Enhanced expansion of tumor-reactive lymphocytes for cancer treatment

Abstract

Provided herein are methods of isolating and expanding a population of tumor reactive lymphocytes (TRLs) from a fluid sample of a subject in need thereof. In some embodiments, methods of isolating and expanding a population of TRLs comprise flowing the TRLs across a magnetic capture zone of a microfluidic device. In some embodiments, expanding a population of TRLs comprises exposing TRLs to a culture condition (e.g., cytokine environment) during expansion that modifies the rapidity or robustness of expansion or the purity, sensitivity (e.g., to cytokines), or memory phenotype of TRLs after expansion. Also provided herein are methods of administering a population of TRLs to a subject in need thereof. Also provided herein are compositions comprising therapeutically enhanced TRLs.

Description

ENHANCED EXPANSION OF TUMOR-REACTIVE LYMPHOCYTES FOR CANCERTREATMENTCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 652,447, filed May 28, 2024, and U.S. Provisional Application No. 63 / 652,433, filed May 28, 2024, which applications are incorporated herein by reference.BACKGROUND

[0002] Adoptive cellular therapy (ACT) is a form of immunotherapy that uses cells from a patient’s immune systems, such as T cells, as a treatment for cancer. ACT involves isolating, expanding, and modifying a patient’s immune cells, and then reinfusing the immune cells into the patient to enhance the immune system’s ability to fight cancer.SUMMARY

[0003] Provided herein is a method of expanding tumor reactive lymphocytes (TRLs), the method comprising: (a) obtaining CD8+CD103+ TRLs from a peripheral blood sample or a processed sample therefrom of a subject, and (b) culturing the CD8+CD103+ TRLs in a cell culture medium that lacks IL-2. In some embodiments, the cell culture medium that lacks IL-2 comprises less than a trace amount of IL-2. In some embodiments, the cell culture medium that lacks IL-2 comprises less than a biologically reactive amount of IL-2. In some embodiments, the cell culture medium that lacks IL-2 comprises less than about 50 lU / mL, less than about 40 lU / mL, less than about 30 lU / mL, less than about 20 lU / mL, less than about 10 lU / mL, less than about 5 lU / mL, less than about 2 lU / mL, or less than about 1 lU / mL of IL-2. In some embodiments, the subject has a cancer, or is suspected of having a cancer. In some embodiments, the cell culture medium comprises IL-7, IL-15, or a combination thereof. In some embodiments, the cell culture medium comprises the IL-7, and wherein the IL-7 is present at a concentration of at least about 20 ng / mL. In some embodiments, the cell culture medium comprises the IL-15, and wherein the IL-15 is present at a concentration of at least about 30 ng / mL. In some embodiments, the method further comprises: harvesting an expanded population of the CD8+CD103+ TRLs, wherein the expanded population is obtained from the culturing of (b). In some embodiments, the CD8+CD103+ TRLs are circulating tumor-reactive lymphocytes (cTRLs). In some embodiments, the culturing of (b) is performed for at least about 14 days. In some embodiments, the culturing of (b) is performed for at least about 7 days. In someembodiments, the culturing of (b) is performed for at least about 5 days. In some embodiments, the culturing of (b) increases a cell number of the CD8+CD103+ TRLs by at least about 1000- fold. In some embodiments, the culturing of (b) increases a cell number of the CD8+CD103+ TRLs by at least about 10-fold greater than that of CD8+CD103+ TRLs cultured in a culture medium comprising a biologically reactive amount of IL-2. In some embodiments, a purity of the expanded population comprises at least about 95% purity. In some embodiments, the expanded population of the CD8+CD103+ TRLs is characterized by having an enhanced IL-2 sensitivity. In some embodiments, an IL-2 sensitivity of the expanded population of the CD8+CD103+ TRLs is at least about 5-fold greater than that of CD8+CD103+ TRLs cultured in a culture medium comprising a biologically reactive amount of IL-2. In some embodiments, the expanded population of the CD8+CD103+ TRLs has at least about 60% increase in CD25+ expression compared to that of CD8+CD103+ TRLs cultured in a culture medium comprising a biologically reactive amount of IL-2. In some embodiments, the expanded population of the CD8+CD103+ TRLs has an enhanced cytotoxicity compared to that of CD8+CD103+ TRLs cultured in a culture medium comprising a biologically reactive amount of IL-2. In some embodiments, at least about 90% of the expanded population of CD8+CD103+ TRLs comprise an effector memory T cell. In some embodiments, the effector memory T cells comprise CCR7- CD45RO+. In some embodiments, the method further comprises enriching the CD8+CD103+ TRLs after the obtaining of (a), wherein the enriching comprises contacting the CD8+CD103+ lymphocytes of (a) with an antibody or antigen -binding fragment specific to CD8 or CD 103, wherein the antibody or antigen-binding fragment is conjugated to a magnetic nanoparticle, and magnetically separating the CD8+CD103+ lymphocytes from other components of the peripheral blood or the processed sample obtained therefrom by flowing the CD8+CD103+ lymphocytes across a magnetic capture zone disposed in a channel of a microfluidic device. In some embodiments, the microfluidic device comprises a plurality of magnetic capture zones, and wherein the plurality of magnetic capture zones is disposed to spatially separate cells with different degrees of magnetization. In some embodiments, the culturing of (b), the harvesting, or a combination thereof occurs in a closed system. In some embodiments, the culturing of (b), the harvesting, or a combination thereof occurs in an open system. In some embodiments, the method further comprises administering the expanded population of the CD8+CD103+ TRLs. In some embodiments, the expanded population of the CD8+CD103+ TRLs further comprises a pharmaceutically acceptable excipient, diluent, or vehicle. In some embodiments, the expanded population of the CD8+CD103+ TRLs further comprises a carbohydrate, an amino acid, a vitamin, a mineral, a pH buffer system, an anticoagulant, or any combination thereof. In someembodiments, the method further comprises modifying a CD8+CD103+ TRLs from the expanded population of the CD8+CD103+ TRLs to obtain an engineered CD8+CD103+ TRL by introducing an exogenous polynucleotide that encodes a therapeutically enhancing polypeptide under conditions sufficient to produce the therapeutically enhancing polypeptide by the at least one cell. In some embodiments, the exogenous polynucleotide encodes a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen binding domain. In some embodiments, the antigen binding domain binds to a tumor antigen. In some embodiments, the exogenous polynucleotide encodes an engineered T-Cell receptor. In some embodiments, the engineered T-Cell receptor comprises an antigen binding domain. In some embodiments, the antigen binding domain binds to a tumor antigen. In some embodiments, the method further comprises expanding the engineered CD8+CD103+ TRL. In some embodiments, the engineered CD8+CD103+ TRL is cultured in a culture medium lacking IL-2. In some embodiments, the engineered CD8+CD103+ TRL is cultured in a culture medium comprising IL- 15, IL-7, and IL- 2. In some embodiments, the cancer comprises a melanoma, a colorectal cancer, a lung cancer, mesothelioma cancer, breast cancer, head and neck cancer, cervical cancer, or ovarian cancer. In some embodiments, the biologically reactive amount of IL-2 comprises an amount sufficient to induce phosphorylation of one or more IL-2 receptor downstream signaling proteins. In some embodiments, the one or more IL-2 receptor downstream signaling proteins comprise STAT5. In some embodiments, the phosphorylation of one or more IL-2 receptor downstream signaling proteins is increased by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2.0-fold or more as compared to that of an unstimulated control. Disclosed herein in some embodiments is a system configured to perform a method disclosed herein. In some embodiments, a system can be configured to produce a composition as disclosed herein. In some embodiments, a system can comprise a composition as disclosed herein. In some embodiments, a system can comprise a computer readable medium operatively coupled to a processor wherein the computer readable medium is configured to perform a method as disclosed herein. In some embodiments, a system can comprise a cell culture apparatus. In some embodiments, a system can comprise a cell culture medium that does not comprise a biologically reactive amount of IL-2.

[0004] Provided herein is a method of treating a human subject with a cancer, the method comprising: administering the expanded population of CD8+CD103+ tumor reactive lymphocytes (TRLs) of the method described herein. Further provided herein is a composition consisting essentially of CD8+CD103+ tumor-reactive lymphocytes (TRLs) and a pharmaceuticallyacceptable excipient, diluent, or vehicle, wherein the CD8+CD103+ TRLs are from an expanded population of CD8+CD103+ TRLs cultured from a culture medium lacking IL-2. Further provided herein is a composition comprising CD8+CD103+ tumor-reactive lymphocytes (TRLs) and a pharmaceutically acceptable excipient, diluent, or vehicle, wherein the CD8+CD103+ TRLs are from an expanded population of CD8+CD103+ TRLs cultured from a culture medium lacking IL- 2. In some embodiments, the culture medium that lacks IL-2 comprises less than a trace amount of IL-2. In some embodiments, the culture medium that lacks IL-2 comprises less than a biologically reactive amount. In some embodiments, the culture medium that lacks IL-2 comprises less than about 50 lU / mL, less than about 40 lU / mL, less than about 30 lU / mL, less than about 20 lU / mL, less than about 10 lU / mL, less than about 5 lU / mL, less than about 2 lU / mL, or less than about 1 lU / mL of IL-2. In some embodiments, the subject has a cancer, or is suspected of having a cancer. In some embodiments, the cell culture medium comprises IL-7, IL-15, or a combination thereof. In some embodiments, the IL-7 is present at a concentration of at least about 20 ng / mL. In some embodiments, the IL-15 is present at a concentration of at least about 30 ng / mL. In some embodiments, the CD8+CD103+ TRLs are circulating tumor-reactive lymphocytes (cTRLs). In some embodiments, the expanded population of the CD8+CD103+ TRLs is characterized by having an enhanced IL-2 sensitivity. In some embodiments, the IL-2 sensitivity of the expanded population of the CD8+CD103+ TRLs is at least about 5-fold greater than that of CD8+CD103+ TRLs cultured in a culture medium comprising a biologically reactive amount of IL-2. In some embodiments, the expanded population of the CD8+CD103+ TRLs has at least about 60% increase in CD25+ expression compared to that of CD8+CD103+ TRLs cultured in a culture medium comprising a biologically reactive amount of IL-2. In some embodiments, the expanded population of the CD8+CD103+ TRLs has an enhanced cytotoxicity compared to that of CD8+CD103+ TRLs cultured in a culture medium comprising a biologically reactive amount of IL-2. In some embodiments, at least about 90% of cells in the expanded population of CD8+CD103+ TRLs comprise an effector memory T cell. In some embodiments, the expanded population of CD8+CD103+ TRLs comprises a population of engineered CD8+CD103+ TRLs, where an engineered CD8+CD103+ TRLs of the population of engineered CD8+CD103+ TRLs is modified by introducing an exogenous polynucleotide that encodes a therapeutically enhancing polypeptide under conditions sufficient to produce the therapeutically enhancing polypeptide by the at least one cell. In some embodiments, the exogenous polynucleotide encodes a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen binding domain. In some embodiments, the antigen binding domain binds to a tumor antigen. In some embodiments, the exogenous polynucleotide encodes an engineered T-Cell receptor. In someembodiments, the engineered T-Cell receptor comprises an antigen binding domain. In some embodiments, the antigen binding domain binds to a tumor antigen. In some embodiments, the cancer comprises a melanoma, a colorectal cancer, a lung cancer, mesothelioma cancer, breast cancer, head and neck cancer, cervical cancer, or ovarian cancer. In some embodiments, the biologically reactive amount of IL-2 comprises an amount sufficient to induce phosphorylation of one or more IL-2 receptor downstream signaling proteins. In some embodiments, the one or more IL-2 receptor downstream signaling proteins comprise STAT5. In some embodiments, the phosphorylation of one or more IL-2 receptor downstream signaling proteins is increased by at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2.0-fold or more as compared to that of an unstimulated control. Disclosed herein in some embodiments is a system configured to perform a method disclosed herein. In some embodiments, a system can be configured to produce a composition as disclosed herein. In some embodiments, a system can comprise a composition as disclosed herein. In some embodiments, a system can comprise a computer readable medium operatively coupled to a processor wherein the computer readable medium is configured to perform a method as disclosed herein. In some embodiments, a system can comprise a cell culture apparatus. In some embodiments, a system can comprise a cell culture medium that does not comprise a biologically reactive amount of IL-2.

[0005] Provided herein is a pharmaceutical formulation comprising the composition described herein. Also provided herein is an engineered circulating tumor-reactive lymphocyte (cTRL), comprising: (a) a cell surface marker, wherein the cell surface marker comprises CD8 and CD 103; and (b) chimeric antigen receptor (CAR), wherein the engineered cTRL is for use in treating cancer in a human subject, wherein the engineered cTRL is cultured in a culture medium lacking IL-2. Disclosed herein in some embodiments is a system configured to perform a method disclosed herein. In some embodiments, a system can be configured to produce a composition as disclosed herein. In some embodiments, a system can comprise a composition as disclosed herein. In some embodiments, a system can comprise a computer readable medium operatively coupled to a processor wherein the computer readable medium is configured to perform a method as disclosed herein. In some embodiments, a system can comprise a cell culture apparatus. In some embodiments, a system can comprise a cell culture medium that does not comprise a biologically reactive amount of IL-2.INCORPORATION BY REFERENCE

[0006] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0007] The novel features of the inventive concepts are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present inventive concepts will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the inventive concepts are utilized, and the accompanying drawings of which:

[0008] FIGs. 1A-1N show the working principle and the results of an experiment in which tumor-reactive lymphocytes (TRLs) in blood circulation are identified and isolated. FIG. 1A shows the working principle of tumor reactivity-mediated microfluidic cell sorting for analysis. Cells were firstly magnetically labeled based on their TCR reactivity with tumor antigen-derived MHC multimers. Then, magnetically labeled cells were separated from their counterparts by microfluidic cell sorting for downstream analysis. FIG. IB shows the workflow of the identification via defined epitope models. CD8+ T cells in PBMC were classified as tumor - reactive and nonreactive populations based on their reactivity with multimers. Classified cells were compared to intratumoral CD8+ tumor-infiltrating lymphocytes (TILs) for clonal analysis. FIG. 1C and FIG. ID show the quantification of OVA tumor-reactive T cells in tumor (FIG. 1C) and blood (FIG. ID). FIG. IE and FIG. IF show HA tumor reactive T-cells in tumor (FIG. IE) and blood (FIG. IF). FIGs. 1G-1N show the quantification of the sorting performance (e.g., purifying tumor reactive T cells) based on antibody and multimer through microfluidic sorting. FIGs. 1G-1I schematically show gating scheme for CD8+ cells at pre-sorting (FIG. 1G), postsorting with CD8 antibodies (FIG. 1H), and post-sorting with multimer (FIG. II). FIG. 1 J shows quantitative analysis of CD8+ cells. FIGs. 1K-1M schematically show gating scheme for HA- reactive T cells at pre-sorting (FIG. IK), post-sorting with CD8 antibody (FIG. IL), and postsorting with multimer (FIG. IM). FIG. IN shows quantitative analysis of HA-reactive T cells.

[0009] FIGs. 2A-2E show a comparison of clonal similarity among OVA reactive TIL (FIG. 2A), circulating Tumor Reactive Lymphocyte (cTRL; FIG. 2B), and peripheral blood monocytes (PBMC; FIG. 2C) by V-J usage profile. FIG. 2D and FIG. 2E show clonal similarities among the coverage of top 50 clones between each population. Unpaired t-test, mean±s.d., each dot represents a biological replicate. FIGs. 2F-2J show a comparison of clonal similarity among HA reactive TIL (FIG. 2F), cTRL (FIG. 2G), and PBMC (FIG. 2H) by V-J usage profile. FIG. 21 and FIG. 2J show clonal similarities among the coverage of top 50 clones between each population. In terms of the TCR repertoire, cTRLs cover 30%-85% of the top 50 clones presented in TILs, which is 3-8 times higher than the coverage of PBMC (-10%). Unpaired t-test, mean±s.d., each dot represents a biological replicate.

[0010] FIGs. 3A-3T show the results of experiments used to characterize the molecular and phenotypic signature of cTRL during and post migration. FIG. 3A shows volcano plots showing the differential expression (DE) of genes when comparing the Fragments Per Kilobase of transcript per Million mapped reads (FPKM0 from PBMC and cTRL identified by multimer-based sorting from the B16 model. FIG. 3B shows that key genes (e.g., TFF7, ILR, LEF1, ITGAE, and ZFP683) for CD8+ T cells are upregulated in cTRLs. FIG. 3C shows a Gene Set Enrichment Analysis (GSEA) analysis of enriched immunological pathways. FIG. 3D shows an overlay of the shared, highly expressed genes (e.g., CRTAM, SIDT1, ITGAE, GGT1, CD8A, and SLC6A19) from cTRLs in B16 and CT26 models. FIGs. 3E-3H show gating scheme to identify tumor- reactive circulating CD8+CD103+ populations by gating on CD8+ (FIG. 3E), CD 103 (FIG. 3F), and OVA-specificity in CD 103- population (FIG. 3G) or CD 103+ population (FIG. 3H). FIG. 31 and FIG. 3 J show the quantification of tumor-reactive fraction in circulating CD8+CD103+ populations in B16 (FIG. 31) and AE17 (FIG. 3 J) models. FIG. 3K and FIG. 3L show representative flow cytometric profiles from the in vivo cTRL migration model bearing a secondary tumor with OVA epitopes (FIG. 3K) and without OVA epitopes (FIG. 3L). cTRLs migrated from donor tumor express CD45.2 while the endogenous TILs from the host express CD45.1. FIG. 3M shows the quantification of the percentage of migrated cTRLs in the in vivo TIL migration model in Bl 6. FIG. 3N shows the quantification of the percentage of migrated cTRLs in the in vivo TIL migration model in AE17. FIGs. 3O-3S show the CyTOF analysis of CD45.2+ cTRLs (FIG. 3P) and CD45.1+ TILs (FIG. 30) for the expression of CD 103 (FIG. 3Q) in AE17 models. As shown in FIG. 3R and FIG. 3S, CD 103+ cells were more predominant in CD45.2+ population (FIG. 3R) than CD451+ population (FIG. 3S). FIG. 3T shows the quantification of CyTOF data for the expression of CD103, CD69 and Programmed cell deathprotein 1 (PD-1) in CD45.2+ cTRLs and CD45.1+ TILs. Unpaired t-test, mean±s.d., each dot represents a biological replicate.

[0011] FIGs. 4A-4X show the results of murine model experiments in which cTRLs were shown to exhibit significant levels of activity against primary and metastasized tumors. FIGs. 4A- 4C show the experimental workflow. cTRL, CD8+CD103- PBMC (FIG. 4A) and TIL (FIG. 4B) were expanded 1-2 weeks in vitro before adoptive cell transfer (FIG. 4C). IL-2 was given daily for the first 3 days post cell transfer to boost lymphocyte proliferation. FIG. 4D and FIG. 4E show the quantification of tumor size (FIG. 4D), and probability of survival (FIG. 4E). FIG. 4F shows the percentage of infiltrated CD8+ cells in s.c. B16 models treated by different T cells (n = 5). FIGs. 4G-4V show the representative bioluminescence images treated by different T cells (n = 6) in induced 4T1 metastasis models: at Day 9 (pre-therapy) of untreated (FIG. 4G), CD 103- PBMC (FIG. 4H), cTRL (FIG. 41), and luminescence scale (FIG. 4 J); at Day 14 (post-therapy) of untreated (FIG. 4K), CD103- PBMC (FIG. 4L), cTRL (FIG. 4M), and luminescence scale (FIG. 4N); at Day 21 of untreated (FIG. 40), CD 103- PBMC (FIG. 4P), cTRL (FIG. 4Q), and luminescence scale (FIG. 4R); at Day 28 of untreated (FIG. 4S), CD103- PBMC (FIG. 4T), cTRL (FIG. 4U), and luminescence scale (FIG. 4 V);. FIGs. 4W-4X show the quantification of the total flux (FIG. 4W) and survival rate (FIG. 4X) in induced 4T1 metastasis models. *p<0.05, **p<0.01, unpaired t-test, mean±s.d., each dot represents a biological replicate.

[0012] FIGs. 5A-5Q show the results of murine model experiments which demonstrate the synergistic effects of cTRLs and ICB / costimulatory molecules. FIGs. 5A-5C show the quantification of tumor size (FIG. 5A), survival rate (FIG. 5B), and percentage of infiltrated CD8+ cells (FIG. 5C) in s.c. MC38 models treated by different therapeutic modalities (n = 5). FIG. 5D is a gene expression analysis showing enriched pathways from upregulated RNAs, which reveal that aPD-1 and cTRLs generate different impacts to the immune responses within the tumor microenvironment in s.c. MC38 models. FIG. 5E and FIG. 5F show the quantification of infiltrated CD4+ T cells (FIG. 5E) and CD208+ dendritic cells (FIG. 5F) post different therapeutic modalities. FIG. 5G and FIG. 5H show rapid tumor rejection (FIG. 5G) and formation of long-lasting TRLs that were observed in cTRL-cured mice (FIG. 5H). FIGs. 5I-5K show the quantification of tumor size (FIG. 51), survival rate (FIG. 5J), and percentage of infiltrated CD8+ cells (FIG. 5K) in s.c. AE17 models treated by different therapeutic modalities (n = 5). FIGs. 5L-5P show the quantification of lymphocyte subpopulations including CD3+ population (FIG. 5L), CD3+CD4+ population (FIG. 5M), CD3+CD8+ (FIG. 5N), CD8+PD-1+ population (FIG. 50), and CD4+CD25+ population (FIG. 5P) at the endpoint of treatment inendogenous (CD45.1+) populations in s.c. AE17 models. FIG. 5Q shows the quantification of CD103 expression in transferred cTRLs (CD45.2+) and endogenous lymphocytes (CD45.1+) in s.c. AE17 models. *p<0.05, **p<0.01, unpaired t-test, mean±s.d., each dot represents a biological replicate.

[0013] FIGs. 6A-6R show the results of experiments investigating the presence of CD 103 cTRLs in human specimens. FIG. 6A shows the workflow of the co-culture assay to study the relationship between tumor-reactivity and CD 103 on human PBMCs and examine the level of tumor specificity of isolated cTRLs. FIGs. 6B-6E show the representative flowcytometric profile of IFN-y secreting populations according to CD 103 expression by gating on CD8+ (FIG. 6B) and CD103+ (FIG. 6C), and then gating for IFN-y in CD103- (FIG. 6D) and CD103+ (FIG. 6E) populations. This specific set of images is from PE71. FIGs. 6F-6I show the quantification of IFN-y secreting populations in CD8+CD103+ and CD8+CD103- cells across the patient cohort: PE69 (FIG. 6F), PE70 (FIG. 6G), PE71 (FIG. 6H), and PE72 (FIG. 61) of malignant pleural effusion (MPE). FIGs. 6J-6L show representative flow cytometric profiles of IFN-y secreting populations in CD8+ alone (FIG. 6J), co-culture with CD8+ population (FIG. 6K) and cocultured with CD8+CD103+ population (FIG. 6L). This specific set of images is from PE86. FIG. 6M and FIG. 6N show the quantification of IFN-y+ cells (FIG. 6M) and fold enrichment of IFN- y+ cells (FIG. 6N) across a set of 18 patient samples. Tumor cells used in a coculture model to induce IFN-y secretion were harvested either from tumor tissue (resected tissue) or malignant pleural effusions (MPE). Fold enrichment was calculated by comparing the percentage of IFNy+ cells in bulk CD8+ and CD8+CD103+ populations post co-culture. FIGs. 6O-6Q show a comparison of clonal similarity among TILs (FIG. 60), cTRLs (FIG. 6P), and CD103-PBMC (FIG. 6Q) by V-J usage profile. cTRLs contains four TIL-derived major clones. FIG. 6R shows an analysis of the coverage of top 50 clones between each population.

[0014] FIGs. 7A-7F show the results of experiments in which human CTRLs were isolated and validated. FIG. 7A and FIG. 7B show a representative flow cytometric profile of IFN-y secreting populations in Solute Carrier Family 6 Member 19 (SLC6A19)+, CD103+, and Systemic RNA Interference Defici ent-1 Transmembrane Family Member 1 (SIDT1)+ populations of CD8+ T cells, wherein the specific set of images is from PE96 (FIG. 7A) and SMARTER (Mesothelioma trail) P29 (FIG. 7B). FIGs. 7C-7F show the quantification of interferon-gamma (IFN-y) secreting populations in SLC6A19+, CD103, and SIDT1+ populations of CD8+ T cells according to gate shown in FIG. 7A and FIG. 7B across the patient cohort: PE96 (FIG. 7C), P29 (FIG. 7D), P30 (FIG. 7E), and P31 (FIG. 7F).

[0015] FIGs. 8A-8E show the immunogenic epitopes that stimulate endogenous immune responses against the implanted tumors. FIG. 8A illustrates a schematic representation of the workflow for identifying Tumor-Resident Lymphocytes (TRLs) and circulating Tumor-Resident Lymphocytes (cTRLs) using defined epitope models. FIG. 8B and FIG. 8C show the quantification of OVA tumor-reactive T cells in tumor (FIG. 8B) and blood (FIG. 8C). FIG. 8D and FIG. 8E show HA tumor reactive T-cells in tumor (FIG. 8D) and blood (FIG. 8E).

[0016] FIGs. 9A-9P show supporting data for the immunogenic epitopes-mediated models. FIGs. 9A-9D show the representative gating strategy used to identify infiltrated T cell in dissociated tumors by gating first on small cells (FIG. 9A), singlets (FIG. 9B), CD8+ cells (FIG. 9C), and then OVA-specific cells (FIG. 9D). FIG. 9E-9J show the representative cytometric profile of B16-OVA (FIG. 9E and FIG. 9F) and B16 (WT; FIG. 9G and FIG. 9H) models. FIG. 91 and FIG. 9 J show quantification of T cell infiltration (FIG. 91) and its impact on tumor weight (FIG. 9 J) at the end point for the B16OVA model. FIG. 9K-9O show the representative cytometric profile of CT26-HA by CD8a (FIG. 9K) and Pentamer (FIG. 9L); and CT26 (WT) by CD8a (FIG. 9N) and Pentamer (FIG. 90). FIG. 9M and FIG. 9P show quantification of T cell infiltration (FIG. 9M) and its impact on tumor weight (FIG. 9P) at the end point for the CT26- HA model.

[0017] FIGs. 10A-10H show the epitope-reactive T cells presented in blood circulation. FIG. 10A-10D show the representative gating strategy used to identify circulating T cells in RBC lysed blood by gating on lymphocytes (FIG. 10A), singlets (FIG. 10B), CD8+ population (FIG. 10C), and then HA-specific population (FIG. 10D). FIG. 10E and FIG. 10F show the representative cytometric profile for the B16-OVA (FIG. 10E) and B 16 (WT; FIG. 10F) models. FIG. 10G and FIG. 10H show the representative cytometric profile for the CT26-HA (FIG. 10G) and CT26 (WT; FIG. 10H) model.

[0018] FIGs. 11A-11J illustrate establishment of the in vivo TIL migration model. FIG. HA illustrates the tumor transplantation model, where the donor mice carry CD45.2 isoform and the host mice carry CD45.1 isoform. FIG. 11B shows a schematic representation of a CD45.1+ C57BL6 mouse carrying two tumors. Circles highlight the location of two tumors. FIG. 11C and FIG. HD shows the representative flow cytometric profile from the in vivo TIL migration with B16-OVA (FIG. 11C) and B16 (WT; FIG. HD). TILs migrated from donor tumor express CD45.2 while the TILs from the host express CD45.1. FIG. HE and FIG. HF show the representative flow cytometric profile from the in vivo cTRL migration model with AE17-OVA(FIG. HE) and AE17 (WT; FIG. HF). cTRLs migrated from donor tumor express CD45.2 while the endogenous TILs from the host express CD45.1. FIG. 11G and FIG. 11H show the quantification of the percentage of migrated cTRLs in the in vivo TIL migration model with B 16- OVA and WT (FIG. 11G) and AE17-0VA and WT (FIG. 11H). FIG. HI and FIG. 11 J show the quantification of CD45.2 / CD45.1 percentage of migrated TILs in the in vivo TIL migration model with B 16-0 VA and WT (FIG. HI) and AE17-0VA and WT (FIG. 11 J).

[0019] FIGs. 12A-12E show microfluidic magnetic cell sorting. FIG. 12A illustrates immunomagnetic labeling workflow. FIG. 12B illustrates the chip design that favors the quantitative capture of cells based on its marker expression. ‘X’-shaped structures were introduced to improve the capture performance through forming low-velocity capture pockets in the microfluidic device. FIG. 12C shows the numerical simulation of the flow velocity profile within the microfluidic device. Capture pockets were formed near the edge of ‘X’-shaped structures, as indicated by the black / dark-red color. FIG. 12D shows the experimental validation of the cell capture profile within the microfluidic device. Almost all cells were captured on the capture pocket, proving the microfluidic device works as designed. FIG. 12E shows photographs of fabricated microfluidic devices along / on magnets. Food dye was used to visualize the channels.

[0020] FIG. 13 shows the representative workflow of microfluidic magnetic cell sorting according to some embodiments herein.

[0021] FIGs. 14A-14F show the comparison of the performance of rare cell isolation based on multimer-labeling among FACS, MACS and microfluidics. FIGs. 14A-14D schematic representations of OVA-specific isolation at pre-sorting (FIG. 14A), after FACS (FIG. 14B), after MACS (FIG. 14C) and after microfluidics (FIG. 14D). FIG. 14E and FIG. 14F show quantifications of isolated OVA-specific cells (FIG. 14E) and recovered OVA-specific T cells (FIG. 14F)

[0022] FIGs. 15A-15G represent enrichment plots of the GSEA data from FIG. 3C. FIG. 15A shows T cell receptor signaling pathway. FIG. 15B shows T-cell activation. FIG. 15C shows positive regulations of T cell receptor signaling pathway. FIG. 15D shows G1 to S cell cycle transition. FIG. 15E shows extension of telomeres. FIG. 15F shows downstream TCR signaling. FIG. 15G shows TCR signaling.

[0023] FIGs. 16A-16B shows the molecular signature of cTRLs from the CT26 model. FIG. 16A shows volcano plots showing the differential expression (DE) of genes when comparing theFPKM from PBMC and cTRLs from the CT26 model. FIG. 16B shows key genes for CD8+ T cells were presented as a heatmap alongside.

[0024] FIGs. 17A-17F illustrate representative gating strategy used to identify cTRLs and endogenous TILs in the in vivo migration model by gating on event length and center (FIG. 17A), width and center (FIG. 17B), and width and residual (FIG. 17C), then selecting DNA+ population (FIG. 17D), CD3+ population (FIG. 17E), and CD8+ populations (FIG. 17F).

