Feeder cell replacement

Abstract

The present disclosure provides methods of expanding immune cells ex vivo or in vitro comprising culturing the immune cells in a medium comprising a feeder cell replacement, e.g., a PCS, comprising a surface-exposed CD3 agonist. In some aspects, the immune cells comprise T cells, TILs, NK cells, Tregs, or any combination thereof. In some aspects, a population of expanded TILs are administered to a subject in need thereof.

Description

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 feedercell 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 thefull 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, Claudin18.2, 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 factorreceptor 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 109TILs.

[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 1010TILs.

[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 theproportion 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 inhibitorcomprises 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 someaspects, 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 illustratedin FIG. 1C. FIG. 8A compares a stemlike phenotype (CD39-CD69-) in CD8+ T cells betweenproducts 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 betweenproducts 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)]. Thetotal 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 105TIL were stimulated with PCS-1 and 5 x 106TIL 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 FSPsupplements. 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 furthercomprises 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 to60.5 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 someaspects, 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 Tregcell. 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 cellculturing 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 ormore 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 particularpopulation 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, atleast 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 OPTMIZERTMPro. In some aspects, the basal media comprises X-VIVOTM15 (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 TRANSACTTMTIL expansion medium. In some aspects, the basal media comprises TIL rapid expansion medium. In some aspects, the basal medium furthercomprises 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 cytokinewith 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 expressedin 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, providesthe 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 leadersequence 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 CD3which together forms the CD3 complex. T-cell receptor- engineered T-cell therapy utilizes the modification of T cells that retain these complexes to specifically target the antigens expressed by particular tumor cells. As used herein, the term "TCR" includes naturally occurring TCRs and engineered TCRs.

[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 nucleobasesequence "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 cancercomprises 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 therapycomprises 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, aneffective 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 furthercomprising 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 106to about 1 X 1010. In some aspects, the amount of reference feeder cells is from about 500 X 106to 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 expansionof 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 106cells. 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 106cells. 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 someaspects, 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, themesoporous 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 150m2 / 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 anycombination 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’)2fragment, 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, theantibody 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 aredescribed 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-CD2antibody. 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-stimulatorymolecule 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 orstoichiometry 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, providedthere 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%, about0.9%, 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) comprisesabout 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. Insome 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 mannerfor 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. Forinstance, 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 reactionsavailable 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 disordersand 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, thesemolecules 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 cellexpansion. 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 Tcell 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 expansionmedium, 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 tumorcomprises 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 comprisesa 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, theTILs 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 1010TILs. 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 about4 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 109TILs are obtained. In some aspects, the initial expansion continues until at least about 2 x 109TILs are obtained. In some aspects, the initial expansion continues until at least about 3 x 109TILs are obtained. In some aspects, the initial expansion continues until at least about 4 x 109TILs are obtained. In some aspects, the initial expansion continues until at least about 5 x 109TILs are obtained. In some aspects, the initial expansion continues until at least about 6 x 109TILs are obtained. In some aspects, the initial expansion continues until at least about 7 x 109TILs are obtained. In some aspects, the initial expansion continues until at least about 8 x 109TILs are obtained. In some aspects, the initial expansion continues until at least about 9 x 109TILs are obtained. In some aspects, the initial expansion continues until at least about 10 x 109TILs are obtained.

[0336] In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 1 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 2 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 3 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 4 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 5 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 6 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 7 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 8 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 9 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 8 days, wherein at least about 10 x 109TILs are obtained.

[0337] In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 1 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 2 x 109TILs are obtained. In some aspects, the initial expansion lasts forless than 9 days, wherein at least about 3 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 4 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 5 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 6 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 7 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 8 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 9 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 9 days, wherein at least about 10 x 109TILs are obtained.

[0338] In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 1 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 2 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 3 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 4 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 5 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 6 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 7 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 8 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 9 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 10 days, wherein at least about 10 x 109TILs are obtained.

[0339] In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 1 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 2 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 3 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 4 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 5 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 6 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 7 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 8 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 9 x 109TILs are obtained. In some aspects, the initial expansion lasts for less than 11 days, wherein at least about 10 x 109TILs 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 leastabout 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 s...

Claims

WHAT IS CLAIMED IS:

1. 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.

2. The method of claim 1, further comprising culturing the immune cells in an additional medium before or after the culturing (“additional expansion”).

3. The method of claim 2, wherein the additional expansion is before the initial expansion and does not comprise any feeder cells or feeder cell replacement.

