medium comprises at least about 70 ng/mL IL-2. In some aspects, the second expansion medium comprises at least about 73.6 ng/mL IL-2. In some aspects, the second expansion medium comprises at least about 75 ng/mL IL-2. In some aspects, the second expansion medium comprises at least about 80 ng/mL IL-2. In some aspects, the second expansion medium comprises at least about 90 ng/mL IL- 2. In some aspects, the second expansion medium comprises at least about 100 ng/mL IL-2. In some aspects, the second expansion medium comprises at least about 200 ng/mL IL-2. In some aspects, the second expansion medium comprises at least about 300 ng/mL IL-2. In some aspects,
the second expansion medium comprises at least about 400 ng/mL IL-2. In some aspects, the second expansion medium comprises at least about 500 ng/mL IL-2. In some aspects, the second expansion medium comprises at least about 600 ng/mL IL-2. [0376] In some aspects, the second expansion medium comprises at least about 500 IU/mL IL-2. In some aspects, the second expansion medium comprises from about 500 IU/mL to about 12,000 IU/mL IL-2. In some aspects, the second expansion medium comprises at least about 500 IU/mL, at least about 600 IU/mL, at least about 700 IU/mL, at least about 800 IU/mL, at least about 900 IU/mL, at least about 1000 IU/mL, at least about 21100 IU/mL, at least about 1200 IU/mL, at least about 1300 IU/mL, at least about 1400 IU/mL, at least about 1500 IU/mL, at least about 1600 IU/mL, at least about 1700 IU/mL, at least about 1800 IU/mL, at least about 1900 IU/mL, at least about 2000 IU/mL, at least about 2100 IU/mL, at least about 2200 IU/mL, at least about 2300 IU/mL, at least about 2400 IU/mL, at least about 2500 IU/mL, at least about 2600 IU/mL, at least about 2700 IU/mL, at least about 2800 IU/mL, at least about 2900 IU/mL, at least about 3000 IU/mL, at least about 3100 IU/mL, at least about 3200 IU/mL, at least about 3300 IU/mL, at least about 3400 IU/mL, at least about 3500 IU/mL, at least about 3600 IU/mL, at least about 3700 IU/mL, at least about 3800 IU/mL, at least about 3900 IU/mL, or at least about 1000 IU/mL. In some aspects, the second expansion medium comprises at least about 1100 IU/mL IL- 2. In some aspects, the second expansion medium comprises at least about 1200 IU/mL IL-2. In some aspects, the second expansion medium comprises at least about 1300 IU/mL IL-2. In some aspects, the second expansion medium comprises at least about 1400 IU/mL IL-2. In some aspects, the second expansion medium comprises at least about 1500 IU/mL IL-2. In some aspects, the second expansion medium comprises at least about 1600 IU/mL IL-2. In some aspects, the second expansion medium comprises at least about 1700 IU/mL IL-2. In some aspects, the second expansion medium comprises at least about 1800 IU/mL IL-2. In some aspects, the second expansion medium comprises at least about 1900 IU/mL IL-2. In some aspects, the second expansion medium comprises at least about 2000 IU/mL IL-2. [0377] In some aspects, the second expansion medium comprises IL-21. In some aspects, the IL-21 is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the second expansion medium comprises at least about 0.1 ng/mL IL-21. In some aspects, the second expansion medium comprises from about 0.1 ng/mL to about 50 ng/mL, about 1 ng/mL to about 50 ng/mL, about 1 ng/mL to about 40 ng/mL, about 1 ng/mL to about 35 ng/mL, about 1 ng/mL to about 30 ng/mL, about 1 ng/mL to about 25 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 10
ng/mL, about 5 ng/mL to about 20 ng/mL, about 5 ng/mL to about 15 ng/mL IL-21, about 5 ng/mL to about 10 ng/mL, about 10 ng/mL to about 20 ng/mL, or about 10 ng/mL to about 15 ng/mL. [0378] In some aspects, the second expansion medium comprises at least about 0.1 ng/mL, at least about 0.5 ng/mL, at least about 1 ng/mL, at least about 2 ng/mL, at least about 3 ng/mL, at least about 4 ng/mL, at least about 5 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about 8 ng/mL, at least about 9 ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least about 12 ng/mL, at least about 13 ng/mL, at least about 14 ng/mL, at least about 15 ng/mL, at least about 16 ng/mL, at least about 17 ng/mL, at least about 18 ng/mL, at least about 19 ng/mL, or at least about 20 ng/mL IL-21. In some aspects, the second expansion medium comprises at least about 1.0 ng/mL IL-21. In some aspects, the second expansion medium comprises at least about 2.0 ng/mL IL-21. In some aspects, the second expansion medium comprises at least about 3.0 ng/mL IL-21. In some aspects, the second expansion medium comprises at least about 4.0 ng/mL IL-21. In some aspects, the second expansion medium comprises at least about 5.0 ng/mL IL-21. In some aspects, the second expansion medium comprises at least about 6.0 ng/mL IL-21. In some aspects, the second expansion medium comprises at least about 7.0 ng/mL IL-21. In some aspects, the second expansion medium comprises at least about 8.0 ng/mL IL-21. In some aspects, the second expansion medium comprises at least about 9.0 ng/mL IL-21. In some aspects, the second expansion medium comprises at least about 10 ng/mL IL-21. In some aspects, the second expansion medium comprises at least about 11 ng/mL IL-21. In some aspects, the second expansion medium comprises at least about 12 ng/mL IL-21. In some aspects, the second expansion medium comprises at least about 13 ng/mL IL-21. In some aspects, the second expansion medium comprises at least about 14 ng/mL IL-21. In some aspects, the second expansion medium comprises at least about 15 ng/mL IL-21. [0379] In some aspects, the second expansion medium comprises between about 10 IU/mL to about 500 IU/mL of IL-21. In some aspects, the second expansion medium comprises about 10 IU/mL, about 20 IU/mL, about 30 IU/mL, about 40 IU/mL, about 50 IU/mL, about 60 IU/mL, about 70 IU/mL, about 80 IU/mL, about 90 IU/mL, about 100 IU/mL, about 125 IU/mL, about 150 IU/mL, about 175 IU/mL, about 200 IU/mL, about 225 IU/mL, about 250 IU/mL, about 275 IU/mL, about 300 IU/mL, about 350 IU/mL, about 400 IU/mL, about 450 IU/mL, or about 500 IU/mL of IL-21. [0380] In some aspects, the second expansion medium does not comprise IL-15. In some aspects, the second expansion medium comprises IL-15. In some aspects, the IL-15 is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In
some aspects, the second expansion medium comprises at least about 0.1 ng/mL IL-15. In some aspects, the second expansion medium comprises from about 0.1 ng/mL to about 20 ng/mL, about 0.1 ng/mL to about 10 ng/mL, about 0.1 ng/mL to about 5 ng/mL, or about 0.1 ng/mL to about 1 ng/mL IL-15. [0381] In some aspects, the second expansion medium comprises at least about 0.1 ng/mL, at least about 0.2 ng/mL, at least about 0.3 ng/mL, at least about 0.4 ng/mL, at least about 0.5 ng/mL, at least about 0.6 ng/mL, at least about 0.7 ng/mL, at least about 0.8 ng/mL, at least about 0.9 ng/mL, at least about 1 ng/mL, at least about 2 ng/mL, at least about 3 ng/mL, at least about 4 ng/mL, at least about 5 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about 8 ng/mL, at least about 9 ng/mL, or at least about 10 ng/mL IL-15. In some aspects, the second expansion medium comprises at least about 0.1 ng/mL IL-15. In some aspects, the second expansion medium comprises at least about 0.2 ng/mL IL-15. In some aspects, the second expansion medium comprises at least about 0.3 ng/mL IL-15. In some aspects, the second expansion medium comprises at least about 0.4 ng/mL IL-15. In some aspects, the second expansion medium comprises at least about 0.5 ng/mL IL-15. In some aspects, the second expansion medium comprises at least about 0.6 ng/mL IL-15. In some aspects, the second expansion medium comprises at least about 0.7 ng/mL IL-15. In some aspects, the second expansion medium comprises at least about 0.8 ng/mL IL-15. In some aspects, the second expansion medium comprises at least about 0.9 ng/mL IL-15. In some aspects, the second expansion medium comprises at least about 1.0 ng/mL IL-15. [0382] In some aspects, the second expansion medium comprises between about 50 IU/mL to about 500 IU/mL of IL-15. In some aspects, the second expansion medium comprises about 50 IU/mL, about 60 IU/mL, about 70 IU/mL, about 80 IU/mL, about 90 IU/mL, about 100 IU/mL, about 125 IU/mL, about 150 IU/mL, about 175 IU/mL, about 200 IU/mL, about 225 IU/mL, about 250 IU/mL, about 275 IU/mL, about 300 IU/mL, about 350 IU/mL, about 400 IU/mL, about 450 IU/mL, or about 500 IU/mL of IL-15. [0383] In some aspects, the second expansion medium does not comprise IL-7. In some aspects, the second expansion medium comprises IL-7. In some aspects, the IL-7 is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the second expansion medium comprises at least about 0.1 ng/mL IL-7. In some aspects, the second expansion medium comprises from about 0.1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about 1 ng/mL to about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11 ng/mL,
about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 2 ng/mL, about 5 ng/mL to about 15 ng/mL, about 5 ng/mL to about 10 ng/mL, about 10 ng/mL to about 20 ng/mL, about 10 ng/mL to about 15 ng/mL, or about 15 ng/mL to about 20 ng/mL IL-7. [0384] In some aspects, the second expansion medium comprises at least about 0.1 ng/mL, at least about 0.5 ng/mL, at least about 1 ng/mL, at least about 1.3 ng/mL, at least about 1.5 ng/mL, at least about 1.7 ng/mL, at least about 2 ng/mL, at least about 2.3 ng/mL, at least about 2.5 ng/mL, at least about 2.7 ng/mL, at least about 3 ng/mL, at least about 3.3 ng/mL, at least about 3.5 ng/mL, at least about 3.7 ng/mL, at least about 4 ng/mL, at least about 4.3 ng/mL, at least about 4.5 ng/mL, at least about 4.7 ng/mL, at least about 5 ng/mL, at least about 5.3 ng/mL, at least about 5.5 ng/mL, at least about 5.7 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about 8 ng/mL, at least about 9 ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least about 12 ng/mL, at least about 13 ng/mL, at least about 14 ng/mL, at least about 15 ng/mL, at least about 16 ng/mL, at least about 17 ng/mL, at least about 18 ng/mL, at least about 19 ng/mL, or at least about 20 ng/mL IL-7. In some aspects, the medium comprises at least about 1.0 ng/mL IL-7. In some aspects, the second expansion medium comprises at least about 2.0 ng/mL IL-7. In some aspects, the second expansion medium comprises at least about 2.3 ng/mL IL-7. In some aspects, the second expansion medium comprises at least about 2.5 ng/mL IL-7. In some aspects, the second expansion medium comprises at least about 2.7 ng/mL IL-7. In some aspects, the second expansion medium comprises at least about 3.0 ng/mL IL-7. In some aspects, the second expansion medium comprises at least about 3.3 ng/mL IL-7. In some aspects, the second expansion medium comprises at least about 3.5 ng/mL IL-7. In some aspects, the second expansion medium comprises at least about 3.7 ng/mL IL-7. [0385] In some aspects, the second expansion medium comprises between about 500 IU/mL to about 1,500 IU/mL of IL-7. In some aspects, the second expansion medium comprises about 500 IU/mL, about 550 IU/mL, about 600 IU/mL, about 650 IU/mL, about 700 IU/mL, about 750 IU/mL, about 800 IU/mL, about 850 IU/mL, about 900 IU/mL, about 950 IU/mL, about 1,000 IU/mL, about 1,050 IU/mL, about 1,100 IU/mL, about 1,150 IU/mL, about 1,200 IU/mL, about 1,250 IU/mL, about 1,300 IU/mL, about 1,350 IU/mL, about 1,400 IU/mL, about 1,450 IU/mL, or about 1,500 IU/mL of IL-7.
[0386] In some aspects, the second expansion medium comprises about 1500 IU/mL IL-2 and about 10 ng/M IL-21, wherein the IL-2, the IL-21, or both are associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the second expansion medium comprises about 1500 IU/mL IL-2 and about 0.4 ng/M IL-15, wherein the IL-2, the IL-15, or both are associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the second expansion medium comprises about 1500 IU/mL IL-2, about 10 ng/M IL-21, and about 0.4 ng/mL IL-15, wherein the IL-2, the IL-21, the IL-15, or any combination thereof is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. II.B.5. Single Expansion Step [0387] In some aspects, the method comprises expanding immune cells, e.g., T cells, NK cells, or TILs, in a single expansion step. In some aspects, the immune cells comprise one or more engineered immune cells, e.g., T cells modified to express a CAR, as disclosed herein. In some aspects, the immune cells comprise TILs. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for about 4 to about 20 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for about 4 to about 19 days, about 4 to about 18 days, about 4 to about 17 days, about 4 to about 16 days, about 4 to about 15 days, about 4 to about 14 days, about 4 to about 13 days, about 4 to about 12 days, about 4 to about 11 days, about 4 to about 10 days, about 4 to about 9 days, about 4 to about 8 days, about 6 to about 18 days, about 6 to about 15 days, about 6 to about 14 days, about 6 to about 12 days, about 6 to about 8 days, about 7 to about 20 days, about 7 to about 18 days, about 7 to about 14 days, or about 7 to about 9 days. [0388] In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for less than about 4 days, for less than about 5 days, for less than about 6 days, for less than about 7 days, for less than about 8 days, for less than about 9 days, for less than about 10 days, for less than about 11 days, for less than about 12 days, for less than about 13 days, for less than about 14 days, for less than about 15 days, for less than about 16 days, for less than about 17 days, for less than about 18 days, for less than about 19 days, for less than about 20 days, for less than about 21 days, for less than about 22 days, for less than about 23 days, or for less than about 24 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for less than about 21 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for less than about
20 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for less than about 19 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for less than about 18 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for less than about 17 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for less than about 16 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for less than about 15 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for less than about 14 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for less than about 13 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for less than about 12 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for less than about 11 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for less than about 10 days. [0389] In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for about 4 days, for about 5 days, for about 6 days, for about 7 days, for about 8 days, for about 9 days, for about 10 days, for about 11 days, for about 12 days, for about 13 days, for about 14 days, for about 15 days, for about 16 days, for about 17 days, for about 18 days, for about 19 days, for about 20 days, for about 21 days, for about 22 days, for about 23 days, or for about 24 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for about 21 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for about 20 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for about 19 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for about 18 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for about 17 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for about 16 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for about 15 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for about 14 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for about 13 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are
subjected to the single expansion step for about 12 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for about 11 days. In some aspects, the immune cells, e.g., T cells, NK cells, or TILs, are subjected to the single expansion step for about 10 days. [0390] In some aspects, the single expansion step is carried out in one or more gas permeable flasks (e.g., G-Rex® flasks). In some aspects, the single expansion step is carried out in static G-Rex®. In some aspects, the single expansion step is carried out in a stirred tank. In some aspects, the single expansion step is carried out in a bioreactor. In some aspects, the single expansion step is carried out in a closed system (e.g., using a G-Rex® closed system). [0391] In some aspects, the cell-support matrix of the medium of the single expansion step comprises a feeder cell replacement disclosed herein, e.g., a PCS, comprising a CD3 agonist, e.g., OKT3. [0392] In some aspects, the medium of the single expansion step comprises from about 1 to about 100 ng/mL OKT3, wherein the OKT3 is associated with the feeder cell replacement, e.g., as a surface cue or a soluble cue, as disclosed herein. In some aspects, the concentration of OKT3 is at least about 1 ng/ml, at least about 5 ng/ml, at least about 10 ng/ml, at least about 15 ng/ml, at least about 20 ng/ml, at least about 25 ng/ml, at least about 30 ng/ml, at least about 35 ng/ml, at least about 40 ng/ml, at least about 45 ng/ml, at least about 50 ng/ml, at least about 60 ng/ml, at least about 70 ng/ml, at least about 80 ng/ml, at least about 90 ng/ml, or at least about 100 ng/ml. In some aspects, the medium of the single expansion step comprises about 10 ng/ml OKT3. In some aspects, the medium of the single expansion step comprises about 15 ng/ml OKT3. In some aspects, the medium of the single expansion step comprises about 20 ng/ml OKT3. In some aspects, the medium of the single expansion step comprises about 25 ng/ml OKT3. In some aspects, the medium of the single expansion step comprises about 30 ng/ml OKT3. In some aspects, the medium of the single expansion step comprises about 35 ng/ml OKT3. In some aspects, the medium of the single expansion step comprises about 40 ng/ml OKT3. In some aspects, the medium of the single expansion step comprises about 45 ng/ml OKT3. In some aspects, the medium of the single expansion step comprises about 50 ng/ml OKT3. [0393] In some aspects, the medium of the single expansion step further comprises 4-1BB ligand. In some aspects, the 4-1BB ligand is associated with the feeder cell replacement, e.g., as a surface cue or a soluble cue, as disclosed herein. In some aspects, the single expansion step comprises contacting the immune cells, e.g., TILs, with 4-1BB ligand on about day 1, about day 2, about day 3, about day 4, about day 5, about day 6, or about day 7 of the single expansion step.
In some aspects, the single expansion step comprises contacting the immune cells, e.g., TILs, with 4-1BB ligand on day 1 of the single expansion step (i.e., at the start of the single expansion step). In some aspects, the single expansion step comprises contacting the immune cells, e.g., TILs, with 4-1BB ligand on day 2 of the single expansion step. In some aspects, the single expansion step comprises contacting the immune cells, e.g., TILs, with 4-1BB ligand on day 3 of the single expansion step. In some aspects, the single expansion step comprises contacting the immune cells, e.g., TILs, with 4-1BB ligand on day 4 of the single expansion step. In some aspects, the single expansion step comprises contacting the TILs with 4-1BB ligand on day 5 of the single expansion step. In some aspects, the single expansion step comprises contacting the immune cells, e.g., TILs, with 4-1BB ligand on day 6 of the single expansion step. In some aspects, the single expansion step comprises contacting the immune cells, e.g., TILs, with 4-1BB ligand on day 7 of the single expansion step. In some aspects, the single expansion step comprises contacting the immune cells, e.g., TILs, with 4-1BB ligand on day 8 of the single expansion step. In some aspects, the single expansion step comprises contacting the immune cells, e.g., TILs, with 4-1BB ligand on day 9 of the single expansion step. In some aspects, the single expansion step comprises contacting the immune cells, e.g., TILs, with 4-1BB ligand on day 10 of the single expansion step. In some aspects, the single expansion step comprises contacting the immune cells, e.g., TILs, with 4-1BB ligand on day 11 of the single expansion step. In some aspects, the single expansion step comprises contacting the immune cells, e.g., TILs, with 4-1BB ligand on day 12 of the single expansion step. II.B.5.i. Cytokines [0394] In some aspects, the medium of the single expansion step comprises IL-2. In some aspects, the IL-2 is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the medium of the single expansion step comprises at least about 50 ng/mL, at least about 60 ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, at least about 90 ng/mL, at least about 100 ng/mL, at least about 110 ng/mL, at least about 120 ng/mL, at least about 130 ng/mL, at least about 140 ng/mL, at least about 150 ng/mL, at least about 160 ng/mL, at least about 170 ng/mL, at least about 180 ng/mL, at least about 190 ng/mL, at least about 200 ng/mL, at least about 210 ng/mL, at least about 220 ng/mL, at least about 230 ng/mL, at least about 240 ng/mL, at least about 250 ng/mL, at least about 260 ng/mL, at least about 270 ng/mL, at least about 280 ng/mL, at least about 290 ng/mL, at least about 300 ng/mL, at least about 310 ng/mL, at least about 320 ng/mL, at least about 330 ng/mL, at least about 340 ng/mL, at least about 350 ng/mL, at least about 360 ng/mL, at least about 370 ng/mL, at least about
380 ng/mL, at least about 390 ng/mL, at least about 400 ng/mL, at least about 410 ng/mL, at least about 420 ng/mL, at least about 430 ng/mL, at least about 440 ng/mL, at least about 450 ng/mL, at least about 460 ng/mL, at least about 470 ng/mL, at least about 480 ng/mL, at least about 490 ng/mL, at least about 500 ng/mL, at least about 510 ng/mL, at least about 520 ng/mL, at least about 530 ng/mL, at least about 540 ng/mL, at least about 550 ng/mL, at least about 560 ng/mL, at least about 570 ng/mL, at least about 580 ng/mL, at least about 590 ng/mL, or at least about 600 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 50 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 60 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 70 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 73.6 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 75 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 80 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 90 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 100 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 200 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 300 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 400 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 500 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 600 ng/mL IL-2. [0395] In some aspects, the medium of the single expansion step comprises at least about 1500 IU/mL IL-2. In some aspects, the medium of the single expansion step comprises from about 1500 IU/mL to about 12,000 IU/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 1500 IU/mL, at least about 1600 IU/mL, at least about 1700 IU/mL, at least about 1800 IU/mL, at least about 1900 IU/mL, at least about 2000 IU/mL, at least about 2100 IU/mL, at least about 2200 IU/mL, at least about 2300 IU/mL, at least about 2400 IU/mL, at least about 2500 IU/mL, at least about 2600 IU/mL, at least about 2700 IU/mL, at least about 2800 IU/mL, at least about 2900 IU/mL, at least about 3000 IU/mL, at least about 3100 IU/mL, at least about 3200 IU/mL, at least about 3300 IU/mL, at least about 3400 IU/mL, at least about 3500 IU/mL, at least about 3600 IU/mL, at least about 3700 IU/mL, at least about 3800 IU/mL, at least about 3900 IU/mL, at least about 4000 IU/mL, at least about 4100 IU/mL, at least about 4200 IU/mL, at least about 4300 IU/mL, at least about 4400 IU/mL, at least about 4500 IU/mL, at least about 4600 IU/mL, at least about 4700 IU/mL, at least about 4800 IU/mL, at least about 4900
IU/mL, at least about 5000 IU/mL, at least about 5100 IU/mL, at least about 5200 IU/mL, at least about 5300 IU/mL, at least about 5400 IU/mL, at least about 5500 IU/mL, at least about 5600 IU/mL, at least about 5700 IU/mL, at least about 5800 IU/mL, at least about 5900 IU/mL, at least about 6000 IU/mL, at least about 6100 IU/mL, at least about 6200 IU/mL, at least about 6300 IU/mL, at least about 6400 IU/mL, at least about 6500 IU/mL, at least about 6600 IU/mL, at least about 6700 IU/mL, at least about 6800 IU/mL, at least about 6900 IU/mL, at least about 7000 IU/mL IL-2, at least about 7100 IU/mL, at least about 7200 IU/mL, at least about 7300 IU/mL, at least about 7400 IU/mL, at least about 7500 IU/mL, at least about 7600 IU/mL, at least about 7700 IU/mL, at least about 7800 IU/mL, at least about 7900 IU/mL, or at least about 8000 IU/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 3000 IU/mL IL- 2. In some aspects, the medium of the single expansion step comprises at least about 3100 IU/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 3200 IU/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 3300 IU/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 3400 IU/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 3500 IU/mL IL-2. [0396] In some aspects, the medium of the single expansion step comprises at least about 50 ng/mL, at least about 60 ng/mL, at least about 70 ng/mL, at least about 80 ng/mL, at least about 90 ng/mL, at least about 100 ng/mL, at least about 110 ng/mL, at least about 120 ng/mL, at least about 130 ng/mL, at least about 140 ng/mL, at least about 150 ng/mL, at least about 160 ng/mL, at least about 170 ng/mL, at least about 180 ng/mL, at least about 190 ng/mL, at least about 200 ng/mL, at least about 210 ng/mL, at least about 220 ng/mL, at least about 230 ng/mL, at least about 240 ng/mL, at least about 250 ng/mL, at least about 260 ng/mL, at least about 270 ng/mL, at least about 280 ng/mL, at least about 290 ng/mL, at least about 300 ng/mL, at least about 310 ng/mL, at least about 320 ng/mL, at least about 330 ng/mL, at least about 340 ng/mL, at least about 350 ng/mL, at least about 360 ng/mL, at least about 370 ng/mL, at least about 380 ng/mL, at least about 390 ng/mL, at least about 400 ng/mL, at least about 410 ng/mL, at least about 420 ng/mL, at least about 430 ng/mL, at least about 440 ng/mL, at least about 450 ng/mL, at least about 460 ng/mL, at least about 470 ng/mL, at least about 480 ng/mL, at least about 490 ng/mL, at least about 500 ng/mL, at least about 510 ng/mL, at least about 520 ng/mL, at least about 530 ng/mL, at least about 540 ng/mL, at least about 550 ng/mL, at least about 560 ng/mL, at least about 570 ng/mL, at least about 580 ng/mL, at least about 590 ng/mL, or at least about 600 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 50 ng/mL IL-2. In some aspects, the
medium of the single expansion step comprises at least about 60 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 70 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 73.6 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 75 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 80 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 90 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 100 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 200 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 300 ng/mL IL-2. In some aspects, the first medium of the single expansion step comprises at least about 400 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 500 ng/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 600 ng/mL IL-2. [0397] In some aspects, the medium of the single expansion step comprises at least about 1500 IU/mL IL-2. In some aspects, the medium of the single expansion step comprises from about 1500 IU/mL to about 12,000 IU/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 1500 IU/mL, at least about 1600 IU/mL, at least about 1700 IU/mL, at least about 1800 IU/mL, at least about 1900 IU/mL, at least about 2000 IU/mL, at least about 2100 IU/mL, at least about 2200 IU/mL, at least about 2300 IU/mL, at least about 2400 IU/mL, at least about 2500 IU/mL, at least about 2600 IU/mL, at least about 2700 IU/mL, at least about 2800 IU/mL, at least about 2900 IU/mL, at least about 3000 IU/mL, at least about 3100 IU/mL, at least about 3200 IU/mL, at least about 3300 IU/mL, at least about 3400 IU/mL, at least about 3500 IU/mL, at least about 3600 IU/mL, at least about 3700 IU/mL, at least about 3800 IU/mL, at least about 3900 IU/mL, at least about 4000 IU/mL, at least about 4100 IU/mL, at least about 4200 IU/mL, at least about 4300 IU/mL, at least about 4400 IU/mL, at least about 4500 IU/mL, at least about 4600 IU/mL, at least about 4700 IU/mL, at least about 4800 IU/mL, at least about 4900 IU/mL, at least about 5000 IU/mL, at least about 5100 IU/mL, at least about 5200 IU/mL, at least about 5300 IU/mL, at least about 5400 IU/mL, at least about 5500 IU/mL, at least about 5600 IU/mL, at least about 5700 IU/mL, at least about 5800 IU/mL, at least about 5900 IU/mL, at least about 6000 IU/mL, at least about 6100 IU/mL, at least about 6200 IU/mL, at least about 6300 IU/mL, at least about 6400 IU/mL, at least about 6500 IU/mL, at least about 6600 IU/mL, at least about 6700 IU/mL, at least about 6800 IU/mL, at least about 6900 IU/mL, at least about 7000 IU/mL IL-2, at least about 7100 IU/mL, at least about 7200 IU/mL, at least about 7300 IU/mL, at least about 7400 IU/mL, at least about 7500 IU/mL, at least about 7600 IU/mL, at least about 7700
IU/mL, at least about 7800 IU/mL, at least about 7900 IU/mL, or at least about 8000 IU/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 3000 IU/mL IL- 2. In some aspects, the medium of the single expansion step comprises at least about 3100 IU/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 3200 IU/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 3300 IU/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 3400 IU/mL IL-2. In some aspects, the medium of the single expansion step comprises at least about 3500 IU/mL IL-2. [0398] In some aspects, the medium of the single expansion step comprises IL-21. In some aspects, the IL-21 is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the medium of the single expansion step comprises at least about 0.1 ng/mL IL-21. In some aspects, the medium of the single expansion step comprises from about 0.1 ng/mL to about 50 ng/mL, about 1 ng/mL to about 50 ng/mL, about 1 ng/mL to about 40 ng/mL, about 1 ng/mL to about 35 ng/mL, about 10 ng/mL to about 40 ng/mL, about 10 ng/mL to about 35 ng/mL, about 10 ng/mL to about 30 ng/mL, about 15 ng/mL to about 40 ng/mL, about 15 ng/mL to about 35 ng/mL, about 15 ng/mL to about 30 ng/mL, about 20 ng/mL to about 40 ng/mL, about 20 ng/mL to about 35 ng/mL, about 20 ng/mL to about 30 ng/mL, about 25 ng/mL to about 35 ng/mL, about 25 ng/mL to about 30 ng/mL, or about 30 ng/mL to about 35 ng/mL IL-21. [0399] In some aspects, the medium of the single expansion step comprises at least about 0.1 ng/mL, at least about 0.5 ng/mL, at least about 1 ng/mL, at least about 2 ng/mL, at least about 3 ng/mL, at least about 4 ng/mL, at least about 5 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about 8 ng/mL, at least about 9 ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least about 12 ng/mL, at least about 13 ng/mL, at least about 14 ng/mL, at least about 15 ng/mL, at least about 16 ng/mL, at least about 17 ng/mL, at least about 18 ng/mL, at least about 19 ng/mL, at least about 20 ng/mL, at least about 25 ng/mL, at least about 30 ng/mL, at least about 35 ng/mL, or at least about 40 ng/mL IL-21. In some aspects, the medium of the single expansion step comprises at least about 1.0 ng/mL IL-21. In some aspects, the medium of the single expansion step comprises at least about 2.0 ng/mL IL-21. In some aspects, the medium of the single expansion step comprises at least about 3.0 ng/mL IL-21. In some aspects, the medium of the single expansion step comprises at least about 4.0 ng/mL IL-21. In some aspects, the medium of the single expansion step comprises at least about 5.0 ng/mL IL-21. In some aspects, the medium of the single expansion step comprises at least about 6.0 ng/mL IL-21. In some aspects, the medium of the single expansion
step comprises at least about 7.0 ng/mL IL-21. In some aspects, the medium of the single expansion step comprises at least about 8.0 ng/mL IL-21. In some aspects, the medium of the single expansion step comprises at least about 9.0 ng/mL IL-21. In some aspects, the medium of the single expansion step comprises at least about 10 ng/mL IL-21. In some aspects, the medium of the single expansion step comprises at least about 15 ng/mL IL-21. In some aspects, the medium of the single expansion step comprises at least about 20 ng/mL IL-21. In some aspects, the medium of the single expansion step comprises at least about 25 ng/mL IL-21. In some aspects, the medium of the single expansion step comprises at least about 30 ng/mL IL-21. In some aspects, the medium of the single expansion step comprises at least about 35 ng/mL IL-21. [0400] In some aspects, the medium of the single expansion step comprises between about 50 IU/mL to about 500 IU/mL of IL-21. In some aspects, the medium of the single expansion step comprises about 50 IU/mL, about 60 IU/mL, about 70 IU/mL, about 80 IU/mL, about 90 IU/mL, about 100 IU/mL, about 125 IU/mL, about 150 IU/mL, about 175 IU/mL, about 200 IU/mL, about 225 IU/mL, about 250 IU/mL, about 275 IU/mL, about 300 IU/mL, about 350 IU/mL, about 400 IU/mL, about 450 IU/mL, or about 500 IU/mL of IL-21. [0401] In some aspects, the medium of the single expansion step does not comprise IL-7. In some aspects, the medium of the single expansion step comprises IL-7. In some aspects, the IL- 7 is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the medium of the single expansion step comprises at least about 0.1 ng/mL IL-7. In some aspects, the medium of the single expansion step comprises from about 0.1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about 1 ng/mL to about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 2 ng/mL, about 5 ng/mL to about 15 ng/mL, about 5 ng/mL to about 10 ng/mL, about 10 ng/mL to about 20 ng/mL, about 10 ng/mL to about 15 ng/mL, or about 15 ng/mL to about 20 ng/mL IL-7. [0402] In some aspects, the medium of the single expansion step comprises at least about 0.1 ng/mL, at least about 0.5 ng/mL, at least about 1 ng/mL, at least about 1.3 ng/mL, at least about 1.5 ng/mL, at least about 1.7 ng/mL, at least about 2 ng/mL, at least about 2.3 ng/mL, at least about 2.5 ng/mL, at least about 2.7 ng/mL, at least about 3 ng/mL, at least about 3.3 ng/mL, at least about 3.5 ng/mL, at least about 3.7 ng/mL, at least about 4 ng/mL, at least about 4.3 ng/mL, at least about
4.5 ng/mL, at least about 4.7 ng/mL, at least about 5 ng/mL, at least about 5.3 ng/mL, at least about 5.5 ng/mL, at least about 5.7 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about 8 ng/mL, at least about 9 ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least about 12 ng/mL, at least about 13 ng/mL, at least about 14 ng/mL, at least about 15 ng/mL, at least about 16 ng/mL, at least about 17 ng/mL, at least about 18 ng/mL, at least about 19 ng/mL, or at least about 20 ng/mL IL-7. In some aspects, the medium comprises at least about 1.0 ng/mL IL-7. In some aspects, the medium of the single expansion step comprises at least about 2.0 ng/mL IL-7. In some aspects, the medium of the single expansion step comprises at least about 2.3 ng/mL IL-7. In some aspects, the medium of the single expansion step comprises at least about 2.5 ng/mL IL-7. In some aspects, the medium of the single expansion step comprises at least about 2.7 ng/mL IL-7. In some aspects, the medium of the single expansion step comprises at least about 3.0 ng/mL IL-7. In some aspects, the medium of the single expansion step comprises at least about 3.3 ng/mL IL-7. In some aspects, the medium of the single expansion step comprises at least about 3.5 ng/mL IL-7. In some aspects, the medium of the single expansion step comprises at least about 3.7 ng/mL IL-7. [0403] In some aspects, the medium of the single expansion step comprises between about 500 IU/mL to about 1,500 IU/mL of IL-7. In some aspects, the medium of the single expansion step comprises about 500 IU/mL, about 550 IU/mL, about 600 IU/mL, about 650 IU/mL, about 700 IU/mL, about 750 IU/mL, about 800 IU/mL, about 850 IU/mL, about 900 IU/mL, about 950 IU/mL, about 1,000 IU/mL, about 1,050 IU/mL, about 1,100 IU/mL, about 1,150 IU/mL, about 1,200 IU/mL, about 1,250 IU/mL, about 1,300 IU/mL, about 1,350 IU/mL, about 1,400 IU/mL, about 1,450 IU/mL, or about 1,500 IU/mL of IL-7. [0404] In some aspects, the medium of the single expansion step does not comprise IL-15. In some aspects, the medium of the single expansion step comprises IL-15. In some aspects, the IL-15 is associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the medium of the single expansion step comprises at least about 0.1 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises from about 0.1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 14 ng/mL, about 1 ng/mL to about 13 ng/mL, about 1 ng/mL to about 12 ng/mL, about 1 ng/mL to about 11 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about 5 ng/mL, about 1 ng/mL to about 4 ng/mL, about 1 ng/mL to about 3 ng/mL, about 1 ng/mL to about 2 ng/mL, about 5 ng/mL to about 15 ng/mL,
about 5 ng/mL to about 10 ng/mL, about 10 ng/mL to about 20 ng/mL, about 10 ng/mL to about 15 ng/mL, or about 15 ng/mL to about 20 ng/mL IL-15. [0405] In some aspects, the medium of the single expansion step comprises at least about 0.1 ng/mL, at least about 0.2 ng/mL, at least about 0.3 ng/mL, at least about 0.4 ng/mL, at least about 0.5 ng/mL, at least about 0.6 ng/mL, at least about 0.7 ng/mL, at least about 0.8 ng/mL, at least about 0.9 ng/mL, at least about 1 ng/mL, at least about 2 ng/mL, at least about 3 ng/mL, at least about 4 ng/mL, at least about 5 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about 8 ng/mL, at least about 9 ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least about 12 ng/mL, at least about 13 ng/mL, at least about 14 ng/mL, at least about 15 ng/mL, at least about 16 ng/mL, at least about 17 ng/mL, at least about 18 ng/mL, at least about 19 ng/mL, or at least about 20 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 0.1 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 0.2 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 0.3 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 0.4 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 0.5 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 0.6 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 0.7 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 0.8 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 0.9 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 1.0 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 2.0 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 3.0 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 4.0 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 5.0 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 6.0 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 7.0 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 8.0 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 9.0 ng/mL IL-15. In some aspects, the medium of the single expansion step comprises at least about 10 ng/mL IL-15. [0406] In some aspects, the medium of the single expansion step comprises between about 50 IU/mL to about 500 IU/mL of IL-15. In some aspects, the medium of the single expansion step comprises about 50 IU/mL, about 60 IU/mL, about 70 IU/mL, about 80 IU/mL, about 90 IU/mL,
about 100 IU/mL, about 125 IU/mL, about 150 IU/mL, about 175 IU/mL, about 200 IU/mL, about 225 IU/mL, about 250 IU/mL, about 275 IU/mL, about 300 IU/mL, about 350 IU/mL, about 400 IU/mL, about 450 IU/mL, or about 500 IU/mL of IL-15. [0407] In some aspects, the medium of the single expansion step comprises about 1500 IU/mL IL-2 and about 30 ng/M IL-21, wherein the IL-2, the IL-21, or both are associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the medium of the single expansion step comprises about 1500 IU/mL IL-2 and about 30 ng/M IL-21, wherein the medium does not comprise IL-7, IL-15, or both IL-7 and IL-15. [0408] In some aspects, the medium of the single expansion step comprises about 3000 IU/mL IL-2 and about 30 ng/M IL-21, wherein the IL-2, the IL-21, or both are associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the medium of the single expansion step comprises about 3000 IU/mL IL-2 and about 30 ng/M IL-21, wherein the medium does not comprise IL-7, IL-15, or both IL-7 and IL-15. [0409] In some aspects, the medium of the single expansion step comprises about 6000 IU/mL IL-2 and about 30 ng/M IL-21, wherein the IL-2, the IL-21, or both are associated with the feeder cell replacement, e.g., PCS, as a surface cue or soluble cue, as described herein. In some aspects, the medium of the single expansion step comprises about 6000 IU/mL IL-2 and about 30 ng/M IL-21, wherein the medium does not comprise IL-7, IL-15, or both IL-7 and IL-15. II.B.5. Rapid Expansion Protocol (REP) [0410] In some aspects, the immune cells are TILs, and the TILs are subjected to an initial expansion and/or an additional expansion as described herein. In some aspects, the TILs are subjected to a single expansion step as described herein. In some aspects, the initial expansion step, the additional expansion step, the single expansion step, or any combination thereof comprises a rapid expansion protocol. The Rapid Expansion Protocol (REP) is a method of rapidly expanding TILs in culture. See, e.g., Dudley, et al., Science 298:850-54 (2002); Dudley, et al., J. Clin. Oncol. 23:2346-57 (2005); Dudley, et al., J. Clin. Oncol. 26:5233-39 (2008); Riddell, et al., Science 257:238-41 (1992); and Dudley, et al., J. Immunother. 26:332-42 (2003), each of which is incorporated by reference herein in its entirety. This section describes various aspects of REP that can be incorporated into the methods disclosed herein. [0411] In some aspects, TILs are rapidly expanded using non-specific T-cell receptor stimulation in the presence of feeder cells and interleukin-2 (IL-2), IL-7, IL-15, IL-21, or combinations thereof. In certain aspects, the TILs are rapidly expanded in the presence of IL-2, IL-
15, and IL-21. In some aspects, the concentration of IL-2 in the media during rapid expansion is lower than the concentration of IL-2 in the media during the initial culture. In some aspects, the concentration of IL-2 during rapid expansion is less than 300 ng/ml. In some aspects, the concentration of IL-2 during rapid expansion is about 50 ng/ml, about 55 ng/ml, about 60 ng/ml, about 65 ng/ml, about 70 ng/ml, about 73.6 ng/ml, about 75 ng/ml, about 80 ng/ml, about 85 ng/ml, about 90 ng/ml, about 95 ng/ml, about 100 ng/ml, about 105 ng/ml, about 110 ng/ml, about 115 ng/ml, about 120 ng/ml, about 125 ng/ml, about 130 ng/ml, about 135 ng/ml, about 140 ng/ml, about 145 ng/ml, about 150 ng/ml, about 175 ng/ml, about 200 ng/ml, about 225 ng/ml, about 250 ng/ml, or about 275 ng/ml. In some aspects, the concentration of IL-2 during rapid expansion is about 50 ng/ml. In some aspects, the concentration of IL-2 during rapid expansion is about 55 ng/ml. In some aspects, the concentration of IL-2 during rapid expansion is about 60 ng/ml. In some aspects, the concentration of IL-2 during rapid expansion is about 65 ng/ml. In some aspects, the concentration of IL-2 during rapid expansion is about 70 ng/ml. In some aspects, the concentration of IL-2 during rapid expansion is about 73.6 ng/ml. In some aspects, the concentration of IL-2 during rapid expansion is about 75 ng/ml. In some aspects, the concentration of IL-2 during rapid expansion is about 80 ng/ml. In some aspects, the concentration of IL-2 during rapid expansion is about 85 ng/ml. In some aspects, the concentration of IL-2 during rapid expansion is about 90 ng/ml. In some aspects, the concentration of IL-2 during rapid expansion is about 95 ng/ml. In some aspects, the concentration of IL-2 during rapid expansion is about 100 ng/ml. [0412] In some aspects, the concentration of IL-21 in the media during rapid expansion is lower than the concentration of IL-21 in the media during the initial culture. In some aspects, the concentration of IL-21 during rapid expansion is less than 30 ng/ml. In some aspects, the concentration of IL-21 during rapid expansion is about 1 ng/ml, about 2 ng/ml, about 3 ng/ml, about 4 ng/ml, about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9 ng/ml, about 10 ng/ml, about 11 ng/ml, about 12 ng/ml, about 13 ng/ml, about 14 ng/ml, about 15 ng/ml, about 16 ng/ml, about 17 ng/ml, about 18 ng/ml, about 19 ng/ml, about 20 ng/ml, about 21 ng/ml, about 22 ng/ml, about 23 ng/ml, about 24 ng/ml, about 25 ng/ml, about 26 ng/ml, about 27 ng/ml, about 28 ng/ml, or about 29 ng/ml. In some aspects, the concentration of IL-21 during rapid expansion is about 5 ng/ml. In some aspects, the concentration of IL-21 during rapid expansion is about 6 ng/ml. In some aspects, the concentration of IL-21 during rapid expansion is about 7 ng/ml. In some aspects, the concentration of IL-21 during rapid expansion is about 8 ng/ml. In some aspects, the concentration of IL-21 during rapid expansion is about 9 ng/ml. In some aspects, the
concentration of IL-21 during rapid expansion is about 10 ng/ml. In some aspects, the concentration of IL-21 during rapid expansion is about 11 ng/ml. In some aspects, the concentration of IL-21 during rapid expansion is about 12 ng/ml. In some aspects, the concentration of IL-21 during rapid expansion is about 13 ng/ml. In some aspects, the concentration of IL-21 during rapid expansion is about 14 ng/ml. In some aspects, the concentration of IL-21 during rapid expansion is about 15 ng/ml. [0413] In some aspects, the concentration of IL-15 in the media during rapid expansion is about 0.1 ng/ml, about 0.2 ng/ml, about 0.3 ng/ml, about 0.4 ng/ml, about 0.5 ng/ml, about 0.6 ng/ml, about 0.7 ng/ml, about 0.8 ng/ml, about 0.9 ng/ml, about 1.0 ng/ml, about 1.1 ng/ml, about 1.2 ng/ml, about 1.3 ng/ml, about 1.4 ng/ml, about 1.5 ng/ml, about 1.6 ng/ml, about 1.7 ng/ml, about 1.8 ng/ml, about 1.9 ng/ml, about 2.0 ng/ml, about 2.25 ng/ml, about 2.5 ng/ml, about 2.75 ng/ml, about 3.0 ng/ml, about 3.5 ng/ml, about 4.0 ng/ml, about 4.5 ng/ml, or about 5.0 ng/ml. In some aspects, the concentration of IL-15 during rapid expansion is about 0.1 ng/ml. In some aspects, the concentration of IL-15 during rapid expansion is about 0.2 ng/ml. In some aspects, the concentration of IL-15 during rapid expansion is about 0.3 ng/ml. In some aspects, the concentration of IL-15 during rapid expansion is about 0.4 ng/ml. In some aspects, the concentration of IL-15 during rapid expansion is about 0.5 ng/ml. In some aspects, the concentration of IL-15 during rapid expansion is about 0.6 ng/ml. In some aspects, the concentration of IL-15 during rapid expansion is about 0.7 ng/ml. In some aspects, the concentration of IL-15 during rapid expansion is about 0.8 ng/ml. In some aspects, the concentration of IL-15 during rapid expansion is about 0.9 ng/ml. In some aspects, the concentration of IL-15 during rapid expansion is about 1.0 ng/ml. [0414] The non-specific T-cell receptor stimulus can include, e.g., OKT3 (e.g., about 30 ng/ml), a mouse monoclonal anti-CD3 antibody (available from Ortho-McNeil®, Raritan, N.J. or Miltenyi Biotec, Bergisch Gladbach, Germany). In some aspects, TILs are rapidly expanded by stimulation of peripheral blood mononuclear cells (PBMC) in vitro with one or more antigens (including antigenic portions thereof, such as epitope(s), or a cell of the cancer, which can be optionally expressed from a vector, such as an human leukocyte antigen A2 (HLA-A2) binding peptide, e.g., approximately 0.3 μM MART-1:26-35 (27 L) or gp100:209-217 (210M)), in the presence of a T-cell growth factor, such as around 200-400 IU/ml of a T-cell growth factor, such as 300 IU/ml IL-2 or IL-15. In some aspects, TILs are expanded by stimulation using TRANSACT™. In some aspects, the in vitro-induced TILs are rapidly expanded by stimulation with the same antigen(s) of the cancer pulsed onto HLA-A2-expressing antigen-presenting cells.
