Compositions and methods of enhancing tumor reactive lymphocytes

Abstract

Provided herein are methods of isolating, providing, 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. Further provided herein are methods of modifying the TRLs by modulating one or more target genes. Also provided herein are methods of administering a population of TRLs or a population of modified TRLs to a subject in need thereof.

Description

COMPOSITIONS AND METHODS OF ENHANCING TUMOR REACTIVE LYMPHOCYTESCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 637,040, filed April 22, 2024, and U.S. Provisional Application No. 63 / 635,539, filed April 17, 2024, each of which is hereby incorporated by reference in its entirety.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 and modifying 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 producing a plurality of modified tumor-reactive lymphocytes (TRLs), the method comprising: (a) isolating a plurality of T cells from a peripheral blood sample from a subject having solid malignant tumor or a processed sample obtained therefrom, wherein the plurality of T cells comprises greater than or equal to about 20% CD8+CD103+ circulating TRLs (cTRLs); (b) genetically modifying the CD8+CD103+ cTRLs to produce a genetically modified CD8+CD103+ cTRL, wherein the genetically modifying comprises introducing into or expressing in the CD8+CD103+ cTRL a gene editing platform comprising a nuclease configured to target at least one target gene, and (c) expanding the genetically modified CD8+CD103+ cTRL thereby producing the plurality of modified TRLs. In some embodiments, the at least one target gene is involved in overcoming a tumor microenvironment (TME). In some embodiments, the at least one target gene comprises PD-1, CTLA-4, TIGIT, LAG-3, TIM-3, TGF-BR, IL-10R, CD39, CD73, FAS, A2AR, DGK, SMAD2, SMAD3, or SMALM. In some embodiments, the at least one target gene is involved in improved effector function. In some embodiments, the at least one target gene comprises CISH, REGNASE- 1, SOCS-1, IKZF3, or TCEB2. In some embodiments, the at least one target gene is involved in T cell fitness and / or exhaustion. In some embodiments, the at least one target gene comprises TET2, DNMT3, SUV39H1, EZH2, BATF, IRF4, NF AT, NF-kB, CBLB, AP-1, FOXO1, FOXO3, EOMES, PRDM1, MED 12, SNX9, ARID 1 A, ARID2, SMARCC1, SMARCD2, RUNX3, BHLHE40, FLU, SNX9, NR4A3, CCNC, HPK1 PTP1B, RASA2, PTPN2, ID3, SOX4, CUL5,TLE4, IKZF2, CD70, ROQUIN-1 DAP5, RGS16, BLIMP-1, T0X1, or TOX2. In some embodiments, the genetically modified CD8+CD103+ cTRL exhibits reduced or eliminated expression level of the at least one target gene compared to that of a control CD8+CD103+ cTRL lacking the polynucleotide-guided endonuclease or the at least one guided RNA. In some embodiments, the expression level is reduced or eliminated by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% as compared to that of the control CD8+CD103+ cTRL. In some embodiments, the genetically modified CD8+CD103+ cTRL exhibits increased expression level of the at least one target gene compared to that of a control CD8+CD103+ cTRL lacking the polynucleotide-guided endonuclease or the at least one guided RNA. In some embodiments, the expression level is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 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 250%, at least about 300%, at least about 400%, or at least about 500% as compared to that of the control CD8+CD103+ cTRL. In some embodiments, the nuclease comprises a polynucleotide guided endonuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, an RNA-binding protein (RBP), a CRISPR-associated RNA binding protein, a derivative thereof, a variant thereof, or a fragment thereof. In some embodiments, the polynucleotide-guided endonuclease comprises a CRISPR-associated (Cas) protein. In some embodiments, the Cas protein is a Cas nickase (nCas) or deactivated Cas (dCas) protein that exhibits reduced or no endonuclease activity. In some embodiments, the polynucleotide-guided endonuclease further comprises a domain that effects one or more activities of the Cas protein. In some embodiments, the domain comprises a deaminase or a reverse transcriptase. In some embodiments, the domain comprises a transcription regulator. In some embodiments, the gene editing platform further comprises at least one guide RNA exhibiting specific binding to a target polynucleotide sequence at or adjacent to the at least one target gene. In some embodiments, the solid malignant tumor is obtained from the subject having a melanoma, a colorectal cancer, a head and neck cancer, a lung cancer, or any combination thereof. In some embodiments, the plurality of T cells comprises greater than or equal to about 50% CD8+CD103+ circulating TRLs (cTRLs). In some embodiments, the plurality of T cells comprises greater than or equal to about 70% CD8+CD103+ circulating TRLs (cTRLs). In some embodiments, the plurality of T cells comprises greater than or equal to about 80% CD8+CD103+ circulating TRLs (cTRLs). In some embodiments, the plurality of T cells comprises greater than or equal to about 90% CD8+CD103+ circulating TRLs (cTRLs). In some embodiments, theplurality of modified TRLs comprise greater than or equal to about 50% CD8+CD103+ modified TRLs. In some embodiments, the plurality of modified TRLs comprise greater than or equal to about 70% CD8+CD103+ modified TRLs. In some embodiments, the plurality of modified TRLs comprise greater than or equal to about 80% CD8+CD103+ modified TRLs. In some embodiments, the plurality of modified TRLs comprise greater than or equal to about 90% CD8+CD103+ modified TRLs. In some embodiments, the CD8+CD103+ TRLs are isolated or obtained by magnetically separating an initial population of CD8+CD103+ TRLs from the peripheral blood sample from the subject having solid malignant tumor or the processed sample obtained therefrom across a magnetic capture zone disposed in a channel of a microfluidic device. In some embodiments, the magnetically separating comprises the peripheral blood sample from the subject having solid malignant tumor or the processed sample obtained therefrom with an antibody capable of binding to a tumor-reactive lymphocyte (TRL) surface protein present on the initial population of CD8+CD103+ cTRLs. In some embodiments, the antibody is further coupled to a magnetic nanoparticle. In some embodiments, the TRL surface protein is CD3, CD4, CD8, CD39, CD103, SLC6A19, SIDT1 or any combination thereof. In some embodiments, the antibody is conjugated to a magnetic nanoparticle. 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 method further comprises 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 of the plurality of modified TRLs. 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 CAR binds to an antigen selected from the group consisting of Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY-SEO-1, tyrosinase, Melan-A / MART, gplOO, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD33, CD123, PD-L1, IGF1R, CD4, CSPG4, B7- H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, Mesothelin, GPC3, MUC1 and CTAG1B. In some embodiments, the method further comprises administering to the subject a composition comprising a therapeutically effective amount of the plurality of modified TRLs.

[0004] Further provided herein is a method of producing a plurality of modified tumor-reactive lymphocytes (TRLs), the method comprising: expanding a genetically modified CD8+CD103+ cTRL thereby producing the plurality of modified TRLs, wherein the genetically modified CD8+CD103+ cTRL is generated by: (a) providing a plurality of T cells isolated from a peripheral blood sample from a subject having solid malignant tumor or a processed sample obtained therefrom, wherein the plurality of T cells comprises greater than or equal to about 20% CD8+CD103+ circulating TRLs (cTRLs); and (b) modifying a CD8+CD103+ cTRL in the plurality of T cells to produce the genetically modified CD8+CD103+ cTRL by introducing into or expressing in the CD8+CD103+ cTRL a gene editing platform comprising a nuclease configured to target at least one target gene. In some embodiments, the at least one target gene is involved in overcoming a tumor microenvironment (TME). In some embodiments, the at least one target gene comprises PD-1, CTLA-4, TIGIT, LAG-3, TIM-3, TGF-BR, IL-10R, CD39, CD73, FAS, A2AR, DGK, SMAD2, SMAD3, or SMAD4. In some embodiments, the at least one target gene is involved in improved effector function. In some embodiments, the at least one target gene comprises CISH, REGNASE-1, SOCS-1, IKZF3, or TCEB2. In some embodiments, the at least one target gene is involved in T cell fitness and / or exhaustion. In some embodiments, the at least one target gene comprises TET2, DNMT3, SUV39H1, EZH2, BATF, IRF4, NFAT, NF-kB, CBLB, AP-1, FOXO1, FOXO3, EOMES, PRDM1, MED12, SNX9, ARID1A, ARID2, SMARCC1, SMARCD2, RUNX3, BHLHE40, FLU, SNX9, NR4A3, CCNC, HPK1 PTP1B, RASA2, PTPN2, ID3, SOX4, CUL5, TLE4, IKZF2, CD70, ROQUIN-1 DAP5, RGS16, BLIMP - 1, TOX1, or TOX2. In some embodiments, the genetically modified CD8+CD103+ cTRL exhibits reduced or eliminated expression level of the at least one target gene compared to that of a control CD8+CD103+ cTRL lacking the polynucleotide-guided endonuclease or the at least one guided RNA. In some embodiments, the expression level is reduced or eliminated by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% as compared to that of the control CD8+CD103+ cTRL. In some embodiments, the genetically modified CD8+CD103+ cTRL exhibits increased expression level of the at least one target gene compared to that of a control CD8+CD103+ cTRL lacking the polynucleotide-guided endonuclease or the at least one guided RNA. In some embodiments, the expression level is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 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 250%, at least about 300%, at least about 400%, or at least about 500% as compared to that of the control CD8+CD103+ cTRL. In some embodiments,the nuclease comprises a polynucleotide guided endonuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, an RNA-binding protein (RBP), a CRISPR-associated RNA binding protein, a derivative thereof, a variant thereof, or a fragment thereof. In some embodiments, the polynucleotide-guided endonuclease comprises a CRISPR-associated (Cas) protein. In some embodiments, the Cas protein is a Cas nickase (nCas) or deactivated Cas (dCas) protein that exhibits reduced or no endonuclease activity. In some embodiments, the polynucleotide-guided endonuclease further comprises a domain that effects one or more activities of the Cas protein. In some embodiments, the domain comprises a deaminase or a reverse transcriptase. In some embodiments, the domain comprises a transcription regulator. In some embodiments, the gene editing platform further comprises at least one guide RNA exhibiting specific binding to a target polynucleotide sequence at or adjacent to the at least one target gene. In some embodiments, the solid malignant tumor is obtained from the subject having a melanoma, a colorectal cancer, a head and neck cancer, a lung cancer, or any combination thereof. In some embodiments, the plurality of T cells comprises greater than or equal to about 50% CD8+CD103+ circulating TRLs (cTRLs). In some embodiments, the plurality of T cells comprises greater than or equal to about 70% CD8+CD103+ circulating TRLs (cTRLs). In some embodiments, the plurality of T cells comprises greater than or equal to about 80% CD8+CD103+ circulating TRLs (cTRLs). In some embodiments, the plurality of T cells comprises greater than or equal to about 90% CD8+CD103+ circulating TRLs (cTRLs). In some embodiments, the plurality of modified TRLs comprise greater than or equal to about 50% CD8+CD103+ modified TRLs. In some embodiments, the plurality of modified TRLs comprise greater than or equal to about 70% CD8+CD103+ modified TRLs. In some embodiments, the plurality of modified TRLs comprise greater than or equal to about 80% CD8+CD103+ modified TRLs. In some embodiments, the plurality of modified TRLs comprise greater than or equal to about 90% CD8+CD103+ modified TRLs. In some embodiments, the CD8+CD103+ TRLs are isolated or obtained by magnetically separating an initial population of CD8+CD103+ TRLs from the peripheral blood sample from the subject having solid malignant tumor or the processed sample obtained therefrom across a magnetic capture zone disposed in a channel of a microfluidic device. In some embodiments, the magnetically separating comprises the peripheral blood sample from the subject having solid malignant tumor or the processed sample obtained therefrom with an antibody capable of binding to a tumor-reactive lymphocyte (TRL) surface protein present on the initial population of CD8+CD103+ cTRLs. In some embodiments, the antibody is further coupled to a magnetic nanoparticle. In some embodiments, the TRL surface protein is CD3, CD4, CD8, CD39, CD103, SLC6A19, SIDT1 or any combination thereof. In some embodiments, the antibodyis conjugated to a magnetic nanoparticle. 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 method further comprises 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 of the plurality of modified TRLs. 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 CAR binds to an antigen selected from the group consisting of Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY-SEO-1, tyrosinase, Melan-A / MART, gplOO, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD33, CD123, PD-L1, IGF1R, CD4, CSPG4, B7- H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, Mesothelin, GPC3, MUC1 and CTAG1B. In some embodiments, the method further comprises administering to the subject a composition comprising a therapeutically effective amount of the plurality of modified TRLs.

[0005] Provided herein is a population of modified tumor reactive lymphocytes (TRLs) comprising: a plurality of CD8+CD103+ tumor reactive lymphocytes (TRLs) comprising one or more modifications in at least one target gene, wherein the at least one target gene is selected from the group consisting of: CD73, A2AR, DGK, REGNASE-1, IKZF3, TCEB2, TET2, DNMT3, SUV39H1, EZH2 IRF4, NFAT, NF-kB, CBLB, AP-1, FOXO1, FOXO3, EOMES MED12, SNX9, ARID1A, ARID2, SMARCC1, SMARCD2, RUNX3, BHLHE40, FLU, SNX9, NR4A3, CCNC, HPK1 PTP1B, RASA2, PTPN2, ID3, SOX4, CUL5, TLE4, IKZF2, CD70, ROQUIN-1, DAP5, RGS16, and BLIMP- 1. In some embodiments, the population of modified TRLs exhibits (a) overcoming tumor microenvironment (TME), (b) improved an effector function, (c) improved T cell fitness and / or exhaustion, or (d) any combination thereof. In some embodiments, expression level of a target gene of the at least one target gene is reduced or eliminated compared to expression level of the target gene in a control population of TRL lacking the one or more modification. In some embodiments, the expression level of a target gene of the at least one target gene is increased compared to expression level of the target gene in a control population of TRL lacking the one or more modification. In some embodiments, the one or more modifications aregenetically edited by a gene editing platform comprising a nuclease configured to target the at least one target gene. In some embodiments, the nuclease comprises a polynucleotide guided endonuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, an RNA-binding protein (RBP), a CRISPR-associated RNA binding protein, a derivative thereof, a variant thereof, or a fragment thereof. In some embodiments, the polynucleotide-guided endonuclease comprises a CRISPR-associated (Cas) protein. In some embodiments, the Cas protein is a Cas nickase (nCas) or deactivated Cas (dCas) protein that exhibits reduced or no endonuclease activity. In some embodiments, the polynucleotide-guided endonuclease further comprises a domain that effects one or more activities of the Cas protein. In some embodiments, the domain comprises a deaminase or a reverse transcriptase. In some embodiments, the domain comprises a transcription regulator. In some embodiments, the gene editing platform further comprises at least one guide RNA exhibiting specific binding to a target polynucleotide sequence at or adjacent to the at least one target gene. In some embodiments, the plurality of CD8+CD103+ TRLs is obtained from a peripheral blood sample from a subject having solid malignant tumor or a processed sample obtained therefrom. In some embodiments, the solid malignant tumor comprises a tumor from melanoma, a colorectal cancer, a head and neck cancer, a lung cancer, or any combination thereof.

