Methods and compositions for engineering t cells

Abstract

The present disclosure provides methods for producing engineered immune cells, engineered immune cell populations, and methods of using the engineered immune cells for treating subjects with cancer and other conditions. The engineered immune cells provided herein can comprise antigen-specific T cells (e.g., neoantigen-specific T cells), TCR-T cells, or CAR-T cells. The engineered immune cells prepared by the methods provided herein can exhibit beneficial phenotypes, such as increased persistence, increased effector function, increased activation function, and / or lower dysfunction.

Description

METHODS AND COMPOSITIONS FOR ENGINEERING T CELLSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 562,533, filed on March 7, 2024, and U.S. Provisional Application Serial No. 63 / 710,396, filed on October 22, 2024, each of which is incorporated by reference herein in its entirety.BACKGROUND

[0002] Adoptive immunotherapy or adoptive cellular therapy (ACT) is the transfer of lymphocytes to a subjectforthetherapy of disease. Adoptive immunotherapy hasyetto realize its potential fortreating a wide variety of diseases including cancer, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency. However, most, if not all adoptive immunotherapy strategies may require T cell activation and expansion steps to generate a clinically effective, therapeutic dose of T cells. Due to the inherent complexity of live cell culture and patient to patient variability, current technologies for generating therapeutic doses of T cells, including engineered T cells, often remain limited by cumbersome T cell manufacturing processes. Existing T cell manufacturing processes are typically not easily scalable, repeatable, reliable, or efficient, and often produce an inferior T cell product that may be prone to exhaustion and loss of effector immune cell function. To date, engineered T cell adoptive immunotherapies have met with only limited success and routinely show variable clinical activity. Therefore, such therapies are generally not suitable for widespread clinical use. Accordingly, there remains a need for developing compositions and methods for expansion and induction of antigen-specific T cells with a favorable phenotype and function.SUMMARY

[0003] Recognized herein is a need for improved methods and compositions for producing antigenspecific T cells with enhanced functional properties such as enhanced persistence, enhanced cytotoxicity, and enhanced proliferation. Provided herein are methods and compositions for T cell manufacturing.

[0004] In one aspect, disclosed herein are methods of producing a population of engineered immune cells, the method comprising: (a) depleting one or more cells selected from the group consisting of CD14+ cells, CD25+ cells, and CD56+ cells from a population of immune cells comprising antigen presenting cells (APCs) and T cells, thereby forming a depleted population of immune cells; and (b)inhibiting expression of an endogenous gene in the depleted population of immune cells, thereby producing the population of engineered immune cells.

[0005] In some embodiments, the endogenous gene comprises one or more genes selected from the group consisting of PRDM1, TNFAIP3, REGNASE-1 (REG-1), SOCS1, PTPN2, CISH, and any combination thereof. In some embodiments, inhibiting the expression of the endogenous gene comprises silencing a gene locus or contactingto the cells or expressing in the cells a miRNA or siRNA that targets an RNA transcript encoding the endogenous gene. In some embodiments, silencing the gene locus comprises gene knockout using a site-specific nuclease, gene knockout using a site-specific nickase, or gene silencing using a site-specific transcriptional or epigenetic regulator. In some embodiments, silencing the gene locus comprises delivering (i) the site-specific nuclease, the sitespecific nickase, or the site-specific epigenetic regulator, or (ii) a nucleic acid encodingthe site-specific nuclease, the site-specific nickase, orthe site-specific epigenetic regulator into the depleted population of immune cells via electroporation, lipid nanoparticles, liposomes, nucleofection, or viral vector. In some embodiments, the endogenous gene comprises PRDM1 and / or REGNASE-1. In some embodiments, inhibiting the expression of endogenous PRDM1 comprises silencing a PRDM1 gene locus or contactingto the cells or expressing in the cells a miRNA or siRNA that targets an RNA transcript encoding PRDM1 . In some embodiments, inhibiting the expression of endogenous PRDM1 comprises expressing in the cells an miRNA or siRNA that targets an RNA transcript encoding PRDM1.

[0006] In some embodiments, inhibiting the expression of endogenous REGNASE-1 comprises silencing a REGNASE-1 gene locus or contactingto the cells or expressing in the cells a miRNA or siRNA that targets an RNA transcript encoding REGNASE-1. In some embodiments, inhibitingthe expression of endogenous REGNASE-1 comprises expressing in the cells an miRNA or siRNA that targets an RNA transcript encoding REGNASE-1.

[0007] In some embodiments, inhibiting the expression of PRDM1 and REGNASE-1 comprises silencing PRDM1 and REGNASE-1 gene loci. In some embodiments, inhibiting the expression of PRDM1 and REGNASE-1 comprises contacting to the cells or expressing in the cells miRNAs or siRNAs that target RNA transcripts encoding PRDM1 and REGNASE-1. In some embodiments, wherein silencing the PRDM1 and / or REGNASE-1 gene locus comprises gene knockout using a sitespecific nuclease, gene knockout using a site-specific nickase, or gene silencing using a site-specific epigenetic regulator.

[0008] In some embodiments, silencing the PRDM1 and / or REGNASE-1 gene locus comprises delivering (i) the site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator, or (ii) a nucleic acid encodingthe site-specific nuclease, the site-specific nickase, orthe site-specific epigenetic regulator into the depleted population of immune cells via electroporation, lipid nanoparticles, liposomes, nucleofection, or viral vector.

[0009] In some embodiments, the endogenous gene comprises TNFAIP3 and / or REGNASE-1 . In some embodiments, inhibiting the expression of endogenous TNFAIP3 comprises silencing a TNFAIP3 gene locus or contacting to the cells or expressing in the cells an miRNA or siRNA that targets an RNA transcript encoding TNFAIP3.

[0010] In some embodiments, inhibiting the expression of endogenous REGNASE-1 comprises silencing a REGNASE-1 gene locus or contacting to the cells or expressing in the cells an miRNA or siRNA that targets an RNA transcript encoding REGNASE-1.

[0011] In some embodiments, inhibiting the expression of TNFAIP3 and REGNASE-1 comprises silencing TNFAIP3 and REGNASE-1 gene loci or contacting to the cells or expressing in the cells miRNAs or siRNAs that target RNA transcripts encoding TNFAIP3 and REGNASE-1 .

[0012] In some embodiments, silencingthe TNFAIP3 and / or REGNASE-1 gene locus comprises gene knockout using a site-specific nuclease, gene knockout using a site-specific nickase, or gene silencing using a site-specific epigenetic regulator.

[0013] In some embodiments, silencing the TNFAIP3 and / or REGNASE-1 gene locus comprises delivering (i) the site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator, or (ii) a nucleic acid encodingthe site-specific nuclease, the site-specific nickase, orthe sitespecific epigenetic regulator into the depleted population of immune cells via electroporation, lipid nanoparticles, liposomes, nucleofection, or viral vector.