In some aspects, the TILs can be stimulated with irradiated, autologous lymphocytes or with irradiated HLA-A2+ allogeneic lymphocytes and IL-2. [0415] In some aspects, the TILs are stimulated during the initial expansion and/or the additional expansion by culturing the cells in a medium comprising TRANSACT™ and optionally 4-1BBL and/or CD27L. In some aspects, the TILs are stimulated during the initial expansion and/or the additional expansion by culturing the cells in a medium comprising TRANSACT™, 4- 1BBL, and CD27L. In some aspects, the TILs are stimulated during the second expansion by culturing the cells in a medium comprising at least about 1:100 TRANSACT™, at least about 1 µg/ml 4-1BBL, and at least about 5 µg/ml CD27L. [0416] In some aspects, one or more TILs are genetically modified before, during, or after TIL expansion. Genetic modification of the TILs can be achieved using any methods known in the art. In some aspects, one or more TILs are modified using a Cas9 endonuclease (CRISPR; see, e.g., US2017067021A1, which is incorporated by reference herein in its entirety), TALEN, a zing- finger endonuclease, site directed mutagenesis, or any combination thereof. In some aspects, one or more TILs are genetically modified to disrupt or ablate expression of human cytokine inducible SH2-containing protein (CISH; see, e.g., US10406177B2, which is incorporated by reference herein in its entirety). In some aspects, one or more TILs is modified using an AAV, e.g., one or more of the TILs comprise an AAV. In some aspects one or more TILs is modified using a lentivirus or a retrovirus. In some aspects, one or more TILs are genetically modified to express an exogenous modified or engineered T cell receptor (TCR). In some aspects, one or more TILs are genetically modified to express chimeric antigen receptor (CAR). In some aspects, one or more TILs are genetically modified to express CD86. In some aspects, one or more TILs are genetically modified to express OX40L. In some aspects, one or more TILs are genetically modified to express 4-1BBL. In some aspects, one or more TILs are genetically modified to express an anti-PD1 antibody. [0417] In some aspects, the TILs are expanded in a culture medium that further comprises a tumor necrosis factor receptor superfamily (TNFRSF) agonist. Any TNFRSF agonist can be used in the methods disclosed herein. Non-limiting examples of TNFRSF agonists can be found, for example, in US20200121719A1, which is incorporated by reference herein in its entirety. In some aspects, the TNFRSF agonist is added after the preliminary culture. In some aspects, the TNFRSF agonist is added during the single expansion step, or the initial expansion and/or the additional expansion.
[0418] In some aspects, the TILs are expanded in a culture medium that further comprises a 4-1BB agonist. Any 4-1BB agonist can be used in the methods disclosed herein. In some aspects, the 4-1BB agonist comprises a 4-1BB antibody. Non-limiting examples of 4-1BB agonists can be found, for example, in US20200032209A1, which is incorporated by reference herein in its entirety. In some aspects, the 4-1BB agonist is added after the preliminary culture. In some aspects, the 4-1BB agonist is added during the single expansion step, or initial expansion and/or the additional expansion. [0419] In some aspects, the TILs are stimulated during the single expansion step, or initial expansion and/or the additional expansion by culturing the cells in a medium comprising TRANSACT™ and optionally 4-1BBL and/or CD27L. In some aspects, the TILs are stimulated during the single expansion step, or initial expansion and/or the additional expansion by culturing the cells in a medium comprising TRANSACT™, 4-1BBL, and CD27L. In some aspects, the TILs are stimulated during the single expansion step, or initial expansion and/or the additional expansion by culturing the cells in a medium comprising at least about 1:100 TRANSACT™, at least about 1 µg/ml 4-1BBL, and at least about 5 µg/ml CD27L. [0420] In some aspects, the TILs are expanded in a culture medium that further comprises an adenosine a2a receptor antagonist. Any adenosine a2a receptor antagonist can be used in the methods disclosed herein. Non-limiting examples of adenosine a2a receptor antagonist can be found, for example, in US20210137930A1, which is incorporated by reference herein in its entirety. In some aspects, the adenosine a2a receptor antagonist comprises vipadenant, CPI-444 (ciforadenant), SCH58261, ZM241385, SCH420814, SYN115, 8-CSC, KW-6002, A2A receptor antagonist 1, ADZ4635, ST4206, KF21213, SCH412348, or 7MMG-49, or pharmaceutically acceptable salts, solvates, hydrates, cocrystals, or prodrugs thereof, or combinations thereof. In some aspects, the adenosine a2a receptor antagonist is added during the preliminary culture. In some aspects, the adenosine a2a receptor antagonist is added during the single expansion step, or initial expansion and/or the additional expansion. [0421] In some aspects, the TILs are expanded in a culture medium that further comprises an AKT pathway inhibitor (AKTi). Any AKTi can be used in the methods disclosed herein. Non- limiting examples of AKTi that can be used in the present disclosure can be found, for example, in WO2020096927, which is incorporated by reference herein in its entirety. In some aspects, the AKTi comprises afuresertib, uprosertib, ipatasertib, AT7867, AT13148, or pharmaceutically acceptable salts, solvates, hydrates, cocrystals, or prodrugs thereof. In some aspects, the AKTi is an mTOR inhibitor, e.g., AZD8055 or pharmaceutically acceptable salts, solvates, hydrates,
cocrystals, or prodrugs thereof. In some aspects, the AKTi is an PI3K inhibitor, e.g., LY294002 or pharmaceutically acceptable salts, solvates, hydrates, cocrystals, or prodrugs thereof. In some aspects, the AKTi is added during the preliminary culture. In some aspects, the AKTi is added during the single expansion step, or initial expansion and/or the additional expansion. [0422] In some aspects, expanded TILs from the additional expansion are subjected to a final expansion. In some aspects, the TILs are transitioned from the additional expansion to the final expansion without opening the closed system (e.g., the G-Rex® closed system). In some aspects, the final expansion step is carried out in one or more gas permeable flasks (e.g., G-Rex® flasks). In some aspects, the additional expansion corresponds with a first phase of the REP protocol (i.e., the REP protocol up until the cells are split), and the final expansion corresponds with the second phase of the REP protocol (i.e., the REP protocol after the cells are split). As such, in some aspects, the additional expansion has a duration of about 3 to 7 days (e.g., about 5 days, about 6 days, or about 7 days), and the final expansion has a duration of about 3 to 7 days (e.g., about 5 days, about 6 days, or about 7 days). [0423] In some aspects, the media during final expansion comprises IL-2, IL-7, IL-15, IL- 21, or combinations thereof. In certain aspects, the media during final expansion comprises IL-2, IL-15, and IL-21. In some aspects, the concentration of IL-2 in the media during final expansion is lower than the concentration of IL-2 in the media during the preliminary culture, the initial expansion, and/or the additional expansion. In some aspects, the concentration of IL-2 during final expansion is less than 300 ng/ml. In some aspects, the concentration of IL-2 during final expansion is about 50 ng/ml, about 55 ng/ml, about 60 ng/ml, about 65 ng/ml, about 70 ng/ml, about 73.6 ng/ml, about 75 ng/ml, about 80 ng/ml, about 85 ng/ml, about 90 ng/ml, about 95 ng/ml, about 100 ng/ml, about 105 ng/ml, about 110 ng/ml, about 115 ng/ml, about 120 ng/ml, about 125 ng/ml, about 130 ng/ml, about 135 ng/ml, about 140 ng/ml, about 145 ng/ml, about 150 ng/ml, about 175 ng/ml, about 200 ng/ml, about 225 ng/ml, about 250 ng/ml, or about 275 ng/ml. In some aspects, the concentration of IL-2 during final expansion is about 50 ng/ml. In some aspects, the concentration of IL-2 during final expansion is about 55 ng/ml. In some aspects, the concentration of IL-2 during final expansion is about 60 ng/ml. In some aspects, the concentration of IL-2 during final expansion is about 65 ng/ml. In some aspects, the concentration of IL-2 during final expansion is about 70 ng/ml. In some aspects, the concentration of IL-2 during final expansion is about 73.6 ng/ml. In some aspects, the concentration of IL-2 during final expansion is about 75 ng/ml. In some aspects, the concentration of IL-2 during final expansion is about 80 ng/ml. In some aspects, the concentration of IL-2 during final expansion is about 85 ng/ml. In some aspects, the concentration
of IL-2 during final expansion is about 90 ng/ml. In some aspects, the concentration of IL-2 during final expansion is about 95 ng/ml. In some aspects, the concentration of IL-2 during final expansion is about 100 ng/ml. [0424] In some aspects, the concentration of IL-21 in the media during final expansion is lower than the concentration of IL-21 in the media during the preliminary culture, the initial expansion, and/or the additional expansion. In some aspects, the concentration of IL-21 during final expansion is less than 30 ng/ml. In some aspects, the concentration of IL-21 during final expansion is about 1 ng/ml, about 2 ng/ml, about 3 ng/ml, about 4 ng/ml, about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9 ng/ml, about 10 ng/ml, about 11 ng/ml, about 12 ng/ml, about 13 ng/ml, about 14 ng/ml, about 15 ng/ml, about 16 ng/ml, about 17 ng/ml, about 18 ng/ml, about 19 ng/ml, about 20 ng/ml, about 21 ng/ml, about 22 ng/ml, about 23 ng/ml, about 24 ng/ml, about 25 ng/ml, about 26 ng/ml, about 27 ng/ml, about 28 ng/ml, or about 29 ng/ml. In some aspects, the concentration of IL-21 during final expansion is about 5 ng/ml. In some aspects, the concentration of IL-21 during final expansion is about 6 ng/ml. In some aspects, the concentration of IL-21 during final expansion is about 7 ng/ml. In some aspects, the concentration of IL-21 during final expansion is about 8 ng/ml. In some aspects, the concentration of IL-21 during final expansion is about 9 ng/ml. In some aspects, the concentration of IL-21 during final expansion is about 10 ng/ml. In some aspects, the concentration of IL-21 during final expansion is about 11 ng/ml. In some aspects, the concentration of IL-21 during final expansion is about 12 ng/ml. In some aspects, the concentration of IL-21 during final expansion is about 13 ng/ml. In some aspects, the concentration of IL-21 during final expansion is about 14 ng/ml. In some aspects, the concentration of IL-21 during final expansion is about 15 ng/ml. [0425] In some aspects, the concentration of IL-15 in the media during final expansion is about 0.1 ng/ml, about 0.2 ng/ml, about 0.3 ng/ml, about 0.4 ng/ml, about 0.5 ng/ml, about 0.6 ng/ml, about 0.7 ng/ml, about 0.8 ng/ml, about 0.9 ng/ml, about 1.0 ng/ml, about 1.1 ng/ml, about 1.2 ng/ml, about 1.3 ng/ml, about 1.4 ng/ml, about 1.5 ng/ml, about 1.6 ng/ml, about 1.7 ng/ml, about 1.8 ng/ml, about 1.9 ng/ml, about 2.0 ng/ml, about 2.25 ng/ml, about 2.5 ng/ml, about 2.75 ng/ml, about 3.0 ng/ml, about 3.5 ng/ml, about 4.0 ng/ml, about 4.5 ng/ml, or about 5.0 ng/ml. In some aspects, the concentration of IL-15 during final expansion is about 0.1 ng/ml. In some aspects, the concentration of IL-15 during final expansion is about 0.2 ng/ml. In some aspects, the concentration of IL-15 during final expansion is about 0.3 ng/ml. In some aspects, the concentration of IL-15 during final expansion is about 0.4 ng/ml. In some aspects, the concentration of IL-15 during final expansion is about 0.5 ng/ml. In some aspects, the
concentration of IL-15 during final expansion is about 0.6 ng/ml. In some aspects, the concentration of IL-15 during final expansion is about 0.7 ng/ml. In some aspects, the concentration of IL-15 during final expansion is about 0.8 ng/ml. In some aspects, the concentration of IL-15 during final expansion is about 0.9 ng/ml. In some aspects, the concentration of IL-15 during final expansion is about 1.0 ng/ml. [0426] In some aspects, the final expansion comprises a stimulation. In some aspects the stimulation is the same as the stimulation used during the secondary expansion. In some aspects, the TILs are stimulated during the second expansion by culturing the cells in an MRM comprising TRANSACT™, 4-1BBL, CD27L, or any combination thereof. In some aspects, the TILs are stimulated during the second expansion by culturing the cells in a MRM comprising TRANSACT™ and optionally 4-1BBL and/or CD27L. In some aspects, the TILs are stimulated during the second expansion by culturing the cells in a MRM comprising at least about 1:100 TRANSACT™, at least about 1 µg/ml 4-1BBL, and at least about 5 µg/ml CD27L. [0427] In some aspects, the final expansion step is carried out in static G-Rex®. In some aspects, the final expansion is carried out in a stirred tank. In some aspects the final expansion step is carried out in a bioreactor. In some aspects, the final expansion is continued until the cell yield in the final TIL media reaches at least about 40x109 to at least about 100x109, at least about 40x109 to at least about 90x109, at least about 40x109 to at least about 80x109, at least about 40x109 to at least about 70x109, at least about 40x109 to at least about 60x109, at least about 40x109 to at least about 50x109, at least about 10x109 to at least about 100x109, at least about 20x109 to at least about 100x109, at least about 30x109 to at least about 100x109, at least about 30x109 to at least about 50x109, or at least about 35x109 to at least about 45x109 cells. In some aspects, the final expansion is continued until the cell yield in the final TIL media reaches at least about 40x109 to at least about 100x109 cells. In some aspects, the final expansion is continued until the cell yield in the final TIL media reaches at least about 40x109, at least about 45x109, at least about 50x109, at least about 55x109, at least about 60x109, at least about 65x109, at least about 70x109, at least about 75x109, at least about 80x109, at least about 85x109, at least about 90x109, at least about 95x109, or at least about 100x109 cells. In some aspects, the final expansion is continued until the cell yield in the final TIL media reaches at least about 40x109 cells. In some aspects, the final expansion is continued until the cell yield in the final TIL media reaches at least about 50x109 cells. In some aspects, the final expansion is continued until the cell yield in the final TIL media reaches at least about 60x109 cells. In some aspects, the final expansion is continued until the cell yield in the final TIL media reaches at least about 70x109 cells. In some aspects, the final expansion is continued
until the cell yield in the final TIL media reaches at least about 80x109 cells. In some aspects, the final expansion is continued until the cell yield in the final TIL media reaches at least about 90x109 cells. In some aspects, the final expansion is continued until the cell yield in the final TIL media reaches at least about 100x109 cells. [0428] In some aspects, the final expansion is continued until the cell yield in the final TIL media for at least about 7 to at least about 21 days. In some aspects, the final expansion is continued until the cell yield in the final TIL media for at least about 7 days. In some aspects, the final expansion is continued until the cell yield in the final TIL media for at least about 8 days. In some aspects, the final expansion is continued until the cell yield in the final TIL media for at least about 9 days. In some aspects, the final expansion is continued until the cell yield in the final TIL media for at least about 10 days. In some aspects, the final expansion is continued until the cell yield in the final TIL media for at least about 11 days. In some aspects, the final expansion is continued until the cell yield in the final TIL media for at least about 12 days. In some aspects, the final expansion is continued until the cell yield in the final TIL media for at least about 13 days. In some aspects, the final expansion is continued until the cell yield in the final TIL media for at least about 14 days. In some aspects, the final expansion is continued until the cell yield in the final TIL media for at least about 15 days. In some aspects, the final expansion is continued until the cell yield in the final TIL media for at least about 16 days. In some aspects, the final expansion is continued until the cell yield in the final TIL media for at least about 17 days. In some aspects, the final expansion is continued until the cell yield in the final TIL media for at least about 18 days. In some aspects, the final expansion is continued until the cell yield in the final TIL media for at least about 19 days. In some aspects, the final expansion is continued until the cell yield in the final TIL media for at least about 20 days. In some aspects, the final expansion is continued until the cell yield in the final TIL media for at least about 21 days. [0429] In some aspects, the full duration of the expansion process (e.g., (i) the preliminary culture process, the secondary expansion process, and the final expansion process; or (ii) the preliminary culture process and the single secondary expansion process) is 22 days or less. Generation of young TILs using shorter expansion processes confers various benefits on the resulting TIL composition. As such, the culture conditions and methods disclosed herein confer additional benefits, e.g., increased stem-like characteristics, expanded clonal diversity, improved cytolytic activity, and/or increased CD8+ cell expansion, on those already identified for young TILs.
II.B.6. Static-REP and Dynamic-REP [0430] In some aspects, the initial expansion and/or the additional expansion comprises a static-REP step followed by a dynamic-REP step. As used herein, "static-REP" refers to an expansion step wherein TILs harvested from a proceeding expansion, e.g., TILs from a preliminary culture for the initial expansion (e.g., first REP step), or TILs from the initial expansion for the additional expansion, as disclosed herein, are further expanded in a medium comprising a CD3 agonist (e.g., OKT3) and a feeder cell replacement, e.g., a PCS, wherein the CD3 agonist is associated with the feeder cell replacement. In some aspects, agitation of the culture is not applied during static-REP. "Dynamic-REP," as used herein, refers to an expansion step wherein TILs from the static-REP are further expanded in a medium until a therapeutic number of TILs is reached, wherein the culture occurs under agitation. In some aspects, the method comprises expanding a population of TILs comprising (a) culturing the TILs in a medium comprising a CD3 agonist (e.g., OKT3) and a feeder cell replacement, e.g., a PCS, wherein the CD3 agonist is associated with the feeder cell replacement (a "static-REP step"); and (b) adding to the TILs from the static-REP step a medium comprising a CD3 agonist (e.g., OKT3) and a feeder cell replacement, e.g., a PCS, wherein the CD3 agonist is associated with the feeder cell replacement, wherein agitation is applied to the culture (a "dynamic-REP step"). In some aspects, no CD3 agonist is added during the dynamic-REP. In some aspects, no feeder cell replacement is added during the dynamic-REP step. In some aspects, no CD3 agonist and no feeder cell replacement are added during the dynamic- REP step. [0431] In some aspects, the TILs obtained during static-REP are added directly to the dynamic-REP. In some aspects, the TILs are not split between the static-REP and the dynamic- REP. In some aspects, the TILs are cryopreserved following the static-REP and prior to the dynamic-REP. [0432] In some aspects, static-REP medium comprising the expanded TILs is applied directly to the dynamic-REP. As such, in some aspects, day 0 of dynamic-REP includes TILs present in a medium that comprises the feeder cell replacement, e.g., PCS, comprising the CD3 agonist, e.g., OKT3, which was carried over from the static-REP to the dynamic-REP with the TILs. However, in some aspects, no additional CD3 agonist or feeder cell replacement are added during the dynamic-REP step. Over time, an concentration of CD3 agonist that had carried over from the static-REP is degraded or otherwise lost, resulting in a decreasing concentration of CD3 agonist during the dynamic-REP step.
[0433] Further, the dynamic-REP step comprises applying agitation to the culture. In some aspects, the agitation comprises rocking the culture. [0434] In some aspects, the dynamic-REP step comprises perfusion. As used herein, "perfusion" refers to a method of culturing cells, e.g., TILs, wherein a portion of the culture medium is constantly replaced with fresh medium without removing the cultured cells, e.g., TILs. The rate of perfusion can be carefully controlled. In some aspects, the perfusion comprises continuous media exchange at a rate of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% of the working volume of the culture vessel every 24 hours. In some aspects, the perfusion comprises continuous media exchange at a rate of about 5% of the working volume of the culture vessel every 24 hours. In some aspects, the perfusion comprises continuous media exchange at a rate of about 10% of the working volume of the culture vessel every 24 hours. In some aspects, the perfusion comprises continuous media exchange at a rate of about 15% of the working volume of the culture vessel every 24 hours. In some aspects, the perfusion comprises continuous media exchange at a rate of about 20% of the working volume of the culture vessel every 24 hours. In some aspects, the perfusion comprises continuous media exchange at a rate of about 25% of the working volume of the culture vessel every 24 hours. In some aspects, the perfusion comprises continuous media exchange at a rate of about 30% of the working volume of the culture vessel every 24 hours. In some aspects, the perfusion comprises continuous media exchange at a rate of about 35% of the working volume of the culture vessel every 24 hours. In some aspects, the perfusion comprises continuous media exchange at a rate of about 40% of the working volume of the culture vessel every 24 hours. In some aspects, the perfusion comprises continuous media exchange at a rate of about 45% of the working volume of the culture vessel every 24 hours. In some aspects, the perfusion comprises continuous media exchange at a rate of about 50% of the working volume of the culture vessel every 24 hours. In some aspects, the perfusion comprises continuous media exchange at a rate of about 55% of the working volume of the culture vessel every 24 hours. In some aspects, the perfusion comprises continuous media exchange at a rate of about 60% of the working volume of the culture vessel every 24 hours. [0435] In some aspects, the perfusion rate is constant for the entirety of the dynamic-REP step. In some aspects, the perfusion rate is varied during the dynamic-REP step. In some aspects, the perfusion comprises continuous media exchange at a rate of about 25% of the working volume of the culture vessel every 24 hours for the first 48 hours of the dynamic-REP culture, and wherein
the perfusion comprises continuous media exchange at a rate of about 50% of the working volume of the culture vessel every 24 hours for the remainder of the dynamic-REP culture. [0436] In some aspects, the volume of the culture medium is held constant for the entirety of the dynamic-REP step. In some aspects, the volume of the culture medium is varied during the dynamic-REP step. In some aspects, the volume of the culture medium is increased during the dynamic-REP step. In some aspects, the perfusion comprises adding more medium to the culture than is removed. In some aspects, the volume of the culture medium is increased from hour 0 to hour 48 of the dynamic-REP step. In some aspects, the volume of the culture medium is increased from hour 0 to hour 48 of the dynamic-REP step, and the volume of the culture is maintained, i.e., neither increased in volume by more than 10% nor decreased in volume by more than 10%, from hour 48 until the completion of the dynamic-REP step. In some aspects, the volume of the medium is increased by 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% every 24 hours for the first 48 hours. In some aspects, the volume of the medium is increased by 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%, or at least about 75% from hour 0 to hour 48 of the dynamic-REP step. In some aspects, the volume of the medium is increased by at least about 25% from hour 0 to hour 48 of the dynamic-REP step. In some aspects, the volume of the medium is increased by at least about 33% from hour 0 to hour 48 of the dynamic-REP step. In some aspects, the volume of the medium is increased by at least about 50% from hour 0 to hour 48 of the dynamic-REP step. In some aspects, the volume of the medium is increased by at least about 65% from hour 0 to hour 48 of the dynamic-REP step. In some aspects, the volume of the medium is increased by at least about 66% from hour 0 to hour 48 of the dynamic-REP step. In some aspects, the volume of the medium is increased by at least about 75% from hour 0 to hour 48 of the dynamic-REP step. [0437] In some aspects, the fresh medium applied during perfusion comprises greater than 4 mM potassium ion, e.g., a MRM disclosed herein. In some aspects, the fresh medium applied during perfusion comprises greater than 4 mM potassium ion, e.g., a MRM disclosed herein, and does not comprise a CD3 agonist, e.g., OKT-3. In some aspects, the fresh medium applied during perfusion comprises greater than 4 mM potassium ion, e.g., a MRM disclosed herein, and does not comprise antigen-presenting cells, e.g., irradiated PBMCs. In some aspects, the fresh medium applied during perfusion comprises greater than 4 mM potassium ion, e.g., a MRM disclosed
herein, and does not comprise a CD3 agonist (e.g., OKT-3) or antigen-presenting cells (e.g., irradiated PBMCs). [0438] As the perfusion comprises continuously replacing a portion of the medium with fresh medium that does not comprise CD3 agonist, the concentration of any CD3 agonist in the starting medium (i.e., at day 0 of the dynamic-REP step) will decrease throughout the dynamic- REP. In some aspects, the concentration of CD3 agonist in the dynamic-REP culture decreases at a rate of about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60% every 24 hours. In some aspects, the rate at which the CD3 agonist decreases in the culture will be linked to the rate of perfusion. In some aspects, the concentration of CD3 agonist in the dynamic-REP culture is less than about 75% that of the concentration of CD3 agonist in the static-REP culture at least about 24 hours after initiation of the dynamic-REP. In some aspects, the concentration of CD3 agonist in the dynamic-REP culture is less than about 45% that of the concentration of CD3 agonist in the static-REP culture at least about 48 hours after initiation of the dynamic-REP. In some aspects, the concentration of CD3 agonist in the dynamic-REP culture is less than about 35% that of the concentration of CD3 agonist in the static-REP culture at least about 72 hours after initiation of the dynamic-REP. In some aspects, the concentration of CD3 agonist in the dynamic-REP culture is less than about 27% that of the concentration of CD3 agonist in the static-REP culture at least about 96 hours after initiation of the dynamic-REP. In some aspects, the concentration of CD3 agonist in the dynamic-REP culture is less than about 21% that of the concentration of CD3 agonist in the static-REP culture at least about 120 hours after initiation of the dynamic-REP. [0439] In some aspects, the duration of the static-REP is about 5 days. In some aspects, the duration of the dynamic-REP is about 9 days to about 13 days. In some aspects, the duration of the dynamic-REP is about 9 days. In some aspects, the duration of the dynamic-REP is about 10 days. In some aspects, the duration of the dynamic-REP is about 11 days. In some aspects, the duration of the dynamic-REP is about 12 days. In some aspects, the duration of the dynamic-REP is about 13 days. In some aspects, duration of the static-REP is about 5 days, and the duration of the dynamic-REP is about 9 days. In some aspects, duration of the static-REP is about 5 days, and the duration of the dynamic-REP is about 10 days. In some aspects, duration of the static-REP is about 5 days, and the duration of the dynamic-REP is about 11 days. In some aspects, duration of the static-REP is about 5 days, and the duration of the dynamic-REP is about 12 days. In some aspects, the TILs are cryopreserved following conclusion of the dynamic-REP step.