[0025] FIGs. 18A-18J show a rapid expansion of cTRLs derived from MC-38 models using a feeder-based protocol. For the sake of visualization, CD8+ T cells from CD45.1+ mice were used as feeder to expand CD45.2+ cTRLs isolated from blood. FIGs. 18A-18D show the representative flow cytometric profile of the CD45.2 / CD45.1 ratio during the process of expansion at Day 0 (FIG. 18A), Day 4 (FIG. 18B), Day7 (FIG. 18C), and Day 10 (FIG. 18D). FIG. 18E and FIG. 18F show the representative flow cytometric profile of the control samples from the CD8+ splenocytes isolated from CD45.1 (FIG. 18E) or CD45.2 (FIG. 18F) mice. FIG. 18G shows the quantification of the fold of expansion. FIGs. 18H-18J show a representative gating by lymphocytes (FIG. 18H), CD8+ population (FIG. 181), and CD45.2 / CD45.1 (FIG. 18J) used for the data presented from FIGs. 18A-18G.

[0026] FIGs. 19A-19G show the rapid expansion of cTRLs derived from KPCY 6419c5 models using a feeder-based protocol. FIGs. 19A-19F illustrate the representative flow cytometric profile of the CD45.2 / CD45.1 ratio during the process of expansion of feeder-only at Day 3 (FIG. 19A), Day6 (FIG. 19B), and Day 10 (FIG. 19C); of CD8+CD103+ with feeder at Day 3 (FIG. 19D), Day6 (FIG. 19E), and Day 10 (FIG. 19F). FIG. 19G shows a quantification of the fold of expansion.

[0027] FIGs. 20A-20I illustrate phenotyping of expanded cTRLs from MC-38 models. FIG. 20A represents the quantification of relative mRNA expression of cTRLs compared to TILs via TaqMan probes. FIG. 20B-20I represents the quantification of protein expression of cTRLs and TILs for essential markers, including CD103 (FIG. 20B), CD69 (FIG. 20C), PD-lhigh(FIG. 20D), TIM3 (FIG. 20E), CD62L (FIG. 20F), TCF7 (FIG. 20G), PD-1+ (FIG. 20H), and 4-1BB (FIG. 201).

[0028] FIGs. 21A-21S show the representative flow cytometric profile of the data used in FIG. 20B-20I. FIGs. 21A-21C show CD 103+ gating schemes for isotypes (FIG. 21 A), cTRL (FIG. 21B), and TIL (FIG. 21C). FIGs. 21D-21F show CD69+ gating schemes for isotypes (FIG.21D), CTRL (FIG. 21E), and TIL (FIG. 21F). FIGs. 21G-21I show TCF7+ gating schemes for isotypes (FIG. 21G), cTRL (FIG. 21H), and TIL (FIG. 211). FIGs. 21 J-21L show 4-1BB+ gating schemes for isotypes (FIG. 21 J), cTRL (FIG. 21K), and TIL (FIG. 21L). FIGs. 21M-21O show TIM3+ gating schemes for isotypes (FIG. 21M), cTRL (FIG. 21N), and TIL (FIG. 210). FIGs. 21P-21R show CD62L+ gating schemes for isotypes (FIG. 21P), cTRL (FIG. 21Q), and TIL (FIG. 21R). FIG. 21S shows gating scheme for lymphocytes.

[0029] FIGs. 22A and 22B show in vitro evaluation of the anti-tumor efficacy of cTRLs and TILs via co-culture killing assay of MC-38 (FIG. 22 A) or KPCY 6419c5 (FIG. 22B) tumors.

[0030] FIGs. 23A-23J illustrate representative pictures and immunohistochemistry images of various tumor models post treatment. FIG. 23A and FIG. 23B show representative pictures (FIG. 23A) and immunochemistry images (FIG. 23B) of B16F10 in WT C57BL6 mice on day 18. FIG. 23C and FIG. 23D show representative pictures (FIG. 23C) and immunochemistry images (FIG. 23D) of LLC-1 in WT C57BL6 mice on day 15. FIG. 23E and FIG. 23F show representative pictures (FIG. 23E) and immunochemistry images (FIG. 23F) of MC-38 in RAG- / - C57BL6 mice on day 18 and 32. FIG. 23G and FIG. 23H show representative pictures (FIG. 23G) and immunochemistry images (FIG. 23H) of MC38 in CD45.1 C57BL6 mice on day 25. FIG. 231 and FIG. 23 J show representative pictures (FIG. 231) and immunochemistry images (FIG. 23 J) of AE17 in CD45.1 C57BL6 mice on day 15.

[0031] FIGs. 24A-24J represent an automatic quantification of infiltrated CD8+ T cells from IHC slides using machine learning and image processing. FIG. 24A and FIG. 24B show a random forest-based tumor (FIG. 24A) classifier trained by user-defined tumor / stroma / glass regions (FIG. 24B) The trained classifier was then applied to perform whole-slide segmentation to identify tumors. Stroma and glass regions were excluded in downstream analysis. FIGs. 24C-24G further illustrate representative decomposed images from the tumors as shown on the original image (FIG. 24C), decoupled (hematoxylin; FIG. 24D), decoupled (CD8a-Red; FIG. 24E), decoupled (Melanin; FIG. 24F), and quantified image (FIG. 24G). Number of CD8+ TILs were quantified by an automated cell counting algorithm using decomposed images. TILs were defined as hematoxylin+ / warp red+. FIGs. 24H-24J show quantification of tumor size (FIG. 24H), percentage of infiltrated CD8+ cells (FIG. 241) and survival rate (FIG. 24J) in s.c. LLC-1 models in WT C57BL6 mice treated by different populations of lymphocytes (n = 5).

[0032] FIGs. 25A-25G illustrate lung metastases in i.v. 4T1 models in nude mice at the endpoint treated by different T cells (n = 6, 6 layers for each animal L: Lung, T: Tumor). FIGs.25A-25C show representative images of untreated (FIG. 25 A), CD 103- PBMC (FIG. 25B), and cTRL (FIG. 25C), and quantitative of lung metastases (FIG. 25D). FIGs. 25E-25G show quantification of tumor size (FIG. 25E), percentage of infiltrated CD8+ cells (FIG. 25F), and survival rate (FIG. 25G) in s.c. MC38 models in RAG- / - C57BL6 mice treated by different therapeutic modalities (n = 5).

[0033] FIGs. 26A-26T show CyTOF analyses of the immune landscape of infiltrated lymphocytes at the endpoint in s.c. AE17 models in CD45.1 C57BL6 mice treated by different therapeutic modalities. FIGs. 26A-26L show the representative flow cytometric profile of the percentage of infiltrated CD3 (FIGs. 26A-26D) of untreated (FIG. 26A), aGITR (FIG. 26B), CTRL (FIG. 26C), and aGITR-cTRL (FIG. 26D); CD3+CD4+ (FIGs. 26E-26H) of untreated (FIG. 26E), aGITR (FIG. 26F), cTRL (FIG. 26G), and aGITR-cTRL (FIG. 26H); and CD3+CD8+ (FIGs. 26I-26L) cells of untreated (FIG. 261), aGITR (FIG. 26J), cTRL (FIG. 26K), and aGITR-cTRL (FIG. 26L). FIGs. 26M-26T show the representative flow cytometric profile of the percentage of PD-1+ cells (FIGs. 26M-26P) of untreated (FIG. 26M), aGITR (FIG. 26N), cTRL (FIG. 260), and aGITR-cTRL (FIG. 26P); and CD25+ cells (FIGs. 26Q-26T) of untreated (FIG. 26Q), aGITR (FIG. 26R), cTRL (FIG. 26S), and aGITR-cTRL (FIG. 26T).

[0034] FIGs. 27A-27C show analysis of CD 103 expression and its correlation with CD8+ T cell infiltration level using the TIMER algorithms. The level of CD8+ T cell infiltration is calculated using three prediction algorithm-CIBERSORT (FIG. 27A), MCPCOUNTER (FIG. 27B) and QUANTISEQ (FIG. 27C). CD 103 positively regulates the CD8+ T cell infiltration in many types of cancer types, including breast invasive carcinoma (BRCA), lung adenocarcinoma (LU AD) and colon adenocarcinoma (COAD).

[0035] FIGs. 28A-28J represent analyses of large-scale patient data using the TIDE algorithm. FIGs. 28A-28D show a quantification of adjusted death risk calculated by TIDE against different markers, such as CD 103 (FIG. 28A), ZNF683 (FIG. 28B), TCF7 (FIG. 28C), and SERPINB9 (FIG. 28D). TCF7 is previously reported as an up-regulator for the improved therapeutic outcome, while SERPINB9 is reported as a down-regulator for therapeutic outcome. Data were visualized by a truncated violin plot and analyzed using an unpaired T-test. FIGs. 28E- 28J show analyses of the relationship between survival benefits and the lymphatic ITGAE expression across multiple cancer types including lung cancer (FIG. 28E), breast cancer (FIG. 28F), ovarian cancer (FIG. 28G), liver cancer (FIG. 28H), cervical cancer (FIG. 281), and bladder cancer (FIG. 28J).

[0036] FIGs. 29A-29K show molecular and phenotypic signatures of cTRL. FIG. 29A shows key gene profiles for CD8+ T cells presented as a heatmap. FIGs. 29B-29G show CyTOF analysis of multimer-binding cTRLs (FIGs. 29B-29C) gating CD3CD103 (FIG. 29B) and multimer (FIG.29C) and expression levels of CD103 (FIG. 29D), CD39 (FIG. 29E), PD-1 (FIG. 29F), and CD69 (FIG. 29G). FIGs. 29H-29K show the quantification of the expression level of CD 103, CD39, PD-1 and CD69 in cTRLs during migration (FIG. 29H) and gating schemes (FIG. 291- 29K).

[0037] FIGs. 30A-30P illustrate CD 103+ cTRL population signature in human PBMC. FIG. 30A schematically illustrates a workflow of the co-culture assay to study the relationship between tumor-reactivity and CD 103 on human PBMCs. FIGs. 30B-30J show the representative flow cytometric profile of IFN-y secreting populations according to CD 103 expression of CD 103 FMO control (FIGs. 30B-30D) gating on CD8+ (FIG. 30B), CD 103+ (FIG. 30C), and CD 103- (FIG. 30D), unstimulated control (FIGs. 30E-30G) gating on CD8+ (FIG. 30E), CD 103+ (FIG. 30F), and CD103- (FIG. 30G), and stimulated MPE (FIGs. 30H-30J) gating on CD103- or CD103+ (FIG. 30H), IFNG+ of CD103- (FIG. 301), and IFNG+ of CD103+ (FIG. 30J) from PE95 patient. FIGs. 30K-30P show quantification of IFN-y secreting populations in CD8+CD103+ and CD8+CD103- cells across the patient cohort: PE69 (FIG. 30K), PE70 (FIG. 30L), PE71 (FIG. 30M), PE72 (FIG. 30N), PE95 (FIG. 300), and PE96 (FIG. 30P) of malignant pleural effusion (MPE).

[0038] FIG. 31A and FIG. 31B illustrate the feeder-cell free expansion of T cells under different culture conditions, using initial seeding concentrations of 1,000 cells (FIG. 31 A) or 10,000 cells (FIG. 31B).

[0039] FIG. 32 depicts a cartoon schematic of a non-limiting exemplary method for analyzing the tumor reactivity of various types of cTRLs.

[0040] FIGs. 33A-33H show that the CD103+CD8+ cTRLs isolated from various colorectal samples show significant tumor reactivity when activated by dissociated tumor cells (T cells + DTCs), as measured by the portion of the cTRLs that show significant expression of IFN-y. Cells treated with eBioscience™ Cell Stimulation Cocktail (T cells + PMA / lono) were used as positive controls for IFN-y expression. CD103+CD8+ cTRLs not incubated with DTCs or PMA / lono were used as negative control. Individual patient samples in which the CD103+CD8+ cTRLs were derived from are listed individually on x-axis (#l-#8). Exemplary FACS analyses of the CD103+CD8+ cTRLs isolated from one exemplary patient sample (#5) are shown (FIGs. 33B- 33D and FIGs. 33F-33H). FIG. 33A-33D shows the tumor reactivity of the CD103+CD8+ cTRLs(FIG. 33A) and gating scheme of T cells (FIG. 33B), T cells and DTCs (FIG. 33C) and T cells and PMA / Iono (FIG. 33D). FIG. 33E-33H show the tumor reactivity of CD 103 -depleted CD8+ cTRLs (FIG. 33E) gating scheme of T cells (FIG. 33F), T cells and DTCs (FIG. 33G) and T cells and PMA / Iono (FIG. 33H). CTRLs were isolated from primary tumors or cancers metastasized to the liver.

[0041] FIG. 34A and FIG. 34B show the performance of the cTRLs isolation methods as described herein with various parameters (flow rates, wash volumes, and magnetic strength). FIG. 34A shows the performance of the methods in isolating PBMCs from the samples of healthy subjects. FIG. 34B shows the performance of the methods in isolating cTRLs from the samples of patients with colorectal cancer.

[0042] FIG. 35A depicts a schematic of a non-limiting exemplary method for cTRL expansion. FIG. 35B and FIG. 35C show the fold-expansion (FIG. 35B) and number of doublings of cTRLs (FIG. 35C), respectively, isolated from various colorectal cancer samples (CRC), using the method of FIG. 33A. Individual cancer sample is listed on the x-axis (labeled as #l-#5). FIGs. 35D-35E show the cell surface marker expression profiles of CTRLs isolated from colorectal cancer samples pre-expansion (FIG. 35D) and post-expansion (FIG. 35E) using the method of FIG. 35A

[0043] FIGs. 36A-36C show CD8 dysfunction (FIG. 36A) and sternness (FIG. 36B) marker expressions of lymphocytes isolated from tumor (e.g., TILs) or blood (cTRLs). FIG. 36C demonstrates that cTRLs have a broader antitumor repertoire when compared to the TILs.

[0044] FIG. 37 describes a high level cTRL manufacturing flow diagram.

[0045] FIG. 38 shows percentage of IFN-y cells of CD8+CD103+ cells isolated from pleural effusions collected from patients of collected from lung metastases of 16 patients with a variety of solid tumors, including colon, melanoma, mesothelioma, lung, and breast tumors.

[0046] FIGs. 39A-39J show tumor reactivity of cTRLs (e.g., CD8+CD103+) collected from peripheral blood from patients. FIG. 39A shows the tumor reactive cTRLs were assessed by measuring% IFN-y secretion against autologous melanoma cancer. FIGs. 39B-39E show gating of CD8+CD103- population of T-cell alone (FIG. 39B) or with Tumor (FIG. 39C), and CD8+CD103+ population of T-cell alone (FIG. 39D) or with tumor (FIG. 39E). FIG. 39F shows the tumor reactive cTRLs were assessed by measuring% IFN-y secretion against autologous colorectal cancer. FIGs. 39G-39J show gating of CD8+CD103- population of T-cell alone (FIG. 39G) or with Tumor (FIG. 39H), and CD8+CD103+ population of T-cell alone (FIG. 391) or with tumor (FIG. 39 J).

[0047] FIGs. 40A-40G demonstrate the effectiveness of cTRLs in recognizing and targeting autologous tumor cells. FIG. 40A shows cTRLs (e.g., CD8+CD103+) cells from patients in the melanoma cohort displayed potent cytolytic activity. FIGs. 40B-40E show gating of CD8+CD103- population of T-cell alone (FIG. 40B) or with Tumor (FIG. 40C), and CD8+CD103+ population of T-cell alone (FIG. 40D) or with tumor (FIG. 40E). FIG. 40F and FIG. 40G show that cTRLs retained anti-tumor reactivity, as assessed by production of multiple cytokines including IFN-y and TNF-a (FIG. 40F), and expression of cytolytic granules and perforin, granzyme, and granulolysin (FIG. 40G).

[0048] FIGs. 41A-41E show robust, rapid (e.g., 5 days) expansion of cTRLs ex vivo in the absence of Interleukin-2 (IL-2). FIG. 41 A shows robust expansion, over 1,000-fold, of melanoma cTRLs in growth medium that did not contain IL-2. FIG. 41B shows that cTRL cells expanded in the absence of IL-2 have over 90% cluster of differentiation 8-positive (CD8+) T-cell identity, wedge (a), after expansion. FIG. 41C-41E show retention of memory phenotype (FIG. 41C), T- cell programmed cell death protein 1 (PDl)-expression (FIG. 41D), and viability of cTRLs expanded in the absence of IL-2 (FIG. 41E), as assessed by percentage of CD3+ and CD8+ cells.

[0049] FIGs. 42A-42C show improved expansion of cTRLs in growth medium containing both Interleukin-7 (IL-7) and Interleukin- 15 (IL- 15), compared to expansion in growth medium containing IL-2. FIG. 42A provides a schematic of a cTRL expansion protocol using a growth medium containing either IL-7 and IL- 15 or IL-2. FIG. 42B provides the cTRL fold expansion after 14 days in a medium containing IL-2. FIG. 42C provides a comparison of the fold expansion of cTRLs grown in media containing either IL-2 or IL-7 and IL- 15, showing that cTRLs have improved expansion in the presence of IL-7 and IL- 15, as compared to in the presence of IL-2.

[0050] FIGs. 43A-43B show improved expansion of cTRLs in growth medium containing higher doses of IL-2. FIG. 43A shows improved expansion of cluster of differentiation 103 (CD103)-enriched cTRLs in growth media containing higher doses of IL-2. FIG. 43B shows improved expansion of CD 103 -depleted cTRLs in growth media containing higher doses of IL-2, with the greatest fold-expansion seen in CD 103 -depleted cTRLs in growth medium containing IL-7 and IL-15.

[0051] FIGs. 44A-44E show improved purity of cTRLs (e.g., melanoma cTRLs) in growth medium containing both Interleukin-7 (IL-7) and Interleukin- 15 (IL- 15), compared to expansion in growth medium containing IL-2. FIG. 44A provides the immune composition of a cTRL population prior to expansion. FIG. 44B and FIG. 44C show the similar immune composition of cTRL populations expanded in the presence of IL-2 (FIG. 44B) or in the presence of IL-7 and IL- 15 (FIG. 44C). FIG. 44D provides the fold-expansion of a melanoma subset of cTRLs expandedin the presence of IL-2 or in the presence of IL-7 and IL-15. FIG. 44E provides the immune composition of a melanoma subset of cTRLs pre-expansion (left bar), expanded in the presence of IL-2 (middle bar), and expanded in the presence of IL-7 and IL-15 (right bar), showing greater purity of the melanoma subset of cTRLs expanded in medium containing IL-7 and IL- 15, compared to those expanded in medium containing IL-2.

[0052] FIGs. 45A-45J show similar memory phenotypes of cTRLs expanded in different cytokine conditions. FIG. 45A provides a schematic of the memory phenotype plots with naive T-cells (TNV) in quadrant 1 enriched in C-C chemokine receptor type 7 (CCR7) and depleted in CD45RO, central memory T-cells (TCM) in quadrant 2 enriched in both CCR7 and CD45RO, effector memory T-cells (TEM) in quadrant 3 depleted in CCR7 and enriched in CD45RO, and terminal effector T-cells (TTE) in quadrant 4 depleted in both CCR7 and CD45RO. FIG. 45B provides the memory phenotype of cTRLs pre-expansion. FIG. 45C provides the memory phenotype of cTRLs grown in a medium containing 50 IL / ml IL-2 after 7 days of expansion. FIG. 45D provides the memory phenotype of cTRLs grown in a medium containing 300 IL / ml IL-2 after 7 days of expansion. FIG. 45E provides the memory phenotype of cTRLs grown in a medium containing 6000 IL / ml IL-2 after 7 days of expansion. FIG. 45F provides the memory phenotype of cTRLs grown in a medium containing 20 ng / ml IL-7 and 30 ng / ml IL- 15 after 7 days of expansion. FIG. 45G provides the memory phenotype of cTRLs grown in a medium containing 50 IL / ml IL-2 after 14 days of expansion. FIG. 45H provides the memory phenotype of cTRLs grown in a medium containing 300 IL / ml IL-2 after 14 days of expansion. FIG. 451 provides the memory phenotype of cTRLs grown in a medium containing 6000 IL / ml IL-2 after 14 days of expansion. FIG. 45J provides the memory phenotype of cTRLs grown in a medium containing 20 ng / ml IL-7 and 30 ng / ml IL- 15 after 14 days of expansion.

[0053] FIGs. 46A-46F show similar memory phenotypes of cTRLs expanded in growth medium containing IL-2 or in growth medium containing IL-7 and IL-15. FIG. 46A and FIG. 46B provide the memory phenotype of cTRLs pre-expansion. FIG. 46C and FIG. 46D provide the memory phenotype of cTRLs expanded in the presence of IL-2. FIG. 46E and FIG. 46F provide the memory phenotype of cTRLs expanded in the presence of IL-7 and IL-15.

[0054] FIG. 47A-47C show an increase in cluster of differentiation 25 (CD25; IL-2 receptor alpha) expression in cTRLs expanded in growth medium containing IL-7 and IL- 15, compared to cTRLs expanded in growth medium containing IL-2. FIG. 47A and FIG. 47B provide the percentage of CD25+ cTRLs expanded in growth medium contain IL-7 and IL-15 (FIG. 47B) compared to cTRLs expanded in growth medium containing IL-2 (FIG. 47A). FIG. 47C provides the percentage of cluster of differentiation 3 -positive and cluster of differentiation 8-positive(CD3+CD8+) cTRLs expanded in growth medium contain IL-7 and IL- 15 (right) compared to cTRLs expanded in growth medium containing IL-2 (left).

[0055] FIG. 48 shows CD25 expression at Day 0 (pre-expansion), Day 7, and Day 14 of cTRLs cultured in 50 lU / mL IL-2, 300 lU / mL IL-2, 6000 lU / mL IL-2, or 20 ng / mL IL-7 + 30 ng / ml IL-15.

[0056] FIGs. 49A-49I show expression of check point inhibitor expression. FIG. 49A shows TIM-3 expression of cTRLs cultured in IL-2. FIG. 49B shows TIM-3 expression of cTRLs cultured in IL-7 and IL-15. FIG. 49C shows TIM-3 frequency of CD3+CD8+ cells of cTRLs cultured in culture media containing IL-2 or IL-7+IL-15. FIG. 49D shows LAG-3 expression of cTRLs cultured in IL-2. FIG. 49E shows LAG-3 expression of cTRLs cultured in IL-7 and IL- 15. FIG. 49F shows LAG-3 frequency of CD3+CD8+ cells of cTRLs cultured in culture media containing IL-2 or IL-7+IL-15. FIG. 49G shows PD-1 expression of cTRLs cultured in IL-2. FIG. 49H shows PD-1 expression of cTRLs cultured in IL-7 and IL-15. FIG. 491 shows PD-1 frequency of CD3+CD8+ cells of cTRLs cultured in culture media containing IL-2 or IL-7+IL- 15.

[0057] FIGs. 50A-50C show expression (%) of TIM3+ (FIG. 50A), LAG3+ (FIG. 50B) and PD1+ (FIG. 50C) expression at Day 0 (pre-expansion), Day 7, and Day 14 of cTRLs cultured in 50 lU / mL IL-2, 300 lU / mL IL-2, 6000 lU / mL IL-2, or 20 ng / mL IL-7 + 30 ng / ml IL-15.DETAILED DESCRIPTION

[0058] Disclosed herein, in some embodiments, are compositions, systems and kits comprising isolated and enriched populations of cells obtained from a sample of a subject, and methods of their isolation, enrichment, expansion, and use for treatment of a disease or a condition disclosed herein. In some embodiments, the sample can comprise a fluid, such as peripheral blood. In some embodiments, the disease or the condition can comprise a cancer. In some embodiments, the subject can be suspected of having a cancer. In some embodiments, the isolated and enriched populations of cells can target and eliminate cancer cells in a subject when administered to the subject as a therapy. In some embodiments, the isolated and enriched populations of cells can be tumor-reactive lymphocytes (TRLs), such as circulating Tumor Reactive Lymphocytes (cTRLs), that can recognize a cancer antigen of the cancer and exhibit anti-cancer activity. In some embodiments, TRLs can be present in the peripheral blood of a subject at a very low frequency, for example, as low as 0.002% of the peripheral blood T cell populations. The isolated and enriched TRLs (e.g., isolated and enriched cTRLs) described herein can be used as an alternative to tumor-infiltrating lymphocytes (TILs) in an adoptive cell therapy. The isolated and enrichedTRLs disclosed herein can secrete interferon-gamma (IFN-y). In some embodiments, the isolated and enriched TRLs can enter the peripheral blood from a primary tumor and accumulate in a secondary tumor. In some embodiments, a TRL can be characterized by having comparable reactivity to a TIL. In some embodiments, a TRL can share a clonotype with a TIL. In some embodiments, a population of TRLs can comprise 30%-85% of the top 50 clones presented in a population of intratumoral TILs. In some embodiments, TRLs described herein may have a tissue- resident-like (Trm-like) phenotype. In some embodiments, the isolated and enriched population of TRLs can be enhanced to generate tumor-specific T cell therapies, such as chimeric antigen receptor (CAR)-T therapy or T-cell receptor (TCR) therapy.

[0059] In some embodiments, a TRL can be CD8+, CD103+, CD3+, CD4+, CD39+, SLC6A19+, SIDT1+, or any combination thereof. In some embodiments, a TRL can be CD103+. In some embodiments, a population of TRLs can comprise a CD 103 signature. In some embodiments, a CD 103 signature can define the population of TRLs. In some embodiments, a TRL can be SLC6A19+, SIDT1+, or a combination thereof. In some embodiments, a TRL can exhibit upregulated expression of TCF7, IL-7R, LEF1, or a combination thereof. In some embodiments, a population of TRLs can comprise a SLC6A19+ and SIDT1+ signature. In some embodiments, a CD8+, CD103+, SLC6A19+, and SIDT1+ signature can define a population of TRLs. In some embodiments, the TRL can be CD3+ or CD4+. In some embodiments, the TRL can be a CD3+ pan T cell. In some embodiments, the TRL can be a CD4+ helper T cell. In some embodiments, a TRL can be CD39+. In some embodiments, the TRL can be CD39+ and CD103+. In some embodiments, a CD39+ and CD103+ signature can define a population of TRLs. In some embodiments, the TRL can be CD39+, CD103+, CD8+, or a combination thereof. In some embodiments, a TRL can be CD8+, CD103+, SLC6A19+, SIDT1+, or a combination thereof. In some embodiments, a TRL can be CD3+, CD103+, SLC6A19+, SIDT1+, or a combination thereof. In some embodiments, a TRL can be CD4+, CD103+, SLC6A19+, SIDT1+, or a combination thereof. In some embodiments, a TRL can be CD4+, CD39+, CD103+, SLC6A19+, SIDT1+, or a combination thereof.

[0060] Also disclosed herein are methods of isolating, enriching, and expanding a population of circulating tumor reactive lymphocytes (cTRLs) from a peripheral blood sample. Described herein are methods of isolating and enriching a population of TRLs that are present in a very low frequency (e.g., less than 0.002%) in the fluid (e.g., peripheral blood) using a microfluidic device or by any methods that allows separation of cells (e.g., cTRLs) and cell populations based on the cell’s expression or expression level of one or more markers, typically cell surface markers, forexample, by incubation with an antibody or binding partner that specifically binds to such markers. In some embodiments, isolating the population of TRLs can comprise magnetically separating a population of TRLs using a microfluidic device. In some embodiments, the microfluidic magnetic cell sorting can rely on the immunomagnetic labeling of the population of TRLs, followed by magnetic separation within the microfluidic device. In some embodiments, the immunoaffmity- based selections can include contacting a sample comprising a population of cells from a fluid sample (e.g., prepared from patient’s peripheral blood) with an antibody or binding partner that specifically binds to the cell surface marker or markers. In some embodiments, the antibody or binding partners can be bound to a solid support or matrix, such as a sphere or bead, for example a nanoparticle, microbeads, nanobeads, including agarose, magnetic bead or paramagnetic beads, to allow for separation of cells for positive and / or negative selection. In some embodiments, a sphere or beads can be coupled to a detectable label (e.g., fluorescent labels). In some embodiments, immunoaffinity chromatography or flow cytometry can be used to isolate the target cells (e.g., cTRLs).

[0061] Further disclosed herein are methods of enhancing a population of the isolated and enriched TRLs comprising CD103+, CD39+, SLC6A19+, and / or SIDT1+ lymphocytes by introducing a nucleotide that expresses a therapeutically enhancing polypeptide under conditions sufficient to produce the enhancing polypeptide by the cell. In some embodiments, the nucleotide can encode a T-cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the TCR or CAR can be configured to recognize an antigen associated with a cancer.

[0062] Disclosed herein are methods of providing a cell therapy (e.g, an adoptive cell therapy) to a subject in need thereof, comprising: (a) obtaining a population of cells (e.g., TRLs) or therapeutically enhanced cells described herein; and (b) administering to the subject the population of cells, thereby providing the cell therapy. Also disclosed herein are compositions comprising a population of TRLs or therapeutically enhanced TRLs (e.g., to express a CAR). In some embodiments, the compositions can comprise a therapeutically effective amount of TRLs produced by any of the methods disclosed herein. In some embodiments, the compositions can be for use in treating a cancer.