4. The method of claim 2, wherein the additional expansion is after the initial expansion and comprises culturing the cells in a second medium.

5. 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.

6. The method of claim 5, wherein the feeder cell replacement in the first medium and the feeder cell replacement in the second medium are different.

7. The method of claim 5 or 6, further comprising culturing the immune cells in an additional medium (“additional expansion”) before or after the culturing in the first medium and / or the second medium.

8. The method of any one of claims 2 to 4 and 6 and 7, wherein the additional expansion is before the first expansion and does not comprise any feeder cells or a feeder cell replacement.

9. The method of claim 5 or 6, further comprising culturing the immune cells in an additional medium (“additional expansion”) between the first expansion and the second expansion.

10. The method of any one of claims 1 to 9, wherein the medium of the initial expansion does not comprise feeder cells.

11. The method of any one of claims 4 to 9, wherein the second medium does not comprise feeder cells.

12. The method of any one of claims 1 to 12, wherein one or more of the media comprise potassium ion at a concentration higher than 4 mM.

13. The method of any one of claims 1 to 9, wherein the medium of the initial expansion further comprises feeder cells.

14. The method of any one of claims 4 to 10 and 13, wherein the second medium further comprises feeder cells.

15. The method of claim 13 or 14, wherein 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.

16. The method of claim 15, wherein 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.

17. The method of claim 15 or 16, wherein the amount of reference feeder cells is between about 1e6 to about 1e10.

18. The method of claim 17, wherein the amount of reference feeder cells is from about 500e6 to about 8e9.

19. The method of any one of claims 1 to 18, wherein the amount of the feeder cell replacement is between about 0.01 to about 1 mg feeder cell replacement per 1e6 cells.

20. The method of any one of claims 1 to 19, wherein 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.

21. The method of claim any one of claims 13 to 17, wherein 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.

22. The method of any one of claims 1 to 21, wherein the PCS comprises (i) a base layer comprising high surface area mesoporous silica micro-rods (MSR); (ii) a continuous, fluid supported lipid bilayer (SLB) layered on the MSR base layer; and (iii) a plurality of surface cues 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").

23. The method of claim 22, wherein the plurality of surface cues is loaded onto the SLB layer.

24. The method of claim 22 or 23, wherein the plurality of surface cues are loaded onto the fluid supported lipid bilayer (SLB).

25. The method of any one of claims 22 to 24, wherein the plurality of surface cues are coated onto the fluid supported lipid bilayer (SLB).

26. The method of any one of claims 22 to 25, wherein the plurality of surface cues are partly embedded onto the fluid supported lipid bilayer (SLB).

27. The method of any one of claims 22 to 26, wherein the plurality of surface cues are loaded onto the mesoporous silica micro-rods (MSR).

28. The method of any one of claims 22 to 27, wherein the CD3 agonist comprises an anti-CD3 antibody or an antigen-binding portion thereof ("anti-CD3 antibody").

29. The method of claim 28, wherein the anti-CD3 antibody comprises OKT3.

30. The method of any one of claims 22 to 29, wherein the CD28 agonist comprises an anti- CD28 antibody or an antigen-binding portion thereof ("anti-CD28 antibody").

31. The method of any one of claims 22 to 30, wherein the plurality of surface cues comprises (i) an anti-CD3 antibody, (ii) an anti-CD28 antibody, and (iii) a 4-1BB agonist.

32. The method of any one of claims 22 to 31, wherein 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.

33. The method of any one of claims 22 to 32, wherein 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.

34. The method of any one of claims 22 to 33, wherein 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.

35. The method of any one of claims 22 to 33, wherein 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.

36. The method of any one of claims 22 to 33, wherein 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.

37. The method of any one of claims 22 to 33, wherein 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.

38. The method of any one of claims 1 to 37, wherein the PCS further comprises a plurality of soluble cues.

39. The method of claim 38, wherein the plurality of surface cues is loaded onto a MSR base layer of the PCS or added directly to the one or more media.

40. The method of claim 38 or 39, wherein the soluble cue is released from the PCS in a controlled-release manner.

41. The method of claim 38 or 39, wherein the soluble cue is released from the PCS in a sustained manner for at least 30 days.

42. The method of any one of claims 38 to 41, wherein 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.

43. The method of any one of claims 38 to 42, wherein the plurality of soluble cues comprises (i) IL-2, an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof and (ii) a second soluble cue comprising IL-7, IL-21, IL-15, IL-15 superagonist, or any combination thereof.