[0440] In some aspects, the medium of the static-REP further comprises IL-2, e.g., recombinant human IL-2. In some aspects, the medium of the static-REP culture comprises at least about 1000 IU, at least about 1100 IU, at least about 1200 IU, at least about 1300 IU, at least about 1400 IU, at least about 1500 IU, at least about 1600 IU, at least about 1700 IU, at least about 1800 IU, at least about 1900 IU, or at least about 2000 IU IL-2. In some aspects, the medium of the static-REP culture comprises about 1000 IU IL-2. In some aspects, the medium of the static-REP culture comprises about 1100 IU IL-2. In some aspects, the medium of the static-REP culture comprises about 1200 IU IL-2. In some aspects, the medium of the static-REP culture comprises about 1300 IU IL-2. In some aspects, the medium of the static-REP culture comprises about 1400 IU IL-2. In some aspects, the medium of the static-REP culture comprises about 1500 IU IL-2. In some aspects, the medium of the static-REP culture comprises about 1600 IU IL-2. In some aspects, the medium of the static-REP culture comprises about 1700 IU IL-2. In some aspects, the medium of the static-REP culture comprises about 1800 IU IL-2. In some aspects, the medium of the static-REP culture comprises about 1900 IU IL-2. In some aspects, the medium of the static- REP culture comprises about 2000 IU IL-2. [0441] In some aspects, the medium of the static-REP further comprises IL-21, e.g., recombinant human IL-21. In some aspects, the medium of the static-REP culture comprises at least about 5 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about 8 ng/mL, at least about 9 ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least about 12 ng/mL, at least about 13 ng/mL, at least about 14 ng/mL, or at least about 15 ng/mL IL-21. In some aspects, the medium of the static-REP culture comprises about 5 ng/mL IL-21. In some aspects, the medium of the static-REP culture comprises about 6 ng/mL IL-21. In some aspects, the medium of the static-REP culture comprises about 7 ng/mL IL-21. In some aspects, the medium of the static-REP culture comprises about 8 ng/mL IL-21. In some aspects, the medium of the static-REP culture comprises about 9 ng/mL IL-21. In some aspects, the medium of the static-REP culture comprises about 10 ng/mL IL-21. In some aspects, the medium of the static-REP culture comprises about 11 ng/mL IL-21. In some aspects, the medium of the static-REP culture comprises about 12 ng/mL IL-21. In some aspects, the medium of the static-REP culture comprises about 13 ng/mL IL-21. In some aspects, the medium of the static-REP culture comprises about 14 ng/mL IL-21. In some aspects, the medium of the static-REP culture comprises about 15 ng/mL IL-21. [0442] In some aspects, the medium of the static-REP culture comprises IL-15, e.g., recombinant human IL-15. In some aspects, the medium of the static-REP culture comprises at least about 0.1 ng/mL, at least about 0.2 ng/mL, at least about 0.3 ng/mL, at least about 0.4 ng/mL,
at least about 0.5 ng/mL, at least about 0.6 ng/mL, at least about 0.7 ng/mL, at least about 0.8 ng/mL, at least about 0.9 ng/mL, or at least about 1 ng/mL IL-15. In some aspects, the medium of the static-REP culture comprises about 0.1 ng/mL IL-15. In some aspects, the medium of the static- REP culture comprises about 0.2 ng/mL IL-15. In some aspects, the medium of the static-REP culture comprises about 0.3 ng/mL IL-15. In some aspects, the medium of the static-REP culture comprises about 0.4 ng/mL IL-15. In some aspects, the medium of the static-REP culture comprises about 0.5 ng/mL IL-15. In some aspects, the medium of the static-REP culture comprises about 0.6 ng/mL IL-15. In some aspects, the medium of the static-REP culture comprises about 0.7 ng/mL IL-15. In some aspects, the medium of the static-REP culture comprises about 0.8 ng/mL IL-15. In some aspects, the medium of the static-REP culture comprises about 0.9 ng/mL IL-15. In some aspects, the medium of the static-REP culture comprises about 1 ng/mL IL-15. [0443] In some aspects, the medium of the static-REP culture comprises IL-2 and IL-21. In some aspects, the static-REP culture comprises about 1500 IU IL-2 and about 10 ng/mL IL-21. [0444] In some aspects, the medium of the static-REP culture comprises IL-2 and IL-15. In some aspects, the medium of the static-REP culture comprises about 1500 IU IL-2 and about 0.4 ng/mL IL-15. [0445] In some aspects, the medium of the static-REP culture comprises IL-2, IL-21, and IL-15. In some aspects, the medium of the static-REP culture comprises about 1500 IU IL-2, about 10 ng/mL IL-21, and about 0.4 ng/mL IL-15. [0446] In some aspects, the perfused fresh medium of the dynamic-REP further comprises IL-2., e.g., recombinant human IL-2. In some aspects, the perfused fresh medium of the dynamic- REP culture comprises at least about 1000 IU, at least about 1100 IU, at least about 1200 IU, at least about 1300 IU, at least about 1400 IU, at least about 1500 IU, at least about 1600 IU, at least about 1700 IU, at least about 1800 IU, at least about 1900 IU, or at least about 2000 IU IL-2. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 1000 IU IL-2. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 1100 IU IL-2. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 1200 IU IL-2. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 1300 IU IL-2. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 1400 IU IL-2. In some aspects, the perfused fresh medium of the dynamic- REP culture comprises about 1500 IU IL-2. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 1600 IU IL-2. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 1700 IU IL-2. In some aspects, the perfused fresh
medium of the dynamic-REP culture comprises about 1800 IU IL-2. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 1900 IU IL-2. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 2000 IU IL-2. [0447] In some aspects, the perfused fresh medium of the dynamic-REP further comprises IL-21, e.g., recombinant human IL-21. In some aspects, the perfused fresh medium of the dynamic- REP culture comprises at least about 5 ng/mL, at least about 6 ng/mL, at least about 7 ng/mL, at least about 8 ng/mL, at least about 9 ng/mL, at least about 10 ng/mL, at least about 11 ng/mL, at least about 12 ng/mL, at least about 13 ng/mL, at least about 14 ng/mL, or at least about 15 ng/mL IL-21. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 5 ng/mL IL-21. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 6 ng/mL IL-21. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 7 ng/mL IL-21. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 8 ng/mL IL-21. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 9 ng/mL IL-21. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 10 ng/mL IL-21. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 11 ng/mL IL-21. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 12 ng/mL IL-21. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 13 ng/mL IL-21. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 14 ng/mL IL-21. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 15 ng/mL IL-21. [0448] In some aspects, the perfused fresh medium of the dynamic-REP culture comprises IL-15, e.g., recombinant human IL-15. In some aspects, the perfused fresh medium of the dynamic- REP culture comprises at least about 0.1 ng/mL, at least about 0.2 ng/mL, at least about 0.3 ng/mL, at least about 0.4 ng/mL, at least about 0.5 ng/mL, at least about 0.6 ng/mL, at least about 0.7 ng/mL, at least about 0.8 ng/mL, at least about 0.9 ng/mL, or at least about 1 ng/mL IL-15. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 0.1 ng/mL IL-15. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 0.2 ng/mL IL-15. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 0.3 ng/mL IL-15. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 0.4 ng/mL IL-15. In some aspects, the perfused fresh medium of the dynamic- REP culture comprises about 0.5 ng/mL IL-15. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 0.6 ng/mL IL-15. In some aspects, the perfused fresh
medium of the dynamic-REP culture comprises about 0.7 ng/mL IL-15. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 0.8 ng/mL IL-15. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 0.9 ng/mL IL-15. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 1 ng/mL IL-15. [0449] In some aspects, the perfused fresh medium of the dynamic-REP culture comprises IL-2 and IL-21. In some aspects, the static-REP culture comprises about 1500 IU IL-2 and about 10 ng/mL IL-21. [0450] In some aspects, the perfused fresh medium of the dynamic-REP culture comprises IL-2 and IL-15. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 1500 IU IL-2 and about 0.4 ng/mL IL-15. [0451] In some aspects, the perfused fresh medium of the dynamic-REP culture comprises IL-2, IL-21, and IL-15. In some aspects, the perfused fresh medium of the dynamic-REP culture comprises about 1500 IU IL-2, about 10 ng/mL IL-21, and about 0.4 ng/mL IL-15. II.B.7. Two-REP Process [0452] In some aspects, the present disclosure includes a method of culturing TILs ex vivo or in vitro for a therapy to a subject in need thereof comprising: (i) culturing a population of cells comprising TILs in a first expansion medium, wherein the first expansion medium comprises a CD3 agonist (a "first REP step"); and (ii) culturing the TILs from the first REP step in a second expansion medium, wherein the second expansion medium comprises a CD3 agonist (a "second REP step"). In some aspects, the population of cells comprising TILs is a dissociated tumor sample, i.e., a single cell suspension. II.B.7.i. First REP Step [0453] In some aspects, the tumor sample (e.g., the dissociated tumor sample, e.g., the single cell suspension) is placed into a first expansion medium, wherein the first expansion medium comprises a CD3 agonist and APCs at a first ratio (a "first REP step"). In some aspects, the TILs are cultured in the first REP step for less than about 14 days. In some aspects, the TILs are cultured in the first REP step for less than about 13 days. In some aspects, the TILs are cultured in the first REP step for less than about 12 days. In some aspects, the TILs are cultured in the first REP step for less than about 11 days. In some aspects, the TILs are cultured in the first REP step for less than about 10 days. In some aspects, the TILs are cultured in the first REP step for less than about
9 days. In some aspects, the TILs are cultured in the first REP step for less than about 8 days. In some aspects, the TILs are cultured in the first REP step for about 8 days. [0454] In some aspects, the TILs are cultured in the first REP step for about 10 days to about 18 days. In some aspects, the TILs are cultured in the first REP step for about 10 days to about 17 days. In some aspects, the TILs are cultured in the first REP step for about 10 days to about 16 days. In some aspects, the TILs are cultured in the first REP step for about 10 days to about 15 days. In some aspects, the TILs are cultured in the first REP step for about 10 days to about 14 days. In some aspects, the TILs are cultured in the first REP step for about 10 days to about 13 days. In some aspects, the TILs are cultured in the first REP step for about 10 days to about 12 days. In some aspects, the TILs are cultured in the first REP step for about 10 days to about 11 days. In some aspects, the TILs are cultured in the first REP step for about 10 days. In some aspects, the TILs are cultured in the first REP step for about 11 days. In some aspects, the TILs are cultured in the first REP step for about 12 days. In some aspects, the TILs are cultured in the first REP step for about 13 days. In some aspects, the TILs are cultured in the first REP step for about 14 days. In some aspects, the TILs are cultured in the first REP step for about 15 days. In some aspects, the TILs are cultured in the first REP step for about 16 days. In some aspects, the TILs are cultured in the first REP step for about 17 days. In some aspects, the TILs are cultured in the first REP step for about 18 days. [0455] In some aspects, the TILs are cultured in the first REP step for a period of time until a certain number of cells is reached. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 10-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 following culture in the first REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 10-fold following culture in the first REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 15-fold following culture in the first REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 20-fold following culture in the first REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 25-fold following culture in the first REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 30-fold following culture in the first REP step. In some aspects, the number of TILs preset in the dissociated tumor sample is increased by at least about 35-fold following culture in the first REP step. In some aspects, the number of TILs present in the
dissociated tumor sample is increased by at least about 40-fold following culture in the first REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 45-fold following culture in the first REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 50-fold following culture in the first REP step. [0456] In some aspects, the number of TILs following culture in the first REP step is at least about 5 x 107, at least about 1 x 108, at least about 2 x 108, at least about 3 x 108, at least about 4 x 108, at least about 5 x 108, at least about 6 x 108, at least about 7 x 108, at least about 8 x 108, at least about 9 x 108, at least about 1 x 109, at least about 2 x 109, at least about 3 x 109, at least about 4 x 109, at least about 5 x 109, at least about 6 x 109, at least about 7 x 109, at least about 8 x 109, at least about 9 x 109, at least about 1 x 1010, or at least about 2 x 1010 TILs. In some aspects, the number of TILs following culture in the first REP step is at least about 1 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 2 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 3 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 4 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 5 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 6 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 7 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 8 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 9 x 109. In some aspects, the number of TILs following culture in the first REP step is at least about 10 x 109. [0457] In some aspects, the first REP step continues until at least about 1 x 109 TILs are obtained. In some aspects, the first REP step continues until at least about 2 x 109 TILs are obtained. In some aspects, the first REP step continues until at least about 3 x 109 TILs are obtained. In some aspects, the first REP step continues until at least about 4 x 109 TILs are obtained. In some aspects, the first REP step continues until at least about 5 x 109 TILs are obtained. In some aspects, the first REP step continues until at least about 6 x 109 TILs are obtained. In some aspects, the first REP step continues until at least about 7 x 109 TILs are obtained. In some aspects, the first REP step continues until at least about 8 x 109 TILs are obtained. In some aspects, the first REP step continues until at least about 9 x 109 TILs are obtained. In some aspects, the first REP step continues until at least about 10 x 109 TILs are obtained. [0458] In some aspects, the first REP step lasts for less than 8 days, wherein at least about 1 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at
least about 2 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at least about 3 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at least about 4 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at least about 5 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at least about 6 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at least about 7 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at least about 8 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at least about 9 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 8 days, wherein at least about 10 x 109 TILs are obtained. [0459] In some aspects, the first REP step lasts for less than 9 days, wherein at least about 1 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 2 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 3 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 4 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 5 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 6 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 7 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 8 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 9 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 9 days, wherein at least about 10 x 109 TILs are obtained. [0460] In some aspects, the first REP step lasts for less than 10 days, wherein at least about 1 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 2 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 3 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 4 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 5 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 6 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 7 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 8 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at
least about 9 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 10 days, wherein at least about 10 x 109 TILs are obtained. [0461] In some aspects, the first REP step lasts for less than 11 days, wherein at least about 1 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 2 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 3 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 4 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 5 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 6 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 7 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 8 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 9 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 11 days, wherein at least about 10 x 109 TILs are obtained. [0462] In some aspects, the first REP step lasts for less than 12 days, wherein at least about 1 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 12 days, wherein at least about 2 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 12 days, wherein at least about 3 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 12 days, wherein at least about 4 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 12 days, wherein at least about 5 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 12 days, wherein at least about 6 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 12 days, wherein at least about 7 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 12 days, wherein at least about 8 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 12 days, wherein at least about 9 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 12 days, wherein at least about 10 x 109 TILs are obtained. [0463] In some aspects, the first REP step lasts for less than 13 days, wherein at least about 1 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 13 days, wherein at least about 2 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 13 days, wherein at least about 3 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 13 days, wherein at least about 4 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 13 days, wherein at least about 5 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 13 days, wherein at least about 6 x 109 TILs are obtained. In
some aspects, the first REP step lasts for less than 13 days, wherein at least about 7 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 13 days, wherein at least about 8 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 13 days, wherein at least about 9 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 13 days, wherein at least about 10 x 109 TILs are obtained. [0464] In some aspects, the first REP step lasts for less than 14 days, wherein at least about 1 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 14 days, wherein at least about 2 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 14 days, wherein at least about 3 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 14 days, wherein at least about 4 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 14 days, wherein at least about 5 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 14 days, wherein at least about 6 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 14 days, wherein at least about 7 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 14 days, wherein at least about 8 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 14 days, wherein at least about 9 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 14 days, wherein at least about 10 x 109 TILs are obtained. [0465] In some aspects, the first REP step lasts for less than 15 days, wherein at least about 1 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 15 days, wherein at least about 2 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 15 days, wherein at least about 3 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 15 days, wherein at least about 4 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 15 days, wherein at least about 5 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 15 days, wherein at least about 6 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 15 days, wherein at least about 7 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 15 days, wherein at least about 8 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 15 days, wherein at least about 9 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 15 days, wherein at least about 10 x 109 TILs are obtained. [0466] In some aspects, the first REP step lasts for less than 16 days, wherein at least about 1 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 16 days, wherein at least about 2 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 16 days, wherein at least about 3 x 109 TILs are obtained. In some aspects, the first REP step lasts for
less than 16 days, wherein at least about 4 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 16 days, wherein at least about 5 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 16 days, wherein at least about 6 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 16 days, wherein at least about 7 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 16 days, wherein at least about 8 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 16 days, wherein at least about 9 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 16 days, wherein at least about 10 x 109 TILs are obtained. [0467] In some aspects, the first REP step lasts for less than 17 days, wherein at least about 1 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 17 days, wherein at least about 2 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 17 days, wherein at least about 3 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 17 days, wherein at least about 4 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 17 days, wherein at least about 5 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 17 days, wherein at least about 6 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 17 days, wherein at least about 7 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 17 days, wherein at least about 8 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 17 days, wherein at least about 9 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 17 days, wherein at least about 10 x 109 TILs are obtained. [0468] In some aspects, the first REP step lasts for less than 18 days, wherein at least about 1 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 18 days, wherein at least about 2 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 18 days, wherein at least about 3 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 18 days, wherein at least about 4 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 18 days, wherein at least about 5 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 18 days, wherein at least about 6 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 18 days, wherein at least about 7 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 18 days, wherein at least about 8 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 18 days, wherein at least about 9 x 109 TILs are obtained. In some aspects, the first REP step lasts for less than 18 days, wherein at least about 10 x 109 TILs are obtained.
[0469] In some aspects, the first expansion medium comprises a CD3 agonist. In some aspects, the CD3 agonist comprises OKT3. In some aspects, the first expansion medium comprises from about 1 to about 100 ng/mL OKT3. In some aspects, the concentration of OKT3 is at least about 1 ng/ml, at least about 5 ng/ml, at least about 10 ng/ml, at least about 15 ng/ml, at least about 20 ng/ml, at least about 25 ng/ml, at least about 30 ng/ml, at least about 35 ng/ml, at least about 40 ng/ml, at least about 45 ng/ml, at least about 50 ng/ml, at least about 60 ng/ml, at least about 70 ng/ml, at least about 80 ng/ml, at least about 90 ng/ml, or at least about 100 ng/ml. In some aspects, the first expansion medium comprises about 10 ng/ml OKT3. In some aspects, the first expansion medium comprises about 15 ng/ml OKT3. In some aspects, the first expansion medium comprises about 20 ng/ml OKT3. In some aspects, the first expansion medium comprises about 25 ng/ml OKT3. In some aspects, the first expansion medium comprises about 30 ng/ml OKT3. In some aspects, the first expansion medium comprises about 35 ng/ml OKT3. In some aspects, the first expansion medium comprises about 40 ng/ml OKT3. In some aspects, the first expansion medium comprises about 45 ng/ml OKT3. In some aspects, the first expansion medium comprises about 50 ng/ml OKT3. [0470] In some aspects, the cells (e.g., TILs) are passed through a strainer following the initial culture. In some aspects, the cells (e.g., TILs) are passed through an at least about 10 µm, an at least about 15 µm, an at least about 20 µm, an at least about 25 µm, an at least about 30 µm, an at least about 35 µm, an at least about 40 µm, an at least about 45 µm, an at least about 50 µm strainer following the first REP step. In some aspects, the cells (e.g., TILs) are passed through an about 40 µm strainer following the first REP step. [0471] In some aspects, the first REP step is carried out in one or more gas permeable flasks (e.g., G-Rex® flasks). In some aspects, the first REP step is carried out in static G-Rex®. In some aspects, the first REP step is carried out in a stirred tank. In some aspects, the first REP step is carried out in a bioreactor. In some aspects, the first REP step is carried out in a closed system (e.g., using a G-Rex® closed system). [0472] In some aspects, the first REP step further comprises contacting the tumor samples or fragments/cells thereof with 4-1BB ligand.4-1BBL (4-1BB ligand, CD137L) is found on APCs (antigen presenting cells) and binds to 4-1BB (also known as CD137), a type 2 transmembrane glycoprotein receptor belonging to the TNF superfamily, which is expressed on activated T Lymphocytes.4-1BB ligand can be used to activate T cells in vitro. In some aspects, the first REP step comprises contacting the tumor samples or fragments thereof with 4-1BB ligand on about day 3, about day 4, about day 5, about day 6, or about day 7 of the first REP step; wherein first REP
step is performed less than about 14 days. In some aspects, the first REP step comprises contacting the tumor samples or fragments thereof with 4-1BB ligand on about day 3, about day 4, about day 5, about day 6, or about day 7 of the first REP step; wherein first REP step is performed less than about 13 days. In some aspects, the first REP step comprises contacting the tumor samples or fragments thereof with 4-1BB ligand on about day 3, about day 4, about day 5, about day 6, or about day 7 of the first REP step; wherein first REP step is performed less than about 12 days. In some aspects, the first REP step comprises contacting the tumor samples or fragments thereof with 4-1BB ligand on about day 3, about day 4, about day 5, about day 6, or about day 7 of the first REP step; wherein first REP step is performed less than about 11 days. In some aspects, the first REP step comprises contacting the tumor samples or fragments thereof with 4-1BB ligand on about day 3, about day 4, about day 5, about day 6, or about day 7 of the first REP step; wherein first REP step is performed less than about 10 days. In some aspects, the first REP step comprises contacting the tumor samples or fragments thereof with 4-1BB ligand on about day 3, about day 4, about day 5, about day 6, or about day 7 of the first REP step; wherein first REP step is performed less than about 9 days. In some aspects, the first REP step comprises contacting the tumor samples or fragments thereof with 4-1BB ligand on about day 3, about day 4, about day 5, about day 6, or about day 7 of the first REP step; wherein first REP step is performed less than about 8 days. [0473] In some aspects, the APCs are not derived from PBMCs. In some aspects, the APCs of the first REP step comprise one or more cancer cell. In some aspects, the APCs of the first REP step are a cancer cell line. In some aspects, the APCs of the first REP step comprise one or more K562 cell. In some aspects, the APCs of the first REP step comprise one or more artificial antigen presenting cells (aAPCs). In some aspects, the APCs of the first REP step comprise one or more genetically engineered human K562 aAPC. In some aspects, the genetically engineered human K562 aAPC expresses one or more of CD70, CD80, CD86, 41BB ligand, and OX40 ligand. In some aspects, the genetically engineered human K562 aAPC comprises a polycistronic lentiviral vector comprising genes encoding one or more of CD70, CD80, CD86, 41BB ligand, and OX40 ligand. In some aspects, the genetically engineered human K562 aAPC comprises a polycistronic lentiviral vector comprising genes encoding CD70, CD80, CD86, 41BB ligand, and OX40 ligand. [0474] In some aspects, the TILs are cryopreserved following the first REP step. In some aspects, the TILs from the first REP step are inoculated directly into a secondary REP step, e.g., as described herein, following the first REP step.
II.B.7.i. Second REP Step [0475] In some aspects, the TILs are subjected to a second REP step, i.e., a secondary expansion step. In some aspects, the secondary expansion step is carried out in one or more gas permeable flasks (e.g., G-Rex® flasks). In some aspects, the TILs are transitioned to the second REP step without opening the closed system. In some aspects, the secondary expansion is carried out in a bioreactor. In some aspects, the secondary expansion comprises two phases, wherein the first phase is carried out in a first container, and the second phase is carried out in a second container, wherein the first container is not a bioreactor, and wherein the second container is a bioreactor. In some aspects, the TILs from the first REP step are screened for tumor-specific cytolytic activity prior to advancing the TILs to the second REP step. In some aspects, the TILs are screened for expression of one or more biomarkers prior to advancing to the second REP step. In some aspects, the biomarker comprises expression of one or more gene typically expressed by more naïve TILs, e.g., CD8+, CD27+, CD3+, CD95+, CD45RA+, CCR7+, CD62L+, TCF7+, or any combination thereof. In some aspects, the TILs are screened for expression of PD-1 prior to advancing to the second REP step. In some aspects, the TILs from the first REP step are not screened prior to advancing the TILs to the second REP step. In some aspects, all TILs obtained in the first REP step are subjected to the second REP step. In some aspects, the TILs from the first REP step are pooled prior to advancement to the second REP step. [0476] In some aspects, the TILs from the first REP step are placed into a second expansion medium, wherein the second expansion medium comprises a CD3 agonist and APCs at a second ratio (a "second REP step"). In some aspects, the TILs are cultured in the second REP step for less than about 14 days. In some aspects, the TILs are cultured in the second REP step for less than about 13 days. In some aspects, the TILs are cultured in the second REP step for less than about 12 days. In some aspects, the TILs are cultured in the second REP step for less than about 11 days. In some aspects, the TILs are cultured in the second REP step for less than about 10 days. In some aspects, the TILs are cultured in the second REP step for less than about 9 days. In some aspects, the TILs are cultured in the second REP step for less than about 8 days. [0477] In some aspects, the TILs are cultured in the second REP step for about 7 days to about 18 days. In some aspects, the TILs are cultured in the second REP step for about 8 days to about 18 days. In some aspects, the TILs are cultured in the second REP step for about 10 days to about 18 days. In some aspects, the TILs are cultured in the second REP step for about 10 days to about 17 days. In some aspects, the TILs are cultured in the second REP step for about 10 days to
about 16 days. In some aspects, the TILs are cultured in the second REP step for about 10 days to about 15 days. In some aspects, the TILs are cultured in the second REP step for about 10 days to about 14 days. In some aspects, the TILs are cultured in the second REP step for about 10 days to about 13 days. In some aspects, the TILs are cultured in the second REP step for about 10 days to about 12 days. In some aspects, the TILs are cultured in the second REP step for about 10 days to about 11 days. In some aspects, the TILs are cultured in the second REP step for about 8 days to about 12 days. In some aspects, the TILs are cultured in the second REP step for about 8 days to about 11 days. In some aspects, the TILs are cultured in the second REP step for about 8 days to about 10 days. In some aspects, the TILs are cultured in the second REP step for about 12 days. In some aspects, the TILs are cultured in the second REP step for about 11 days. In some aspects, the TILs are cultured in the second REP step for about 10 days. In some aspects, the TILs are cultured in the second REP step for about 9 days. In some aspects, the TILs are cultured in the second REP step for about 8 days. In some aspects, the TILs are cultured in the second REP step for about 7 days. [0478] In some aspects, the TILs are cultured in the second REP step for a period of time until a certain number of cells is reached. In some aspects, the number of TILs present following the first REP step is increased by at least about 10-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 following culture in the second REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 10-fold following culture in the second REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 15-fold following culture in the second REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 20-fold following culture in the second REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 25-fold following culture in the second REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 30-fold following culture in the second REP step. In some aspects, the number of TILs preset in the dissociated tumor sample is increased by at least about 35-fold following culture in the second REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 40-fold following culture in the second REP step. In some aspects, the number of TILs present in the dissociated tumor sample is increased by at least about 45-fold following culture in the second REP step. In some aspects,
the number of TILs present in the dissociated tumor sample is increased by at least about 50-fold following culture in the second REP step. [0479] In some aspects, the number of TILs following culture in the second REP step is at least about 1 x 109, at least about 2 x 109, at least about 3 x 109, at least about 4 x 109, at least about 5 x 109, at least about 6 x 109, at least about 7 x 109, at least about 8 x 109, at least about 9 x 109, at least about 1 x 1010, at least about 2 x 1010, at least about 3 x 1010, at least about 4 x 1010, at least about 5 x 1010, at least about 6 x 1010, at least about 7 x 1010, at least about 8 x 1010, or at least about 9 x 1010 TILs. In some aspects, the number of TILs following culture in the second REP step is at least about 5 x 109. In some aspects, the number of TILs following culture in the second REP step is at least about 1 x 1010. In some aspects, the number of TILs following culture in the second REP step is at least about 2 x 1010. In some aspects, the number of TILs following culture in the second REP step is at least about 3 x 1010. In some aspects, the number of TILs following culture in the second REP step is at least about 4 x 1010. In some aspects, the number of TILs following culture in the second REP step is at least about 5 x 1010. In some aspects, the number of TILs following culture in the second REP step is at least about 6 x 1010. In some aspects, the number of TILs following culture in the second REP step is at least about 7 x 1010. In some aspects, the number of TILs following culture in the second REP step is at least about 8 x 1010. In some aspects, the number of TILs following culture in the second REP step is at least about 9 x 1010. In some aspects, the number of TILs following culture in the second REP step is at least about 10 x 1010. [0480] In some aspects, the second REP step continues until at least about 5 x 109 TILs are obtained. In some aspects, the second REP step continues until at least about 1 x 1010 TILs are obtained. In some aspects, the second REP step continues until at least about 2 x 1010 TILs are obtained. In some aspects, the second REP step continues until at least about 3 x 1010 TILs are obtained. In some aspects, the second REP step continues until at least about 4 x 1010 TILs are obtained. In some aspects, the second REP step continues until at least about 5 x 1010 TILs are obtained. In some aspects, the second REP step continues until at least about 6 x 1010 TILs are obtained. In some aspects, the second REP step continues until at least about 7 x 1010 TILs are obtained. In some aspects, the second REP step continues until at least about 8 x 1010 TILs are obtained. In some aspects, the second REP step continues until at least about 9 x 1010 TILs are obtained. In some aspects, the second REP step continues until at least about 10 x 1010 TILs are obtained. [0481] In some aspects, the second REP step lasts for less than 8 days, wherein at least about 1 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 8 days,
wherein at least about 2 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 8 days, wherein at least about 3 x 100 TILs are obtained. In some aspects, the second REP step lasts for less than 8 days, wherein at least about 4 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 8 days, wherein at least about 5 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 8 days, wherein at least about 6 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 8 days, wherein at least about 7 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 8 days, wherein at least about 8 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 8 days, wherein at least about 9 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 8 days, wherein at least about 10 x 1010 TILs are obtained. [0482] In some aspects, the second REP step lasts for less than 9 days, wherein at least about 1 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 2 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 3 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 4 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 5 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 6 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 7 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 8 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 9 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 9 days, wherein at least about 10 x 1010 TILs are obtained. [0483] In some aspects, the second REP step lasts for less than 10 days, wherein at least about 1 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 10 days, wherein at least about 2 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 10 days, wherein at least about 3 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 10 days, wherein at least about 4 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 10 days, wherein at least about 5 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 10 days, wherein at least about 6 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 10 days, wherein at least about 7 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 10 days, wherein at least about 8 x 1010 TILs are obtained. In some aspects, the second REP step
lasts for less than 10 days, wherein at least about 9 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 10 days, wherein at least about 10 x 1010 TILs are obtained. [0484] In some aspects, the second REP step lasts for less than 11 days, wherein at least about 1 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about 2 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about 3 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about 4 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about 5 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about 6 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about 7 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about 8 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about 9 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 11 days, wherein at least about 10 x 1010 TILs are obtained. [0485] In some aspects, the second REP step lasts for less than 12 days, wherein at least about 1 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 12 days, wherein at least about 2 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 12 days, wherein at least about 3 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 12 days, wherein at least about 4 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 12 days, wherein at least about 5 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 12 days, wherein at least about 6 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 12 days, wherein at least about 7 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 12 days, wherein at least about 8 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 12 days, wherein at least about 9 x 1010 TILs are obtained. In some aspects, the second REP step lasts for less than 12 days, wherein at least about 10 x 1010 TILs are obtained. [0486] In some aspects, the TILs are cultured in the first REP step for 7 to 10 days, and the TILs are cultured in the second REP step for 7 to 10 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. In some aspects, the TILs are cultured in the first REP step for 7 days, and the TILs are cultured in the second REP step for 7 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. In some aspects, the TILs are cultured in the first REP step for 10 days or less, and the TILs are cultured in the second REP step for 10 days or less, wherein the total number of TILs
following culture in the second REP step is at least about 1 x 1010 TILs. In some aspects, the TILs are cultured in the first REP step for 8 days, and the TILs are cultured in the second REP step for 8 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. [0487] In some aspects, the TILs are cultured in the first REP step for 10 to 18 days, and the TILs are cultured in the second REP step for 10 to 11 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. In some aspects, the TILs are cultured in the first REP step for 10 days, and the TILs are cultured in the second REP step for 10 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. In some aspects, the TILs are cultured in the first REP step for 10 days, and the TILs are cultured in the second REP step for 11 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. [0488] In some aspects, the TILs are cultured in the first REP step for 11 days, and the TILs are cultured in the second REP step for 10 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. In some aspects, the TILs are cultured in the first REP step for 11 days, and the TILs are cultured in the second REP step for 11 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. [0489] In some aspects, the TILs are cultured in the first REP step for 12 days, and the TILs are cultured in the second REP step for 10 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. In some aspects, the TILs are cultured in the first REP step for 12 days, and the TILs are cultured in the second REP step for 11 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. [0490] In some aspects, the TILs are cultured in the first REP step for 13 days, and the TILs are cultured in the second REP step for 10 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. In some aspects, the TILs are cultured in the first REP step for 13 days, and the TILs are cultured in the second REP step for 11 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. [0491] In some aspects, the TILs are cultured in the first REP step for 14 days, and the TILs are cultured in the second REP step for 10 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. In some aspects, the TILs are cultured
in the first REP step for 14 days, and the TILs are cultured in the second REP step for 11 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. [0492] In some aspects, the TILs are cultured in the first REP step for 15 days, and the TILs are cultured in the second REP step for 10 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. In some aspects, the TILs are cultured in the first REP step for 15 days, and the TILs are cultured in the second REP step for 11 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. [0493] In some aspects, the TILs are cultured in the first REP step for 16 days, and the TILs are cultured in the second REP step for 10 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. In some aspects, the TILs are cultured in the first REP step for 16 days, and the TILs are cultured in the second REP step for 11 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. [0494] In some aspects, the TILs are cultured in the first REP step for 17 days, and the TILs are cultured in the second REP step for 10 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. In some aspects, the TILs are cultured in the first REP step for 17 days, and the TILs are cultured in the second REP step for 11 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. [0495] In some aspects, the TILs are cultured in the first REP step for 18 days, and the TILs are cultured in the second REP step for 10 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. In some aspects, the TILs are cultured in the first REP step for 18 days, and the TILs are cultured in the second REP step for 11 days, wherein the total number of TILs following culture in the second REP step is at least about 1 x 1010 TILs. [0496] In some aspects, the second expansion medium comprises a CD3 agonist. In some aspects, the CD3 agonist comprises OKT3. In some aspects, the second expansion medium comprises from about 1 to about 100 ng/mL OKT3. In some aspects, the concentration of OKT3 is at least about 1 ng/ml, at least about 5 ng/ml, at least about 10 ng/ml, at least about 15 ng/ml, at least about 20 ng/ml, at least about 25 ng/ml, at least about 30 ng/ml, at least about 35 ng/ml, at least about 40 ng/ml, at least about 45 ng/ml, at least about 50 ng/ml, at least about 55 ng/ml, at
least about 60 ng/ml, at least about 65 ng/ml, at least about 70 ng/ml, at least about 75 ng/ml, at least about 80 ng/ml, at least about 90 ng/ml, or at least about 100 ng/ml. In some aspects, the second expansion medium comprises about 10 ng/ml OKT3. In some aspects, the second expansion medium comprises about 15 ng/ml OKT3. In some aspects, the second expansion medium comprises about 20 ng/ml OKT3. In some aspects, the second expansion medium comprises about 25 ng/ml OKT3. In some aspects, the second expansion medium comprises about 30 ng/ml OKT3. In some aspects, the second expansion medium comprises about 35 ng/ml OKT3. In some aspects, the second expansion medium comprises about 40 ng/ml OKT3. In some aspects, the second expansion medium comprises about 45 ng/ml OKT3. In some aspects, the second expansion medium comprises about 50 ng/ml OKT3. In some aspects, the second expansion medium comprises about 55 ng/ml OKT3. In some aspects, the second expansion medium comprises about 60 ng/ml OKT3. In some aspects, the second expansion medium comprises about 65 ng/ml OKT3. In some aspects, the second expansion medium comprises about 70 ng/ml OKT3. In some aspects, the second expansion medium comprises about 75 ng/ml OKT3. [0497] In some aspects, the cells (e.g., TILs) are passed through a strainer following the second expansion. In some aspects, the cells (e.g., TILs) are passed through an at least about 10 µm, an at least about 15 µm, an at least about 20 µm, an at least about 25 µm, an at least about 30 µm, an at least about 35 µm, an at least about 40 µm, an at least about 45 µm, an at least about 50 µm strainer following the second REP step. In some aspects, the cells (e.g., TILs) are passed through an about 40 µm strainer following the second REP step. [0498] In some aspects, the second REP step is carried out in one or more gas permeable flasks (e.g., G-Rex® flasks). In some aspects, the second REP step is carried out in static G-Rex®. In some aspects, the second REP step is carried out in a stirred tank. In some aspects, the second REP step is carried out in a bioreactor. In some aspects, the second REP step is carried out in a closed system (e.g., using a G-Rex® closed system). [0499] In some aspects, the second REP step comprises two phases. In some aspects the second REP comprises culturing the TILs in a first phase medium followed by culturing the TILs in a second phase medium. In some aspects, the TILs are cultured in the first phase medium for about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 50 hours, about 62 hours, about 74 hours, about 86 hours, or about 98 hours before the TILs are cultured in the second phase medium. In some aspects, the TILs are cultured in the first phase medium for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, or about 6 days before the TILs are cultured in the second phase medium. In some aspects, the TILs are cultured in the first phase medium for about
1 day before the TILs are cultured in the second phase medium. In some aspects, the TILs are cultured in the first phase medium for about 2 days before the TILs are cultured in the second phase medium. In some aspects, the TILs are cultured in the first phase medium for about 3 days before the TILs are cultured in the second phase medium. In some aspects, the TILs are cultured in the first phase medium for about 4 days before the TILs are cultured in the second phase medium. In some aspects, the TILs are cultured in the first phase medium for about 5 days before the TILs are cultured in the second phase medium. [0500] In some aspects, the first phase medium comprises culturing the TILs in a metabolic reprogramming medium (MRM) disclosed herein. In some aspects, the first phase medium comprises about 45 mM potassium ion. In some aspects, the first phase medium comprises about 50 mM potassium ion. In some aspects, the first phase medium comprises about 55 mM potassium ion. In some aspects, the first phase medium comprises about 60 mM potassium ion. In some aspects, the first phase medium comprises about 65 mM potassium ion. In some aspects, the first phase medium comprises about 70 mM potassium ion. In some aspects, the first phase medium comprises about 75 mM potassium ion. In some aspects, the first phase medium comprises about 80 mM potassium ion. In some aspects, the first phase medium comprises about 85 mM potassium ion. In some aspects, the first phase medium comprises about 90 mM potassium ion. [0501] In some aspects, the contents of the first phase medium culture are transferred to a second phase medium, wherein the first phase medium is diluted by fresh medium to arrive at the second phase medium. In some aspects, the first phase medium is diluted by the fresh medium at a ratio of 1:1, respectively, i.e., a 50% solution. In some aspects, the first phase medium is diluted by the fresh medium at a ratio of 1:2, respectively, i.e., a 33% solution. In some aspects, the first phase medium is diluted by the fresh medium at a ratio of 1:3, respectively, i.e., a 25% solution. In some aspects, the first phase medium is diluted by the fresh medium at a ratio of 3:1, respectively, i.e., a 75% solution. In some aspects, the fresh medium does not comprise greater than about 5% potassium ion. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 75% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 70% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 65% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 60% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 55% MRM in the second phase medium. In some aspects, the
MRM of the first phase medium culture is diluted to a final concentration of about 50% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 45% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 40% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 35% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 30% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 25% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 20% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 15% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 10% MRM in the second phase medium. In some aspects, the MRM of the first phase medium culture is diluted to a final concentration of about 5% MRM in the second phase medium. In some aspects, culturing the TILs in a medium comprising less MRM (i.e., in the second phase medium) allows the TILs to recover prior to administration to a subject. [0502] In some aspects, the fresh medium does not comprise a CD3 agonist. In some aspects, the fresh medium does not comprise additional antigen-presenting cells (e.g., irradiated PBMCs). As a result, the number of viable antigen-presenting cells present in the first phase medium (i.e., at day 0 of the second REP step) will decrease throughout the second REP step. [0503] In some aspects, the duration of the first phase of the second REP step is about 1 day. In some aspects, the duration of the first phase of the second REP step is about 2 days. In some aspects, the duration of the first phase of the second REP step is about 3 days. In some aspects, the duration of the first phase of the second REP step is about 4 days. In some aspects, the duration of the first phase of the second REP step is about 5 days. [0504] In some aspects, the duration of the second phase of the second REP step is about 3 days to about 13 days. In some aspects, the duration of the second phase of the second REP step is about 4 days to about 13 days. In some aspects, the duration of the second phase of the second REP step is about 5 days to about 13 days. In some aspects, the duration of the second phase of the second REP step is about 6 days to about 13 days. In some aspects, the duration of the second phase of the second REP step is about 7 days to about 13 days. In some aspects, the duration of the second phase of the second REP step is about 9 days to about 13 days. In some aspects, the duration
of the second phase of the second REP step is about 5 days. In some aspects, the duration of the second phase of the second REP step is about 6 days. In some aspects, the duration of the second phase of the second REP step is about 7 days. In some aspects, the duration of the second phase of the second REP step is about 8 days. In some aspects, the duration of the second phase of the second REP step is about 9 days. In some aspects, the duration of the second phase of the second REP step is about 10 days. In some aspects, the duration of the second phase of the second REP step is about 11 days. In some aspects, the duration of the second phase of the second REP step is about 12 days. In some aspects, the duration of the second phase of the second REP step is about 13 days. [0505] In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 7 to about 13 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 7 to about 12 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 7 to about 11 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 7 to about 10 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 7 to about 9 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 8 to about 12 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 8 to about 11 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 8 to about 10 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 9 to about 12 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 9 to about 11 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 9 to about 10 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 10 to about 13 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 10 to about 12 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 10 to about 11 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 8 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the
second REP step is about 9 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 10 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 11 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 12 days. In some aspects, the total duration of the first phase of the second REP step and the second phase of the second REP step is about 13 days. [0506] In some aspects, duration of the first phase of the second REP step is about 3 days, and the duration of the second phase of the second REP step is about 7 days. In some aspects, duration of the first phase of the second REP step is about 3 days, and the duration of the second phase of the second REP step is about 8 days. In some aspects, duration of the first phase of the second REP step is about 3 days, and the duration of the second phase of the second REP step is about 9 days. In some aspects, duration of the first phase of the second REP step is about 2 days, and the duration of the second phase of the second REP step is about 8 days. In some aspects, duration of the first phase of the second REP step is about 2 days, and the duration of the second phase of the second REP step is about 9 days. In some aspects, duration of the first phase of the second REP step is about 2 days, and the duration of the second phase of the second REP step is about 10 days. In some aspects, duration of the first phase of the second REP step is about 2 days, and the duration of the second phase of the second REP step is about 11 days. In some aspects, duration of the first phase of the second REP step is about 4 days, and the duration of the second phase of the second REP step is about 6 days. In some aspects, duration of the first phase of the second REP step is about 4 days, and the duration of the second phase of the second REP step is about 7 days. In some aspects, duration of the first phase of the second REP step is about 4 days, and the duration of the second phase of the second REP step is about 8 days. In some aspects, duration of the first phase of the second REP step is about 4 days, and the duration of the second phase of the second REP step is about 9 days. In some aspects, duration of the first phase of the second REP step is about 5 days, and the duration of the second phase of the second REP step is about 5 days. In some aspects, duration of the first phase of the second REP step is about 5 days, and the duration of the second phase of the second REP step is about 6 days. In some aspects, duration of the first phase of the second REP step is about 5 days, and the duration of the second phase of the second REP step is about 7 days. In some aspects, duration of the first phase of the second REP step is about 5 days, and the duration of the second phase of the second REP step is about 8 days. In some aspects, duration of the first phase of the second REP step is about 5 days, and the duration of the second phase of the second REP step is about 9 days. In some aspects,
duration of the first phase of the second REP step is about 5 days, and the duration of the second phase of the second REP step is about 10 days. In some aspects, duration of the first phase of the second REP step is about 5 days, and the duration of the second phase of the second REP step is about 11 days. In some aspects, duration of the first phase of the second REP step is about 5 days, and the duration of the second phase of the second REP step is about 12 days. In some aspects, the TILs are cryopreserved following conclusion of the second phase of the second REP step. [0507] In some aspects, the duration of the first REP plus the duration of the second REP is about 18 days. In some aspects, the duration of the first REP plus the duration of the second REP is about 19 days. In some aspects, the duration of the first REP plus the duration of the second REP is about 20 days. In some aspects, the duration of the first REP plus the duration of the second REP is about 21 days. In some aspects, the duration of the first REP plus the duration of the second REP is about 22 days. [0508] In some aspects, the TILs are cryopreserved following the second REP step. In some aspects, the TILs from the second REP step are administered directly to a subject. [0509] In some aspects, the duration of the first REP step is about 8 days, and the duration of the second REP step is about 8 days. In some aspects, the duration of the first REP step is about 9 days, and the duration of the second REP step is about 9 days. In some aspects, the duration of the first REP step is about 10 days, and the duration of the second REP step is about 10 days. [0510] In some aspects, the duration of the first REP step is about 10-18 days, and the duration of the second REP step is about 10-18 days. In some aspects, the duration of the first REP step is about 10-18 days, and the duration of the second REP step is about 10-17 days. In some aspects, the duration of the first REP step is about 10-18 days, and the duration of the second REP step is about 10-16 days. In some aspects, the duration of the first REP step is about 10-18 days, and the duration of the second REP step is about 10-15 days. In some aspects, the duration of the first REP step is about 10-18 days, and the duration of the second REP step is about 10-14 days. In some aspects, the duration of the first REP step is about 10-18 days, and the duration of the second REP step is about 10-13 days. In some aspects, the duration of the first REP step is about 10-18 days, and the duration of the second REP step is about 10-12 days. In some aspects, the duration of the first REP step is about 10-18 days, and the duration of the second REP step is about 10-11 days. In some aspects, the duration of the first REP step is about 10-18 days, and the duration of the second REP step is about 10 days. In some aspects, the duration of the first REP step is about 10-18 days, and the duration of the second REP step is about 11 days.