[0063] Disclosed herein, in some embodiments, are compositions, systems, and kits for producing or utilizing therapeutically active cells described herein (e.g., enhanced TRLs). Compositions disclosed herein can comprise, in some embodiments, microfluidic devices for separating the population of TRLs from a fluid sample disclosed herein. In some embodiments,the compositions can comprise engineered T cell receptors (TCRs) and nucleic acid molecules encoding TCRs. In some embodiments, the compositions can comprise engineered chimeric antigen receptors (CARs) and nucleic acid molecules encoding CARs. In some embodiments, the fluid compositions disclosed herein can be obtained from the subject in need of a treatment with the therapeutically active cells (e.g, autologous).I. COMPOSITIONS

[0064] Disclosed herein are isolated and enriched populations of cells (e.g, T-cells) from the fluid (e.g., peripheral blood) of a subject (e.g., an individual with cancer) that can comprise tumor- reactive lymphocytes (TRLs) capable of targeting and eliminating cancer cells. Also disclosed herein are enhanced TRLs for use in therapeutic applications, such as synthetic chimeric antigen receptor (CAR) targeting tumor specific antigen. The compositions disclosed herein may be or comprise a polynucleotide encoding one or more components of the compositions disclosed herein, e.g., chimeric antigen receptor. The compositions disclosed herein may comprise in a pharmaceutical formulation, for example, in a formulation for administration to a subject disclosed herein. In some embodiments, a microfluidic device disclosed herein can be used to separate the population of TRLs from a fluid sample disclosed herein. In some embodiments, isolating and enriching tumor-reactive lymphocytes can involve a process that can comprise magnetic separation of lymphocytes from the sample. This magnetic separation may involve flowing the lymphocytes through a microfluidic device channel that contains one or multiple magnetic capture zones.A. Tumor Reactive Lymphocytes

[0065] Disclosed herein are compositions comprising isolated and enriched tumor-reactive lymphocytes (TRLs) derived from a fluid sample (e.g., peripheral blood) that express Solute Carrier Family 6 Member 19 (SLC6A19) (NCBI Entrez Gene: 340024; UniProtKB / Swiss-Prot: Q695T7), Systemic RNA Interference Defective (SID) Protein 1 Transmembrane family member 1 (SIDT1) (NCBI Entrez Gene: 54847; UniProtKB / Swiss-Prot: Q9NXL6), Cluster of Differentiation 103 (CD103) (NCBI Entrez Gene: 3682; UniProtKB / Swiss-Prot: P38570), Cluster of Differentiation 39 (CD39) (NCBI Entrez Gene: 953; UniProtKB / Swiss-Prot: P49961) or any combination of thereof. In some embodiments, TRLs can be circulating tumor-reactive lymphocytes (cTRLs). In some embodiments, a population of TRLs (e.g., cTRLs) can comprise CD103 expressing cells. In some embodiments, a population of TRLs can comprise a CD103+ signature. In some embodiments, a population of TRLs can comprise CD39 expressing cells. Insome embodiments, a population of TRLs can comprise a CD39+ signature. In some embodiments, a CD 103+ signature can define the population of TRLs. In some embodiments, TRLs can express SLC6A19, SIDT1, or a combination thereof. In some embodiments, a population of TRLs can comprise a SLC6A19+ and / or SIDT1+ signature. In some embodiments, a SLC6A19+ and / or SIDT1+ signature can define the population of TRLs. In some embodiments, TRLs can express CD8+ and CD103+, but lacking CD39 (CD39-). For example, in some embodiments, a CD8+, CD103+, and CD39- signature can define a population of TRLs. In some embodiments, a population of TRLs can be characterized by having CD8+, CD103+, and CD39- , wherein at most about 60%, at most about 55%, at most about 50%, at most about 45%, at most about 40%, at most about 35%, at most about 30%, at most about 25%, at most about 20%, at most about 15%, at most about 10%, or at most about 5% of the population of TRLs express CD39 (CD39+).

[0066] In some embodiments, the isolated and enriched TRLs may comprise about 100 to about 20,000 CD103+ (e.g., CD103 expression) cells per 10 million PBMCs (e.g., peripheral blood mononuclear cells). In some embodiments, the isolated and enriched TRLs may comprise about 100 cells to about 500 cells, from about 100 cells to about 1,000 cells, from about 100 cells to about 2,000 cells, from about 100 cells to about 3,000 cells, from about 100 cells to about 4,000 cells, from about 100 cells to about 5,000 cells, from about 100 cells to about 6,000 cells, from about 100 cells to about 8,000 cells, from about 100 cells to about 10,000 cells, from about 100 cells to about 15,000 cells, from about 100 cells to about 20,000 cells, from about 500 cells to about 1,000 cells, from about 500 cells to about 2,000 cells, from about 500 cells to about 3,000 cells, from about 500 cells to about 4,000 cells, from about 500 cells to about 5,000 cells, from about 500 cells to about 6,000 cells, from about 500 cells to about 8,000 cells, from about 500 cells to about 10,000 cells, from about 500 cells to about 15,000 cells, from about 500 cells to about 20,000 cells, from about 1,000 cells to about 2,000 cells, from about 1,000 cells to about 3,000 cells, from about 1,000 cells to about 4,000 cells, from about 1,000 cells to about 5,000 cells, from about 1,000 cells to about 6,000 cells, from about 1,000 cells to about 8,000 cells, from about 1,000 cells to about 10,000 cells, from about 1,000 cells to about 15,000 cells, from about 1,000 cells to about 20,000 cells, from about 2,000 cells to about 3,000 cells, from about 2,000 cells to about 4,000 cells, from about 2,000 cells to about 5,000 cells, from about 2,000 cells to about 6,000 cells, from about 2,000 cells to about 8,000 cells, from about 2,000 cells to about 10,000 cells, from about 2,000 cells to about 15,000 cells, from about 2,000 cells to about 20,000 cells, from about 3,000 cells to about 4,000 cells, from about 3,000 cells to about 5,000 cells, fromabout 3,000 cells to about 6,000 cells, from about 3,000 cells to about 8,000 cells, from about 3,000 cells to about 10,000 cells, from about 3,000 cells to about 15,000 cells, from about 3,000 cells to about 20,000 cells, from about 4,000 cells to about 5,000 cells, from about 4,000 cells to about 6,000 cells, from about 4,000 cells to about 8,000 cells, from about 4,000 cells to about 10,000 cells, from about 4,000 cells to about 15,000 cells, from about 4,000 cells to about 20,000 cells, from about 5,000 cells to about 6,000 cells, from about 5,000 cells to about 8,000 cells, from about 5,000 cells to about 10,000 cells, from about 5,000 cells to about 15,000 cells, from about 5,000 cells to about 20,000 cells, from about 6,000 cells to about 8,000 cells, from about 6,000 cells to about 10,000 cells, from about 6,000 cells to about 15,000 cells, from about 6,000 cells to about 20,000 cells, from about 8,000 cells to about 10,000 cells, from about 8,000 cells to about 15,000 cells, from about 8,000 cells to about 20,000 cells, from about 10,000 cells to about 15,000 cells, from about 10,000 cells to about 20,000 cells, or from about 15,000 cells to about 20,000 CD103+cells per 10 million PBMCs.

[0067] In some embodiments, the isolated and enriched TRLs may comprise about 100 cells, about 500 cells, about 1,000 cells, about 2,000 cells, about 3,000 cells, about 4,000 cells, about 5,000 cells, about 6,000 cells, about 8,000 cells, about 10,000 cells, about 15,000 cells, or about 20,000 CD103+cells per 10 million PBMCs. In some embodiments, the isolated and enriched TRLs may comprise at least about 100 cells, about 500 cells, about 1,000 cells, about 2,000 cells, about 3,000 cells, about 4,000 cells, about 5,000 cells, about 6,000 cells, about 8,000 cells, about 10,000 cells, or about 15,000 CD103+ cells per 10 million PBMCs. In some embodiments, the isolated and enriched TRLs may comprise at most about 500 cells, about 1,000 cells, about 2,000 cells, about 3,000 cells, about 4,000 cells, about 5,000 cells, about 6,000 cells, about 8,000 cells, about 10,000 cells, about 15,000 cells, or about 20,000 CD103+ cells per 10 million PBMCs.

[0068] In some embodiments, the isolated and enriched TRLs may comprise about 100 cells to about 20,000 SLA6A19+ and / or SIDT1+ (e.g., SLA6A19 and / or SIDT1 expressing) cells per 10 million PBMCs (e.g., peripheral blood mononuclear cells). In some embodiments, the isolated and enriched TRLs may comprise about 100 cells to about 500 cells, from about 100 cells to about 1,000 cells, from about 100 cells to about 2,000 cells, from about 100 cells to about 3,000 cells, from about 100 cells to about 4,000 cells, from about 100 cells to about 5,000 cells, from about 100 cells to about 6,000 cells, from about 100 cells to about 8,000 cells, from about 100 cells to about 10,000 cells, from about 100 cells to about 15,000 cells, from about 100 cells to about 20,000 cells, from about 500 cells to about 1,000 cells, from about 500 cells to about 2,000 cells, from about 500 cells to about 3,000 cells, from about 500 cells to about 4,000 cells, from about500 cells to about 5,000 cells, from about 500 cells to about 6,000 cells, from about 500 cells to about 8,000 cells, from about 500 cells to about 10,000 cells, from about 500 cells to about 15,000 cells, from about 500 cells to about 20,000 cells, from about 1,000 cells to about 2,000 cells, from about 1,000 cells to about 3,000 cells, from about 1,000 cells to about 4,000 cells, from about 1,000 cells to about 5,000 cells, from about 1,000 cells to about 6,000 cells, from about 1,000 cells to about 8,000 cells, from about 1,000 cells to about 10,000 cells, from about 1,000 cells to about 15,000 cells, from about 1,000 cells to about 20,000 cells, from about 2,000 cells to about 3,000 cells, from about 2,000 cells to about 4,000 cells, from about 2,000 cells to about 5,000 cells, from about 2,000 cells to about 6,000 cells, from about 2,000 cells to about 8,000 cells, from about 2,000 cells to about 10,000 cells, from about 2,000 cells to about 15,000 cells, from about 2,000 cells to about 20,000 cells, from about 3,000 cells to about 4,000 cells, from about 3,000 cells to about 5,000 cells, from about 3,000 cells to about 6,000 cells, from about 3,000 cells to about 8,000 cells, from about 3,000 cells to about 10,000 cells, from about 3,000 cells to about 15,000 cells, from about 3,000 cells to about 20,000 cells, from about 4,000 cells to about 5,000 cells, from about 4,000 cells to about 6,000 cells, from about 4,000 cells to about 8,000 cells, from about 4,000 cells to about 10,000 cells, from about 4,000 cells to about 15,000 cells, from about 4,000 cells to about 20,000 cells, from about 5,000 cells to about 6,000 cells, from about 5,000 cells to about 8,000 cells, from about 5,000 cells to about 10,000 cells, from about 5,000 cells to about 15,000 cells, from about 5,000 cells to about 20,000 cells, from about 6,000 cells to about 8,000 cells, from about 6,000 cells to about 10,000 cells, from about 6,000 cells to about 15,000 cells, from about 6,000 cells to about 20,000 cells, from about 8,000 cells to about 10,000 cells, from about 8,000 cells to about 15,000 cells, from about 8,000 cells to about 20,000 cells, from about 10,000 cells to about 15,000 cells, from about 10,000 cells to about 20,000 cells, or from about 15,000 cells to about 20,000 SLA6A19+ and / or SIDT1+ cells per 10 million PBMCs.

[0069] In some embodiments, the isolated and enriched TRLs may comprise about 100 cells, about 500 cells, about 1,000 cells, about 2,000 cells, about 3,000 cells, about 4,000 cells, about 5,000 cells, about 6,000 cells, about 8,000 cells, about 10,000 cells, about 15,000 cells, or about 20,000 SLA6A19+ and / or SIDT1+ cells per 10 million PBMCs (e.g., peripheral blood mononuclear cells). In some embodiments, the isolated and enriched TRLs may comprise at least about 100 cells, about 500 cells, about 1,000 cells, about 2,000 cells, about 3,000 cells, about 4,000 cells, about 5,000 cells, about 6,000 cells, about 8,000 cells, about 10,000 cells, or about 15,000 cells per 10 million PBMCs. In some embodiments, the isolated and enriched TRLs may comprise at most about 500 cells, about 1,000 cells, about 2,000 cells, about 3,000 cells, about4,000 cells, about 5,000 cells, about 6,000 cells, about 8,000 cells, about 10,000 cells, about 15,000 cells, or about 20,000 SLA6A19+ and / or SIDT1+ cells per 10 million PBMCs.

[0070] In some embodiments, the purity of isolated and enriched TRLs (e.g., CD39+, CD103+, SLC6A19+ and / or SIDT1+ cells) described herein can be about 10% to about 95%. In some embodiments, the purity of isolated and enriched TRLs (e.g., CD103+, SLC6A19+ and / or SIDT1+ cells) described herein can be about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 60%, about 15% to about 70%, about 15% to about 80%, about 15% to about 90%, about 15% to about 95%, about 20% to about 25%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 95%, about 25% to about 30%, about 25% to about 40%, about 25% to about 50%, about 25% to about 60%, about 25% to about 70%, about 25% to about 80%, about 25% to about 90%, about 25% to about 95%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 95%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 95%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 95%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 80% to about 90%, about 80% to about 95%, or about 90% to about 95%. In some embodiments, the purity of isolated and enriched TRLs (e.g., CD39+, CD103+, SLC6A19+ and / or SIDT1+ cells) described herein can be about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. In some embodiments, the purity of isolated and enriched TRLs (e.g., CD39+, CD103+, SLC6A19+ and / or SIDT1+ cells) described herein can be at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In some embodiments, the purity of isolated and enriched TRLs (e.g., CD39+, CD103+, SLC6A19+ and / or SIDT1+ cells) described herein can be at most about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%.

[0071] In some embodiments, the purity of isolated and enriched TRLs e.g., CD39+, CD103+, SLC6A19+, or SIDT1+ cells) described herein can be about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%. In some embodiments, the purity of isolated and enriched TRLs described herein at least about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, or about 98%. In some embodiments, the purity of isolated and enriched TRLs described herein can be at most about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0072] In some embodiments, the isolated and enriched TRLs described herein may comprise at least about 100 cells with a purity of at least about 90% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 500 cells with a purity of at least about 90% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 1,000 cells with a purity of at least about 90% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 2,000 cells with a purity of at least about 90% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 3,000 cells with a purity of at least about 90% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 4,000 cells with a purity of at least about 90% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 5,000 cells with a purity of at least about 90% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 6,000 cells with a purity of at least about 90% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 8,000 cells with a purity of at least about 90% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells about 10,000 cells with a purity of at least about 90% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 15,000 cells with a purity of at least about 90% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, or about 20,000 cells with a purity of at least about 90% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells per 10 million peripheral blood mononuclear cells (PBMCs).

[0073] In some embodiments, the isolated and enriched TRLs described herein may comprise at least about 100 cells with a purity of at least about 80% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 500 cells with a purity of at least about 80% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 1,000 cells with a purity of at least about 80% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 2,000 cells with a purity of at least about 80% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 3,000 cells with a purity of at least about 80% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 4,000 cells with a purity of at least about 80% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 5,000 cells with a purity of at least about 80% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 6,000 cells with apurity of at least about 80% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 8,000 cells with a purity of at least about 80% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 10,000 cells with a purity of at least about 80% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 15,000 cells with a purity of at least about 80% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, or about 20,000 cells with a purity of at least about 80% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, per 10 million PBMCs.

[0074] In some embodiments, the isolated and enriched TRLs described herein may comprise at least about 100 cells with a purity of at least about 70% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 500 cells with a purity of at least about 70% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 1,000 cells with a purity of at least about 70% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 2,000 cells with a purity of at least about 70% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 3,000 cells with a purity of at least about 70% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 4,000 cells with a purity of at least about 70% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 5,000 cells with a purity of at least about 70% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 6,000 cells with a purity of at least about 70% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 8,000 cells with a purity of at least about 70% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 10,000 cells with a purity of at least about 70% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 15,000 cells with a purity of at least about 70% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, or about 20,000 cells with a purity of at least about 70% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, per 10 million PBMCs.

[0075] In some embodiments, the isolated and enriched TRLs described herein may comprise at least about 100 cells with a purity of at least about 60% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 500 cells with a purity of at least about 60% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 1,000 cells with a purity of at least about 60% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 2,000 cells with a purity of at least about 60% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 3,000 cells with a purity of at least about 60% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 4,000 cells with a purity of at least about 60% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 5,000 cells with a purity of at least about 60% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 6,000 cells with a purity of at least about 60% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 8,000 cells with a purity of at least about 60% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 10,000 cells with a purity of at least about 60% of CD39+, CD103+, SLC6A19+, and / orSIDT1+ cells, about 15,000 cells with a purity of at least about 60% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, or about 20,000 cells with a purity of at least about 60% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, per 10 million PBMCs.

[0076] In some embodiments, the isolated and enriched TRLs described herein may comprise at least about 100 cells with a purity of at least about 50% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 500 cells with a purity of at least about 50% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 1,000 cells with a purity of at least about 50% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 2,000 cells with a purity of at least about 50% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 3,000 cells with a purity of at least about 50% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 4,000 cells with a purity of at least about 50% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 5,000 cells with a purity of at least about 50% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 6,000 cells with a purity of at least about 50% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 8,000 cells with a purity of at least about 50% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 10,000 cells with a purity of at least about 50% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 15,000 cells with a purity of at least about 50% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, or about 20,000 cells with a purity of at least about 50% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, per 10 million PBMCs.

[0077] In some embodiments, the isolated and enriched TRLs described herein may comprise at least about 100 cells with a purity of at least about 40% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 500 cells with a purity of at least about 40% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 1,000 cells with a purity of at least about 40% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 2,000 cells with a purity of at least about 40% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 3,000 cells with a purity of at least about 40% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 4,000 cells with a purity of at least about 40% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 5,000 cells with a purity of at least about 40% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 6,000 cells with a purity of at least about 40% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 8,000 cells with a purity of at least about 40% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 10,000 cells with a purity of at least about 40% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 15,000 cells with a purity of at least about 40% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, or about 20,000 cells with a purity of at least about 40% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, per 10 million PBMCs.

[0078] In some embodiments, the isolated and enriched TRLs described herein may comprise at least about 100 cells with a purity of at least about 30% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 500 cells with a purity of at least about 30% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 1,000 cells with a purity of at least about 30% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 2,000 cells with a purity of at least about 30% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 3,000 cells with a purity of at least about 30% of CD39+, CD103+, SLC6A19+, and / or SIDT1+, about 4,000 cells with a purity of at least about 30% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 5,000 cells with a purity of at least about 30% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 6,000 cells with a purity of at least about 30% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 8,000 cells with a purity of at least about 30% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 10,000 cells with a purity of at least about 30% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, about 15,000 cells with a purity of at least about 30% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, or about 20,000 cells with a purity of at least about 30% of CD39+, CD103+, SLC6A19+, and / or SIDT1+ cells, per 10 million PBMCs.

[0079] In some embodiments, the population of TRLs can be CD3+, CD4+, CD8+, CD39+, CD103+ or any combination thereof. For example, the isolated and enriched TRLs can be CD3 + and CD 103+ expressing cells. In some embodiments, the isolated and enriched TRLs can be CD4+ and CD 103+ expressing cells. In some embodiments, the isolated and enriched TRLs can be CD8+ and CD 103+ expressing cells. In some embodiments, the isolated and enriched TRLs can be CD39+ and CD103+ expressing cells. In some embodiments, the isolated and enriched TRLs can be CD3+, CD39+, and CD103+ expressing cells. In some embodiments, the isolated and enriched TRLs can be CD4+, CD39+, and CD103+ expressing cells. In some embodiments, the isolated and enriched TRLs can be CD8+, CD39+, and CD103+ expressing cells. In some embodiments, the isolated and enriched TRLs can be CD3+, SLC6A19+, and SIDT1+ expressing cells. In some embodiments, the isolated and enriched TRLs can be CD4, SLC6A19, and SIDT1 expressing cells. In some embodiments, the isolated and enriched TRLs can be CD8+, SLC6A19+, and SIDT1+ expressing cells. In some embodiments, the isolated and enriched TRLs can be CD3+, CD103+, and SLC6A19+ expressing cells. In some embodiments, the isolated and enriched TRLs can be CD4+, CD103+, and SLC6A19+ expressing cells. In some embodiments, the isolated and enriched TRLs can be CD8+, CD103+, and SLC6A19+ expressing cells. In some embodiments, the isolated and enriched TRLs can be CD3+, CD103+, SLC6A19+, and SIDT1+ expressing cells. In some embodiments, the isolated and enriched TRLs can be CD4+, CD103+, SLC6A19+, andSIDT1+ expressing cells. In some embodiments, the isolated and enriched TRLs can be CD8+, CD103+, SLC6A19+, and SIDT1+ expressing cells.

[0080] In some embodiments, a population of tumor reactive lymphocytes can comprise CD8+CD103+ tumor reactive lymphocytes (e.g., a population of isolated, enriched, and / or expanded tumor reactive lymphocytes), wherein the population of tumor reactive lymphocytes lack CD39 expression (e.g., CD8+CD103+CD39- tumor reactive lymphocytes). In some embodiments, the CD8+CD103+ tumor reactive lymphocytes described herein can comprise at most about 1%, at most about 2%, at most about 3%, at most about 4%, at most about 5%, at most about 6%, at most about 7%, at most about 8%, at most about 9%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50% of tumor reactive lymphocytes that are CD39+ (e.g., CD8+CD103+CD39+). In some embodiments, the CD8+CD103+ tumor reactive lymphocytes described herein can comprise at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% of tumor reactive lymphocytes that are CD39+ (e.g., CD8+CD103+CD39+). In some embodiments, the CD8+CD103+ tumor reactive lymphocytes described herein can comprise at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 72%, at least about 74%, at least about 76%, at least about 78%, at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, 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% of tumor reactive lymphocytes that lack CD39 expression (e.g., CD8+CD103+CD39-). In some embodiments, CD8+CD103+ tumor reactive lymphocytes can be sorted (e.g., positive selection or negative selection) to exclude CD39+ cells from the tumor reactive lymphocytes.

[0081] In some embodiments, a population of tumor-reactive lymphocytes (TRLs) may be found in a fluid sample of a patient having cancer. In some embodiments, a fluid sample may comprise a peripheral blood sample. In some embodiments, a fluid sample may be other biofluids, such as pleural effusion, ascites, and the like. In some embodiments, a fluid sample also be cord blood, bone marrow, lymph nodes, liver pleural effusion, thorax, abdominal cavity, synovial fluid, peritoneum, retroperitoneal space, thymus, and tumor.

[0082] In some embodiments, the population of TRLs described herein can exhibit enhanced therapeutic activity. In some embodiments, the enhanced therapeutic activity of TRLs can be characterized by having less-dysfunctional phenotypes, anti-exhaustion phenotypes, enhanced killing potency, and / or sternness.

[0083] In some embodiments, the population of TRLs can have less-dysfunctional phenotypes compared to that of a population of tumor infiltrating lymphocytes (TILs). In some embodiments, one or more dysfunction markers e.g., PD-1, LAG3 or TIM3) of the population of TRLs can be reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% compared to that of the population of TILs. In some embodiments, one or more dysfunction markers of the population of TRLs can be reduced by at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95% compared to that of the population of TILs. In some embodiments, one or more dysfunction markers of the population of TRLs can be reduced by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% compared to that of the population of TILs.

[0084] In some embodiments, the population of TRLs can exhibit an increase in sternness phenotype (e.g., measured by TCF7, IL-7R, SELL, CCR7, or CD62L) compared to that of a population of tumor infiltrating lymphocytes (TILs). In some embodiments, one or more sternness phenotypes can be increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% compared to that of the population of TILs. In some embodiments, one or more sternness phenotypes can be increased by at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about90%, at most about 95% compared to that of the population of TILs. In some embodiments, one or more sternness phenotypes can be increased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% compared to that of the population of TILs.

[0085] In some embodiments, the population of TRLs can exhibit an increase in cytotoxicity compared to that of a population of tumor infiltrating lymphocytes (TILs). In some embodiments, cytotoxicity of TRLs can be increased by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% compared to that of the population of TILs. In some embodiments, cytotoxicity of TRLs can be increased by at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 35%, at most about 40%, at most about 45%, at most about 50%, at most about 55%, at most about 60%, at most about 65%, at most about 70%, at most about 75%, at most about 80%, at most about 85%, at most about 90%, at most about 95% compared to that of the population of TILs. In some embodiments, cytotoxicity of TRLs can be increased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% compared to that of the population of TILs.B. Enhanced TRLs

[0086] Disclosed herein, in some embodiments, are compositions comprising a population of isolated and enriched TRLs (e.g., isolated and enriched cTRLs) that are therapeutically enhanced (e.g., to express a CAR or a TCR). In some embodiments, genetic materials encoding either a cloned TCR or a synthetic chimeric antigen receptor (CAR) targeting tumor specific antigen can be introduced to the isolated and enriched TRLs described herein.

[0087] Generally, CARs are engineered fusion proteins constructed from antigen recognition, signaling, and costimulatory domains that may be expressed in T cells to reprogram the T cells to specifically target tumor cells. In some embodiments, a CAR can be a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain comprising a functional signaling domain derived from astimulatory molecule. Exogenous T cell receptors can be similar to CARs in that they may be engineered to recognize an antigen (e.g., tumor antigen). In some embodiments the TCR can be a recombinant polypeptide.

[0088] In some embodiments, the extracellular antigen binding domain can be an antigen binding fragment of an antibody, or a functional portion thereof (e.g., an scFv) or a functional variant thereof. The specificity of the antigen binding domain may be modified to treat a variety of different disorders and may be mono-valent or multi-valent (e.g., di-valent, tri-valent). In some embodiments, the antigen binding domain can comprise an scFv, and multivalent binding can be provided by tandem addition of multiple scFvs bearing different antigen specificities. In some embodiments, the specificity and intended indication of the antigen binding can match that of any of the CAR-T constructs in contemporary clinical trials. For example, the specificity may include anti-CD19 (e.g., axicabtageneciloleucel for R / R diffuse large cell lymphoma; or Tisagenlecleucel, for R / R B cell ALL and non-Hodgkin lymphoma), anti-CD22 (e.g., for R / RB-ALL), anti- CD19 / CD22 dual targeted (e.g., for R / R ALL), anti-CAIX (carbonic anhydrase 9), anti-PSMA (a.k.a FOLH1, e.g., for renal cell carcinoma), anti-MUCl (e.g., for seminal vesicle carcinoma), anti-CD33(e.g., for acute myeloid leukemia), anti-mesothelin mRNA (e.g., for adenocarcinoma and pleural mesothelioma), anti-FOLRl (e.g., for metastatic ovarian cancer), anti- carcinoembryonic antigen (a.k.a. CEA, e.g., for CEA-expressing adenocarcinoma liver metastases), anti-IL13RA2 (e.g., for glioblastoma), anti-HER2 (e.g., for sarcoma), or any combination thereof. In some embodiments, one or more of the following antigens may be bound by the CAR-T construct: 1-40-P-amyloid, 4-1BB, 5 AC, 5T4, 707-AP, A kinase anchor protein 4 (AKAP-4), activin receptor type-2B (ACVR2B), activin receptor-like kinase 1 (ALK1), adenocarcinoma antigen, adipophilin, adrenoceptor P 3 (ADRB3), AGS-22M6, a folate receptor, a-fetoprotein (AFP), AIM-2, anaplastic lymphoma kinase (ALK), androgen receptor, angiopoietin 2, angiopoietin 3, angiopoi etin-binding cell surface receptor 2 (Tie 2), anthrax toxin, AOC3 (VAP- 1), B cell maturation antigen (BCMA), B7-H3 (CD276), Bacillus anthracis anthrax, B-cell activating factor (BAFF), B-lymphoma cell, bone marrow stromal cell antigen 2 (BST2), Brother of the Regulator of Imprinted Sites (BORIS), C242 antigen, C5, CA-125, cancer antigen 125 (CA- 125 or MUC16), Cancer / testis antigen 1 (NY-ESO-1), Cancer / testis antigen 2 (LAGE-la), carbonic anhydrase 9 (CA-IX), Carcinoembryonic antigen (CEA), cardiac myosin, CCCTC- Binding Factor (CTCF), CCL11 (eotaxin-1), CCR4, CCR5, CD11, CD123, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD40L), CD171, CD179a, CD18, CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD24, CD25 (a chain of IL-2receptor), CD27,CD274, CD28, CD3, CD3 e, CD30, CD300 molecule-like family member f (CD300LF), CD319 (SLAMF7), CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44 v7, CD44 v8, CD44 v6, CD5, CD51, CD52, CD56, CD6, CD70, CD72, CD74, CD79A, CD79B, CD80, CD97, CEA- related antigen, CFD, ch4D5, chromosome X open reading frame 61 (CXORF61), claudin 18.2 (CLDN18.2), claudin 6 (CLDN6), Clostridium difficile, clumping factor A, CLCA2, colony stimulating factor 1 receptor (CSF1R), CSF2, CTLA-4, C-type lectin domain family 12 member A (CLEC12A), C-type lectin-like molecule- 1 (CLL-1 or CLECL1), C-X-C chemokine receptor type 4, cyclin Bl, cytochrome P4501B1 (CYP1B1), cyp-B, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4, DPP4, DR5, E. coli shiga toxin type-1, E. coli shiga toxin type- 2, ecto-ADP- ribosyltransferase 4 (ART4), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), EGF-like-domain multiple 7 (EGFL7), elongation factor 2 mutated (ELF2M), endotoxin, Ephrin A2, Ephrin B2, ephrin type-A receptor 2, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), episialin, epithelial cell adhesion molecule (EpCAM), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), ERBB2, ERBB3, ERBB4, ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), Escherichia coli, ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML), F protein of respiratory syncytial virus, FAP, Fc fragment of IgA receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor, fibrin II P chain, fibroblast activation protein a (FAP), fibronectin extra domain-B, FGF-5, Fms-Like Tyrosine Kinase 3 (FLT3), folate binding protein (FBP), folate hydrolase, folate receptor 1, folate receptor a, folate receptor P, Fos-related antigen 1, Frizzled receptor, Fucosyl GM1, G250, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), ganglioside G2 (GD2), GD3 ganglioside, glycoprotein 100 (gplOO), glypican-3 (GPC3), GMCSF receptor a-chain, GPNMB, GnT-V, growth differentiation factor 8, GUCY2C, heat shock protein 70-2 mutated (mut hsp70-2), hemagglutinin, Hepatitis A virus cellular receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, hexasaccharide portion of globoH glycoceramide (GloboH), HGF, HHGFR, high molecular weight -melanoma-associated antigen (HMW-MAA), histone complex, HIV-1, HLA-DR, HNGF, Hsp90, HST-2 (FGF6), human papilloma virus E6 (HPV E6), human papilloma virus E7 (HPV E7), human scatter factor receptor kinase, human Telomerase reverse transcriptase (hTERT), human TNF, ICAM-1 (CD54), iCE, IFN-a, IFN-P, IFN-y, IgE, IgE Fc region, IGF-1, IGF-1 receptor, IGHE, IL- 12, IL- 13, IL- 17, IL-17A, IL-17F, IL-ip, IL-20, IL-22, IL-23, IL-31, IL-3 IRA, IL-4, IL-5, IL-6, IL-6 receptor, IL- 9, immunoglobulin lambda-like polypeptide 1 (IGLL1), influenza A hemagglutinin, insulin-like growth factor 1 receptor (IGF-I receptor), insulin-like growth factor 2 (ILGF2), integrin a4p7,integrin P2, integrin a2, integrin a4, integrin a5pi, integrin a7p7, integrin allbp3, integrin avP3, interferon a / p receptor, interferon y-induced protein, Interleukin 11 receptor a (IL-l lRa), Interleukin- 13 receptor subunit a-2 (IL-13Ra2 or CD213A2), intestinal carboxyl esterase, kinase domain region (KDR), KIR2D, KIT (CD117), Ll-cell adhesion molecule (Ll-CAM), legumain, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis-Y antigen, LFA-1 (CDl la), LINGO-1, lipoteichoic acid, L0XL2, L-selectin (CD62L), lymphocyte antigen 6 complex, locus K 9 (LY6K), lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), lymphotoxin-a (LT-a) or Tumor necrosis factor-P (TNF-P), macrophage migration inhibitory factor (MIF or MMIF), M-CSF, mammary gland differentiation antigen (NY-BR-1), MCP-1, melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), melanoma inhibitor of apoptosis (ML-IAP), melanoma-associated antigen 1 (MAGE-A1), mesothelin, mucin 1, cell surface associated (MUC1), MUC-2, mucin CanAg, myelin-associated glycoprotein, myostatin, N-Acetyl glucosaminyl -transferase V (NA17), NCA-90 (granulocyte antigen), nerve growth factor (NGF), neural apoptosis-regulated proteinase 1, neural cell adhesion molecule (NCAM), neurite outgrowth inhibitor (e.g., NOGO-A, NOGO-B, NOGO-C), neuropilin-1 (NRP1), N-glycolylneuraminic acid, NKG2D, Notch receptor, o-acetyl-GD2 ganglioside (0AcGD2), olfactory receptor 51E2 (OR51E2), oncofetal antigen (h5T4), oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), Oryctolagus cuniculus, OX-40, oxLDL, p53 mutant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), pannexin 3 (PANX3), phosphatesodium co-transporter, phosphatidylserine, placenta-specific 1 (PLAC1), platelet-derived growth factor receptor a (PDGF-R a), platelet-derived growth factor receptor p (PDGFR-P), poly sialic acid, proacrosin binding protein sp32 (OY-TES1), programmed cell death protein 1 (PD-1), proprotein convertase subtilisin / kexin type 9 (PCSK9), prostase, prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI), P15, P53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostatic acid phosphatase (PAP), prostatic carcinoma cells, prostein, Protease Serine 21 (Testisin or PRSS21), Proteasome (Prosome, Macropain) Subunit, P Type, 9 (LMP2), Pseudomonas aeruginosa, rabies virus glycoprotein, RAGE, Ras Homolog Family Member C (RhoC), receptor activator of nuclear factor kappa-B ligand (RANKL), Receptor for Advanced Glycation Endproducts (RAGE-1), receptor tyrosine kinase-like orphan receptor 1 (R0R1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), respiratory syncytial virus, Rh blood group D antigen, Rhesus factor, sarcoma translocation breakpoints, sclerostin (SO ST), selectin P, sialylLewis adhesion molecule (sLe), sperm protein 17 (SPA17), sphingosine- 1 -phosphate, squamous cell carcinoma antigen recognized by T Cells 1, 2, and 3 (SART1, SART2, and SART3), stagespecific embryonic antigen -4 (S SEA-4), Staphylococcus aureus, STEAP1, surviving, syndecan 1 (SDC1)+A314, SOXIO, survivin, surviving-2B, synovial sarcoma, X breakpoint 2 (SSX2), T-cell receptor, TCR Alternate Reading Frame Protein (TARP), telomerase, TEM1, tenascin C, TGF- P e.g., TGF-P 1, TGF-P 2, TGF-P 3), thyroid stimulating hormone receptor (TSHR), tissue factor pathway inhibitor (TFPI), Tn antigen ((Tn Ag) or (GalNAca-Ser / Thr)), TNF receptor family member B cell maturation (BCMA), TNF -a, TRAIL-R1, TRAIL-R2, TRG, transglutaminase 5 (TGS5), tumor antigen CTAA16.88, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tumor protein p53 (p53), tumor specific glycosylation of MUC1, tumor-associated calcium signal transducer 2, tumor-associated glycoprotein 72 (TAG72), tumor-associated glycoprotein 72 (TAG-72)+A327, TWEAK receptor, tyrosinase, tyrosinase-related protein 1 (TYRP1 or glycoprotein 75), tyrosinase-related protein 2 (TYRP2), uroplakin 2 (UPK2), vascular endothelial growth factor (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), vimentin, v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), von Willebrand factor (VWF), Wilms tumor protein (WT1), X Antigen Family, Member 1 A (XAGE1), P-amyloid, and K-light chain.