[0014] In some embodiments, the endogenous gene comprises PRDM1 and / or TNFAIP3. In some embodiments, inhibiting the expression of endogenous PRDM1 comprises silencing a PRDM1 gene locus or contacting to the cells or expressing in the cells a miRNA or siRNA that targets an RNA transcript encoding PRDM1. In some embodiments, inhibiting the expression of endogenous TNFAIP3 comprises silencing a TNFAIP3 gene locus or contacting to the cells or expressing in the cells a miRNA or siRNA that targets an RNA transcript encoding TNFAIP3. In some embodiments, inhibiting the expression of PRDM1 and TNFAIP3 comprises silencing PRDM1 and TNFAIP3 gene loci or contacting to the cells or expressing in the cells miRNAs or siRNAs that target RNA transcripts encoding PRDM1 and TNFAIP3. In some embodiments, silencing the PRDM1 and / or TNFAIP3 gene locus comprises gene knockout using a site-specific nuclease, gene knockout using a site-specific nickase, or gene silencing using a site-specific epigenetic regulator. In some embodiments, silencing the PRDM1 and / or TNFAIP3 gene locus comprises delivering (i) the site-specific nuclease, the sitespecific nickase, orthe site-specific epigenetic regulator, or (ii) a nucleic acid encodingthe site-specificnuclease, the site-specific nickase, or the site-specific epigenetic regulator into the depleted population of immune cells via electroporation, lipid nanoparticles, liposomes, nucleofection, or viral vector.

[0015] In some embodiments, the depleted population of immune cells comprises a first population of APCs and T cells, and the method further comprises (c) incubating the first population of APCs and T cells for a first time period in the presence of (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, incubating the first population of APCs and T cells for the first time period further comprises incubating in the presence of (i) FLT3L, and (ii) (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encodingthe polypeptide, thereby forming a population of engineered immune cells comprising stimulated T cells. In some embodiments, the polypeptide comprises a cancer protein or a fragment thereof comprising at least one tumor antigen epitope sequence expressed by cancer cells. In some embodiments, incubating in (c) is performed prior to or subsequent to inhibiting in (b).

[0016] In some embodiments, the method further comprises, subsequent to (b) and (c), expanding the stimulated T cells, thereby forming an expanded population of cells comprisingtumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells that are specific to a complex comprising (1) the at least one tumor antigen epitope sequence from step (c)(ii), and, (2) an MHC protein expressed by the cancer cells, or APCs of the human subject of (c)(ii). In some embodiments, inhibiting in (b) comprises inhibiting expression of endogenous REGNASE-1 in the depleted population of immune cells. In some embodiments, inhibiting the expression of endogenous REGNASE-1 comprises silencing REGNASE-1 gene locus or comprises expression of an exogenous miRNA or an exogenous siRNA that targets REGNASE-1 .

[0017] In another aspect, disclosed herein are methods of producing a population of engineered immune cells, the method comprising: (a) inhibiting expression of an endogenous gene in a population of immune cells, thereby producingthe population of engineeredimmunecells, wherein the population of immune cells comprises T cells; and (b) depleting one or more cells selected from the group consisting of CD14+ cells, CD25+ cells, and CD56+ cells in the population of immune cells to obtain a depleted population of immune cells.

[0018] In some embodiments, the population of immune cells further comprises antigen presenting cells (APCs). In some embodiments, the depleted population of immune cells comprises a first population of APCs and T cells, and the method further comprises (c) incubating the first population of APCs and T cells for a first time period in the presence of FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, incubating the first population of APCs and T cells for the first time period further comprises incubating in the presence of (i) FLT3L, and (ii) (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subjectwith cancer, or (B) a polynucleotide encoding the polypeptide, thereby forming a population of engineered immune cells comprising stimulated T cells. In some embodiments, wherein (c) is performed subsequent to depleting in (b).

[0019] In some embodiments, the method further comprises, subsequent to (b) and (c), expanding the stimulated T cells, thereby formingan expanded population of cells comprisingtumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells that are specific to a complex comprising (1) the at least one tumor antigen epitope sequence from step (c)(ii), and, (2) an MHC protein expressed by the cancer cells, or APCs of the human subject of (c)(ii).

[0020] In another aspect, disclosed herein are methods of producing a population of engineered immune cells, the method comprising: (a) inhibiting expression of an endogenous gene in a population of immune cells, wherein the population of immune cells comprises T cells; and (b) incubating the population of immune cells from (a) for a first time period in the presence of FMS-like tyrosine kinase 3 receptor ligand (FLT3L).

[0021] In some embodiments, the method further comprises depleting one or more cells selected from the group consisting of CD14+ cells, CD25+ cells, and CD56+ cells in the population of immune cells after (a).

[0022] In some embodiments, the method further comprises depleting one or more cells selected from the group consisting of CD14+ cells, CD25+ cells, and CD56+ cells in the population of immune cells after (a) and prior to (b). In some embodiments, the population of immune cells in (a) have been depleted of one or more cells selected from the group consisting of CD 14+ cells, CD25+ cells, and CD56+ cells. In some embodiments, depleting further comprises depleting CD19+ cells from the population of immune cells. In some embodiments, depleting further comprises depleting CDllb+ cells from the population of immune cells. In some embodiments, the endogenous gene comprises one or more genes selected from the group consisting of PRDM1, TNFAIP3, REGNASE-1, SOCS1, PTPN2, CISH, and any combination thereof. In some embodiments, inhibiting the expression of the endogenous gene comprises silencing a gene locus or contacting to the cells or expressing in the cells a miRNA or siRNA that targets an RNA transcript encoding the endogenous gene. In some embodiments, silencing the gene locus comprises gene knockout using a site-specific nuclease, gene knockout using a site-specific nickase, or gene silencing using a site-specific epigenetic regulator. In some embodiments, silencingthe gene locus comprises delivering (i) the site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator, or (ii) a nucleic acid encoding the sitespecific nuclease, the site-specific nickase, or the site-specific epigenetic regulator into the depleted population of immune cells via electroporation, lipid nanoparticles, liposomes, nucleofection, or viralvector. In some embodiments, the endogenous gene comprises PRDM1 and / or TNFAIP3. In some embodiments, the endogenous gene comprises PRDM1 and / or REGNASE-1.

[0023] In some embodiments, the endogenous gene comprises TNFAIP3 and / or REGNASE-1 .

[0024] In some embodiments, inhibiting in (a) comprises inhibiting in the population of immune cells expression of endogenous REGNASE-1 . In some embodiments, inhibiting REGNASE-1 comprises inhibiting the expression of endogenous REGNASE-1 comprises silencing REGNASE-1 gene locus to eliminate expression of endogenous REGNASE- 1 or comprises expression of an exogenous miRNA or an exogenous siRNA that targets REGNASE-1.