44. The method of any one of claims 38 to 43, wherein the plurality of soluble cues comprises (i) IL-2, an agonist thereof, a mimetic thereof, a variant thereof, a functional fragment thereof, or a combination thereof, (ii) a second soluble cue comprising IL-7, IL-21, IL-15, IL-15 superagonist, or any combination thereof, and (iii) a third soluble cue comprising IL-7, IL-21, IL-15, IL-15 superagonist, or any combination thereof.

45. The method of any one of claims 38 to 44, wherein the plurality of soluble cues comprises an N-terminal IL-2 fragment comprising the first 30 amino acids of IL-2 (pl-30), an IL-2 superkine peptide, an IL-2 partial agonist peptide, or a combination thereof.

46. The method of any one of claims 1 to 45, wherein the PCS further comprises a recruitment compound comprising granulocyte macrophage-colony stimulating factor (GM-CSF), chemokine (C-C motif) ligand 21 (CCL-21), chemokine (C-C motif) ligand 19 (CCL-19), Chemokine (C-X- C Motif) ligand 12 (CXCL12), interferon gamma (IFNy), a FMS-like tyrosine kinase 3 (Flt-3) ligand, or any combination thereof.

47. The method of claim 46, wherein the recruitment compound comprises granulocyte macrophage colony stimulating factor (GM-CSF).

48. The method of any one of claims 1 to 47, wherein the PCS comprises about 0.005% to about 1% anti-CD3 antibody, e.g., OKT3.

49. The method of any one of claims 1 to 48, wherein 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.

50. The method of any one of claims 1 to 49, wherein the PCS comprises about 0.05% anti- CD3 antibody, e.g., OKT3.

51. The method of any one of claims 1 to 50, wherein the PCS comprises about 0.005% to about 1% anti-CD28 antibody.

52. The method of any one of claims 1 to 51, wherein 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.

53. The method of any one of claims 1 to 52, wherein the PCS comprises about 0.01% anti- CD28 antibody.

54. The method of any one of claims 1 to 53, wherein the PCS comprises about 0.005% to about 1% r4-1BBL.

55. The method of any one of claims 1 to 54, wherein 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.

56. The method of any one of claims 1 to 55, wherein the PCS comprises about 0.15% r4- 1BBL.

57. The method of any one of claims 1 to 56, wherein the PCS comprises about 0.005% to about 1% anti-CD2 antibody.

58. The method of any one of claims 1 to 57, wherein 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.

59. The method of any one of claims 1 to 58, wherein the PCS comprises about 0.01% anti- CD2 antibody.

60. The method of any one of claims 2 to 4 and 7 to 59, wherein the PCS of the initial expansion and the PCS of the additional expansion are the same.

61. The method of any one of claims 2 to 4 and 7 to 59, wherein the PCS of the initial expansion and the PCS of the additional expansion are different.

62. The method of any one of claims 1 to 61, wherein the CD3 agonist comprises an antibody or an antigen-binding portion thereof that specifically binds CD3 ("anti-CD3 antibody").

63. The method of claim 62, wherein the anti-CD3 antibody comprises OKT3.

64. The method of any one of claims 1 to 63, wherein the medium further comprises a CD28 agonist.

65. The method of claim 64, wherein the CD28 agonist comprises an antibody or an antigen- binding portion thereof that specifically binds CD28 ("anti-CD28 antibody").

66. The method of any one of claims 1 to 65, wherein one or more of the media does not comprise a CD3 agonist and / or a CD28 agonist that is not part of the PCS.

67. The method of any one of claims 1 to 66, wherein the immune cells comprise T cells, TILs, NK cells, TILs, Tregs, or any combination thereof.