[0089] In some embodiments, the transmembrane domain of the CAR can be a domain that localizes the CAR to the correct membrane location and stabilizes its structure. Suitable transmembrane domains can include the transmembrane region(s) of alpha, beta or zeta chain of the T-cell receptor; or a transmembrane region from CD28, CD3 epsilon, CD3(^, CD45, CD4, CD5, CD8alpha, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 or a functional portion or functional variant thereof. Alternatively, the transmembrane domain can be synthetic, and can comprise hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine is found at one or both termini of a synthetic transmembrane domain. Optionally, a short oligonucleotide or polypeptide linker, in some embodiments, between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of a CAR. In some embodiments, the linker can be a glycine-serine linker. In some embodiments, the CAR can comprise a cytoplasmic signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In some embodiments, the stimulatory molecule is a stimulatory receptor molecule. In some embodiments, the stimulatory receptor molecule is a stimulatory receptormolecule of an adaptive immune cell. In some embodiments, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In some embodiments, the stimulatory molecule is e.g., FCER1G, Fc gamma Rlla, FcR beta (Fc Epsilon Rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP 10, or DAP1, or a functional portion or functional variant thereof. In some embodiments, the intracellular signaling domain can comprise one or more functional signaling domains derived from at least one costimulatory molecule. In some embodiments, the costimulatory molecule can comprise 4-1BB (i.e., CD137), CD27, CD28 CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, a ligand that binds to CD83, ICAM-1, LFA-1 (CD1 Ia / CD18), ICOS, a functional portion or functional variant thereof, or a combination thereof. In some embodiments, the CAR can comprise a leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In some embodiments, the CAR can comprise a signal peptide sequence at the N-terminus of the extracellular antigen recognition domain, wherein the signal peptide sequence is optionally cleaved from the antigen recognition domain (e.g., a scFv), or a functional portion or functional variant thereof during cellular processing and localization of the CAR to the cellular membrane.

[0090] In some embodiments, a CAR disclosed herein can be a first-, second-, third-, or fourth-generation CAR system, a functional variant thereof, or a combination thereof. In some embodiments, a first-generation CAR can comprise an antigen binding domain with specificity for a particular antigen (e.g., an antibody or antigen -binding fragment thereof, such as an scFv, a Fab fragment, a VHH domain, or a VH domain of a heavy-chain only antibody), a transmembrane domain derived from an adaptive immune receptor (e.g., the transmembrane domain from the CD28 receptor or a functional portion or functional variant thereof), and a signaling domain derived from an adaptive immune receptor (e.g., the three ITAM domains derived from the intracellular region of the CD3 C, receptor or FcsRIy or a functional portion or functional variant thereof). In some embodiments, a second-generation CAR construct can comprise the elements of the first-generation CAR and an addition of a co-stimulatory domain to the intracellular signaling domain portion of the CAR (e.g., derived from co-stimulatory receptors that act alongside T-cell receptors such as CD28, CD137 / 4-1BB, and CD134 / OX40 or a functional portion or functional variant thereof). In some embodiments, the co-stimulatory domain can abrogate the need for administration of IL-2 alongside a first-generation CAR. In some embodiments, a third-generation CAR can comprise the elements of a first-generation CAR with the addition of multiple co- stimulatory domains to the intracellular signaling domain portion of the CAR (e.g., CD3(^-CD28- 0X40, or CD3(^-CD28-41BB or a functional portion or functional variant thereof). In someembodiments, fourth-generation CAR can comprise the elements of a second- or third-generation CARs with the addition of an activating cytokine (e.g., IL-12, IL-23, or IL-27 or a functional portion or functional variant thereof) to the intracellular signaling portion of the CAR (typically between one or more of the costimulatory domains and the CD3(^ IT AM domain or a functional portion or functional variant thereof) or under the control of a CAR-induced promoter (e.g., the NFAT / IL-2 minimal promoter or a functional portion or functional variant thereof).

[0091] The isolated and enriched TRLs may be manufactured to express a CAR by variety approaches known to those skilled in the art, which generally include the following steps: isolating a subject’s T cells, activating said T cells, transducing said T cells with a CAR transgene, and expanding said transduced T Cells for the required number for cell therapy. In some embodiments, the T cells (e.g., isolated and enriched TRLs such as CD103, SLC6A19 and / or SIDT1 expressing cells) can be isolated using any of the methods disclosed herein. In some embodiments, transducing a cell with a CAR transgene can comprise introducing the cell to a nucleotide that expresses a CAR under conditions sufficient to produce the CAR by the cell. Methods for introducing genetically engineered components, such as CARs, to T-cells are well known to skilled artisans and may be used to produce the CARs disclosed herein. Exemplary methods for transferring a nucleic encoding the CAR, can include viral transduction, e.g., via retroviral transduction or lentiviral transduction, transposon, and electroporation-mediated methods.

[0092] Also provided are polynucleotides encoding the compositions disclosed herein. In some embodiments, the vector can comprise a backbone and the polynucleotides encoding a chimeric antigen receptor (CAR), wherein the CAR can comprise (a) antigen binding domain; (b) a transmembrane domain; (c) a costimulatory signaling domain (e.g, 4- IBB or CD28, or both); and / or (d) a CD3 zeta signaling domain. In some embodiments, a vector can comprise one or more of the polynucleotides disclosed herein. In some embodiments, the vector can be a plurality of vectors. In some embodiments, the polynucleotides encoding the CAR can be transferred to a TRL (e.g, cTRL) using a lentiviral vector. In some embodiments, the nucleotide encoding the CAR can be transferred to a TRL using a retroviral vector. In some embodiments, the vector can be a non-viral vector. In some embodiments, the non-viral vector can be a Sleeping Beauty transposon. In some embodiments, the vector can comprise a plasmid. In some embodiments, each vector described herein can comprise an expression plasmid.

[0093] In some embodiments, the polynucleotides encoding the CAR can be cloned into a vector comprising lentiviral backbone components. Exemplary backbone components caninclude, but are not limited to, pFUGW, and pSMPUW. The pFUGW lentiviral vector backbone is a self-inactivating (SIN) lentiviral vector backbone and has unnecessary HIV-1 viral sequences removed resulting in reduced potential for the development of neoplasia, harmful mutations, and regeneration of infectious particles. In some embodiments, the CAR can be under the control of an inducible promoter. In some embodiments, an inducible promoter can be a small molecule ligand-inducible two polypeptide ecdysone receptor-based gene switch. In some embodiments, the CAR can be under the control of a constitutive promoter.

[0094] Provided herein is a system for expressing a CAR in a TRL (e.g., cTRLs), wherein the system can comprise one or more vectors encoding polynucleotides disclosed herein. In some embodiments, the system can further comprise a nucleic acid encoding at least one additional gene. In some embodiments, the additional gene can comprise a cytokine. In some embodiments, the cytokine can comprise at least one of IL-2, IL-15, IL-12, IL-21, and a fusion of IL-15 and IL- 15Ra, or a functional portion or functional variant thereof. In some embodiments, the cytokine can be in secreted form. In some embodiments, the cytokine can be in membrane bound form.C. Pharmaceutical Formulations

[0095] Described herein are pharmaceutical formulations comprising the TRLs (e.g., cTRLs) or the enhanced TRLs (e.g., enhanced cTRLs) described herein. In some embodiments, a pharmaceutical formulation can comprise TRLs or enhanced TRLs comprising a population of SLC6A19+ lymphocytes, SIDT1+ lymphocytes, CD103+ lymphocytes, CD39+ lymphocytes, or combination thereof. In some embodiments, the pharmaceutical formulations further can comprise a pharmaceutically acceptable: carrier, excipient, diluent, or nebulized inhalant.

[0096] In some embodiments, the pharmaceutical formulations can include one or more active agents, or one or more therapeutic agents as disclosed herein. In some embodiments, the one or more active agents can be contained in a single dosage unit such as, for example, when the TRL or the enhanced TRLs (e.g., CAR or TCR) can comprise or can be administered with one or more therapeutic agents. In some embodiments, the one or more active agents can be contained in separate dosage units such as when the TRLs or the enhanced TRLs (e.g., CAR or TCR) can be administered separately from an additional therapeutic agent or adjuvant.

[0097] In some embodiments, the active agents or the additional therapeutic agent can be a chemotherapeutic agent, cytotoxic agent, cytokine, growth-inhibitory agent, anti-hormonal agent, anti-angiogenic agent, cardio protectant, an immunosuppressant, an antifungal and / or checkpoint inhibitor. Non-limiting checkpoint inhibitor includes IMP321 / Eftilagimod alpha (Immutep),Relatlimab BMS-986016, Ipilimumab (Yervoy), Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), Ipilimumab (Yervoy), LAG525, MK-4280, Irinotecan, Oxaliplatin, REGN3767, TSR- 033, BI754111, Sym022, FS118 (abi-specific anti-LAG3 / PD-Ll antagonistic mAb), MGD013 (a bi-specific anti-LAG3 / PD-l antagonistic mAb), TSR-022, Niraparib, Bevacizumab, MBG453, Decitabine, Spartalizumab, Sym023, INCAGN2390, LY3321367, Ramucirumab, Abemaciclib, Merestinib, BMS-986258, SHR-1702, Camrelizumab, MK-7684, Etigilimab / OMP-313 M32, Tiragolumab / MTIG7192A / RG-6058, BMS-986207, AB-154, ASP-8374, JNJ-61610588, CA- 170d, Enoblituzumab / MGA271, MGD009, I-8H9 / omburtamab, Trastuzumab, MGD013 (Anti- PD-1, anti-LAG-3 dual checkpoint inhibitor), BGB-A1217, CM-24 (MK-6018), BMS 986178, MEDI6469, PF -04518600, GSK3174998, MOXR0916, Utomilimab (PF-05082566), Urelumab (BMS-663513) ES101, BMS-986156, TRX-518, AMG 228, JTX-2011, GSK3359609, BMS- 986226, MEDI-570, or Varlilumab (CDX-1127).

[0098] In some embodiments, a non-limiting immunosuppressant can comprise at least one member selected from the group consisting of cyclophosphamide, mechlorethamine, chlorambucil, melphalan, ifosfamide, thiotepa, hexamethylmelamine, busulfan, fludarabine, nitrosoureas, platinum, methotrexate, azathioprine, mercaptopurine, procarbazine, dacarbazine, temozolomide, carmustine, lomustine, streptozocin, fluorouracil, dactinomycin, anthracycline, mitomycin C, bleomycin, and mithramycin.

[0099] In some embodiments, a cyclophosphamide can be administered from about 40 mg / kg to about 50 mg / kg of a subject. In some embodiments, a cyclophosphamide can be administered to a subj ect over at least about 2 days to about 15 days. In some embodiments, a cyclophosphamide can be administered from about 10 mg / kg to about 15 mg / kg of a subject. In some embodiments, cyclophosphamide can be administered to a subject over at least about 7 days to about 10 days. In some embodiments, cyclophosphamide can be administered from about 3 mg per kg to about 5 mg per kg of a subject. In some embodiments, cyclophosphamide can be administered from about 50 mg per kg to about 80 mg / kg of a subject. In some embodiments, cyclophosphamide can be administered in excess of 50 mg per kg. In some embodiments, cyclophosphamide can be administered at about 60 mg per kg. In some embodiments, fludarabine can be administered from about 20 mg / m2to about 30 mg / m2of body surface area of a subject. In some embodiments, fludarabine can be administered at about 25 mg / m2of body surface area of a subject. In some embodiments, a preparative regime can comprise partial or complete immunosuppression. In some embodiments, an antifungal can be selected from a group consisting of: polyene, azole, allylamine, and echinocandin. An antifungal can be an azole. An azole can be selected from the groupconsisting of: bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propi conazole, ravuconazole, terconazole, and voriconazole. An antifungal that is an azole can be fluconazole. In some embodiments, fluconazole can be administered from about 100 mg to about 800 mg. In some embodiments, fluconazole can be administered at 400 mg. An antifungal can be administered concurrently or sequentially with TRLs (e.g., cTRLs). An antifungal can be administered from about day 0 to about day 4 after TRLs. In some embodiments, an antibiotic can comprise at least one of: a bacterial wall targeting agent, a cell membrane targeting agent, a bacterial enzyme interfering agent, a bactericidal agent, a protein synthesis inhibitor, or a bacteriostatic agent. In some embodiments, an antibiotic can comprise a bactericidal agent. A bactericidal agent can be cephalosporin or quinolone. In some embodiments, an antibiotic can comprise a bacteriostatic agent. A bacteriostatic agent can be administered prophylactically. In some embodiments, a bacteriostatic agent can be trimethoprim, sulfamethoxazole, or pentamidine Trimethoprim, sulfamethoxazole, or pentamidine can be administered from about 100 mg to about 1000 mg. In some embodiments, trimethoprim can be administered at 160 mg. In some embodiments, sulfamethoxazole can be administered at 800 mg. In some embodiments, pentamidine can be administered at 300 mg.

[0100] In some embodiments, the additional therapeutic agent or a bactericidal agent can be administered prior to TRLs (e.g., cTRLs), concurrent with TRLs (e.g., cTRLs), or after TRLs (e.g., cTRLs). In some embodiments, the additional therapeutic agent or a bactericidal agent can be administered from about 10 days prior to administration of TRLs to about 6 months after administration of TRLs (e.g., cTRLs). In some embodiments, the additional therapeutic agent or a bactericidal agent can be administered from about 10 days prior to said TRLs to at least 4 days after TRLs (e.g., cTRLs).

[0101] The pharmaceutical formulations described herein may be formulated for administration to a subject by appropriate administration routes, including but not limited to, intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal administration routes. The composition described herein may include, but not limited to, aqueous liquid dispersions, selfemulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended-release formulations, pulsatilerelease formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

[0102] The pharmaceutical formulations including a therapeutic agent may be manufactured in a conventional manner such as, by way of example only, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or compression processes.

[0103] The pharmaceutical formulations may include at least an exogenous therapeutic agent as an active ingredient in free-acid or free-base form, or in a pharmaceutically acceptable salt form. In addition, the methods and compositions described herein can include the use of N-oxides (if appropriate), crystalline forms, amorphous phases, as well as active metabolites of these compounds having the same type of activity. In some embodiments, therapeutic agents can exist in unsolvated form or in solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the therapeutic agents can be also considered to be disclosed herein.

[0104] In some embodiments, pharmaceutical formulations provided herein can include one or more preservatives to inhibit microbial activity. Suitable preservatives can include mercury- containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0105] In some embodiments, pharmaceutical formulations described herein can benefit from antioxidants, metal chelating agents, thiol containing compounds and other general stabilizing agents. Examples of such stabilizing agents, include, but are not limited to: (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, I about 0.01% to about 2% w / v ascorbic acid, (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v. polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (1) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

[0106] The pharmaceutical formulations described herein can be formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled releaseformulations. In one aspect, a therapeutic agent as discussed herein, e.g., therapeutic agent can be formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection. In one aspect, formulations suitable for intramuscular, subcutaneous, or intravenous injection can include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for rehydration into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles can include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, formulations suitable for subcutaneous injection also can contain additives such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the growth of microorganisms may be ensured by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and the like. In some embodiments, it is desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the use of agents delaying absorption such as aluminum monostearate and gelatin.

[0107] For intravenous injections or drips or infusions, a pharmaceutical formulation described herein can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. For other parenteral injections, appropriate formulations can include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients. Such excipients are known.

[0108] Parenteral injections may involve bolus injection or continuous infusion. Pharmaceutical formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative. The composition described herein may be in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. In one aspect, the active ingredient is in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0109] For administration by inhalation, a therapeutic agent can be formulated for use as an aerosol, a mist or a powder. Pharmaceutical formulations described herein can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or nebulizers, withthe use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of such as, by way of example only, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the therapeutic agent described herein and a suitable powder base such as lactose or starch. Formulations that include a composition can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, fluorocarbons, and / or other solubilizing or dispersing agents known in the art. Preferably these compositions and formulations can be prepared with suitable nontoxic pharmaceutically acceptable ingredients. The choice of suitable carriers can be dependent upon the exact nature of the nasal dosage form desired, e.g., solutions, suspensions, ointments, or gels. Nasal dosage forms generally can contain large amounts of water in addition to the active ingredient. Minor amounts of other ingredients such as pH adjusters, emulsifiers or dispersing agents, preservatives, surfactants, gelling agents, or buffering and other stabilizing and solubilizing agents can be optionally present. Preferably, the nasal dosage form can be isotonic with nasal secretions.

[0110] Pharmaceutical preparations for oral use can be obtained by mixing one or more solid excipients with one or more of the compositions described herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients can include, for example, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate. If desired, disintegrating agents can be added such as the cross linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. In some embodiments, dyestuffs or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active therapeutic agent doses.[OHl] In some embodiments, the pharmaceutical formulations of the exogenous therapeutic agents can be in the form of a capsules, including push fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. The push fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active therapeutic agent can be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In some embodiments, stabilizers canbe added. A capsule may be prepared, for example, by placing the bulk blend of the formulation of the therapeutic agent inside of a capsule. In some embodiments, the formulations (non-aqueous suspensions and solutions) can be placed in a soft gelatin capsule. In other embodiments, the formulations can be placed in standard gelatin capsules or non-gelatin capsules such as capsules comprising HPMC. In other embodiments, the formulation can be placed in a sprinkle capsule, wherein the capsule is swallowed whole, or the capsule is opened, and the contents sprinkled on food prior to eating.

[0112] Pharmaceutical formulations for oral administration can be in dosages suitable for such administration. In one aspect, solid oral dosage forms can be prepared by mixing a composition with one or more of the following: antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents. In some embodiments, the solid dosage forms disclosed herein can be in the form of a tablet, (including a suspension tablet, a fast-melt tablet, a bite-disintegration tablet, a rapid-disintegration tablet, an effervescent tablet, or a caplet), a pill, a powder, a capsule, solid dispersion, solid solution, bioerodible dosage form, controlled release formulations, pulsatile release dosage forms, multiparticulate dosage forms, beads, pellets, granules. In other embodiments, the composition can be in the form of a powder. Compressed tablets can be solid dosage forms prepared by compacting the bulk blend of the formulations described above. In various embodiments, tablets comprise one or more flavoring agents. In other embodiments, the tablets can comprise a film surrounding the final compressed tablet. In some embodiments, the film coating may provide a delayed release of a therapeutic agent from the formulation. In other embodiments, the film coating can aid in patient compliance. Film coatings typically can range from about 1% to about 3% of the tablet weight. In some embodiments, solid dosage forms, e.g., tablets, effervescent tablets, and capsules, can be prepared by mixing particles of a therapeutic agent with one or more pharmaceutical excipients to form a bulk blend composition. The bulk blend can be readily subdivided into equally effective unit dosage forms such as tablets, pills, and capsules. In some embodiments, the individual unit dosages include film coatings. These formulations can be manufactured by conventional formulation techniques.

[0113] In another aspect, dosage forms can include microencapsulated formulations. In some embodiments, one or more other compatible materials can be present in the microencapsulation material. Non-limiting example of materials can include pH modifiers, erosion facilitators, antifoaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.

[0114] Liquid formulation dosage forms for oral administration can be optionally aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. In addition to therapeutic agent, the liquid dosage forms optionally can include additives such as: (a) disintegrating agents; (b) dispersing agents; (c) wetting agents; (d) at least one preservative, (e) viscosity enhancing agents, (f) at least one sweetening agent, and (g) at least one flavoring agent. In some embodiments, the aqueous dispersions further include a crystal-forming inhibitor.

[0115] In some embodiments, the pharmaceutical formulations described herein can be selfemulsifying drug delivery systems (SEDDS). Emulsions can be dispersions of one immiscible phase in another, usually in the form of droplets. Generally, emulsions can be created by vigorous mechanical dispersion. SEDDS, as opposed to emulsions or microemulsions, spontaneously form emulsions when added to an excess of water without any external mechanical dispersion or agitation. An advantage of SEDDS can be that only gentle mixing is required to distribute the droplets throughout the solution. Additionally, water or the aqueous phase can be optionally added just prior to administration, which ensures stability of an unstable or hydrophobic active ingredient. Thus, the SEDDS provides an effective delivery system for oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS can provide improvements in the bioavailability of hydrophobic active ingredients.

[0116] Buccal formulations can be administered using a variety of formulations known in the art. In addition, the buccal dosage forms described herein may further include a bioerodible (hydrolysable) polymeric carrier that also serves to adhere the dosage form to the buccal mucosa. For buccal or sublingual administration, the compositions may take the form of tablets, lozenges, or gels formulated in a conventional manner.

[0117] For intravenous injections, a pharmaceutical formulation can be optionally formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated can be used in the formulation. For other parenteral injections, appropriate formulations include aqueous or nonaqueous solutions, preferably with physiologically compatible buffers or excipients.

[0118] Parenteral injections optionally involve bolus injection or continuous infusion. Formulations for injection can be optionally presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative. In some embodiments, a composition described herein is in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending,stabilizing and / or dispersing agents. The compositions for parenteral administration include aqueous solutions of an agent that modulates the activity of a carotid body in water soluble form. Additionally, suspensions of an agent that modulates the activity of a carotid body can be optionally prepared as appropriate, e.g., oily injection suspensions.

[0119] Conventional formulation techniques include, e.g., one or a combination of methods: (1) dry mixing, (2) direct compression, (3) milling, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion. Other methods include, e.g., spray drying, pan coating, melt granulation, granulation, fluidized bed spray drying or coating (e.g., wurster coating), tangential coating, top spraying, tableting, extruding and the like.

[0120] In some embodiments, the compositions can be provided that include particles of a therapeutic agent and at least one dispersing agent or suspending agent for oral administration to a subject. The formulations may be a powder and / or granules for suspension, and upon admixture with water, a substantially uniform suspension is obtained.

[0121] Furthermore, the pharmaceutical formulations optionally include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and tris-hydroxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers can be included in an amount required to maintain pH of the composition in an acceptable range.

[0122] Additionally, the pharmaceutical formulations optionally include one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0123] Other pharmaceutical formulations can include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0124] In one embodiment, the aqueous suspensions and dispersions described herein remain in a homogenous state for at least 4 hours. In one embodiment, an aqueous suspension is resuspended into a homogenous suspension by physical agitation lasting less than 1 minute. In still another embodiment, no agitation is necessary to maintain a homogeneous aqueous dispersion.

[0125] An aerosol formulation for nasal administration is generally an aqueous solution designed to be administered to the nasal passages in drops or sprays. Nasal solutions may be similar to nasal secretions in that they can be generally isotonic and slightly buffered to maintain a pH of about 5.5 to about 6.5, although pH values outside of this range may additionally be used. Antimicrobial agents or preservatives may also be included in the formulation.

[0126] An aerosol formulation for inhalations and inhalants may be designed so that the agent or combination of agents is carried into the respiratory tree of the subject when administered by the nasal or oral respiratory route. Inhalation solutions may be administered, for example, by a nebulizer. Inhalations or insufflations, comprising finely powdered or liquid drugs, may be delivered to the respiratory system as a pharmaceutical aerosol of a solution or suspension of the agent or combination of agents in a propellant, e.g., to aid in disbursement. Propellants may be liquefied gases, including halocarbons, for example, fluorocarbons such as fluorinated chlorinated hydrocarbons, hydrochlorofluorocarbons, and hydrochlorocarbons, as well as hydrocarbons and hydrocarbon ethers.

[0127] Halocarbon propellants may include fluorocarbon propellants in which all hydrogens can be replaced with fluorine, chlorofluorocarbon propellants in which all hydrogens can be replaced with chlorine and at least one fluorine, hydrogen-containing fluorocarbon propellants, and hydrogen-containing chlorofluorocarbon propellants. Hydrocarbon propellants useful include, for example, propane, isobutane, n-butane, pentane, isopentane and neopentane. A blend of hydrocarbons may also be used as a propellant. Ether propellants include, for example, dimethyl ether as well as the ethers. An aerosol formulation may also comprise more than one propellant. For example, the aerosol formulation can comprise more than one propellant from the same class such as two or more fluorocarbons; or more than one, more than two, more than three propellants from different classes such as a fluorohydrocarbon and a hydrocarbon. The compositions of the present disclosure may also be dispensed with a compressed gas, e.g., an inert gas such as carbon dioxide, nitrous oxide or nitrogen.

[0128] Aerosol formulations may also include other components, for example, ethanol, isopropanol, propylene glycol, as well as surfactants or other components such as oils and detergents. These components may serve to stabilize the formulation and / or lubricate valve components.

[0129] The aerosol formulation may be packaged under pressure and may be formulated as an aerosol using solutions, suspensions, emulsions, powders and semisolid preparations. For example, a solution aerosol formulation can comprise a solution of an agent such as a transporter, carrier, or ion channel inhibitor in (substantially) pure propellant or as a mixture of propellant andsolvent. The solvent may be used to dissolve the agent and / or retard the evaporation of the propellant. Solvents may include, for example, water, ethanol and glycols. Any combination of suitable solvents may be used, optionally combined with preservatives, antioxidants, and / or other aerosol components.

[0130] An aerosol formulation may be a dispersion or suspension. A suspension aerosol formulation can comprise a suspension of an agent or combination of agents, e.g., a transporter, carrier, or ion channel inhibitor, and a dispersing agent. Dispersing agents may include, for example, sorbitan trioleate, oleyl alcohol, oleic acid, lecithin and corn oil. A suspension aerosol formulation may also include lubricants, preservatives, antioxidant, and / or other aerosol components.