[0511] In some aspects, the duration of the first REP step is about 10 days, and the duration of the second REP step is about 10 days. In some aspects, the duration of the first REP step is about 10 days, and the duration of the second REP step is about 11 days. [0512] In some aspects, the duration of the first REP step is about 11 days, and the duration of the second REP step is about 10 days. In some aspects, the duration of the first REP step is about 11 days, and the duration of the second REP step is about 11 days. [0513] In some aspects, the duration of the first REP step is about 12 days, and the duration of the second REP step is about 10 days. In some aspects, the duration of the first REP step is about 12 days, and the duration of the second REP step is about 11 days. [0514] In some aspects, the duration of the first REP step is about 13 days, and the duration of the second REP step is about 10 days. In some aspects, the duration of the first REP step is about 13 days, and the duration of the second REP step is about 11 days. [0515] In some aspects, the duration of the first REP step is about 14 days, and the duration of the second REP step is about 10 days. In some aspects, the duration of the first REP step is about 14 days, and the duration of the second REP step is about 11 days. [0516] In some aspects, the duration of the first REP step is about 15 days, and the duration of the second REP step is about 10 days. In some aspects, the duration of the first REP step is about 15 days, and the duration of the second REP step is about 11 days. [0517] In some aspects, the duration of the first REP step is about 16 days, and the duration of the second REP step is about 10 days. In some aspects, the duration of the first REP step is about 16 days, and the duration of the second REP step is about 11 days. [0518] In some aspects, the duration of the first REP step is about 17 days, and the duration of the second REP step is about 10 days. In some aspects, the duration of the first REP step is about 17 days, and the duration of the second REP step is about 11 days. [0519] In some aspects, the duration of the first REP step is about 18 days, and the duration of the second REP step is about 10 days. In some aspects, the duration of the first REP step is about 18 days, and the duration of the second REP step is about 11 days. II.B.8. Harvest and Cryopreservation [0520] In some aspects, the expanded immune cells, e.g., T cells, NK cells, and/or TILs are harvested. The immune cells, e.g., T cells, NK cells, and/or TILs, can be harvested using any method, including by centrifugation. In some aspects, the immune cells, e.g., T cells, NK cells,
and/or TILs, are harvested using an automated system. Cell harvesters and/or cell processing systems are commercially available from a variety of sources, and any cell-based harvester can be used in the methods disclosed herein. In some aspects, the cell harvester and/or cell processing systems is a membrane-based cell harvester. In some aspects, the cell harvesting is conducted using a cell processing system, e.g., the LOVO system (Fresenius Kabi). In some aspects, the cell harvester and/or cell processing system can perform cell separation, washing, fluid-exchange, concentration, and/or other cell processing steps in a closed, sterile system. [0521] In some aspects, the harvest is performed from a closed system bioreactor. In some aspects, a closed system is employed for the cell expansion. In some aspects, a single bioreactor is employed. In some aspects, the closed system bioreactor is a single bioreactor. Examples of methods of expanding TILs ex vivo in open and closed systems can be found, for example, in US Patent No.10,166,257, which is incorporated by reference herein in its entirety. [0522] In some aspects, the expanded immune cells, e.g., T cells, NK cells, and/or TILs, are cryopreserved. The immune cells, e.g., T cells, NK cells, and/or TILs, can be cryopreserved using any methods. Various methods of cryopreserving mammalian cells, including TILs, have been described, e.g., by (i) General Protocol for the Cryopreservation of Mammalian Cells, UNC (2007), available at unclineberger.org/tissueculture/protocols/general-protocol-for-the- cryopreservation-of-mammalian-cells/; and (ii) Clarke et al., Improved post-thaw recovery of peripheral blood stem/progenitor cells using a novel intracellular-like cryopreservation solution, Cytotherapy 2009-6-6, available at sigmaaldrich.com/catalog/papers/19499402; each of which is incorporated by reference herein in its entirety. II.C. Metabolic Reprogramming Media [0523] Some aspects of the present disclosure are directed to methods of culturing immune cells, e.g., T cells, NK cells, and/or TILs, comprising contacting immune cells with programmable cell-signaling scaffolds (PCS) in a culture condition (e.g., medium), wherein the culture condition (e.g., certain ion concentrations, tonicity of the media, cytokines, and/or any combination thereof) is capable of reducing, limiting or preventing the differentiation of the immune cells, e.g., T cells, NK cells, and/or TILs, thereby affecting or improving their use in cell therapy, e.g., adoptive cell therapy. In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, are contacted with PCS and cultured in a metabolic reprogramming media (MRM) disclosed herein. In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, contacted with PCS and cultured in MRM have a higher proportion of stem-like cells as compared to cells cultured using conventional
methods, e.g., in a medium having less than 5 mM potassium ion and not comprising PCS. In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, contacted with PCS and cultured in MRM have a higher proportion of effector-like cells as compared to cells cultured using conventional methods, e.g., in a medium having less than 5 mM potassium ion. In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, contacted with PCS and cultured in MRM have a higher proportion of both stem-like and effector-like cells as compared to cells cultured using conventional methods, e.g., in a medium having less than 5 mM potassium ion. In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, contacted with PCS and cultured in MRM have a higher proliferative potential as compared to cells cultured using conventional methods, e.g., in a medium having less than 5 mM potassium ion. [0524] In some aspects, the method is capable of preserving a stem-like phenotype (e.g., minimal differentiation) of the cultured cells. In some aspects, the method is capable of preserving a stem-like phenotype (e.g., minimal differentiation) of the cultured T cells. In some aspects, the cultured cells have more stem-like phenotypes (e.g., less differentiated) than cells grown in a medium having a lower potassium concentration. In some aspects, the medium further comprises interleukin (IL)-2, IL-21, IL-7, IL-15, or any combination thereof. In some aspects, the medium further comprises sodium ion (e.g., NaCl), calcium ion, glucose, or any combination thereof. [0525] In some aspects, a population of immune cells, e.g., T cells, NK cells, and/or TILs, cultured using the methods disclosed herein, exhibits an increased number of stem-like cells relative to a population of cells cultured using conventional methods, e.g., in a medium having less than 5 mM potassium ion. In some aspects, a population of immune cells, e.g., T cells, NK cells, and/or TILs, cultured using the methods disclosed herein, exhibits an increased number of stem- like T cells relative to a population of immune cells, e.g., T cells, NK cells, and/or TILs, cultured using conventional methods, e.g., in a medium having less than 5 mM potassium ion. In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, exhibit increased expression of markers characteristic of stem-like cells relative to the starting population of immune cells (i.e., prior to the culturing). In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, exhibit increased expression of markers characteristic of stem-like cells relative to the starting population of T cells (i.e., prior to the culturing). In some aspects, the starting population of immune cells comprises immune cells, e.g., T cells, NK cells, and/or TILs, obtained from a subject. In some aspects, the starting population of immune cells comprises T cells obtained from a human subject. In some aspects, the starting population of immune T cells comprises TN cells, TSCM cells, TCM cells, TEM cells, or any combination thereof. In some aspects, the starting population of
immune cells comprises T cells prior to transfection/modification with a construct encoding a ligand binding protein as described herein. [0526] Increased cell multipotency can be measured using any methods known in the art. In some aspects, cell stemness is measured by antibody staining followed by gated flow cytometry. In some aspects, the cell stemness is measured by autophagy flux. In some aspects, the cell stemness is measured by glucose uptake. In some aspects, the cell stemness is measured by fatty acid uptake. In some aspects, the cell stemness is measured by mitochondrial biomass. In some aspects, the cell stemness is measured by RNA quantification/expression analysis (e.g., microarray, qPCR (taqman), RNA-Seq., single-cell RNA-Seq., or any combinations thereof). In some aspects, the cell stemness is measured by transcripts that are linked to a metabolism assay (e.g., a seahorse metabolism assay, analysis of extracellular acidification rate (ECAR); analysis of oxygen consumption rate (OCR); analysis of spare respiratory capacity; and/or analysis of mitochondrial membrane potential). In some aspects, stemness is measured using one or more in vivo or in vitro functional assays (e.g., assaying cell persistence, antitumor capacity, antitumor clearance, viral clearance, multipotency, cytokine release, cell killing, or any combination thereof). [0527] In some aspects, the differentiation status of the immune cells, e.g., T cells, NK cells, and/or TILs, is characterized by increased numbers of cells expressing markers typical of less differentiated cells. In some aspects, the differentiation status of the T cells is characterized by increased numbers of cells expressing markers typical of less differentiated T cells. In some aspects, an increase in the number of stem-like cells is characterized by increased numbers of T cells or TILs expressing markers typical of TN and/or TSCM cells. In some aspects, an increase in the number of stem-like T cells or TILs is characterized by increased numbers of cells expressing markers typical of TSCM cells. In some aspects, the T cell or TIL population exhibits an increased number of cells that express CD45RA. In some aspects, the T cell or TIL population exhibits an increased number of cells that express CCR7. In some aspects, the T cell or TIL population exhibits an increased number of cells that express CD62L. In some aspects, the T cell or TIL population exhibits an increased number of cells that express CD28. In some aspects, the Tcell population exhibits an increased number of cells that express CD95. In some aspects, the cells are CD45ROlow. In some aspects, the cells do not express CD45RO. In some aspects, the cell population exhibits an increased number of cells that are CD45RA+, CCR7+, and CD62L+. In some aspects, the cell population exhibits an increased number of cells that are CD95+, CD45RA+, CCR7+, and CD62L+. In some aspects, the cell population exhibits an increased number of cells that express TCF7. In some aspects, the T cell or TIL population exhibits an increased number of cells that are CD45RA+,
CCR7+, CD62L+, and TCF7+. In some aspects, the T cell or TIL population exhibits an increased number of cells that are CD95+, CD45RA+, CCR7+, CD62L+, and TCF7+. In some aspects, the T cell or TIL population exhibits an increased number of cells that are CD3+, CD45RA+, CCR7+, CD62L+, and TCF7+. In some aspects, the T cellpopulation exhibits an increased number of cells that are CD3+, CD95+, CD45RA+, CCR7+, CD62L+, and TCF7+. In some aspects, the cells express CD27. In some aspects, the T cell or TIL population exhibits an increased number of cells that are CD27+, CD3+, CD45RA+, CCR7+, CD62L+, and TCF7+. In some aspects, the T cell or TIL population exhibits an increased number of cells that are CD27+, CD3+, CD95+, CD45RA+, CCR7+, CD62L+, and TCF7+. In some aspects, the T cell or TIL population exhibits an increased number of cells that are CD39- and CD69-. In some aspects the T cell or TIL population exhibits an increased number of cells that are TCF7+ and CD39-. In some aspects, the cell population exhibits an increased number of TSCM cells. In some aspects, the cell population exhibits an increased number of TN cells. In some aspects, the cell population exhibits an increased number of TSCM and TN cells. In some aspects, the cell population exhibits an increased number of stem-like T cells. In some aspects the T cells or TILs are CD4+ cells; in some aspects the T cells or TILs are CD8+ cells. [0528] In some aspects, the number of stem-like cells in the culture is increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, relative to the number of stem-like cells prior to culture with MRM. In some aspects, the number of stem-like cells in the culture is increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5- fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, or at least about 20-fold, relative to the number of stem-like cells prior to culture with MRM. [0529] In some aspects, following culture of T cells or TILs according to the methods disclosed herein, stem-like T cells or TILs constitute at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, or at least about 15% of the total number of CD8+ T cells or TILs in the culture. In some aspects, following culture of T cells or TILs according to the methods disclosed herein, stem-like T cells or TILs constitute at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, or at least about 15% of the total number of CD4+ T cells or TILs in the culture.
[0530] In some aspects, following culture of T cells or TILs according to the methods disclosed herein, stem-like T cells or TILs constitute at least about 10% to at least about 70% of the total number of T cells or TILs in the culture. In some aspects, following culture of T cells or TILs according to the methods disclosed herein, stem-like T cells or TILs constitute at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% of the total number of CD8+ T cells or TILs in the culture. In some aspects, following culture of T cells or TILs according to the methods disclosed herein, stem-like T cells or TILs constitute at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% of the total number of CD4+ T cells or TILs in the culture. [0531] In some aspects, following culture of T cells or TILs according to the methods disclosed herein, at least about 10% to at least about 40% of the total number of T cells or TILs in the culture are CD39-/CD69- T cells. In some aspects, following culture of T cells or TILs according to the methods disclosed herein, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, or at least about 40% of the total number of T cells or TILs in the culture are CD39-/CD69- T cells. [0532] In some aspects, following culture of T cells or TILs according to the methods disclosed herein, at least about 10% to at least about 70% of the total number of T cells or TILs in the culture are CD39-/TCF7+ T cells. In some aspects, following culture of T cells or TILs according to the methods disclosed herein, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, or at least about 40% of the total number of T cells or TILs in the culture are CD39-/TCF7+ T cells. In some aspects the T cells or TILs are CD4+ T cells. In some aspects the T cells or TILs are CD8+ T cells. [0533] In some aspects, engineered immune cells (e.g., T cells and/or NK cells) of the present disclosure, cultured according to the methods disclosed herein, exhibit increased transduction efficiency. In some aspects, the engineered T cells cultured according to the methods disclosed herein, exhibit increased transduction efficiency. In some aspects, a greater percentage of cells express a target transgene, e.g., encoding a ligand binding protein, following transduction, wherein the cells are cultured according to the methods disclosed herein as compared to cells similarly transduced and cultured using conventional methods, (e.g., in media containing less than 5 mM K+). In certain aspects, a greater percentage of cells cultured according to the methods disclosed herein express a ligand binding protein following lentiviral transduction of the cells, as compared to similarly transduced cells cultured using conventional methods., e.g., in media
containing less than 5 mM K+. In some aspects, transduction efficiency is increased at least about 1.5-fold relative to similarly transduced cells cultured using conventional methods., e.g., in media containing less than 5 mM K+. In some aspects, transduction efficiency is increase at least about 2-fold relative to similarly transduced cells cultured using conventional methods., e.g., in media containing less than 5 mM K+. [0534] In some aspects, the immune cells, e.g., T cells and/or NK cells, are transduced before culturing according to the methods disclosed herein. In some aspects, the immune cells, e.g., T cells and/or NK cells, are transduced after culturing according to the methods disclosed herein. In some aspects, the immune cells, e.g., T cells and/or NK cells, are cultured according to the methods disclosed herein, e.g., by contacting the immune cells with an APC-MS in a medium comprising at least 5 mM potassium ion, prior to, during, and after transduction. [0535] In certain aspects, the immune cells are transduced using a viral vector. In some aspects, the vector comprises a lentiviral vector, adenoviral vector, adeno-associated viral vector, vaccinia vector, herpes simplex viral vector, and Epstein-Barr viral vector. In some aspects, the viral vector comprises a retrovirus. In some aspects, the viral vector comprises a lentivirus. In some aspects, the viral vector comprises an AAV. [0536] In some aspects, the immune cells are transduced using a non-viral method. In some aspects, the non-viral method includes the use of a transposon. In some aspects, use of a non-viral method of delivery permits reprogramming of immune cells, e.g., T cells and/or NK cells, and direct infusion of the cells into the subject. In some aspects, the polynucleotide can be inserted into the genome of a target cell (e.g., a T cell) or a host cell (e.g., a cell for recombinant expression of the encoded proteins) by using CRISPR/Cas systems and genome edition alternatives such as zinc- finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases (MNs). [0537] In some aspects, upon adoptive transfer of the immune cells, e.g., T cells, NK cells, and/or TILs, optionally expressing a ligand binding protein, cultured according to the methods disclosed herein, the transferred cells exhibit decreased cell exhaustion, as compared to cells cultured using conventional methods, e.g., in media containing less than 5 mM K+. In some aspects, upon adoptive transfer of the immune cells, optionally expressing a ligand binding protein, cultured according to the methods disclosed herein, the transferred immune cells exhibit decreased cell exhaustion, as compared to immune cells cultured using conventional methods, e.g., in media containing less than 5 mM K+. In some aspects, upon adoptive transfer of the cells cultured according to the methods disclosed herein, the transferred cells persist for a longer period of time
in vivo, as compared to cells cultured using conventional methods, e.g., in media containing less than 5 mM K+. In some aspects, the transferred cells, e.g., immune cells and/or NK cells, have a greater in vivo efficacy, e.g., tumor-killing activity, as compared to cells cultured using conventional methods, e.g., in media containing less than 5 mM K+. In some aspects, a lower dose of the cells cultured according to the methods disclosed herein is needed to elicit a response, e.g., decreased tumor volume, in a subject as compared to cells cultured using conventional methods, e.g., in media containing less than 5 mM K+. [0538] In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, are cultured according to the methods disclosed herein, e.g., by contacting the immune cells with PCS in a medium comprising at least 5 mM potassium ion, immediately upon isolation from a subject. In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, are cultured according to the methods disclosed herein during expansion of the cells. In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, are cultured according to the methods disclosed herein during engineering of the cells, e.g., during transduction with a construct encoding a transgene, e.g., a ligand binding protein. In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, are cultured according to the methods disclosed herein following engineering of the cells, e.g., following transduction with a construct encoding a transgene., e.g., a ligand binding protein. In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, are cultured according to the methods disclosed herein throughout expansion and engineering. In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, are cultured according to the methods disclosed herein throughout viral genetic engineering. In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, are cultured according to the methods disclosed herein throughout non-viral genetic engineering. In some aspects, the immune cells, e.g., T cells and/or NK cells, are cultured according to the methods disclosed herein during introduction of ligand binding proteins to the immune cell (e.g., T cells and/or NK cells) to allow for tumor specific targeting (e.g., a CAR, TCR, or a TCR mimic). In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, are cultured according to the methods disclosed herein throughout introduction of one or more endogenous genes that improve immune cell function. In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, are cultured according to the methods disclosed herein throughout introduction of one or more synthetic genes that improve cell function. [0539] In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, are cultured according to the methods disclosed herein, e.g., by contacting the immune cells with PCS in a medium comprising at least 5 mM potassium ion, e.g., from the time the immune cells, e.g., T
cells, NK cells, and/or TILs, are isolated from a subject, through growing, expansion, optional engineering, and until administration into a subject in need of adoptive cell therapy. In some aspects, the T cells are cultured according to the methods disclosed herein, e.g., by contacting the immune cells with PCS in a medium comprising at least 5 mM potassium ion, for the entirety of ex vivo culture, e.g., from the time the T cells are isolated from a subject, through growing, expansion, engineering, and until administration into a subject in need of adoptive cell therapy. In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, are cultured according to the methods disclosed herein, e.g., by contacting the immune cells with PCS in a medium comprising at least 5 mM potassium ion, for the duration of expansion. In some aspects, the immune cells, e.g., T cells and/or NK cells, are cultured according to the methods disclosed herein, e.g., by contacting the immune cells with PCS in a medium comprising at least 5 mM potassium ion, until the total number of viable immune cells, e.g., T cells, NK cells, and/or TILs, is at least about 104, at least about 5 x 104, at least about 105, at least about 5 x 105, at least about 106, or at least about 5 x 106, at least about 1 x 107, at least about 5 x 107, at least about 1 x 108, at least about 5 x 108, at least about 1 x 109, at least about 5 x 109, at least about 1 x 1010, at least about 5 x 1010, at least about 1 x 1011, at least about 5 x 1011, at least about 1 x 1012, or at least about 5 x 1012 total cells. In some aspects, the T cells are cultured according to the methods disclosed herein until the total number of viable T cells is at least about 104, at least about 5 x 104, at least about 105, at least about 5 x 105, at least about 106, or at least about 5 x 106, at least about 1 x 107, at least about 5 x 107, at least about 1 x 108, at least about 5 x 108, at least about 1 x 109, at least about 5 x 109, at least about 1 x 1010, at least about 5 x 1010, at least about 1 x 1011, at least about 5 x 1011, at least about 1 x 1012, or at least about 5 x 1012 total T cells. [0540] In some aspects, the medium further comprises a cell expansion agent. As used herein, a "cell expansion agent" refers to an agent, e.g., small molecule, polypeptide, or any combination thereof, that promotes the in vitro and/or ex vivo growth and proliferation of cultured cells, e.g., immune cells (e.g., T cells, NK cells, and/or TILs). In some aspects, the cell expansion agent comprises a PI3K inhibitor. In some aspects, the medium further comprises an AKT inhibitor. In some aspects, the medium further comprises a PI3K inhibitor and an AKT inhibitor. In some aspects, the PI3K inhibitor comprises LY294002. In some aspects, the PI3K inhibitor comprises IC87114. In some aspects, the PI3K inhibitor comprises idelalisib (see, e.g., Peterson et al., Blood Adv. 2(3):210-23 (2018)). In some aspects, the medium further comprises a GSK3B inhibitor. In some aspects, the GSK3B inhibitor comprises TWS119. In some aspects, the medium further comprises an ACLY inhibitor. In some aspects, the ACLY inhibitor comprises potassium
hydroxycitrate tribasic monohydrate. In some aspects, the PI3K inhibitor comprises hydroxyl citrate. In some aspects, the PI3K inhibitor comprises pictilisib. In some aspects, the PI3K inhibitor comprises CAL-101. In some aspects, the AKT inhibitor comprises MK2206, A443654, or AKTi- VIII (CAS 612847-09-3). In some aspects, the cell expansion agent is linked to or associated with the PCS. [0541] In some aspects, the metabolic reprogramming media comprises a mitochondrial fuel. In some aspects, the metabolic reprogramming media comprises O-Acetyl-L-carnitine hydrochloride. In some aspects, the metabolic reprogramming media comprises at least about 0.1 mM, at least about 0.5 mM, at least about 1.0 mM, at least about 5 mM, or at least about 10 mM O-Acetyl-L-carnitine hydrochloride. In some aspects, the metabolic reprogramming media comprises at least about 1.0 mM O-Acetyl-L-carnitine hydrochloride. In some aspects, the mitochondrial fuel is linked to or associated with the PCS. [0542] In some aspects, the metabolic reprogramming media further comprises one or more of (i) one or more cell expansion agents, (ii) sodium ion (e.g., NaCl), (iii) one or more saccharides, (iv) calcium ion, and (v) one or more cytokines. II.C.1. Potassium [0543] In some aspects, an expansion medium disclosed herein comprise potassium ion. In some aspects, an expansion medium disclosed herein comprise more than 4 mM potassium ion. In some aspects, the concentration of potassium ion is at least about 30 mM to at least about 100 mM. In some aspects, the concentration of potassium ion is 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, at least about 80 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 concentration of potassium ion is at least about 50 mM. In some aspects, the concentration of potassium ion is about 40 mM. In some aspects, the concentration of potassium ion is about 45 mM. In some aspects, the concentration of potassium ion is about 50 mM. [0544] In some aspects, the concentration of potassium ion is 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, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, or at least about 100 mM, at least about 105 mM, at least about 110 mM, at least about 115 mM, at least about 120 mM. In some aspects, the concentration of potassium ion is about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100
mM, about 105 mM, about 110 mM, about 115 mM, about 120 mM. In some aspects, the concentration of potassium ion is about 55 mM. In some aspects, the concentration of potassium ion is about 60 mM. In some aspects, the concentration of potassium ion is about 65 mM. In some aspects, the concentration of potassium ion is about 70 mM. In some aspects the concentration of potassium ion is about 40 mM to about 90 mM. [0545] In some aspects, the concentration of potassium ion is about 40 mM to about 90 mM. In some aspects, the concentration of potassium ion is 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 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. [0546] In some aspects, the concentration of potassium ion is about 50 mM to about 100 mM. In some aspects, the concentration of potassium ion is 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. [0547] In some aspects, the concentration of potassium ion is about 55 mM to about 100 mM. In some aspects, the concentration of potassium ion is 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. [0548] In some aspects, the concentration of potassium ion is about 60 mM to about 100 mM. In some aspects, the concentration of potassium ion is 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. [0549] In some aspects, the concentration of potassium ion is about 65 mM to about 100 mM. In some aspects, the concentration of potassium ion is 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.
[0550] In some aspects, the concentration of potassium ion is about 70 mM to about 100 mM. In some aspects, the concentration of potassium ion is 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. [0551] In some aspects, the concentration of potassium ion is about 75 mM to about 100 mM. In some aspects, the concentration of potassium ion is 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. [0552] In some aspects, the concentration of potassium ion is about 80 mM to about 100 mM. In some aspects, the concentration of potassium ion is 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. [0553] In some aspects, the concentration of potassium ion is about 85 mM to about 100 mM. In some aspects, the concentration of potassium ion is about 85 mM to about 100 mM, about 85 mM to about 95 mM, or about 85 mM to about 90 mM. [0554] In some aspects, the concentration of potassium ion is about 90 mM to about 100 mM. In some aspects, the concentration of potassium ion is about 90 mM to about 95 mM. [0555] In some aspects, the concentration of potassium ion is about 95 mM to about 100 mM. [0556] In some aspects, the concentration of potassium ion 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. In some aspects, the medium is hypertonic. In some aspects, the medium is isotonic. In some aspects, the 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. [0557] In some aspects, the concentration of potassium ion is about 50 mM to about 55 mM. 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 60 mM to about 65 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 70 mM to about 75 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 80 mM to about 85 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 90 mM to about 95 mM. 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 100 mM to about 105 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 110 mM to about 115 mM. In some aspects, the concentration of potassium ion is about 115 mM to about 120 mM. [0558] In some aspects, the concentration of potassium ion is about 40 mM to about 90 mM. In some aspects, the concentration of potassium ion is about 40 mM to about 80 mM. In some aspects, the concentration of potassium ion is about 40 mM to about 70 mM. In some aspects, the concentration of potassium ion 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 50 mM to about 70 mM. In some aspects, the concentration of potassium ion is about 55 mM to about 90 mM. In some aspects, the concentration of potassium ion is about 55 mM to about 80 mM. In some aspects, the concentration of potassium ion is about 55 mM to about 70 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 60 mM to about 70 mM. In some aspects, the concentration of potassium ion is about 65 mM to about 90 mM. In some aspects, the concentration of potassium ion is about 65 mM to about 80 mM. In some aspects, the concentration of potassium ion is about 65 mM to about 70 mM. [0559] In some aspects, the concentration of potassium ion is higher than about 40 mM. In some aspects, the concentration of potassium ion is about 40 mM. In some aspects, the concentration of potassium ion is higher than about 41 mM. In some aspects, the concentration of potassium ion is about 41 mM. In some aspects, the concentration of potassium ion is higher than about 42 mM. In some aspects, the concentration of potassium ion is about 42 mM. In some aspects, the concentration of potassium ion is higher than about 43 mM. In some aspects, the concentration of potassium ion is about 43 mM. In some aspects, the concentration of potassium ion is higher than about 44 mM. In some aspects, the concentration of potassium ion is about 44 mM. In some aspects, the concentration of potassium ion is higher than about 45 mM. In some aspects, the concentration of potassium ion is about 45 mM. In some aspects, the concentration of potassium ion is higher than about 46 mM. In some aspects, the concentration of potassium ion is
about 46 mM. In some aspects, the concentration of potassium ion is higher than about 47 mM. In some aspects, the concentration of potassium ion is about 47 mM. In some aspects, the concentration of potassium ion is higher than about 48 mM. In some aspects, the concentration of potassium ion is about 48 mM. In some aspects, the concentration of potassium ion is higher than about 49 mM. In some aspects, the concentration of potassium ion is about 49 mM. [0560] In some aspects, the concentration of potassium ion is higher than about 50 mM. In some aspects, the concentration of potassium ion is about 50 mM. In some aspects, the concentration of potassium ion is higher than about 51 mM. In some aspects, the concentration of potassium ion is about 51 mM. In some aspects, the concentration of potassium ion is higher than about 52 mM. In some aspects, the concentration of potassium ion is about 52 mM. In some aspects, the concentration of potassium ion is higher than about 53 mM. In some aspects, the concentration of potassium ion is about 53 mM. In some aspects, the concentration of potassium ion is higher than about 54 mM. In some aspects, the concentration of potassium ion is about 54 mM. In some aspects, the concentration of potassium ion is higher than about 55 mM. In some aspects, the concentration of potassium ion is about 55 mM. In some aspects, the concentration of potassium ion is higher than about 56 mM. In some aspects, the concentration of potassium ion is about 56 mM. In some aspects, the concentration of potassium ion is higher than about 57 mM. In some aspects, the concentration of potassium ion is about 57 mM. In some aspects, the concentration of potassium ion is higher than about 58 mM. In some aspects, the concentration of potassium ion is about 58 mM. In some aspects, the concentration of potassium ion is higher than about 59 mM. In some aspects, the concentration of potassium ion is about 59 mM. [0561] In some aspects, the concentration of potassium ion is higher than about 60 mM. In some aspects, the concentration of potassium ion is about 60 mM. In some aspects, the concentration of potassium ion is higher than about 61 mM. In some aspects, the concentration of potassium ion is about 61 mM. In some aspects, the concentration of potassium ion is higher than about 62 mM. In some aspects, the concentration of potassium ion is about 62 mM. In some aspects, the concentration of potassium ion is higher than about 63 mM. In some aspects, the concentration of potassium ion is about 63 mM. In some aspects, the concentration of potassium ion is higher than about 64 mM. In some aspects, the concentration of potassium ion is about 64 mM. In some aspects, the concentration of potassium ion is higher than about 65 mM. In some aspects, the concentration of potassium ion is about 65 mM. In some aspects, the concentration of potassium ion is higher than about 66 mM. In some aspects, the concentration of potassium ion is about 66 mM. In some aspects, the concentration of potassium ion is higher than about 67 mM. In
some aspects, the concentration of potassium ion is about 67 mM. In some aspects, the concentration of potassium ion is higher than about 68 mM. In some aspects, the concentration of potassium ion is about 68 mM. In some aspects, the concentration of potassium ion is higher than about 69 mM. In some aspects, the concentration of potassium ion is about 69 mM. [0562] In some aspects, the concentration of potassium ion is higher than about 70 mM. In some aspects, the concentration of potassium ion is about 70 mM. In some aspects, the concentration of potassium ion is higher than about 71 mM. In some aspects, the concentration of potassium ion is about 71 mM. In some aspects, the concentration of potassium ion is higher than about 72 mM. In some aspects, the concentration of potassium ion is about 72 mM. In some aspects, the concentration of potassium ion is higher than about 73 mM. In some aspects, the concentration of potassium ion is about 73 mM. In some aspects, the concentration of potassium ion is higher than about 74 mM. In some aspects, the concentration of potassium ion is about 74 mM. In some aspects, the concentration of potassium ion is higher than about 75 mM. In some aspects, the concentration of potassium ion is about 75 mM. In some aspects, the concentration of potassium ion is higher than about 76 mM. In some aspects, the concentration of potassium ion is about 76 mM. In some aspects, the concentration of potassium ion is higher than about 77 mM. In some aspects, the concentration of potassium ion is about 77 mM. In some aspects, the concentration of potassium ion is higher than about 78 mM. In some aspects, the concentration of potassium ion is about 78 mM. In some aspects, the concentration of potassium ion is higher than about 79 mM. In some aspects, the concentration of potassium ion is about 79 mM. [0563] In some aspects, the concentration of potassium ion is higher than about 80 mM. In some aspects, the concentration of potassium ion is about 80 mM. In some aspects, the concentration of potassium ion is higher than about 81 mM. In some aspects, the concentration of potassium ion is about 81 mM. In some aspects, the concentration of potassium ion is higher than about 82 mM. In some aspects, the concentration of potassium ion is about 82 mM. In some aspects, the concentration of potassium ion is higher than about 83 mM. In some aspects, the concentration of potassium ion is about 83 mM. In some aspects, the concentration of potassium ion is higher than about 84 mM. In some aspects, the concentration of potassium ion is about 84 mM. In some aspects, the concentration of potassium ion is higher than about 85 mM. In some aspects, the concentration of potassium ion is about 85 mM. In some aspects, the concentration of potassium ion is higher than about 86 mM. In some aspects, the concentration of potassium ion is about 86 mM. In some aspects, the concentration of potassium ion is higher than about 87 mM. In some aspects, the concentration of potassium ion is about 87 mM. In some aspects, the
concentration of potassium ion is higher than about 88 mM. In some aspects, the concentration of potassium ion is about 88 mM. In some aspects, the concentration of potassium ion is higher than about 89 mM. In some aspects, the concentration of potassium ion is about 89 mM. [0564] In some aspects, the concentration of potassium ion is higher than about 90 mM. In some aspects, the concentration of potassium ion is about 90 mM. In some aspects, the concentration of potassium ion is higher than about 91 mM. In some aspects, the concentration of potassium ion is about 91 mM. In some aspects, the concentration of potassium ion is higher than about 92 mM. In some aspects, the concentration of potassium ion is about 92 mM. In some aspects, the concentration of potassium ion is higher than about 93 mM. In some aspects, the concentration of potassium ion is about 93 mM. In some aspects, the concentration of potassium ion is higher than about 94 mM. In some aspects, the concentration of potassium ion is about 94 mM. In some aspects, the concentration of potassium ion is higher than about 95 mM. In some aspects, the concentration of potassium ion is about 95 mM. In some aspects, the concentration of potassium ion is higher than about 96 mM. In some aspects, the concentration of potassium ion is about 96 mM. In some aspects, the concentration of potassium ion is higher than about 97 mM. In some aspects, the concentration of potassium ion is about 97 mM. In some aspects, the concentration of potassium ion is higher than about 98 mM. In some aspects, the concentration of potassium ion is about 98 mM. In some aspects, the concentration of potassium ion is higher than about 99 mM. In some aspects, the concentration of potassium ion is about 99 mM. [0565] In some aspects, the concentration of potassium ion is higher than about 100 mM. In some aspects, the concentration of potassium ion is about 100 mM. [0566] In some aspects, the concentration of potassium ion 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. [0567] In some aspects, the concentration of potassium ion 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 mM to about 85 mM. 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 mM to about 80 mM. 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 mM to about 45 mM. 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 less than 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. [0568] In some aspects, the concentration of potassium ion is higher than about 40 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 40 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 41 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 41 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 42 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 42 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 43 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 43 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 44 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 44 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 45 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 45 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 46 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 46 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the
concentration of potassium ion is higher than about 47 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 47 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 48 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 48 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 49 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 49 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. [0569] In some aspects, the concentration of potassium ion is higher than about 50 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 50 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 51 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 51 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 52 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 52 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 53 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 53 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 54 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 54 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 55 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and
140 mM. In some aspects, the concentration of potassium ion is about 55 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 56 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 56 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 57 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 57 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 58 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 58 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 59 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 59 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. [0570] In some aspects, the concentration of potassium ion is higher than about 60 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 60 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 61 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 61 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 62 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 62 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 63 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 63 mM, wherein the total concentration of potassium ion and NaCl in the
medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 64 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 64 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 65 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 65 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 66 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 66 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 67 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 67 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 68 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 68 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 69 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 69 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. [0571] In some aspects, the concentration of potassium ion is higher than about 70 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 70 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 71 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 71 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 72 mM, wherein the total concentration of
potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 72 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 73 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 73 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 74 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 74 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 75 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 75 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 76 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 76 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 77 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 77 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 78 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 78 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 79 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 79 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. [0572] In some aspects, the concentration of potassium ion is higher than about 80 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 80 mM, wherein the total
concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 81 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 81 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 82 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 82 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 83 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 83 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 84 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 84 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 85 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 85 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 86 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 86 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 87 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 87 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 88 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 88 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 89 mM, wherein the total concentration of potassium ion and NaCl in the medium
is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 89 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. [0573] In some aspects, the concentration of potassium ion is higher than about 90 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 90 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 91 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 91 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 92 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 92 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 93 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 93 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 94 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 94 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 95 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 95 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 96 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 96 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 97 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 97 mM, wherein the total concentration of potassium ion
and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 98 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 98 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 99 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 99 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. [0574] In some aspects, the concentration of potassium ion is higher than about 100 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 100 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 101 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 101 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 102 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 102 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 103 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 103 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 104 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 104 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 105 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 105 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 106 mM,
wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 106 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 107 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 107 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 108 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 108 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 109 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 109 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. [0575] In some aspects, the concentration of potassium ion is higher than about 110 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 110 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 111 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 111 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 112 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 112 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 113 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 113 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 114 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is
about 114 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 115 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 115 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 116 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 116 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 117 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 117 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 118 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 118 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 119 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 119 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. [0576] In some aspects, the concentration of potassium ion is higher than about 120 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 120 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 121 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 121 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 122 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 122 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of
potassium ion is higher than about 123 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 123 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 124 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 124 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 125 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 125 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 126 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 126 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 127 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 127 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 128 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 128 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 129 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 129 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is higher than about 130 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. In some aspects, the concentration of potassium ion is about 130 mM, wherein the total concentration of potassium ion and NaCl in the medium is between 110 mM and 140 mM. [0577] In some aspects, the medium comprising potassium ion can be 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 hexafluoronickelate, 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 nitrosodisulfonate, 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, and potassium hydroxycitrate tribasic monohydrate. In some aspects, the potassium salt comprises potassium chloride (KCl). In some aspects, the potassium salt comprises potassium gluconate. In certain aspects, the potassium salt comprises potassium citrate. In certain aspects, the potassium salt comprises potassium hydroxycitrate. In some aspects, the potassium salt comprises a combination of any of the potassium salts disclosed herein. II.C.2. Cell Expansion Agents [0578] In some aspects, an expansion medium disclosed herein further comprises a cell expansion agent. As used herein, a "cell expansion agent" refers to an agent, e.g., small molecule, polypeptide, or any combination thereof, that promotes the in vitro and/or ex vivo growth and proliferation of cultured immune cells, e.g., T cells, NK cells, and/or TILs. In some aspects, the cell expansion agent comprises a PI3K inhibitor. In some aspects, the medium further comprises an AKT inhibitor. In some aspects, the medium further comprises a PI3K inhibitor and an AKT
inhibitor. In some aspects, the PI3K inhibitor comprises LY294002. In some aspects, the PI3K inhibitor comprises IC87114. In some aspects, the PI3K inhibitor comprises idelalisib (see, e.g., Peterson et al., Blood Adv.2(3):210-23 (2018)). In some aspects, the medium further comprises a GSK3B inhibitor. In some aspects, the GSK3B inhibitor comprises TWS119. In some aspects, the medium further comprises an ACLY inhibitor. In some aspects, the ACLY inhibitor comprises potassium hydroxycitrate tribasic monohydrate. In some aspects, the PI3K inhibitor comprises hydroxyl citrate. In some aspects, the PI3K inhibitor comprises pictilisib. In some aspects, the PI3K inhibitor comprises CAL-101. In some aspects, the AKT inhibitor comprises MK2206, A443654, or AKTi-VIII (CAS 612847-09-3). II.C.3. Sodium [0579] In some aspects, an expansion medium disclosed herein further comprises sodium ion (e.g., NaCl). In some aspects, the metabolic reprogramming media comprises sodium ion (e.g., NaCl) at a concentration of less than about 115 mM. In some aspects the metabolic reprogramming media, comprises sodium ion (e.g., NaCl) at a concentration of 40 mM to about 80 mM. [0580] In some aspects, the target concentration of sodium is reached by starting with a basal medium comprising a higher concentration of sodium ion (e.g., NaCl) and diluting the solution to reach the target concentration of sodium ion (e.g., NaCl). In some aspects, the target concentration of sodium is reached by raising the concentration of sodium ion (e.g., NaCl) by adding one or more sodium salts (e.g., more NaCl). 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, and any combination thereof. In some aspects, the sodium salt comprises sodium chloride (NaCl). In some aspects, the sodium salt comprises sodium gluconate. In certain aspects, the sodium salt comprises sodium bicarbonate. In certain aspects, the sodium salt comprises sodium hydroxycitrate. In certain aspects, the sodium salt comprises sodium phosphate. II.C.4. Saccharides [0581] In some aspects, an expansion medium disclosed herein comprises a saccharide. 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. [0582] In some aspects, the concentration of the saccharide, e.g., glucose, is about 10 mM to about 30 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 10 mM to about 24 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is less than about 24 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is more than about 10 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 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 30 mM, from about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 10 mM to about 5 mM, 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 30 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. [0583] 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, or about 30 mM. [0584] 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 about 6 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 7 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 8 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 9 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 10 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 10.5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 11 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 11.5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 12 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 12.5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 13 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 13.5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 14 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 14.5 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 15 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 15.4 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 15.9 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 16.3 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 16.8 mM. In some aspects, the concentration of the saccharide,
e.g., glucose, is about 17.2 mM. In some aspects, the concentration of the saccharide, e.g., glucose, is about 17.7 mM. II.C.5. Calcium [0585] In some aspects, an expansion medium disclosed herein comprises calcium ion. 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 more calcium 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, and any combination thereof. In some aspects, the calcium salt comprises calcium chloride (CaCl2). In some aspects, the calcium salt comprises calcium gluconate. [0586] 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. [0587] 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 2.8 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 about 1.2 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, about 1.2 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 about 1.8 mM, or about 1.7 to about 1.8 mM. [0588] In some aspects, the concentration of calcium ion is from about 0.5 mM to about 2.8 mM. In some aspects, the concentration of calcium ion is from about 0.8 mM to about 1.8 mM. In some aspects, the concentration of calcium ion is from about 0.9 mM to about 1.8 mM. In some aspects, the concentration of calcium ion is from about 1.0 mM to about 1.8 mM. In some aspects, the concentration of calcium ion is from about 1.1 mM to about 1.8 mM. In some aspects, the concentration of calcium ion is from about 1.2 mM to about 1.8 mM. In some aspects, the concentration of calcium ion is from about 0.8 mM to about 1.8 mM. In some aspects, the concentration of calcium ion is from about 0.8 mM to about 0.9 mM. In some aspects, the concentration of calcium ion is from about 0.9 mM to about 1.0 mM. In some aspects, the concentration of calcium ion is from about 1.0 mM to about 1.1 mM. In some aspects, the concentration of calcium ion is from about 1.1 mM to about 1.2 mM. In some aspects, the concentration of calcium ion is from about 1.2 mM to about 1.3 mM. In some aspects, the concentration of calcium ion is from about 1.3 mM to about 1.4 mM. In some aspects, the concentration of calcium ion is from about 1.4 mM to about 1.5 mM. In some aspects, the
concentration of calcium ion is from about 1.5 mM to about 1.6 mM. In some aspects, the concentration of calcium ion is from about 1.7 mM to about 1.8 mM. [0589] In some aspects, the concentration of calcium ion is about 0.5 mM, 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, about 2.0 mM, about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, about 2.5 mM, about 2.6 mM, about 2.7 mM, or about 2.8 mM. In some aspects, the concentration of calcium ion is about 0.5 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, the concentration 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. In some aspects, the concentration of calcium ion is about 1.9 mM. In some aspects, the concentration of calcium ion is about 2 mM. In some aspects, the concentration of calcium ion is about 2.1 mM. In some aspects, the concentration of calcium ion is about 2.2 mM. In some aspects, the concentration of calcium ion is about 2.3 mM. In some aspects, the concentration of calcium ion is about 2.4 mM. In some aspects, the concentration of calcium ion is about 2.5 mM. In some aspects, the concentration of calcium ion is about 2.6 mM. In some aspects, the concentration of calcium ion is about 2.7 mM. In some aspects, the concentration of calcium ion is about 2.8 mM. II.C.6. Additional Components [0590] In some aspects, an expansion medium disclosed herein further comprises one or more essential amino acids. In some aspects, the basal media comprises one or more essential amino acids selected form L-arginine, L-cystine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-histidine, L-tyrosine, L-valine, and L-glutamine, or any combination thereof. In some aspects, the basal media comprises L-glutamine. [0591] In some aspects, an expansion medium disclosed herein comprises at least about 0.01 mM of one or more essential amino acids. In some aspects, an expansion medium disclosed
herein comprises about 0.01 mM to about 10 mM of one or more essential amino acids. In some aspects, the MRM comprises about 0.01 mM to about 10 mM, about 0.01 mM to about 9 mM, about 0.01 mM to about 8 mM, about 0.01 mM to about 7 mM, about 0.01 mM to about 6 mM, about 0.01 mM to about 5 mM, about 0.01 mM to about 4 mM, about 0.01 mM to about 3 mM, about 0.01 mM to about 2 mM, about 0.01 mM to about 1 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 9 mM, about 0.1 mM to about 8 mM, about 0.1 mM to about 7 mM, about 0.1 mM to about 6 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 1 mM to about 10 mM, about 1 mM to about 9 mM, about 1 mM to about 8 mM, about 1 mM to about 7 mM, about 1 mM to about 6 mM, about 1 mM to about 5 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, or about 1 mM to about 2 mM of one or more essential amino acids. [0592] In some aspects, an expansion medium disclosed herein comprises at least about 0.01 mM, at least about 0.1 mM, at least about 0.5 mM, at least about 1.0 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least about 12 mM, at least about 13 mM, at least about 14 mM, or at least about 15 mM or at least about 50 mM of one or more essential amino acids. [0593] In some aspects, an expansion medium disclosed herein comprises about 0.01 mM, about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 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, about 2.0 mM, about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, about 2.5 mM, about 2.6 mM, about 2.7 mM, about 2.8 mM, about 2.9 mM, about 3.0 mM, about 3.1 mM, about 3.2 mM, about 3.3 mM, about 3.4 mM, about 3.5 mM, about 3.6 mM, about 3.7 mM, about 3.8 mM, about 3.9 mM, about 4.0 mM, about 4.1 mM, about 4.2 mM, about 4.3 mM, about 4.4 mM, about 4.5 mM, about 4.6 mM, about 4.7 mM, about 4.8 mM, about 4.9 mM, about 5.0 mM, about 5.1 mM, about 5.2 mM, about 5.3 mM, about 5.4 mM, about 5.5 mM, about 5.6 mM, about 5.7 mM, about 5.8 mM, about 5.9 mM, about 6.0 mM, about 6.1 mM, about 6.2 mM, about 6.3 mM, about 6.4 mM, about 6.5 mM, about 6.6 mM, about 6.7 mM, about 6.8 mM, about 6.9 mM, or about 7.0 mM of one or more essential amino acids. [0594] In some aspects, an expansion medium disclosed herein comprises L-glutamine. In some aspects, first expansion medium, the second expansion medium, or both comprises at least about 0.01 mM L-glutamine. In some aspects, an expansion medium disclosed herein comprises
about 0.01 mM to about 10 mM L-glutamine. In some aspects, an expansion medium disclosed herein comprises about 0.01 mM to about 10 mM, about 0.01 mM to about 9 mM, about 0.01 mM to about 8 mM, about 0.01 mM to about 7 mM, about 0.01 mM to about 6 mM, about 0.01 mM to about 5 mM, about 0.01 mM to about 4 mM, about 0.01 mM to about 3 mM, about 0.01 mM to about 2 mM, about 0.01 mM to about 1 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 9 mM, about 0.1 mM to about 8 mM, about 0.1 mM to about 7 mM, about 0.1 mM to about 6 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 1 mM to about 10 mM, about 1 mM to about 9 mM, about 1 mM to about 8 mM, about 1 mM to about 7 mM, about 1 mM to about 6 mM, about 1 mM to about 5 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, or about 1 mM to about 2 mM L-glutamine. [0595] In some aspects, an expansion medium disclosed herein comprises at least about 0.01 mM, at least about 0.1 mM, at least about 0.5 mM, at least about 1.0 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least about 12 mM, at least about 13 mM, at least about 14 mM, or at least about 15 mM or at least about 50 mM L-glutamine. [0596] In some aspects, an expansion medium disclosed herein comprises about 0.01 mM, about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 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, about 2.0 mM, about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, about 2.5 mM, about 2.6 mM, about 2.7 mM, about 2.8 mM, about 2.9 mM, about 3.0 mM, about 3.1 mM, about 3.2 mM, about 3.3 mM, about 3.4 mM, about 3.5 mM, about 3.6 mM, about 3.7 mM, about 3.8 mM, about 3.9 mM, about 4.0 mM, about 4.1 mM, about 4.2 mM, about 4.3 mM, about 4.4 mM, about 4.5 mM, about 4.6 mM, about 4.7 mM, about 4.8 mM, about 4.9 mM, about 5.0 mM, about 5.1 mM, about 5.2 mM, about 5.3 mM, about 5.4 mM, about 5.5 mM, about 5.6 mM, about 5.7 mM, about 5.8 mM, about 5.9 mM, about 6.0 mM, about 6.1 mM, about 6.2 mM, about 6.3 mM, about 6.4 mM, about 6.5 mM, about 6.6 mM, about 6.7 mM, about 6.8 mM, about 6.9 mM, or about 7.0 mM L-glutamine. In some aspects, the MR first expansion medium, the second expansion medium, or both M comprises about 1.7 mM L-glutamine. In some aspects, an expansion medium disclosed herein comprises about 1.68 mM L-glutamine.