[0006] Further provided herein is a method of treating a cancer in a subject in need thereof, the method comprising: administering to the subject a composition comprising a therapeutically effective amount of the population of modified TRLs described herein. In some embodiments, the population of modified TRLs comprises a plurality of CD8+CD103+ tumor reactive lymphocytes (TRLs) comprising one or more modifications in at least one target gene, wherein the at least one target gene is selected from the group consisting of: CD73, A2AR, DGK, REGNASE-1, IKZF3, TCEB2, TET2, DNMT3, SUV39H1, EZH2 IRF4, NFAT, NF-kB, CBLB, AP-1, FOXO1, FOXO3, EOMES MED 12, SNX9, ARID 1 A, ARID2, SMARCC1, SMARCD2, RUNX3, BHLHE40, FLU, SNX9, NR4A3, CCNC, HPK1 PTP1B, RASA2, PTPN2, ID3, SOX4, CUL5, TLE4, IKZF2, CD70, ROQUIN-1, DAP5, RGS16, and BLIMP- 1. In some embodiments, the population of modified TRLs exhibits (a) overcoming tumor microenvironment (TME), (b) improved an effector function, (c) improved T cell fitness and / or exhaustion, or (d) any combination thereof. In some embodiments, expression level of a target gene of the at least one target gene is reduced or eliminated compared to expression level of the target gene in a control population of TRL lacking the one or more modification. In some embodiments, expression level of a target gene of the at least one target gene is increased compared to expression level of the target gene in a control population of TRL lacking the one or more modification. In someembodiments, the one or more modifications are genetically edited by a polynucleotide-guided polypeptide mediated gene editing platform. In some embodiments, the polynucleotide-guided polypeptide mediated gene editing platform comprises: (i) a polynucleotide-guided endonuclease; and (ii) at least one guide RNA exhibiting specific binding to a target polynucleotide sequence at or adjacent to the at least one target gene. In some embodiments, the polynucleotide-guided endonuclease is a Cas protein. In some embodiments, the Cas protein is a Cas nickase (nCas) or deactivated Cas (dCas) protein that exhibits reduced or no endonuclease activity. In some embodiments, the polynucleotide-guided endonuclease further comprises a domain that effects one or more activities of the Cas protein. In some embodiments, the domain comprises a deaminase or a reverse transcriptase. In some embodiments, the domain comprises a transcription regulator. In some embodiments, the plurality of CD8+CD103+ TRLs is obtained from a peripheral blood sample from a subject having solid malignant tumor or a processed sample obtained therefrom. In some embodiments, the solid malignant tumor comprises a tumor from melanoma, a colorectal cancer, a head and neck cancer, a lung cancer, or any combination thereof.INCORPORATION BY REFERENCE

[0007] 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

[0008] 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:

[0009] 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 bymicrofluidic 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. FIGs. 1C and ID show the quantitation of OVA tumor-reactive T cells in tumor (FIG. 1C) and blood (FIG. ID). FIGs. IE and IF show HA tumor reactive T-cells in tumor (FIG. IE) and blood (FIG. IF). FIGs. 1G-1N show the quantitation 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), post-sorting with CD8 antibodies (FIG.1H), and post-sorting with multimer (FIG. II). FIG. 1J shows quantitative analysis of CD8+ cells. FIGs. 1K-1M schematically show gating scheme for HA-reactive T cells at pre- sorting (FIG.1K), post-sorting with CD8 antibody (FIG.1L), and post-sorting with multimer (FIG. IM). FIG. IN shows quantitative analysis of HA-reactive T cells.

[0010] FIGs. 2A-2E show a comparison of clonal similarity among OVA reactive TIL (FIG. 2A), circulating TRL (cTRL; FIG. 2B), and peripheral blood monocyte (PBMC; FIG. 2C) by V-J usage profile. FIGs. 2D and 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.

[0011] 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. FIGs. 21 and 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.

[0012] 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 mul timer-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). FIGs. 31 and 3 J show the quantitation of tumor-reactive fraction in circulating CD8+CD103+ populations in B16 (FIG. 31) and AE17 (FIG. 3 J) models. FIGs. 3K and 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 quantitation of the percentage of migrated cTRLs in the in vivo TIL migration model in B16. FIG. 3N shows the quantitation 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 CD103 (FIG. 3Q) in AE17 models. As shown in FIGs. 3R and 3S, CD 103+ cells were more predominant in CD45.2+ population (FIG. 3R) than CD451+ population (FIG. 3S). FIG. 3T shows the quantitation of CyTOF data for the expression of CD 103, CD69 and Programmed cell death protein 1 (PD-1) in CD45.2+ cTRLs and CD45.1+ TILs. Unpaired t-test, mean+s.d., each dot represents a biological replicate.

[0013] 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. FIGs. 4D and 4E show the quantitation 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), CD 103- 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 quantitation 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.

[0014] 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 quantitation 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. FIGs. 5E and 5F show the quantitation of infiltrated CD4+ T cells (FIG. 5E) and CD208+ dendritic cells (FIG. 5F) post different therapeutic modalities. FIGs. 5G and 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 quantitation 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 quantitation 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 in endogenous (CD45.1+) populations in s.c. AE17 models. FIG. 5Q shows the quantitation of CD 103 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.

[0015] 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 quantitation 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 co-cultured with CD8+CD103+ population (FIG. 6L). This specific set of images is from PE86. FIG. 6M and FIG. 6N show the quantitation 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.

[0016] FIGs. 7A-7F show the results of experiments in which human CTRLs were isolated and validated. FIGs. 7A and 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 Deficient-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 quantitation of interferon-gamma (IFN-y) secreting populations in SLC6A19+, CD103, and SIDT1+ populations of CD8+ T cells according to gate shown in FIGs. 7A and 7B across the patient cohort: PE96 (FIG. 7C), P29 (FIG. 7D), P30 (FIG. 7E), and P31 (FIG. 7F).

[0017] 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. FIGs. 8B and 8C show the quantitation of OVA tumor-reactive T cells in tumor (FIG. 8B) and blood (FIG. 8C). FIGs. 8D and 8E show HA tumor reactive T-cells in tumor (FIG. 8D) and blood (FIG. 8E).

[0018] 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 (FIGs. 9E and 9F) and B 16 (WT; FIGs. 9G and 9H) models. FIGs. 91 and 9 J show quantitation 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). FIGs. 9M and 9P show quantitation of T cell infiltration (FIG. 9M) and its impact on tumor weight (FIG. 9P) at the end point for the CT26-HA model.

[0019] 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 10F show the representative cytometric profile for the B16-OVA (FIG. 10E) and B 16 (WT; FIG. 10F) models. FIG. 10G andFIG. 10H show the representative cytometric profile for the CT26-HA (FIG. 10G) and CT26 (WT; FIG. 10H) model.

[0020] FIGs. 11A-11J illustrate establishment of the in vivo TIL migration model. FIG. 11A 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. 11D show the representative flow cytometric profile from the in vivo TIL migration with B16-OVA (FIG. 11C) and B16 (WT; FIG. 11D). 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. FIGs. HG and HH show the quantitation of the percentage of migrated cTRLs in the in vivo TIL migration model with B16-OVA and WT (FIG. HG) and AE17-OVA and WT (FIG. HH). FIG. HI and FIG. 11 J show the quantitation of CD45.2 / CD45.1 percentage of migrated TILs in the in vivo TIL migration model with B16- OVA and WT (FIG. HI) and AE17-OVA and WT (FIG. HJ).

[0021] 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.

[0022] FIG. 13 shows the representative workflow of microfluidic magnetic cell sorting according to some embodiments herein.

[0023] 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), afterMACS (FIG. 14C) and after microfluidics (FIG. 14D). FIGs. 14E and 14F show quantifications of isolated OVA-specific cells (FIG. 14E) and recovered OVA-specific T cells (FIG. 14F).

[0024] 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.

[0025] FIGs. 16A-16B shows the molecular signature of cTRLs from the CT26 model. FIG. 16A shows volcano plots show the differential expression (DE) of genes when comparing the FPKM from PBMC and cTRLs from the CT26 model. FIG. 16B shows key genes for CD8+ T cells were presented as a heatmap alongside.

[0026] 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).

[0027] 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 a 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). FIGs. 18E and 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 quantitation 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.

[0028] 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 quantitation of the fold of expansion.

[0029] FIGs. 20A-20I illustrate phenotyping of expanded cTRLs from MC-38 models. FIG. 20A represents the quantitation of relative mRNA expression of cTRLs compared to TILs via TaqMan probes. FIG. 20B-20I represents the quantitation 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).

[0030] 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.

[0031] 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. 22A) or KPCY 6419c5 (FIG. 22B) tumors.

[0032] FIGs. 23A-23J illustrate representative pictures and immunohistochemistry images of various tumor models post treatment. FIGs. 23A and 23B show representative pictures (FIG. 23A) and immunochemistry images (FIG. 23B) of B16F10 in WT C57BL6 mice on day 18. FIGs. 23C and 23D show representative pictures (FIG. 23C) and immunochemistry images (FIG. 23D) of LLC-1 in WT C57BL6 mice on day 15. FIGs. 23E and 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. FIGs. 231 and 23J show representative pictures (FIG. 231) and immunochemistry images (FIG. 23 J) of AE17 in CD45.1 C57BL6 mice on day 15.

[0033] FIGs. 24A-24J represent an automatic quantitation 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-24Gfurther illustrate representative decomposed images from the tumors as shown on 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).

[0034] 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 quantitation 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).

[0035] 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).

[0036] 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).

[0037] FIGs. 28A-28J represent analyses of large-scale patient data using the TIDE algorithm. FIGs. 28A-28D shows a quantitation of adjusted death risk calculated by TIDE against different markers, such as CD 103 (FIG. 28 A), 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 lymphocytic 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).

[0038] 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 quantitation of the expression level of CD 103, CD39, PD-1 and CD69 in cTRLs during migration (FIG. 29H) and gating schemes (FIG. 291- 29K) for CD 103 vs CD39 (FIG. 291) CD 103 vs PD-1 (FIG. 291) and CD 103 vs CD69 (FIG. 29K).

[0039] 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), CD103+ (FIG. 30C), and CD103- (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 quantitation 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 PE72 (FIG. 30P) of malignant pleural effusion (MPE).

[0040] FIGs. 31A and 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)

[0041] FIG. 32 depicts a schematic of a non-limiting exemplary method for analyzing the tumor reactivity of various types of cTRLs.

[0042] 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 / lono (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 / lono (FIG. 33H). CTRLs were isolated from primary tumors or cancers metastasized to the liver.

[0043] FIG. 34 A 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.

[0044] FIG. 35A depicts a schematic of a non-limiting exemplary method for cTRL expansion. FIGs. 35B and 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). FIG. 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

[0045] 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.

[0046] FIG. 37 describes a high level cTRL manufacturing flow diagram.

[0047] FIGs. 38A-38C show phenotype of expanded cTRLs from patients. FIG. 38A shows frequency of CD3+CD8+T-cell subtypes (e.g., T effector memory cells). FIG. 38B shows PD-1 level of expanded cTRLs. FIG. 38C shows viability of the expanded cTRLs.

[0048] FIG. 39 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.

[0049] FIGs. 40A-40J show tumor reactivity of cTRLs (e.g., CD8+CD103+) collected from peripheral blood from patients. FIG. 40A shows the tumor reactive cTRLs were assessed by measuring % IFN-y secretion against autologous melanoma cancer. 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 shows the tumor reactive cTRLs were assessed by measuring % IFN-y secretion against autologous colorectal cancer. FIGs. 40G-40J show gating of CD8+CD103- population of T-cell alone (FIG. 40G) or with Tumor (FIG. 40H), and CD8+CD103+ population of T-cell alone (FIG. 401) or with tumor (FIG. 40 J).

[0050] FIGs. 41A-41G demonstrate the effectiveness of cTRLs in recognizing and targeting autologous tumor cells. FIG. 41A shows cTRLs (e.g., CD8+CD103+) cells from patients in the melanoma cohort displayed potent cytolytic activity. FIGs. 42B-42E show gating of CD8+CD103- population of T-cell alone (FIG. 41B) or with Tumor (FIG. 41C), and CD8+CD103+ population of T-cell alone (FIG. 41D) or with tumor (FIG. 41E). FIG. 41F and FIG. 41G show that cTRLs retained anti-tumor reactivity, as assessed by production of multiple cytokines including IFN-y and TNF-a (FIG. 41F), and expression of cytolytic granules and perforin, granzyme, and granulolysin (FIG. 41G).DETAILED DESCRIPTION

[0051] Disclosed herein, in some embodiments, are compositions, systems and kits comprising isolated and enriched populations of cells obtained from the 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 betumor-reactive lymphocytes (TRLs), such as circulating TRLs (cTRLs), that can recognize a cancer antigen of the cancer and exhibit anti -cancer activity. In some cases, 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 enriched TRLs disclosed herein can secrete interferongamma (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 tumorspecific T cell therapies, such a modified cTRL or an enhanced cTRLs for chimeric antigen receptor (CAR)-T therapy or T-cell receptor (TCR) therapy.

[0052] 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, IL7R, 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. For example, the TRL can be CD8+ and CD103+. In some embodiments, the TRL can be CD8+ CD 103+ and CD39+. In some embodiments, the TRL can be CD8+CD103+ and CD39-. 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 combinationthereof. In some embodiments, a TRL can be CD4+, CD39+, CD103+, SLC6A19+, SIDT1+, or a combination thereof.

[0053] 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, for example, 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 immunoaffinity- 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).

[0054] Further disclosed herein are methods of modulating one or more genes in TRL (e.g., a modified cTRL) from a population of the isolated and / or enriched TRLs comprising CD8+, CD103+, CD39+, SLC6A19+, and / or SIDT1+ lymphocytes (e.g., CD8+CD103+ circulating TRLs) via a gene editing platform described herein.

[0055] 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.

[0056] 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.

[0057] 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

[0058] 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. Disclosed herein are modified TRLs (e.g., modified TRLs with one or more target genes modulated (e.g., increased or decreased)). 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

[0059] 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. In some 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.

[0060] 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 about1,000 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 CD103+cells per 10 million PBMCs.

[0061] 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.

[0062] 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 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, 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.

[0063] 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 leastabout 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, 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.

[0064] 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 %.

[0065] 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%.

[0066] 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).

[0067] 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 a purity 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.

[0068] 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.

[0069] 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 about60% 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 / or SIDT1+ 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.

[0070] 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.

[0071] 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 8,000cells 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.

[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 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.

[0073] In some embodiments, the population of TRLs can be CD3+, CD4+, CD8+, CD39+, 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+, and SIDT1+ expressing cells. In some embodiments, the isolated and enriched TRLs can be CD8+, CD103+, SLC6A19+, and SIDT1+ expressing cells.

[0074] In some embodiments, a composition described herein (e.g., an isolated and / or enriched population of tumor reactive lymphocytes from a sample e.g., a peripheral blood)) can comprise greater than or equal to about 5% CD8+CD103+ circulating TRLs (cTRLs), greater than or equal to about 10% CD8+CD103+ circulating TRLs (cTRLs), greater than or equal to about 15% CD8+CD103+ circulating TRLs (cTRLs), greater than or equal to about 20% CD8+CD103+ circulating TRLs (cTRLs), greater than or equal to about 25% CD8+CD103+ circulating TRLs (cTRLs), greater than or equal to about 30% CD8+CD103+ circulating TRLs (cTRLs), greater than or equal to about 35% CD8+CD103+ circulating TRLs (cTRLs), greater than or equal to about 40% CD8+CD103+ circulating TRLs (cTRLs), greater than or equal to about 45% CD8+CD103+ circulating TRLs (cTRLs), greater than or equal to about 50% CD8+CD103+ circulating TRLs (cTRLs), greater than or equal to about 60% CD8+CD103+ circulating TRLs (cTRLs), greater than or equal to about 70% CD8+CD103+ circulating TRLs (cTRLs), greater than or equal to about 80% CD8+CD103+ circulating TRLs (cTRLs), greater than or equal to about 90% CD8+CD103+ circulating TRLs (cTRLs), or greater than or equal to about 95% CD8+CD103+ circulating TRLs (cTRLs) of a plurality of T cells from a peripheral blood sample (e.g., a total isolated T cells form the sample).

[0075] In some embodiments, a population of tumor reactive lymphocytes can be CD8+CD103+, wherein the population of tumor reactive lymphocytes substantially lack CD39 expression (e.g., CD8+CD103+CD39-). In some embodiments, the CD8+CD 103+ tumor reactive lymphocytes 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 co-express CD39 (e.g., CD8+CD103+CD39+). In some embodiments, the CD8+CD103+ tumor reactive lymphocytes 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 cells 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.

[0076] 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.

[0077] 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.

[0078] 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 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 ofTILs. 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%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% compared to that of the population of TILs.

[0079] In some embodiments, the population of TRLs can exhibit an increase in sternness phenotype (e.g., measured by TCF7, IL7R, 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 about 90%, 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.