[0131] An aerosol formulation may similarly be formulated as an emulsion. An emulsion aerosol formulation may include, for example, an alcohol such as ethanol, a surfactant, water and a propellant, as well as an agent or combination of agents, e.g., a transporter, carrier, or ion channel. The surfactant used may be nonionic, anionic or cationic. One example of an emulsion aerosol formulation can comprise, for example, ethanol, surfactant, water and propellant. Another example of an emulsion aerosol formulation can comprise, for example, vegetable oil, glyceryl monostearate and propane.II. METHODS

[0132] Disclosed herein are methods of isolating, enriching, expanding, and using the tumor reactive lymphocytes (TRLs) or the enhanced TRLs of the present disclosure. Also provided are methods of producing the enhanced TRLs from the TRLs of the present disclosure. Disclosed herein are methods of isolating and expanding a population of tumor reactive lymphocytes (TRLs) from a fluid sample (e.g., peripheral blood) of a subject. In some embodiments, the subject may have a cancer or may be suspected of having cancer. For example, the subject may have a cancer in a breast tissue, a renal tissue, a cervical tissue, a lung tissue, an ovarian tissue, or a skin tissue. The resulting TRLs may be used, for example, in an adoptive cell therapy. In some embodiments, methods disclosed herein comprise isolating TRLs from the peripheral blood of a subject, wherein the TRL population can comprise CD8+CD103+ expressing lymphocytes (CD 103+ lymphocytes). In some embodiments, methods described herein can comprise isolating from the peripheral blood of a subject a population of TRLs expressing CD103, CD39, SLC6A19, and / or SIDTl. In some embodiments, isolating the population of TRLs can comprise magnetically separating a population of TRLs. In some embodiments, the methods of isolating the population of TRLs can comprise magnetically separating the population of TRLs using a microfluidic device. In some embodiments, the microfluidic magnetic cell sorting can rely on theimmunomagnetic labeling of the population of TRLs, followed by magnetic separation within the microfluidic device.

[0133] Also disclosed herein are methods of producing a population of enhanced TRLs. In some embodiments, methods of enhancing a population of TRLs comprise isolating and expanding population of CD103, CD39, SLC6A19 and / or SIDT1 expressing lymphocytes to a nucleotide that expresses a therapeutically enhancing polypeptide under conditions sufficient to produce the enhancing polypeptide by the cell. In some embodiments, the nucleotide encodes a chimeric antigen receptor (CAR). In some embodiments, the CAR can be configured to recognize an antigen associated with a cancer.

[0134] Also disclosed herein are methods of providing a cell therapy (e.g., an adoptive cell therapy) comprising the TRLs or the enhanced TRLs of the present disclosed to a subject in need thereof. In some embodiments, the methods comprise (a) obtaining a population of cells or therapeutically enhanced cells described herein; and (b) administering to the subject the population of cells, thereby providing the cell therapy. In some embodiments, the isolated TRLs can be expanded using any of the methods disclosed herein. In some embodiments, autologous cells can be preferred over allogeneic cells because of their inherent heterogeneity that maximizes the tumor-recognizing T cell receptors (TCRs) while minimizing off-tissue effects. In some embodiments, the subject has a cancer.A. Methods of Isolating Tumor Reactive Lymphocytes

[0135] Disclosed herein, in some embodiments, is a method of isolating a population of TRLs (e.g., cTRLs) comprising obtaining a peripheral blood sample from a subject comprising lymphocytes; and separating from the sample a population of CD103, CD39, SLC6A19 and / or SIDT1 expressing TRLs. In some embodiments, the sample can be a peripheral blood sample which is then subject to leukapheresis to separate the red blood cells and platelets and to isolate immune cells (e.g., CD8+ cells). In some embodiments, the sample can be a leukopak from which immune cells can be isolated or enriched. In some embodiments, separating from the sample a population of CD103, CD39, SLC6A19 and / or SIDT1 expressing TRLs may comprise magnetically separating from the sample. In some embodiments, magnetically separating from the sample a population of CD103, CD39, SLC6A19 and / or SIDT1 expressing lymphocytes may comprise immunomagnetically labeling the population of TRLs followed by magnetic separation within the microfluidic device.

[0136] In some embodiments, immunomagnetically labeling a population of TRLs can comprise attaching-directly or indirectly-a magnetic label to a surface maker of at least one of the TRLs. In some embodiments, the surface marker can be CD103, CD39, SLC6A19, SIDT1 or a combination thereof. In some embodiments, the surface marker can further comprise CD3, CD4, CD8 or a combination thereof. In some embodiments, the surface marker can be CD 103. In some embodiments, the surface marker can be CD39. In some embodiments, the surface marker can be SLC6A19. In some embodiments, the surface marker can be SIDT1. In some embodiments, the surface marker can comprise CD3, CD4, CD8, CD39, CD103, SLC6A19, SIDT1, or a combination thereof. In some embodiments, the TRLs can be labeled by an antibody targeting a surface marker of interest (e.g., an anti-CD103 antibody). In some embodiments, the cells can be labeled by a multimer targeting a surface marker of interest (e.g., an MHC multimer). In some embodiments, the antibody or multimer can be conjugated to a magnetic micro or nano particle (MNP). In some embodiments, the antibody or multimer can be labeled by a secondary antibody conjugated with an MNP. In some embodiments, at least one of the TRLs can be labeled with one or more antibodies or one or more multimers sequentially. In some embodiments, at least one of the TRLs can be labeled with one or more antibodies or one or more multimers concurrently. In some embodiments, after labeling, the TRLs can obtain a level of magnetization as a function of the expression level of the surface marker recognized by the antibody or multimer.

[0137] In some embodiments, the microfluidic device can be configured to isolate the population of TRLs from a population of non-TRLs based on the levels of magnetization exhibited by the TRLs. For example, the microfluidic device may comprise a sorting chamber comprising several separated zones with varying heights. In some embodiments, in each zone, microstructures can be patterned to generate capture pockets that create low-velocity zones for trapping magnetically labeled TRLs. In some embodiments, during operation, the microfluidic device can be sandwiched by arrays of magnets that generate constant magnetic fields in the sorting chamber and can be connected to a syringe pump for fluidic processing. Without being bound by any theory, when the cells can be added to the device, they experience two major forces: the magnetic force generated by the interaction between MNPs and constant magnetic field, and a fluidic drag force which is defined by the fluidic velocity in a specific zone. When the magnetic force overcomes the drag force, a cell can acquire enough trapping force to stay in a specific zone. Otherwise, a cell would be flush into the next zone with a lower drag force, and eventually into the syringe if it cannot be captured by any zone. After sorting, captured cells at each zone can be recovered by removing the external magnets.

[0138] In some embodiments, the microfluidic device can exhibit about 20% to about 98% capture efficiency when capturing a population of rare cells (e.g., a population of TRLs). In some embodiments, the microfluidic device can exhibit about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 92%, about 20% to about 94%, about 20% to about 96%, about 20% to about 98%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 92%, about 30% to about 94%, about 30% to about 96%, about 30% to about 98%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 92%, about 40% to about 94%, about 40% to about 96%, about 40% to about 98%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 92%, about 50% to about 94%, about 50% to about 96%, about 50% to about 98%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 92%, about 60% to about 94%, about 60% to about 96%, about 60% to about 98%, about 70% to about 80%, about 70% to about 90%, about 70% to about 92%, about 70% to about 94%, about 70% to about 96%, about 70% to about 98%, about 80% to about 90%, about 80% to about 92%, about 80% to about 94%, about 80% to about 96%, about 80% to about 98%, about 90% to about 92%, about 90% to about 94%, about 90% to about 96%, about 90% to about 98%, about 92% to about 94%, about 92% to about 96%, about 92% to about 98%, about 94% to about 96%, about 94% to about 98%, or about 96% to about 98% capture efficiency when capturing a population of rare cells (e.g., a population of TRLs). In some embodiments, the microfluidic device can exhibit about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 92%, about 94%, about 96%, or about 98% capture efficiency when capturing a population of rare cells (e.g., a population of TRLs). In some embodiments, the microfluidic device can exhibit at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 92%, about 94%, or about 96% capture efficiency when capturing a population of rare cells (e.g., a population of TRLs). In some embodiments, the microfluidic device can exhibit at most about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 92%, about 94%, about 96%, or about 98% capture efficiency when capturing a population of rare cells (e.g., a population of TRLs.

[0139] In some embodiments, the methods of isolating a population of TRLs described herein can achieve a higher cell recovery than a standard cell-sorting approach (e.g., fluorescenceactivated cell sorting, or MACS) performed on the same sample. In some embodiments, the microfluidic approach to cell sorting described herein can achieve a higher cell recovery than a standard cell-sorting approach, while retaining similar purity.

[0140] In some embodiments, the microfluidic approach to cell sorting described herein can achieve a higher cell recovery by at least about 0.5 fold to about 50 fold as compared to a standard cell-sorting approach. In some embodiments, the microfluidic approach to cell sorting described herein can achieve a higher cell recovery by about 0.5 fold to about 1-fold, about 0.5 fold to about 2-fold, about 0.5 fold to about 2.5-fold, about 0.5 fold to about 5-fold, about 0.5 fold to about 7.5- fold, about 0.5 fold to about 10-fold, about 0.5 fold to about 20-fold, about 0.5 fold to about 30- fold, about 0.5 fold to about 40-fold, about 0.5 fold to about 50-fold, about 1 fold to about 2-fold, about 1 fold to about 2.5-fold, about 1 fold to about 5-fold, about 1 fold to about 7.5-fold, about 1 fold to about 10-fold, about 1 fold to about 20-fold, about 1 fold to about 30-fold, about 1 fold to about 40-fold, about 1 fold to about 50-fold, about 2 fold to about 2.5-fold, about 2 fold to about 5-fold, about 2 fold to about 7.5-fold, about 2 fold to about 10-fold, about 2 fold to about 20-fold, about 2 fold to about 30-fold, about 2 fold to about 40-fold, about 2 fold to about 50-fold, about 2.5 fold to about 5-fold, about 2.5 fold to about 7.5-fold, about 2.5 fold to about 10-fold, about 2.5 fold to about 20-fold, about 2.5 fold to about 30-fold, about 2.5 fold to about 40-fold, about 2.5 fold to about 50-fold, about 5 fold to about 7.5-fold, about 5 fold to about 10-fold, about 5 fold to about 20-fold, about 5 fold to about 30-fold, about 5 fold to about 40-fold, about 5 fold to about 50-fold, about 7.5 fold to about 10-fold, about 7.5 fold to about 20-fold, about 7.5 fold to about 30-fold, about 7.5 fold to about 40-fold, about 7.5 fold to about 50-fold, about 10 fold to about 20-fold, about 10 fold to about 30-fold, about 10 fold to about 40-fold, about 10 fold to about 50- fold, about 20 fold to about 30-fold, about 20 fold to about 40-fold, about 20 fold to about 50-fold, about 30 fold to about 40-fold, about 30 fold to about 50-fold, or about 40 fold to about 50 fold as compared to a standard cell-sorting approach. In some embodiments, the microfluidic approach to cell sorting described herein can achieve a higher cell recovery by about 0.5-fold, about 1-fold, about 2-fold, about 2.5-fold, about 5-fold, about 7.5-fold, about 10-fold, about 20-fold, about 30- fold, about 40-fold, or about 50 fold as compared to a standard cell-sorting approach. In some embodiments, the microfluidic approach to cell sorting described herein can achieve a higher cell recovery by at least about 0.5-fold, about 1-fold, about 2-fold, about 2.5-fold, about 5-fold, about 7.5-fold, about 10-fold, about 20-fold, about 30-fold, or about 40 fold as compared to a standard cell-sorting approach. In some embodiments, the microfluidic approach to cell sorting described herein can achieve a higher cell recovery by at most about 1-fold, about 2-fold, about 2.5-fold,about 5-fold, about 7.5-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, or about 50 fold as compared to a standard cell-sorting approach.

[0141] In some embodiments, methods described herein may comprise isolating from a peripheral blood sample a population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes by treating the sample with a plurality of major histocompatibility complex (MHC) multimers mimicking a defined tumor epitope. Generally, T-cells express surface T-cell receptors (TCR) which enable T-cells to recognize peptide antigens bound to major histocompatibility complex (MHC) molecules, and TCR recognition of MHC -peptide complexes can result in T-cell activation, clonal expansion and differentiation of the T-cells into effector, memory and regulatory T-cells. MHC -multimers can comprise multiple copies of MHC-peptide complexes. In some embodiments, MHC multimers can exhibit increased T-cell affinity, compared to a monomer of the same complex. In some embodiments, the MHC molecules can be human MHC molecules. In some embodiments, the MHC molecules can be murine MHC molecules. In some embodiments, the MHC molecules can be class 1 MHC molecules. In some embodiments, the MHC Class 1 molecules can be human HLA-A, HLA-B, or HLA-C molecules. In some embodiments, the MHC class I molecules can be murine H-2K, H-2D or H-2L molecules. In some embodiments, the molecules can be class 2 MHC molecules. In some embodiments, an MHC multimer can comprise at least 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 MHC molecules. In some embodiments, an MHC multimer can comprise about 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 MHC molecules. In some embodiments, an MHC multimer can comprise an MHC dimer comprising two MHC molecules. In some embodiments, an MHC multimer can comprise an MHC tetramer comprising four MHC molecules. In some embodiments, an MHC multimer can comprise an MHC pentamer comprising five MHC molecules. In some embodiments, an MHC multimer can comprise a dextramer, comprising 10 or more MHC molecules.

[0142] In some embodiments, an MHC multimer may comprise a peptide. In some embodiments, an MHC multimer may comprise a peptide-MHC complex. In some embodiments, the peptide can be any natural or non-natural peptide capable of being presented by an MHC molecule. In some embodiments, the peptide-MHC complex can be one to which a TRL has reactivity. In some embodiments, the peptide-MHC complex can be one to which a CD8 and CD 103 expressing lymphocyte has reactivity. In some embodiments, the peptide-MHC complex can be one to which a CD39 and CD103 expressing lymphocyte has reactivity. In some embodiments, the peptide-MHC complex can mimic a defined tumor epitope. In someembodiments, the peptide can comprise an epitope from influenza A hemagglutinin. In some embodiments, the peptide can comprise amino acid residues 533-541 of influenza A hemagglutinin. In some embodiments, the peptide can comprise an epitope from chicken ovalbumin. In some embodiments, the peptide can comprise amino acid residues 257-264 of chicken ovalbumin. In some embodiments, an MHC multimer can comprise a plurality of peptide- MHC complexes. In some embodiments, each of the peptide-MHC complexes can be associated with a multimerization domain. In some embodiments, the peptide-MHC complex can comprise the MC-38-derived peptide of SIIVFNLL sequence and an H-2Kb molecule. In some embodiments, the MHC multimer can be operatively linked to a magnetic nanoparticle. In some embodiments, the magnetic nanoparticle can be joined to the MHC multimer via fluorophore linker.

[0143] In some embodiments, the linker can comprise a polymer linker, such as an amino acid linker, biotin linker, and the like. In some embodiments, the linker can be cleavable. In some embodiments, the linker cannot be cleavable.Microfluidic devices for cell sorting

[0144] In some embodiments, methods described herein can comprise separating from a peripheral blood sample a population of TRLs. In some embodiments, a microfluidic device disclosed herein can be used to separate the population of TRLs (e.g., CD103+ lymphocytes) from the peripheral blood sample. In some embodiments, isolated TRLs may comprise CD 103+ lymphocytes. In some embodiments, the isolated CD 103+ lymphocytes may be CD8+CD103+ lymphocytes, CD3+CD103+ lymphocytes, CD4+CD103+ lymphocytes, or CD39+CD103+ lymphocytes. In some embodiments, the isolated CD 103+ lymphocytes may be CD8+CD39+CD103+ lymphocytes, CD3+ CD39+CD103+ lymphocytes, or CD4+ CD39+CD103+ lymphocytes. In some embodiments, the isolated CD103+ lymphocytes may comprise CD8+CD103+ SLC6A19+ lymphocytes, CD3+CD103+ SLC6A19+ lymphocytes, or CD4+CD103+ SLC6A19+ lymphocytes. In some embodiments, the isolated CD 103+ lymphocytes may comprise CD8+CD103+ SLC6A19+SIDT1+ lymphocytes, CD3+CD103+SLC6A19+SIDT1+ lymphocytes, or CD4+CD103+SLC6A19+SIDT1+ lymphocytes.

[0145] In some embodiments, any of the microfluidic devices disclosed in PCT Publication No. WO 2014 / 166000, the contents of which can be incorporated by reference herein, can be used to separate the TRLs from the peripheral blood sample. In some embodiments, once the TRLshave been separated, the TRLs may be eluted from the microfluidic device by removing an attractant acting on the TRLs (e.g., via removal of a magnetic field). In some embodiments, the captured TRLs can then be expanded, enhanced, or a combination thereof, by any of the methods disclosed herein.

[0146] Microfluidic devices disclosed herein can be configured to magnetically sort a population of cells. In some embodiments, the population of cells may comprise a population of TRLs. In some embodiments, the population of TRLs may be labeled with magnetic nanoparticles. In some embodiments, each of the magnetic nanoparticles can be about 0-50 nm in diameter, 51- 100 nm in diameter, 100-150 nm in diameter, or 150-200 nm in diameter. In some embodiments, each of the magnetic nanoparticles can be about 50 nm in diameter. In some embodiments, compared with conventional microbeads, magnetic nanoparticles can have improved colloidal stability, which may be useful for processing larger samples. In some embodiments, cells labeled with magnetic nanoparticles may be difficult to capture because their orders-of-magnitude lower magnetic susceptibilities, compared to microbeads, result in lower capture efficiencies. Therefore, in some embodiments, the microfluidic devices disclosed herein can include flow rate-reducing structures that give rise to localized regions of lower flow rate, as a sample comprising the cells can be flowed through the device. In some embodiments, the presence of such low flow velocity regions can enable capture of the magnetically labeled cells.

[0147] In some embodiments, a microfluidic device disclosed herein can comprise a microfluidic chip. In some embodiments, the microfluidic chip can comprise a sorting chamber. In some embodiments, the sorting chamber can be etched or molded into the chip. In some embodiments, the sorting chamber can be in communication with a flow inlet and a flow outlet. In some embodiments, the flow inlet can be configured to receive a sample, e.g., a peripheral blood sample comprising a population of TRLs suspended in a fluid medium, and the outlet can be configured for delivering the fluid medium depleted of said TRLs. In some embodiments, tubing can be connected to the inlet such that the fluid medium can be delivered into the inlet through the tubing. In some embodiments, tubing can be connected to the outlet such that the fluid medium can be received from the outlet through the tubing. In some embodiments, the tubing can be silicone tubing. In some embodiments, the microfluidic device can comprise a syringe pump capable of controlling the flow rate of fluid medium at the inlet.

[0148] In some embodiments, the sorting chamber can comprise at least one magnetic capture zone. In some embodiments, the sorting chamber can comprise a plurality of magnetic capturezones. In some embodiments, the sorting chamber can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 magnetic capture zones. In some embodiments, the sorting chamber can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 magnetic capture zones. In some embodiments, at least two of the magnetic capture zones can vary in height. In some embodiments, the magnetic capture zones can range from 50-800 pm in height. In some embodiments, sorting chamber can comprise three magnetic capture zones, one about 100 pm in height, one about 200 pm in height, and one about 400 pm in height.

[0149] In some embodiments, the microfluidic device can comprise at least one array of magnets positioned on the outer surface of the microfluidic chip, disposed above or below the sorting chamber, such that a magnetic field can be created in the magnetic capture zones by the at least one array of magnets. In some embodiments, the microfluidic device can comprise two arrays of magnetics positioned on two outer surfaces of the microfluidic chip above and below the sorting chamber such that a magnetic field can be created in the magnetic capture zones by the two arrays of magnets. In some embodiments, magnets can be positioned in two arrays, with alternating polarities on opposing sides of the sorting chamber. In some embodiments, the at least one array of magnets can produce a magnetic field strength between .1-.5 T, .5-1 T, or 1-1 5T in the magnetic capture zones. In some embodiments, the at least one array of magnets can produce a magnetic field strength between .5-1 T in the magnetic capture zones. In some embodiments, the magnets can comprise neodymium magnets. In some embodiments, the magnets can comprise N52 Nd FeB magnets.

[0150] In some embodiments, a magnetic capture zone can comprise a plurality of microstructures. In some embodiments, the microstructures can be flow rate-reducing structures configured to improve capture cells labeled with magnetic nanoparticles in the flow. In some embodiments, the microstructures can produce localized regions of lower flow rate, which may allow for capture of the particles (e.g., the reduced flow rate may allow the magnetic force to overcome the drag force on the particles). In some embodiments, the structures can be designed to avoid trapping of non -target particles. For example, despite being lower in flow rate, the regions of lower flow rate may still have enough flow velocity (that is, the flow rate may be at least nonzero) for non-target particles to be washed from the device, while target particles may be trapped in the low flow rate region. In some embodiments, the microstructures can be X-shaped.

[0151] In some embodiments, the device can comprise a plurality of magnetic capture zones, with a first zone comprising the inlet of the sorting chamber, a final magnetic capture zonecomprising the outlet of the sorting chamber, and a plurality of magnetic capture zones disposed between the first magnetic capture zone and the final magnetic capture zone. In some embodiments, the size or pattern of the microstructures can vary among the capture zones. In some embodiments, the heights can vary among the magnetic capture zones. In some embodiments, the first zone can exhibit the highest linear velocity and thus can retain cells with high magnetic content because the retaining magnetic force can overcome the drag force exerted by the locally high flow velocity. In some embodiments, the other magnetic capture zones can exhibit gradually reduced linear velocities, with the final magnetic capture zone exhibiting the lowest velocity. This design can allow cells with high levels of magnetization to be captured in the first zone of the device, whereas cells with lower magnetization can become sorted in later zones according to level of magnetization.

[0152] In some methods disclosed herein, a population of TRLs suspended in fluid can be propelled through the inlet, across the magnetic capture zones and through the outlet. In some embodiments, magnetically labelled cells can be captured in the capture zones if the magnetic force exerted on the cells can be sufficient to overcome the drag force compelling the cell to flow through the capture zone. Otherwise, a cell would be flush into the next zone with a lower drag force, and eventually into the syringe if it cannot be captured by any zone. After sorting, captured cells at each zone can be recovered by removing the external magnets.

[0153] In some methods disclosed herein, a population of cells can be loaded into the microfluidic device, through the inlet, at a flow rate of at least 1 milliliters per hour, 3 mL / h, 6 mL / h, 9 mL / h, 12 mL / h, 15 mL / h, 18 mL / h, 21 mL / h, 24 mL / h, 27 mL / h, 30 mL / h, 35 mL / h, 40 mL / h, 45 mL / h, or 50 mL / h. The magnetic force exerted on the cells can be determined by, for example, the size of the magnetic nanoparticle, the number of magnetic nanoparticles attached to the cell, the size of the cell, and the strength of the applied magnetic field. In some embodiments, the relationship between drag force and linear flow velocity in a microfluidic device capable of magnetically capturing particles by leveraging rate reducing microstructures can be disclosed in PCT Publication No. WO 2014 / 166000, the contents of which can be incorporated by reference herein.

[0154] In some embodiments, the microfluidic approaches to cell sorting disclosed herein can yield a population of CD39, CD103, SLC6A19, and / or SIDT1 expressing lymphocytes about 2 fold to about 20 fold higher than a population of CD39, CD103, SLC6A19, and / or SIDT1 expressing lymphocytes separated from a peripheral blood sample from the subject usingfluorescence activated cell sorting (FACS). In some embodiments, the microfluidic approaches to cell sorting described herein can yield a population of CD39, CD103, SLC6A19, and / or SIDT1 expressing lymphocytes about 2 fold to about 4-fold, about 2 fold to about 7-fold, about 2 fold to about 8-fold, about 2 fold to about 10-fold, about 2 fold to about 12-fold, about 2 fold to about 14-fold, about 2 fold to about 16-fold, about 2 fold to about 17-fold, about 2 fold to about 18-fold, about 2 fold to about 19-fold, about 2 fold to about 20-fold, about 4 fold to about 7-fold, about 4 fold to about 8-fold, about 4 fold to about 10-fold, about 4 fold to about 12-fold, about 4 fold to about 14-fold, about 4 fold to about 16-fold, about 4 fold to about 17-fold, about 4 fold to about 18-fold, about 4 fold to about 19-fold, about 4 fold to about 20-fold, about 7 fold to about 8-fold, about 7 fold to about 10-fold, about 7 fold to about 12-fold, about 7 fold to about 14-fold, about 7 fold to about 16-fold, about 7 fold to about 17-fold, about 7 fold to about 18-fold, about 7 fold to about 19-fold, about 7 fold to about 20-fold, about 8 fold to about 10-fold, about 8 fold to about 12-fold, about 8 fold to about 14-fold, about 8 fold to about 16-fold, about 8 fold to about 17-fold, about 8 fold to about 18-fold, about 8 fold to about 19-fold, about 8 fold to about 20-fold, about 10 fold to about 12-fold, about 10 fold to about 14-fold, about 10 fold to about 16-fold, about 10 fold to about 17-fold, about 10 fold to about 18-fold, about 10 fold to about 19-fold, about 10 fold to about 20-fold, about 12 fold to about 14-fold, about 12 fold to about 16-fold, about 12 fold to about 17-fold, about 12 fold to about 18-fold, about 12 fold to about 19-fold, about 12 fold to about 20-fold, about 14 fold to about 16-fold, about 14 fold to about 17-fold, about 14 fold to about 18-fold, about 14 fold to about 19-fold, about 14 fold to about 20-fold, about 16 fold to about 17-fold, about 16 fold to about 18-fold, about 16 fold to about 19-fold, about 16 fold to about 20-fold, about 17 fold to about 18-fold, about 17 fold to about 19-fold, about 17 fold to about 20-fold, about 18 fold to about 19-fold, about 18 fold to about 20-fold, or about 19 fold to about 20 fold higher than a population of CD39, CD103, SLC6A19, and / or SIDT1 expressing lymphocytes separated from a peripheral blood sample from the subject using fluorescence activated cell sorting. In some embodiments, the microfluidic approaches to cell sorting described herein can yield a population of CD39, CD103, SLC6A19, and / or SIDT1 expressing lymphocytes about 2-fold, about 4-fold, about 7-fold, about 8-fold, about 10-fold, about 12-fold, about 14-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20 fold higher than a population of CD39, CD103, SLC6A19, and / or SIDT1 expressing lymphocytes separated from a peripheral blood sample from the subject using fluorescence activated cell sorting. In some embodiments, the microfluidic approaches to cell sorting described herein can yield a population of CD39, CD103, SLC6A19, and / or SIDT1 expressing lymphocytes at least about 2-fold, about 4-fold, about 7-fold, about 8-fold, about 10-fold, about 12-fold, about 14-fold, about 16-fold,about 17-fold, about 18-fold, or about 19 fold higher than a population of CD39, CD103, SLC6A19, and / or SIDT1 expressing lymphocytes separated from a peripheral blood sample from the subject using fluorescence activated cell sorting. In some embodiments, the microfluidic approaches to cell sorting described herein can yield a population of CD39, CD103, SLC6A19, and / or SIDT1 expressing lymphocytes at most about 4-fold, about 7-fold, about 8-fold, about 10- fold, about 12-fold, about 14-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20 fold higher than a population of CD39, CD103, SLC6A19, and / or SIDT1 expressing lymphocytes separated from a peripheral blood sample from the subject using fluorescence activated cell sorting.

[0155] In some embodiments, the microfluidic approaches to cell-sorting disclosed herein can achieve a percent recovery of about 40% to about 99% of the CD39, CD103, SLC6A19, or SIDT1 lymphocytes. In some embodiments, the microfluidic approaches to cell-sorting disclosed herein can achieve a percent recovery of about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 95%, about 40% to about 96%, about 40% to about 97%, about 40% to about 98%, about 40% to about 99%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 50% to about 96%, about 50% to about 97%, about 50% to about 98%, about 50% to about 99%, about 60% to about 70%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 96%, about 60% to about 97%, about 60% to about 98%, about 60% to about 99%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 96%, about 70% to about 97%, about 70% to about 98%, about 70% to about 99%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 96%, about 80% to about 97%, about 80% to about 98%, about 80% to about 99%, about 85% to about 90%, about 85% to about 95%, about 85% to about 96%, about 85% to about 97%, about 85% to about 98%, about 85% to about 99%, about 90% to about 95%, about 90% to about 96%, about 90% to about 97%, about 90% to about 98%, about 90% to about 99%, about 95% to about 96%, about 95% to about 97%, about 95% to about 98%, about 95% to about 99%, about 96% to about 97%, about 96% to about 98%, about 96% to about 99%, about 97% to about 98%, about 97% to about 99%, or about 98% to about 99% of the 103+ lymphocytes. In some embodiments, the microfluidic approaches to cell-sorting disclosed herein can achieve a percent recovery of about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of theCD39, CD 103, SLC6A19, or SIDT1 lymphocytes. In some embodiments, the microfluidic approaches to cell-sorting disclosed herein can achieve a percent recovery at least about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, or about 98% of the CD39, CD 103, SLC6A19, or SIDT1 lymphocytes. In some embodiments, the microfluidic approaches to cell-sorting disclosed herein can achieve a percent recovery at most about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the CD39, CD103, SLC6A19, or SIDT1 lymphocytes.

[0156] In some embodiments, the purity of isolated cells using methods described herein can be about 10% to about 99% or essentially pure (e.g., 100%). In some embodiments, the purity of isolated cells using methods of described herein can be about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 99%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 60%, about 15% to about 70%, about 15% to about 80%, about 15% to about 90%, about 15% to about 99%, about 20% to about 25%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 99%, about 25% to about 30%, about 25% to about 40%, about 25% to about 50%, about 25% to about 60%, about 25% to about 70%, about 25% to about 80%, about 25% to about 90%, about 25% to about 99%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 99%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 99%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 99%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 99%, about 70% to about 80%, about 70% to about 90%, about 70% to about 99%, about 80% to about 90%, about 80% to about 99%, or about 90% to about 99%. In some embodiments, the purity of isolated cells using methods of described herein can be about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99%. In some embodiments, the purity of isolated cells using methods of described herein can be at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about90%. In some embodiments, the purity of isolated cells using methods of described herein can be at most about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99%.B. Culturing

[0157] Disclosed in some embodiments herein are methods of enriching and expanding a population of TRLs (e.g., cTRLs). In some embodiments, the methods can comprise magnetically separating a population of TRLs described using any of the methods disclosed herein and culturing the magnetically separated population of TRLs.