[0025] In some embodiments, the population of immune cells is from a biological sample from a human subject. In some embodiments, the population of immune cells is from the same human subject the cancer cells are obtained. In some embodiments, the expanded population of cells comprises at least about 1x106total cells. In some embodiments, the expanded population of cells comprises atleast about IxlO7total cells. In some embodiments, the expanded population of cells comprises at least about 1x108total cells. In some embodiments, the expanded population of cells comprises from about lxl08to about IxlO11total cells. In some embodiments, the expanded population of cells comprises from about 0.75xl08to about 1.25xl010total cells. In some embodiments, the expanded population of cells comprises from about 5x108to about IxlO10total cells, about 5xl08to about IxlO9total cells, or from about 5x108to about 2xl09total cells. In some embodiments, the depleting comprises depleting CD14+ cells and / or CD25+ cells directly from a washed and / or cryopreserved peripheral blood mononuclear cell (PBMC) sample from a human subject. In some embodiments, incubating and expanding are performed in less than 28 days. In some embodiments, the fraction of CD8+ antigenspecific T cells of the total number of CD8+ T cells in the expanded population of cells is at least twofold higher than the fraction of CD8+ antigen-specific T cells of the total number of CD8+ T cells in the depleted population of immune cells. In some embodiments, the fraction of CD4+ antigen-specific T cells of the total number of CD4+ T cells in the expanded population of cells is at least two-fold higher than the fraction of CD4+ antigen-specific T cells of the total number of CD4+ T cells in the depleted population of immune cells. In some embodiments, atleast 0.1% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from naive CD8+ T cells. In some embodiments, wherein at least 0.1% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from naive CD4+ T cells.

[0026] In some embodiments, expanding comprises (A) contacting the population of cells comprising stimulated T cells with a second population of mature APCs, wherein the second population of mature APCs (i) have been incubated with FLT3L and (ii) present the peptide consisting of the epitope sequence from the protein expressed by the cancer cells of the human subject; and (B) expanding thepopulation of cells comprising stimulated T cells for a second time period, thereby forming an expanded population of T cells. In some embodiments, the second population of mature APCs have been incubated with FLT3L for at least 1 day prior to contacting the population of cells comprising stimulated T cells with the second population of mature APCs. In some embodiments, depleting one or more cells selected from the group consisting of CD14+ cells, CD25+ cells, and CD56+ cells from the population of immune cells comprises contacting the population of immune cells with a CD 14 binding agent, a CD25 binding agent, and / or a CD56 binding agent. In some embodiments, the percentage of CD3+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 40%, at least 50% or at least 60% of the total cell population. In some embodiments, the percentage of CD 107a+ cells in the expanded population of cells comprisingtumor antigen-specific T cells is at least 10% of the tumor antigen-specific T cell population. In some embodiments, the percentage of TNFa+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 5% of the tumor antigen-specific T cell population. In some embodiments, the percentage of IFNy+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 15% of the tumor antigen-specific T cell population. In some embodiments, the percentage of TNFa+and IFNy+ cells in the expanded population of cells comprising tumor antigenspecific T cells is at least 2% of the tumor antigen-specific T cell population. In some embodiments, the percentage of TNFa+ and CD 107a+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 0.5% of the tumor antigen-specific T cell population. In some embodiments, the percentage of IFNy+ and CD107a+ cells in the expanded population of cells comprisingtumor antigen-specific T cells is at least 5% of the tumor antigen-specific T cell population. In some embodiments, the percentage of TNFa+ and IFNy+ and CD107a+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 0.1% of the tumor antigenspecific T cell population. In some embodiments, the percentage of CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are naive T cells (CD62L+ and CD45RA+) is at most 15%. In some embodiments, the percentage of CD4+ T cells in the expanded population of cells comprisingtumor antigen-specific T cells that are effector memory T cells (CD62L- and CD45RA-) is at least 60%. In some embodiments, the percentage of CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector T cells (CD62L- and CD45RA+) is at most 5%. In some embodiments, the percentage of CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are central memory T cells (CD62L+ and CD45RA-) is at least 10%. In some embodiments, the percentage of CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are naive T cells (CD62L+ and CD45RA+) is at most 25%. In some embodiments, the percentage of CD8+ T cells in the expandedpopulation of cells comprisingtumor antigen-specific T cells that are effector memory T cells (CD62L- and CD45RA-) is at least 60%. In some embodiments, the percentage of CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector T cells (CD62L- and CD45RA+) is at most 10%. In some embodiments, the percentage of CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are central memory T cells (CD62L+ and CD45RA-) is at least 15%.

[0027] In some embodiments, the percentage of CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are CD127+ is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90%.

[0028] In some embodiments, the expanded population of cells comprising tumor antigen-specific T cells produce cytokines and cause degranulation upon recognition of target cells. In some embodiments, depleting comprises depleting CD14+ cells and CD25+ cells from a peripheral blood mononuclear cell (PBMC) sample from a human subject thathas notbeen subject to a step of monocyte maturation into mature dendritic cells (DCs). In some embodiments, depleting further comprises depleting CD1 lb+ cells from the peripheral blood mononuclear cell (PBMC) sample from the human subject that has notbeen subject to a step of monocyte maturation into mature dendritic cells (DCs). In some embodiments, the fraction of CD8+ tumor antigen-specific T cells of the total number of CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells is at least two-fold higher than the fraction of CD8+ tumor antigen-specific T cells of the total number of CD8+ T cells in the biological sample. In some embodiments, the fraction of CD4+ tumor antigen-specific T cells of the total number of CD4+ T cells in the expanded population of cells comprisingtumor antigenspecific T cells is at least two-fold higher than the fraction of CD4+ tumor antigen-specific T cells of the total number of CD4+ T cells in the biological sample. In some embodiments, at least 0.1% of the CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells are CD8+ tumor antigen-specific T cells derived from naive CD8+ T cells. In some embodiments, at least 0.1% of the CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells are CD4+ tumor antigen-specific T cells derived from naive CD4+ T cells.

[0029] In some embodiments, expanding comprises (A) contacting the population of cells comprising stimulated T cells with a second population of mature APCs, wherein the second population of mature APCs (i) have been incubated with FLT3L and (ii) present the at least one tumor antigen epitope sequence; and (B) expanding the population of cells comprising stimulated T cells for a second time period, thereby forming an expanded population of T cells. In some embodiments, the second population of mature APCs have been incubated with FLT3L for at least 1 day prior to contacting the population of cells comprising stimulated T cells with the second population of mature APCs. In someembodiments, the biological sample is a peripheral blood sample, a leukapheresis sample or an apheresis sample.