[0080] 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%, about70%, about 75%, about 80%, about 85%, about 90%, about 95% compared to that of the population of TILs.B. Modified TRLs

[0081] Described herein are modified TRLs comprising one or more genomic modifications resulting in an increased or reduced expression and / or function of one or more endogenous target genes involved in overcoming tumor microenvironment, improving effector function, and / or T cell fitness / exhaustions.

[0082] In some embodiments, a modified TRL (e.g., a modified cTRL) can comprise one or more modifications (e.g., insertions, deletions, or mutations of one or more nucleic acids) in a genomic DNA sequence of one or more endogenous target genes resulting in an increased or reduced expression and / or function of the endogenous gene. In some embodiments, the modified TRL (e.g., the modified cTRL) can comprise the gene editing platform described herein further comprising a partner (e.g., dCas coupled to a partner such as a transcriptional effector, a deaminase, a reverse transcriptase, or an epigenetic modulator) described herein that can modulate (e.g., increase or decrease) one or more target genes (e.g., genes involved in overcoming tumor microenvironment, improving effector function, and / or T cell fitness / exhaustions) in the genomic DNA sequence of one or more endogenous target genes resulting in an increased or reduced expression and / or function of the endogenous gene. In some embodiments, the modified TRL (e.g., the modified cTRL) can comprise a gene editing platform comprising an epigenetic modifier described herein that can modulate (e.g., increase or decrease) one or more target genes (e.g., genes involved in overcoming tumor microenvironment, improving effector function, fitness / exhaustions) in the genomic DNA sequence of one or more endogenous target genes resulting in an increased or reduced expression and / or function the endogenous gene. In some embodiments, the modified TRL (e.g., the modified cTRL) can comprise a gene editing platform comprising a transcriptional effector (e.g., transcriptional activator or transcriptional repressor) described herein that can modulate (e.g., increase or decrease) one or more target genes (e.g., genes involved in overcoming tumor microenvironment, improving effector function, fitness / exhaustions) in the genomic DNA sequence of one or more endogenous target genes resulting in an increased or reduced expression and / or function the endogenous gene. In some embodiments, the modified TRL (e.g., the modified cTRL) can comprise a gene editing platform comprising a deaminase (e.g., cytidine deaminase or adenosine deaminase) described herein that can modify (insertion, deletion, or mutations) of one or more nucleic acids in the genomic DNA sequence of one or more endogenous target genes resulting in an increased or reduced expressionand / or function the endogenous gene. In some embodiments, the modified TRL (e.g., the modified cTRL) can comprise a gene editing platform comprising a reverse transcriptase (e.g., prime editing) described herein that can modify, by introducing new genetic information, the genomic DNA sequence of one or more endogenous target genes resulting in an increased or reduced expression and / or function the endogenous gene.

[0083] In some embodiments, the modified TRL (e.g., the modified cTRL) described herein can demonstrate an increase in one or more immune cell effector functions. As used herein, the term “effector function” can refer to functions of an immune cell related to the generation, maintenance, and / or enhancement of an immune response against a target cell or target antigen. In some embodiments, the modified TRL described herein can demonstrate one or more of the following characteristics compared to an unmodified TRL: increased infiltration or migration in to a tumor, increased proliferation, increased or prolonged cell viability, increased resistance to inhibitory factors in the surrounding microenvironment such that the activation state of the cell is prolonged or increased, increased production of pro-inflammatory immune factors (e.g., pro- inflammatory cytokines, chemokines, and / or enzymes), increased cytotoxicity, and / or increased resistance to exhaustion. In some embodiments, the modified TRL can demonstrate one or more of the following enhanced efficacy characteristics compared to unmodified TRL: preservation of the TCR repertoire, stimulation-independent growth, in vivo expansion.

[0084] In some embodiments, the modified TRL described herein can demonstrate increased infiltration into a tumor compared to an unmodified TRL. In some embodiments, increased tumor infiltration by modified TRL can be an increase the number of modified TRL infiltrating into a tumor during a given period of time compared to the number of unmodified TRL that infiltrate into a tumor during the same period of time. In some embodiments, the modified TRL can demonstrate at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 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 fold 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 foldincrease in tumor filtration compared to an unmodified TRL. Tumor infiltration can be measured by isolating one or more tumors from a subject and assessing the number of modified immune cells in the sample by flow cytometry, immunohistochemistry, and / or immunofluorescence.

[0085] In some embodiments, a modified TRL described herein can demonstrate an increase in cell proliferation compared to an unmodified TRL. In these embodiments, the result can be an increase in the number of modified TRL present compared to unmodified TRL after a given period of time. For example, in some embodiments, a modified TRL can demonstrate increased rates of proliferation compared to unmodified TRLs, wherein the modified TRL divide at a more rapid rate than unmodified TRLs. In some embodiments, a modified TRL can demonstrate at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 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 fold 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 increase in the rate of proliferation compared to an unmodified TRL.

[0086] In some embodiments, a modified TRL can demonstrate prolonged periods of proliferation compared to unmodified TRLs, wherein the modified TRL and unmodified TRL divide at similar rates, but wherein the modified TRL can maintain the proliferative state for a longer period of time. In some embodiments, a modified TRL can maintain a proliferative state for at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 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 fold 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 longer than an unmodified TRL.

[0087] In some embodiments, a modified TRL described herein can demonstrate increased or prolonged cell viability compared to an unmodified TRL. In such embodiments, the result can be an increase in the number of the modified TRL or present compared to unmodified TRL after a given period of time. For example, in some embodiments, modified TRL described herein can remain viable and persist for at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 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 fold 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 longer than an unmodified TRL.

[0088] In some embodiments, the modified TRL described herein can demonstrate an increased resistance to inhibitory factors compared to an unmodified TRL. Exemplary inhibitory factors can include signaling by immune checkpoint molecules (e.g., PD1, PDL1, CTLA4, LAG3, IDO) and / or inhibitory cytokines (e.g., IL-10, TGFP).

[0089] In some embodiments, the modified T cells described herein can demonstrate an increased resistance to T cell exhaustion compared to an unmodified T cell. T cell exhaustion can be a state of antigen-specific T cell dysfunction characterized by decreased effector function and leading to subsequent deletion of the antigen-specific T cells. In some embodiments, exhausted T cells can lack the ability to proliferate in response to antigen, demonstrate decreased cytokine production, and / or demonstrate decreased cytotoxicity against target cells such as tumor cells. In some embodiments, exhausted T cells can be identified by altered expression of cell surface markers and transcription factors, such as decreased cell surface expression of CD 122 and CD 127; increased expression of inhibitory cell surface markers such as PD1, LAGS, CD244, CD 160, TIM3, and / or CTLA4; and / or increased expression of transcription factors such as Blimpl, NF AT, and / or BATF. In some embodiments, exhausted T cells can demonstrate altered sensitivity tocytokine signaling, such as increased sensitivity to TGFP signaling and / or decreased sensitivity to IL-7 and IL- 15 signaling. In some embodiments, a T cell exhaustion can be determined, for example, by co-culturing the T cells with a population of target cells and measuring T cell proliferation, cytokine production, and / or lysis of the target cells.

[0090] In some embodiments, the modified TRL described herein can be co-cultured with a population of target cells (e.g., autologous tumor cells or cell lines that have been engineered to express a target tumor antigen) and effector cell proliferation, cytokine production, and / or target cell lysis can be measured. These results can be then compared to the results obtained from coculture of target cells with a control population of immune cells (such as unmodified TRL or immune effector cells that have a control modification).

[0091] In some embodiments, a resistance to T cell exhaustion can be demonstrated by increased production of one or more cytokines (e.g., IFNy, TNFa, or IL-2) from the modified TRL compared to the cytokine production observed from the control population of immune cells. In some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 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 fold 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 increase in cytokine production from the modified TRL compared to the cytokine production from the control population of immune cells is indicative of an increased resistance to T cell exhaustion.

[0092] In some embodiments, a resistance to T cell exhaustion can be demonstrated by increased proliferation of the modified TRL compared to the proliferation observed from the control population of immune cells. In some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 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 fold about 15 fold, at least about 20 fold, at least about 30 fold, atleast 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 increase in proliferation of the modified TRL compared to the proliferation of the control population of immune cells is indicative of an increased resistance to T cell exhaustion.

[0093] In some embodiments, a resistance to T cell exhaustion can be demonstrated by increased target cell lysis by the modified TRL compared to the target cell lysis observed by the control population of immune cells. In some embodiments at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 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 fold 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 increase in target cell lysis by the modified TRL compared to the target cell lysis by the control population of immune cells is indicative of an increased resistance to T cell exhaustion.

[0094] In some embodiments, the modified TRL described herein can demonstrate an increased expression or production of pro-inflammatory immune factors compared to an unmodified TRL. Examples of pro-inflammatory immune factors can include cytolytic factors, such as granzyme B, perforin, and granulysin; and pro-inflammatory cytokines such as interferons (IFNa, IFNP, IFNy), TNFa, IL-ip, IL-12, IL-2, IL-17, CXCL8, and / or IL-6.

[0095] In some embodiments, the modified TRL described herein can demonstrate an increased cytotoxicity against a target cell compared to an unmodified TRL. In some embodiments, the modified TRL demonstrate at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 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 fold 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 increase in cytotoxicity against a target cell compared to an unmodified TRL.

[0096] In some embodiments, the modified TRL described herein can demonstrate an increased preservation of the TCR repertoire compared to an unmodified TRL. In some embodiments, the modified TRL demonstrate at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 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 fold 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 increase in preservation of the TCR repertoire compared to an unmodified TRL.

[0097] In some embodiments, the modified TRL described herein can demonstrate an increased stimulation-independent growth compared to an unmodified TRL. In some embodiments, the modified TRL demonstrate at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 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 fold 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 leastabout 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 increase in stimulation-independent growth compared to an unmodified TRL.

[0098] In some embodiments, the modified TRL described herein can demonstrate an increased in vivo expansion compared to an unmodified TRL. In some embodiments, the modified TRL demonstrate at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 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 fold 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 increase in in vivo expansion compared to an unmodified TRL.C. Enhanced TRLs

[0099] 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. 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 a stimulatory 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.

[0100] 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 a, 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, sialyl Lewis 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-cellreceptor, 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.

[0101] 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 receptor molecule 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 functionalsignaling 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.

[0102] 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 some embodiments, 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(^ ITAM 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).

[0103] 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.

[0104] 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 cases, 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 cases, each vector described herein can comprise an expression plasmid.

[0105] In some embodiments, the polynucleotides encoding the CAR can be cloned into a vector comprising lentiviral backbone components. Exemplary backbone components can include, 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 cases, an inducible promoter can be a small molecule ligandinducible two polypeptide ecdysone receptor-based gene switch. In some embodiments, the CAR can be under the control of a constitutive promoter.

[0106] 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 cases, 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.D. Pharmaceutical Formulations

[0107] Described herein are pharmaceutical formulations comprising the TRLs (e.g., cTRLs), the modified TRLs (e.g., modified cTRL), or the enhanced TRLs (e.g., enhanced cTRLs) described herein. In some embodiments, a pharmaceutical formulation can comprise TRLs, modified 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.

[0108] 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 TRLs, the modified TRLs, 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, the modified TRLs, or the enhanced TRLs (e.g., CAR or TCR) can be administered separately from an additional therapeutic agent or adjuvant.

[0109] 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).

[0110] 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.[OHl] 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 subject over at least about 2 days to about 15 days. In some cases, a cyclophosphamide can be administered from about 10 mg / kg to about 15 mg / kg of a subject. In some cases, cyclophosphamide can be administered to a subject over at least about 7 days to about 10 days. In some cases, cyclophosphamide can be administered from about 3 mg per kg to about 5 mg per kg of a subject. In some cases, cyclophosphamide can be administered from about 50 mg per kg to about 80 mg / kg of a subject. In some cases, cyclophosphamide can be administered in excess of 50 mg per kg. In some cases, cyclophosphamide can be administered at about 60 mg per kg. In some cases, fludarabine can be administered from about 20 mg / m2to about 30 mg / m2of body surface area of a subject. In some cases, fludarabine can be administered at about 25 mg / m2of body surface area of a subject. In some cases, 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 group consisting of: bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, albaconazole, efinaconazole, epoxiconazole, fluconazole, isavuconazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole, and voriconazole. An antifungal that is an azole can be fluconazole. In some cases, fluconazole can be administered from about 100 mg to about 800 mg. In some cases, fluconazole can be administered at 400 mg. An antifungal can be administered concurrently orsequentially with TRLs (e.g., cTRLs). An antifungal can be administered from about day 0 to about day 4 after TRLs. In some cases, 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 cases, an antibiotic can comprise a bactericidal agent. A bactericidal agent can be cephalosporin or quinolone. In some cases, an antibiotic can comprise a bacteriostatic agent. A bacteriostatic agent can be administered prophylactically. In some cases, 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 cases, trimethoprim can be administered at 160 mg. In some cases, sulfamethoxazole can be administered at 800 mg. In some cases, pentamidine can be administered at 300 mg.

[0112] 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).

[0113] 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, pulsatile release formulations, multiparticulate formulations, and mixed immediate and controlled release formulations.

[0114] 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.

[0115] 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 saltform. 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.

[0116] 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.

[0117] 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.

[0118] 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 release formulations. 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 oliveoil) 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.

[0119] 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.

[0120] 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.

[0121] 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, with the use of a suitable propellant, e.g., dichlorodifluoromethane, tri chlorofluoromethane, 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 thesecompositions 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.

[0122] 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.

[0123] 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 can be 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] Other pharmaceutical formulations optionally 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.

[0136] 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.

[0137] 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.

[0138] 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 anebulizer. 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.

[0139] 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.

[0140] 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.

[0141] 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 and solvent. 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.

[0142] 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 aerosolformulation may also include lubricants, preservatives, antioxidants, and / or other aerosol components.

[0143] 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

[0144] Disclosed herein are methods of isolating, enriching, expanding, and using the tumor reactive lymphocytes (TRLs). Disclosed herein are methods of modulating one or more target genes of a TRL (e.g., an isolated TRL, an enriched TRL, or an expanded TRL), expanding the modified TRL, and using (e.g., treating) the modified TRLs. Further disclosed herein are methods of engineering a TRL or a modified TRL to generate the enhanced TRLs (e.g., CART) of the present disclosure. Also provided are methods of producing the modified TRLs or 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) or tumor 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 can comprise isolating TRLs from the peripheral blood or tumor of a subject, wherein the TRL population can comprise CD8+CD103+ 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 the immunomagnetic labeling of the population of TRLs, followed by magnetic separation within the microfluidic device.

[0145] Also disclosed herein are methods of producing a population of enhanced TRLs. In some embodiments, methods of enhancing a population of TRLs comprise isolating andexpanding 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.

[0146] 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

[0147] 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.

[0148] 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 someembodiments, the surface marker can be CD39. In some embodiments, the surface marker can be SLC6A19. In some embodiments, the surface marker can be SIDTl. In some embodiments, the surface marker can comprise CD3, CD4, CD8, CD39, CD103, SLC6A19, SIDTl, 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.

[0149] 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.

[0150] 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).

[0151] 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., fluorescence activated 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.

[0152] 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, about2.5 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, about7.5 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.

[0153] In some embodiments, methods described herein may comprise isolating from a peripheral blood sample a population of CD103, CD39, SLC6A19, and / or SIDT1 expressinglymphocytes 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.

[0154] 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 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 instances, 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.

[0155] 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

[0156] 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+CD 103+ 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.

[0157] 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 then be expanded, enhanced, or a combination thereof, by any of the methods disclosed herein.

[0158] 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.

[0159] 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 instances, 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.

[0160] 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 can vary in height. In some embodiments, the magnetic capture zones can range from 50 - 800 pm in height. In some embodiments, sortingchamber 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.

[0161] 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.

[0162] 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.

[0163] 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 can retain cells with high magneticcontent 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.

[0164] 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.

[0165] 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-1, 6 mL h-1, 9 mL h-1, 12 mL h-1, 15 mL h-1, 18 mL h-1, 21 mL h-1, 24 mL h-1, 27 mL h-1, 30 mL h-1, 35 mL h-1, 40 mL h-1, 45 mL h-1, or 50 mL h-1. 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.