[0158] In some embodiments, an initial cell population comprising about 1,000 to about 20,000 magnetically separated CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes can be seed in the culture system described herein. In some embodiments, an initial cell population comprising about 1,000 to about 2,000, about 1,000 to about 3,000, about 1,000 to about 4,000, about 1,000 to about 5,000, about 1,000 to about 6,000, about 1,000 to about 7,000, about 1,000 to about 8,000, about 1,000 to about 9,000, about 1,000 to about 10,000, about 1,000 to about 15,000, about 1,000 to about 20,000, about 2,000 to about 3,000, about 2,000 to about 4,000, about 2,000 to about 5,000, about 2,000 to about 6,000, about 2,000 to about 7,000, about 2,000 to about 8,000, about 2,000 to about 9,000, about 2,000 to about 10,000, about 2,000 to about 15,000, about 2,000 to about 20,000, about 3,000 to about 4,000, about 3,000 to about 5,000, about 3,000 to about 6,000, about 3,000 to about 7,000, about 3,000 to about 8,000, about 3,000 to about 9,000, about 3,000 to about 10,000, about 3,000 to about 15,000, about 3,000 to about 20,000, about 4,000 to about 5,000, about 4,000 to about 6,000, about 4,000 to about 7,000, about 4,000 to about 8,000, about 4,000 to about 9,000, about 4,000 to about 10,000, about 4,000 to about 15,000, about 4,000 to about 20,000, about 5,000 to about 6,000, about 5,000 to about 7,000, about 5,000 to about 8,000, about 5,000 to about 9,000, about 5,000 to about 10,000, about 5,000 to about 15,000, about 5,000 to about 20,000, about 6,000 to about 7,000, about 6,000 to about 8,000, about 6,000 to about 9,000, about 6,000 to about 10,000, about 6,000 to about 15,000, about 6,000 to about 20,000, about 7,000 to about 8,000, about 7,000 to about 9,000, about 7,000 to about 10,000, about 7,000 to about 15,000, about 7,000 to about 20,000, about 8,000 to about 9,000, about 8,000 to about 10,000, about 8,000 to about 15,000, about 8,000 to about 20,000, about 9,000 to about 10,000, about 9,000 to about 15,000, about 9,000 to about 20,000, about 10,000 to about 15,000, about 10,000 to about 20,000, or about 15,000 to about 20,000 magnetically separated CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes can be seed in the culture systemdescribed herein. In some embodiments, an initial cell population comprising about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 15,000, or about 20,000 magnetically separated CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes can be seed in the culture system described herein. In some embodiments, an initial cell population comprising at least about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, or about 15,000 magnetically separated CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes can be seed in the culture system described herein. In some embodiments, an initial cell population comprising at most about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 15,000, or about 20,000 magnetically separated CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes can be seed in the culture system described herein.

[0159] In some embodiments, culturing the magnetically separated CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes can comprise growing the CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes in a cell culture to expand the population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes. In some embodiments, the population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes can be expanded at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14- fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or at least 20-fold. In some embodiments, the population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes can be expanded at least 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold, 1700-fold, 1800- fold, 1900-fold, 2000-fold, 2100-fold, 2200-fold, 2300-fold, 2400-fold, 2500-fold, 2600-fold, 2700-fold, 2800-fold, 2900-fold, 3000-fold, 3100-fold, 3200-fold, 3300-fold, 3400-fold, 3500- fold, 3600-fold, 3700-fold, 3800-fold, 3900-fold, 4000-fold, 4100-fold, 4200-fold, 4300-fold, 4400-fold, 4500-fold, 4600-fold, 4700-fold, 4800-fold, 4900-fold, or 5000-fold.

[0160] In some embodiments, the rapid expansion protocol disclosed in Dudley, M. E., Wunderlich, J. R., Shelton, T. E., Even, J. & Rosenberg, S. A., Generation of Tumor -Infiltrating Lymphocyte Cultures for Use in Adoptive Transfer Therapy for Melanoma Patients'. J. Immunother. 26, 332-342 (2003), which can be incorporated by reference in its entirety herein, can be used to culture the magnetically separated lymphocytes. In some embodiments, the culturing can be achieved in a culture flask or other container known in the art using feeder cells, T-cell growth factors, and monoclonal antibodies capable of inducing T-cell activation. In someembodiments, the cells can be co-cultured with feeder cells. In some embodiments, the cells can be cultured without feeder cells. In some embodiments, the cells can be cultured with one or more T-cell growth factors. In some embodiments, the cells can be cultured without one or more T-cell growth factors. In some embodiments, the cells can be cultured with one or more monoclonal antibodies capable of inducing T-cell activation. In some embodiments, the cells can be cultured without one or more monoclonal antibodies capable of inducing T-cell activation. In some embodiments, the culturing can be performed using a culture flask or container known to those of skill in the art. In some embodiments, culturing can be performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11, 12, 13, or 14 days (e.g., a predetermined expansion period).

[0161] In some embodiments, culturing can be performed for about 1 day to about 20 days. In some embodiments, culturing can be performed for about 1 day to about 2 days, about 1 day to about 3 days, about 1 day to about 4 days, about 1 day to about 5 days, about 1 day to about 6 days, about 1 day to about 7 days, about 1 day to about 8 days, about 1 day to about 9 days, aboutI day to about 10 days, about 1 day to about 11 days, about 1 day to about 12 days, about 2 days to about 3 days, about 2 days to about 4 days, about 2 days to about 5 days, about 2 days to about 6 days, about 2 days to about 7 days, about 2 days to about 8 days, about 2 days to about 9 days, about 2 days to about 10 days, about 2 days to about 11 days, about 2 days to about 12 days, about3 days to about 4 days, about 3 days to about 5 days, about 3 days to about 6 days, about 3 days to about 7 days, about 3 days to about 8 days, about 3 days to about 9 days, about 3 days to about 10 days, about 3 days to about 11 days, about 3 days to about 12 days, about 4 days to about 5 days, about 4 days to about 6 days, about 4 days to about 7 days, about 4 days to about 8 days, about 4 days to about 9 days, about 4 days to about 10 days, about 4 days to about 11 days, about4 days to about 12 days, about 5 days to about 6 days, about 5 days to about 7 days, about 5 days to about 8 days, about 5 days to about 9 days, about 5 days to about 10 days, about 5 days to aboutI I days, about 5 days to about 12 days, about 6 days to about 7 days, about 6 days to about 8 days, about 6 days to about 9 days, about 6 days to about 10 days, about 6 days to about 11 days, about 6 days to about 12 days, about 7 days to about 8 days, about 7 days to about 9 days, about 7 days to about 10 days, about 7 days to about 11 days, about 7 days to about 12 days, about 8 days to about 9 days, about 8 days to about 10 days, about 8 days to about 11 days, about 8 days to about 12 days, about 9 days to about 10 days, about 9 days to about 11 days, about 9 days to about 12 days, about 10 days to about 11 days, about 10 days to about 12 days, or about 11 days to about 12 days. In some embodiments, culturing can be performed for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, or about 20 days. In some embodiments, culturing can be performed for at least about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, or about 19 days. In some embodiments, culturing can be performed for at most about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, or about 20 days.

[0162] In some embodiments, the culture can be grown in the presence of a T-cell growth factor. In some embodiments, the culture medium can comprise a T-cell growth factor. In some embodiments, the T-cell growth factor can include IL-2, IL-7, IL-9 or IL-15. In some embodiments, the T-cell growth factor can comprise IL-2. In some embodiments, the culture medium can comprise at least about 6,000 HJ / mL of IL-2.

[0163] In some embodiments, the culture medium can comprise no IL-2. In some embodiments, the culture medium can comprise at least about 0-5 HJ / mL, 5-10 HJ / mL, 10-15 lU / mL, 15-20 lU / mL 20-25 lU / mL, 25-30 lU / mL, 30-35 lU / mL, 35-40 lU / mL 40-45 lU / mL, 45- 50 lU / mL, 50-500 lU / mL, 500-1000 lU / mL, 1000-1500 lU / mL, about 1500-2000 lU / mL, about 2000-2500 lU / mL, about 2500-3000 lU / mL, about 3000-3500 lU / mL, about 3500-4000 lU / mL, about 4000-4500 lU / mL, about 4500-5000 lU / mL, about 5000-5500 lU / mL, about 5500-6000 lU / mL, about 6000-6500 lU / mL, about 6500-7000 lU / mL, about 7000-7500 lU / mL, about 7500- 8000 lU / mL or about 8000-8500 lU / mL IL-2. In some embodiments, the culture medium can comprise at most about 0.5 lU / mL, about 1 lU / mL, 1.5 lU / mL, 2 lU / mL, 3 lU / mL, 4 lU / mL, 5 lU / mL, 5-10 lU / mL, 10-15 lU / mL, 15-20 lU / mL 20-25 lU / mL, 25-30 lU / mL, 30-35 lU / mL, 35- 40 lU / mL 40-45 lU / mL, 45-50 lU / mL, 50-500 lU / mL, 500-1000 lU / mL, 1000-1500 lU / mL, about 1500-2000 lU / mL, about 2000-2500 lU / mL, about 2500-3000 lU / mL, about 3000-3500 lU / mL, about 3500-4000 lU / mL, about 4000-4500 lU / mL, about 4500-5000 lU / mL, about 5000-5500 lU / mL, about 5500-6000 lU / mL, about 6000-6500 lU / mL, about 6500-7000 lU / mL, about 7000- 7500 lU / mL, about 7500-8000 lU / mL or about 8000-8500 lU / mL IL-2.

[0164] In some embodiments, the culture medium can comprise at least about 0-5 ng / mL, 5- 10 ng / mL, 10-15 ng / mL, 15-20 ng / mL 20-25 ng / mL, 25-30 ng / mL, 30-35 ng / mL, 35-40 ng / mL 40-45 ng / mL, 45-50 ng / mL, 50-500 ng / mL, 500-1000 ng / mL, 1000-1500 ng / mL, about 1500- 2000 ng / mL, about 2000-2500 ng / mL, about 2500-3000 ng / mL, about 3000-3500 ng / mL, about3500-4000 ng / mL, about 4000-4500 ng / mL, about 4500-5000 ng / mL, about 5000-5500 ng / mL, about 5500-6000 ng / mL, about 6000-6500 ng / mL, about 6500-7000 ng / mL, about 7000-7500 ng / mL, about 7500-8000 ng / mL or about 8000-8500 ng / mL IL-7.

[0165] In some embodiments, the culture medium can comprise at least about 0-5 ng / mL, 5- 10 ng / mL, 10-15 ng / mL, 15-20 ng / mL 20-25 ng / mL, 25-30 ng / mL, 30-35 ng / mL, 35-40 ng / mL 40-45 ng / mL, 45-50 ng / mL, 50-500 ng / mL, 500-1000 ng / mL, 1000-1500 ng / mL, about 1500- 2000 ng / mL, about 2000-2500 ng / mL, about 2500-3000 ng / mL, about 3000-3500 ng / mL, about 3500-4000 ng / mL, about 4000-4500 ng / mL, about 4500-5000 ng / mL, about 5000-5500 ng / mL, about 5500-6000 ng / mL, about 6000-6500 ng / mL, about 6500-7000 ng / mL, about 7000-7500 ng / mL, about 7500-8000 ng / mL or about 8000-8500 ng / mL IL-15.

[0166] In some embodiments, TRLs described herein can be grown in a culture medium that lacks a T-cell growth factor. In some embodiments, a culture medium that lacks a T-cell growth factor can comprise less than a trace amount of the T-cell growth factor. As used herein, the term “less than a trace amount” can comprise an amount that is insufficient to produce a biological effect, that cannot be detected by standard detection methods, or a combination thereof. In some embodiments, standard detection methods can comprise an immunoassay, an Alamar Blue assay, an enzyme-linked immunosorbent assay (ELISA), an enzyme-linked immunosorbent spot (ELISpot), or a combination thereof. In some embodiments, “less than a trace amount” can comprise substantially none of a substance being present. In some embodiments, a T-cell growth factor can comprise a cytokine. In some embodiments, a T-cell growth factor can comprise IL-2, IL-7, IL-9, IL- 15, or a combination thereof. In some embodiments, a culture medium that lacks one or more T-cell growth factors can comprise a culture medium that lacks IL-2, IL-7, IL-9, or IL-15. In some embodiments, a culture medium can lack IL-2.

[0167] In some embodiments, a culture medium that lacks IL-2 can comprise less than a trace amount of IL-2. In some embodiments, a culture medium that comprises less than a trace amount of IL-2 can comprise an amount of IL-2 that is insufficient to trigger IL-2 mediated cell signaling in a TRL. In some embodiments, an amount of IL-2 that is insufficient to trigger IL-2 mediated cell signaling can comprise an amount of IL-2 that is lower than a threshold necessary to trigger IL-2 mediated cell signaling in a TRL. In some embodiments, a culture medium that lacks IL-2 can comprise IL-2, but less than a biologically reactive amount (e.g., measured by T-cell proliferation, apoptosis, or activation level) or sufficient amount to trigger IL-2 signaling in TRLs (e.g., measured by phosphorylation level of one or more downstream protein STAT5). In some embodiments, a biologically reactive amount of IL-2 can comprise a sufficient amount to trigger IL-2 receptor signaling, as evidenced by an increase in the phosphorylation level (e.g., at leastabout 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, or more) of one or more downstream signaling proteins, e.g., phosphorylated STAT5 (pSTAT5), relative to the phosphorylation level of T cells cultured in culture media lacking IL-2 or relative to an unstimulated control. The phosphorylation may be measured by flow cytometry, western blotting, or other suitable methods. In some embodiments, a biologically reactive amount of IL-2 can induce at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2.0-fold or greater increase in phosphorylation of the downstream proteins (e.g., STAT5) within 15 minutes to 4 hours of exposure.

[0168] In some embodiments, the isolated TRLs can be cultured in a culture medium, wherein the culture medium is free of one or more exogenous T-cell growth factors (e.g., IL-2, IL-7, IL-9, or IL- 15). In some embodiments, the culture medium can be free of exogenous IL-2. In some embodiments, the culture medium can be free of exogenous IL-7. In some embodiments, the culture medium can be free of exogenous IL-9. In some embodiments, the culture medium can be free of exogenous IL-15. In some embodiments, the culture medium can be free of at least two exogenous T-cell growth factors. For example, the culture medium can be free of exogenous IL- 2 and IL-7, IL-2 and IL-9, or IL-2 and IL-15. In some embodiments, the culture medium can be free of exogenous IL-7 and IL-9, or IL-7 and IL-15. In some embodiments, the culture medium can be free of exogenous IL-9 and IL-15. In some embodiments, the culture medium can be free of at least three exogenous T-cell growth factors. For example, the culture medium can be free of exogenous IL-2, IL-7, and IL-9. In some embodiments, the culture medium can be free of exogenous IL-2, IL-7, and IL-15. In some embodiments, the culture medium can be free of exogenous IL-2, IL-9, and IL-15. In some embodiments, the culture medium can be free of exogenous IL-7, IL-9, and IL-15. In some embodiments, the culture medium can be free of at least four exogenous T-cell growth factors. For example, the culture medium can be free of exogenous IL-2, IL-7, IL-9, and IL-15. In some embodiments, the TRLs cultured (e.g., over a predetermined expansion period) in the absence of one or more T-cell growth factors (e.g, IL-2, IL-7, IL-9, and IL- 15) can exhibit enhanced proliferation compared to the TRLs cultured in the presence of one or more T-cell growth factors. In some embodiments, the TRLs cultured in the absence of exogenous IL-2 can exhibit enhanced proliferation compared to the TRLs cultured in the presence of exogenous IL-2. In some embodiments, proliferation of the TRLs cultured in the absence of exogenous IL-2 can be enhanced by at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 500%, at least about 600%, at least about 700%, at least about 800%, at least about 900%, at least about 1000%, at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11-fold, at least about 12-fold, at least about 13 -fold, at least about 14, at least about 15 -fold, at least about 20-fold, at least about 30- fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 250-fold, at least about 300-fold, at least about 350-fold, at least about 400-fold, at least about 500-fold, at least about 600-fold, at least about 700-fold, at least about 800-fold, at least about 900-fold, at least about 1000-fold, at least about 1500-fold, at least about 2000-fold, or at least about 3000 fold greater than the TRLs cultured in the presence of exogenous IL-2.

[0169] In some embodiments, proliferation of the TRLs cultured in the absence of exogenous IL-2 can be enhanced by at most about 1%, at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, at most about 90%, at most about 100%, at most about 110%, at most about 120%, at most about 130%, at most about 140%, at most about 150%, at most about 160%, at most about 170%, at most about 180%, at most about 190%, at most about 200%, at most about 250%, at most about 300%, at most about 350%, at most about 400%, at most about 500%, at most about 600%, at most about 700%, at most about 800%, at most about 900%, at most about 1000%, at most about 1.1-fold, at most about 1.2-fold, at most about 1.3- fold, at most about 1.4-fold, at most about 1.5-fold, at most about 1.6-fold, at most about 1.7-fold, at most about 1.8-fold, at most about 1.9-fold, at most about 2-fold, at most about 3-fold, at most about 4-fold, at most about 5-fold, at most about 6-fold, at most about 7-fold, at most about 8- fold, at most about 9-fold, at most about 10-fold, at most about 11 -fold, at most about 12-fold, at most about 13 -fold, at most about 14, at most about 15 -fold, at most about 20-fold, at most about 30-fold, at most about 40-fold, at most about 50-fold, at most about 60-fold, at most about 70- fold, at most about 80-fold, at most about 90-fold, at most about 100-fold, at most about 150-fold,at most about 200-fold, at most about 250-fold, at most about 300-fold, at most about 350-fold, at most about 400-fold, at most about 500-fold, at most about 600-fold, at most about 700-fold, at most about 800-fold, at most about 900-fold, at most about 1000-fold, at most about 1500-fold, at most about 2000-fold, or at most about 3000 fold greater than the TRLs cultured in the presence of exogenous IL-2.

[0170] In some embodiments, proliferation of the TRLs cultured in the absence of exogenous IL-2 can be enhanced by about 1%, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 250%, about 300%, about 350%, about 400%, about 500%, about 600%, about 700%, about 800%, about 900%, about 1000%, about 1.1 -fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9- fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8- fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14, about 15- fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 500-fold, about 600-fold, about 700-fold, about 800-fold, about 900-fold, about 1000-fold, about 1500-fold, about 2000-fold, or about 3000 fold greater than the TRLs cultured in the presence of exogenous IL-2.

[0171] In some embodiments, the isolated TRLs can be cultured in a culture medium comprising one or more exogenous T-cell growth factors (e.g., IL-2, IL-7, IL-9, or IL-15). In some embodiments, there is a positive correlation between the concentration of an exogenous T-cell growth factor (e.g., the concentration of IL-2, IL-7, IL-9, or IL-15), and the proliferation of the TRLs during expansion.

[0172] In some embodiments, the TRLs expanded in the absence of one or more exogenous T-cell growth factors (e.g., IL-2, IL-7, IL-9, and IL-15) can exhibit greater purity compared to pre-expansion TRLs or TRLs expanded in the presence of one or more T-cell growth factors, leading to a more uniform cell population. For example, the absence of exogenous IL-2 in the expansion medium can selectively enhance the expansion of the desired cell populations (e.g., CD8+ CD103+ cells) while minimizing the expansion of undesired cells (e.g., NK cells, CD3- cells). In some embodiments, the purity of TRLs (e.g., the percentage of the population of TRLs of a specific subtype) expanded by the methods and systems described herein can be at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at leastabout 90% greater than the pre-expansion TRLs. In some embodiments, the purity of TRLs expanded by the methods and systems described herein can be at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, or at most about 90% greater than the pre-expansion TRLs. In some embodiments, the purity of TRLs expanded by the methods and systems described herein can be about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% greater than the preexpansion TRLs.

[0173] In some embodiments, the purity of TRLs expanded in the absence of IL-2 can be at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 0.2-fold, at least about 0.4-fold, at least about 0.6-fold, at least about 0.8-fold, at least about 1.0-fold, at least about 1.1-fold, at least about 1.2- fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11 -fold, at least about 12-fold, at least about 13-fold, at least about 14, at least about 15-fold, or at least about 20 fold greater than that of the TRLs expanded in the presence of IL-2. In some embodiments, the purity of TRLs expanded in the absence of IL-2 can be at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, at most about 90%, at most about 0.2-fold, at most about 0.4-fold, at most about 0.6-fold, at most about 0.8-fold, at most about 1.0-fold, at most about 1.1-fold, at most about 1.2-fold, at most about 1.3-fold, at most about 1.4-fold, at most about 1.5-fold, at most about 1.6-fold, at most about 1.7-fold, at most about 1.8-fold, at most about 1.9-fold, at most about 2-fold, at most about 3-fold, at most about 4-fold, at most about 5-fold, at most about 6-fold, at most about 7-fold, at most about 8-fold, at most about 9-fold, at most about 10-fold, at most about 11 -fold, at most about 12-fold, at most about 13 -fold, at most about 14, at most about 15 -fold, or at most about 20 fold greater than that of the TRLs expanded in the presence of IL-2. In some embodiments, the purity of TRLs expanded in the absence of IL-2 can be about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 0.2-fold, about 0.4- fold, about 0.6-fold, about 0.8-fold, about 1.0-fold, about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9- fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14, about 15-fold, or about 20 fold greater than that of the TRLs expanded in the presence of IL-2.

[0174] In some embodiments, the manufacturing time of TRLs expanded in the absence of one or more T-cell growth factors (e.g., IL-2, IL-7, IL-9, and IL-15) can shorter than the manufacturing time of TRLs expanded in the presence of the growth factor. For example, TRLs expanded in the absence of IL-2 can have greater purity or better expansion (e.g., increased proliferation) than TRLs expanded in the presence of IL-2, reducing manufacturing time. In some embodiments, the manufacturing time of TRLs expanded in the absence of IL-2 can be at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 0.2-fold, at least about 0.4-fold, at least about 0.6- fold, at least about 0.8-fold, at least about 1.0-fold, at least about 1.1-fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 11 -fold, at least about 12-fold, at least about 13-fold, at least about 14, at least about 15-fold, or at least about 20 fold less than that of the TRLs expanded in the presence of IL-2. In some embodiments, the manufacturing time of TRLs expanded in the absence of IL-2 can be at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 25%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, at most about 90%, at most about 0.2-fold, at most about 0.4-fold, at most about 0.6-fold, at most about 0.8-fold, at most about 1.0-fold, at most about 1.1-fold, at most about 1.2-fold, at most about 1.3-fold, at most about 1.4-fold, at most about 1.5-fold, at most about 1.6-fold, at most about 1.7- fold, at most about 1.8-fold, at most about 1.9-fold, at most about 2-fold, at most about 3-fold, at most about 4-fold, at most about 5-fold, at most about 6-fold, at most about 7-fold, at most about 8-fold, at most about 9-fold, at most about 10-fold, at most about 11 -fold, at most about 12-fold, at most about 13-fold, at most about 14, at most about 15-fold, or at most about 20 fold less than that of the TRLs expanded in the presence of IL-2. In some embodiments, the manufacturing time of TRLs expanded in the absence of IL-2 can be about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 0.2-fold, about 0.4-fold, about 0.6-fold, about 0.8-fold, about 1.0-fold, about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7- fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14, about 15-fold, or about 20 fold less than that of the TRLs expanded in the presence of IL-2.

[0175] For example, the TRLs cultured in the absence of exogenous IL-2 can have at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% greater CD8+CD103+T cells population compared to pre-expansion TRLs or TRLs cultured in the presence of exogenous IL-2. In some embodiments, the TRLs cultured in the absence of exogenous IL-2 can have at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, at most about 90%, at most about 100%, at most about 150%, at most about 200%, at most about 300%, at most about 400%, or at most about 500% greater CD8+CD103+T cells population compared to pre-expansion TRLs or TRLs cultured in the presence of exogenous IL-2. In some embodiments, the TRLs cultured in the absence of exogenous IL-2 can have about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%, about 400%, or about 500% greater CD8+CD103+T cells population compared to pre-expansion TRLs or TRLs cultured in the presence of exogenous IL-2.

[0176] In some embodiments, the TRLs cultured in the absence of exogenous IL-2 can have at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 300%, at least about 400%, or at least about 500% reduction in undesired cells populations (e.g., NK cells or CD3- cells) compared to pre-expansion TRLs or TRLs cultured in the presence of exogenous IL-2. In some embodiments, the TRLs cultured in the absence of exogenous IL-2 can have at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, at most about 90%, at most about 100%, at most about 150%, at most about 200%, at most about 300%, at most about 400%, or at most about 500% reduction in undesired cells populations (e.g., NK cells or CD3- cells) compared to pre-expansion TRLs or TRLs cultured in the presence of exogenous IL-2. In some embodiments, the TRLs cultured in the absence of exogenous IL-2 can have about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 300%,about 400%, or about 500% reduction in undesired cells populations (e.g., NK cells or CD3- cells) compared to pre-expansion TRLs or TRLs cultured in the presence of exogenous IL-2.

[0177] In some embodiments, TRLs expanded in the absence of one or more T-cell growth factors (e.g., IL-2, IL-7, IL-9, and IL- 15) can exhibit a higher IL-2 receptor density (cluster of differentiation 25, CD25) when compared to TRLs expanded in the presence of one or more T- cell growth factors or when compared to pre-expansion TRLs. In some embodiments, the TRLs expanded in the absence of IL-2 can exhibit a higher IL-2 receptor density that is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% greater than the pre-expansion TRLs. In some embodiments, the TRLs expanded in the absence of IL-2 can exhibit a higher IL-2 receptor density that is at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, or at most about 90% greater than the pre-expansion TRLs. In some embodiments, the TRLs expanded in the absence of IL-2 can exhibit a higher IL-2 receptor density that is about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% greater than the pre-expansion TRLs. In some embodiments, the TRLs expanded in the absence of IL-2 can exhibit a higher IL-2 receptor density that is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 0.2-fold, about 0.4-fold, about 0.6-fold, about 0.8-fold, about 1.0-fold, about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8- fold, about 1.9-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7- fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14, about 15-fold, or about 20 fold higher than that of pre-expansion TRLs. In some embodiments, the TRLs expanded in the absence of IL-2 can exhibit a higher IL-2 receptor density that is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% greater than that of TRLs expanded in the presence of IL-2. In some embodiments, the TRLs expanded in the absence of IL-2 can exhibit a higher IL-2 receptor density that is at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, or at most about 90% greater than the pre-expansion TRLs. In some embodiments, the TRLs expanded in the absence of IL-2 can exhibit a higher IL-2 receptor density that is about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,or about 90% greater than that of TRLs expanded in the presence of IL-2. In some embodiments, the TRLs expanded in the absence of IL-2 can exhibit a higher IL-2 receptor density that is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 0.2-fold, about 0.4-fold, about 0.6-fold, about 0.8- fold, about 1.0-fold, about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 3-fold, about 4- fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11- fold, about 12-fold, about 13-fold, about 14, about 15-fold, or about 20 fold higher than that of TRLs expanded in the presence of IL-2.

[0178] In some embodiments, TRLs expanded in the absence of one or more T-cell growth factors (e.g., IL-2, IL-7, IL-9, and IL-15) can exhibit a higher IL-2 sensitivity when compared to TRLs expanded in the presence of one or more T-cell growth factors or when compared to preexpansion TRLs. In some embodiments, the TRLs expanded in the absence of IL-2 can exhibit an IL-2 sensitivity that is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% greater than that of the pre-expansion TRLs. In some embodiments, the TRLs expanded in the absence of IL-2 can exhibit an IL-2 sensitivity that is at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, or at most about 90% greater than that of TRLs pre-expansion. In some embodiments, the TRLs expanded in the absence of IL-2 can exhibit an IL-2 sensitivity that is about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% greater than that of the pre-expansion TRLs. In some embodiments, the TRLs expanded in the absence of IL-2 can exhibit an IL-2 sensitivity that is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 0.2-fold, about 0.4-fold, about 0.6-fold, about 0.8-fold, about 1.0-fold, about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14, about 15-fold, or about 20 fold higher than that of pre-expansion TRLs. In some embodiments, the TRLs expanded in the absence of IL-2 can exhibit an IL-2 sensitivity that is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% greater than that of TRLs expanded in the presence of IL-2. In someembodiments, the TRLs expanded in the absence of IL-2 can exhibit an IL-2 sensitivity that is at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, or at most about 90% greater than that of TRLs expanded in the presence of IL-2. In some embodiments, the TRLs expanded in the absence of IL-2 can exhibit an IL-2 sensitivity that is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 0.2-fold, about 0.4-fold, about 0.6-fold, about 0.8-fold, about 1.0-fold, about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14, about 15-fold, or about 20 fold higher than that of TRLs expanded in the presence of IL-2.

[0179] In some embodiments, TRLs expanded in the absence of one or more T-cell growth factors (e.g., IL-2, IL-7, IL-9, and IL-15) can exhibit an enrichment of Effector Memory T-cells (TEM) when compared to TRLs expanded in the presence of one or more T-cell growth factors or when compared to pre-expansion TRLs. In some embodiments, the TRLs expanded in the absence of IL-2 can have a percentage of TEM cells that is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% greater than that of the pre-expansion TRLs. In some embodiments, the TRLs expanded in the absence of IL-2 can have a percentage of TEM cells that is at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, or at most about 90% greater than that of TRLs preexpansion. In some embodiments, the TRLs expanded in the absence of IL-2 can have a percentage of TEM cells that is about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% greater than that of the preexpansion TRLs. In some embodiments, TRLs expanded in the absence of IL-2 can have a percentage of TEM cells that is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 0.2-fold, about 0.4-fold, about 0.6-fold, about 0.8-fold, about 1.0-fold, about 1.1-fold, about 1.2-fold, about 1.3- fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 3 -fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14, about 15-fold, or about 20 fold higher than that of pre-expansion TRLs. In some embodiments, the TRLsexpanded in the absence of IL-2 can have a percentage of TEM cells that is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% greater than that of TRLs expanded in the presence of IL-2. In some embodiments, the TRLs expanded in the absence of IL-2 can have a percentage of TEM cells that is at most about 5%, at most about 10%, at most about 15%, at most about 20%, at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 70%, at most about 80%, or at most about 90% greater than that of TRLs expanded in the presence of IL-2. In some embodiments, TRLs expanded in the absence of IL-2 can have a percentage of TEM cells that is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 0.2-fold, about 0.4-fold, about 0.6-fold, about 0.8-fold, about 1.0- fold, about 1.1-fold, about 1.2-fold, about 1.3-fold, about 1.4-fold, about 1.5-fold, about 1.6-fold, about 1.7-fold, about 1.8-fold, about 1.9-fold, about 2-fold, about 3-fold, about 4-fold, about 5- fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 11-fold, about 12-fold, about 13-fold, about 14, about 15-fold, or about 20 fold higher than that of TRLs expanded in the presence of IL-2.

[0180] In some embodiments, TRLs expanded in the absence of one or more T-cell growth factors (e.g., IL-2, IL-7, IL-9, and IL-15) can exhibit a similar memory phenotype (e.g., percentage of TEM) when compared to TRLs expanded in the presence of one or more T-cell growth factors. For example, TRLs expanded in the absence of IL-2 can have a similar percentage of TEM cells to that of TRLs expanded in the presence of IL-2.