[0030] In some embodiments, the method further comprises harvesting the expanded population of cells comprising tumor antigen-specific T cells, cry opreserving the expanded population of cells comprising tumor antigen-specific T cells or preparing a pharmaceutical composition containing the expanded population of cells comprising tumor antigen-specific T cells. In some embodiments, incubating comprises incubatingthe depleted population of immune cells comprisingafirstpopulation of APCs and T cells for a first time period in the presence of FLT3L and an RNA encoding the polypeptide. In some embodiments, the population of engineered immune cells does not comprise a cell expressing a chimeric antigen receptor (CAR). In some embodiments, the population of engineered immune cells exhibit enhanced T cell activation, enhanced effector function, enhanced persistence, enhanced proliferation, or enhanced avoidance of dysfunction, or any combination thereof compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, the enhanced T cell activation comprises enhanced T cell priming, enhanced antigen recognition, enhanced cytokine sensing, or enhanced costimulation properties, or any combination thereof. In some embodiments, the enhanced effector function comprises enhanced tumor infiltration, enhancedmetabolic changes, enhanced proliferation, enhanced bystander activation, enhanced cytokine secretion, or enhanced cytotoxicity, or any combination thereof. In some embodiments, the population of engineered immune cells has a decreased proportion of PD-1+ T cells, TIM3+ T cells, TIGIT+ T cells, Lag-3+ T cells, CD39+ T cells, and / or TOX+ T cells compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, the percentage of PD-1 + T cells, TIM3+ T cells, TIGIT+ T cells, Lag-3+ T cells, CD39+ T cells, and / or TOX+ T cells is at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% less than the percentage of PD-1 + T cells, TIM3+ T cells, TIGIT+ T cells, Lag-3+ T cells, CD39+ T cells, and / or TOX+ T cells in an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, the population of engineered immune cells has an increased cytokine release compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, the cytokine comprises IL-2, IFNg, TNF- a, Perforin, IL- 18, or any combination thereof. In some embodiments, the increased cytokine release comprises atleast about 0.5 -fold, 1 -fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 100-fold, 150-fold, or more than a cytokine release of an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, the enhanced persistencecomprises enhancedhomeostatic cytokinerelease, enhanced memory function, enhanced proliferation, enhanced sternness, or enhanced metabolism, or any combination thereof. In some embodiments, the population of engineered immune cells has an increased fold expansion compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, a fold expansion is atleast about 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 2.5- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, or more than a fold expansion of an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene after culturing for a period of time. In some embodiments, a fold expansion is at least about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8- fold, 1.9-fold, 2-fold, 2.1 -fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9- fold, 3 -fold, 3.1 -fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5 -fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4-fold, 4.1-fold, 4.2-fold, 4.3-fold, 4.4-fold, 4.5 -fold, 4.6-fold, 4.7-fold, 4.8 -fold, 4.9-fold, 5-fold, 5.5-fold, 6- fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, or more than a 13-fold expansion of an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene after culturing for a period of time.

[0031] In some embodiments, the period of time is at least about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more. In some embodiments, the population of engineered immune cells has an increased persistence or expansion in a peripheral blood sample compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene(s) after being administered into a subject.

[0032] In some embodiments, the population of engineered immune cells has an increased proportion of central memory T cells compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, the increased proportion of central memory T cells comprises at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% more than the proportion of central memory T cells in an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, the increased proportion of central memory T cells comprises atleast about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% more than the proportion of central memory T cells in an otherwise identical population ofengineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, the enhanced avoidance of dysfunction comprises reduced overactivation, reduced AICD expression, reduced exhaustion, reduced apoptosis, reduced toxicity, reduced antigen escape, reduced immunosuppression, or enhanced homeostasis, or any combination thereof. In some embodiments, the population of engineered immune cells has an increased suppression of tumor growth compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, a tumor volume in a subject administered with the population of engineered immune cells is at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% less than that of an identical subject administered with an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, a tumor volume in a subject administered with the population of engineered immune cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% less than that of an identical subject administered with an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, the enhanced cytotoxicity comprises enhanced suppression of tumor cell growth compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, a rate of tumor cell growth is reduced by a factor of 1 .2-fold, 1.5-fold, 2-fold, 3 -fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold ormore compared to a rate of tumor cell growth in the presence of an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, the population of engineered immune cells with reduced expression of the endogenous gene(s) has an increased proportion of CD127+ expressing cells compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene(s). In some embodiments, the population of engineered immune cells has an increased percent frequency of TexPr°82cells or LylO8+CD69- cells in a population of antigen-specific CD8+ cells compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene(s).

[0033] In some embodiments, the increased percent frequency of TexPro§2cells orLyl08+CD69- cells in the population of engineered immune cells is at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to that of an identical population of engineered immune cells not having the reduced expression of the endogenous gene(s). In some embodiments, the increased percent frequency of Texf’ros2cells or Ly 108+CD69- cells in the population of engineered immune cells is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% compared to that of an identical population of engineered immune cells not having the reduced expression of the endogenous gene(s).

[0034] In some embodiments, the method further comprises preparing a pharmaceutical composition comprising the population of engineered immune cells.

[0035] In some embodiments, the method further comprises administering the pharmaceutical composition in a subject in need thereof. In some embodiments, the subject is the same subject from which the biological sample is obtained.

[0036] In another aspect, disclosed herein are engineered immune cells comprising: a nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising a target binding domain, a transmembrane domain and an intracellular signaling domain; and an agent for reducing expression of an endogenous gene, wherein the engineered immune cell has reduced expression of the endogenous gene compared to an otherwise identical engineered immune cell not having the agent.

[0037] In some embodiments, the endogenous gene comprises one or more genes selected from the group consisting of PRDM1, TNFAIP3, REGNASE-1, SOCS1, PTPN2, CISH, and any combination thereof. In some embodiments, the endogenous gene comprises PRDM1 and / or TNFAIP3. In some embodiments, the endogenous gene comprises PRDM1 and TNFAIP3. In some embodiments, the endogenous gene comprises REGNASE- 1. In some embodiments, the agent is a nucleic acid encoding a component for gene silencing, or the agent is a miRNA or siRNA that targets an RNA transcript encodingthe endogenous gene. In some embodiments, the endogenous gene comprises PRDM1 and / or REGNASE-1. In some embodiments, the endogenous gene comprises PRDM1 and REGNASE-1.

[0038] In some embodiments, the endogenous gene comprises TNFAIP3 and / or REGNASE-1 . In some embodiments, the endogenous gene comprises TNFAIP3 and REGNASE-1.