[0166] 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 using fluorescence 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, CD 103, 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, about16 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.

[0167] 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 the CD39, CD103, 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, CD103, SLC6A19, or SIDT1 lymphocytes. In some embodiments, the microfluidic approaches to cellsorting 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.

[0168] 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 about 90 %. 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

[0169] Disclosed in some embodiments herein are methods of enriching and expanding a population of TRLs (e.g., cTRLs). Further disclosed herein in some embodiments are methods of expanding a modified TRL or an enhanced TRL. 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.

[0170] 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 system described 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.

[0171] 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.

[0172] 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 some embodiments, 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).

[0173] In some embodiments, the culture (comprising the isolated TRLs, the enriched TRLs, the modified TRLs, or the enhanced TRLs) can be grown in the presence of culture medium. 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-7 and 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 IU / mL of IL-2. In some embodiments, the culture medium can comprise at least about 5-50 IU / mL, 50 - 500 IU / mL, 500 - 1000 IU / mL, 1000 - 1500 IU / mL, about 1500 - 2000 IU / mL, about 2000 - 2500 IU / mL, about 2500 -3000 IU / mL, about 3000 - 3500 IU / mL, about 3500 - 4000 IU / mL, about 4000 - 4500 IU / mL, about 4500 -5000 IU / mL, about 5000 - 5500 IU / mL, about 5500 - 6000 IU / mL, about 6000 - 6500 IU / mL, about 6500 - 7000 IU / mL, about 7000 - 7500 IU / mL, about 7500 -8000 IU / mL or about 8000 - 8500 IU / mL IL-2. In some embodiments, the T-Cell growth factor can comprise IL-15. In some embodiments, the culture medium can comprise at least about 6,000 IU / mL of IL-15. In some embodiments, the culture medium can comprise at least about 5-50 IU / mL, 50 - 500 IU / mL, 500 - 1000 IU / mL, 1000 - 1500 IU / mL, about 1500 - 2000 IU / mL, about 2000 - 2500 IU / mL, about 2500 -3000 IU / mL, about 3000 - 3500 IU / mL, about 3500 - 4000 IU / mL, about 4000 - 4500 IU / mL, about 4500 -5000 IU / mL, about 5000 - 5500 IU / mL, about 5500 - 6000 IU / mL, about 6000 - 6500 IU / mL, about 6500 - 7000 IU / mL, about 7000 - 7500 IU / mL, about 7500 -8000 IU / mL or about 8000 - 8500 IU / mL IL-15. In some embodiments, the T-Cell growth factor can comprise IL-7. In some embodiments, the culture medium can comprise at least about 6,000 IU / mL of IL-7. In some embodiments, the culture medium can comprise at least about 5-50 IU / mL, 50 - 500 IU / mL, 500 - 1000 IU / mL, 1000 - 1500 IU / mL, about 1500 - 2000 IU / mL, about 2000 - 2500 IU / mL, about 2500 -3000 IU / mL, about 3000 - 3500 IU / mL, about 3500 - 4000 IU / mL, about 4000 - 4500 IU / mL, about 4500 -5000 IU / mL, about 5000 - 5500 IU / mL, about 5500 - 6000 IU / mL, about 6000 - 6500 IU / mL, about 6500 - 7000 IU / mL, about 7000 - 7500 IU / mL, about 7500 -8000 IU / mL or about 8000 - 8500 IU / mL IL-7. In some embodiments, the T-Cell growth factor can comprise IL-9. In some embodiments, theculture medium can comprise at least about 6,000 IU / mL of IL-9. In some embodiments, the culture medium can comprise at least about 5-50 IU / mL, 50 - 500 IU / mL, 500 - 1000 IU / mL, 1000 - 1500 IU / mL, about 1500 - 2000 IU / mL, about 2000 - 2500 IU / mL, about 2500 -3000 IU / mL, about 3000 - 3500 IU / mL, about 3500 - 4000 IU / mL, about 4000 - 4500 IU / mL, about 4500 -5000 IU / mL, about 5000 - 5500 IU / mL, about 5500 - 6000 IU / mL, about 6000 - 6500 IU / mL, about 6500 - 7000 IU / mL, about 7000 - 7500 IU / mL, about 7500 -8000 IU / mL or about 8000 - 8500 IU / mL IL-9.

[0174] 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, 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.

[0175] 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.

[0176] 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 aliquoted 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, IL-2 can be 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 5 to 50 lU / mL. In some embodiments, IL-2 can be added to the cultureflask 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 / AIM V containing 6000 lU / mL IL-2. 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 with human albumin. In some embodiments, the resulting cell populations can be suitable for administration to a subject in need thereof.C. Method of Modifying Target gene

[0177] Disclosed in some embodiments are methods of producing a population of modified TRLs (e.g., modified cTRLs). In some embodiments, the methods can comprise isolating a population of TRLs described using any of the methods disclosed herein, and introducing a gene editing platform described herein configured to modulate one or more target genes described herein. In some embodiments, methods described herein can further comprise culturing the modified TRLs using any of the culturing methods disclosed herein. As used herein, the term “gene editing platform” generally refers to molecules introduced into a cell to induce a genetic change in a nucleic acid of the cell.

[0178] In various aspects of the present disclosure, the gene editing platform, as disclosed herein, can be utilized for binding a target gene, such as an endogenous target gene (e.g., a chromosomal DNA sequence). The gene editing platform can comprise a nuclease, such as an endonuclease (e.g., a heterologous endonuclease). Suitable nucleases include, but are not limited to, CRISPR-associated (Cas) proteins or Cas nucleases including type I CRISPR-associated (Cas) polypeptides, type II CRISPR-associated (Cas) polypeptides, type III CRISPR-associated (Cas) polypeptides, type IV CRISPR-associated (Cas) polypeptides, type V CRISPR-associated (Cas) polypeptides, and type VI CRISPR-associated (Cas) polypeptides; zinc finger nucleases (ZFN); transcription activator-like effector nucleases (TALEN); meganucleases; RNA-binding proteins (RBP); CRISPR-associated RNA binding proteins; recombinases; flippases; transposases; Argonaute (Ago) proteins (e.g., prokaryotic Argonaute (pAgo), archaeal Argonaute (aAgo), andeukaryotic Argonaute (eAgo)); any derivative thereof; any variant thereof and any fragment thereof.

[0179] In some embodiments, the gene editing platform can comprise a DNA nuclease such as an engineered (e.g., programmable or targetable) DNA nuclease that can be nuclease-deficient. In some embodiments, the gene editing platform can comprise a nuclease-null DNA binding protein derived from a DNA nuclease that does not induce transcriptional activation or repression of a target DNA sequence unless it is present in a complex with one or more heterologous gene effectors of the disclosure. In some embodiments, the gene editing platform can comprise a nuclease-null DNA binding protein derived from a DNA nuclease that can induce transcriptional activation or repression of a target DNA sequence (e.g., which can be altered or augmented by the presence of a heterologous gene effector of the disclosure).

[0180] In some embodiments, the gene editing platform can comprise an RNA nuclease such as an engineered (e.g., programmable or targetable) RNA nuclease. In some embodiments, the gene editing platform can comprise a nuclease-null RNA binding protein derived from an RNA nuclease that does not induce transcriptional activation or repression of a target RNA sequence unless it is present in a complex with one or more heterologous gene effectors of the disclosure. In some embodiments, the gene editing platform can comprise a nuclease-null RNA binding protein derived from a RNA nuclease that can induce transcriptional activation or repression of a target RNA sequence (e.g., which can be altered or augmented by the presence of a heterologous gene effector of the disclosure).

[0181] In some embodiments, the gene editing platform can comprise a nucleic acid-guided targeting system. In some embodiments, the gene editing platform can comprise a DNA-guided targeting system. In some embodiments, the gene editing platform can comprise an RNA-guided targeting system. The nucleic acid-guided targeting system can comprise and utilize, for example, a guide nucleic acid sequence that facilitates specific binding of a CRISPR-Cas system (e.g., a nuclease deficient form thereof, such as dCas9 or dCasl4; or a reduced nuclease activity, such as nCas9) to a target gene (e.g., target endogenous gene) or target gene regulatory sequence. Binding specificity can be determined by use of a guide nucleic acid, such as a single guide RNA (sgRNA) or a part thereof. In some embodiments, the use of different sgRNAs allows the compositions and methods of the disclosure to be used with (e.g., targeted to) different target genes (e.g., target endogenous genes) or target gene regulatory sequences.

[0182] Prokaryotic CRISPR-Cas (Clustered regularly interspaced short palindromic repeats- CRISPR associated) systems, for example, Class II CRISPR-Cas systems such as Cas9 and Cast 2a, can be repurposed as a tool for regulation of gene expression, epigenome editing, andchromatin looping in compositions and methods of the disclosure. Nuclease-deactivated Cas (dCas) proteins complexed with heterologous gene effectors can allow for regulation of expression of target genes (e.g., target endogenous genes) adjacent to a site bound by the dCas.

[0183] In some embodiments, the gene editing platform can comprise a CRISPR-associated (Cas) protein or a Cas nuclease that functions in a non-naturally occurring CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated) system. In bacteria, this system can provide adaptive immunity against foreign DNA.

[0184] In a wide variety of organisms including diverse mammals, animals, plants, microbes, and yeast, a CRISPR / Cas system (e.g., modified and / or unmodified) can be utilized as a genome engineering tool, or can be modified to direct specific binding of engineered proteins to target loci as disclosed herein. A CRISPR / Cas system can comprise a guide nucleic acid such as a guide RNA (gRNA) complexed with a Cas protein for targeted regulation of gene expression and / or activity or nucleic acid binding. An RNA-guided Cas protein (e.g., a Cas nuclease such as a Cas9 nuclease) can specifically bind a target polynucleotide (e.g., DNA) in a sequence-dependent manner. The Cas protein, if possessing nuclease activity, can cleave the DNA.

[0185] In some cases, the Cas protein can be mutated and / or modified to yield a nuclease deficient protein or a protein with decreased nuclease activity relative to a wild-type Cas protein. A nuclease deficient protein can retain the ability to bind DNA, but may lack or have reduced nucleic acid cleavage activity.

[0186] In some embodiments, the gene editing platform can comprise a Cas protein that forms a complex with a guide nucleic acid, such as a guide RNA or a part thereof. In some embodiments, the gene editing platform can comprise a Cas protein that forms a complex with a single guide nucleic acid, such as a single guide RNA (sgRNA). In some embodiments, the gene editing platform can comprise an RNA-binding protein (RBP) optionally complexed with a guide nucleic acid, such as a guide RNA (e.g., sgRNA), which is able to form a complex with a Cas protein. In some embodiments, the gene editing platform can comprise a nuclease-null DNA binding protein derived from a DNA nuclease that can induce transcriptional activation or repression of a target DNA sequence. In some embodiments, the gene editing platform can comprise a nuclease-null RNA binding protein derived from an RNA.

[0187] A guide nucleic acid used in compositions and methods of the disclosure can comprise a spacer sequence that can bind to an endogenous target gene described herein. The spacer sequence can be, for example, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at-n-least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, or at least 40 nucleotides.

[0188] In some embodiments, a spacer sequence of a guide nucleic acid used in compositions and methods of the disclosure can be at most at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, or at most 40 nucleotides.

[0189] In some embodiments, a spacer sequence of a guide nucleic acid used in compositions and methods of the disclosure can be between about 8 and about 40 nucleotides, between about 10 and about 40 nucleotides, between about 11 and about 40 nucleotides, between about 12 and about 40 nucleotides, between about 13 and about 40 nucleotides, between about 14 and about 40 nucleotides, between about 15 and about 40 nucleotides, between about 16 and about 40 nucleotides, between about 17 and about 40 nucleotides, between about 18 and about 40 nucleotides, between about 19 and about 40 nucleotides, between about 20 and about 40 nucleotides, between about 22 and about 40 nucleotides, between about 24 and about 40 nucleotides, between about 26 and about 40 nucleotides, between about 28 and about 40 nucleotides, between about 30 and about 40 nucleotides, between about 8 and about 30 nucleotides, between about 10 and about 30 nucleotides, between about 11 and about 30 nucleotides, between about 12 and about 30 nucleotides, between about 13 and about 30 nucleotides, between about 14 and about 30 nucleotides, between about 15 and about 30 nucleotides, between about 16 and about 30 nucleotides, between about 17 and about 30 nucleotides, between about 18 and about 30 nucleotides, between about 19 and about 30 nucleotides, between about 20 and about 30 nucleotides, between about 22 and about 30 nucleotides, between about 24 and about 30 nucleotides, between about 26 and about 30 nucleotides, between about 28 and about 30 nucleotides, between about 8 and about 25 nucleotides, between about 10 and about 25 nucleotides, between about 11 and about 25 nucleotides, between about 12 and about 25 nucleotides, between about 13 and about 25 nucleotides, between about 14 and about 25 nucleotides, between about 15 and about 25 nucleotides, between about 16 and about 25 nucleotides, between about 17 and about 25 nucleotides, between about 18 and about 25 nucleotides, between about 19 and about 25 nucleotides, between about 20 and about 25 nucleotides, between about 22 and about 25 nucleotides, between about 24 and about 25 nucleotides, between about 8 and about 20 nucleotides, between about 10 and about 20 nucleotides, between about 11 and about 20nucleotides, between about 12 and about 20 nucleotides, between about 13 and about 20 nucleotides, between about 14 and about 20 nucleotides, between about 15 and about 20 nucleotides, between about 16 and about 20 nucleotides, between about 17 and about 20 nucleotides, between about 18 and about 20 nucleotides, between about 19 and about 20 nucleotides, between about 8 and about 18 nucleotides, between about 10 and about 18 nucleotides, between about 11 and about 18 nucleotides, between about 12 and about 18 nucleotides, between about 13 and about 18 nucleotides, between about 14 and about 18 nucleotides, between about 15 and about 18 nucleotides, between about 16 and about 18 nucleotides, between about 8 and about 16 nucleotides, between about 10 and about 16 nucleotides, between about 11 and about 16 nucleotides, between about 12 and about 16 nucleotides, between about 13 and about 16 nucleotides, between about 14 and about 16 nucleotides, or between about 15 and about 16 nucleotides. In some embodiments, a guide nucleic acid can be a guide RNA or a part thereof.

[0190] Any suitable CRISPR / Cas system can be used. A CRISPR / Cas system can be referred to using a variety of naming systems. A CRISPR / Cas system can be a type I, a type II, a type III, a type IV, a type V, a type VI system, or any other suitable CRISPR / Cas system. A CRISPR / Cas system as used herein can be a Class 1, Class 2, or any other suitably classified CRISPR / Cas system. Class 1 or Class 2 determination can be based upon the genes encoding the effector module. Class 1 systems generally have a multi-subunit crRNA-effector complex, whereas Class 2 systems generally have a single protein, such as Cas9, Cast 2a, C2cl, C2c2, C2c3 or a crRNA- effector complex. A Class 1 CRISPR / Cas system can use a complex of multiple Cas proteins to effect regulation. A Class 1 CRISPR / Cas system can comprise, for example, type I (e.g., I, IA, IB, IC, ID, IE, IF, IU), type III (e.g. III, IIIA, IIIB, IIIC, IIID), and type IV (e g, IV, IVA, IVB) CRISPR / Cas type. A Class 2 CRISPR / Cas system can use a single large Cas protein to effect regulation. A Class 2 CRISPR / Cas systems can comprise, for example, type II (e.g, II, IIA, IIB) and type V CRISPR / Cas type. CRISPR systems can be complementary to each other, and / or can lend functional units in trans to facilitate CRISPR locus targeting.

[0191] When a gene editing platform comprises a Cas protein or derivative thereof, the Cas protein or derivative thereof can be a Class 1 or a Class 2 Cas protein. A Cas protein can be a type I, type II, type III, type IV, type V Cas protein, or type VI Cas protein. A Cas protein can comprise one or more domains. Non-limiting examples of domains can include, guide nucleic acid recognition and / or binding domain, nuclease domains (e.g, DNase or RNase domains, RuvC, HNH), DNA binding domain, RNA binding domain, helicase domains, protein-protein interaction domains, and dimerization domains. A guide nucleic acid recognition and / or binding domain caninteract with a guide nucleic acid. A nuclease domain can comprise catalytic activity for nucleic acid cleavage. A nuclease domain can lack catalytic activity to prevent nucleic acid cleavage. A Cas protein can be a chimeric Cas protein or fragment thereof that can be fused to other proteins or polypeptides. A Cas protein can be a chimera of various Cas proteins, for example, comprising domains from different Cas proteins.