[0597] In some aspects, an expansion medium disclosed herein comprises about 0.14 mM L-glutamine. In some aspects, an expansion medium disclosed herein comprises about 0.15 mM L-glutamine. In some aspects, an expansion medium disclosed herein comprises about 1.76 mM L-glutamine. In some aspects, an expansion medium disclosed herein comprises about 1.83 mM L-glutamine. In some aspects, an expansion medium disclosed herein comprises about 1.84 mM L-glutamine. In some aspects, an expansion medium disclosed herein comprises about 1.97 mM L-glutamine. In some aspects, an expansion medium disclosed herein comprises about 2 mM L- glutamine. In some aspects, an expansion medium disclosed herein comprises about 2.11 mM L- glutamine. In some aspects, an expansion medium disclosed herein comprises about 2.18 mM L- glutamine. In some aspects, an expansion medium disclosed herein comprises about 5.41 mM L- glutamine. In some aspects, an expansion medium disclosed herein comprises about 5.47 mM L- glutamine. In some aspects, an expansion medium disclosed herein comprises about < 0.10 mM L-glutamine. [0598] In some aspects, an expansion medium disclosed herein comprises L-glutamic acid. In some aspects, an expansion medium disclosed herein comprises at least about 0.01 mM L- glutamic acid. In some aspects, an expansion medium disclosed herein comprises about 0.01 mM to about 10 mM L-glutamic acid. In some aspects, an expansion medium disclosed herein comprises about 0.01 mM to about 10 mM, about 0.01 mM to about 9 mM, about 0.01 mM to about 8 mM, about 0.01 mM to about 7 mM, about 0.01 mM to about 6 mM, about 0.01 mM to about 5 mM, about 0.01 mM to about 4 mM, about 0.01 mM to about 3 mM, about 0.01 mM to about 2 mM, about 0.01 mM to about 1 mM, about 0.1 mM to about 10 mM, about 0.1 mM to about 9 mM, about 0.1 mM to about 8 mM, about 0.1 mM to about 7 mM, about 0.1 mM to about 6 mM, about 0.1 mM to about 5 mM, about 0.1 mM to about 4 mM, about 0.1 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.1 mM to about 1 mM, about 1 mM to about 10 mM, about 1 mM to about 9 mM, about 1 mM to about 8 mM, about 1 mM to about 7 mM, about 1 mM to about 6 mM, about 1 mM to about 5 mM, about 1 mM to about 4 mM, about 1 mM to about 3 mM, or about 1 mM to about 2 mM L-glutamic acid. [0599] In some aspects, an expansion medium disclosed herein comprises at least about 0.01 mM, at least about 0.1 mM, at least about 0.5 mM, at least about 1.0 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least about 12 mM, at least about 13 mM, at least about 14 mM, or at least about 15 mM or at least about 50 mM L-glutamic acid.
[0600] In some aspects, an expansion medium disclosed herein comprises about 0.01 mM, about 0.05 mM, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 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, about 2.0 mM, about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, about 2.5 mM, about 2.6 mM, about 2.7 mM, about 2.8 mM, about 2.9 mM, about 3.0 mM, about 3.1 mM, about 3.2 mM, about 3.3 mM, about 3.4 mM, about 3.5 mM, about 3.6 mM, about 3.7 mM, about 3.8 mM, about 3.9 mM, about 4.0 mM, about 4.1 mM, about 4.2 mM, about 4.3 mM, about 4.4 mM, about 4.5 mM, about 4.6 mM, about 4.7 mM, about 4.8 mM, about 4.9 mM, about 5.0 mM, about 5.1 mM, about 5.2 mM, about 5.3 mM, about 5.4 mM, about 5.5 mM, about 5.6 mM, about 5.7 mM, about 5.8 mM, about 5.9 mM, about 6.0 mM, about 6.1 mM, about 6.2 mM, about 6.3 mM, about 6.4 mM, about 6.5 mM, about 6.6 mM, about 6.7 mM, about 6.8 mM, about 6.9 mM, or about 7.0 mM L-glutamic acid. [0601] In some aspects, an expansion medium disclosed herein comprises about 0.15 mM L-glutamic acid. In some aspects, an expansion medium disclosed herein comprises about 0.17 mM L-glutamic acid. In some aspects, an expansion medium disclosed herein comprises about 0.18 mM L-glutamic acid. In some aspects, an expansion medium disclosed herein comprises about 0.19 mM L-glutamic acid. In some aspects, an expansion medium disclosed herein comprises about 0.85 mM L-glutamic acid. In some aspects, an expansion medium disclosed herein comprises about 0.86 mM L-glutamic acid. In some aspects, an expansion medium disclosed herein comprises about 0.9 mM L-glutamic acid. In some aspects, an expansion medium disclosed herein comprises about 0.95 mM L-glutamic acid. In some aspects, an expansion medium disclosed herein comprises about 1.06 mM L-glutamic acid. In some aspects, an expansion medium disclosed herein comprises about 1.09 mM L-glutamic acid. In some aspects, an expansion medium disclosed herein comprises about < 0.10 mM L-glutamic acid. [0602] In some aspects, an expansion medium disclosed herein comprises a dipeptide. In some aspects, an expansion medium disclosed herein comprises glutamine-glutamine (Gln-Gln). In some aspects, an expansion medium disclosed herein comprises alanyl-glutamine (Ala-Gln). [0603] In some aspects, an expansion medium disclosed herein comprises at least about 0.1 mM dipeptide (e.g., Ala-Gln). In some aspects, an expansion medium disclosed herein comprises about 0.1 mM to about 50 mM dipeptide (e.g., Ala-Gln). In some aspects, an expansion medium disclosed herein comprises about 0.1 mM to about 40 mM, about 0.1 mM to about 35 mM, about 0.1 mM to about 30 mM, about 0.1 mM to about 25 mM, about 0.1 mM to about 20
mM, about 1 mM to about 20 mM, about 2 mM to about 20 mM, about 3 mM to about 20 mM, about 4 mM to about 20 mM, about 5 mM to about 20 mM, about 6 mM to about 20 mM, about 7 mM to about 20 mM, about 8 mM to about 20 mM, about 9 mM to about 20 mM, about 10 mM to about 20 mM, about 1 mM to about 10 mM, about 2 mM to about 10 mM, about 3 mM to about 10 mM, about 4 mM to about 10 mM, about 5 mM to about 10 mM, about 6 mM to about 10 mM, about 7 mM to about 10 mM, about 8 mM to about 10 mM, or about 9 mM to about 10 mM dipeptide (e.g., Ala-Gln). [0604] In some aspects, an expansion medium disclosed herein comprises at least about 0.1 mM, at least about 1.0 mM, at least about 2 mM, at least about 3 mM, at least about 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, at least about 10 mM, at least about 11 mM, at least about 12 mM, at least about 13 mM, at least about 14 mM, at least about 15 mM, at least about 16 mM, at least about 17 mM, at least about 18 mM, at least about 19 mM, at least about 20 mM, at least about 25 mM, at least about 30 mM, or at least about 50 mM dipeptide (e.g., Ala-Gln). [0605] In some aspects, an expansion medium disclosed herein comprises about 1 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 dipeptide (e.g., Ala-Gln). In some aspects, the basal medium comprises about 1.7 mM dipeptide (e.g., Ala-Gln). In some aspects, an expansion medium disclosed herein comprises about 1.68 mM dipeptide (e.g., Ala-Gln). [0606] In some aspects, an expansion medium disclosed herein comprises about 6 mM, about 6.1 mM, about 6.2 mM, about 6.3 mM, about 6.4 mM, about 6.5 mM, about 6.6 mM, about 6.7 mM, about 6.8 mM, about 6.9 mM, about 7.0 mM, about 7.1 mM, or about 7.2 mM dipeptide (e.g., Ala-Gln). In some aspects, an expansion medium disclosed herein comprises about 6.8 mM dipeptide (e.g., Ala-Gln). In some aspects, an expansion medium disclosed herein comprises about 6.81 mM dipeptide (e.g., Ala-Gln). In some aspects, an expansion medium disclosed herein comprises about 6.9 mM dipeptide (e.g., Ala-Gln). In some aspects, an expansion medium disclosed herein comprises about 6.96 mM dipeptide (e.g., Ala-Gln). In some aspects, an expansion medium disclosed herein comprises about 7.0 mM dipeptide (e.g., Ala-Gln). [0607] In some aspects, an expansion medium disclosed herein comprises less than about 5 mM ammonia (NH3). In some aspects, an expansion medium disclosed herein comprises less than about 4 mM, less than about 3.5 mM, less than about 3 mM, less than about 2.5 mM, less than about 2 mM, less than about 1.5 mM, less than about 1 mM, less than about 0.5 mM, less than about 0.4 mM, less than about 0.3 mM, less than about 0.2 mM, or less than about 0.1 mM
ammonia. In some aspects, an expansion medium disclosed herein comprises about 0.01 mM ammonia to less than about 2 mM ammonia, about 0.01 mM ammonia to less than about 1.9 mM ammonia, about 0.01 mM ammonia to less than about 1.8 mM ammonia, about 0.01 mM ammonia to less than about 1.7 mM ammonia, about 0.01 mM ammonia to less than about 1.6 mM ammonia, about 0.01 mM ammonia to less than about 1.5 mM ammonia, about 0.01 mM ammonia to less than about 1.4 mM ammonia, about 0.01 mM ammonia to less than about 1.3 mM ammonia, about 0.01 mM ammonia to less than about 1.2 mM ammonia, about 0.01 mM ammonia to less than about 1.1 mM ammonia, about 0.01 mM ammonia to less than about 1 mM ammonia, about 0.01 mM ammonia to less than about 0.9 mM ammonia, about 0.01 mM ammonia to less than about 0.8 mM ammonia, about 0.01 mM ammonia to less than about 0.7 mM ammonia, about 0.01 mM ammonia to less than about 0.6 mM ammonia, about 0.01 mM ammonia to less than about 0.5 mM ammonia, about 0.01 mM ammonia to less than about 0.4 mM ammonia, about 0.01 mM ammonia to less than about 0.3 mM ammonia, about 0.01 mM ammonia to less than about 0.2 mM ammonia, or about 0.01 mM ammonia to less than about 0.1 mM ammonia. In some aspects, an expansion medium disclosed herein comprises about 1.2 mM ammonia. In some aspects, an expansion medium disclosed herein comprises about 1.25 mM ammonia. In some aspects, an expansion medium disclosed herein comprises about 1.259 mM ammonia. In some aspects, an expansion medium disclosed herein comprises about 1.28 mM ammonia. In some aspects, an expansion medium disclosed herein comprises about 1.3 mM ammonia. In some aspects, an expansion medium disclosed herein comprises about 0.3 mM ammonia. In some aspects, an expansion medium disclosed herein comprises about 0.34 mM ammonia. In some aspects, an expansion medium disclosed herein comprises about 0.35 mM ammonia. In some aspects, an expansion medium disclosed herein comprises about 0.36 mM ammonia. In some aspects, an expansion medium disclosed herein comprises about 0.37 mM ammonia. In some aspects, an expansion medium disclosed herein comprises less than about 0.3 mM ammonia. In some aspects, an expansion medium disclosed herein comprises less than about 0.29 mM ammonia. In some aspects, an expansion medium disclosed herein comprises less than about 0.28 mM ammonia. In some aspects, an expansion medium disclosed herein comprise less than about 0.278 mM ammonia. In some aspects, an expansion medium disclosed herein do not comprise ammonia. [0608] In some aspects, an expansion medium disclosed herein comprises lactate. In some aspects, an expansion medium disclosed herein does not comprise lactate. [0609] In some aspects, an expansion medium disclosed herein is modified from a basal medium selected from 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), M199, OpTmizer™ CTS™ T-Cell Expansion Basal Medium (ThermoFisher), OPTMIZER™ Complete, IMMUNOCULT™ XF (STEMCELL™ Technologies), IMMUNOCULT™ XF, AIM V, TEXMACS™ medium, and any combination thereof. In some aspects, the basal medium is serum free. 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 particular aspects, the basal media comprises OPTMIZER™ complete. In some aspects, suitable basal medium includes Click's medium, OpTimizer® (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. [0610] In some aspects, an expansion medium disclosed herein further comprises about 2.5% serum supplement (CTS™ Immune Cell SR, Thermo Fisher), 2 mM L-glutamine, 2 mM L- glutamax, MEM Non-Essential Amino Acids Solution, Pen-strep, 20μg/ml FUNGIN™, Sodium pyruvate, or any combination thereof. In some aspects, an expansion medium disclosed herein further comprises O-Acetyl-L-carnitine hydrochloride. In some aspects, an expansion medium disclosed herein further comprises a kinase inhibitor. II.D. Cells II.D.1. T Cells and NK Cells [0611] The immune cells, e.g., T cells and/or NK cells, that are placed in the medium can be cells that are collected and/or isolated from a subject in need of a therapy. In some aspects, the immune cells, e.g., T cells and/or NK cells, that are placed in the medium have been engineered prior to the culturing. In some aspects, the immune cells, e.g., T cells and/or NK cells, that are placed in the medium have been expanded. The immune cells, e.g., T cells and/or NK cells, that are placed in the medium can be referred to as starting (initial, i.e., patient sample, apheresis sample, buffy coat) cells. The immune cells, e.g., T cells and/or NK cells, that result from culturing
them in the metabolic reprogramming media disclosed herein can be referred to as resulting (cultured or expanded) cells. [0612] In some aspects, the T cells and/or NK cells comprise helper T-cells (CD4+ T cells), cytotoxic T-cells (CD8+ T cells), memory T-cells (CD45RO+ T cells), suppressor T-cells (Ts cells), regulatory T-cells (Tregs), natural killer T-cells (NKT cells), gamma delta T cells, (γδ T cells), or any combination thereof. In some aspects, the T cells are γδ T cells. In some aspects, the T cells are αβ T cells. In some aspects, the T cells are NKT cells. In some aspects, the T cells are Tregs. In some aspects, the T cells are Ts cells. In some aspects, the T cells are memory T-cells. In some aspects, the T cells are cytotoxic T-cells. In some aspects, the T cells are helper T-cells. [0613] The methods disclosed herein provide culture conditions that promote a less- differentiated phenotype for cultured immune cells, e.g., T cells and/or NK cells. In some aspects, the starting immune cells, e.g., T cells and/or NK cells, are isolated from a human subject. In some aspects, the starting immune cells, e.g., T cells and/or NK cells, are isolated from a human subject for allogeneic cell therapy. In some aspects, the starting immune cells, e.g., T cells and/or NK cells, are isolated from a human subject for autologous cell therapy. In some aspects, the immune cells are T cells. In some aspects, the immune cells are NK cells. In some aspects, the immune cells are TILs. In some aspects, the immune cells are Tregs. In some aspects, the immune cells, e.g., T cells and/or NK cells, are isolated from a human subject. In some aspects, the immune cells are tumor- infiltrating T cells or tumor-infiltrating NK cells. In certain aspects, the immune cells, e.g., T cells and/or NK cells, are engineered. In some aspects, the immune cells, e.g., T cells and/or NK cells, are engineered to comprise a chimeric antigen receptor (CAR). In some aspects, the immune cells, e.g., T cells and/or NK cells, are engineered to comprise an engineered T cell receptor (TCR). [0614] In some aspects, the cells, e.g., T cells, NK cells, and/or TILs, are engineered before culturing according to the methods disclosed herein. In some aspects, the cells, e.g., T cells, NK cells, and/or TILs, are engineered after culturing according to the methods disclosed herein. In some aspects, the cells, e.g., T cells, NK cells, and/or TILs, are cultured according to the methods disclosed herein, e.g., in a hypotonic or isotonic medium comprising at least 5 mM potassium ion, prior to, during, and after cell engineering. In some aspects, the cells, e.g., T cells, NK cells, and/or TILs, are engineered to express a chimeric antigen receptor (CAR). In some aspects, the cells, e.g., T cells, NK cells, and/or TILs, are engineered to express an engineered T cell receptor (TCR). In certain aspects, culturing the cells, e.g., T cells, NK cells, and/or TILs, under the conditions disclosed herein, e.g., in a hypotonic or isotonic medium comprising at least about 5 mM potassium ion, results in higher transduction efficiency. In some aspects, transduction efficiency is at least
about 2-fold greater in cells, e.g., T cells, NK cells, and/or TILs, cultured in hypotonic or isotonic medium comprising at least about 60 mM potassium ion, according to the methods disclosed herein, as compared to cells, e.g., T cells, NK cells, and/or TILs, cultured in medium comprising 4 mM potassium ion or less. In some aspects, transduction efficiency is at least about 2.5-fold greater in cells, e.g., T cells, NK cells, and/or TILs, cultured in hypotonic or isotonic medium comprising at least about 65 mM potassium ion, according to the methods disclosed herein, as compared to cells, e.g., T cells, NK cells, and/or TILs, cultured in medium comprising 4 mM potassium ion or less. [0615] In some aspects, the cell comprises a construct expressing an antigen receptor and/or another additional polypeptide. In some aspects, the antigen receptor comprises an antibody, an engineered antibody such as scFv, a CAR, an engineered TCR, a TCR mimic (e.g., an antibody- T cell receptor (abTCR) or a chimeric antibody-T cell receptor (caTCR)), or a chimeric signaling receptor (CSR). By way of example, a TCR can comprise an engineered TCR in which the antigen- binding domain of a TCR (e.g., an alpha/beta TCR or a gamma/delta TCR) has been replaced by that of an antibody (with or without the antibody’s constant domains); the engineered TCR then becomes specific for the antibody’s antigen while retaining the TCR’s signaling functions. A chimeric signaling receptor can comprise (1) an extracellular binding domain (e.g., natural/modified receptor extracellular domain, natural/modified ligand extracellular domain, scFv, nanobody, Fab, DARPin, and affibody), (2) a transmembrane domain, and (3) an intracellular signaling domain (e.g., a domain that activates transcription factors, or recruits and/or activates JAK/STAT, kinases, phosphatases, and ubiquitin; SH3; SH2; and PDZ). See, e.g., EP340793B1, WO 2017/070608, WO 2018/200582, WO 2018/200583, WO 2018/200585, and Xu et al., Cell Discovery (2018) 4:62. [0616] In some aspects, the construct expressing an antigen receptor and/or another additional polypeptide comprises a regulatory element, and wherein a vector comprises the exogenous polynucleotide. In some aspects, the vector is a polycistronic expression vector. In some aspects, the regulatory element comprises a promoter. In some aspects, the promoter comprises a dl587rev primer-binding site substituted (MND) promoter, EF1a promoter, ubiquitin promoter, or combinations thereof. In some aspects, the vector comprises a viral vector, a mammalian vector, or a bacterial vector. In some aspects, the vector comprises an adenoviral vector, a lentivirus, a Sendai virus vector, a baculoviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, a hybrid vector, or an adeno associated virus (AAV) vector. In some aspects, the vector is a lentivirus.
[0617] In some aspects, the antigen receptor targets an antigen of interest (e.g., a tumor antigen or an antigen of a pathogen). The antigens can include, without limitation, AFP (alpha- fetoprotein), αvβ6 or another integrin, BCMA, B7-H3, B7-H6, Braf, 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, CD123, CD138, 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-α (fibroblast activation protein α), fetal AchR (fetal acetylcholine receptor), FBP (a folate binding protein), FCRL5, FR-α (folate receptor alpha), GCC (guanyl cyclase C), GD2, GD3, GPC2 (glypican-2), GPC3, gp100 (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)-A1, 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, 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), ROR1, ROR2, SIRPα (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. [0618] In certain aspects, the antigen receptor targets hTERT. In some aspects, the antigen receptor targets KRAS. In some aspects, the antigen receptor targets Braf. In some aspects, the antigen receptor targets TGFβRII. In some aspects, the antigen receptor targets MAGE A10/A4.
In some aspects, the antigen receptor targets AFP. In some aspects, the antigen receptor targets PRAME. In some aspects, the antigen receptor targets MAGE A1. In some aspects, the antigen receptor targets WT-1. In some aspects, the antigen receptor targets NY-ESO. In some aspects, the antigen receptor targets PRAME. In some aspects, the antigen receptor targets NY-ESO. In some aspects, the antigen receptor targets CD19. [0619] In some aspects, the antigen receptor targets BCMA. In some aspects, the antigen receptor targets CD147. In some aspects, the antigen receptor targets CD19. In some aspects, the antigen receptor targets CD19 and CD22. In some aspects, the antigen receptor targets CD19 and CD28. In some aspects, the antigen receptor targets CD20. In some aspects, the antigen receptor targets CD20 and CD19. In some aspects, the antigen receptor targets CD22. In some aspects, the antigen receptor targets CD30. In some aspects, the antigen receptor targets CEA. In some aspects, the antigen receptor targets DLL3. In some aspects, the antigen receptor targets EGFRvIII. In some aspects, the antigen receptor targets GD2. In some aspects, the antigen receptor targets HER2. In some aspects, the antigen receptor targets IL-1RAP. In some aspects, the antigen receptor targets mesothelin. In some aspects, the antigen receptor targets methothelin. In some aspects, the antigen receptor targets NKG2D. In some aspects, the antigen receptor targets PSMA. In some aspects, the antigen receptor targets TnMUC1. [0620] In some aspects, the cells, e.g., T cells and/or NK cells, are engineered before culturing according to the methods disclosed herein. In some aspects, the cells, e.g., T cells and/or NK cells, are engineered after culturing according to the methods disclosed herein. In some aspects, the cells, e.g., T cells and/or NK cells, are cultured according to the methods disclosed herein, e.g., in a hypotonic or isotonic medium comprising at least 5 mM potassium ion, prior to, during, and after cell engineering. In some aspects, the cells, e.g., T cells and/or NK cells, are engineered to express a chimeric antigen receptor (CAR). In some aspects, the cells, e.g., T cells and/or NK cells, are engineered to express an engineered T cell receptor (TCR). In certain aspects, culturing the cells, e.g., T cells and/or NK cells, under the conditions disclosed herein, e.g., in a hypotonic or isotonic medium comprising at least about 5 mM potassium ion, results in higher transduction efficiency. In some aspects, transduction efficiency is at least about 2-fold greater in cells, e.g., T cells and/or NK cells, cultured in hypotonic or isotonic medium comprising at least about 60 mM potassium ion, according to the methods disclosed herein, as compared to cells, e.g., T cells and/or NK cells, cultured in medium comprising 4 mM potassium ion or less. In some aspects, transduction efficiency is at least about 2.5-fold greater in cells, e.g., T cells and/or NK cells, cultured in hypotonic or isotonic medium comprising at least about 65 mM potassium ion,
according to the methods disclosed herein, as compared to cells, e.g., T cells and/or NK cells, cultured in medium comprising 4 mM potassium ion or less. [0621] Primary immune cells, including primary T cells, can be obtained from a number of tissue sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and/or tumor tissue. Leukocytes, including PBMCs, can be isolated from other blood cells by well-known techniques, e.g., FICOLL™ separation and leukapheresis. Leukapheresis products typically contain lymphocytes (including T and B cells), monocytes, granulocytes, and other nucleated white blood cells. T cells are further isolated from other leukocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of T cells, such as CD3+, CD25+, CD28+, CD4+, CD8+, CD45RA+, GITR+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques (e.g., using fluorescence-based or magnetic-based cell sorting). For example, T cells can be isolated by incubation with any of a variety of commercially available antibody-conjugated beads, such as Dynabeads®, CELLectionTM, DETACHaBEADTM (Thermo Fisher) or MACS® cell separation products (Miltenyi Biotec), for a time period sufficient for positive selection of the desired T cells or negative selection for removal of unwanted cells. [0622] In some instances, autologous T cells are obtained from a cancer patient directly following cancer treatment. It has been observed that following certain cancer treatments, in particular those that impair the immune system, the quality of T cells collected shortly after treatment can have an improved ability to expand ex vivo and/or to engraft after being engineered ex vivo. II.D.1.i. Chimeric Antigen Receptor (CAR) [0623] In some aspects, the cell, e.g., human immune cell, e.g., T cell (αβ T cell or γδ T cell) and/or NK cell, comprises a CAR. In some aspects, the cell that can be prepared to express a CAR (e.g., a CAR T cell) is, e.g., a CD8+ T cell or CD4+ T cell. In some aspects, a CAR-expressing cell disclosed herein is a CAR T cell, e.g., a mono CAR T cell, a genome-edited CAR T cell, a dual CAR T cell, or a tandem CAR T cell. Examples of such CAR T cells are provided in International Application No. PCT/US2019/044195. [0624] In some aspects, the CAR is designed as a standard CAR, a split CAR, an off-switch CAR, an on-switch CAR, a first-generation CAR, a second-generation CAR, a third-generation CAR, or a fourth-generation CAR. In some aspects, the CAR comprises antigen-binding domain,
a transmembrane domain, a costimulatory domain, an intracellular signaling domain, or combinations thereof. [0625] In some aspects, the CAR specifically binds (i.e., target) one or more antigens expressed on a tumor cell, such as a malignant B cell, a malignant T cell, or a malignant plasma cell. [0626] In some aspects, the CAR specifically binds to (i.e., targets) an antigen comprising AFP (alpha-fetoprotein), αvβ6 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, CD123, CD138, 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-α (fibroblast activation protein α), fetal AchR (fetal acetylcholine receptor), FBP (a folate binding protein), FCRL5, FR-α (folate receptor alpha), GCC (guanyl cyclase C), GD2, GD3, GPC2 (glypican-2), GPC3, gp100 (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)-A1, 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, KLK2, 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), ROR1, ROR2, SIRPα (signal-regulatory protein alpha), SLIT, SLITRK6 (NTRK-like protein 6), STEAP1 (six transmembrane epithelial antigen of the prostate 1), STEAP2, survivin, TAG72 (tumor-associated glycoprotein 72), TPBG (trophoblast
glycoprotein), Trop-2, VEGFR1 (vascular endothelial growth factor receptor 1), VEGFR2, or antigens from HIV, HBV, HCV, HPV, or other pathogens, or any combination thereof. [0627] In some aspects, the CAR specifically binds ROR1. An exemplary anti-ROR1 CAR that can be expressed in an immune cell described herein is described in Hudecek, et al., Clin. Cancer Res. 19.12(2013):3153-64, which is incorporated herein by reference in its entirety. In some aspects, an immune cell modified to comprise an anti-ROR1 CAR is generated as described in Hudecek et al. (for example, as described in Hudecek et al. at page 3155, first full paragraph, incorporated herein by reference in its entirety). In some aspects, the spacer disclosed in Hudecek has been replaced by a different spacer (e.g., such as those described herein). In some aspect, an anti-ROR1 CAR useful for the present disclosure comprises an antibody or fragment thereof, which comprises the VH and/or VL sequences of the 2A2, R11, and R12 anti-ROR1 monoclonal antibodies described in Hudecek et al. at paragraph bridging pages 3154-55; Baskar et al. MAbs 4(2012):349-61; and Yang et al. PLoS ONE 6(2011):e21018, each of which is incorporated herein by reference in their entirety. [0628] In some aspects, the CAR specifically binds GPC2. [0629] 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, OX40, DAP10, or any combination thereof. In some aspects, the costimulatory domain comprises a 4-1BB/CD137 costimulatory domain. [0630] In some aspects, the transmembrane domain comprises a transmembrane domain of KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, 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, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, NKG2C, CD19, or any combination thereof. In some aspects, the transmembrane domain comprises a CD28 transmembrane domain. [0631] In some aspects, the intracellular signaling domain comprises an intracellular signaling domain derived from CD3 zeta, FcR gamma, common FcR gamma (FCER1G), Fc
gamma RIIa, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD22, CD79a, CD79b, CD278 (“ICOS”), FcεRI, CD66d, CD32, DAP10, DAP12, or any combination thereof. In some aspects, the intracellular signaling domain comprises a CD3 zeta intracellular signaling domain. II.D.1.ii. T Cell Receptor-Engineered (TCR) Cells [0632] In some aspects, an immune cell, e.g., a T cell (αβ T cell or γδ T cell) and/or an NK cell, disclosed herein comprises a T cell receptor (TCR), e.g., an engineered TCR. In some aspects, the TCR specifically binds to a tumor antigen. As used herein, the term "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, NK cells, and/or TILs. In some aspects, the TCR specifically binds a neoantigen identified from a cancer patient. [0633] In some aspects, the TCR specifically binds (i.e., targets) one or more antigens expressed on a tumor cell, such as a malignant B cell, a malignant T cell, or a malignant plasma cell. In some aspects, the TCR specifically binds a tumor antigen/MHC complex. In some aspects, the tumor antigen is derived from AFP, CD19, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL- 13Ra2, mesothelin, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WTl, NY-ESO-1, LAGE-la, MAGE-Al, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT- 2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA- 1/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, 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, CD3ε, CD4, CD5, CD7, the extracellular portion of the APRIL protein, neoantigen, or any combinations thereof. In some aspects, the TCR specifically binds (i.e., targets) a tumor antigen derived from NY-ESO-1. [0634] In certain aspects, an engineered cell of the present disclosure can express a T cell receptor (TCR) targeting an antigen. In some aspects, the TCR is an αβ TCR. In some aspects, the TCR is a γδ T cell). In some aspects, the TCR engineered cells can target main types: shared tumor- associated antigens (shared TAAs) and unique tumor-associated antigens (unique TAAs), or tumor-specific antigens. The former can include, without any limitation, cancer-testis (CT) antigens, overexpressed antigens, and differentiation antigens, while the latter can include, without any limitation, neoantigens and oncoviral antigens. Human papillomavirus (HPV) E6 protein and HPV E7 protein belong to the category of oncoviral antigens. [0635] In some aspects, the TCR engineered cells can target a CT antigen, e.g., melanoma- associated antigen (MAGE) including, but not limited to, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A8, MAGE-A9.23, MAGE-A10, and MAGE-A12. In some aspects, the TCR engineered cells can target glycoprotein (gp100), melanoma antigen recognized by T cells (MART-1), and/or tyrosinase, which are mainly found in melanomas and normal melanocytes. In some aspects, the TCR engineered cells can target Wilms tumor 1 (WT1), i.e., one kind of overexpressed antigen that is highly expressed in most acute myeloid leukemia (AML), acute lymphoid leukemia, almost every type of solid tumor and several critical tissues, such as heart tissues. In some aspects, the TCR engineered cells can target mesothelin, another kind of overexpressed antigen that is highly expressed in mesothelioma but is also present on mesothelial cells of several tissues, including trachea. [0636] In some aspects, the TCR engineered cells can target any neoantigen, which can be formed by random somatic mutations specific to individual tumors. In some aspects, the TCR specifically binds to (i.e., targets) a cancer antigen comprising AFP, Braf, CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL- 13Ra2, mesothelin, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2/neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2,
TARP, WTl, NY-ESO-1, LAGE-la, MAGE-Al, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT- 2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA- 1/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY- TES1, LCK, AKAP-4, SSX2, RAGE-1, 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, CD3ε, CD4, CD5, CD7, the extracellular portion of the APRIL protein, or any combinations thereof. [0637] In certain aspects, the TCR specifically binds (i.e., targets) hTERT. In some aspects, the TCR specifically binds (i.e., targets) KRAS. In some aspects, the TCR specifically binds (i.e., targets) Braf. In some aspects, the TCR specifically binds (i.e., targets) TGFβRII. In some aspects, the TCR specifically binds (i.e., targets) MAGE A10/A4. In some aspects, the TCR specifically binds (i.e., targets) AFP. In some aspects, the TCR specifically binds (i.e., targets) PRAME. In some aspects, the TCR specifically binds (i.e., targets) MAGE A1. In some aspects, the TCR specifically binds (i.e., targets) WT-1. In some aspects, the TCR specifically binds (i.e., targets) NY-ESO. In some aspects, the TCR specifically binds (i.e., targets) PRAME. In some aspects, the TCR specifically binds (i.e., targets) NY-ESO. In some aspects, the TCR specifically binds (i.e., targets) CD19. In certain aspects, the TCR specifically binds a neoantigen identified from a cancer patient. [0638] In some aspects, the TCR comprises an intracellular gamma/delta domain. In some aspects, the TCR is an antibody-T-cell receptor (AbTCR) (see, e.g., Xu et al., Cell Discovery 4:62 (2018), which is incorporated by reference herein in its entirety. II.D.1.iii. T Cell Receptor Mimics (TCRm) [0639] In some aspects, an immune cell, e.g., a T cell (αβ T cell or γδ T cell) and/or an NK cell, disclosed herein comprises a T cell receptor mimic (TCRm), also known as a TCR-like antibody. TCRm are a type of antibody that recognize epitopes comprising both the peptide and the MHC-I molecule, similar to the recognition of such complexes by the TCR on T cells (see, e.g., Traneska et al., Front. Immunol.8(1001):1-12 (2017), which is incorporated by reference herein in its entirety). In some aspects, the TCRm specifically binds to a tumor antigen. In certain aspects, the TCRm specifically binds a neoantigen identified from a cancer patient.