[0039] In some embodiments, the component for gene silencing comprises a guide RNA. In some embodiments, the target binding domain is an antigen binding domain. In some embodiments, the target binding domain comprises an antibody, an antibody fragment, an scFv, an Fv, a Fab, a (Fab')2, a single domain antibody (SdAb), a VH or VL domain, or a camelid VHH domain. In some embodiments, the transmembrane domain is fused to an extracellular domain comprising the target binding domain. In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2Rbeta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6, CD49f, ITGAD, CDIId, ITGAE, CD103, ITGAL, CDIIa, LFA-1, ITGAM, CDIIb, ITGAX, CDIIc, ITGBI, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGLI, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C, a synthetic material, or a functional variant thereof. In some embodiments, the intracellular signaling domain comprises a co-stimulatory domain. In some embodiments, the intracellular signaling domain comprises a CD3-zeta signaling domain. In some embodiments, wherein the intracellular signaling domain comprises a CD3-zeta signaling domain and a costimulatory signaling domain.

[0040] In some embodiments, the CAR comprises multiple costimulatory domains. In some embodiments, the one or more costimulatory domains comprise CD28, CD137 (4-1BB), TNFR, CD134 (0X40), CD278, or any combination thereof. In some embodiments, the CAR further comprises a CD8a hinge domain. In some embodiments, the hinge domain forms a link between the transmembrane domain and the extracellular domain. In some embodiments, the CAR further comprises a fusion protein. In some embodiments, the fusion protein further comprises a linker, a signal peptide, or any combination thereof.

[0041] In yet another aspect, disclosed herein are engineered immune cells comprising: a nucleic acid molecule encoding a T-cell receptor (TCR), wherein the TCR binds to KRAS, PRAME, or GATA3; and an agent for reducing expression of an endogenous gene, wherein the engineered immune cell has reduced expression of the endogenous gene compared to an otherwise identical engineered immune cell not having the agent.

[0042] In some embodiments, the endogenous gene comprises one or more genes selected from the group consisting of PRDM1, TNFAIP3, REGNASE-1, S0CS1, PTPN2, CISH, and any combinationthereof. In some embodiments, the endogenous gene comprises PRDM1 and / or TNFAIP3. In some embodiments, the endogenous gene comprises PRDM1 and TNFAIP3. In some embodiments, the endogenous gene comprises REGNASE- 1. In some embodiments, the agent is a nucleic acid encoding a component for gene silencing, or the agent is a miRNA or siRNA that targets an RNA transcript encoding the endogenous gene. In some embodiments, the endogenous gene comprises REGNASE-1.

[0043] In some cases, the endogenous gene can comprise PRDM1 and / or REGNASE-1. In some embodiments, the endogenous gene comprises PRDM1 and REGNASE-1 . In some embodiments, the endogenous gene comprises TNFAIP3 and / or REGNASE-1 . In some embodiments, the endogenous gene comprises TNFAIP3 and REGNASE-1 . In some embodiments, the component for gene silencing comprises a guide RNA. In some embodiments, the TCR binds to a PRAME epitope in complex with an MHC encoded by an HLA-A02 :01 allele. In some embodiments, the PRAME epitope comprises an amino acid sequence of SEQ ID NO : 116. In some embodiments, the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 105. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 103 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 104. In some embodiments, the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence setforth in SEQ ID NO: 100, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 101, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 102.

[0044] In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence setforth in SEQ ID NO: 113. In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence setforth in SEQ ID NO: 124, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 124, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 123, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 123. In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 126, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 126, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 125, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 125. In some embodiments, the TCR binds to a complex comprising (i) an epitope from human RAS comprising a mutation G12V and (ii) an MHC protein encoded by an HLA- A11 :01 allele. In some embodiments, the TCRbindsto the epitope comprising an amino acid sequenceof SEQ ID NO: 51, 52, 53, 55 or 56 in complex with an MHC encoded by an HLA-A11 :01 allele. In some embodiments, the TCR comprises a TCRbeta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) havingan amino acid sequence of SEQ ID NO: 6. In some embodiments, the TCRbeta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 4 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 1, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 2, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 13. In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 17, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 14.

[0045] In some embodiments, the TCRbinds to a complex comprising (i) an epitope from human RAS comprising a mutation G12V and (ii) an MHC protein encoded by an HLA-A68:01 allele. In some embodiments, the TCR binds to the epitope comprising an amino acid sequence of SEQ ID NO: 51 in complex with an MHC encoded by an HLA-A68:01 allele. In some embodiments, the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 132. In some embodiments, the TCRbeta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 134. In some embodiments, the TCRbeta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 130 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 131. In some embodiments, the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 127, the CDR2 has anamino acid sequence set forth in SEQ ID NO: 128, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 129. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 133.

[0046] In some embodiments, the engineered immune cell exhibits enhanced T cell activation, enhanced effector function, enhanced persistence, enhanced proliferation, or enhanced avoidance of dysfunction, or any combination thereof compared to an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene. In some embodiments, the enhanced T cell activation comprises enhanced T cell priming, enhanced antigen recognition, enhanced cytokine sensing, or enhanced costimulation properties, or any combination thereof. In some embodiments, the enhanced effector function comprises enhanced tumor infiltration, enhanced metabolic changes, enhanced proliferation, enhanced bystander activation, enhanced cytokine secretion, or enhanced cytotoxicity, or any combination thereof. In some embodiments, the engineered immune cell has an increased cytokine release compared to an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene. In some embodiments, the cytokine comprises IL-2, IFNg, TNF-a, Perforin, IL-18, or any combination thereof. In some embodiments, the increased cytokine release comprises at least about 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 100-fold, 150-fold, or more than a cytokine release of an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene. In some embodiments, the enhanced persistence comprises enhanced homeostatic cytokine release, enhanced memory function, enhanced proliferation, enhanced sternness, or enhanced metabolism, or any combination thereof. In some embodiments, the engineered immune cell has an increased fold expansion compared to an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene. In some embodiments, a fold expansion is at least about 1. 1-fold, 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 11 -fold, 12-fold, 13-fold, or morethan a fold expansion ofan otherwise identical engineered immune cell not having a reduced expression of the endogenous gene after culturing for a period of time. In some embodiments, the period of time is at least about2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more. In some embodiments, the engineered immune cell comprises a population of engineered immune cells, and whereinthe population of engineered immune cells has an increased persistence as measured by cell count of TCR+ / CD3+ cells compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, the engineered immune cell comprises a population of engineered immune cells, and wherein the population of engineered immune cells has an increasedpersistence as measured by cell count of TCR+ / CD3+ cells in a sample from a subject administered with the population of engineered immune cells compared to that of a sample from a subject administered with an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, the TCR+ / CD3+ cell count is increased by 1-fold, 10-fold, 100-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, ormore compared to the amount of TCR+ / CD3+ in the sample from the subject administered with an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, the TCR+ / CD3+ cell count in a peripheral blood sample from a subject administered with the population of engineered immune cells is increased by 1-fold, 10-fold, 100-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, or more compared to the amount of TCR+ / CD3+ in a peripheral blood sample from a subject administered with an otherwise identical population of engineered immune cells in the peripheral blood sample not having a reduced expression of the endogenous gene. In some embodiments, the TCR+ / CD3+ cell count in a peripheral blood sample from a subject administered with the population of engineered immune cells is increased by 1- fold, 10-fold, 100-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, or more compared to the amount of TCR+ / CD3+ in a peripheral blood sample from a subject administered with an otherwise identical population of engineered immune cells in the peripheral blood sample not having a reduced expression of the endogenous gene, after at least 8 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days or more post adoptive cell transfer of the population of engineered immune cells.