[0192] Non-limiting examples of Cas proteins can include c2cl, C2c2, c2c3, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cash, Cas6e, Cas6f, Cas7, Cas8a, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, CaslO, CaslOd, CasF, CasG, CasH, Casl2a, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cul966, Casl3a, Casl3b, Casl3c, Casl3d, Casl3X, Casl3Y, Casl4 (e.g., Casl4 variants, such as Casl4a, Casl4b, Casl4c, etc.) and homologs or modified versions thereof.

[0193] A Cas protein or fragment or derivative thereof can be from any suitable organism. Non-limiting examples can include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromo genes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas nap hthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Leptotrichia shahii, and Francisella novicida. In some aspects, the organism can be Streptococcus pyogenes (S. pyogenes). In some aspects, the organism can be Staphylococcus aureus (S. aureus). In some aspects, the organism can be Streptococcus thermophilus (S. therm ophilus).

[0194] A Cas protein can be derived from a variety of bacterial species including, but not limited to, Veillonella atypical, Fusobacterium nucleatum, Filifactor alocis, Solobacterium moorei, Coprococcus catus, Treponema denticola, Peptoniphilus duerdenii, Catenibacterium mitsuokai, Streptococcus mutans, Listeria innocua, Staphylococcus pseudintermedius, Acidaminococcus intestine, Olsenella uli, Oenococcus kitaharae, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus gasseri, Finegoldia magna, Mycoplasma mobile, Mycoplasma gallisepticum, Mycoplasma ovipneumoniae, Mycoplasma canis, Mycoplasma synoviae, Eubacterium rectale, Streptococcus thermophilus, Eubacterium dolichum, Lactobacillus coryniformis subsp. Torquens, Ilyobacter polytropus, Ruminococcus albus, Akkermansia muciniphila, Acidothermus cellulolyticus, Bifidobacterium longum, Bifidobacterium dentium, Cory neb acterium diphtheria, Elusimicrobium minutum, Nitratifractorsalsuginis, Sphaerochaeta globus, Fibrobacter succinogenes subsp. Succinogenes, Bacteroides fragilis, Capnocytophaga ochracea, Rhodopseudomonas palustris, Prevotella micans, Prevotella ruminicola, Flavobacterium columnare, Aminomonas paucivorans, Rhodospirillum rubrum, Candidatus Puniceispirillum marinum, Verminephrobacter eiseniae, Ralstonia syzygii, Dinoroseobacter shibae, Azospirillum, Nitrobacter hamburgensis, Bradyrhizobium, Wolinellasuccinogenes, Campylobacter jejuni subsp. Jejuni, Helicobacter mustelae, Bacillus cereus, Acidovorax ebreus, Clostridium perfringens, Parvibaculum lavamentivorans, Roseburia intestinalis, Neisseria meningitidis, Pasteurella multocida subsp. Multocida, Sutterella wadsworthensis, proteob acterium, Legionella pneumophila, Parasutterella excrementihominis, Wolinella succinogenes, and Francisella novicida.

[0195] A Cas protein as used herein can be a wildtype or a modified form of a Cas protein. A Cas protein can be an active variant, inactive variant, or fragment of a wild type or modified Cas protein. A Cas protein can comprise an amino acid change such as a deletion, insertion, substitution, variant, mutation, fusion, chimera, or any combination thereof relative to a wild-type version of the Cas protein (e.g., a wild-type version of Cas9). A Cas protein can be a polypeptide with at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity or sequence similarity to a wild type Cas protein. A Cas protein can be a polypeptide with at most about 5%, at most about 10%, 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%, or at most about 100% sequence identity and / or sequence similarity to a wildtype exemplary Cas protein. Variants or fragments can comprise at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity or sequence similarity to a wild type or modified Cas protein or a portion thereof. Variants or fragments can be targeted to a nucleic acid locus in complex with a guide nucleic acid while lacking nucleic acid cleavage activity.

[0196] In some embodiments, a Cas protein can comprise a nuclease domain. In some embodiments, a nuclease domain can comprise a DNase domain. In some embodiments, a Cas9 protein can comprise a RuvC-like nuclease domain and / or an HNH-like 20 nuclease domain. In some embodiments, a nuclease domain can comprise a nuclease active form of Cas9, RuvC and HNH domains that can each cut a different strand of double-stranded DNA to make a doublestranded break in the DNA. In some embodiments, a Cas protein can comprise only one nuclease domain (e.g., Casl2a can comprise RuvC domain but lacks HNH domain). In some embodiments, nuclease domains can be absent. In some embodiments, nuclease domains can be present but inactive or have reduced or minimal activity. In some embodiments, nuclease domains can be present and active.

[0197] One or a plurality of the nuclease domains (e.g., RuvC, HNH) of a Cas protein can be deleted or mutated so that they are no longer functional or comprise reduced nuclease activity. For example, in a Cas protein comprising at least two nuclease domains (e.g., Cas9), if one of the nuclease domains is deleted or mutated, the resulting Cas protein, known as a nickase, can generate a single-strand break at a CRISPR RNA (crRNA) recognition sequence within a doublestranded DNA but not a double-strand break. Such a nickase can cleave the complementary strand or the non-complementary strand, but may not cleave both. If all of the nuclease domains of a Cas protein (e.g., both RuvC and HNH nuclease domains in a Cas9 protein; RuvC nuclease domain in a Cas 12a protein) are deleted or mutated, the resulting Cas protein can have a reduced or no ability to cleave both strands of a double-stranded DNA. An example of a mutation that can convert a Cas9 protein into a nickase is a D10A (aspartate to alanine at position 10 of Cas9) mutation in the RuvC domain of Cas9 from S. pyogenes. H939A (histidine to alanine at amino acid position 839) or H840A (histidine to alanine at amino acid position 840) in the HNH domain of Cas9 from S. pyogenes can convert the Cas9 into a nickase. An example of a mutation that can convert a Cas9 protein into a dead Cas9 is a D10A (aspartate to alanine at position 10 of Cas9) mutation in theRuvC domain and H939A (histidine to alanine at amino acid position 839) or H840A (histidine to alanine at amino acid position 840) in the HNH domain of Cas9 from S. pyogenes.

[0198] A nuclease dead Cas protein can comprise one or more mutations relative to a wildtype version of the protein. The mutation can result in no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, or no more than 1% of the nucleic acidcleaving activity in one or more of the plurality of nucleic acid-cleaving domains of the wild-type Cas protein. The mutation can result in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the complementary strand of the target nucleic acid but reducing its ability to cleave the non-complementary strand of the target nucleic acid. The mutation can result in one or more of the plurality of nucleic acid-cleaving domains retaining the ability to cleave the non-complementary strand of the target nucleic acid but reducing its ability to cleave the complementary strand of the target nucleic acid. The mutation can result in one or more of the plurality of nucleic acid-cleaving domains lacking the ability to cleave the complementary strand and the non-complementary strand of the target nucleic acid. The residues to be mutated in a nuclease domain can correspond to one or more catalytic residues of the nuclease. For example, residues in the wild type exemplary S. pyogenes Cas9 polypeptide such as Asp 10, His840, Asn854 and Asn856 can be mutated to inactivate one or more of the plurality of nucleic acid-cleaving domains (e.g., nuclease domains). The residues to be mutated in a nuclease domain of a Cas protein can correspond to residues Asp 10, His840, Asn854 and Asn856 in the wild type S. pyogenes Cas9 polypeptide, for example, as determined by sequence and / or structural alignment.

[0199] A Cas protein can comprise an amino acid sequence having at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity or sequence similarity to a nuclease domain (e.g., RuvC domain, HNH domain) of a wild-type Cas protein.

[0200] A Cas protein, variant or derivative thereof can be modified to enhance regulation of gene expression by compositions and methods of the disclosure, e.g., as part of a complex disclosed herein. A Cas protein can be modified to increase or decrease nucleic acid binding affinity, nucleic acid binding specificity, enzymatic activity, and / or binding to other factors, such as heterodimerization or oligomerization domains and induce ligands. Cas proteins can also be modified to change any other activity or property of the protein, such as stability. For example,one or more nuclease domains of the Cas protein can be modified, deleted, or inactivated, or a Cas protein can be truncated to remove domains that can be not essential for the desired function of the protein or complex. A Cas protein can be modified to modulate (e.g., enhance or reduce) the activity of the Cas protein for regulating gene expression by a complex of the disclosure that can comprise a heterologous gene effector.

[0201] For example, a Cas protein can be coupled (e.g., fused, covalently coupled, or non- covalently coupled) to a heterologous gene effector (e.g., an epigenetic modification domain, a transcriptional activation domain, and / or a transcriptional repressor domain). A Cas protein can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to an oligomerization or dimerization domain as disclosed herein (e.g., a heterodimerization domain). A Cas protein can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to a heterologous polypeptide that provides increased or decreased stability. A Cas protein can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to a sequence that can facilitate degradation of the Cas protein or a complex containing the Cas protein, for example, a degron, such as an inducible degron (e.g., auxin inducible).

[0202] A Cas protein can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to any suitable number of partners, for example, at least one, at least two, at least three, at least four, or at least five, at least six, at least seven, or at least 8 partners. In some embodiments, a Cas protein of the disclosure can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to at most two, at most three, at most four, at most five, at most six, at most seven, at most eight, or at most ten partners. In some embodiments, a Cas protein of the disclosure can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to 1 - 5, 1 - 4, 1 - 3, 1 - 2, 2 - 5, 2 - 4, 2 - 3, 3 - 5, 3 -4, or 4 - 5 partners. In some embodiments, a Cas protein of the disclosure can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to one partner. In some embodiments, a Cas protein of the disclosure can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to two partners. In some embodiments, a Cas protein of the disclosure can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to three partners. In some embodiments, a Cas protein of the disclosure can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to four partners. In some embodiments, a Cas protein of the disclosure can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to five partners. In some embodiments, a Cas protein of the disclosure can be coupled (e.g., fused, covalently coupled, or non-covalently coupled) to six partners.

[0203] A Cas protein can be a fusion protein, e.g., a fusion comprising the Cas protein and one or more of the partners as disclosed herein. The fused domain or heterologous polypeptidecan be located at the N-terminus, the C-terminus, or internally within the Cas protein. In some embodiments, one or more of the partners disclosed herein can be a deaminase (an adenosine deaminase or a cytidine deaminase) or a reverse transcriptase (e.g., as in a prime editor). In some embodiments, a deaminase can be APOBEC (e.g., APOBEC1, APOBEC3A), PmCDAl, TadA (e.g., TadA-8e), an engineered variant thereof, or a modified variant thereof.

[0204] A partner of the Cas protein (e.g., covalently or non-covalently coupled to a dCas or nCas protein as disclosed herein) can be a transcriptional effector (e.g., a transcriptional activator or a transcriptional repressor). The transcriptional effector can be heterologous to the cell as provided herein.

[0205] In some embodiments, the Cas protein and the transcriptional effector (e.g., transcriptional activator) can be fused in a single polypeptide sequence. The Cas protein and the transcriptional effector can be fused directly to one another. Alternatively, the Cas protein and the transcriptional effector can be fused via a peptide linker (or an amino acid linker) that can be heterologous to the Cas protein and the transcriptional activator. The peptide linker can be derived from a natural polypeptide sequence. Alternatively, the peptide linker can be a synthetic sequence. The peptide linker can have a length of at least or up to about 1 amino acid residue, at least or up to about 2 amino acid residues, at least or up to about 3 amino acid residues, at least or up to about 4 amino acid residues, at least or up to about 5 amino acid residues, at least or up to about 10 amino acid residues, at least or up to about 15 amino acid residues, at least or up to about 20 amino acid residues, at least or up to about 25 amino acid residues, at least or up to about 30 amino acid residues, at least or up to about 35 amino acid residues, at least or up to about 40 amino acid residues, at least or up to about 45 amino acid residues, at least or up to about 50 amino acid residues, at least or up to about 60 amino acid residues, at least or up to about 70 amino acid residues, at least or up to about 80 amino acid residues, at least or up to about 90 amino acid residues, or at least or up to about 100 amino acid residues. In some cases, the peptide linker can be a GS linker.

[0206] The term “GS linker” or “GS linker sequence,” as used interchangeably herein, generally refers to a peptide linker that mainly can comprise glycine and serine residues. Particularly, at least or up to about 60%, at least or up to about 65%, at least or up to about 70%, at least or up to about 75%, at least or up to about 80%, at least or up to about 85%, at least or up to about 90%, at least or up to about 95% or substantially about 100% of the amino acid residues in the GS linker sequence can be selected from glycine and serine residues. The GS linker sequence according to the present invention can, for example, comprise from about 1 to about 50 amino acid residues, from about 1 to about 45 amino acid residues, from about 1 to about 40 aminoacid residues, from about 1 to about 35 amino acid residues, or from about 1 to about 30 amino acid residues, in total. In some cases, the GS linker sequence may not comprise about 10, about 5, about 4, about 3, about 2 or about 1 amino acid residue (s) other than glycine or serine.

[0207] In some embodiments, the transcriptional effector can be a histone epigenetic modifier (or a histone modifier). In some cases, the histone epigenetic modifier can modulate histones through methylation (e.g., a histone methylation modifier, such as an amino acid methyltransferase, e.g., KRAB). In some cases, the histone epigenetic modifier can modulate histones through acetylation. In some cases, the histone epigenetic modifier can modulate histones through phosphorylation. In some cases, the histone epigenetic modifier can modulate histones through ADP-ribosylation. In some cases, the histone epigenetic modifier can modulate histones through glycosylation. In some cases, the histone epigenetic modifier can modulate histones through SUMOylation. In some cases, the histone epigenetic modifier can modulate histones through ubiquitination. In some cases, the histone epigenetic modifier can modulate histones by remodeling histone structure, e.g., via an ATP hydrolysis-dependent process.

[0208] In some embodiments, the transcriptional effector can be a gene epigenetic modifier (or a gene modifier). In some cases, a gene modifier can modulate genes through methylation (e.g., a gene methylation modifier, such as a DNA methyltransferase or DNMT). In some cases, a gene modifier can modulate genes through acetylation.

[0209] In some embodiments, the transcriptional effector can be from a family of related histone acetyltransferases. Non-limiting examples of histone acetyltransferases include GNAT subfamily, MYST subfamily, p300 / CBP subfamily, HAT1 subfamily, GCN5, PCAF, Tip60, MOZ, MORF, MOF, HBO1, p300, CBP, HAT1, ATF-2, SRC1, and TAFII250.

[0210] In some embodiments, the transcriptional effector can be from a histone epigenetic modifier (e.g., a histone lysine methyltransferase, a histone lysine demethylase, or a DNA methylase). Non-limiting examples of histone epigenetic modifier include EZH subfamily, NonSET subfamily, Other SET subfamily, PRDM subfamily, SET1 subfamily, SET2 subfamily, SUV39 subfamily, SYMD subfamily, ASH1L, EHMT1, EHMT2, EZH1, EZH2, MLL, MLL2, MLL3, MLL4, MLL5, NSD1, NSD2, NSD3, PRDM1, PRDM10, PRDM11, PRDM12, PRDM13, PRDM14, PRDM15, PRDM16, PRDM2, PRDM4, PRDM5, PRDM6, PRDM7, PRDM8, PRDM9, SET1, SET1L, SET2L, SETD2, SETD3, SETD4, SETD5, SETD6, SETD7, SETD8, SETDB1, SETDB2, SETMAR, SUV39H1, SUV39H2, SUV420H1, SUV420H2, SYMD1, SYMD2, SYMD3, SYMD4, and SYMD5.