[0181] In some embodiments, TRLs expanded in the absence of one or more T-cell growth factors (e.g., IL-2, IL-7, IL-9, and IL- 15) can exhibit greater homing to a tumor site than do TRLs expanded in the presence of one or more T-cell growth factors or than do TRLs pre-expansi on. For example, TRLs expanded in the absence of IL-2 can exhibit greater homing to a tumor site than do TRLs expanded in the presence of IL-2. TRLs expanded in the absence of IL-2 can exhibit greater homing to a tumor site than do pre-expansion TRLs.

[0182] In some embodiments, the culture medium can comprise an antibody (e.g., monoclonal antibody) capable of inducing T-cell activation. In some embodiments, the culture medium can comprise an OKT-3 antibody. In some embodiments, the culture medium can comprise about 30 ng / mL of OKT-3 antibody. In some embodiments, the culture medium can comprise an antibody (e.g., monoclonal antibody) specific to CD2, CD3, CD28, or any combination thereof. In some embodiments, the culture medium can comprise a plurality of the antibodies disclosed herein, such as anti-OKT-3 antibodies, anti-CD2 antibodies, anti-CD3 antibodies, and anti-CD28 antibodies,-n-or any combinations thereof. In some embodiments, the culture medium can be from about 0.1-5 ng / mL, about 5-10 ng / mL, about 10-15 ng / mL, about 15-20 ng / mL, about 20-25 ng / mL, about 25- 30 ng / mL, about 30-35 ng / mL, about 35-40 ng / mL, about 40-45 ng / mL, or about 45-50 ng / mL of one or more, or each, the antibodies.

[0183] In some embodiments, the feeder cells can be allogeneic. In some embodiments, the feeder cells can be peripheral blood mononuclear cells (PBMCs). In some embodiments, the PBMCs can be obtained from standard whole blood from donors. In some embodiments, the ratio of TRL to feeder cells can be about 1 :25, about 1 :50, about 1 : 100, about 1 : 125, about 1 : 150, about 1 : 175, about 1 :200, about 1 :225, about 1 :250, about 1 :275, about 1 :300, about 1 :325, about 1 :350, about 1 :375 or about 1 : 400. In some embodiments, TRLs can be cultured without feeder cells.

[0184] In some embodiments, TRL populations retrieved from the magnetic sorting methods described herein can be cultured using an expansion protocol described herein. In some embodiments, irradiated PBMC feeder cells, OKT3 antibody, and the TRL can be combined, mixed, and ali quoted to a tissue culture flask or a tissue culture plate. In some embodiments, TRL populations can be cultured in a feeder-free system. In some embodiments, the flask can be incubated upright at 37°C in 5% CO2. In some embodiments, no IL-2 is added to the culture flask on day 2. In some embodiments, IL-2 is added to the culture flask at 6000 lU / mL on day 2. In some embodiments, IL-2 can be added to the culture flask at a concentration ranging from 0.001 to 50 lU / mL. In some embodiments, IL-2 can be added to the culture flask at a concentration ranging from 5 to 6000 lU / mL. In some embodiments, on day 5, culture supernatant can be removed by aspiration and the culture media can be replaced with a 1 : 1 mixture of CM / AIMV containing no IL-2. In some embodiments, on day 5, culture supernatant can be removed by aspiration and the culture media can be replaced with a 1 : 1 mixture of CM / AIMV containing 6000 lU / mL IL-2. In some embodiments, on day 6 and every day thereafter, cell concentration can be determined and cells are split into additional flasks or transferred to culture bags with additional medium in the absences of IL -2 to maintain cell densities around 1 x 106cells / mL. In some embodiments, on day 6 and every day thereafter, cell concentration can be determined and cells can be split into additional flasks or transferred to culture bags with additional medium containing 6000 lU / mL IL-2 as needed to maintain cell densities around 1 x 106 cells / mL. In some embodiments, about 14 days after initiation of the culture, cells can be harvested from culture bags. In some embodiments, harvesting can be accomplished using a Baxter / Fenwal continuous centrifuge cell harvester system. In some embodiments, the harvested cells can be washed in sodium chloride. In some embodiments, the cells can be resuspended in sodium chloride withhuman albumin. In some embodiments, the resulting cell populations can be suitable for administration to a subject in need thereof.C. Methods of Producing Enhanced TRL Cells

[0185] Disclosed in some embodiments are methods of producing a population of enhanced TRLs (e.g., cTRLs). In some embodiments, the methods can comprise isolating a population of TRLs described using any of the methods disclosed herein, culturing the isolated TRLs using any of the methods disclosed herein, and introducing a cell from the cultured TRLs to a nucleotide that expresses a therapeutically enhancing polypeptide under conditions sufficient to produce the enhancing polypeptide by the cell. In some embodiments, the nucleotide can encode a chimeric antigen receptor (CAR). In some embodiments, methods described herein can comprise culturing the therapeutically enhanced cells using any of the culturing methods disclosed herein.

[0186] In some embodiments, at least one cell from a population of TRLs disclosed herein can be enhanced to express a CAR or an exogenous T cell receptor (TCR). In some embodiments, at least one cell from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 5, 10, 50, 100, 500, 1000, 2000, 5000, 10,000, 15,000, 20,000, or 25,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes disclosed herein can be used to produce CAR-T cells.

[0187] In some embodiments, at least one cell from an enriched population of CD 103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 90% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 5 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 90% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 10 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 90% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 50 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 90% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 100 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 90% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 500 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 90% disclosed herein can be used to produce a CAR-Tcell. In some embodiments, at least 1,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 90% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 2,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 90% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 5,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 90% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 10,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 90% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 15,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 90% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 20,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 90% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 25,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 90% disclosed herein can be used to produce a CAR-T cell.

[0188] In some embodiments, at least one cell from an enriched population of CD 103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 80% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 5 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 80% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 10 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 80% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 50 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 80% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 100 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 80% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 500 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 80% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 1,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 80% disclosed hereincan be used to produce a CAR-T cell. In some embodiments, at least 2,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 80% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 5,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 80% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 10,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 80% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 15,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 80% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 20,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 80% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 25,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 80% disclosed herein can be used to produce a CAR-T cell.

[0189] In some embodiments, at least one cell from an enriched population of CD 103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 70% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 5 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 70% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 10 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 70% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 50 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 70% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 100 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 70% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 500 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 70% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 1,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 70% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 2,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purityof at least 70% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 5,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 70% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 10,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 70% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 15,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 70% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 20,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 70% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 25,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 70% disclosed herein can be used to produce a CAR-T cell.

[0190] In some embodiments, at least one cell from an enriched population of CD 103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 60% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 5 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 60% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 10 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 60% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 50 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 60% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 100 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 60% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 500 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 60% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 1,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 60% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 2,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 60% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 5,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1expressing lymphocytes having a purity of at least 60% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 10,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 60% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 15,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 60% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 20,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 60% disclosed herein can be used to produce a CAR-T cell. In some embodiments, at least 25,000 cells from an enriched population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes having a purity of at least 60% disclosed herein can be used to produce a CAR-T cell.

[0191] In some embodiments, the TRLs described herein can be engineered to express a T- cell Receptor (TCR) or a chimeric antigen receptor (CAR), wherein the cell exhibits an antitumor property. In some embodiments, a TRL can be transformed with the TCR or CAR and the at least part of the TCR or CAR can be expressed on the cell surface. In some embodiments, the TRLs may be transduced with a viral vector encoding a TCR or CAR. In some embodiments, the viral vector can be a retroviral vector. In some embodiments, the viral vector can be a lentiviral vector. In some such embodiments, the cell may stably express the TCR or CAR. In another embodiment, the TRLs can be transfected with a nucleic acid, e.g., mRNA, cDNA, DNA, encoding a TCR or a CAR. In some such embodiments, the cell may transiently express the TCR or the CAR. In one aspect, the antigen binding domain of the TCR or CAR can comprise a murine (e.g., rat or mouse) antibody or antibody fragment.

[0192] In some embodiments, the TRLs can be engineered to express a chimeric antigen receptor (e.g., CART), wherein the cell (e.g., “CART”) exhibits an antitumor property. In some embodiments, the methods disclosed herein can comprise a recombinant DNA construct comprising sequences encoding a CAR, wherein the CAR comprises an antigen binding domain (e.g., antibody, antibody fragment) that binds to an antigen of interest (e.g., tumor antigen). The intracellular signaling domain can comprise a costimulatory signaling domain and / or a primary signaling domain, e.g., a zeta chain. The costimulatory signaling domain can refer to a portion of the CAR comprising at least a portion of the intracellular domain of a costimulatory molecule.

[0193] In some embodiments, the enhanced TRLs can be an allogenic immune effector cell lacking expression of a functional T cell receptor (TCR) and / or human leukocyte antigen (HLA),e.g., HLA class I and / or HLA class II. An enhanced TRL lacking a functional TCR can be, e.g., engineered such that it does not express any functional TCR on its surface, engineered such that it does not express one or more subunits that comprise a functional TCR or engineered such that it produces very little functional TCR on its surface. Alternatively, the TRL can express a substantially impaired TCR, e.g., by expression of mutated or truncated forms of one or more of the subunits of the TCR. The term “substantially impaired TCR” means that this TCR would not elicit an adverse immune reaction in a host. Such cells can be created through the use of one or more gene editing systems. An enhanced TRL described herein can be, e.g., engineered such that it does not express a functional HLA on its surface. For example, an enhanced TRL described herein, can be engineered such that cell surface expression HLA, e.g., HLA class 1 and / or HLA class II, is downregulated. Such cells can be created through the use of one or more gene editing systems as described herein. In some embodiments, the gene editing system targets a sequence encoding a component of one or more HLA molecules. In some embodiments, the gene editing system can target a sequence encoding a factor which affects the expression of one or more HLA molecules. In some embodiments, the gene editing system can target a regulator of MHC class I expression, for example a sequence encoding beta-2 microglobulin (B2M). In some embodiments, the gene editing system can target a sequence encoding a regulator of MHC class II molecule expression, for example, CIITA. In some embodiments, gene editing systems can target both a regulator of MHC class I expression (for example, B2M) and a regulator of MHC class II molecule expression (e.g., CIITA), such that at least MHC class I molecule and at least one MHC class II molecule expression can be downregulated. Modified T cells that lack expression of a functional TCR and / or HLA can be obtained by any suitable means, including a knockout or knock down of one or more subunit of TCR or HLA. For example, the T cell can include a knock down of TCR and / or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR) transcription-activator like effector nuclease (TALEN), or zinc finger endonuclease (ZFN).

[0194] Provided herein are delivery systems (e.g., viral -based systems or non-viral based systems), in which a polynucleotide encoding a TCR, or a CAR disclosed herein can be inserted. Representative viral expression vectors can include, but are not limited to, the adenovirus-based vectors (e.g., the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands)), lentivirus-based vectors (e.g., the lentiviral-based pLPI from Life Technologies (Carlsbad, Calif.)) and retroviral vectors (e.g., the pFB-ERV plus pCFB-EGSH), herpes viruses. In some embodiments, the viral vector can be a lentivirus vector. Vectors derived fromretroviruses such as the lentivirus can be used to achieve long-term gene transfer. In some embodiments, the lentivirus can transduce non-proliferating cells. In some embodiments, the lentivirus can have a low immunogenicity. In some embodiments, a suitable vector can comprise an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. Other suitable vectors may include integrating expression vectors, which may randomly integrate into the host cell's DNA, or may include a recombination site to enable the specific recombination between the expression vector and the host cell's chromosome. Such integrating expression vectors may utilize the endogenous expression control sequences of the host cell's chromosomes to effect expression of the desired protein. Examples of vectors that integrate in a site specific manner can include, for example, components of the flp-in system from Invitrogen (Carlsbad, Calif.) (e.g., pcDNA™5 / FRT), or the cre-lox system, such as can be found in the pExchange-6 Core Vectors from Stratagene (La Jolla, Calif). Examples of vectors that randomly integrate into host cell chromosomes can include, for example, pcDNA3.1 (when introduced in the absence of T- antigen) from Invitrogen (Carlsbad, Calif), and pCI or pFNIOA (ACT) FLEXI™ from Promega (Madison, Wis.). Additional promoter elements, e.g., enhancers, can regulate the frequency of transcriptional initiation. Non-limiting example of a suitable promoter can be the cytomegalovirus (CMV) promoter sequence, which can constitutively drive high levels of expression of any polynucleotide sequence operatively linked thereto. Another non-limiting example of a suitable promoter can include human elongation growth factor 1 alpha 1 (hEFlal). In some embodiments, the vector construct comprising a CAR described herein can comprise hEFlal functional variants. In some embodiments, the vector may comprise a constitutive promoter sequences including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the systems described herein should not be limited to the use of constitutive promoters. In some embodiments, inducible promoters can be used. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0195] In some embodiments, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infectedthrough viral vectors. In some embodiments, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Useful selectable markers include, for example, antibiotic-resistance genes, such as neomycin resistance gene (neo) and ampicillin resistance gene and the like. In some embodiments, a truncated epidermal growth factor receptor (HERlt or HERlt-1) tag can be used as a selectable marker gene. Reporter genes can be used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene can be a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Suitable reporter genes include genes encoding luciferase, beta- galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene.

[0196] In some embodiments, the polynucleotide encoding a TCR, or a CAR described herein can also be introduced into TRLs using a non-viral based delivery system, such as the “Sleeping Beauty (SB) Transposon System,” which refers a synthetic DNA transposon system for introducing DNA sequences into the chromosomes of vertebrates. The Sleeping Beauty transposon system can be composed of a Sleeping Beauty (SB) transposase and a SB transposon. In some embodiments, the Sleeping Beauty transposon system can include the SB 11 transposon system, the SB 100X transposon system, or the SB 110 transposon system.

[0197] In some embodiments, non-viral based delivery systems (e.g., delivery vehicle) can include lipid-based delivery systems, polymeric delivery systems, inorganic compound-based nanoparticles, or extracellular vesicle-based delivery. In some embodiments, an exemplary delivery vehicle can be a liposome. Lipid formulations can be used for the introduction of the polynucleotide into a host cell (in vitro, ex vivo, or in vivo). In some embodiments, the polynucleotide may be associated with a lipid. The polynucleotide associated with a lipid can be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure.

[0198] In some embodiments, an exemplary delivery vehicle can be a lipid nanoparticle (LNP). In some embodiments, polynucleotides encoding the compositions disclosed herein can beincorporated into or associated with one or more LNPs. In some embodiments, the LNP can comprise l,2-dioleoyl-3 -trimethylammonium propane (DOTAP), dioleoylphosphatidylethanolamine (DOPE), a cationic cholesterol derivative mixed with dimethylaminoethane-carbamoyl (DC-Chol), phosphatidylcholine (PC), triolein (glyceryl trioleate), and l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(poly ethylene glycol)-2000] (DSPE-PEG), l,2-dimyristoyl-rac-glycero-3-methoxypoly ethyleneglycol -2000 (DMG-PEG 2K), and 1,2 distearoyl-sn-glycero-3 phosphocholine (DSPC) and / or comprising of one or more molecules selected from polyethylenimine (PEI) and poly(lactic-co-glycolic acid) (PLGA), and N-Acetylgalactosamine (GalNAc). The LNP can comprise one or more of a structural lipid (e.g., DSPC), a PEG-conjugated lipid (CDM-PEG), a cationic lipid (MC3), cholesterol, and a targeting ligand (e.g., GalNAc). In some embodiments, a nanoparticle described herein can be a particle having a diameter of less than about 1000 nm. In some embodiments, nanoparticles can have a greatest dimension (e.g., diameter) of about 500 nm or less, or about 400 nm or less, or about 300 nm or less, or about 200 nm or less, or about 100 nm or less. In some embodiments, nanoparticles described herein can have a greatest dimension ranging between about 50 nm and about 150 nm, or between about 70 nm and about 130 nm, or between about 80 nm and about 120 nm, or between about 90 nm and about 110 nm. In some embodiments, the nanoparticles described herein can have a greatest dimension (e.g., a diameter) of about 100 nm.

[0199] In some embodiments, TRLs transduced with a nucleic acid encoding a TCR or a CAR can be expanded, e.g., by a method described herein. In some embodiments, the enhanced TRLs (e.g., TRLs transduced with a nucleic acid encoding a CAR) can be expanded in culture for a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days). In one embodiment, the cells can be expanded for a period of 4 to 9 days. In one embodiment, the cells can be expanded for a period of 8 days or less, e.g., 7, 6 or 5 days.

[0200] Conditions appropriate for T cell culture (e.g., enhanced TRLs) may include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFP, and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added aminoacids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g, penicillin and streptomycin, can be included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells can be maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2).

[0201] In one embodiment, the enhanced TRLs can be expanded in an appropriate media (e.g., media described herein) that includes one or more interleukin that result in at least a 200-fold (e.g., 200-fold, 250-fold, 300-fold, 350-fold) increase in cells over a 14-day expansion period, e.g., as measured by a method described herein such as flow cytometry. In some embodiments, the enhanced TRLs can be expanded in the presence of IL-15 and / or IL-7 (e.g., IL-15 and IL-7).

[0202] Once a CAR (e.g., enhanced TRL) is constructed, various assays can be used to evaluate the activity of the molecule, such as but not limited to, the ability to expand T cells following antigen stimulation, sustain T cell expansion in the absence of re-stimulation, and anticancer activities in appropriate in vitro and animal models. For example, western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers and dimers. In vitro expansion of the enhanced CAR cells following antigen stimulation can be measured by flow cytometry. Animal models can also be used to measure a CART activity. For example, imaging technologies can be used to evaluate specific trafficking and proliferation of CARs in tumor-bearing animal models.D. Methods of Treatment

[0203] Disclosed herein are methods of treating a disease or a condition in a subject by administering to the subject a composition or pharmaceutical formulation disclosed herein. In some embodiments, the composition can comprise the TRLs (e.g., cTRLs) or enhanced TRLs (e.g., enhanced cTRLs) disclosed herein. In some embodiments, a plurality of the TRLs can be therapeutically enhanced to express a chimeric antigen receptor (CAR) having antigenic specificity for a cancer antigen, e.g, of any of the cancers disclosed herein. In some embodiments, the cells can be isolated from the peripheral blood of a subject using any of the methods disclosed herein. In some embodiments, cells can be autologous to the subject. In some embodiments, the cells can be isolated from the peripheral blood of the subject using any of the methods of isolating TRLs disclosed herein. In some embodiments, the isolated TRLs can be expanded using any of the methods disclosed herein. In some embodiments, autologous cells can be preferred overallogeneic cells because of their inherent heterogeneity that maximizes the tumor-recognizing T cell receptors (TCRs) while minimizing off-tissue effects.

[0204] In some embodiments, the TRLs (e.g., enhanced TRLs) can be injected into a body part of a subject (e.g., a vein, a marrow, etc. of a patient). In some embodiments, the cells can be administered by intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavemous, and / or intravitreal injection.

[0205] In some embodiments, administration of a populations of cells disclosed herein can be by a single administration, at continuous intervals, or at distinct intervals, and can be readily determined by a person skilled in the art. In some embodiments, the subject can be injected with a dose of the cells at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some embodiments, the subject can be injected with a dose of the cells at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 time. In some embodiments, the subject can be injected with a dose of the cells at a frequency of at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 60, 90, 180, 360, or more days. In some embodiments, the subject can be injected with a dose of the cells at a frequency of at most once every 360, 180, 90, 60, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, the subject can be injected with a dose of at least about 1 x 104cells / kg, 1 x 105cells / kg, 1 x 106cells / kg, 1 x 107cells / kg, 1 x 108cells / kg, 1 x 109cells / kg, 1 x 1010cells / kg, 1 x 1011cells / kg or more. In some embodiments, the subject may be injected with at most a dose of about at least about 1 x 104cells, 1 x 105cells, 1 x 106cells, 1x107cells, 1x108cells, 1x109cells, 1x1010cells, 1x10ncells, 1x1012cells, or more.

[0206] In some embodiments, the cells may be useful in a variety of applications, including, but not limited to, immunotherapy to treat diseases and disorders. Diseases and disorders that can be treated using the cells of the present disclosure include, but are not limited to, inflammatory conditions, cancer, infectious diseases, autoimmune diseases, and neurodegenerative diseases. In some embodiments, the cell therapies disclosed herein can be used to treat cancer. In some embodiments, the cell therapy can be used to treat a tumor that is not optimal source material for tumor-infiltrating lymphocyte (TIL) isolation. In some embodiments, a cell therapy described herein, can be used to treat a tumor wherein large resectable lesion (e.g., less than 3 cm in diameter) is not readily accessible. In some embodiments, a cell therapy described herein can be used to treat a patient for which excisional surgery is not an option for patients (e.g., due to a substantial risk or rapid tumor progression). In some embodiments, a cell therapy described herein can be used to treat a tumor which exhibited a reduced response rate to a TIL-based adoptive celltherapy. In some embodiments, the cell therapy can be used to treat solid tumors, such as renal carcinoma, cervical cancer, and breast cancer, which have been demonstrated, in some embodiments, to be less responsive to TIL therapy. In some embodiments, the cells of the present disclosure can be used to treat cancer. In some embodiments, the cancer can be in a tissue. In some embodiments, the tissue can comprise a bone tissue, a muscle tissue, a breast tissue, an epithelial tissue, a connective tissue, a brain tissue, a lung tissue, a kidney tissue, a liver tissue, a pancreatic tissue, a prostate tissue, a lymphoid tissue, a myeloid tissue, or a bladder tissue. Non-limiting examples of cancer include: Non-limiting examples of cancer include: Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS- related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic T-cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Brown tumor, Burkitt’s lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site, Carcinosarcoma, Castleman’s Disease, Central Nervous System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T-cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy-associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing’s sarcoma, Extracranial Germ Cell Tumor, Extragonadal GermCell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget’s disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin’s lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi’s sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic Disease, Myelodysplastic Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, NonHodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget’s disease of the breast, Pancoast tumor,Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastoma, Polyembryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter’s transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom’s macroglobulinemia, Warthin’s tumor, Wilms’ tumor, and combinations thereof.

[0207] In some embodiments, compositions and methods disclosed herein are administered in combination with other cancer immunotherapies or with a chemotherapy. In some embodiments, a method of providing a cell therapy to a subject in need thereof further comprises administering at least one additional therapy. In some embodiments, administering at least one additional therapy comprises administering to the subject an additional therapeutic agent. In some embodiments, the additional therapeutic agent can be an immunosuppressive drug. In some embodiments, the immunosuppressive drugs can comprise a monoclonal antibody. In some embodiments, the monoclonal antibody can deplete endogenous lymphocytes. In some embodiments, anti-CD3, anti-CD2, and / or anti-CD52 can be used to deplete endogenouslymphocytes. In some embodiments, the additional therapeutic agent is an anti -oncologic agent. In some embodiments, the anti -oncologic agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is a lymphocyte depleting chemotherapeutic agent. In some embodiments, the anti-oncologic agent is an immunotherapy agent. In some embodiments, the anti -oncologic agent is an immune checkpoint inhibitor, a chemotherapeutic agent, or any combination thereof. In some embodiments, the anti-oncologic agent is a costimulatory molecule. In some embodiments, the costimulatory molecule is Glucocorticoid-Induced Tumor Necrosis Factor Receptor (GITR). In some embodiments, the costimulatory molecule can comprise CD28, CD137 (4-1BB), CD134 (0X40), Inducible T cell costimulatory (ICOS), CD27 or any combination thereof. In some embodiments, the ani-oncologic agent can be a cytokine that stimulates survival, proliferation and activation. In some embodiments, the cytokine can be IL-2, IL-7, IL-15, IL-21, or any combination thereof. In some embodiments, the anti-oncologic agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PDl antibody, an anti-PD-Ll antibody, or a variant or functional fragment thereof. In some embodiments, the anti-PDl antibody is selected from pidilizumab, BMS-936559, nivolumab, pembrolizumab, or a variant or functional fragment thereof. In some embodiments, the anti-PD- Ll antibody is selected from atezolizumab, avelumab, durvalumab, MDX-1105, or a variant or functional fragment thereof.

[0208] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody, or a variant or functional fragment thereof. Anti-CTLA-4 antibodies can bind human CTLA-4 to interfere with the interaction between CTLA-4 and human B7 receptors. Since the interaction between CTLA-4 and B7 transforms a signal leading to the inactivation of T-cells carrying the CTLA-4 receptor, interfering with the interaction effectively induces the activation of these T cells. An exemplary clinical anti-CTLA-4 Ab is human mAb 10D1 (now known as ipilimumab and marketed as YERVOY®). In some embodiments, the anti-CTLA-4 Ab can be a mAb. In some embodiments, the anti-CTLA-4 antibody can be a chimeric, humanized or human antibody. In an embodiment, the anti-CTLA-4 antibody can be ipilimumab.

[0209] In some embodiments, the additional therapeutic agent can be administered before, during (e.g., co-administration), and / or after administration of the cell population. For example, anti-CD3 can be administered before administration of the TRLs (e.g., enhanced TRLs) to deplete endogenous lymphocytes. In some embodiments, the additional therapeutic agent (e.g., a recombinant IL-2) can be co-administered with the cell population described herein. In some embodiments, the additional therapeutic agent (e.g., a recombinant IL-2) can be administered afteradministration of the cell population described herein. For example, a recombinant IL-2 can be co-administered or administered after administration of the TRLs (e.g., enhanced TRLs) to stimulate expansion (e.g., cell proliferation, activation, survival) of the administered TRLs. In some embodiments, the additional therapeutic agent can be administered concurrently with administration of the cell population. In some embodiments, the additional therapeutic agent can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times (e.g., daily administration). In some embodiments, the additional therapeutic agent can be administered at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 time. In some embodiments, the additional therapeutic agent can be administered at a frequency of at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 60, 90, 180, 360, or more days. In some embodiments, the additional therapeutic agent can be administered at a frequency of at most once every 360, 180, 90, 60, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, one or more therapeutic agents can be administered 20 days, 19 days, 18 days, 17 days, 16 days, 15 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, or 5 minutes before or after administration of TRLs or the enhanced TRLs as described herein.

[0210] The additional therapeutic agent can be IL-2 (e.g, a recombinant IL-2) or aldesleukin. IL-2 (or aldesleukin) can be administered at low or high doses. A high-dose IL-2 (or aldesleukin) regimen can involve intravenous IL-2 (or aldesleukin) administration at up to about 14 doses of about 0.037 mg / kg (600,000 lU / kg) every 8 hours, as long as it is tolerated. Immunostimulatory agents (e.g, aldesleukin) can be administered within 24 hours after cell administration. IL-2 (or aldesleukin) can be administered as an infusion over about 15 minutes about every 8 hours for up to about 4 days after cell infusion. IL-2 (or aldesleukin) can be administered about 100,000 lU / kg, 200,000 lU / kg, 300,000 lU / kg, 400,000 lU / kg, 500,000 lU / kg, 600,000 lU / kg, 700, 000 lU / kg, 800,000 lU / kg, 900,000 lU / kg or up to about 1,000,000 lU / kg. In some embodiments, IL-2 (or aldesleukin) can be administered about 100,000 lU / kg to 300,000 lU / kg, 300,000 lU / kg to 500,000 lU / kg, 500,000 lU / kg to 700,000 lU / kg, 700,000 lU / kg to a dose of about 1,000,000 lU / kg. In some embodiments, IL-2 (or aldesleukin) can be administered in 1 dose to about 14 doses. IL-2 (or aldesleukin) can be administered at least about 1 dose, 2 doses, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses, 10 doses, 11 doses, 12 doses, 13 14 doses, 15 doses, 16 doses, 17 doses, 18 doses, 19 doses or up to about 20 doses. In some embodiments, IL-2 (or aldesleukin), can be administered at about 1-3 doses, 3-5 doses, 5-8 doses, 8-10 doses, 10-14 doses, 14-20 doses. In some embodiments, IL-2 (or aldesleukin) can be administered in excess of20 doses. In some embodiments, IL-2 (or aldesleukin) can be administered sequentially or concurrently with the tumor reactive lymphocytes administration. In some embodiments, IL-2 (or aldesleukin) can be administered on days 0-4 after administration of the cell population. In some embodiments, IL-2 (or aldesleukin) can be administered over a period of about 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, or up to about 3 hours. In some embodiments, IL-2 (or aldesleukin) can be administered from about 24 hours before administration of the tumor reactive cells described herein to about 4 days after administration of the tumor reactive cells described herein.

[0211] In some embodiments, the tumor reactive lymphocytes described herein can be administered without administering the additional therapeutic agent (e.g., IL-2). For example, in some embodiments, the tumor reactive lymphocytes described herein can be administered to a subject without administering IL-2. In some embodiments, the tumor reactive lymphocytes described herein can be administered to a subject without administering high or low dose of IL-2 before the administration of the TRLs. In some embodiments, the tumor reactive lymphocytes described herein can be administered to a subject without co-administering high or low dose of IL-2. In some embodiments, the tumor reactive lymphocytes described herein can be administered to a subject without administering high or low dose of IL-2 after the administration of the TRLs.

[0212] In practicing the methods of treatment or use provided herein, therapeutically effective amounts of pharmaceutical formulations described herein can be administered to a mammal having a disease, disorder, or condition to be treated, e.g., cancer. In some embodiments, the mammal can be a human. A therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent used and other factors. In some embodiments, pharmaceutical formulations described herein, may be used singly or in combination with one or more therapeutic agents as components of mixtures.III. SYSTEMS

[0213] Described herein, in some embodiments, are systems comprising one or more compositions or devices disclosed herein. In some embodiments, the system can comprise a microfluidic device and instructions for how to use the microfluidic device to magnetically separate to separate tumor reactive lymphocytes (TRLs) from a fluid sample from a subject. In some embodiments, the microfluidic device can comprise a magnetic capture zone disposed in a channel of the microfluidic device, wherein the microfluidic device can be configured tomagnetically separate the population of cells from the fluid sample. In some embodiments, the system can comprise the fluid sample obtained from a subject, wherein the fluid sample can comprise a population of cells comprising (i) CD103 lymphocytes, (ii) CD39 lymphocytes, (iii) SLC6A19+ lymphocytes, (iv) SIDT1+ lymphocytes, or (v) any combination of (i) to (iv). In some embodiments, the system can comprise an enhanced TRL provided in Section I (Compositions) of this disclosure. In some embodiments, the system can comprise a kit provided in Section IV (Kits) of this disclosure.