68. The method of claim 67, wherein the TILs are obtained from a human tumor sample.

69. The method of claim 68, wherein the tumor sample is dissociated prior to the first expansion.

70. The method of claim 69, wherein the dissociated tumor sample is a single cell suspension.

71. The method of any one of claims 1 to 70, wherein the immune cells express a chimeric antigen receptor (CAR).

72. The method of claim 71, wherein 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.

73. The method of any one of claims 1 to 72, wherein the cells immune cells express an engineered T cell receptor (TCR).

74. The method of claim 73, wherein 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.

75. The method of any one of claims 1 to 74, wherein 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.

76. The method of claim 75, wherein the one or more cytokines comprises IL-2 and IL-21.

77. The method of claim 74 or 75, wherein the one or more cytokines comprises IL-2, IL-15, and IL-21.

78. The method of any one of claims 1 to 77, wherein the PCS further comprises an antigen.

79. The method of claim 78, wherein the antigen comprises a tumor antigen.

80. The method of claim 79, wherein the tumor antigen is adenomatous polyposis coli protein (APC), adenosine deaminase-binding protein (AD Abp), a-fetoprotein, AFP (alpha-fetoprotein), AIM-2, AIM-3, and WT1), ART1, ART4, B7-H3, B7-H6, BAGE, BCMA, B-cyclin, BMI1, Braf, brain glycogen phosphorylase, BRAP, C13orf24, C6orfl53, C9orf 112, CA-125, CA9 (carbonic anhydrase 9), CASP-8, cathepsin B, Cav-1, CCL-1 (C-C motif chemokine ligand 1), CD123, 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, Claudin18.2, 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 factorreceptor 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.

81. The method of any one of claims 22 to 80, wherein 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.

82. The method of any one of claims 23 to 81, wherein 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.

83. The method of any one of claims 23 to 82, wherein the mesoporous silica microrod-lipid bilayer (MSR-SLB) scaffold retains a continuous, fluid architecture for at least 14 days.

84. The method of any one of claims 23 to 83, wherein 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.

85. The method of any one of claims 1 to 84, wherein the initial expansion lasts about 5-19 days.

86. The method of any one of claims 68 to 70 and 75 to 85, wherein the TILs are subjected to the initial expansion for less than 11 days.

87. The method of claim 86, wherein the TILs are subjected to the initial expansion for less than 10 days.

88. The method of claim 86 or 87, wherein the TILs are subjected to the initial expansion for less than 9 days.

89. The method of any one of claims 86 to 88, wherein the TILs are subjected to the initial expansion for about 8 days.

90. The method of any one of claims 86 to 89, wherein the TILs are subjected to the initial expansion for at least about 14 days.

91. The method of any one of claims 86 to 90, wherein 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.

92. The method of any one of claims 86 to 91, wherein the number of TILs present in the dissociated tumor sample is increased by at least about 20-fold following the first expansion.

93. The method of any one of claims 86 to 92, wherein 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 109TILs.

94. The method of any one of claims 86 to 93, wherein the TILs from the initial expansion are subjected to the additional expansion for less than 11 days.

95. The method of claim 94, wherein the TILs are subjected to the additional expansion for less than 10 days.

96. The method of claim 94 or 95, wherein the TILs are subject to the additional expansion for less than 9 days.

97. The method of any one of claims 94 to 96, wherein the TILs are subjected to the additional expansion for less than 8 days.

98. The method of any one of claims 94 to 97, wherein 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.

99. The method of any one of claims 94 to 98, wherein the number of TILs present following the initial expansion is increased by at least about 20-fold following the additional expansion.

100. The method of any one of claims 94 to 99, wherein 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 1010TILs.

101. The method of any one of claims 68 to 70 and 75 to 100, wherein 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.

102. The method of claim 101, wherein 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.

103. The method of claim 101 or 102, wherein 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.

104. The method of any one of claims 66 to 70 and 75 to 103, wherein 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.

105. The method of claim 104, wherein 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.

106. The method of claim 104 or 105, wherein 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.

107. The method of any one of claims 68 to 70 and 75 to 106, wherein the tumor sample is obtained from a subject that has previously received one or more therapy for treating the tumor.

108. The method of claim 107, wherein the subject is relapsed or refractory to one or more prior therapy for treating the tumor.

109. The method of claim 107 or 108, wherein the one or more therapy for treating the tumor is selected from a chemotherapy, an immunotherapy, a radiotherapy, a surgery, or any combination thereof.

110. The method of any one of claims 68 to 70 and 75 to 109, wherein the tumor is refractory to a checkpoint inhibitor.

111. The method of claim 110, wherein 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.

112. The method of claim 111, wherein 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.

113. The method of any one of claims 68 to 70 and 75 to 112, wherein the tumor is metastatic.

114. The method of any one of claims 68 to 70 and 75 to 113, wherein the tumor comprises a solid tumor.

115. The method of claim 114, wherein 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.

116. The method of any one of claims 12 to 115, wherein 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.

117. The method of any one of claims 12 to 116, wherein the concentration of potassium ion in the one or more media is about 55 mM to about 90 mM.