[0211] Examples of proteins (or fragments thereof) that can be used as a fusion partner to increase transcription include but are not limited to: transcriptional activators such as VP16, VP64,VP48, VP160, p65 subdomain (e.g., from NFkB), and activation domain of EDLL and / or TAL activation domain (e.g., for activity in plants); and histone epigenetic modifier such as SET1A, SET1B, MLL1 to 5, ASH1, SYMD2, NSD1, JHDM2a / b, UTX, JMJD3, GCN5, PCAF, CBP, p300, TAF1, TIP60 / PLIP, M0ZMYST3, M0RFMYST4, SRC1, ACTR, PI 60, CLOCK, Ten- Eleven Translocation (TET) dioxygenase 1 (TET1CD), TET1, DME, DML1, DML2, ROS1, and the like. An additional example of such gene activating modulator can be VP64-p65-Rta fusion polypeptide (VPR).

[0212] Examples of proteins (or fragments thereof) that can be used as a fusion partner to decrease transcription include but are not limited to: transcriptional repressors such as the Kruppel associated box (KRAB or SKD); K0X1 repression domain; the Mad mSIN3 interaction domain (SID); the ERF repressor domain (ERD), the SRDX repression domain (e.g., for repression in plants), and the like; histone lysine methyltransferases such as Pr-SET7 / 8, SUV4- 20H1, RIZ1, and the like; histone lysine demethylases such as JMJD2A / JHDM3 A, JMJD2B, JMJD2C / GASC1, JMJD2D, J ARID 1 A / RBP2, JARID1B / PLU-1, J ARID 1C / SMCX, JARIDID / SMCY, and the like; histone lysine deacetylases such as HDAC1, HDAC2, HDAC3, HDAC8, HDAC4, HDAC5, HDAC7, HDAC9, SIRT1, SIRT2, HD AC 11, and the like; DNA methylases such as Hhal DNA m5c-methyltransferase (M.Hhal), DNA methyltransferase 1 (DNMT1), DNA methyltransferase 3a (DNMT3a), DNA methyltransferase 3b (DNMT3b), METI, DRM3 (plants), ZMET2, CMT1, CMT2 (plants), and the like; and periphery recruitment elements such as Lamin A, Lamin B, and the like.

[0213] A Cas protein can be provided in any form. For example, a Cas protein can be provided in the form of a protein, such as a Cas protein alone or complexed with a guide nucleic acid as a ribonucleoprotein. A Cas protein can be provided in a complex, for example, complexed with a guide nucleic acid and / or one or more heterologous gene effectors of the disclosure. A Cas protein can be provided in the form of a nucleic acid encoding the Cas protein, such as an RNA (e.g., messenger RNA (mRNA)), or DNA. The nucleic acid encoding the Cas protein can be codon optimized for efficient translation into protein in a particular cell or organism.

[0214] Nucleic acids encoding Cas proteins, fragments, or derivatives thereof can be stably integrated in the genome of a cell. Nucleic acids encoding Cas proteins can be operably linked to a promoter, for example, a promoter that is constitutively or inducibly active in the cell. Nucleic acids encoding Cas proteins can be operably linked to a promoter in an expression construct. Expression constructs can include any nucleic acid constructs capable of directing expression of a gene or other nucleic acid sequence of interest (e.g., a Cas gene) and which can transfer such a nucleic acid sequence of interest to a target cell.

[0215] In some embodiments, a Cas protein, variant or derivative thereof can be a nuclease dead Cas (dCas) protein. A dead Cas protein can be a protein that lacks nucleic acid cleavage activity.

[0216] A Cas protein can comprise a modified form of a wild type Cas protein. The modified form of the wild type Cas protein can comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the Cas protein. For example, the modified form of the Cas protein can have no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, or no more than 1% of the nucleic acid-cleaving activity of the wild-type Cas protein (e.g., Cas9 from S. pyogenes). The modified form of Cas protein can have no substantial nucleic acid-cleaving activity. When a Cas protein can be a modified form that has no substantial nucleic acid-cleaving activity, it can be referred to as enzymatically inactive, “deactivated” and / or “dead” (abbreviated by “d”). A dead Cas protein (e.g., dCas, dCas9, dCasl4) can bind to a target polynucleotide but may not cleave or minimally cleaves the target polynucleotide. In some aspects, a dead Cas protein can be a dead Casl4 protein. In some aspects, a dead Cas protein can be not a dead Cas 14 protein.

[0217] A dCas polypeptide (e.g., dCasl4 polypeptide) can associate with a single guide RNA (sgRNA) to activate or repress transcription of a target gene (e.g., target endogenous gene), for example, in combination with heterologous gene effector(s) disclosed herein. sgRNAs can be introduced into cells expressing the Cas or variant thereof, as provided herein. In some cases, such cells can contain one or more different sgRNAs that target the same target gene (e.g., target endogenous gene) or target gene regulatory sequence. In other cases, the sgRNAs can target different nucleic acids in the cell (e.g., different target genes, different target gene regulatory sequences, or different sequences within the same target gene or target gene regulatory sequence).

[0218] Enzymatically inactive can refer to a nuclease that can bind to a nucleic acid sequence in a polynucleotide in a sequence-specific manner, but will not cleave a target polynucleotide or will cleave it at a substantially reduced frequency. An enzymatically inactive guide moiety can comprise an enzymatically inactive domain (e.g. nuclease domain). Enzymatically inactive can refer to no activity. Enzymatically inactive can refer to substantially no activity. Enzymatically inactive can refer to essentially no activity. Enzymatically inactive can refer to an activity no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, or no more than 10% activity compared to a comparable wild-type activity (e.g., nucleic acid cleaving activity, wild-type Cas9 or wildtype Cas 14 activity).

[0219] In some embodiments, the gene editing platform as disclosed herein does not contain a nucleic acid-guided targeting system. For example, the gene editing platform can include proteins that bind to a target gene (e.g., target endogenous gene) or target gene regulatory sequence based on protein structural features, such as certain nucleases disclosed herein.

[0220] In some embodiments, the wild-type Cas protein that the engineered Cas protein can be a modification of has a native amino acid sequence with a length of less than 800 amino acids (e.g., Cas9 or a variant thereof). This relatively small size can provide several advantages to the provided engineered Cas protein. For example, the small size can allow the Cas protein to be delivered to a host cell, e.g., a cell of a human patient, via a single adeno-associated virus delivery system that would be otherwise incapable of delivering a larger protein. The native amino acid sequence can have a length that is, for example, between 500 amino acids and 700 amino acids, e.g., between 500 amino acids and 620 amino acids, between 540 amino acids and 660 amino acids, between 560 amino acids and 680 amino acids, or between 580 amino acids and 700 amino acids. In terms of upper limits, the native amino acid sequence can have a length that is less than 700 amino acids, e.g., less than 680 amino acids, less than 660 amino acids, less than 640 amino acids, less than 620 amino acids, less than 600 amino acids, less than 580 amino acids, less than 560 amino acids, less than 540 amino acids, or less than 520 amino acids. In terms of lower limits, the native amino acid sequence can have an length that is greater than 500 amino acids, e.g., greater than 520 amino acids, greater than 540 amino acid, greater than 560 amino acids, greater than 580 amino acids, greater than 600 amino acids, greater than 620 amino acids, greater than 640 amino acids, greater than 660 amino acids, or greater than 700 amino acids. Larger lengths, e.g., greater than 700 amino acids, and smaller lengths, e.g., less than 500 amino acids, are also contemplated.

[0221] In some embodiments, according to any of the Cas protein systems described herein, the target nucleic acid can be dsDNA. In such embodiments, dsDNA-targeting specificity can be determined, at least in part, by two parameters: the gRNA spacer targeting a protospacer in the target dsDNA (the sequence in the target dsDNA corresponding to the gRNA spacer on the non- complementary DNA strand) and a short sequence, the protospacer-adjacent motif (PAM), located immediately 5' (upstream) or 3’ (downstream) of the protospacer on the non-complementary DNA strand. In some embodiments, the PAM can be 5’-TTTG-3’ or 5’-TTTA-3’. In some embodiments, the PAM can be 5’-TTTG-3’. In some embodiments, the PAM can be 5’-TTTA- 3’. In some embodiments, a modified Cas can be utilized such that no specific PAM sequence can be needed.

[0222] In some embodiments, according to any of the Cas protein systems described herein, the target nucleic acid can be RNA. In such embodiments, RNA-targeting specificity can be determined, at least in part, by the gRNA spacer targeting a protospacer-like sequence in the target RNA (the sequence in the target RNA complementary to the gRNA spacer), and can be independent of the sequence located immediately 5’ (upstream) or 3 ’(downstream) of the protospacer-like sequence. In some embodiments, the Cas protein system can be also capable of targeting a dsDNA molecule, wherein the gRNA spacer can be selected such that it targets a protospacer in the target dsDNA molecule having a PAM selected from 5'-TTTG-3' and 5'-TTTA- 3'. In other embodiments, the Cas protein system can be incapable of targeting a dsDNA molecule, wherein the gRNA spacer can be selected such that any protospacers in the dsDNA molecule targeted by the gRNA spacer do not have a PAM selected from 5'-TTTG-3' and 5'-TTTA-3'.

[0223] In some embodiments, a gene editing platform can comprise a zinc finger nuclease (ZFN) or a variant, fragment, or derivative thereof. ZFN can refer to a fusion between a cleavage domain, such as a cleavage domain of Fokl, and at least one zinc finger motif (e.g., at least 2, at least 3, at least 4, or at least 5 zinc finger motifs) which can bind polynucleotides such as DNA and RNA. In some embodiments, a ZFN can be used in a targeting moiety of the disclosure to bind a polynucleotide (e.g., target gene or target gene regulatory sequence), but the ZFN does not cleave or substantially does not cleave the polynucleotide, e.g., a nuclease dead ZFN. A ZFN or a variant, fragment, or derivative thereof can be fused to or associated with one or more heterologous gene effectors to form a complex of the disclosure.

[0224] The heterodimerization at certain positions in a polynucleotide of two individual ZFNs in certain orientation and spacing can lead to cleavage of the polynucleotide in nuclease-active ZFN. For example, a ZFN binding to DNA can induce a double-strand break in the DNA. In order to allow two cleavage domains to dimerize and cleave DNA, two individual ZFNs can bind opposite strands of DNA with their C-termini at a certain distance apart. In some cases, linker sequences between the zinc finger domain and the cleavage domain can require the 5' edge of each binding site to be separated by about 5-7 base pairs. In some cases, a cleavage domain can be fused to the C-terminus of each zinc finger domain.

[0225] In some embodiments, the cleavage domain of a gene editing platform comprising a ZFN can comprise a modified form of a wild type cleavage domain. The modified form of the cleavage domain can comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the cleavage domain. For example, the modified form of the cleavage domain can have no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, nomore than 10%, no more than 5%, or no more than 1% of the nucleic acid-cleaving activity of the corresponding wild-type cleavage domain. The modified form of the cleavage domain can have no substantial nucleic acid-cleaving activity. In some embodiments, the cleavage domain can be enzymatically inactive.

[0226] In some embodiments, a gene editing platform can comprise a “TALEN” or “TAL- effector nuclease” or a variant, fragment, or derivative thereof. TALENs refer to engineered transcription activator-like effector nucleases that generally contain a central domain of DNA- binding tandem repeats and a cleavage domain. TALENs can be produced by fusing a TAL effector DNA binding domain to a DNA cleavage domain. In some cases, a DNA-binding tandem repeat can comprise 33-35 amino acids in length and contains two hypervariable amino acid residues at positions 12 and 13 that can recognize at least one specific DNA base pair. A transcription activator-like effector (TALE) protein can be fused to a nuclease such as a wild-type or mutated Fokl endonuclease or the catalytic domain of Fokl. In some embodiments, a TALEN can be used in a targeting moiety of the disclosure to bind a polynucleotide (e.g., target gene or target gene regulatory sequence), but the TALEN does not cleave or substantially does not cleave the polynucleotide, e.g., a nuclease dead TALEN. A TALEN or a variant, fragment, or derivative thereof can be fused to or associated with one or more heterologous gene effectors to form a complex of the disclosure.

[0227] In some embodiments, a TALEN can be engineered for reduced nuclease activity. In some embodiments, the nuclease domain of a TALEN can comprise a modified form of a wild type nuclease domain. The modified form of the nuclease domain can comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces the nucleic acid-cleaving activity of the nuclease domain. For example, the modified form of the nuclease domain can have no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, or no more than 1% of the nucleic acid-cleaving activity of the wild-type nuclease domain. The modified form of the nuclease domain can have no substantial nucleic acid-cleaving activity. In some embodiments, the nuclease domain can be enzymatically inactive. A TALEN or a variant, fragment, or derivative thereof can be fused to or associated with one or more heterologous gene effectors to form a complex of the disclosure.

[0228] Several mutations to Fokl have been made for its use in TALENs, which, for example, improve cleavage specificity or activity. Such TALENs can be engineered to bind any desired DNA sequence. TALENs can be used to generate gene modifications (e.g., nucleic acid sequenceediting) by creating a double-strand break in a target DNA sequence, which in turn, undergoes NHEJ or HDR.

[0229] In some embodiments, a TALE or a variant, fragment, or derivative thereof can be fused to or associated with one or more heterologous gene effectors to form a complex of the disclosure. In some embodiments, the transcription activator-like effector (TALE) protein can be fused to a heterologous gene effector and does not comprise a nuclease. In some embodiments, a TALEN does not cleave or substantially does not cleave the polynucleotide, e.g., a nuclease dead TALE. A TALE or a variant, fragment, or derivative thereof can be fused to or associated with one or more heterologous gene effectors to form a complex of the disclosure.

[0230] In some embodiments, the complex of the transcription activator-like effector (TALE) protein and the heterologous gene effector can be designed to function as a transcriptional activator. In some embodiments, the complex of the transcription activator-like effector (TALE) protein and the heterologous gene effector can be designed to function as a transcriptional repressor. For example, the DNA-binding domain of the transcription activator-like effector (TALE) protein can be fused (e.g., linked) to one or more heterologous gene effectors that comprise transcriptional activation domains, or to one or more heterologous gene effectors that comprise transcriptional repression domains.

[0231] In some embodiments, a gene editing platform can comprise a meganuclease. Meganucleases generally refer to rare-cutting endonucleases or homing endonucleases that can be highly sequence specific. Meganucleases can recognize DNA target sites ranging from at least 12 base pairs in length, e.g., from 12 to 40 base pairs, 12 to 50 base pairs, or 12 to 60 base pairs in length. Meganucleases can be modular DNA-binding nucleases such as any fusion protein comprising at least one catalytic domain of an endonuclease and at least one DNA binding domain or protein specifying a nucleic acid target sequence. The DNA-binding domain can contain at least one motif that recognizes single- or double-stranded DNA. A nuclease-active meganuclease can generate a double-stranded break. In some embodiments, a meganuclease can be used in a targeting moiety of the disclosure to bind a polynucleotide (e.g., target gene or target gene regulatory sequence), but the meganuclease does not cleave or substantially does not cleave the polynucleotide, e.g., a nuclease dead meganuclease. A meganuclease or a variant, fragment, or derivative thereof can be fused to or associated with one or more heterologous gene effectors to form a complex of the disclosure.

[0232] The meganuclease can be monomeric or dimeric. In some embodiments, the meganuclease can be naturally-occurring (found in nature) or wild-type, and in other instances, the meganuclease can be non-natural, artificial, engineered, synthetic, rationally designed, or man-made. In some embodiments, the meganuclease of the present disclosure can include an I-Crel meganuclease, I-Ceul meganuclease, I-Msol meganuclease, I-Scel meganuclease, variants thereof, derivatives thereof, and fragments thereof.

[0233] In some embodiments, the nuclease domain of a meganuclease can comprise a modified form of a wild type nuclease domain. The modified form of the nuclease domain can comprise an amino acid change (e.g., deletion, insertion, or substitution) that reduces or eliminates the nucleic acid-cleaving activity of the nuclease domain. For example, the modified form of the nuclease domain can have no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 5%, or no more than 1% of the nucleic acid-cleaving activity of the wildtype nuclease domain. The modified form of the nuclease domain can have no substantial nucleic acid-cleaving activity. In some embodiments, the nuclease domain can be enzymatically inactive. In some embodiments, a meganuclease can bind DNA but cannot cleave the DNA. In some embodiments, a nuclease-inactive meganuclease can be fused to or associated with one or more heterologous gene effectors to generate a complex of the disclosure.