[0047] In some embodiments, the TCR+ / CD3+ cell count is increased by 1-fold, 10-fold, 100-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, or more compared to the amount of TCR+ / CD3+ in an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene. In some embodiments, the enhanced avoidance of dysfunction comprises reduced overactivation, reduced AICD expression, reduced exhaustion, reduced apoptosis, reduced toxicity, reduced antigen escape, reduced immunosuppression, or enhanced homeostasis, or any combination thereof. In some embodiments the engineered immune cell has an increased suppression of tumor growth compared to an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene. In some embodiments, a tumor volume in a subject administered with the engineered immune cell is at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% less than that of an identical subject administered with an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene. In some embodiments, the enhanced cytotoxicity comprises enhanced suppression of tumor cell growth compared to an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene. In some embodiments, a rate oftumor cell growth is reduced by a factor of 1 .2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7- fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold or more comparedto arate of tumor cell growth in the presence of an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene.

[0048] In some embodiments, a population of engineered immune cells comprises the engineered immune cells previously described herein.

[0049] In yet another aspect, disclosed herein is a population of engineered immune cells, wherein expression of an endogenous gene is inhibited in the population of engineered immune cells; and wherein the population of engineered immune cells is from a biological sample from a subject and the percentage of CD14+ cells and / or CD25+ cells and / or CD56+ cells in the population of engineered immune cells is lower compared to the percentage of CD 14+ cells and / or CD25+ cells and / or CD56+ cells in the biological sample.

[0050] In some embodiments of the population of engineered immune cells, the endogenous gene comprises one or more genes selected from the group consisting of PRDM1, TNFAIP3, REGNASE- 1, SOCS1, PTPN2, CISH, and any combination thereof. In some embodiments of the population of engineered immune cells, the endogenous gene comprises PRDM1 and / or TNFAIP3. In some embodiments, the endogenous gene comprises PRDM1 and TNFAIP3. In some embodiments, the endogenous gene comprises REGNASE-1. In some embodiments, the endogenous gene comprises PRDM1 and / or REGNASE-1 . In some embodiments, the endogenous gene comprises PRDM1 and REGNASE-1 . In some embodiments, the endogenous gene comprises TNFAIP3 and / or REGNASE- 1. In some embodiments, the endogenous gene comprises TNFAIP3 and REGNASE-1. In some embodiments, the agent is a nucleic acid encoding a component for gene silencing, or the agent is a miRNAor siRNA th at targets an RNA transcript encodingthe endogenous gene. In someembodiments, the component for gene silencing comprises a guide RNA. In some embodiments, the population of engineered immune cells comprises at least 1x10A5 cells. In some embodiments, the engineered immune cells in the population of engineered immune cells further comprise a chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

[0051] In yet another aspect, disclosed herein are methods of preparing an engineered immune cell expressing a chimeric antigen receptor (CAR), the method comprising: (a) introducing into an immune cell a nucleic acid sequence encoding the CAR comprising a target binding domain, a transmembrane domain and an intracellular signaling domain; and (b) prior to, sub sequent to, or concurrently to (a), inhibiting in the immune cell an expression of an endogenous gene, wherein the endogenous gene comprises one or more genes selected from the group consisting of PRDM1, TNFAIP3, REGNASE- 1, SOCS1, PTPN2, CISH, and any combination thereof.

[0052] In some embodiments, the endogenous gene comprises PRDM1 and / or TNFAIP3. In some embodiments, the endogenous gene comprises PRDM1 and TNFAIP3. In some embodiments, the endogenous gene comprises REGNASE-1. In some embodiments, the endogenous gene comprises PRDM1 and / or REGNASE-1 . In some embodiments, the endogenous gene comprises PRDM1 and REGNASE-1 . In some embodiments, the endogenous gene comprises TNFAIP3 and / or REGNASE- 1. In some embodiments, the endogenous gene comprises TNFAIP3 and REGNASE-1. In some embodiments, inhibiting the expression of the endogenous gene comprises silencing a gene locus or contacting to the cells or expressing in the cells a miRNA or siRNA that targets an RNA transcript encoding the endogenous gene. In some embodiments, the CAR comprises a target binding domain, a transmembrane domain, and a T cell signaling domain. In some embodiments, the target binding domain is an antigen binding domain. In some embodiments, the target binding domain comprises an antibody, an antibody fragment, an scFv, an Fv, a Fab, a (Fab')2, a single domain antibody (sdAb), a VH or VL domain, or a VHH domain. In some embodiments, the transmembrane domain is fused to an extracellular domain. In some embodiments, the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIId, ITGAE, CD 103, ITGAL, CDIIa, LFA-1, ITGAM, CDIIb, ITGAX, CDIIc, ITGBI, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGLI, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, andNKG2C, a synthetic material, or a functional variant thereof . In some embodiments, the intracellular signaling domain comprises a co-stimulatory domain. In some embodiments, the intracellular signaling domain comprises a CD3-zeta signaling domain. In some embodiments, the intracellular signaling domain comprises a CD3-zeta signaling domain and a costimulatory signaling domain. In some embodiments, the CAR comprises multiple costimulatory domains. In some embodiments, the one or more costimulatory domains comprise CD28, CD137 (4-1BB), TNFR, CD134 (0X40), CD278, or any combination thereof. In some embodiments, the CARfurther comprises a CD8a hinge domain. In some embodiments, the hinge domain forms a link between the transmembrane domain and the extracellular domain. In some embodiments, the CAR further comprises a fusion protein. In some embodiments, the fusion protein further comprises a linker, a signal peptide, or any combination thereof.

[0053] In yet another aspect, disclosed herein are methods of preparing an engineered immune cell expressing a T-cell receptor (TCR) targeting KRAS, PRAME or GAT A3, the method comprising: (a) introducing into an immune cell a nucleic acid sequence encoding the TCR, wherein the TCR binds to KRAS, PRAME or GAT A3; and (b) prior to, sub sequent to, or concurrently to (a), inhibiting in the immune cell an expression of an endogenous gene, wherein the endogenous gene comprises one or more genes selected from the group consisting of PRDM1, TNFAIP3, REGNASE-1, SOCS1, PTPN2, CISH, and any combination thereof.