[0214] In some embodiments, the system may comprise a population of isolated and enriched tumor-reactive lymphocytes derived from a sample (e.g., peripheral blood) that express SLC6A19, SIDT1, CD103, CD39, or any combination of thereof. In some embodiments, a TRL can express CD103. In some embodiments, a population of TRLs can comprise a CD103 signature. In some embodiments, a CD 103 signature can define the population of TRLs. In some embodiments, a TRL can express CD39. In some embodiments, a population of TRLs can comprise a CD39 signature. In some embodiments, a CD39 signature can define the population of TRLs. In some embodiments, a TRL can express SLC6A19, SIDT1, or a combination thereof. In some embodiments, a population of TRLs can comprise a SLC6A19+, SIDT1+ signature. In some embodiments, the population of TRLs can further comprise CD3, CD4, CD8 or any combination thereof. For example, the isolated and enriched TRLs can be CD3 and CD 103 cells. In some embodiments, the isolated and enriched TRLs can be CD4 and CD 103 cells. In some embodiments, the isolated and enriched TRLs can be CD8 and CD 103 cells. In some embodiments, the isolated and enriched TRLs can be CD39 and CD103 cells. In some embodiments, the isolated and enriched TRLs can be CD3, CD39, and CD103 cells. In some embodiments, the isolated and enriched TRLs can be CD4, CD39, and CD103 cells. In some embodiments, the isolated and enriched TRLs can be CD8, CD39, and CD103 cells. In some embodiments, the isolated and enriched TRLs can be CD3, SLC6A19, and SIDT1 cells. In some embodiments, the isolated and enriched TRLs can be CD4, SLC6A19, and SIDT1 cells. In some embodiments, the isolated and enriched TRLs can be CD8, SLC6A19, and SIDT1 cells. In some embodiments, the isolated and enriched TRLs can be CD3, CD103, and SLC6A19 cells. In some embodiments, the isolated and enriched TRLs can be CD4, CD103, and SLC6A19 cells. In some embodiments, the isolated and enriched TRLs can be CD8, CD103, and SLC6A19 cells. In some embodiments, the isolated and enriched TRLs can be CD3, CD103, SLC6A19, and SIDT1 cells. In some embodiments, the isolated and enriched TRLs can be CD4, CD103, SLC6A19, and SIDT1 cells. In some embodiments, the isolated and enriched TRLs can be CD8, CD103, SLC6A19, and SIDT1 cells.

[0215] In some embodiments, a population of tumor-reactive lymphocytes (TRLs) may be found in a fluid sample of a patient having cancer. In some embodiments, a fluid sample may comprise a peripheral blood sample. In some embodiments, a fluid sample may be other biofluids, such as pleural effusion, ascites, and the like. In some embodiments, a fluid sample also can be cord blood, bone marrow, lymph nodes, liver pleural effusion, thorax, abdominal cavity, synovial fluid, peritoneum, retroperitoneal space, thymus, and tumor.

[0216] In some embodiments, a system described herein can comprise isolating or enriching a population of CD103, CD39, SLC6A19, and / or SIDT1 expressing lymphocytes from a peripheral blood sample by treating the sample with a plurality of major histocompatibility complex (MHC) multimers mimicking a defined tumor epitope. Generally, T-cells can express surface T-cell receptors (TCR) which enable T-cells to recognize peptide antigens bound to major histocompatibility complex (MHC) molecules, and TCR recognition of MHC -peptide complexes result in T-cell activation, clonal expansion and differentiation of the T-cells into effector, memory and regulatory T-cells. MHC-multimers can comprise multiple copies of MHC-peptide complexes. In some embodiments, MHC multimers can exhibit increased T-cell affinity, compared to a monomer of the same complex. In some embodiments, the MHC molecules can be human MHC molecules. In some embodiments, the MHC molecules can be murine MHC molecules. In some embodiments, the MHC molecules can be class 1 MHC molecules. In some embodiments, the MHC Class 1 molecules can be human HLA-A, HLA-B, or HLA-C molecules. In some embodiments, the MHC class I molecules can be murine H-2K, H-2D or H-2L molecules. In some embodiments, the molecules can be class 2 MHC molecules. In some embodiments, an MHC multimer can comprise at least 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 MHC molecules. In some embodiments, an MHC multimer can comprise about 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 MHC molecules. In some embodiments, an MHC multimer can comprise an MHC dimer comprising two MHC molecules. In some embodiments, an MHC multimer can comprise an MHC tetramer comprising four MHC molecules. In some embodiments, an MHC multimer can comprise an MHC pentamer comprising five MHC molecules. In some embodiments, an MHC multimer can comprise a dextramer, comprising 10 or more MHC molecules.

[0217] In some embodiments, an MHC multimer can comprise a peptide. In some embodiments, an MHC multimer can comprise a peptide-MHC complex. In some embodiments, the peptide can be any natural or non-natural peptide capable of being presented by an MHC molecule. In some embodiments, the peptide-MHC complex can be one to which a TRL hasreactivity. In some embodiments, the peptide-MHC complex can be one to which a CD8+CD103+ lymphocyte has reactivity. In some embodiments, the peptide-MHC complex can be one to which a CD103+CD39+ lymphocyte has reactivity. In some embodiments, the peptide-MHC complex can be one to which a CD8+CD103+CD39+ lymphocyte has reactivity. In some embodiments, the peptide-MHC complex can mimic a defined tumor epitope. In some embodiments, the peptide can comprise an epitope from influenza A hemagglutinin. In some embodiments, the peptide can comprise amino acid residues 533-541 of influenza A hemagglutinin. In some embodiments, the peptide can comprise an epitope from chicken ovalbumin. In some embodiments, the peptide can comprise amino acid residues 257-264 of chicken ovalbumin. In some embodiments, an MHC multimer can comprise a plurality of peptide-MHC complexes. In some embodiments, each of the peptide-MHC complexes can be associated with a multimerization domain. In some embodiments, the peptide-MHC complex can comprise the MC-38-derived peptide of SIIVFNLL sequence and an H-2Kb molecule. In some embodiments, the MHC multimer can be operatively linked to a magnetic nanoparticle. In some embodiments, the magnetic nanoparticle can be joined to the MHC multimer via fluorophore linker.

[0218] In some embodiments, the linker can comprise a polymer linker, such as an amino acid linker, biotin linker, and the like. In some embodiments, the linker can be cleavable. In some embodiments, the linker can be not cleavable.

[0219] In some embodiments, a system described herein may comprise separating, isolating or enriching from a peripheral blood sample a population of TRLs. In some embodiments, a microfluidic device disclosed herein can be used to separate the population of TRLs from the peripheral blood sample. In some embodiments, any of the microfluidic devices disclosed in PCT Publication No. WO 2014 / 166000, the contents of which can be incorporated by reference herein, can be used to separate the TRLs from the peripheral blood sample. In some embodiments, once the TRLs have been separated, the TRLs may be eluted from the microfluidic device by removing an attractant acting on the TRLs (e.g., via removal of a magnetic field). In some embodiments, the captured TRLs can be then expanded, enhanced, or a combination thereof, by any of the methods disclosed herein.

[0220] Microfluidic devices disclosed herein can be configured to magnetically sort a population of cells. In some embodiments, the microfluidic device can be provided in United States Patent No. 10,073,079, which is hereby incorporated by reference in its entirety. In some embodiments, the population of cells can comprise a population of TRLs. In some embodiments,the population of TRLs can be labeled with magnetic nanoparticles. In some embodiments, each of the magnetic nanoparticles can be about 0-50 nanometer (nm) in diameter, 51-100 nm in diameter, 100-150 nm in diameter, or 150-200 nm in diameter. In some embodiments, each of the magnetic nanoparticles can be about 50 nm in diameter. In some embodiments, compared with conventional microbeads, magnetic nanoparticles have improved colloidal stability, which may be useful for processing larger samples. In some embodiments, cells labeled with magnetic nanoparticles can be difficult to capture because their orders-of-magnitude lower magnetic susceptibilities, compared to microbeads, result in lower capture efficiencies. Therefore, in some embodiments, the microfluidic devices disclosed herein can include flow rate-reducing structures that give rise to localized regions of lower flow rate, as a sample comprising the cells can be flowed through the device. In some embodiments, the presence of such low flow velocity regions can enable capture of the magnetically labeled cells.

[0221] In some embodiments, a microfluidic device disclosed herein can comprise a microfluidic chip. In some embodiments, the microfluidic chip can comprise a sorting chamber. In some embodiments, the sorting chamber can be etched or molded into the chip. In some embodiments, the sorting chamber can be in communication with a flow inlet and a flow outlet. In some embodiments, the flow inlet can be configured to receive a sample, e.g., a peripheral blood sample comprising a population of TRLs suspended in a fluid medium, and the outlet can be configured for delivering the fluid medium depleted of said TRLs. In some embodiments, tubing can be connected to the inlet such that the fluid medium can be delivered into the inlet through the tubing. In some embodiments, tubing can be connected to the outlet such that the fluid medium can be received from the outlet through the tubing. In some embodiments, the tubing can be silicone tubing. In some embodiments, the microfluidic device can comprise a syringe pump capable of controlling the flow rate of fluid medium at the inlet.

[0222] In some embodiments, the sorting chamber can comprise at least one magnetic capture zone. In some embodiments, the sorting chamber can comprise a plurality of magnetic capture zones. In some embodiments, the sorting chamber can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 magnetic capture zones. In some embodiments, the sorting chamber can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 magnetic capture zones. In some embodiments, at least two of the magnetic capture zones vary in height. In some embodiments, the magnetic capture zones range from 50 - 800 pm in height. In some embodiments, sorting chamber can comprise three magnetic capture zones, one about 100 pm in height, one about 200 pm in height, and one about 400 pm in height.

[0223] In some embodiments, the microfluidic device can comprise at least one array of magnets positioned on the outer surface of the microfluidic chip, disposed above or below the sorting chamber, such that a magnetic field is created in the magnetic capture zones by the at least one array of magnets. In some embodiments, the microfluidic device can comprise two arrays of magnetics positioned on two outer surfaces of the microfluidic chip above and below the sorting chamber such that a magnetic field is created in the magnetic capture zones by the two arrays of magnets. In some embodiments, magnets can be positioned in two arrays, with alternating polarities on opposing sides of the sorting chamber. In some embodiments, the at least one array of magnets produces a magnetic field strength between .1 - .5 Tesla (T), .5 - 1 T, or 1 - 1.5 T in the magnetic capture zones. In some embodiments, the at least one array of magnets can produce a magnetic field strength between .5 - 1 T in the magnetic capture zones. In some embodiments, the magnets can comprise neodymium magnets. In some embodiments, the magnets can comprise N52 Nd FeB magnets.

[0224] In some embodiments, a magnetic capture zone can comprise a plurality of microstructures. In some embodiments, the microstructures can be flow rate-reducing structures configured to improve capture cells labeled with magnetic nanoparticles in the flow. In some embodiments, the microstructures can produce localized regions of lower flow rate, which may allow for capture of the particles (e.g., the reduced flow rate may allow the magnetic force to overcome the drag force on the particles). In some embodiments, the structures can be designed to avoid trapping of non -target particles. For example, despite being lower in flow rate, the regions of lower flow rate may still have enough flow velocity (that is, the flow rate may be at least nonzero) for non-target particles to be washed from the device, while target particles may be trapped in the low flow rate region. In some embodiments, the microstructures can be X-shaped.

[0225] In some embodiments, the device can comprise a plurality of magnetic capture zones, with a first zone comprising the inlet of the sorting chamber, a final magnetic capture zone comprising the outlet of the sorting chamber, and a plurality of magnetic capture zones disposed between the first magnetic capture zone and the final magnetic capture zone. In some embodiments, the size or pattern of the microstructures can vary among the capture zones. In some embodiments, the heights can vary among the magnetic capture zones. In some embodiments, the first zone can exhibit the highest linear velocity and thus retains cells with high magnetic content because the retaining magnetic force overcomes the drag force exerted by the locally high flow velocity. In some embodiments, the other magnetic capture zones can exhibit gradually reduced linear velocities, with the final magnetic capture zone exhibiting the lowest velocity. This designcan allow cells with high levels of magnetization to be captured in the first zone of the device, whereas cells with lower magnetization become sorted in later zones according to level of magnetization.

[0226] In some methods disclosed herein, a population of TRLs suspended in fluid can be propelled through the inlet, across the magnetic capture zones and through the outlet. In some embodiments, magnetically labelled cells can be captured in the capture zones if the magnetic force exerted on the cells is sufficient to overcome the drag force compelling the cell to flow through the capture zone. Otherwise, a cell would be flush into the next zone with a lower drag force, and eventually into the syringe if it cannot be captured by any zone. After sorting, captured cells at each zone can be recovered by removing the external magnets.

[0227] In some methods disclosed herein, a population of cells can be loaded into the microfluidic device, through the inlet, at a flow rate of at least 1 milliliter per hour (mL / h), 3 mL / h, 6 mL / h, 9 mL / h, 12 mL / h, 15 mL / h, 18 mL / h, 21 mL / h, 24 mL / h, 27 mL / h, 30 mL / h, 35 mL / h, 40 mL / h, 45 mL / h, or 50 mL / h. The magnetic force exerted on the cells can be determined by, for example, the size of the magnetic nanoparticle, the number of magnetic nanoparticles attached to the cell, the size of the cell, and the strength of the applied magnetic field. In some embodiments, the relationship between drag force and linear flow velocity in a microfluidic device capable of magnetically capturing particles by leveraging rate reducing microstructures is disclosed in United States Patent No. 10,073,079, which is hereby incorporated by reference in its entirety.

[0228] Disclosed in some embodiments herein are systems of producing a population of enhanced TRLs. In some embodiments, the systems can comprise isolating a population of TRLs described using any of the systems disclosed herein, culturing the isolated TRLs using any of the systems disclosed herein, and introducing a cell from the cultured TRLs to a nucleotide that expresses a therapeutically enhancing polypeptide under conditions sufficient to produce the enhancing polypeptide by the cell. In some embodiments, the nucleotide encodes a chimeric antigen receptor (CAR). In some embodiments, the systems described herein can comprise culturing the therapeutically enhanced cells using any of the culturing systems disclosed herein.

[0229] Described herein are pharmaceutical formulations comprising the enhanced TRLs or the compositions described herein. In some embodiments, the pharmaceutical formulations can further comprise a pharmaceutically acceptable: carrier, excipient, diluent, or nebulized inhalant.

[0230] In some embodiments, the pharmaceutical formulations can include two or more active agents, or two or more therapeutic agents as disclosed herein. In some embodiments, the two ormore active agents can be contained in a single dosage unit such as, for example, when the enhanced TRLs (e.g., CAR or TCR) comprises two or more therapeutic agents. In some embodiments, the two or more active agents can be contained in separate dosage units such as when the enhanced TRLs (e.g., CAR or TCR) is administered separately from an additional therapeutic agent or adjuvant. In some embodiments, the active agents that may be, in some embodiments, the additional therapeutic agent include a chemotherapeutic agent, cytotoxic agent, cytokine, growth-inhibitory agent, anti-hormonal agent, anti-angiogenic agent, cardio protectant, and / or checkpoint inhibitor.IV. KITS

[0231] Disclosed herein, in some embodiments, are kits comprising the compositions or systems disclosed herein, with instructions for how to produce or use the compositions or systems. In some embodiments, the kit can comprise the microfluidic device disclosed herein, with instructions for how to use the microfluidic device to isolate TRL’s from a fluid sample obtained from a subject. In some embodiments, the kit further can comprise reagents for isolating, enriching or expanding the TRLs from the fluid sample. Such reagents may include antibodies (e.g., magnetic nanoparticles), buffers, and / or conditioned medium.

[0232] In some embodiments, the kit comprises a vector comprising polynucleotides encoding the CAR components described herein. The kit can include multiple vectors each encoding different proteins or subsets of proteins. These vectors can be viral, non-viral, episomal, or integrating. In some embodiments, the vectors are transposons. In some embodiments, the kit further comprises reagents or devices for TRL transfection or transduction, such as calcium phosphate DNA coprecipitation, DEAE-dextran, electroporation, cationic liposome-mediated transfection, tungsten particle-facilitated microparticle bombardment, and strontium phosphate DNA co- precipitation.

[0233] In some embodiments, the instructions further comprise instructions for how to engineer the TRLs to produce enhanced TRLs. Instructions may also include instructions for cry opreserving, lyophilizing, or cryo-hibernating the compositions disclosed herein during storage and / or transport. Instructions may also include instructions for thawing or otherwise reviving the biological activity of the compositions disclosed herein prior to administration to a subject. In some embodiments, the instructions may direct a medical laboratory to separate TRLs from a fluid sample disclosed herein (e.g., peripheral blood) utilizing the system components disclosed herein (e.g., microfluidic device). For example, the instructions may include methods comprising separating from a fluid sample of a subject a population of SLC6A19+SIDT1+ lymphocytes. Insome embodiments, the instructions may include methods comprising magnetically separating from a fluid sample of a subject a population of CD 103 lymphocytes, wherein the magnetically separating comprises flowing the CD 103 lymphocytes across a magnetic capture zone disposed in a channel of a microfluidic device. In some embodiments, instructions can comprise methods for administering to the subject the pharmaceutical formulation disclosed herein or the engineered TRL disclosed herein. In some embodiments, the methods can f...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of expanding tumor reactive lymphocytes (TRLs), the method comprising:(a) providing CD8+CD103+ TRLs from a subject, and(b) culturing the CD8+CD103+ TRLs ex vivo in a cell culture medium that lacks IL-2.

2. A method of expanding tumor reactive lymphocytes (TRLs), the method comprising: culturing CD8+CD103+ TRLs ex vivo in a cell culture medium that lacks IL-2, wherein the CD8+CD103+ TRLs are from a subject.

3. The method of claim 1 or 2, wherein the cell culture medium that lacks IL-2 comprises less than a trace amount of IL-2.

4. The method of claim 1 or 2, wherein the cell culture medium that lacks IL-2 comprises less than a biologically reactive amount of IL-2.

5. The method of claim 1 or 2, wherein the cell culture medium that lacks IL-2 comprises less than about 50 lU / mL, less than about 40 lU / mL, less than about 30 lU / mL, less than about 20 lU / mL, less than about 10 lU / mL, less than about 5 lU / mL, less than about 2 lU / mL, or less than about 1 lU / mL of IL-2.

6. The method of any one of claims 1-5, wherein the subject has a cancer, or is suspected of having a cancer, wherein the cancer comprises a melanoma, a colorectal cancer, a lung cancer, a mesothelioma cancer, a breast cancer, a head and neck cancer, a cervical cancer, or an ovarian cancer.

7. The method of any one of claims 1-6, wherein the sample is from a peripheral blood sample, a tumor sample, or a processed sample therefrom of the subject.

8. The method of any one of claims 1-7, wherein the cell culture medium comprises IL-7, IL- 15, or a combination thereof.

9. The method of claim 8, wherein the cell culture medium comprises the IL-7, and wherein the IL-7 is present at a concentration of at least about 20 ng / mL.

10. The method of claim 8, wherein the cell culture medium comprises the IL-15, and wherein the IL-15 is present at a concentration of at least about 30 ng / mL.

11. The method of any one of claims 1-10, further comprising: harvesting an expanded population of the CD8+CD103+ TRLs, wherein the expanded population is obtained from the culturing of (b).

12. The method of any one of claims 1-11, wherein the CD8+CD103+ TRLs are circulating tumor-reactive lymphocytes (cTRLs).

13. The method of any one of claims 1-12, wherein the culturing of (b) is performed for at least about 14 days.

14. The method of any one of claims 1-12, wherein the culturing of (b) is performed for at least about 7 days.

15. The method of any one of claims 1-12, wherein the culturing of (b) is performed for at least about 5 days.

16. The method of any one of claims 1-15, wherein the culturing of (b) increases a cell number of the CD8+CD103+ TRLs by at least about 1000-fold.

17. The method of any one of claims 1-16, wherein the culturing of (b) increases a cell number of the CD8+CD103+ TRLs by at least about 10-fold greater than that of CD8+CD103+ TRLs cultured in a culture medium comprising a biologically reactive amount of IL-2.

18. The method of any one of claims 11-17, wherein a purity of the expanded population comprises at least about 95% purity.

19. The method of any one of claims 11-18, wherein the expanded population of the CD8+CD103+ TRLs is characterized by having an enhanced IL-2 sensitivity.

20. The method of any one of claims 11-19, wherein an IL-2 sensitivity of the expanded population of the CD8+CD103+ TRLs is at least about 5-fold greater than that of CD8+CD103+ TRLs cultured in a culture medium comprising the biologically reactive amount of IL-2.

21. The method of any one of claims 11-20, wherein the expanded population of the CD8+CD103+ TRLs has at least about 60% increase in CD25+ expression compared to that of CD8+CD103+ TRLs cultured in a culture medium comprising a biologically reactive amount of IL-2.

22. The method of any one of claims 11-21, wherein the expanded population of the CD8+CD103+ TRLs has an enhanced cytotoxicity compared to that of CD8+CD103+ TRLs cultured in a culture medium comprising a biologically reactive amount of IL-2.

23. The method of any one of claims 11-22, wherein at least about 90% of the expanded population of CD8+CD103+ TRLs comprise an effector memory T cell.

24. The method of claim 23, wherein the effector memory T cells comprise CCR7-CD45RO+.

25. The method of any one of claims 1-24, further comprising: enriching the CD8+CD103+ TRLs after the obtaining of (a), wherein the enriching comprises contacting the CD8+CD103+ lymphocytes of (a) with an antibody or antigenbinding fragment specific to CD8 or CD 103, wherein the antibody or antigen -binding fragment is conjugated to a magnetic nanoparticle, andmagnetically separating the CD8+CD103+ lymphocytes from other components of the peripheral blood or the processed sample obtained therefrom by flowing the CD8+CD103+ lymphocytes across a magnetic capture zone disposed in a channel of a microfluidic device.

26. The method of claim 25, wherein the microfluidic device comprises a plurality of magnetic capture zones, and wherein the plurality of magnetic capture zones is disposed to spatially separate cells with different degrees of magnetization.

27. The method of any one of claims 11-26, wherein the culturing of (b), the harvesting, or a combination thereof occurs in a closed system.

28. The method of any one of claims 11-27, wherein the culturing of (b), the harvesting, or a combination thereof occurs in an open system.

29. The method of any one of claims 11-28, further comprising administering the expanded population of the CD8+CD103+ TRLs to a subject in need thereof.

30. The method of any one of claims 11-29, wherein the expanded population of the CD8+CD103+ TRLs further comprises a pharmaceutically acceptable excipient, diluent, or vehicle.

31. The method of claim 30, wherein the expanded population of the CD8+CD103+ TRLs further comprises a carbohydrate, an amino acid, a vitamin, a mineral, a pH buffer system, an anticoagulant, or any combination thereof.

32. The method of any one of claims 11-31, further comprising modifying a CD8+CD103+ TRLs from the expanded population of the CD8+CD103+ TRLs to obtain an engineered CD8+CD103+ TRL by introducing an exogenous polynucleotide that encodes a therapeutically enhancing polypeptide under conditions sufficient to produce the therapeutically enhancing polypeptide by the at least one cell.

33. The method of claim 32, wherein the exogenous polynucleotide encodes a chimeric antigen receptor (CAR).

34. The method of claim 33, wherein the CAR comprises an antigen binding domain.

35. The method of claim 33, wherein the antigen binding domain binds to a tumor antigen.

36. The method of any one of claims 33-35, wherein the exogenous polynucleotide encodes an engineered T-Cell receptor.

37. The method of claim 36, wherein the engineered T-Cell receptor comprises an antigen binding domain.

38. The method of claim 37, wherein the antigen binding domain binds to a tumor antigen.

39. The method of any one of claims 32-38, further comprising expanding the engineered CD8+CD103+ TRL.

40. The method of claim 39, wherein the engineered CD8+CD103+ TRL is cultured in a culture medium lacking IL-2.

41. The method of claim 39, wherein the engineered CD8+CD103+ TRL is cultured in a culture medium comprising IL- 15, IL-7, and IL-2.

42. A method of treating a human subject with a cancer, the method comprising: administering the expanded population of CD8+CD103+ tumor reactive lymphocytes (TRLs) of the method of any one of claims 11-41 to a subject in need thereof.

43. The method of claim 42, wherein the subject has a cancer, or is suspected of having a cancer, wherein the cancer comprises a melanoma, a colorectal cancer, a lung cancer, a mesothelioma cancer, a breast cancer, a head and neck cancer, a cervical cancer, or an ovarian cancer.

44. The method of any one of claims 4-43, wherein the biologically reactive amount of IL-2 comprises an amount sufficient to induce phosphorylation of one or more IL-2 receptor downstream signaling proteins.

45. The method of claim 44, wherein the one or more IL-2 receptor downstream signaling proteins comprise STAT5.

46. The method of claim 44 or 45, wherein the phosphorylation of one or more IL-2 receptor downstream signaling proteins is increased by at least about 1.1 -fold, at least about 1.2- fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2.0-fold or more as compared to that of an unstimulated control.

47. A composition consisting essentially of CD8+CD103+ tumor-reactive lymphocytes (TRLs) and a pharmaceutically acceptable excipient, diluent, or vehicle, wherein the CD8+CD103+ TRLs are from an expanded population of CD8+CD103+ TRLs cultured in a culture medium lacking IL-2.

48. A composition comprising CD8+CD103+ tumor-reactive lymphocytes (TRLs) and a pharmaceutically acceptable excipient, diluent, or vehicle, wherein the CD8+CD103+ TRLs are from an expanded population of CD8+CD103+ TRLs cultured in a culture medium lacking IL-2.

49. The composition of claim 47 or 48, wherein the culture medium that lacks IL-2 comprises less than a trace amount of IL-2.

50. The composition of claim 47 or 48, wherein the culture medium that lacks IL-2 comprises less than a biologically reactive amount of IL-2.

51. The composition of claim 47 or 48, wherein the culture medium that lacks IL-2 comprises less than about 50 HJ / mL, less than about 40 HJ / mL, less than about 30 lU / mL, less than about 20 HJ / mL, less than about 10 lU / mL, less than about 5 lU / mL, less than about 2 lU / mL, or less than about 1 lU / mL of IL-2.

52. The composition of any one of claims 47-51, wherein the subject has a cancer, or is suspected of having a cancer.

53. The composition of any one of claims 47-52, wherein the cell culture medium comprises IL-7, IL-15, or a combination thereof.

54. The composition of claim 53, wherein the IL-7 is present at a concentration of at least about 20 ng / mL.

55. The composition of claim 53, wherein the IL-15 is present at a concentration of at least about 30 ng / mL.

56. The composition of any one of claims 47-55, wherein the CD8+CD103+ TRLs are circulating tumor-reactive lymphocytes (cTRLs).

57. The composition of any one of claims 47-56, wherein the expanded population of the CD8+CD103+ TRLs is characterized by having an enhanced IL-2 sensitivity.

58. The composition of any one of claims 47-57, wherein the IL-2 sensitivity of the expanded population of the CD8+CD103+ TRLs is at least about 5-fold greater than that of CD8+CD103+ TRLs cultured in a culture medium comprising a biologically reactive amount of IL-2.

59. The composition of any one of claims 47-58, wherein the expanded population of the CD8+CD103+ TRLs has at least about 60% increase in CD25+ expression compared to that of CD8+CD103+ TRLs cultured in a culture medium comprising a biologically reactive amount of IL-2.

60. The composition of any one of claims 47-59, wherein the expanded population of the CD8+CD103+ TRLs has an enhanced cytotoxicity compared to that of CD8+CD103+ TRLs cultured in a culture medium comprising a biologically reactive amount of IL-2.

61. The composition of any one of claims 47-60, wherein at least about 90% of cells in the expanded population of CD8+CD103+ TRLs comprise an effector memory T cell.

62. The composition of any one of claims 47-61, wherein the expanded population of CD8+CD103+ TRLs comprises a population of engineered CD8+CD103+ TRLs, wherein an engineered CD8+CD103+ TRL of the population of engineered CD8+CD103+ TRLsis modified by introducing an exogenous polynucleotide that encodes a therapeutically enhancing polypeptide under conditions sufficient to produce the therapeutically enhancing polypeptide by the at least one cell.

63. The composition of claim 62, wherein the exogenous polynucleotide encodes a chimeric antigen receptor (CAR).

64. The composition of claim 63, wherein the CAR comprises an antigen binding domain.

65. The composition of claim 64, wherein the antigen binding domain binds to a tumor antigen.

66. The composition of any one of claims 62-65, wherein the exogenous polynucleotide encodes an engineered T-Cell receptor.

67. The composition of claim 66, wherein the engineered T-Cell receptor comprises an antigen binding domain.

68. The composition of claim 67, wherein the antigen binding domain binds to a tumor antigen.

69. The composition of any one of claims 52-68, wherein the cancer comprises a melanoma, a colorectal cancer, a lung cancer, mesothelioma cancer, breast cancer, head and neck cancer, cervical cancer, or ovarian cancer.

70. The composition of any one of claims 50-69, wherein the biologically reactive amount of IL-2 comprises an amount sufficient to induce phosphorylation of one or more IL-2 receptor downstream signaling proteins.

71. The composition of claim 70, wherein the one or more IL-2 receptor downstream signaling proteins comprise STAT5.

72. The composition of claim 70 or 71, wherein the phosphorylation of one or more IL-2 receptor downstream signaling proteins is increased by at least about 1.1 -fold, at least about 1.2-fold, at least about 1.3-fold, at least about 1.4-fold, at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2.0-fold or more as compared to that of an unstimulated control.

73. A pharmaceutical formulation comprising the composition of any one of claims 47-72.

74. An engineered circulating tumor-reactive lymphocyte (cTRL), comprising:(a) a cell surface marker, wherein the cell surface marker comprises CD8 and CD 103; and(b) a chimeric antigen receptor (CAR), wherein the engineered cTRL is for use in treating cancer in a human subject, wherein the engineered cTRL is cultured in a culture medium lacking IL-2.

75. A system configured to perform the method of any one of claims 1-46.

76. The system of claim 75, wherein the system is configured to produce the composition of any one of claims 47-74.

77. The system of claim 75 or 76, wherein the system comprises the composition of any one of claims 47-74.

78. The system of any one of claims 75-77, wherein the system comprises a computer readable medium operatively coupled to a processor wherein the computer readable medium is configured to perform the method of any one of claims 1-46.

79. The system of any one of claims 75-78, wherein the system comprises a cell culture apparatus.

80. The system of any one of claims 75-79, wherein the system comprises a cell culture medium that does not comprise a biologically reactive amount of IL-2.

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