[0234] In some embodiments, the gene editing platform (e.g., and / or a complex comprising the gene editing platform) can regulate expression and / or activity of a target gene (e.g., target endogenous gene) when expressed in the cell (e.g., cTRL). In some embodiments, the gene editing platform and / or a complex thereof can edit the sequence of a nucleic acid (e.g., a gene and / or gene product). A nuclease-active Cas protein can edit a nucleic acid sequence by generating a doublestranded break or single-stranded break in a target polynucleotide.

[0235] In some embodiments, the gene editing platform (e.g., and / or a complex comprising the heterologous polypeptide) can generate a double-strand break in a target polynucleotide, such as DNA. A double-strand break in DNA can result in DNA break repair which allows for the introduction of gene modification(s) (e.g., nucleic acid editing). In some embodiments, a nuclease induces site-specific single-strand DNA can break or nick, thus resulting in HDR.

[0236] A double-strand break in DNA can result in DNA break repair which allows for the introduction of gene modification(s) (e.g., nucleic acid editing). DNA break repair can occur via non-homologous end joining (NHEJ) or homology-directed repair (HDR). In HDR, a donor DNA repair template or template polynucleotide that contains homology arms flanking sites of the target DNA can be provided.

[0237] In some embodiments, the gene editing platform (e.g., and / or a complex comprising the gene editing platform) does not generate a double-strand break in a target polynucleotide, such as DNA. Binding of the heterologous polypeptide (e.g., Cas) of the complex comprising the geneediting platform (e.g., a complex comprising a dCas-effector and a guide RNA) without a nucleic acid break can be sufficient to regulate expression (e.g., enhance or suppress) of a target gene (e.g., endogenous target gene).Target gene

[0238] The disclosure provides compositions, methods, and systems for modulating expression of target genes. The target genes can be one or more endogenous target genes that is involved in overcoming tumor microenvironment, improving effector function, and / or fitness / exhaustions. For example, disclosed herein are complexes that comprise a gene editing platform (e.g., a guide moiety and one or more Cas molecule) that can increase or decrease an activity or expression level of a target gene.

[0239] In some embodiments, one or more target genes can be modulated (e.g., increased or decreased). In some embodiments, the systems and methods described herein can modulate at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten genes can be targeted. Non-limiting target genes are listed in Table 16.

[0240] In some embodiments, systems and methods described herein can target at least one gene involved in overcoming tumor microenvironment (e.g., PD-1, CTLA-4, TIGIT, LAG-3, TIM-3, TGF-BR, IL-10R, CD39, CD73, FAS, A2AR, DGK, SMAD2, SMAD3, or SMAD4), and at least one gene involved in improving effector function (e.g., CISH, REGNASE-1, SOCS-1, IKZF3, or TCEB2).

[0241] In some embodiments, systems and methods described herein can target at least one gene involved in overcoming tumor microenvironment (e g., PD-1, CTLA-4, TIGIT, LAG-3, TIM-3, TGF-BR, IL-10R, CD39, CD73, FAS, A2AR, DGK, SMAD2, SMAD3, or SMAD4), and at least one gene involved in T cell fitness and / or exhaustion (e.g., TET2, DNMT3, SUV39H1, EZH2, BATF, IRF4, NF AT, NF-kB, CBLB, AP-1, FOXO1, FOXO3, EOMES, PRDM1, MED12, SNX9, ARID1A, ARID2, SMARCC1, SMARCD2, RUNX3, BHLHE40, FLU, SNX9, NR4A3, CCNC, HPK1 PTP1B, RASA2, PTPN2, ID3, SOX4, CUL5, TLE4, IKZF2, CD70, ROQUIN-1 DAP5, RGS16, BLIMP- 1, TOX1, or TOX2).

[0242] In some embodiments, systems and methods described herein can target at least one gene involved in improved effector function (e.g., CISH, REGNASE-1, SOCS-1, IKZF3, or TCEB2), and at least one gene involved in improving T cell fitness and / or exhaustion (e.g., TET2, DNMT3, SUV39H1, EZH2, BATF, IRF4, NF AT, NF-kB, CBLB, AP-1, FOXO1, FOXO3, EOMES, PRDM1, MED 12, SNX9, ARID 1 A, ARID2, SMARCC1, SMARCD2, RUNX3,BHLHE40, FLU, SNX9, NR4A3, CCNC, HPK1 PTP1B, RASA2, PTPN2, ID3, SOX4, CUL5, TLE4, IKZF2, CD70, ROQUIN-1 DAP5, RGS16, BLIMP-1, T0X1, or T0X2)Table 16: Non-limiting example of target genes

[0243] In some embodiments, a modified cTRLs (e.g., a CTRL with one or more target gene modulated by the gene editing platform described herein) can exhibit preservation of less differentiated phenotype. For example, a modified cTRLs reduced upregulation of terminal differentiation, exhaustion, or dysfunctional markers.

[0244] In some embodiments, the genetically engineered T cells can comprise one or more mutated genes involved in cell self-renewal, one or more disrupted genes involved in apoptosis, and / or one or more disrupted genes involved in cell exhaustion. Such T cells may be generated via gene editing (including genomic editing), a type of genetic engineering in which nucleotide(s) / nucleic acid(s) is / are inserted, deleted, and / or substituted in a DNA sequence, such as in the genome of a targeted cell. Targeted gene editing can enable insertion, deletion, and / or substitution at pre-selected sites in the genome of a targeted cell (e.g., in a targeted gene or targeted DNA sequence). When a sequence of an endogenous gene is edited, for example by deletion, insertion or substitution of nucleotide(s) / nucleic acid(s), the endogenous gene comprising the affected sequence may be knocked-out or knocked-down due to the sequence alteration. Therefore, targeted editing may be used to disrupt endogenous gene expression. “Targeted integration” refers to a process involving insertion of one or more exogenous sequences, with or without deletion of an endogenous sequence at the insertion site. Targeted integration can result from targeted gene editing when a donor template containing an exogenous sequence can be present.

[0245] In some embodiments, a target gene or regulatory sequence thereof can be endogenous to a cell, for example, present in the cell’s genome, or endogenous to a subject, for example,present in the subject’s genome. In some embodiments, a target gene or regulatory sequence thereof can be not part of an engineered reporter system.

[0246] In some embodiments, the systems and methods as disclosed herein can modulate (e.g., increase or decrease) expression of a target gene (e.g., upon introducing a complex comprising the heterologous polypeptide into a cell or population of cells). In some embodiments, an expression level can comprise an RNA expression level. In some embodiments, an RNA expression level can be measured by, for example, RNAseq, qPCR, microarray, gene array, FISH, etc. In some embodiments, an expression level can comprise a protein expression level. In some embodiments, a protein expression level can be measured by Western Blot, ELISA, multiplex immunoassay, mass spectrometry, NMR, proteomics, flow cytometry, mass cytometry, or any combination thereof.

[0247] In some embodiments, the systems and methods as disclosed herein can modulate (e.g., increase or decrease) expression of a target gene (e.g., upon introducing a complex comprising the gene editing platform into a cell or population of cells) by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2 fold, at least about 3 fold, at least about 4 fold, at least about 5 fold, at least about 6 fold, at least about 7 fold, at least about 8 fold, at least about 9 fold, at least about 10 fold, at least about 11 fold, at least about 12 fold, at least about 13 fold, at least about 14 fold, at least about 15 fold, at least about 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.

[0248] In some embodiments, the systems and methods as disclosed herein can modulate (e.g., increase or decrease) expression of a target gene (e.g., upon introducing a complex comprising the gene editing platform into a cell or population of cells) by at most about 50%, at most about 60%, at most about 70%, at most about 80%, at most about 90%, 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 fold 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 mostabout 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, at most about 3000 fold, at most about 5000 fold, or at most about 10000 fold.

[0249] In some embodiments, the systems and methods as disclosed herein can modulate (e.g., increase or decrease) expression of a target gene (e.g., upon introducing a complex comprising the gene editing platform into a cell or population of cells) about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, 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, about 3000 fold, about 5000 fold, or about 10000 fold.

[0250] In some embodiments, the systems and methods as disclosed herein can modulate (e.g., increase or decrease) expression of a target gene (e.g., upon introducing a complex comprising the gene editing platform into a cell or population of cells) from below a limit of detection to a detectable level.

[0251] In some embodiments, the system and method as disclosed herein can modulate (e.g., increase or decrease) an activity level of a target gene (e.g., upon introducing a gene editing platform into a cell or population of cells). An activity level can be determined by a suitable functional assay for the target gene in question depending on the functional characteristics of the target gene. For example, an activity level of a target gene that is a mitogen could be determined by measuring cell proliferation; an activity level of a target gene that induces apoptosis could be measured by an annexin V assay or other suitable cell death assay; an activity level of an antiinflammatory cytokine could be measured by an LPS-induced cytokine release assay.

[0252] In some embodiments, the system and method as disclosed herein can modulate (e.g., increase or decrease) the activity of the target gene by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 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 leastabout 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.

[0253] In some embodiments, the system and method as disclosed herein can modulate (e.g., increase or decrease) the activity of the target gene by at most 50%, at most 60%, at most 70%, at most 80%, at most 90%, 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, at most about 3000 fold, at most about 5000 fold, or at most about 10000 fold.

[0254] In some embodiments, the system to method can increase the expression of the endogenous target gene encoding the target protein by at least about 0.01 fold to about 5,000 fold. In some embodiments, the system to method can increase the expression of the endogenous target gene encoding the target protein by at least about 0.01 fold to about 0.05 fold, about 0.01 fold to about 0.1 fold, about 0.01 fold to about 0.5 fold, about 0.01 fold to about 1 fold, about 0.01 fold to about 5 fold, about 0.01 fold to about 10 fold, about 0.01 fold to about 50 fold, about 0.01 fold to about 100 fold, about 0.01 fold to about 500 fold, about 0.01 fold to about 1,000 fold, about 0.01 fold to about 5,000 fold, about 0.05 fold to about 0.1 fold, about 0.05 fold to about 0.5 fold, about 0.05 fold to about 1 fold, about 0.05 fold to about 5 fold, about 0.05 fold to about 10 fold, about 0.05 fold to about 50 fold, about 0.05 fold to about 100 fold, about 0.05 fold to about 500 fold, about 0.05 fold to about 1,000 fold, about 0.05 fold to about 5,000 fold, about 0.1 fold to about 0.5 fold, about 0.1 fold to about 1 fold, about 0.1 fold to about 5 fold, about 0.1 fold to about 10 fold, about 0.1 fold to about 50 fold, about 0.1 fold to about 100 fold, about 0.1 fold to about 500 fold, about 0.1 fold to about 1,000 fold, about 0.1 fold to about 5,000 fold, about 0.5 fold to about 1 fold, about 0.5 fold to about 5 fold, about 0.5 fold to about 10 fold, about 0.5 fold to about 50 fold, about 0.5 fold to about 100 fold, about 0.5 fold to about 500 fold, about 0.5 fold to about1,000 fold, about 0.5 fold to about 5,000 fold, about 1 fold to about 5 fold, about 1 fold to about 10 fold, about 1 fold to about 50 fold, about 1 fold to about 100 fold, about 1 fold to about 500 fold, about 1 fold to about 1,000 fold, about 1 fold to about 5,000 fold, about 5 fold to about 10 fold, about 5 fold to about 50 fold, about 5 fold to about 100 fold, about 5 fold to about 500 fold, about 5 fold to about 1,000 fold, about 5 fold to about 5,000 fold, about 10 fold to about 50 fold, about 10 fold to about 100 fold, about 10 fold to about 500 fold, about 10 fold to about 1,000 fold, about 10 fold to about 5,000 fold, about 50 fold to about 100 fold, about 50 fold to about 500 fold, about 50 fold to about 1,000 fold, about 50 fold to about 5,000 fold, about 100 fold to about 500 fold, about 100 fold to about 1,000 fold, about 100 fold to about 5,000 fold, about 500 fold to about 1,000 fold, about 500 fold to about 5,000 fold, or about 1,000 fold to about 5,000 fold. In some embodiments, the system to method can increase the expression of the endogenous target gene encoding the target protein by at least about 0.01 fold, about 0.05 fold, about 0.1 fold, about 0.5 fold, about 1 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 500 fold, about 1,000 fold, or about 5,000 fold. In some embodiments, the system to method can increase the expression of the endogenous target gene encoding the target protein by at least at least about 0.01 fold, about 0.05 fold, about 0.1 fold, about 0.5 fold, about 1 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 500 fold, or about 1,000 fold. In some embodiments, the system to method can increase the expression of the endogenous target gene encoding the target protein by at least at most about 0.05 fold, about 0.1 fold, about 0.5 fold, about 1 fold, about 5 fold, about 10 fold, about 50 fold, about 100 fold, about 500 fold, about 1,000 fold, or about 5,000 fold.

[0255] The systems and methods of the present disclosure can, in some cases, elicit changes in expression and / or activity level of a target gene (e.g., target endogenous gene) that persists for longer than can be achieved with alternative compositions and methods (e.g., suppression via RNAi, e.g., using siRNA). In some embodiments, persistent modulation of gene expression can be advantageous as compared to transient modulation.

[0256] In some embodiments, the systems and methods as disclosed herein can modulate (e.g., increase or decrease) expression and / or activity level of a target gene for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 18 hours, at least about 20 hours, at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 14 days, at least about 21 days, at least about 28 days,at least about 5 weeks, at least about 6 weeks, at least about 7 weeks, at least about 8 weeks, at least about 9 weeks, at least about 10 weeks, at least about 12 weeks, at least about 14 weeks, at least about 18 weeks, at least about 20 weeks, at least about 26 weeks, or at least about 5 months, at least about 6 months, at least about 9 months, at least about 12 months, or more.

[0257] In some embodiments the systems and methods as disclosed herein can modulate (e.g., increase or decrease) expression and / or activity level of a target gene (e.g., target endogenous gene) to above a certain threshold for at most about 1 hour, at most about 2 hours, at most about 3 hours, at most about 4 hours, at most about 5 hours, at most about 6 hours, at most about 7 hours, at most about 8 hours, at most about 9 hours, at most about 10 hours, at most about 12 hours, at most about 14 hours, at most about 18 hours, at most about 20 hours, at most about 1 day, at most about 2 days, at most about 3 days, at most about 4 days, at most about 5 days, at most about 6 days, at most about 7 days, at most about 8 days, at most about 9 days, at most about 10 days, at most about 14 days, at most about 21 days, at most about 28 days, at most about 5 weeks, at most about 6 weeks, at most about 7 weeks, at most about 8 weeks, at most about 9 weeks, at most about 10 weeks, at most about 12 weeks, at most about 14 weeks, at most about 18 weeks, at most about 20 weeks, at most about 26 weeks, or at most about 5 months, at most about 6 months, at most about 9 months, at most about 12 months, or more.

[0258] In some embodiments, the systems and methods as disclosed herein can modulate (e.g., increase or decrease) expression and / or activity level of a target gene (e.g., target endogenous gene) to above a certain threshold for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 12 hours, about 14 hours, about 18 hours, about 20 hours, 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 14 days, about 21 days, about 28 days, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 12 weeks, about 14 weeks, about 18 weeks, about 20 weeks, about 26 weeks, about 5 months, about 6 months, about 9 months, or about 12 months.Composition

[0259] In some aspects, the present disclosure provides a composition comprising at least a portion of a system as described. In some embodiments, a system can comprise a gene editing platform, a heterologous polynucleotide encoding components of a gene editing platform, a guide nucleic acid, a heterologous polynucleotide encoding a guide nucleic acid, or any combination thereof. In some embodiments, a composition can be usable for modifying a cell in vitro, ex vivo,or in vivo. In some embodiments, a composition can be usable for treating or enhancing a condition (e.g., Cancer) of a subject, as disclosed herein.