[0640] In some aspects, the TCRm specifically binds (i.e., target) one or more antigens expressed on a tumor cell, such as a malignant B cell, a malignant T cell, or a malignant plasma cell. In some aspects, the TCRm is a monoclonal antibody. In some aspects, the TCRm specifically binds to WT1. In some aspects, the TCRm specifically binds to a fragment of WT1. In some aspects, the TCRm comprises ESK1 (see, e.g., Ataie et al., J. Mol. Biol. 428(1):194-205 (2016), which is incorporated by reference herein in its entirety). In some aspects, the TCRm specifically binds to MAGE-A1. In some aspects, the TCRm specifically binds to p68 RNA helicase/HLA- A*02:01. In some aspects, the TCRm specifically binds to hCG-b/HLAA*02:01. In some aspects, the TCRm specifically binds to Her2-E75/HLA-A*02:01. In some aspects, the TCRm specifically binds to PR-1 in context of HLA-A*02:01 (see, e.g., Oncoimmunology 5(1):e1049803 (June 2015), which is incorporated by reference herein in its entirety). In some aspects, the TCRm specifically binds to the survivin-2B-derived nonamer peptide, AYACNTSTL (SV2B80-88), presented on HLA-A*24 (SV2B80-88/HLA-A*24) (see, e.g., Kurosawa et al., Nature Scientific Reports 9(9827):1- (2019), which is incorporated by reference herein in its entirety). In some aspects, the TCRm specifically binds one or more tumor-associated PRAME peptide/HLA-I antigens (see, e.g., J Clin Invest.127(7):2705-18 (2017), which is incorporated by reference herein in its entirety). In some aspects, the TCRm specifically binds to tyrosinase. In some aspects, the TCRm specifically binds telomerase catalytic subunit. In some aspects, the TCRm specifically binds to glycoprotein 100 (gp100). In some aspects, the TCRm specifically binds to mucin 1 (MUC1). In some aspects, the TCRm specifically binds to human telomerase reverse transcriptase (hTERT). In some aspects, the TCRm specifically binds to NYESO-1. In some aspects, the TCRm specifically binds to MART-1. In some aspects, the TCRm specifically binds to PRAME. [0641] In some aspects, the TCRm specifically binds to a viral antigen. In some aspects, the TCRm specifically binds to Env183/A2 (Hep B/HLA-A*02:01). In some aspects, the TCRm specifically binds to KP14/1 and KP15/11 (HIV envelope gp160/HLAA*02:01). In some aspects, the TCRm specifically binds to RL36A (West Nile Virus/mouse H-2Db). In some aspects, the TCRm specifically binds to a viral epitope derived from HTLV. In some aspects, the TCRm specifically binds to a viral epitope derived from influenza. In some aspects, the TCRm specifically binds to a viral epitope derived from CMV. In some aspects, the TCRm specifically binds to a viral epitope derived from HIV.
II.D.2. TILs [0642] The TILs cultured according to the methods disclosed herein can be TILs that are collected and/or isolated from a subject in need of a therapy. In some aspects, the TILs that are placed in the medium have been expanded prior to being subjected to the methods disclosed herein. The TILs that are placed in the initial expansion medium can be referred to as starting (initial, i.e., patient sample, apheresis sample, buffy coat) TILs. The TILs that result from culturing them according to the methods disclosed herein can be referred to as resulting (cultured) TILs. [0643] In some aspects, the proportion of CD8+ TILs to non-CD8+ TILs (e.g., the proportion of CD8+ TILs to CD4+ TILs) is increased following the culture in the initial expansion medium, as compared to the proportion of CD8+ TILs to non-CD8+ TILs prior to the culture in the initial expansion medium. In some aspects, the proportion of CD8+ TILs to non-CD8+ TILs (e.g., the proportion of CD8+ TILs to CD4+ TILs) is increased following the culture in the initial expansion medium, as compared to the proportion of CD8+ TILs to non-CD8+ TILs following an culture in the initial expansion medium in a basal medium or a medium that does not comprise an increased concentration of potassium ion (control medium). In some aspects, the proportion of CD8+ TILs is increased by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 45-fold, or at least about 50-fold. In some aspects, the proportion of CD8+ TILs is increased by at least about 40-fold. In some aspects, the proportion of CD8+ TILs is increased by at least about 50-fold. [0644] In some aspects, the proportion of CD8+ TILs is increased by at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500%. In some aspects, the proportion of CD8+ TILs is increased by at least about 20%. In some aspects, the proportion of CD8+ TILs is increased by at least about 40%. In some aspects, the proportion of CD8+ TILs is increased by at least about 60%. In some aspects, the proportion of CD8+ TILs is increased by at least about 80%. In some aspects, the proportion of CD8+ TILs is increased by at least about 100%.
[0645] In some aspects, the proportion of CD8+ TILs is increased to 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 96%, at least about 97%, at least about 98%, or at least about 99% of the total number of TILs in the culture. In some aspects, the proportion of CD8+ TILs is increased to at least about 20% of the total number of TILs in the culture. In some aspects, the proportion of CD8+ TILs is increased to at least about 30% of the total number of TILs in the culture. In some aspects, the proportion of CD8+ TILs is increased to at least about 40% of the total number of TILs in the culture. In some aspects, the proportion of CD8+ TILs is increased to at least about 50% of the total number of TILs in the culture. In some aspects, the proportion of CD8+ TILs is increased to at least about 60% of the total number of TILs in the culture. In some aspects, the proportion of CD8+ TILs is increased to at least about 70% of the total number of TILs in the culture. In some aspects, the proportion of CD8+ TILs is increased to at least about 75% of the total number of TILs in the culture. In some aspects, the proportion of CD8+ TILs is increased to at least about 80% of the total number of TILs in the culture. In some aspects, the proportion of CD8+ TILs is increased to at least about 90% of the total number of TILs in the culture. [0646] In some aspects, the number of tumor-reactive cells in the culture is increased by about 2-fold to about 500-fold, about 2-fold to about 250-fold, about 2-fold to about 200-fold, about 2-fold to about 150-fold, about 2-fold to about 100-fold, about 2-fold to about 90-fold, about 2-fold to about 80-fold, about 2-fold to about 70-fold, about 2-fold to about 60-fold, about 2-fold to about 50-fold, about 2-fold to about 40-fold, about 2-fold to about 30-fold, about 2-fold to about 20-fold, about 2-fold to about 10-fold, about 5-fold to about 200-fold, about 5-fold to about 150- fold, about 5-fold to about 100-fold, about 5-fold to about 90-fold, about 5-fold to about 80-fold, about 5-fold to about 70-fold, about 5-fold to about 60-fold, about 5-fold to about 50-fold, about 5-fold to about 40-fold, about 5-fold to about 30-fold, about 5-fold to about 20-fold, about 5-fold to about 10-fold, about 10-fold to about 150-fold, about 10-fold to about 100-fold, about 10-fold to about 90-fold, about 10-fold to about 80-fold, about 10-fold to about 70-fold, about 10-fold to about 60-fold, about 10-fold to about 50-fold, about 10-fold to about 40-fold, about 10-fold to about 30-fold, or about 10-fold to about 20-fold following the culture methods disclosed herein, as compared to the number of tumor-reactive cells prior to the culture in the initial expansion medium. In some aspects, the number of tumor-reactive cells in the culture is increased by at least about 2- fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least
about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-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, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 125-fold, at least about 150-fold, at least about 175-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 450-fold, or at least about 500-fold following the culture methods disclosed herein, as compared to the number of tumor-reactive cells prior to the culture in the initial expansion medium. In some aspects, the number of tumor-reactive cells in the culture is increased by at least about 2- fold following the culture methods disclosed herein, as compared to the number of tumor-reactive cells prior to the culture in the initial expansion medium. In some aspects, the number of tumor- reactive cells in the culture is increased by at least about 3-fold following the culture methods disclosed herein, as compared to the number of tumor-reactive cells prior to the culture in the initial expansion medium. In some aspects, the number of tumor-reactive cells in the culture is increased by at least about 4-fold following the culture methods disclosed herein, as compared to the number of tumor-reactive cells prior to the culture in the initial expansion medium. In some aspects, the number of tumor-reactive cells in the culture is increased by at least about 5-fold following the culture methods disclosed herein, as compared to the number of tumor-reactive cells prior to the culture in the initial expansion medium. In some aspects, the number of tumor-reactive cells in the culture is increased by at least about 10-fold following the culture methods disclosed herein, as compared to the number of tumor-reactive cells prior to the culture in the initial expansion medium. [0647] In some aspects, the number of tumor-reactive cells in the culture is increased by about 2-fold to about 500-fold, about 2-fold to about 250-fold, about 2-fold to about 200-fold, about 2-fold to about 150-fold, about 2-fold to about 100-fold, about 2-fold to about 90-fold, about 2-fold to about 80-fold, about 2-fold to about 70-fold, about 2-fold to about 60-fold, about 2-fold to about 50-fold, about 2-fold to about 40-fold, about 2-fold to about 30-fold, about 2-fold to about 20-fold, about 2-fold to about 10-fold, about 5-fold to about 200-fold, about 5-fold to about 150- fold, about 5-fold to about 100-fold, about 5-fold to about 90-fold, about 5-fold to about 80-fold, about 5-fold to about 70-fold, about 5-fold to about 60-fold, about 5-fold to about 50-fold, about 5-fold to about 40-fold, about 5-fold to about 30-fold, about 5-fold to about 20-fold, about 5-fold to about 10-fold, about 10-fold to about 150-fold, about 10-fold to about 100-fold, about 10-fold to about 90-fold, about 10-fold to about 80-fold, about 10-fold to about 70-fold, about 10-fold to about 60-fold, about 10-fold to about 50-fold, about 10-fold to about 40-fold, about 10-fold to
about 30-fold, or about 10-fold to about 20-fold following the culture methods disclosed herein, as compared to the number of tumor-reactive cells following expansion using control methods . In some aspects, the number of tumor-reactive cells in the culture is increased by at least about 2- fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-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, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 125-fold, at least about 150-fold, at least about 175-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 450-fold, or at least about 500-fold following the culture methods disclosed herein, as compared to the number of tumor-reactive cells following expansion using control methods. In some aspects, the number of tumor-reactive cells in the culture is increased by at least about 2-fold following the culture methods disclosed herein, as compared to the number of tumor-reactive cells following expansion using control methods. In some aspects, the number of tumor-reactive cells in the culture is increased by at least about 3-fold following the culture methods disclosed herein, as compared to the number of tumor-reactive cells following expansion using control methods. In some aspects, the number of tumor-reactive cells in the culture is increased by at least about 4-fold following the culture methods disclosed herein, as compared to the number of tumor-reactive cells following expansion using control methods. In some aspects, the number of tumor-reactive cells in the culture is increased by at least about 5-fold following the culture methods disclosed herein, as compared to the number of tumor-reactive cells following expansion using control methods. In some aspects, the number of tumor-reactive cells in the culture is increased by at least about 10- fold following the culture methods disclosed herein, as compared to the number of tumor-reactive cells following expansion using control methods. [0648] In some aspects, the tumor sample is isolated from a human subject. In some aspects, the starting tumor sample isolated from a human subject, and the TILs therein are expanded for an allogeneic cell therapy. In some aspects, the tumor sample is isolated from a human subject, and the TILs therein are expanded for an autologous cell therapy.
III. Compositions of the Disclosure III.A. Cell Compositions [0649] Certain aspects of the present disclosure are directed to a cell composition comprising a population of immune cells (e.g., T cells, NK cells, and/or TILs) cultured according to the methods disclosed herein. Cell populations cultured according to the methods and/or in a metabolic reprogramming medium disclosed herein have an increased number of less- differentiated cells as compared to comparable cells cultured according to conventional methods. In some aspects, the cells cultured according to the methods disclosed herein exhibit increased expression of one or more marker typical of a stem-like phenotype. In some aspects, cell populations cultured according to the methods and/or in a metabolic reprogramming medium disclosed herein have an increased number of effector-like cells as compared to comparable cells cultured according to conventional methods. In some aspects, cell populations cultured according to the methods and/or in a metabolic reprogramming medium disclosed herein have both an increased number of stem-like and effector-like cells as compared to comparable cells cultured according to conventional methods. In some aspects, the cells cultured according to the methods disclosed herein exhibit greater proliferative potential compared to cells cultured according to conventional methods. In some aspects, the cells cultured according to the methods disclosed herein exhibit increased transduction efficiency. In some aspects, the cells cultured according to the methods disclosed herein exhibit increased in vivo viability upon transplantation in a subject. In some aspects, the cells cultured according to the methods disclosed herein exhibit increased cell potency. In some aspects, the cells cultured according to the methods disclosed herein exhibit decreased cell exhaustion. In some aspects, the cells cultured according to the methods disclosed herein exhibit increased in vivo persistence upon transplantation in a subject. In some aspects, the cells cultured according to the methods disclosed herein exhibit increased in vivo activity upon transplantation in a subject. In some aspects, the cells cultured according to the methods disclosed herein exhibit a more durable in vivo response upon transplantation in a subject. In some aspects, the subject is a human. [0650] In some aspects, at least about 5% of the cells in the cell composition have a stem- like phenotype. In some aspects, at least about 10% of the cells in the cell composition have a stem- like phenotype. In some aspects, at least about 15% of the cells in the cell composition have a stem- like phenotype. In some aspects, at least about 20% of the cells in the cell composition have a stem- like phenotype. In some aspects, at least about 25% of the cells in the cell composition have a stem-
like phenotype. In some aspects, at least about 30% of the cells in the cell composition have a stem- like phenotype. In some aspects, at least about 35% of the cells in the cell composition have a stem- like phenotype. In some aspects, at least about 40% of the cells in the cell composition have a stem- like phenotype. In some aspects, at least about 45% of the cells in the cell composition have a stem- like phenotype. In some aspects, at least about 50% of the cells in the cell composition have a stem- like phenotype. In some aspects, at least about 55% of the cells in the cell composition have a stem- like phenotype. In some aspects, at least about 60% of the cells in the cell composition have a stem- like phenotype. In some aspects, at least about 65% of the cells in the cell composition have a stem- like phenotype. In some aspects, at least about 70% of the cells in the cell composition have a stem- like phenotype. [0651] In some aspects, following culture of the immune cells (e.g., T cells, NK cells, and/or TILs) according to the methods disclosed herein, stem-like immune cells (e.g., T cells, NK cells, and/or TILs) constitute at least about 10% to at least about 70% of the total number of immune cells in the culture. In some aspects, following culture of the immune cells (e.g., T cells, NK cells, and/or TILs) according to the methods disclosed herein, stem-like immune cells (e.g., T cells, NK cells, and/or TILs) constitute at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% of the total number of CD8+ cells, e.g., CD8+ T cells, in the culture. In some aspects, following culture of the immune cells (e.g., T cells, NK cells, and/or TILs) according to the methods disclosed herein, stem-like immune cells (e.g., T cells, NK cells, and/or TILs) constitute at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% of the total number of CD4+ cells, e.g., CD4+ T cells, in the culture. [0652] In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 1.5-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 2.0-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 2.5-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 3.0-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 3.5-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the
number of cells having a stem-like phenotype in the cell composition is increased at least about 4.0-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 4.5-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 5.0-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 5.5-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 6.0-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 6.5-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 7.0-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 7.5-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 8.0-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 9.0-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 10-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 15-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 20-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 30-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 40-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 50-fold as
compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 75-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 100-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 500-fold as compared to the number of cells in the cell composition prior to the culture. In some aspects, the number of cells having a stem-like phenotype in the cell composition is increased at least about 1000-fold as compared to the number of cells in the cell composition prior to the culture. [0653] In some aspects, following culture of immune cells (e.g., T cells, NK cells, and/or TILs) according to the methods disclosed herein, at least about 10% to at least about 70% of the total number of immune cells in the culture are CD39-/TCF7+ cells. In some aspects, following culture of immune cells (e.g., T cells, NK cells, and/or TILs) according to the methods disclosed herein, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, or at least about 40% of the total number of immune cells in the culture are CD39-/TCF7+ cells. In some aspects the immune cells are CD4+ cells, e.g., CD4+ T cells. In some aspects the immune cells are CD8+ cells, e.g., CD8+ T cells. [0654] In some aspects, the cell composition comprises immune cells, e.g., T cells, NK cells, and/or TILs. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express CD95. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which do not express CD45R0. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express CD45RA. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express CCR7. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express CD62L. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express TCF7. In some aspects, the cell composition comprises an in the increase percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express CD3. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express CD27. In some aspects, the cell composition comprises an in the increase percent of immune cells, e.g., T cells, NK cells,
and/or TILs, which express CD95 and CD45RA. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express CD45RA and CCR7. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express CD95, CD45RA, and CCR7. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express CD45RA, CCR7, and CD62L. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express CD95, CD45RA, CCR7, and CD62L. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express CD45RA, CCR7, CD62L, and TCF7. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express CD95, CD45RA, CCR7, CD62L, and TCF7. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express CD45RA, CCR7, CD62L, TCF7, and CD27. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express CD95, CD45RA, CCR7, CD62L, TCF7, and CD27. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express, CD45RA, CCR7, CD62L, TCF7, and CD27, and which do not express CD45RO or which are CD45ROlow. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express CD95, CD45RA, CCR7, CD62L, TCF7, and CD27, and which do not express CD45RO or which are CD45ROlow. [0655] In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which do not express CD39 and CD69. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express CD8, and which do not express CD39 and CD69. In some aspects, following culture of immune cells according to the methods disclosed herein, at least about 10% to at least about 40% of the total number of immune cells in the culture are CD39-/CD69- cells, e.g., CD39-/CD69- T cells. In some aspects, following culture of immune cells according to the methods disclosed herein, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, or at least about 40% of the total number of T cells in the culture are CD39-/CD69- cells, e.g., CD39-/CD69- T cells. [0656] In some aspects, the cell composition comprises an increased percentage of immune cells, e.g., T cells, NK cells, and/or TILs, which express both (i) one or more stem-like markers
and (ii) one or more effector-like markers. In some aspects, the cell composition comprises an increased percentage of immune cells, e.g., T cells, NK cells, and/or TILs, which express at least two stem-like markers and one or more effector-like markers. In some aspects, the cell composition comprises an increase percent of immune cells, e.g., T cells, NK cells, and/or TILs, which express at least three stem-like markers and one or more effector-like markers. In some aspects, the cell composition comprises an increased percentage of immune cells, e.g., T cells, NK cells, and/or TILs, which express at least four stem-like markers and one or more effector-like markers. In some aspects, the cell composition comprises an increased percentage of immune cells, e.g., T cells, NK cells, and/or TILs, which express one or more stem-like markers and at least two effector-like markers. [0657] In some aspects, the stem-like markers are selected from CD45RA+, CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF1+, TCF7+, and any combination thereof. In some aspects the stem-like markers comprise CD45RA+, CD62L+, CCR7+, and TCF7+, or any combination thereof. In some aspects, the cell expresses CD45ROlow. In some aspects, the stem-like markers comprise one or more genes listed herein as part of a gene-signature (see supra; see, e.g., Gattinoni, L., et al., Nat Med 17(10): 1290-97 (2011) or Galletti et al. Nat Immunol 21, 1552-62 (2020)). [0658] In some aspects, the stem-like markers comprise a gene expressed in the WNT signaling pathway. In some aspects, the stem-like markers comprise one or more genes selected from GNG2, PSMC3, PSMB10, PSMC5, PSMB8, PSMB9, AKT1, MYC, CLTB, PSME1, DVL2, PFN1, H2AFJ, LEF1, CTBP1, MOV10, HIST1H2BD, FZD3, ITPR3, PARD6A, LRP5, HIST2H4A, HIST2H3C, HIST1H2AD, HIST2H2BE, HIST3H2BB, DACT1, and any combination thereof. In some aspects, the stem-like markers comprise one or more genes selected from MYC, AKT1, LEF1, and any combination thereof. [0659] In some aspects, the effector-like markers are selected from pSTAT5+, STAT5+, pSTAT3+, STAT3+, and any combination thereof. In some aspects, the effector-like marker comprises a STAT target comprising 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, IL11RA, 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, SOS1, SOS2, SPRED1, SPRED2, SPRY1, SPRY2, SPRY3, SPRY4, STAM, STAM2, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, STAT6, TPO, TSLP, TYK2, or any combination thereof. [0660] In some aspects, the effector-like markers are effector memory-associated genes that comprise one or more genes selected from TBCD, ARL4C, KLF6, LPGAT1, LPIN2, WDFY1, PCBP4, PIK343, FAS, LLGL2, PPP2R2B, TTC39C, GGA2, LRP8, PMAIP1, MVD, IL15RA, FHOD1, EML4, PEA15, PLEKHA5, WSB2, PAM, CD68, MSC, TLR3, S1PR5, KLRB1, CYTH3, RAB27B, SCD5, and any combination thereof. In some aspects, the effector-like markers comprise one or more genes selected from KLF6, FAS, KLRB1, TLR3, and any combination thereof. [0661] In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, that are CD45RA+, STAT5+, and STAT3+. In some aspects, the cell composition comprises an increase in the percent of immune cells e.g., T cells and/or NK cells, that are CD62L+, STAT5+, and STAT3+. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, that are TCF7+, STAT5+, and STAT3+. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, that are CD45RA+, CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF1+, TCF7+, STAT5+, and STAT3+. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, that are CD45RA+, CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF1+, TCF7+, pSTAT5+, STAT5+, pSTAT3+, and STAT3+. In some aspects, the cell composition comprises an increase in the percent of immune cells, e.g., T cells, NK cells, and/or TILs, that are CD45RA+, CD45RO-, CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF1+, TCF7+, pSTAT5+, STAT5+, pSTAT3+, and STAT3+. [0662] In some aspects, an immune cell, e.g., T cells, NK cells, and/or TILs, comprises one or more markers selected from CD45RA+, CD62L+, CCR7+, CD27+, CD28+, BACH2+, LEF1+, TCF7+, and any combination thereof and one or more markers selected from pSTAT5+, STAT5+, pSTAT3+, STAT3+, and any combination thereof. In some aspects, the immune cell, e.g., T cells, NK cells, and/or TILs, expresses CD45ROlow. In some aspects, an immune cell, e.g., T cells, NK cells, and/or TILs, comprises one or more markers selected from CD45RA+, CD62L+, CCR7+,
CD27+, CD28+, BACH2+, LEF1+, TCF7+, and any combination thereof and one or more effector-like markers. In some aspects, an immune cell, e.g., T cells, NK cells, and/or TILs, comprises one or more stem-like markers and one or more markers selected from pSTAT5+, STAT5+, pSTAT3+, STAT3+, and any combination thereof. In some aspects, the immune cell, e.g., T cells and/or NK cells, expresses CD45ROlow. [0663] Some aspects of the present disclosure are directed to a cell composition comprising a population of immune cells, wherein the population of immune cells comprises (i) a first sub- population of immune cells expressing one or more stem-like markers (e.g., stem-like immune cells) and (ii) a second sub-population of immune cells expressing one or more effector-like marker (e.g., effector-like immune cells), wherein the population of immune cells comprises a higher percentage (i.e., the number of stem-like immune cells/the total number of immune cells) of the first sub-population of immune cells expressing one or more stem-like markers, as compared to a population of immune cells cultured using conventional methods, e.g., in a medium having less than 5 mM potassium ion. In some aspects the immune cells are T cells. In some aspects the immune cells are NK cells. In some aspects, the immune cells, e.g., T cells, NK cells, and/or TILs, cultured according to the methods disclosed herein result in these cell compositions. [0664] In some aspects, immune cells, e.g., T cells, NK cells, and/or TILs, cultured according to the methods disclosed herein have increased expression, e.g., a higher percentage of immune cells, e.g., T cells, NK cells, and/or TILs, that express, GZMB, MHC-II, LAG3, TIGIT, and/or NKG7, and decreased expression, e.g., a lower percentage of immune cells, e.g., T cells, NK cells, and/or TILs, that express, IL-32. Cells highest for NKG7 have been shown to be better killers (Malarkannan et al.2020 Nat. Immuno.), whereas cells higher in IL-32 have been shown to have activation-induced cell death (Goda et al., 2006 Int. Immunol). In some aspects the immune cells, e.g., T cells, NK cells, and/or TILs, with higher expression of GZMB, MHC-II, LAG3, TIGIT, and/or NKG7 are CD8+ T cells expressing effector-like markers. In some aspects the immune cells, e.g., T cells, NK cells, and/or TILs, with lower expression of IL-32 are CD8+ T cells expressing effector-like markers. [0665] In some aspects, the cell composition comprises one or more immune cell, e.g., T cells and/or NK cells, which is genetically engineered. In some aspects, the cell composition comprises one or more immune cell, e.g., T cells and/or NK cells, which is engineered to express a chimeric antigen receptor (CAR). Any CAR disclosed herein can be used in the cells of the cell composition.
[0666] In some aspects, the cell composition comprises one or more immune cell, e.g., T cells and/or NK cells, which is engineered to express a T cell receptor (TCR), e.g., an engineered TCR. Any TCR disclosed herein can be used in the cells of the cell composition. [0667] In some aspects, the cell composition comprises one or more immune cell, e.g., T cells and/or NK cells, which is engineered to express a TCRm. Any TCRm disclosed herein can be used in the cells of the cell composition. [0668] In some aspects, the cell composition, obtained by any method described herein (e.g., the yield of the final cell product for use as a therapy), comprises at least about 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, or 5 x 109 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 1 x 103, 5 x 103, 1 x 104, 5 x 104, 1 x 105, 5 x 105, 1 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108, 5 x 108, 1 x 109, or 5 x 109 stem-like cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 5 x 109, 6 x 109, 7 x 109, 8 x 109, 9 x 109, 1 x 1010, 2 x 1010, 3 x 1010, 4 x 1010, 5 x 1010, 6 x 1010, 7 x 1010, 8 x 1010, 9 x 1010, 10 x 1010, 11 x 1010, 12 x 1010, 13 x 1010, 14 x 1010, or 15 x 1010 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 1 x 106 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 1 x 106 stem-like cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 1 x 1010 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 2 x 1010 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 3 x 1010 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 4 x 1010 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 5 x 1010 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 6 x 1010 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 7 x 1010 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 8 x 1010 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 9 x 1010 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 10 x 1010 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 11 x 1010 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 12 x 1010 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least
about 13 x 1010 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 14 x 1010 cells. In some aspects, the cell composition, obtained by any method described herein, comprises at least about 15 x 1010 cells. In some aspects, cell yield represents the total number of CD3+ cells. [0669] In some aspects, the methods disclosed herein yield a composition comprising at least about 1 x 1010, at least about 1.1 x 1010, at least about 1.2 x 1010, at least about 1.3 x 1010, at least about 1.4 x 1010, at least about 1.5 x 1010, at least about 1.6 x 1010, at least about 1.7 x 1010, at least about 1.8 x 1010, at least about 1.9 x 1010, or at least about 2.0 x 1010 cells by at least about day 10 of culturing in the presently disclosed medium. In some aspects, the methods disclosed herein yield a composition comprising at least about 1.8 x 1010 cells by at least about day 10 of culturing in the presently disclosed medium. [0670] In some aspects, the cell composition comprises at least about 1 x 1010, at least about 1.1 x 1010, at least about 1.2 x 1010, at least about 1.3 x 1010, at least about 1.4 x 1010, at least about 1.5 x 1010, at least about 1.6 x 1010, at least about 1.7 x 1010, at least about 1.8 x 1010, at least about 1.9 x 1010, or at least about 2.0 x 1010 stem-like cells. In some aspects, the methods disclosed herein yield a composition comprising at least about 1 x 1010, at least about 1.1 x 1010, at least about 1.2 x 1010, at least about 1.3 x 1010, at least about 1.4 x 1010, at least about 1.5 x 1010, at least about 1.6 x 1010, at least about 1.7 x 1010, at least about 1.8 x 1010, at least about 1.9 x 1010, or at least about 2.0 x 1010 stem-like cells by at least about day 10 of culture. In some aspects, the methods disclosed herein yield a composition comprising at least about 1.8 x 1010 stem-like cells by at least about day 10 of culturing in the presently disclosed medium. [0671] In some aspects, the methods disclosed herein yield a composition comprising immune cells that are at least about 80%, at least about 85%, at least about 90%, 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% viable. In some aspects, the methods disclosed herein yield a composition comprising at least about 1.8 x 1010 stem-like cells with at least about 94% cell viability. [0672] Some aspects of the present disclosure are directed to a population of TILs cultured according to any of the methods disclosed herein. In some aspects, the TILs are tumor-infiltrating T cells. In some aspects, the TILs comprise both CD4+ T cells and CD8+ T cells. In some aspects, the TILs comprise CD8+ T cells. In some aspects, the TILs are enriched for tumor reactive (e.g., tumor specific) TILs. In some aspects, the TILs are enriched for stem-like TILs. [0673] In some aspects, the TILs comprise tumor reactive TILs and non-tumor reactive TILs, wherein the proportion of tumor reactive TILs to non-tumor reactive TILs in the population
of expanded TILs is higher than the proportion of tumor reactive TILs to non-tumor reactive TILs in the tumor sample. In some aspects, the proportion of tumor reactive TILs to non-tumor reactive TILs in the population of expanded TILs is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, 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 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-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 higher than the proportion of tumor reactive TILs to non-tumor reactive TILs in the tumor sample. In some aspects, the proportion of tumor reactive TILs to non-tumor reactive TILs in the population of expanded TILs is at least about 2-fold higher than the proportion of tumor reactive TILs to non-tumor reactive TILs in the tumor sample. the proportion of tumor reactive TILs to non-tumor reactive TILs in the population of expanded TILs is at least about 3-fold higher than the proportion of tumor reactive TILs to non-tumor reactive TILs in the tumor sample. the proportion of tumor reactive TILs to non-tumor reactive TILs in the population of expanded TILs is at least about 4-fold higher than the proportion of tumor reactive TILs to non-tumor reactive TILs in the tumor sample. the proportion of tumor reactive TILs to non-tumor reactive TILs in the population of expanded TILs is at least about 5-fold higher than the proportion of tumor reactive TILs to non- tumor reactive TILs in the tumor sample. the proportion of tumor reactive TILs to non-tumor reactive TILs in the population of expanded TILs is at least about 6-fold higher than the proportion of tumor reactive TILs to non-tumor reactive TILs in the tumor sample. the proportion of tumor reactive TILs to non-tumor reactive TILs in the population of expanded TILs is at least about 7- fold higher than the proportion of tumor reactive TILs to non-tumor reactive TILs in the tumor sample. the proportion of tumor reactive TILs to non-tumor reactive TILs in the population of expanded TILs is at least about 8-fold higher than the proportion of tumor reactive TILs to non- tumor reactive TILs in the tumor sample. the proportion of tumor reactive TILs to non-tumor reactive TILs in the population of expanded TILs is at least about 9-fold higher than the proportion of tumor reactive TILs to non-tumor reactive TILs in the tumor sample. the proportion of tumor reactive TILs to non-tumor reactive TILs in the population of expanded TILs is at least about 10- fold higher than the proportion of tumor reactive TILs to non-tumor reactive TILs in the tumor sample.
[0674] In some aspects, the TILs comprise CD8+ TILs and CD4+ TILs, wherein the proportion of CD8+ TILs to CD4+ TILs in the population of expanded TILs is higher than the proportion of CD8+ TILs to CD4+ TILs in the tumor sample. In some aspects, the proportion of CD8+ TILs to CD4+ TILs in the population of expanded TILs is at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, 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 16-fold, at least about 17-fold, at least about 18-fold, at least about 19-fold, at least about 20-fold, at least about 21-fold, at least about 22-fold, at least about 23-fold, at least about 24-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 higher than the proportion of CD8+ TILs to CD4+ TILs in the tumor sample. In some aspects, the proportion of CD8+ TILs to CD4+ TILs in the population of expanded TILs is at least about 2-fold higher than the proportion of CD8+ TILs to CD4+ TILs in the tumor sample. In some aspects, the proportion of CD8+ TILs to CD4+ TILs in the population of expanded TILs is at least about 3-fold higher than the proportion of CD8+ TILs to CD4+ TILs in the tumor sample. In some aspects, the proportion of CD8+ TILs to CD4+ TILs in the population of expanded TILs is at least about 4-fold higher than the proportion of CD8+ TILs to CD4+ TILs in the tumor sample. In some aspects, the proportion of CD8+ TILs to CD4+ TILs in the population of expanded TILs is at least about 5-fold higher than the proportion of CD8+ TILs to CD4+ TILs in the tumor sample. In some aspects, the proportion of CD8+ TILs to CD4+ TILs in the population of expanded TILs is at least about 6-fold higher than the proportion of CD8+ TILs to CD4+ TILs in the tumor sample. In some aspects, the proportion of CD8+ TILs to CD4+ TILs in the population of expanded TILs is at least about 7-fold higher than the proportion of CD8+ TILs to CD4+ TILs in the tumor sample. In some aspects, the proportion of CD8+ TILs to CD4+ TILs in the population of expanded TILs is at least about 8-fold higher than the proportion of CD8+ TILs to CD4+ TILs in the tumor sample. In some aspects, the proportion of CD8+ TILs to CD4+ TILs in the population of expanded TILs is at least about 9-fold higher than the proportion of CD8+ TILs to CD4+ TILs in the tumor sample. In some aspects, the proportion of CD8+ TILs to CD4+ TILs in the population of expanded TILs is at least about 10-fold higher than the proportion of CD8+ TILs to CD4+ TILs in the tumor sample.
III.B. Immune Cell Culture Medium [0675] Some aspects of the present disclosure are directed to a culture medium, e.g., an immune cell culture medium, comprising one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the medium comprises a basal medium modified to further comprise one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the basal medium is 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), M199, OpTmizer™ CTS™ T-Cell Expansion Basal Medium (ThermoFisher), OPTMIZER™ Complete, IMMUNOCULT™ XF (STEMCELL™ Technologies), IMMUNOCULT™ XF, AIM V, TEXMACS™ medium, or any combination thereof. In particular aspects, the basal media comprises OPTMIZER™ complete. In some aspects, suitable basal medium includes Click's medium, OpTimizer® (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. [0676] In some aspects, the culture medium, e.g., an immune cell culture medium, comprising one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1, is serum free. In some aspects, the culture medium, e.g., an immune cell culture medium, comprising one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1, further comprises immune cell serum replacement (ICSR). For example, in some aspects, the medium comprises OPTMIZER™ Complete supplemented with ICSR and one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the medium comprises AIM V supplemented with ICSR and one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the medium comprises, IMMUNOCULT™ XF supplemented with ICSR and one or more of CCL5, CXCL10, IL-18, IL- 8, and SLAMF-1. In some aspects, the medium comprises RPMI supplemented with ICSR and one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the medium comprises TEXMACS™ supplemented with ICSR and one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the medium comprises an MRM disclosed herein, supplemented with one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1.