118. The method of any one of claims 12 to 117, wherein 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.

119. The method of any one of claims 12 to 118, wherein 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.

120. The method of any one of claims 1 to 119, wherein one or more of the media further comprise sodium ion, calcium ion, glucose, or any combination thereof.

121. The method of any one of claims 1 to 120, wherein the one or more media further comprise sodium ion.

122. The method of claim 121, wherein the concentration of the sodium ion is from about 25 mM to about 100 mM.

123. The method of claim 121 or 122, wherein 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.

124. The method of any one of claims 121 to 123, wherein 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.

125. The method of any one of claims 121 to 124, wherein the concentration of the sodium ion is about 55 mM.

126. The method of any one of claims 121 to 125, wherein the concentration of the sodium ion is about 60 mM.

127. The method of any one of claims 121 to 124, wherein the concentration of the sodium ion is about 65 mM.

128. The method of any one of claims 1 to 127, wherein the one or more media further comprise a cell expansion agent.

129. The method of claim 128, wherein the cell expansion agent comprises a GSK3B inhibitor, an ACLY inhibitor, a PI3K inhibitor, an AKT inhibitor, or any combination thereof.

130. The method of claim 129, wherein the PI3K inhibitor comprises LY294002, pictilisib, CAL101, IC87114, or any combination thereof.

131. The method of claim 129, wherein the AKT inhibitor comprises MK2206, A443654, AKTi- VIII, or any combination thereof.

132. The method of any one of claims 1 to 131, wherein the one or more media further comprise glucose.

133. The method of claim 132, wherein the concentration of glucose is more than about 10 mM.

134. The method of claim 132 or 133, wherein 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.

135. The method of any one of claims 132 to 134, wherein the concentration of glucose is about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM.

136. The method of any one of claims 1 to 135, wherein the one or more media further comprise calcium ion.

137. The method of claim 136, wherein the concentration of calcium ion is more than about 0.4 mM.

138. The method of claim 136 or 137, wherein 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.

139. The method of any one of claims 136 to 138, wherein the concentration of calcium ion is about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, or about 2.0 mM.

140. The method of any one of claims 1 to 139, wherein 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).

141. A population of expanded immune cells prepared by the method of any one of claims 1 to 140.

142. A pharmaceutical composition comprising the population of expanded immune cells of claim 141, and a pharmaceutically acceptable carrier.

143. A method of killing target cells, comprising contacting the target cells with the population of expanded immune cells of claim 141 or the pharmaceutical composition of claim 142 under conditions that allow killing of the target cells by the immune cells.

144. A method of treating a patient in need thereof, comprising administering the population of expanded TILs of claim 138 or the pharmaceutical composition of claim 139 to the patient.

145. Use of the population of human expanded TILs or claim 141 for the manufacture of a medicament for treating a patient in need thereof.

146. A culture medium, comprising one or more of CCL5, CXCL10, IL-18, IL-8, and SLAMF- 1.

147. The culture medium of claim 146, comprising a modified basal medium, wherein the basal medium further comprises one or more of the CCL5, CXCL10, IL-18, IL-8, and SLAMF-1.

148. The culture medium of claim 147, wherein the basal medium is a balanced salt solution (e.g., PBS, DPBS, HBSS, EBSS), Dulbecco's Modified Eagle's Medium (DMEM), Click’smedium, 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.

149. The culture medium of claim 146 or 147, which is serum free.

150. The culture medium of claim 146 or 147, further comprising immune cell serum replacement (ICSR).

151. The culture medium of any one of claims 146 to 150, wherein the medium comprises a MRM.

152. The culture medium of any one of claims 146 to 151, comprising CCL5.

153. The culture medium of any one of claims 146 to 152, comprising CXCL10.

154. The culture medium of any one of claims 146 to 153, comprising IL-18.

155. The culture medium of any one of claims 146 to 154, comprising IL-8.

156. The culture medium of any one of claims 146 to 155, comprising SLAMF-1.

157. The culture medium of any one of claims 146 to 156, wherein the medium comprises two, three, or four of CCL5, CXCL10, IL-18, IL-8, and SLAMF-1.

158. The culture medium of any one of claims 146 to 157, wherein the medium comprises CCL5, CXCL10, IL-18, IL-8, and SLAMF-1.

159. The culture medium of any one of claims 146 to 157, wherein the medium further comprises one or more of IL-2, IL-17, IL-15, and IL-21, in any combination.

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