[0260] The composition as disclosed herein can comprise an active ingredient (e.g., (i) the gene editing platform or a heterologous polynucleotide encoding components of the gene editing platform, and (ii) the guide nucleic acid or a heterologous polynucleotide encoding the guide nucleic acid, as disclosed herein and optionally an additional ingredient (e.g., excipient). If necessary and / or desirable, the composition can be divided, shaped and / or packaged into a desired single- or multi-dose unit or single-or multi-implantation unit.

[0261] In some embodiments, the composition can comprise one or more heterologous polynucleotides encoding the active ingredients as disclosed herein. When there are different members within the active ingredients, each member can be encoded by a different heterologous polynucleotide. Alternatively, two or more (e.g., all of) the ingredients can be encoded by a single heterologous polynucleotide.

[0262] The one or more heterologous polynucleotides can further comprise one or more promoters (or one or more transcriptional control elements, as used interchangeably herein). Different active ingredients encoded by the one or more heterologous polynucleotides can be under the control of the same promoter or different promoters. A promoter as disclosed herein can be active in a eukaryotic, mammalian, non -human mammalian or human cell. The promoter can be an inducible or constitutively active promoter. Alternatively or additionally, the promoter can be tissue or cell specific. Non-limiting examples of suitable eukaryotic promoters (i.e. promoters functional in a eukaryotic cell) can include those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40, long terminal repeats (LTRs) from retrovirus, human elongation factor-1 promoter (EFl), a hybrid construct comprising the cytomegalovirus (CMV) enhancer fused to the chicken beta-active promoter (CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase- 1 locus promoter (PGK) and mouse metallothionein-I. The promoter can be a fungi promoter. The promoter can be a plant promoter. A database of plant promoters can be found (e.g., PlantProm). The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression. In some cases, a promoter as disclosed herein can be a promoter specific for any of the tissues provided herein, or a promoter specific for any of the cell types provided herein.

[0263] A heterologous polynucleotide of the one or more heterologous polynucleotides (e.g., the single heterologous polynucleotide) can have a size of at least or up to about 2.5 kilobases, at least or up to about 2.6 kilobases, at least or up to about 2.7 kilobases, at least or up to about 2.8kilobases, at least or up to about 2.9 kilobases, at least or up to about 3.0 kilobases, at least or up to about 3.1 kilobases, at least or up to about 3.2 kilobases, at least or up to about 3.3 kilobases, at least or up to about 3.4 kilobases, at least or up to about 3.5 kilobases, at least or up to about 3.6 kilobases, at least or up to about 3.7 kilobases, at least or up to about 3.8 kilobases, at least or up to about 3.9 kilobases, at least or up to about 4.0 kilobases, at least or up to about 4.1 kilobases, at least or up to about 4.2 kilobases, at least or up to about 4.3 kilobases, at least or up to about 4.4 kilobases, at least or up to about 4.5 kilobases, at least or up to about 4.6 kilobases, at least or up to about 4.7 kilobases, at least or up to about 4.8 kilobases, at least or up to about 4.9 kilobases, at least or up to about 5.0 kilobases, at least or up to about 5.5 kilobases, at least or up to about 6.0 kilobases, at least or up to about 6.5 kilobases, at least or up to about 7.0 kilobases, at least or up to about 7.5 kilobases, at least or up to about 8.0 kilobases, at least or up to about 9.0 kilobases, or at least or up to about 10 kilobases. In some cases, the heterologous polynucleotide of the one or more heterologous polynucleotides (e.g., the single heterologous polynucleotide) can have a size of between about 3 kilobases and about 5 kilobases, between about 3 kilobases and about 4.8 kilobases, between about 3 kilobases and about 4.6 kilobases, between about 3 kilobases and about 4.4 kilobases, between about 3 kilobases and about 4.2 kilobases, between about 3 kilobases and about 4.0 kilobases, between about 3 kilobases and about 3.5 kilobases, between about 3.5 kilobases and about 5 kilobases, between about 3.5 kilobases and about 4.8 kilobases, between about 3.5 kilobases and about 4.6 kilobases, between about 3.5 kilobases and about 4.4 kilobases, between about 3.5 kilobases and about 4.2 kilobases, between about 3.5 kilobases and about 4 kilobases, between about 4 kilobases and about 5 kilobases, between about 4 kilobases and about 4.9 kilobases, between about 4 kilobases and about 4.8 kilobases, between about 4 kilobases and about 4.7 kilobases, between about 4 kilobases and about 4...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of producing a plurality of modified tumor-reactive lymphocytes (TRLs), the method comprising:(a) isolating a plurality of T cells from a peripheral blood sample from a subject having solid malignant tumor or a processed sample obtained therefrom, wherein the plurality of T cells comprises greater than or equal to about 20% CD8+CD103+ circulating TRLs (cTRLs);(b) genetically modifying the CD8+CD103+ cTRLs to produce a genetically modified CD8+CD103+ cTRL, wherein the genetically modifying comprises introducing into or expressing in the CD8+CD103+ cTRL a gene editing platform comprising a nuclease configured to target at least one target gene, and(c) expanding the genetically modified CD8+CD103+ cTRL thereby producing the plurality of modified TRLs.

2. A method of producing a plurality of modified tumor-reactive lymphocytes (TRLs), the method comprising: expanding a genetically modified CD8+CD103+ cTRL thereby producing the plurality of modified TRLs, wherein the genetically modified CD8+CD103+ cTRL is generated by:(a) providing a plurality of T cells isolated from a peripheral blood sample from a subject having solid malignant tumor or a processed sample obtained therefrom, wherein the plurality of T cells comprises greater than or equal to about 20% CD8+CD103+ circulating TRLs (cTRLs); and(b) modifying a CD8+CD103+ cTRL in the plurality of T cells to produce the genetically modified CD8+CD103+ cTRL by introducing into or expressing in the CD8+CD103+ cTRL a gene editing platform comprising a nuclease configured to target at least one target gene.

3. The method of claim 1 or 2, wherein the at least one target gene is involved in overcoming a tumor microenvironment (TME).

4. The method of claim 3, wherein the at least one target gene comprises PD-1, CTLA-4, TIGIT, LAG-3, TIM-3, TGF-BR, IL-10R, CD39, CD73, FAS, A2AR, DGK, SMAD2, SMAD3, or SMAD4.

5. The method of claim 1 or 2, wherein the at least one target gene is involved in improved effector function.

6. The method of claim 5, wherein the at least one target gene comprises CISH, REGNASE- 1, SOCS-1, IKZF3, or TCEB2.

7. The method of claim 1 or 2, wherein the at least one target gene is involved in T cell fitness and / or exhaustion.

8. The method of claim 7, wherein the at least one target gene comprises TET2, DNMT3, SUV39H1, EZH2, BATF, IRF4, NFAT, NF-kB, CBLB, AP-1, FOXO1, FOXO3, EOMES, PRDM1, MED12, SNX9, ARID1A, ARID2, SMARCC1, SMARCD2, RUNX3, BHLHE40, FLU, SNX9, NR4A3, CCNC, HPK1 PTP1B, RASA2, PTPN2, ID3, SOX4, CUL5, TLE4, IKZF2, CD70, ROQUIN-1 DAP5, RGS16, BLIMP-1, T0X1, or TOX2.

9. The method of any one of claims 1-8, wherein the genetically modified CD8+CD103+ cTRL exhibits reduced or eliminated expression level of the at least one target gene compared to that of a control CD8+CD103+ cTRL lacking the polynucleotide-guided endonuclease or the at least one guided RNA.

10. The method of claim 9, wherein expression level is reduced or eliminated by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% as compared to that of the control CD8+CD103+ cTRL.

11. The method of any one of claims 1-8, wherein the genetically modified CD8+CD103+ cTRL exhibits increased expression level of the at least one target gene compared to that of a control CD8+CD103+ cTRL lacking the polynucleotide-guided endonuclease or the at least one guided RNA.

12. The method of claim 11, wherein expression level is increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 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 250%, at least about 300%, at least about 400%, or at least about 500% as compared to that of the control CD8+CD103+ cTRL.

13. The method of any one of claims 1-12, wherein the nuclease comprises a polynucleotide guided endonuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, an RNA-binding protein (RBP), a CRISPR-associated RNA binding protein, a derivative thereof, a variant thereof, or a fragment thereof.

14. The method of claim 13, wherein the polynucleotide-guided endonuclease comprises a CRISPR-associated (Cas) protein.

15. The method of claim 14, wherein the Cas protein is a Cas nickase (nCas) or deactivated Cas (dCas) protein that exhibits reduced or no endonuclease activity.

16. The method of claim 14 or 15, wherein the polynucleotide-guided endonuclease further comprises a domain that effects one or more activities of the Cas protein.

17. The method of claim 16, wherein the domain comprises a deaminase or a reverse transcriptase.

18. The method of claim 16, wherein the domain comprises a transcription regulator.

19. The method of any one of claims 1-18, wherein the gene editing platform further comprises at least one guide RNA exhibiting specific binding to a target polynucleotide sequence at or adjacent to the at least one target gene.

20. The method of any one of claims 1-19, wherein the solid malignant tumor is obtained from the subject having a melanoma, a colorectal cancer, a head and neck cancer, a lung cancer, or any combination thereof.

21. The method of any one of claims 1-20, wherein the plurality of T cells comprises greater than or equal to about 50% CD8+CD103+ circulating TRLs (cTRLs).

22. The method of any one of claims 1-20, wherein the plurality of T cells comprises greater than or equal to about 70% CD8+CD103+ circulating TRLs (cTRLs).

23. The method of any one of claims 1-20, wherein the plurality of T cells comprises greater than or equal to about 80% CD8+CD103+ circulating TRLs (cTRLs).

24. The method of any one of claims 1-20, wherein the plurality of T cells comprises greater than or equal to about 90% CD8+CD103+ circulating TRLs (cTRLs).

25. The method of any one of claims 1-24, wherein the plurality of modified TRLs comprises greater than or equal to about 50% CD8+CD103+ modified TRLs.

26. The method of any one of claims 1-24, wherein the plurality of modified TRLs comprises greater than or equal to about 70% CD8+CD103+ modified TRLs.

27. The method of any one of claims 1-24, wherein the plurality of modified TRLs comprises greater than or equal to about 80% CD8+CD103+ modified TRLs.

28. The method of any one of claims 1-24, wherein the plurality of modified TRLs comprises greater than or equal to about 90% CD8+CD103+ modified TRLs.

29. The method of any one of claims 1-28, wherein the CD8+CD103+ TRLs are isolated or obtained by magnetically separating an initial population of CD8+CD103+ TRLs from the peripheral blood sample from the subject having solid malignant tumor or the processed sample obtained therefrom across a magnetic capture zone disposed in a channel of a microfluidic device.

30. The method of claim 29, wherein the magnetically separating comprises the peripheral blood sample from the subject having solid malignant tumor or the processed sample obtained therefrom with an antibody capable of binding to a tumor-reactive lymphocyte (TRL) surface protein present on the initial population of CD8+CD103+ cTRLs.

31. The method of claim 30, wherein the antibody is further coupled to a magnetic nanoparticle.

32. The method of claim 30 or 31, wherein the TRL surface protein is CD3, CD4, CD8, CD39, CD 103, SLC6A19, SIDT1, or any combination thereof.

33. The method of any one of claims 30-32, wherein the antibody is conjugated to a magnetic nanoparticle.

34. The method of any one of claims 29-32, 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.

35. The method of any one of claims 1-34, wherein the method further comprises 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 of the plurality of modified TRLs.

36. The method of claim 35, wherein the exogenous polynucleotide encodes a chimeric antigen receptor (CAR).

37. The method of claim 36, wherein the CAR 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 35-38, wherein the exogenous polynucleotide encodes an engineered T-Cell receptor.

40. The method of claim 39, wherein the engineered T-Cell receptor comprises an antigen binding domain.

41. The method of claim 40, wherein the antigen binding domain binds to a tumor antigen.

42. The method of claim 41, wherein the CAR binds to an antigen selected from the group consisting of Her-2, B7-H3, GPC2, GD2, CD19, CD20, CD22, MAGE, BAGE, CAGE, GAGE, HAGE, LAGE, PAGE, PRAME, NY-ESO-1, NY-SEO-1, tyrosinase, Melan-A / MART, gplOO, TRP-1, TRP-2, CD30, EGFR, EGFRvIII, FAP, CD33, CD123, PD-L1, IGF1R, CD4, CSPG4, B7- H4, NKG2D, CS1, CD138, EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, CEA, CD52, MUC5AC, c-Met, FAB, WT-1, PSMA, AFP, BCMA, Mesothelin, GPC3, MUC1 and CTAG1B.

43. The method of any one of claims 1-42, the method further comprises administering to the subject a composition comprising a therapeutically effective amount of the plurality of modified TRLs.

44. A population of modified tumor reactive lymphocytes (TRLs) comprising: a plurality of CD8+CD103+ tumor reactive lymphocytes (TRLs) comprising one or more modifications in at least one target gene,wherein the at least one target gene is selected from the group consisting of: CD73, A2AR, DGK, REGNASE-1, IKZF3, TCEB2, TET2, DNMT3, SUV39H1, EZH2 IRF4, NF AT, NF-kB, CBLB, AP-1, FOXO1, FOXO3, EOMES MED 12, SNX9, ARID 1 A, ARID2, SMARCC1, SMARCD2, RUNX3, BHLHE40, FLU, SNX9, NR4A3, CCNC, HPK1 PTP1B, RASA2, PTPN2, ID3, SOX4, CUL5, TLE4, IKZF2, CD70, ROQUIN-1, DAP5, RGS16, and BLIMP-1.

45. The population of modified TRLs of claim 44, wherein the population of modified TRLs exhibits (a) overcoming tumor microenvironment (TME), (b) an improved effector function, (c) an improved T cell fitness and / or exhaustion, or (d) any combination thereof.

46. The population of modified TRLs of claim 44 or 45, wherein expression level of a target gene of the at least one target gene is reduced or eliminated compared to expression level of the target gene in a control population of TRL lacking the one or more modifications.

47. The population of modified TRLs of claim 44 or 45, wherein expression level of a target gene of the at least one target gene is increased compared to expression level of the target gene in a control population of TRL lacking the one or more modifications.

48. The population of modified TRLs of any one of claims 44-47, wherein the one or more modifications are genetically edited by a gene editing platform comprising a nuclease configured to target the at least one target gene.

49. The population of modified TRLs of claim 48, wherein the nuclease comprises a polynucleotide guided endonuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), a meganuclease, an RNA-binding protein (RBP), a CRISPR- associated RNA binding protein, a derivative thereof, a variant thereof, or a fragment thereof.

50. The population of modified TRLs of claim 49, wherein the polynucleotide-guided endonuclease comprises a CRISPR-associated (Cas) protein.

51. The population of modified TRLs of claim 50, wherein the Cas protein is a Cas nickase (nCas) or deactivated Cas (dCas) protein that exhibits reduced or no endonuclease activity.

52. The population of modified TRLs of claim 50 or 51, wherein the polynucleotide-guided endonuclease further comprises a domain that effects one or more activities of the Cas protein.

53. The population of modified TRLs of claim 52, wherein the domain comprises a deaminase or a reverse transcriptase.

54. The population of modified TRLs of claim 52, wherein the domain comprises a transcription regulator.

55. the population of modified TRLs of any one of claims 48-54, wherein the gene editing platform further comprises at least one guide RNA exhibiting specific binding to a target polynucleotide sequence at or adjacent to the at least one target gene.

56. The population of modified TRLs of any one of claims 44-55, wherein the plurality of CD8+CD103+ TRLs is obtained from a peripheral blood sample from a subject having a solid malignant tumor or a processed sample obtained therefrom.

57. The population of modified TRLs of claim 56, wherein the solid malignant tumor comprises a tumor from a melanoma, a colorectal cancer, a head and neck cancer, a lung cancer, or any combination thereof.

58. A method of treating a cancer in a subject in need thereof, the method comprising: administering to the subject a composition comprising a therapeutically effective amount of the population of modified TRLs of any one of claims 44-57.

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