[0677] In some aspects, the medium comprises CCL5. In some aspects, the medium comprises CXCL10. In some aspects, the medium comprises IL-18. In some aspects, the medium comprises IL-8. In some aspects, the medium comprises SLAMF-1. [0678] In some aspects, the medium comprises CCL5, CXCL10, IL-18, IL-8, and SLAMF- 1. In some aspects, the medium comprises CCL5 and CXCL10. In some aspects, the medium comprises CCL5, CXCL10, and IL-18. In some aspects, the medium comprises CCL5, CXCL10, IL-18, and IL-8. In some aspects, the medium comprises CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the medium comprises CCL5 and IL-18. In some aspects, the medium comprises CCL5, IL-18, and IL-8. In some aspects, the medium comprises CCL5, IL-18, IL-8, and SLAMF-1. In some aspects, the medium comprises CCL5, CXCL10, and IL-8. In some aspects, the medium comprises CCL5, CXCL10, IL-8, and SLAMF-1. In some aspects, the medium comprises CCL5 and IL-8. In some aspects, the medium comprises CCL5, IL-8, and SLAMF-1. In some aspects, the medium comprises CCL5 and SLAMF-1. In some aspects, the medium comprises CXCL10, and IL-18. In some aspects, the medium comprises CXCL10, IL-18, and IL- 8. In some aspects, the medium comprises CXCL10, IL-18, IL-8, and SLAM-1. In some aspects, the medium comprises CXCL10, IL-18, and SLAMF-1. In some aspects, the medium comprises CXCL10, IL-8, and SLAMF-1. In some aspects, the medium comprises CXCL10 and SLAMF-1. In some aspects, the medium comprises IL-18, and IL-8. In some aspects, the medium comprises IL-18 and SLAMF-1. In some aspects, the medium comprises IL-8 and SLAMF-1. [0679] In some aspects, the medium comprises CCL5, CXCL10, IL-18, IL-8, and SLAMF- 1. [0680] In some aspects, the medium comprises an MRM disclosed herein, modified to further comprise one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the modified MRM comprises CCL5 and CXCL10. In some aspects, the modified MRM comprises CCL5, CXCL10, and IL-18. In some aspects, the modified MRM comprises CCL5, CXCL10, IL- 18, and IL-8. In some aspects, the modified MRM comprises CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. In some aspects, the modified MRM comprises CCL5, and IL-18. In some aspects, the modified MRM comprises CCL5, IL-18, and IL-8. In some aspects, the modified MRM comprises CCL5, IL-18, IL-8, and SLAMF-1. In some aspects, the modified MRM comprises CCL5, CXCL10, and IL-8. In some aspects, the modified MRM comprises CCL5, CXCL10, IL-8, and SLAMF-1. In some aspects, the modified MRM comprises CCL5 and IL-8. In some aspects, the modified MRM comprises CCL5, IL-8, and SLAMF-1. In some aspects, the modified MRM comprises CCL5 and SLAMF-1. In some aspects, the modified MRM comprises CXCL10, and
IL-18. In some aspects, the modified MRM comprises CXCL10, IL-18, and IL-8. In some aspects, the modified MRM comprises CXCL10, IL-18, IL-8, and SLAM-1. In some aspects, the modified MRM comprises CXCL10, IL-18, and SLAMF-1. In some aspects, the modified MRM comprises CXCL10, IL-8, and SLAMF-1. In some aspects, the modified MRM comprises CXCL10 and SLAMF-1. In some aspects, the modified MRM comprises IL-18, and IL-8. In some aspects, the modified MRM comprises IL-18 and SLAMF-1. In some aspects, the modified MRM comprises IL-8 and SLAMF-1. [0681] In some aspects, the modified MRM comprises CCL5, CXCL10, IL-18, IL-8, and SLAMF-1. IV. Methods of Treatment [0682] In some aspects, a composition comprising a population of immune cells, e.g., expanded T cells, NK cells, and/or TILs, disclosed herein, is administered to a subject in need thereof. In some aspects the immune cells, e.g., expanded T cells, NK cells, and/or TILs, prepared using the methods disclose herein are administered to a subject to treat a cancer, e.g., a tumor. In some aspects, the method of treating comprises administering to the subject an effective amount of an immune cell composition of the disclosure, e.g., a composition comprising a population of immune cells, e.g., T cells, NK cells, and/or TILs, prepared according to the methods disclosed herein. Non-limiting examples of tumors that can be treated are further described elsewhere in the present disclosure. In some aspects, the tumor is refractory to a checkpoint inhibitor. Non-limiting examples of checkpoint inhibitors include a PD-1 antagonist (e.g., anti-PD-1 antibody or anti-PD- L1 antibody), a CTLA-4 antagonist (e.g., anti-CTLA-4 antibody), a TIM3 antagonist (e.g., anti- TIM3 antibody), a GITR antagonist (e.g., anti-GITR antibody), a KIR antagonist (e.g., anti-KIR antibody), a LAG3 antagonist (e.g., anti-LAG3 antibody), or any combination thereof. In some aspects, the tumor is relapsed. In some aspects, the tumor is metastatic. In some aspects, the tumor that can be treated with the present disclosure is refractory to a checkpoint inhibitor and relapsed. In some aspects, the tumor is refractory to a checkpoint inhibitor and metastatic. In some aspects, the tumor is refractory to a checkpoint inhibitor, relapsed, and metastatic. [0683] The present disclosure also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue in a subject, comprising administering an effective amount of a composition of the disclosure, e.g., a population of immune cells, e.g., T cells, NK cells, and/or TILs, prepared according to the methods disclosed herein, e.g., a population of TILs enriched for CD8+ TILs.
[0684] In some aspects, the population of immune cells, e.g., expanded T cells, NK cells, and/or TILs, administered in the cell composition of the disclosure comprises autologous immune cells, e.g., autologous T cells, NK cells, and/or TILs. [0685] In some aspects, the method comprises administering at least about 1 x 104, at least about 5 x 104, at least about 1 x 105, at least about 5 x 105, at least about 1 x 106, at least about 2 x 106, at least about 3 x 106, at least about 4 x 106, at least about 5 x 106, at least about 6 x 106, at least about 7 x 106, at least about 8 x 106, at least about 9 x 106, at least about 1 x 107, at least about 5 x 107, at least about 1 x 108 cells to the subject. In some aspects, the method comprises administering at least about 1 x 104, at least about 5 x 104, at least about 1 x 105, at least about 5 x 105, at least about 1 x 106, at least about 2 x 106, at least about 3 x 106, at least about 4 x 106, at least about 5 x 106, at least about 6 x 106, at least about 7 x 106, at least about 8 x 106, at least about 9 x 106, at least about 1 x 107, at least about 5 x 107, at least about 1 x 108, at least about 1 x 109, at least about 2 x 109, at least about 3 x 109, at least about 4 x 109, at least about 5 x 109, at least about 6 x 109, at least about 7 x 109, at least about 8 x 109, at least about 9 x 109, at least about 1 x 1010 , at least about 10 x 1010, at least about 15 x 1010, at least about 20 x 1010, at least about 25 x1010, or at least about 30 x 1010 cells to the subject. In some aspects, the cells are TILs. In some aspects, the TILs are CD8+ TILs. [0686] In some aspects, the method comprises administering at least about 5 x 109, 10 x 109, at least about 20 x 109, at least about 30 x 109, at least about 40 x 109, at least about 50 x 109, at least about 60 x 109, at least about 70 x 109, at least about 80 x 109, at least about 90 x 109, or at least about 100 x 109 cells. In some aspects, the method comprises administering about 10 x 109 to about 100 x 109, e.g., about 10 x 109, about 20 x 109, about 30 x 109, about 40 x 109, about 50 x 109, about 60 x 109, about 70 x 109, about 80 x 109, about 90 x 109, or about 100 x 109 cells. In some aspects, the method comprises administering about 10 x 109 to about 20 x 109 cells, about 20 x 109 to about 30 x 109 cells, about 30 x 109 to about 40 x 109 cells, about 40 x 109 to about 50 x 109 cells, about 50 x 109 to about 60 x 109 cells, about 60 x 109 to about 70 x 109 cells, about 70 x 109 to about 80 x 109 cells, about 80 x 109 to about 90 x 109 cells, or about 90 x 109 to about 100 x 109 cells. In some aspects, the method comprises administering about 10 x 109 to about 20 x 109 cells. In some aspects, the method comprises administering about 20 x 109 to about 30 x 109 cells. In some aspects, the method comprises administering about 30 x 109 to about 40 x 109 cells. In some aspects, the method comprises administering about 40 x 109 to about 50 x 109 cells. In some aspects, the method comprises administering about 50 x 109 to about 60 x 109 cells. In some aspects, the method comprises administering about 60 x 109 to about 70 x 109 cells. In some
aspects, the method comprises administering about 70 x 109 to about 80 x 109 cells. In some aspects, the method comprises administering about 80 x 109 to about 90 x 109 cells. In some aspects, the method comprises administering about 90 x 109 to about 100 x 109 cells. In some aspects, the TILs are CD8+ TILs. In some aspects, the method comprises administering about 5 x 109 to about 8 x 109 cells. In some aspects, the method comprises administering about 10 x 109 to about 30 x 109 cells. In some aspects, the method comprises administering about 10 x 109 to about 40 x 109 cells. In some aspects, the method comprises administering about 40 x 109 to about 80 x 109 cells In some aspects, the method comprises administering about 50 x 109 to about 80 x 109 cells. In some aspects, the method comprises administering about 90 x 109 to about 110 x 109 cells. [0687] In some aspects, administering the cell composition of the disclosure reduces a tumor volume in the subject compared to a reference tumor volume. In some aspects, the reference tumor volume is the tumor volume in the subject prior to the administration of the engineered cell. In further aspects, the reference tumor volume is the tumor volume in a corresponding subject that did not receive the administration. In some aspects, the tumor volume in the subject is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after the administration compared to the reference tumor volume. [0688] In some aspects, treating a tumor comprises reducing a tumor weight in the subject. In some aspects, administering the cell composition of the disclosure (e.g., comprising a population of TILs prepared according to the methods disclosed herein can reduce the tumor weight in a subject when administered to the subject. In some aspects, the tumor weight is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% after the administration compared to a reference tumor weight. In some aspects, the reference tumor weight is the tumor weight in the subject prior to the administration of the cell composition of the disclosure. In further aspects, the reference tumor weight is the tumor weight in a corresponding subject that did not receive the administration. [0689] In some aspects, administering the cell composition of the disclosure to a subject, e.g., suffering from a tumor, can increase the number and/or percentage of TILs (e.g., CD8+ TILs) in a tumor and/or a tumor microenvironment (TME) of the subject. In some aspects, the number and/or percentage of TILs (e.g., CD8+ TILs) in a tumor and/or TME 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 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 210%, at least 220%, at least about 230%, at least about 240%, at least about 250%, at least about 260%, at least about 270%, at least about 280%, at least about 290%, or at least about 300% or more compared to a reference (e.g., corresponding value in a subject that did not receive the cell composition of the present disclosure or the same subject prior to the administration of the cell composition of the present disclosure). [0690] In some aspects, administering the cell composition of the disclosure to a subject, e.g., suffering from a tumor, can increase the duration of an immune response in a subject relative to the duration of an immune response in a subject administered a similar cell therapy comprising cells prepared according to conventional methods, e.g., cultured in a medium not comprising a potassium ion concentration of at least about 40 mM to at least about 90 mM, e.g., at least 50 mM. In some aspects, the duration of the immune response is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, or at least about 1000% or more compared to a reference (e.g., a subject administered a similar cell therapy comprising cells prepared according to conventional methods, e.g., cultured in a medium not comprising a potassium ion concentration of at least 50 mM). In some aspects, the duration of the immune response is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold or more compared to a reference (e.g., a subject administered a similar cell therapy comprising cells prepared according to conventional methods, e.g., cultured in a medium not comprising a potassium ion concentration of at least about 40 mM to at least about 90 mM, e.g., at least 50 mM). [0691] In addition to the above, administering the cell composition of the disclosure can have other effects which are conducive for the treatment of a tumor. [0692] As described herein, the cell composition of the disclosure can be used to treat variety of cancer types, e.g., a tumor derived from a cancer comprising a breast cancer, head and neck cancer, uterine cancer, brain cancer, skin cancer, renal cancer, lung cancer, colorectal cancer, prostate cancer, liver cancer, bladder cancer, kidney cancer, pancreatic cancer, thyroid cancer, esophageal cancer, eye cancer, stomach (gastric) cancer, gastrointestinal cancer, ovarian cancer,
carcinoma, sarcoma, leukemia, lymphoma, myeloma, or a combination thereof. In some aspects, the cancer comprises a solid tumor. In some aspects, the cancer comprises a solid tumor derived from a melanoma, a colon cancer, a lung cancer, a cervical cancer, a gastrointestinal cancer, a breast cancer, a prostate cancer, a liver cancer, bone cancer, a pancreatic cancer, a small cell carcinoma of the head and neck, lung squamous cell carcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, head and neck squamous cell carcinoma, testicular germ cell tumors, stomach adenocarcinoma, skin cutaneous melanoma, mesothelioma, kidney renal clear cell carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, esophageal carcinoma, bladder urothelial carcinoma, breast invasive carcinoma, kidney renal papillary cell carcinoma, colon adenocarcinoma, or any combination thereof. In some aspects, the cancer comprises a melanoma. In some aspects, the cancer comprises colorectal cancer. In some aspects, the cancer comprises a colon cancer. In some aspects, the cancer comprises pancreatic cancer. In some aspects, the cancer comprises head and neck cancer. In some aspects, the cancer comprises cervical cancer. In some aspects, the cancer comprises ovarian cancer. In some aspects, the cancer comprises a lung cancer. In some aspects, the cancer comprises a gastrointestinal cancer. In some aspects, the cancer comprises a breast cancer. In some aspects, the cancer comprises a prostate cancer. In some aspects, the cancer comprises a liver cancer. In some aspects, the cancer comprises bone cancer. In some aspects, the cancer comprises a small cell carcinoma of the head and neck. In some aspects, the cancer comprises lung squamous cell carcinoma. In some aspects, the cancer comprises lung adenocarcinoma. In some aspects, the cancer comprises pancreatic adenocarcinoma. In some aspects, the cancer comprises head and neck squamous cell carcinoma. In some aspects, the cancer comprises a testicular germ cell tumor. In some aspects, the cancer comprises stomach adenocarcinoma. In some aspects, the cancer comprises skin cutaneous melanoma. In some aspects, the cancer comprises mesothelioma. In some aspects, the cancer comprises kidney renal clear cell carcinoma. In some aspects, the cancer comprises cervical squamous cell carcinoma. In some aspects, the cancer comprises endocervical adenocarcinoma. In some aspects, the cancer comprises esophageal carcinoma. In some aspects, the cancer comprises bladder urothelial carcinoma. In some aspects, the cancer comprises breast invasive carcinoma. In some aspects, the cancer comprises kidney renal papillary cell carcinoma. In some aspects, the cancer comprises colon adenocarcinoma. In some aspects, the cancer comprises a uterine cancer. In some aspects, the cancer comprises a brain. In some aspects, the cancer comprises a thyroid cancer. In some aspects, the cancer comprises an esophageal cancer. In some aspects, the cancer comprises an eye cancer. In some aspects, the cancer comprises a stomach (gastric) cancer. In some
aspects, the cancer comprises a gastrointestinal cancer. In some aspects, the cancer comprises a sarcoma. In some aspects, the cancer comprises a leukemia. In some aspects, the cancer comprises a lymphoma. In some aspects, the cancer comprises a myeloma. [0693] As such, some aspects of the present disclosure are directed to methods of treating a melanoma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a colorectal cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a colon cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating pancreatic cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating head and neck cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating cervical cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating ovarian cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a lung cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a gastrointestinal cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a breast cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a prostate cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a liver cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating bone cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a small cell carcinoma of the head and neck in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating lung squamous
cell carcinoma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating lung adenocarcinoma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating pancreatic adenocarcinoma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating head and neck squamous cell carcinoma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a testicular germ cell tumor in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating stomach adenocarcinoma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating skin cutaneous melanoma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating mesothelioma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating kidney renal clear cell carcinoma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating cervical squamous cell carcinoma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating endocervical adenocarcinoma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating esophageal carcinoma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating bladder urothelial carcinoma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating breast invasive carcinoma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating kidney renal papillary cell carcinoma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating colon adenocarcinoma in a subject in need thereof, comprising administering to the
subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a uterine cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a brain tumor in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating an esophageal cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a thyroid cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating an eye cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a stomach (gastric) cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a gastrointestinal cancer in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a sarcoma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a leukemia in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a lymphoma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. Some aspects of the present disclosure are directed to methods of treating a myeloma in a subject in need thereof, comprising administering to the subject a cell composition disclosed herein. [0694] In some aspects, the cell composition of the disclosure can be used in combination with other therapeutic agents (e.g., anti-cancer agents and/or immunomodulating agents). Accordingly, in some aspects, a method of treating a tumor disclosed herein comprises administering the cell composition of the disclosure in combination with one or more additional therapeutic agents. [0695] In some aspects, the cell composition of the disclosure can be used in combination with one or more anti-cancer agents, such that multiple elements of the immune pathway can be targeted. In some aspects, an anti-cancer agent comprises an immune checkpoint inhibitor (i.e., blocks signaling through the particular immune checkpoint pathway).
[0696] Non-limiting examples of immune checkpoint inhibitors that can be used in the present methods comprise a CTLA-4 antagonist (e.g., anti-CTLA-4 antibody), PD1 antagonist (e.g., anti-PD1 antibody, anti-PD-L1 antibody), TIM-3 antagonist (e.g., anti-TIM-3 antibody), or combinations thereof. Non-limiting examples of such antagonists are provided elsewhere in the present disclosure. In some aspects, the checkpoint inhibitor is a PD1 antagonist. In some aspects, the checkpoint inhibitor is an anti-PD1 antibody. A comprehensive and non-limiting list of combination treatment is disclosed in detail elsewhere in this application. [0697] In some aspects, the cell composition of the disclosure is administered to the subject prior to or after the administration of the additional therapeutic agent. In other aspects, the cell composition of the disclosure is administered to the subject concurrently with the additional therapeutic agent. In some aspects, the cell composition of the disclosure and the additional therapeutic agent can be administered concurrently as a single composition in a pharmaceutically acceptable carrier. In other aspects, the cell composition of the disclosure and the additional therapeutic agent are administered concurrently as separate compositions. [0698] In some aspects, the subject is a nonhuman animal such as a rat or a mouse. In some aspects, the subject is a human. [0699] In some aspects, a cell composition disclosed herein can be used in combination with other therapeutic agents (e.g., anti-cancer agents and/or immunomodulating agents). Accordingly, in some aspects, a method of treating a tumor disclosed herein comprises administering a cell composition of the present disclosure in combination with one or more additional therapeutic agents to a subject. Such agents can include, for example, chemotherapeutic drug, targeted anti-cancer therapy, oncolytic drug, cytotoxic agent, immune-based therapy, cytokine, surgical procedure, radiation procedure, activator of a costimulatory molecule, immune checkpoint inhibitor, a vaccine, a cellular immunotherapy, or any combination thereof. [0700] In some aspects, a cell composition disclosed herein can be used in combination with a standard of care treatment (e.g., surgery, radiation, and chemotherapy). Methods described herein can also be used as a maintenance therapy, e.g., a therapy that is intended to prevent the occurrence or recurrence of tumors. [0701] In some aspects, a cell composition of the present disclosure can be used in combination with one or more anti-cancer agents, such that multiple elements of the immune pathway can be targeted. Non-limiting of such combinations include: a therapy that enhances tumor antigen presentation (e.g., dendritic cell vaccine, GM-CSF secreting cellular vaccines, CpG oligonucleotides, imiquimod); a therapy that inhibits negative immune regulation e.g., by
inhibiting CTLA-4 and/or PD1/PD-L1/PD-L2 pathway and/or depleting or blocking Tregs or other immune suppressing cells (e.g., myeloid-derived suppressor cells); a therapy that stimulates positive immune regulation, e.g., with agonists that stimulate the CD-137, OX-40, and/or CD40 or GITR pathway and/or stimulate T cell effector function; a therapy that increases systemically the frequency of anti-tumor T cells; a therapy that depletes or inhibits Tregs, such as Tregs in the tumor, e.g., using an antagonist of CD25 (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion; a therapy that impacts the function of suppressor myeloid cells in the tumor; a therapy that enhances immunogenicity of tumor cells (e.g., anthracyclines); an additional adoptive T cell or NK cell transfer including genetically engineered cells, e.g., cells engineered to express a chimeric antigen receptor (CAR-T therapy); a therapy that inhibits a metabolic enzyme such as indoleamine dioxigenase (IDO), dioxigenase, arginase, or nitric oxide synthetase; a therapy that reverses/prevents T cell anergy or exhaustion; a therapy that triggers an innate immune activation and/or inflammation at a tumor site; administration of immune stimulatory cytokines; blocking of immuno repressive cytokines; or any combination thereof. [0702] In some aspects, an anti-cancer agent comprises an immune checkpoint inhibitor (i.e., blocks signaling through the particular immune checkpoint pathway). Non-limiting examples of immune checkpoint inhibitors that can be used in the present methods comprise a CTLA-4 antagonist (e.g., anti-CTLA-4 antibody), PD1 antagonist (e.g., anti-PD1 antibody, anti-PD-L1 antibody), TIM-3 antagonist (e.g., anti-TIM-3 antibody), or combinations thereof. Non-limiting examples of such immune checkpoint inhibitors include the following: anti-PD1 antibody (e.g., nivolumab (OPDIVO®), pembrolizumab (KEYTRUDA®; MK-3475), pidilizumab (CT-011), PDR001, MEDI0680 (AMP-514), TSR-042, REGN2810, JS001, AMP-224 (GSK-2661380), PF- 06801591, BGB-A317, BI 754091, SHR-1210, and combinations thereof); anti-PD-L1 antibody (e.g., atezolizumab (TECENTRIQ®; RG7446; MPDL3280A; RO5541267), durvalumab (MEDI4736, IMFINZI®), BMS-936559, avelumab (BAVENCIO®), LY3300054, CX-072 (Proclaim-CX-072), FAZ053, KN035, MDX-1105, and combinations thereof); and anti-CTLA-4 antibody (e.g., ipilimumab (YERVOY®), tremelimumab (ticilimumab; CP-675,206), AGEN-1884, ATOR-1015, and combinations thereof). Additional description of immune checkpoint inhibitors that are useful for the present disclosure are provided below. [0703] In some aspects, the cell composition of the present disclosure is administered to a subject in combination with a PD1 antagonist. In some aspects, the cell composition of the present disclosure is administered to a subject in combination with an anti-PD1 antibody. In some aspects, the cell composition of the present disclosure is administered to a subject in combination with
nivolumab. In some aspects, the cell composition of the present disclosure is administered to a subject in combination with pembrolizumab. [0704] In some aspects, the cell composition of the present disclosure is administered to a subject in combination with a PD-L1 antagonist. In some aspects, the cell composition of the present disclosure is administered to a subject in combination with an anti-PD-L1 antibody. In some aspects, the cell composition of the present disclosure (e.g., comprising a population of TILs prepared according to the methods disclosed herein (e.g., enriched for CD8+ TILs, tumor-specific TILs, and/or naïve TILs)) is administered to a subject in combination with atezolizumab. In some aspects, the cell composition of the present disclosure (e.g., comprising a population of TILs prepared according to the methods disclosed herein (e.g., enriched for CD8+ TILs, tumor-specific TILs, and/or naïve TILs)) is administered to a subject in combination with durvalumab. In some aspects, the cell composition of the present disclosure (e.g., comprising a population of TILs prepared according to the methods disclosed herein (e.g., enriched for CD8+ TILs, tumor-specific TILs, and/or naïve TILs)) is administered to a subject in combination with avelumab. [0705] In some aspects, the cell composition of the present disclosure is administered to a subject in combination with a CTLA-4 antagonist. In some aspects, the cell composition of the present disclosure is administered to a subject in combination with an anti- CTLA-4 antibody. In some aspects, the cell composition of the present disclosure is administered to a subject in combination with ipilimumab. In some aspects, the cell composition of the present disclosure is administered to a subject in combination with tremelimumab. [0706] In some aspects, an anti-cancer agent comprises an immune checkpoint activator (i.e., promotes signaling through the particular immune checkpoint pathway). In some aspects, immune checkpoint activator comprises OX40 agonist (e.g., anti-OX40 antibody), LAG-3 agonist (e.g. anti-LAG-3 antibody), 4-1BB (CD137) agonist (e.g., anti-CD137 antibody), GITR agonist (e.g., anti-GITR antibody), TIM3 agonist (e.g., anti-TIM3 antibody), or combinations thereof. In some aspects, the additional therapeutic agent comprises a cytokine. In some aspects, the cytokine comprises IL-2, IL-21, IL-7, IL-15, or any combination thereof. [0707] In some aspects, a cell composition disclosed herein is administered to the subject prior to or after the administration of the additional therapeutic agent. In other aspects, cell composition disclosed herein is administered to the subject concurrently with the additional therapeutic agent. In some aspects, the cell composition disclosed herein and the additional therapeutic agent can be administered concurrently as a single composition in a pharmaceutically acceptable carrier. In other aspects, the cell composition disclosed herein and the additional
therapeutic agent are administered concurrently as separate compositions. In some aspects, the additional therapeutic agent and the cell composition disclosed herein are administered sequentially. [0708] In some aspects, a cell composition disclosed herein is administered to the subject in combination with a checkpoint inhibitor (e.g., an anti-PD1 antibody). In some aspects, the cell composition is administered before the checkpoint inhibitor (e.g., an anti-PD1 antibody). In some aspects, the cell composition is administered after the checkpoint inhibitor (e.g., an anti-PD1 antibody). [0709] In some aspects, the subject is administered a lymphodepleting therapy prior to receiving the cell composition. Any lymphodepleting therapy can be used in the method disclosed herein. In some aspects, the lymphodepleting therapy comprises a chemotherapy. In some aspects, the lymphodepleting therapy comprises cyclophosphamide. In some aspects, the lymphodepleting therapy comprises fludarabine. In some aspects, the lymphodepleting therapy comprises cyclophosphamide and fludarabine. In some aspects, the lymphodepleting therapy is administered at least about 3 days, at least about 4 days, at least about 5 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days prior to the cell composition. [0710] As described herein, a cell composition disclosed herein can be used to treat various types of tumors. As it is generally known in the art, certain tumors are less responsive to currently available anti-cancer treatments (e.g., such as those described above). For instance, some tumors are refractory to a checkpoint inhibitor. As used herein, the term "refractory" refers to a disease or condition that does not respond to treatment. Where the term is used to describe a tumor (e.g., "refractory tumor"), the term refers to tumors that do not respond to treatment and includes circumstances where the tumor is resistant at the beginning of treatment or the tumor becomes resistant during treatment. [0711] Accordingly, some aspects of the present disclosure are related to a method of treating a tumor in a subject in need thereof, comprising administering to the subject any of the cell compositions described herein, wherein the tumor is refractory to one or more checkpoint inhibitors. In some aspects, the checkpoint inhibitor comprises a PD-1 antagonist, a CTLA-4 antagonist, a TIM3 antagonist, a GITR antagonist, a KIR antagonist, a LAG3 antagonist, or any combination thereof. In some aspects, the checkpoint inhibitor comprises an anti-PD1 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-GITR antibody, an anti-KIR antibody, an anti-LAG3 antibody, or any combination thereof. Accordingly, in some aspects, a tumor that can
be treated with the present disclosure is refractory to a PD-1 antagonist (e.g., anti-PD-1 antibody). In some aspects, the tumor is refractory to a CTLA-4 antagonist (e.g., anti-CTLA-4 antibody). In some aspects, the tumor is refractory to a PD-1 antagonist (e.g., anti-PD-1 antibody) and a CTLA- 4 antagonist (e.g., anti-CTLA-4 antibody). In some aspects, the tumor is refractory to a TIM3 antagonist (e.g., anti-TIM3 antibody). In some aspects, the tumor is refractory to a GITR antagonist (e.g., anti-GITR antibody). In some aspects, the tumor is refractory to a KIR antagonist (e.g., anti- KIR antibody). In some aspects, the tumor is refractory to a LAG3 antagonist (e.g., anti-LAG3 antibody). In some aspects, the tumor is refractory to a PD-1 antagonist, a B-raf enzyme inhibitor, and a MEK protein inhibitor. In some aspects, the tumor is refractory to one or more PD-1 antagonists, radiation, a B-raf enzyme inhibitor, and/or a MEK protein inhibitor. In some aspects, the tumor is refractory to one or more PD-1 antagonists, radiation and/or an oncolytic virus. [0712] In some aspects, provided herein is a method of treating a tumor in a subject in need thereof, comprising administering to the subject any of the cell compositions described herein, wherein the tumor is refractory to a PD-1 antagonist. [0713] In some aspects, the PD-1 antagonist is an anti-PD-1 antibody. In some aspects, the PD-1 antagonist includes any known anti-PD1 antibodies in the art. Antibodies (e.g., human antibodies) that bind specifically to PD-1 with high affinity have been disclosed in U.S. Patent Nos.8,008,449 and 8,779,105, each of which is hereby incorporated by reference. Other anti-PD- 1 mAbs have been described in, for example, U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757 and 8,354,509, and PCT Publication No. WO 2012/145493, each of which is hereby incorporated by reference. Additional examples of anti-PD-1 antibodies include lambrolizumab (MK-3475) described in WO 2008/156712, and AMP-514 described in WO 2012/145493, the teachings of which are hereby incorporated by reference. Further known anti-PD-1 antibodies and other PD-1 inhibitors include those described in WO 2009/014708, WO 03/099196, WO 2009/114335 and WO 2011/161699, the teachings of which are hereby incorporated by reference. In some aspects, anti-PD-1 antibodies include monoclonal antibodies 5C4 (referred to herein as Nivolumab or BMS-936558), 17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in WO 2006/121168, the teachings of which are hereby incorporated by reference, can be used. In some aspects, the anti- PD-1 antibody is pidilizumab (CT-011). Antibodies or antigen binding fragments thereof that compete with any of these antibodies or inhibitors are also relevant for the present disclosure. [0714] In some aspects, the anti-PD-1 antibody cross-competes with nivolumab. In some aspects, the anti-PD-1 antibody binds to the same epitope as nivolumab. In some aspects, the anti- PD-1 antibody has the same CDRs as nivolumab. In some aspects, the anti-PD-1 antibody is
nivolumab. Accordingly, some aspects of the present disclosure is related to a method of treating a tumor which is refractory to nivolumab in a subject in need thereof, wherein the method comprises administering to the subject any of the cell compositions described herein to the subject. [0715] In some aspects, the anti-PD-1 antibody cross-competes with pembrolizumab. In some aspects, the anti-PD-1 antibody binds to the same epitope as pembrolizumab. In some aspects, the anti-PD-1 antibody has the same CDRs as pembrolizumab. In some, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also known as "Keytruda®", lambrolizumab, and MK-3475) is a humanized monoclonal IgG4 antibody directed against human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587; see also worldwideweb.cancer.gov/drugdictionary?cdrid=695789 (last accessed: May 25, 2017), each of which is hereby incorporated by reference. Accordingly, some aspects of the present disclosure is related to a method of treating a tumor which is refractory to pembrolizumab in a subject in need thereof, wherein the method comprises administering to the subject any of the cell compositions described herein to the subject. [0716] In some aspects, the anti-PD-1 antibody cross-competes with MEDI0608. In some aspects, the anti-PD-1 antibody binds to the same epitope as MEDI0608. In some aspects, the anti- PD-1 antibody has the same CDRs as MEDI0608. In soem aspects, the anti-PD-1 antibody is MEDI0608 (formerly AMP-514), which is a monoclonal antibody. MEDI0608 is described, for example, in U.S. Patent No. 8,609,089 or in worldwideweb.cancer.gov/drugdictionary?cdrid=756047 (last accessed May 25, 2017), each of which is hereby incorporated by reference. Accordingly, some aspects of the present disclosure is related to a method of treating a tumor which is refractory to MEDI0608 in a subject in need thereof, wherein the method comprises administering to the subject any of the cell compositions described herein to the subject. [0717] In some aspects, the anti-PD-1 antibody cross-competes with BGB-A317. In some aspects, the anti-PD-1 antibody binds the same epitope as BGB-A317. In some aspects, the anti- PD-1 antibody has the same CDRs as BGB-A317. In some aspects, the anti-PD-1 antibody is BGB- A317, which is a humanized monoclonal antibody. BGB-A317 is described in U.S. Publ. No. 2015/0079109, which is hereby incorporated by reference. Accordingly, some aspects of the present disclosure is related to a method of treating a tumor which is refractory to BGB-A317 in a subject in need thereof, wherein the method comprises administering to the subject any of the cell compositions described herein to the subject.
[0718] In some aspects, the PD-1 antagonist is an anti-PD-L1 antibody. In some aspects, the PD-1 antagonist includes any known anti-PD-L1 antibodies in the art. In some aspects, human anti-PD-L1 antibodies disclosed in U.S. Pat. No. 7,943,743, the contents of which are hereby incorporated by reference, are within the scope of the present disclosure. Such anti-PD-L1 antibodies include 3G10, 12A4 (also referred to as BMS-936559), 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4. Other art recognized anti-PD-L1 antibodies include those described in, for example, U.S. Pat. Nos.7,635,757 and 8,217,149, U.S. Publication No.2009/0317368, and PCT Publication Nos. WO 2011/066389 and WO 2012/145493, the teachings of which also are hereby incorporated by reference. Additional examples of an anti-PD-L1 antibody include atezolizumab (TECENTRIQ®; RG7446), or durvalumab (IMFINZI®; MEDI4736). Antibodies or antigen binding fragments thereof that compete with any of these art-recognized antibodies or inhibitors for binding to PD-L1 are also relevant for the present disclosure. [0719] In some aspects, the anti-PD-L1 antibody cross-competes with BMS-936559 (formerly 12A4 or MDX-1105) (see, e.g., U.S. Patent No.7,943,743; WO 2013/173223, both of which are hereby incorporated by reference). In some aspects, the anti-PD-L1 antibody binds to the same epitope as BMS-936559. In some aspects, the anti-PD-L1 antibody has the same CDRs as BMS-936559. In some aspects, the anti-PD-L1 antibody is BMS-936559. Accordingly, some aspects of the present disclosure is related to a method of treating a tumor which is refractory to BMS-936559 in a subject in need thereof, wherein the method comprises administering to the subject any of the cell compositions described herein to the subject. [0720] In some aspects, the anti-PD-L1 antibody cross-competes with MPDL3280A (also known as RG7446 and atezolizumab) (see, e.g., Herbst et al.2013 J Clin Oncol 31(suppl):3000; U.S. Patent No. 8,217,149, both of which are hereby incorporated by reference), MEDI4736 (Khleif, 2013, In: Proceedings from the European Cancer Congress 2013; September 27-October 1, 2013; Amsterdam, The Netherlands. Abstract 802, which is hereby incorporated by reference). In some aspects, the anti-PD-L1 antibody binds to the same epitope as MPDL3280A. In some aspects, the anti-PD-L1 antibody has the same CDRs as MPDL3280A. In some aspects, the anti- PD-L1 antibody is MPDL3280A. Accordingly, some aspects of the present disclosure is related to a method of treating a tumor which is refractory to MPDL3280A in a subject in need thereof, wherein the method comprises administering to the subject any of the cell compositions described herein to the subject. [0721] In some aspects, the anti-PD-L1 antibody cross-competes with MSB0010718C (also called Avelumab; see US 2014/0341917, which is hereby incorporated by reference). In some
aspects, the anti-PD-L1 antibody binds to the same epitope as MSB0010718C. In some aspects, the anti-PD-L1 antibody has the same CDRs as MSB0010718C. In some aspects, the anti-PD-L1 antibody is MSB0010718C. Accordingly, some aspects of the present disclosure is related to a method of treating a tumor which is refractory to MSB0010718C in a subject in need thereof, wherein the method comprises administering to the subject any of the cell compositions described herein to the subject. [0722] In some aspects, provided herein is a method of treating a tumor in a subject in need thereof, comprising administering to the subject any of the cell compositions described herein, wherein the tumor is refractory to a CTLA-4 antagonist. [0723] In some aspects, the CTLA-4 antagonist is an anti-CTLA-4 antibody. In some aspects, the CTLA-4 antagonist includes any known anti-CTLA-4 antibodies in the art. For instance, human antibodies that bind specifically to CTLA-4 with high affinity have been disclosed in U.S. Patent Nos.6,984,720 and 7,605,238, each of which is hereby incorporated by reference. Additional examples of anti-CTLA-4 antibodies are described in, for example, U.S. Patent Nos. 5,977,318, 6,051,227, 6,682,736, and 7,034,121, each of which is hereby incorporated by reference. Other non-limiting examples of anti-CTLA-4 antibodies that are within the scope of the present disclosure include: MK-1308 (Merck) and AGEN-1884 (Agenus Inc.; see WO 2016/196237). Anti-CTLA-4 antibodies relevant for the present disclosure also include isolated antibodies that bind specifically to human CTLA-4 and cross-compete for binding and/or binding to the same epitope region as one or more of the anti-CTLA-4 antibodies disclosed herein (e.g., ipilimumab, tremelimumab, MK-1308, or AGEN-1884). [0724] In some aspects, the anti-CTLA-4 antibody cross-competes with ipilimumab. In some aspects, the anti-CTLA-4 antibody binds to the same epitope as ipilimumab. In some aspects, the anti-CTLA-4 antibody has the same CDRs as ipilimumab. In some aspects, the anti-CTLA-4 antibody is ipilimumab. Ipilimumab (also known as 10D1 and marketed as YERVOY®) is a fully human, IgG1 monoclonal antibody that blocks the binding of CTLA-4 to its B7 ligands. Ipilimumab is further described in U.S. Patent No. 6,984,720, which is hereby incorporated by reference. Accordingly, some aspects of the present disclosure is related to a method of treating a tumor which is refractory to ipilimumab in a subject in need thereof, wherein the method comprises administering to the subject any of the cell compositions described herein to the subject. [0725] As is apparent from at least the above disclosure, in some aspects, provided herein is a method of treating a tumor that is refractory to a TIM3 antagonist (e.g., anti-TIM3 antibody) in a subject in need thereof, comprising administering to the subject any of the cell compositions
described herein. In some aspects, provided herein is a method of treating a tumor that is refractory to a GITR antagonist (e.g., anti-GITR antibody) in a subject in need thereof, comprising administering to the subject any of the cell compositions described herein. In some aspects, provided herein is a method of treating a tumor that is refractory to a KIR antagonist in a subject in need thereof, comprising administering to the subject any of the cell compositions described herein. In some aspects, provided herein is a method of treating a tumor that is refractory to a LAG3 antagonist in a subject in need thereof, comprising administering to the subject any of the cell compositions described herein. [0726] For any of the treatment methods described above, in some aspects, the tumor is relapsed. In some aspects, the tumor is metastatic. In some aspects, the tumor is both relapsed and metastatic. EXAMPLES Example 1. Preparation of PCS [0727] In the studies described herein, different PCS formulations were assessed for feeder cell replacement in a TIL manufacturing protocol incorporating metabolic reprogramming media (MRM). Different signal molecules in combination with a polyclonal TCR stimulus (aCD3) were evaluated, specifically those involved in co-stimulation and adhesion. To this end, PCS formulations were designed with different combinations, densities, and stoichiometries of surface- bound signal molecules. See Table 1 for exemplary PCS formulations. These formulations were used to stimulate TIL in either the REP2 step only or both the REP1 and REP2 steps (feeder-free) of a two-REP expansion process, and characterized the TIL products based on fold expansion, phenotype, and clonality. In brief, stimulation in REP2 with PCS formulations presenting aCD3/aCD28/r41bbL/aCD2 resulted in greater TIL proliferation after 8 days compared to feeder cell stimulation. In addition, the results showed that this critical combination of signaling molecules on PCS-1 outperformed PCS variants without one or more of these signals, as well as formulations with other signal substitutions or combinations. It was observed that an optimized aCD3/aCD28/r41bbL/aCD2 PCSformulation (PCS-1) produced TIL with similar stem-like phenotypic characteristics, clonality, and retention of putative tumor-reactive clones as feeder- expanded cells. Furthermore, the feasibility of using PCS-1 for both REP1 and REP2 stimulation in a completely feeder-free process in the two-REP expansion process was demonstrated, with similar total fold expansion and TIL product quality to feeder cells following two stimulations
using PCS-1 observed. Overall, these data demonstrate that PCS may be used as a fully synthetic replacement for feeder cells in the TIL manufacturing process to achieve comparable or improved TIL yield while maintaining favorable product quality attributes. [0728] A sterile solution of PCS Blocking Buffer was prepared by dissolving 250 mg of BSA in 100 mL of PBS, filtered through a 0.22 μm membrane and stored at 4°C for up to 1 month. The Lyophilization Buffer was prepared by dissolving 2 g trehalose dihydrate in 20 mL of PBS. The solution was mixed thoroughly using a vortex and then filtered through a 0.22 μm membrane to be used on the same day. [0729] Approximately 2.5 mg of the desired lipid(s) was obtained from a chloroform- dissolved lipid solution using gastight glass syringe(s) and transferred into glass culture tube(s). The desired mol% of biotinylated lipid can be achieved by adding the appropriate amount of lipophilic payloads to the lipid mixture (e.g., perflubron). A nitrogen gas stream passing through an in-line 0.2 μm filter was used to evaporate the chloroform solvent leaving behind a translucent lipid film at the bottom of the glass tube. Residual chloroform was evaporated using a vacuum chamber overnight. The lipid film-containing glass culture tube was then transferred to a vacuum chamber to remove residual chloroform. The lipid film was then resuspended in PBS at 2.5 mg lipid per mL PBS while ensuring that the solution is brought above the temperature of the highest Tm among lipid mixture. The lipid suspension was vigorously mixed for 30 seconds (or until no visible lipid film remained on the bottom of the tube) using a vortex set at max speed. This was repeated every 10 minutes over an hour (7 mixes total). The mini-extruder glass syringes were rinsed with 10 aspiration/dispense cycles in isopropyl alcohol or ethanol followed by 10 aspiration/dispense cycles in distilled water and 3 aspiration/dispense cycles in PBS. The extruder apparatus was assembled according to manufacturing instructions. Approximately 500 μL sterile PBS was aspirated into a syringe and pushed through the extrusion apparatus into the opposite syringe. Spent PBS was then discarded. [0730] The lipid suspension was thoroughly mixed using a vortex and aspirated into a pre- wet extruder syringe. The syringes were inserted into the extrusion apparatus. The suspension were forced out by pushing the lipid suspension through the extruder from one syringe to the other for a total of 21 passes. The syringe containing the extruded liposome suspension was removed from the extruder and the suspension was transferred to an appropriately sized vessel (e.g., 1.5 mL protein lo-bind microcentrifuge tube). The liposome suspension was stored at 4°C for up to two weeks.