[0054] In some embodiments, the endogenous gene comprises PRDM1 and / or TNFAIP3. In some embodiments, the endogenous gene comprises PRDM1 and TNFAIP3. In some embodiments, the endogenous gene comprises REGNASE-1. In some embodiments, the endogenous gene comprises PRDM1 and / or REGNASE-1 . In some embodiments, the endogenous gene comprises PRDM1 and REGNASE-1 . In some embodiments, the endogenous gene comprises TNFAIP3 and / or REGNASE- 1. In some embodiments, the endogenous gene comprises TNFAIP3 and REGNASE-1. In some embodiments, inhibiting the expression of the endogenous gene comprises silencing a gene locus or contacting to the cells or expressing in the cells a miRNA or siRNA that targets an RNA transcript encodingthe endogenous gene. In some embodiments, the TCRbinds to a PRAME epitope in complex with an MHC encoded by an HLA-A02:01 allele. In some embodiments, the PRAME epitope comprises an amino acid sequence of SEQ ID NO: 116. In some embodiments, the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 105. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115. In some embodiments, the TCRbeta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 103 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 104. In some embodiments, the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence setforthin SEQ ID NO: 100, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 101, and the CDR3 has an amino acid sequence SEQ ID NO: 102. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 113. In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence setforthin SEQ ID NO: 124, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 124, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 123, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 123. In some embodiments, theTCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 126, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 126, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 125, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 125. In some embodiments, the TCR binds to a complex comprising (i) an epitope from human RAS comprising a mutation G12V and (ii) an MHC protein encoded by an HLA- A11 :01 allele. In some embodiments, the TCR binds to a K-RAS epitope in complex with an MHC encoded by an HLA-Al l :01 allele. In some embodiments, the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence SEQ ID NO: 6. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 12. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 4 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 5. In some embodiments, the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 1, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 2, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 3. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 9. In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 13. In some embodiments, the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 17, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 14. In some embodiments, the TCR binds to a K-RAS epitope in complex with an MHC encoded by an HLA-A68:01 allele. In some embodiments, the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence SEQ ID NO: 132. In some embodiments, the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 134. In some embodiments, the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 130 and a complementaritydetermining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 131. In some embodiments, the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence setforth in SEQ ID NO: 127, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 128, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 129. In some embodiments, the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence setforth in SEQ ID NO: 133.

[0055] In another aspect, disclosedherein are pharmaceutical compositions comprisingthe population of engineered immune cells produced according to the previously disclosed methods, the engineered immune cells of any of the previously disclosed methods, or the populations of engineered immune cells previously disclosed, and a pharmaceutically acceptable carrier.

[0056] In yet another aspect, disclosed hereinis a method of treating a disease or condition in a subject in need thereof comprising administering the pharmaceutical compositions disclosed herein. In some aspects, the subject is the same subject from which the biological sample is obtained.

[0057] In yet another aspect, disclosedherein are uses of the engineered immune cells disclosed herein or the populations of engineered immune cells disclosed herein in the manufacture of a medicament for treating cancer in a subject.INCORPORATION BY REFERENCE

[0058] 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.BRIEF DESCRIPTION OF THE DRAWINGS

[0059] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0060] FIG. 1A depicts a general schematic of the CRISPR Cas9-mediated gene editing process for indel formation.

[0061] FIG. IB depicts a general schematic of CRISPR Cas9-mediated gene perturbation.

[0062] FIG. 2 is a schematic depicting example steps of a T cell CRISPR Cas9-mediated gene editing and screening method.

[0063] FIG. 3A depicts a CRISPR screening platform utilizing sgRNA libraries for loss-of-function and / or gain-of-function screening. The platform can include sgRNA libraries, screening systems, model systems, and phenotypic readouts.

[0064] FIG. 3B depicts example workflow of screening. The screen can use CRISPR-Cas9 system in different model systems and readouts. Validation can be done across more donors and functional readouts. The identified genes can be further validated in cell therapy models and / or in vivo models. Mechanism-of-action (MoA) analysis can be established. The identified genes can be down-regulated in cells using various gene silencing methods described herein for cell therapies.

[0065] FIG. 4 is a schematic depicting a phenotypic testing method for sternness and proliferation in a population of T cells, as well as a positive selection methodforresistanceto exhaustion upon repeated antigen stimulation for edited T cells.

[0066] FIG. 5A and FIG. 5B depict schematics of a screening method involving evaluating sgRNA to determine if genetic knockout is beneficial to a population of interest. FIG. 5A depicts a process of sgRNA analysis evaluating factors in comparing two cell populations, including initial guide distribution, determination of gene essentiality, determination of sternness, proliferation determination, and determination of resistance to repeated stimulation in each population. FIG. 5B depicts the sgRNA log-fold change color key, whether values of sgRNA log-fold change result in sgRNA depletion or enrichment, and whether gene knockout is beneficial or detrimental for a population based on sgRNA log-fold change values.

[0067] FIG. 6 depicts information concerning different exemplary CRISPR screens, including library size, donor information, phenotypic assay type, and model system type.

[0068] FIG. 7A depicts summary data of screening analysis conducted on target knockout genes of interest, including gene z-scores identified through screening analysis of CD3 / CD28-activated T cells across different contrasts including CD4 sternness, CD8 sternness, CD4 proliferation, CD8 proliferation, CD4 repeated stimulation, and CD8 repeated stimulation.

[0069] FIG. 7B depicts data concerning exemplary analysis of sgRNAenrichmentfortargetknockout genes of interest across CD4+ cell sternness in CD3+ / CD28+ stimulated cell populations.

[0070] FIG. 8 A depicts information concerning sgRNA enrichment for target knockout genes of interest across CD4+ cell sternness and proliferation contrast in CD3+ / CD28- stimulated cell populations.

[0071] FIG. 8B depicts information concerning analysis of sgRNA enrichment for target knockout genes of interest across CD8+ cell sternness and proliferation contrasts in CD3+ / CD28- stimulated cell populations.

[0072] FIG.8C depicts data concerning analysis of sgRNA enrichment across T cell sternness contrast in NEOSTIM cell populations with labeled target knockout genes of interest in donors HD 110 and HD138.

[0073] FIG.9 A depicts data concerning aggregated z-score analysis of sgRNA enrichment in PRAME TCR T-cell repeated stimulation assays for multiple tumor cell lines.

[0074] FIG. 9B depicts information concerning analysis of sgRNA enrichment in PRAME TCR T- cell repeated stimulation assays for multiple tumor cell populations in various donors HD315, HD317, and HD318.

[0075] FIG. 10 depicts a schematic of a validation method including initial hit validation, shortlisted target validation, target validation in cell therapy models, evaluating anti-tumor efficacy in vivo, and establishing mode of action, and implementation.