[0731] The extrusion apparatus was disassembled. All glass and Teflon components were thoroughly washed using isopropyl alcohol or ethanol followed by distilled water. Syringes and the Teflon filter support were rinsed with 10 aspiration/dispense cycles in isopropyl alcohol or ethanol followed by 10 aspiration/dispense cycles in distilled water. The rubber o-rings were washed with water and a mild soap or detergent. Metal components (e.g., casing, retainer nut, heating block) were wiped with 140-proof ethanol as needed [0732] The appropriate number of lyophilized liposome vials (5 mg of lipid per vial) were obtained from the -20°C and transferred into the biosafety cabinet (BSC) and allowed to warm to room temperature. The liposomes were then reconstituted by adding 1 mL of sterile distilled water, effectively bringing the liposome suspension to 5 mg/mL in 1x PBS (liposomes lyophilized in 1x PBS). The suspension was swirled to reconstitute any residual dry lipid on the walls of the vial and kept at room temperature for 5 minutes. Next, 1 mL sterile PBS was added to the vial, effectively bringing the liposome suspension to 2.5 mg/mL in 1x PBS. The suspension was gently mixed using a hand pipet and transferred to an appropriate vessel (e.g., 1.5 mL protein lo-bind microcentrifuge tube). Example 2. Assembly of PCS [0733] In the BSC, the appropriate amount of MSRs was weighed out in a 1.5 mL microcentrifuge tube (or appropriately sized tube) using a sterile spatula and an analytical balance. The weight of the MSRs was recorded by taking the difference between the weight of the tube before and after. [0734] The MSRs were resuspended in sterile WFI water at 25 mg/mL before being submerged in a bath sonicator for 2-5 seconds. The MSR suspension was gently mixed using a 1 mL pipette tip or appropriately sized serological pipette. The sonication / mixing steps were repeated until the MSRs were fully resuspended and the suspension can be pipetted through a standard 1 mL pipette tip without clogging. This typically took 3-4 cycles. [0735] If using resuspended dry MSRs, the MSR suspension was gently mixed using a 1 mL pipette tip or appropriately sized serological pipette before transferring an appropriate amount of MSR suspension to a 1.5 mL microcentrifuge tube (or appropriately sized tube). [0736] Appropriately sized aliquots of soluble cue(s) from the -80°C freezer were retrieved and thawed on ice. Sterile WFI water was added to an appropriately sized tube, ideally treated to minimize non-specific protein adsorption (referred to as the assembly tube). Soluble cue(s) were transferred to the assembly tube and mixed using a pipette before adding the MSR
suspension. The assembly tube was then gently mixed using a pipette. The soluble payloads were incubated for an hour at room temperature with gentle mixing using a hand pipet every 10 minutes (7x mixes total). Liposomes (2.5 mg/mL in PBS) were added to the assembly tube and gently mixed (when soluble cues were not loaded in the PCS formulation, the protocol was started here). The lipid were allowed to coat the MSRs for an hour at room temperature with gentle mixing using a hand pipet every 10 minutes (7x mixes total). Each mg of MSRs was treated with 0.5 mg of lipid(i.e., a lipid-to-MSR mass ratio = 0.5). This lipid-to-MSR ratio was used for all formulations. [0737] The material suspension mixture was diluted by adding PBS to the assembly tube and gently mixed using a hand pipet. It was then centrifuged at 700 x g for 3 minutes at room temperature. The supernatant was aspirated using a hand pipette without disturbing the pellet. The pellet was resuspended in PBS and centrifuged at 700 x g for 3 minutes at room temperature. The aspiration, resuspension in PBS, and centrifugation steps were repeated once more. The pellet was then resuspended in PCS blocking buffer for 10 minutes at room temperature. An appropriately sized aliquot of streptavidin retrieved from the -20°C freezer and thaw on ice. Biotinylated surface cue cocktail was prepared in a lo-bind 1.5 mL microcentrifuge tube for desired formulation. The material suspension was gently mixed using a hand pipet before an appropriate amount of streptavidin was added and mixed in the assembly tube. Streptavidin and the biotinylated lipids were incubated for 10 minutes at room temperature with gentle mixing using a hand pipet every 2 minutes (5x mixes total). Surface cue cocktail was then added to the assembly tube, mixed, and incubated for 40 minutes at room temperature with gentle mixing using a hand pipet every 10 minutes (5x mixes total). Following that, the material suspension was diluted by adding and mixing PCS blocking buffer before centrifuging at 700 x g for 3 minutes at room temperature. The supernatant was aspirated and the pellet was resuspended in PBS. These steps were repeated twice more. After the third time, the pellet was resuspended in an appropriate solvent at desired concentration (e.g., for frozen storage, 10 mg PCS per mL formulation buffer (sterile-filtered 10% w/v trehalose in PBS)). PCS was divided into appropriately sized aliquots and flash froze in liquid nitrogen. [0738] Accuracy of final volume is important and it very difficult to fully remove residual solvent. Especially if final resuspension is to be performed at a high concentration, recommendation is to resuspend PCS pellet in a lower volume than the target volume. The resulting resuspension volume should then be measured using a pipet, and the appropriate additional volume added to bring total final volume as close to target volume as possible.
Table 1. Mass ratio details for exemplary PCS formulations % m
[0739] Materials for TIL production Table 2. Complete OpTmizer “TCM” media
[0740] MRM was made by combining the Complete OpTmizer “TCM” media (Table 2) and the Complete 2X MCM media at 1:1 ratio. Exemplary cytokines useful for TIL expansion
protocols are listed in Table 3. Various source samples for TIL used in the analyses are listed in Table 4. Table 3. Cytokine information
Table 4. TIL from tumors assessed
Example 3. Tumor Processing [0741] Tumor resections (see, e.g., Table 4) were processed to a single cell suspension [0742] TIL expansion was assessed following feeder replacement in REP2. In initial assays, single cell suspensions from tumors were expanded in REP1 of the MRM process according to the scheme illustrated in FIG.1B. Following REP1 expansion with feeder cells, 250,000 cells were seeded in a 24 W TC plate with 0.1 mg of PCS (REP2 Day 0) in 2 mL of Complete MRM media at 37 C in a 5% CO2 controlled cell culture incubator. On REP2 Day 4, the cell culture was mixed gently by pipetting up and down, counted, and transferred to a 24 W G-Rex®. A total of 6
mL of Complete MRM media was added to each well to bring the total culture volume to 8 mL. On REP2 Day 7, half the media was exchanged in each well of the 24 W G-Rex® by carefully aspirating and discarding the top 4 mL of each well and replacing with 4 mL of fresh Complete MRM media. On REP2 Day 8, TIL were counted and fold expansion was calculated as total viable cells/original cell seeding number. In addition, samples were collected for phenotypic analysis via flow cytometry and TCR sequencing, and the remaining TIL product was cryopreserved (described in further detail below). See, e.g., FIG.1B. In further studies, additional feeder replacement assays were performed using longer REP1 and REP2 expansion phases, i.e., a 10-11 days for REP1 and REP2. After Day 10 or Day 11 in REP2, the TIL were counted and fold expansion was calculated as total viable cells/original cell seeding number. See, e.g., FIG.1C. [0743] TIL expansion also was assessed following feeder replacement in both REP1 and REP2 expansion phases. See e.g., FIG. 1B. Single cell suspensions from processed tumors were stimulated. TIL stimulation was carried out in two ways: (1) 250,000 cells were immediately seeded in a 24 W TC plate with 0.1 mg of PCS (REP1 Day 0) in 2 mL of Complete MRM media, or (2) cells were cultured for 4 days in Pre-REP media, and then on day 4, 250,000 cells were seeded in a 24 W TC plate with 0.1 mg of PCS (REP1 Day 0) in 2 mL of Complete MRM media. Cells were cultured at 37C in a 5% CO2 controlled cell culture incubator and split into multiple 24 W TC plate wells when media (containing phenol red) changed color. Cells were cultured until REP1 Day 8 in a 24 W plate, at which point the cells were pooled (if cultures were split) and restimulated with PCS for REP2: 250,000 cells were seeded in a 24 W TC plate with 0.1 mg of PCS (REP2 Day 0) in 2 mL of Complete MRM media at 37 C in a 5% CO2 controlled cell culture incubator. On REP2 Day 4, the cell culture was mixed gently by pipetting up and down, counted, and transferred to a 24 W G-Rex®. 6 mL of Complete MRM media was added to each well to bring the total culture volume to 8 mL. On REP2 Day 7, half the media was exchanged in each well of the 24 W G-Rex® by carefully aspirating and discarding the top 4 mL of each well and replacing with 4 mL of fresh Complete MRM media. On REP2 Day 8, TIL were counted and fold expansion was calculated as total viable cells/original cell seeding number. In addition, samples were collected for phenotypic analysis via flow cytometry and TCR sequencing, and the remaining TIL product was cryopreserved. Table 5. Stemness panel staining information
[0744] At the end of the MRM REP2 process, TIL were counted.500,000-800,000 viable TILs were collected for flow cytometry. TIL were washed 1x with PBS, then stained with surface antibodies for 20 minutes at 4 C. Finally, they were fixed/permeabilized using the FOXP3 Transcription Factor Staining Buffer Set following the manufacturers protocol and stained with TCF-1 for 30 min. Cells were washed and visualized on the Cytek flow cytometer. [0745] Following REP2 expansion, 2.5 M TIL were collected and washed 1x in DPBS. After the wash, the supernatant was decanted and the cell pellet was frozen in the -80C. Following thaw, DNA was extracted from the pellets using the Qiagen DNAeasy Blood and Tissue kit following the manufacturer’s protocol. DNA samples were subjected to bulk TCR sequencing (Adaptive Biotechnologies). In some instances, RNA samples were collected and subjected to TCR sequencing (Takara). [0746] After sampling for phenotypic analysis and TCR sequencing, leftover TIL were cryopreserved at 10e6/ml in CryoStor CS10 (Stemcell Technologies 07930) using a Corning CoolCell LX cell freezing container (Fisher Scientific 07-210-001). After at least 3 days in the - 80C, cells were transferred to liquid nitrogen for long term storage. Example 4. The effect of anti-CD3/28 and r41BBL density on PCS TIL expansion in REP2 [0747] These studies aimed to use the PCS platform to artificially mimic the functions of antigen presenting cells (APCs), which are likely to be the primary contributors to the T cell
activation activity of feeder cells (APCs comprise a subset of the feeder cell mixture). When APCs activate T cells, a specialized structure at the interface of the APC and the T cell called the immunological synapse is formed. We hypothesized that the critical signals to activate TIL would be comprised of (1) TCR stimulation, (2) co-stimulation, and (3) adhesion. An initial screen was performed on feeder APCs to determine what surface signals were present and might contribute to the activation functionality. Irradiated feeder cells, pooled from three healthy donors, were analyzed using flow cytometry to assess expression of potential T-cell stimulating ligands. Dimensional reduction (UMAP) and clustering (FlowSOM) analytical tools were applied to identify populations of putative APCs in the bulk feeder cell population. APCs were identified based on high HLA-DR expression and low CD3 expression. The expression of various putative activation signaling molecules in the identified feeder subsets was determined. See FIG.1A. Based on this screen, several candidate signaling molecules were selected for inclusion in PCS formulations for potential feeder replacement. [0748] To test PCS formulations for feeder cell replacement, feeder cells were replaced with PCS in either the REP2 step only, or both the REP1 and REP2 steps (feeder-free). Because of limited cell material, arrays of PCS formulations were assessed in an iterative screening approach (i.e., a signal molecule titration was performed and the best performing formulation would be tested in a subsequent study with a different tumor cell sample along with new formulations). To assess the activity of the PCS formulations, the formulations were first ranked based on their ability to mediate TIL expansion and promote a high CD8+ frequency in the TIL product. These studies aimed to identify PCS formulations that can expand TIL and maintain a CD8+ frequency comparable to or greater than feeder cells. TIL products produced using these PCS formulations were then compared to feeder cell TIL products from the same tumor sample and production run based on their stemness phenotype and retention of putative tumor-reactive clones. This study aimed to identify PCS formulations that can generate TIL products that are comparable to feeder cell TIL products with respect to these attributes. The overall workflow for assessing PCS for replacing feeder cells in the two-REP step MRM process is described in FIGs.1B and 1C. The TIL assessed herein in the initial assays were expanded according to the scheme in Fig. 1B. TIL characterized in additional assays were expanded according to the scheme in FIG. 1C where explicitly noted. [0749] TIL were activated with various PCS formulations presenting anti-CD3, anti-CD28, and r41bbL at different densities and stoichiometries, during the REP2 step of the two-REP step MRM process. PCS formulations are described in Example 1 and Example 2. TIL were cultured
according to the methods described in Example 3. At the end of production, the TIL were counted and the total expansion from day 0 was determined based on the total viable cells in the product. Based on this screening effort, which varied the number of molecules of aCD3, aCD28, and r41BBL on the surface of PCS, PCS-12 was found to have increased or maintained the expansion seen when feeder cells were used in the REP2 process (FIG.2). Example 5. Addition of costimulation/adhesion improves the expansion of PCS-12 in REP2 [0750] Various formulations with aCD2 and rICAM were tested at varying densities and stoichiometries onto the PCS-12 base formulation. The most dramatic increase in expansion was observed with aCD2-containing formulations PCS-1 to PCS-3, highlighting the importance of CD2 signaling in TIL expansion (FIG. 3). Less dramatic increases were observed with the ICAM containing formulations. CD2-containing formulations were further assessed. [0751] Additional costimulatory ligands (CD70 dimer trimer, OX40L, SLAMF-6) and cytokines (rIL-21) were added to increase expansion onto the PCS-1 base (FIG.4). However, these ligands did not increase TIL expansion compared to PCS-1. [0752] To test the quality of the expanded product, TIL products were phenotyped via flow cytometry. In TIL product, the makeup of CD8+ vs CD4+ cells in the T cell compartment is very important given CD8+ cells are cytotoxic and are essential in tumor elimination. (See Raskov et al. (2021). Cytotoxic CD8+ T cells in cancer and cancer immunotherapy. British journal of cancer, 124(2), 359–367). FIG.5A illustrates results of an expansion assay of select PCS formulations to specifically determine relative expansion specifically of the CD8+ subset of TIL, for both melanoma (open circles) and NSCLC (open squares) of TIL. The TIL in these assays were expanded according to the schemes illustrated in either FIG. 1B or FIG.1C in which the typical feeder cells were replaced with the indicated PCS formulations in the REP2 stage. In this assay, rICAM did not contribute to increased CD8+ TIL expansion, but aCD40, aOX40, CD70, and aSLAMF6 had some measurable positive effect. [0753] Inclusion of aCD2 and r4-1BB signals appeared to have the most favorable effect on TIL expansion. The PCS-1, PCS2, and PCS-3 all increased the total fold expansion of TIL compared to feeders in the REP2 stage. FIG. 5B illustrates results of a titration experiment to determine an optimal aCD2 signal intensity for a PCS-based feeder replacement, indicating that a relatively low aCD2 signal intensity in the PCS formulation results in higher CD8+ skew in the expanded cultures. Notably, this trend was consistent in another assay when the CD8+ frequency from PCS-1, PCS-2, and PCS-3 were compared to feeder cells, demonstrating that the CD8+
frequency in the PCS-1 product remained consistent with the CD8+ population in feeder cell expanded-product (FIG.6A). Further, increased mol percentages of aCD2 caused a drop in CD8+ frequency. In additional analyses, melanoma TIL were expanded using PCS-1 instead of feeder cells in a longer REP2 (see FIG.1C). FIGs.6B and 6C illustrate the fold expansion and % CD8+ in paired melanoma TIL samples from parallel PCS-1 and feeder control conditions, where the TIL were expanded according to the scheme illustrated in FIG. 1C. These results demonstrate that replacement of feeder cells in at least the REP2 process results in equivalent expansion and CD8+ skew during production. Example 6. REP2 PCS-expanded TIL exhibit similar stem-like properties and similar diversity of clones compared to feeder-expanded TIL [0754] TIL products activated with PCS-1 in REP2 were phenotyped and compared to REP2 feeder-expanded TIL from the same tumor that were expanded in parallel. Overall, compared to feeder-expanded cells, PCS-expanded cells showed comparable stem-like phenotype in the CD8+ T cell compartment for all tumor types tested. Comparing PCS-1-stimulated CD8 T cells to feeder-expanded CD8 T cells in the TIL product showed there are no significant differences in the CD39-/CD69- population, which serve as a regenerative source of non-differentiated T cells (FIGs. 7A-7D). See Krishna et al. (2020) Stem-like CD8 T cells mediate response of adoptive cell immunotherapy against human cancer. Science, 370(6522), 1328–1334. Moreover, there were no differences in the CD27+ population between feeder-expanded TIL and PCS-expanded TIL. CD27+ cells serve as an activatable population for tumor cell elimination and CD8 T cell survival (FIGs.7E-7H). See Martin et al. (2018). Defining memory CD8 T cell. Frontiers in Immunology. Lastly, stratifying this population into CD27+/CD62L+ showed there were no significant differences in PCS-expanded TIL compared to feeder-expanded TIL (FIGs. 7I-7L). The flow cytometry gating strategy and output for a representative melanoma sample is illustrated in FIGs. 9A-9F. Overall, these results suggest that the PCS-1 formulation preserves the stemness of the CD8+ TIL from a broad variety of tumor types during expansion and does not significantly alter the stem-like-phenotype compared to feeder-expanded TIL. FIGs. 8A-8D illustrate the results additional phenotypical analyses of paired melanoma-derived TIL expanded in PCS-1 or feeder cells in the REP2 expansion stage according to the scheme of FIG. 1C. These data confirm that replacement of feeder cells with PCS results in similarly robust stemness profiles in the expanded TIL product. [0755] TIL products that were activated with PCS-1 in REP2 were processed and underwent bulk TCR sequencing, as described in Example 3. Simpson Clonality is a measure of
the diversity of TCR clones in the TIL product, where a lower value denotes a more diverse TCR repertoire. On the other hand, a higher Simpson Clonality represents dominance of one TCR clone in the TIL product. PCS-1-expanded TIL products showed similarly low Simpson Clonality as the feeder-expanded TIL products (FIGs.10A-10D). These results demonstrate that PCS-1 activation does not bias toward any particular clone, which is an important factor in producing a diverse TIL product. These results were confirmed in additional analyses of melanoma-derived TIL cultured in a process with a longer REP2 phase. The TIL were expanded using a 10-day REP2 expansion stage with either PCS-1 or feeder cells. FIG. 10E illustrates the Simpson Clonality scores of paired samples for TIL expanded according to the scheme of FIG. 1C, showing that the PCS-based expansion results in equivalent diversity in the expanded TIL. It is noted that a few individual instances were observed where the PCS-based expansion resulted in more diversity (i.e., lower Simpson Clonality score) compared to the paired feeder-expanded product. Example 7. The top 200 clones from the original tumor are maintained at a comparable levels between PCS and feeder-expanded TIL in REP2 [0756] TIL products that were activated with PCS-1 in REP2 were processed and underwent bulk TCR sequencing, as described in Example 3. The most prevalent clones (e.g., top 200) from the original tumor are an important subset of TIL that have been shown to be enriched in putative tumor-reactive clones. The distribution of the top 200 clones is similar when comparing the PCS and feeder-expanded TIL in the REP2 process (FIGs. 11A-11D). These results were confirmed in additional analyese of melanoma-derived TIL expanded in a longer REP2 phase. FIG. 11E shows the estimated number of putative tumor reactive cells in TIL populations that were expanded with either PCS-1 or feeder cells according to the scheme of FIG. 1C. There was no difference observed between the paired samples. There is no single dominant clone comprising a majority of the product and the top clone in the tumor is generally preserved in the TIL and feeder- expanded product. The sum productive frequencies of the top 200 clones (i.e., the percent of all TCRs in the expanded product that are in the top 200 from the original tumor) are similar in the PCS-1 product compared to the feeder product for all histologies (± 5%) (FIGs.12A-12D). Minor differences between PCS and feeder-expanded TIL in terms of top 200 clones are expected as the scale of experiments are not the same. Lastly, a similar number of the top 200 clones from the tumor are detectable in the PCS-1 product compared to the feeder product for all histologies (± 20%). Minor differences in the sum productive frequencies and absolute numbers of the top 200
clones retained between the PCS-1 and feeder products are expected because of differences in the production (250k for PCS-1 vs ≥ 1M for feeder). [0757] FIGs.13A and 13B are tables summarizing metrics used to evaluate TIL products that were produced using PCS-1 in REP2 (according to the general scheme in FIG. 1B). The relative expansion and CD8+ expansion, stemness profile, and retention of top 200 clones from tumor relative to the feeder-expanded products are summarized. FIG.13B specifically represents assays with additional costim combinations used in the REP1 expansion phase. These data demonstrate that PCS-1 replacement of feeder cells in the REP2 stage result in equivalent or improved expansion of stemlike TIL while retaining the diversity of putative tumor reactive cells. Example 8. Replacing feeder cells completely with PCS demonstrates comparable overall expansion in the MRM two-REP step process [0758] PCS-1 was tested as a complete replacement for feeder cells in the MRM two-REP step process. When using PCS-1 for T cell stimulation in both REP1 and REP2 (methods described in Example 3), an increased expansion in REP1 was observed compared to the feeder control arm (FIG. 14A). When PCS-1 was used again in REP2 (following REP1 stimulation with PCS-1) a modest decrease in expansion compared to feeder cells (FIG. 14B) was observed, potentially because the cells had expanded considerably more in REP1. However, evaluating the total expansion in the PCS feeder-free process with REP1 and REP2 combined, we observed improved total expansion compared to the feeder control arm (FIG.14C). Example 9. Replacing feeder cells completely with PCS demonstrates comparable overall CD8 percentages in the MRM two-REP step process [0759] To assess the quality of the PCS feeder free TIL product, TIL were phenotyped via flow cytometry. As discussed, given CD8+ cells are cytotoxic and are particularly potent at mediating tumor killing, the makeup of CD8+ vs CD4+ cells in the T cell compartment of the TIL product is important (see Raskov et al. (2021). Cytotoxic CD8+ T cells in cancer and cancer immunotherapy. British journal of cancer, 124(2), 359–367). At the end of both REP1 and REP2, we observed similar CD8+ percentages in the Stim-R and feeder arms suggesting that the complete replacement of feeder cells with PCS-1 does not alter the frequency of CD8+ T cells in the TIL throughout the production process or in the final product (FIGs.15A and 15B).
Example 10. TIL expanded in a feeder free process demonstrates comparable stem-like phenotypes compared to feeder-expanded TIL [0760] TIL were expanded with PCS in a feeder-free process and TIL products were phenotyped after REP2 Day 8 (8 days of REP1 and 8 days of REP2) along with a feeder cell control. On day 8 of REP2, the TIL products were stained for surface markers related to T cell phenotype and stemness as described in Example 3. The TIL products were compared to feeder- expanded TIL from the same tumor and production run. [0761] Overall, PCS cells showed comparable stem-like phenotype in the CD8 T cell compartment compared to feeder-expanded cells (FIGs.16A-16C). Comparing PCS-1-stimulated CD8 T cells to feeder-expanded CD8+ T cells, no loss in the CD39-CD69- population were observed, which serve as a regenerative source of non-differentiated T cells (FIG. 16A). See Krishna et al. (2020) Stem-like CD8 T cells mediate response of adoptive cell immunotherapy against human cancer. Science, 370(6522), 1328–1334. Moreover, there were no differences in the CD27+ population between feeder-expanded TIL and PCS-expanded TIL. CD27+ cells serve as an activatable population for tumor cell elimination and CD8+ T cell survival (FIG.16B) (Martin et al. (2018). Defining memory CD8 T cell. Frontiers in Immunology). Lastly, stratifying this population into CD27+CD62L+ there were no differences in PCS-expanded TIL compared to feeder-expanded TIL (FIG. 16C). Overall, these results suggest a feeder free process using PCS can produce TIL with similar stem-like phenotype as TIL expanded with feeder cells. Example 11. PCS presenting a combination of aCD3/aCD28/aCD2 and r41BBL showed superior TIL expansion compared to PCS presenting only aCD3/aCD28 [0762] TILs were activated using PCS-1, which presents a combination of aCD3, aCD28, aCD2 and r4-1BBL, or using PCS-18, which presents only aCD3 and aCD28 at the same molar densities as PCS-1, during the REP2 step of the MRM two-REP process. PCS formulations are described in Example 1. TILs were cultured according to the methods described in Example 3. TILs activated using PCS-1 showed significantly more robust expansion compared to using PCS- 18 (FIG.17). The expansion achieved using PCS-18 is likely insufficient to generate a clinically relevant dose-level. This data suggests that stimulation using aCD3 and aCD28 alone is not sufficient to robustly expand TILs.
Example 12. Utilizing PCS to Reduce Live Feeder Cells in the Tumor Infiltrating Lymphocyte Culture Process [0763] In the studies described herein, PCS was further assessed as a synthetic replacement for live feeder cells during the MRM P2 process (FIG.18). Irradiated feeder cells were analyzed using flow cytometry to assess the expression of T-cell stimulating ligands. Based on these data, PCS formulations were designed to present ligands expressed by feeder cells in different combinations, densities, and stoichiometries (as previously described). These formulations were utilized to replace feeder cells, as described above. PCS-1 meditated the highest CD8+ TIL expansion. Deeper characterization of TIL products produced from 12 melanoma tumor samples using either PCS-1 or feeder cells was performed. The TIL products were evaluated for the expression of stemness markers (CD62L+CD27+ and CD39-CD69-) using flow cytometry, and retention of putative tumor-reactive clones, assessed as the highest frequency clones from the starting tumor material, using T cell receptor (TCR) sequencing. [0764] PCS-1 mediated comparable TIL expansion as feeder cells in the MRM two-REP process at research scale (FIG. 19). TIL products generated with PCS-1 retained similar proportions of stem-like CD8 T cells and tumor reactive clones as TIL products generated with feeder cells. [0765] TIL products generated from 12 melanoma samples at research scale using PCS-1 exhibited similar expansion, expression of stemness markers, and retention of putative tumor- reactive clones as feeder cells. These results support implementation of PCS as a synthetic replacement for feeder cells as a solution to a major bottleneck in TIL manufacturing processes. Example 13. Large Scale TIL Production with PCS-Based Feeder Replacement, with Additional Formulation and Media Optimizations to Expand Low-Growing TIL [0766] As described in the above Examples, PCS-based feeder replacement formulations were developed that can reduce or eliminate entirely the feeder cell component from TIL production protocols. Using PCS formulations, e.g., PCS-1, TIL expanded at similar levels and maintained advantageous stemness profiles, CD8+ skew profiles, and numbers of putative tumor reactive T cells. As initial production protocols were conducted at small research-scale (e.g., 2.5x105 seeded into 24 well tissue culture (“24 W TC”) plates), TIL production was conducted at larger scale in the 6M G-Rex® platform seeding with initial 2.5x106 cells to determine if expansion
using the PCS feeder replacement platform could be practically applied for clinical-scale production. [0767] Briefly, 2.5x106 post-REP1 TIL were seeded in the G-Rex® platform in 50 mLs of Complete MRM media, including IL-2, IL-15 and IL-21 (see, e.g., Table 3, above), with 0.25 mg of PCS. After 4 or 5 days, 50 mLs of Complete OpTmizer “TCM” media, also including IL-2, IL- 15 and IL-21, was slowly added to the 6M wells to avoid disturbing the cell. Cells were harvested 10 days post- seeding and analyzed with assays listed in Example 3. [0768] TIL obtained from NSCLC, melanoma, and CRC/CRLM tumors were cultured in modes for comparison: the benchmark MRM two-REP process that incorporates feeder cells in the REP1 and REP2 stages, the large-scale 6M format with PCS-1 feeder replacement in REP2, and the research-scale 24W tissue culture format with PCS-1 feeder replacement in REP2. FIGs.20A- 20C compare the REP2 expansion of TIL in the different formats for the respective TIL. While three of the six NSCLC-derived TIL demonstrated superior expansion in REP2 compared to the feeder benchmark protocol, it was noted that the other three NSCLC-derived TIL that were culture did not expand in the 6M feeder replacement format. In contrast, the melanoma and CRC/CRLM- derived TIL did expand in this format, at times better than in the research scale format. The presence of a few TIL sample (specifically from NSCLC) that did not expand indicated that some tumors have low growing TIL in a feeder-free context and further optimization would be required for these TIL to more precisely replicate the positive effects of feeder cells. Without being limited to any particular theory, it was hypothesized that one or more soluble cues and/or surface cues typically presented by feeder cells may be missing from current feeder replacement formulations that would be beneficial to support expansion of these low-growing TIL. [0769] To further characterize the practical utility of TIL expanded using feeder replacement, we assessed reactivity from the TIL grown with PCS-1 based on preservation of putative tumor reactive clones (via T cell repertoire sequencing) and gMACs (tumor) coculture reactivity. Based on sequencing analysis, we first determined the identity of the top 20% of TCR clones in the input cell population. We subsequently observed a slight drop in the fraction of the aforementioned top 20% of clones in the expanded TIL product after REP2 in PCS-1 feeder replacement compared to a feeder control (FIGs. 21A and 21B for NSCLC and melanoma, respectively). Notwithstanding the slight drop in representation of these clones, when we estimated the putative tumor reactivity of a final clinical-scale product we observed no difference in the amount of putative tumor reactive cells in a patient dose when replacing feeder cells with PCS
(FIGs. 21C and 21D for NSCLC and melanoma, respectively), which is likely due to superior overall expansion of the putative tumor reactive cells. [0770] Tumor reactivity of a TIL product can also be measured by assaying the upregulation of activation markers by the TIL when co-cultured with the originating tumor. In this assay, we evaluated the tumor reactivity of TIL products from three tumors made using PCS-based feeder replacement and a feeder-based control process by co-culturing the TIL products with CD45-depleted tumor samples. We assessed reactivity via upregulation of 4-1BB (CD137) in the TIL (FIGs.22A-22C). In two of the three tumors, TIL product reactivity to the tumor was at least equivalent if not better (within 2% of 4-1BB expression). The tumor that saw reduced reactivity in the product the was expanded from an NSCLC sample with an established low activation status (CD3+PD-1lowCD39low) and established low level of TIL infiltration. Overall, these observations demonstrate that the TIL product generated with PCS-based feeder replacement retains putative tumor reactivity (based on retained T cell clone repertoire) in the product and these cells are remain reactive to the original tumor cells, as comparable to the feeder controls. It is noted that these conclusions, in particular the coculture reactivity, were made from a preliminary data set with a small set of samples. Further tumor coculture experiments with more replicates will bolster the conclusions and strengthen the argument for tumor reactivity equivalence between PCS-based feeder replacement and feeder-based T cell expansion protocols. [0771] To address potential soluble cues in the Stim-R/coculture we cultured feeder cells (2.5 million feeder cells/mL) in MRM media for 48 hours. After 48 hours we pelleted the cells and collected the media for proteomics analysis. Combining proteomics analysis with gene ontology data we identified 5 proteins, CCL5, CXCL10, IL-18, IL-8, and SLAMF-1, secreted by feeder cells (referred to as “feeder-secreted proteins” or “FSPs”) that may have a role in promoting T cell expansion. We performed TIL expansion in the benchmark MRM two-REP process that incorporates feeder cells in the REP1 and REP2 stages, as described above, and in the large-scale 6M format using PCS-1 feeder replacement with or without adding the FSPs into the REP2 media (10ng/mL). We observed an increase in TIL expansion using PCS-1 feeder replacement with the FSPs while also maintaining the CD8 percentage of the final product compared to feeder-based expansion. This favorable expansion and maintenance of CD8 component were observed regardless of whether the TIL were from fresh or frozen samples (FIGs.23A-23D). [0772] To explore whether there were any additional surface cues from feeder cells that could further promote expansion of the observed low-growing TIL, we profiled TIL after REP1 stage of expansion. Specifically, we applied a costim panel using flow cytometry to understand
what receptors are present on the cultured TIL cells that could be targetable. We observed increased expression of ICOS on CD8+ T cells. Accordingly, we added an anti-ICOS antibody onto the PCS- 1 base formulation to create the formula designated PCS-21 (see Table 6). Table 6. Mass ratio details for PCS formulation PCS-21.
[0773] Combining the FSPs described above with PCS-21, we observed increased expansion compared to both PCS-1 with FSPs and feeder-based MRM two replication stage protocol (with no supplemented FSPs) (FIGs.24A-24D). As shown, TIL generally expanded better when utilizing the PCS-21 feeder replacement formulation in REP2 compared to the PCS-1 feeder replacement formulation and uniformly better than the feeder-based MRM control expansion protocol. Further, the PCS-21 and PCS-1 feeder replacement formulations resulted in promising expansion even for identified lowly activated tumors (based on initial CD39 and PD-1 positivity). Both formulations were confirmed to maintain the CD8+ proportion of the expanded product, indicating that critical characteristics of the expanded TIL are unchanged from benchmark feeder- based protocols. To further investigate the phenotypes of the TIL expanded with PCS-based feeder replacement in REP2, the stemness profiles of the TIL product were assayed by flow cytometry. As shown in FIGs. 25A-25D, no differences in the stemlike qualities of the TIL product were observed when expanding with the PCS-21 feeder replacement formulation compared to the PCS- 1 feeder replacement formulation or the feeder-based MRM two-REP process expansion protocol control. [0774] Paramount to the efficacy of TIL is the preservation of putative tumor reactive T cells in the expanded product. TIL expanded with PCS-21 and the FSPs were on average 14% less populated with the top 20% clones from the original tumor (FIG.26A) compared to a feeder-based control process. However, when projecting out to a clinical dose of TIL given to patients,
significantly more putative tumor reactive T cells are predicted in a product made using the PCS- 21 feeder replacement + FSPs compared to the control feeder-based P2 process (FIG.26B). [0775] These results demonstrate that the PCS platform for feeder replacement can successfully facilitate TIL expansion at large (e.g., clinical) scales while maintaining favorable T cell phenotypes, including stemness profiles and CD8+ skew. Furthermore, these studies identified additional signals that can be readily incorporated into the feeder replacement platform to specifically facilitate expansion of TIL that are particularly difficult to grow in the large-scale format (i.e., “low-growing”). The additional cues include an anti-ICOS binder that can be attached as a surface cue in the PCS formulation and that engages ICOS on the TIL. The study also identified five feeder-secreted proteins (FSPs) that can be added as media supplements or soluble cues in the PCS formulation itself to facilitate expansion of the TIL. Ultimately, this work provides a feeder replacement platform that is practically scalable and tunable to facilitate expansion of high quality T cells, e.g., TIL, NK cells, Treg cells, gamma delta T cells, etc., that obviates the need for large numbers of, or indeed any, feeder cells. Furthermore, use of a defined PCS formulation allows for a consistent activation reagent for expansion of the cells that is not susceptible to batch-to-batch variation as are feeder cells, which need to be continually sourced from multiple healthy donors. Thus, the disclosed PCS feeder replacement platform can be incorporated into manufacturing protocols to produce T cells appropriate for adoptive cell therapies. Example 14. TIL Production Characteristics with Increased anti-ICOS Component in the PCS-Based Feeder Replacement [0776] To further evaluate the effect of ICOS stimulation, we increased the density of the aICOS molecule on the surface of PCS-21 and evaluated growth and CD8 frequency as described in Example 13. [0777] The new formulation, i.e., PCS-22, is the same as PCS-21 but has 0.05 mol percentage of aICOS compared to the 0.01 on PCS-21. See Table 7. Table 7. Mass ratio details for PCS formulation PCS-22.
[0778] As illustrated in FIGs.27A and 27B, the increased ICOS stimulation provided by PCS-22 resulted in increased growth and expansion of TIL in the REP2 stage compared to PCS- 21 and feeder cells. The proportion of the CD8+ cells after the REP2 stage was slightly lower using the increased ICOS component compared to the PCS-21 formulation and feeder cell control, it remained above 70%. This demonstrates that anti-ICOS component of a PCS formulation can be modified, e.g., increased, to tune the PCS formulation to achieve desired TIL expansion characteristics without need for feeder cells during the expansion stage. Example 15. Optimization of Feeder Secreted Protein (FSP) Components for TIL Expansion [0779] Example 13 described the discovery of several feeder secreted proteins (FSPs) that can be incorporated into the PCS formulation or expansion media to promote expansion of even difficult to grow TIL. The individual contributions of each of the individual FSPs on TIL growth can be titrated to optimize the signaling and resultant expansion of the TIL. In one assay, the contributions of each individual FSP would be measured by singly eliminating each factor from both Melanoma and NSCLC TIL experiments as described in Example 13. Briefly, PCS-21 would be utilized in conjunction with an “all-but-one” combination of FSPs (e.g., CCL5, CXCL10, IL- 18, IL-8) evaluating cell proliferation and CD8 skew compared to TIL expanded with PCS-21 and the full set of FSPs and feeder cells. Critical factors could then be identified by dramatic drops in proliferation or CD8 skew, whereas extraneous FSPs could be identified by no loss of proliferation or CD8 skew compared to the controls. [0780] Once the critical, minimal set of FSPs is identified, optimal concentrations of the FSPs for TIL culture could be identified by titrating each protein individually starting with a saturating concentration (i.e., EC100) and evaluating the same readouts as described above on both Melanoma and NSCLC TIL cultures. *** [0781] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary embodiments of the present disclosure as contemplated by the inventor(s), and thus, are not intended to limit the present disclosure and the appended claims in any way.
[0782] The present disclosure has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed. [0783] The foregoing description of the specific embodiments will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance. [0784] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents. [0785] The contents of all cited references (including literature references, U.S. or foreign patents or patent applications, and websites) that are cited throughout this application are hereby expressly incorporated by reference as if written herein in their entireties for any purpose, as are the references cited therein. Where any inconsistencies arise, material literally disclosed herein controls.