[0076] FIG. 11 depicts a schematic of a phenotypic validation method, including RNP -mediated editing, performance of a plurality of phenotypic assays, and generating various types of results from the plurality of phenotypic assays.

[0077] FIG. 12 depicts data concerning TNFAIP3 knockout efficiency for donor HD315 for an initial TNFAIP3 -targeted sgRNA and an optimized TNFAIP3 -targeted sgRNA.

[0078] FIGs. 13A-13O depict various validation assay results ensuing from targeting of various knockout genes of interest including PRDM1, TNFAIP3, and REGNASE-1 in a neoantigen-specific cell model. FIG. 13A depicts editing efficiency percentage for sgRNA knockout of genes of interest PRDM1, TNFAIP3, and REGNASE-1 for cells from various donors on day 14 after NEOSTIM. FIG. 13B depicts total cell counts for sgRNA knockout of genes of interest PRDM1, TNFAIP3, and REGNASE-1 for various donors on day 14 after NEOSTIM. FIG. 13C depicts data concerning MART-1 antigen-specific cell frequency (e.g., pMHC+cell frequency -MART-1) in CD8+ cells for cells with various knockout genes of interests and obtained from various donors. FIG. 13D depicts information concerning GLI3 antigen-specific cell frequency (e.g., pMHC+ cell frequency - GLI3) in CD8+ cells for cells with various knockout genes of interests and obtained from various donors. BIG. 13E depicts data concerning total cell counts of MART-1 antigen-specific cells (e.g., pMHC+ total cell counts - MART-1) for cells with various knockout genes of interests and obtained from various donors. FIG. 13F depicts information concerning total cell counts of GLI3 antigen-specific cells (e.g, pMHC+ total cell counts - GLI3) for cells with various knockout genes of interests and obtained from various donors. FIG. 13G depicts data concerning CD 127+ cell percentage in a MART-1 specific cell population for cells with various knockout genes of interests and obtained from various donors. BIG. 13H depicts information concerning CD 127+ cell percentage in a GLI3 specific cell population for cells with various knockout genes of interests and obtained from various donors. FIG. 131 and FIG.13 J depict data relating to the percentage of different types of cells (e.g., T memory stem (Tscm) cells, T central memory (Tern), T effector memory (Tern), and effector T cells (Teff)) for each knockout gene of interest in a MART-1 specific cell population engineered with cells obtained from various donors. FIG. 13K and FIG. 13L depict data relating to the percentage of different types of cells (e.g, T memory stem (Tscm) cells, T central memory (Tern), T effector memory (Tern), and effector T cells (Teff)) for each knockout gene of interest in a GLI3 specific cell population engineered with cells obtained from various donors. FIG. 13M depicts data concerning percentage of cells that are PD1 + for various knockout genes of interest in a MART-1 specific cell population forvarious donors. BIG. 13N depicts data concerning percentage of cells that are PD1+ forvarious knockout genes of interest in a GLI3 specific cell population for various donors. FIG. 130 depicts an example of a 2D cytotoxicity assay for cells with each knockout gene of interest.

[0079] FIG. 14 depicts a representation of the evaluation of genetic knockouts of interest in the cells manufactured using NEOSTIM process, including knockout percentage for a cell population, expansion, frequency, and whether the knockouts caused an increase or decrease in number of CD 127+ cells and central memory T cells in the cell population.

[0080] FIGs. 15A-15D depict data concerning values forvarious cell populations manufactured using NEOSTIM process with various knockout genes of interest when the cells were edited on day 0. FIG. 15A depicts frequency of MART-1 specific cells in the CD8+ cells for cells with PRDM1 knockout and obtained from various donors. FIG. 15B depicts data concerning total fold expansion for cells with PRDM1 knockout and obtained from various donors. FIG. 15C depicts data relating to gene editing knockout efficiency in a bulk cell population for knockout gene of interest PRDM1 in cells obtained from various donors. FIG. 15D depicts data concerning editing efficiency of MART-1 specific cells in the cells obtained from various donors by measuring CD47 markers.

[0081] FIGs. 16A-16D depict data concerning values forvarious cell populations manufactured using NEOSTIM process with various knockoutgenes ofinterestwhen the cells were edited on day 12. FIG. 16A depicts frequency of MART-1 specific cells in the CD8+ cells for cells with PRDM1 knockout and obtained from various donors. FIG. 16B depicts data concerning total fold expansion for cells with PRDM1 knockout and obtained from various donors. FIG. 16C depicts data relating to gene editing knockout efficiency in a bulk cell population for knockout gene of interest PRDM1 in cells obtained from various donors. FIG. 16D depicts data concerning editing efficiency of MART-1 specific cells in the cells obtained from various donors by measuring CD47 marker.

[0082] FIG. 17A depicts flow cytometry data of cells manufactured usingNEOSTIM and edited on day 0 for central memory T cells, naive T cells, effective memory T cells, and effector T cells for a control cell population and a PRDM1 target gene knockout population of cells, measuring CCR7markers and CD45RA markers. FIG. 17B depicts data of cells manufactured using NEOSTIM and edited on day 0 concerning percentage of a MART-1 + cell population comprising central memory T cells, naive T cells, effective memory T cells, and effector T cells for various donors.

[0083] FIG. 18A depicts flow cytometry data of cells manufactured usingNEOSTIM and edited on day 12 for central memory T cells, naive T cells, effective memory T cells, and effector T cells for a control cell population and a PRDM1 target gene knockout population of cells, measuring CCR7 markers and CD45RA markers.

[0084] FIG. 18B depicts data of cells manufactured usingNEOSTIM and edited on day 12 concerning percentage of a MART- 1+ cell population comprising central memory T cells, naive T cells, effective memory T cells, and effector T cells for various donors.

[0085] FIG. 19 depicts a schematic illustrating validation methods of knockout genes of interest in PRAME or KRAS TCR-T cells.

[0086] FIGs. 20A-20B depict data relating to functionality of KRAS T cell receptor (TCR) T cell models. FIG. 20A depicts percent editing efficiency for knockout genes of interest for multiple subjects HD127, HD140, and HD141. FIG. 20B depicts cell expansion on day 9 of KRAS TCR-T cells for knockout genes of interest for multiple subjects HD 140 and HD141.

[0087] FIG. 21A illustrates an example of phenotypic characterization assays using KRAS TCR-T cells. FIG. 21B and FIG. 21C depict data concerning 3D repetitive killing assays in KRAS TCR-T cells for various knockout genes of interest. FIG. 21D and FIG. 21E depict data concerning cytokine secretion by KRAS TCR-T cells for various knockout genes of interest after four restimulations. BIG. 21 F depicts data concerning the percentage of TCR+ central memory T cells having a knockout of each gene of interest after the first restimulation. FIG. 21G depicts data concerning the percentage of TCR+ CD127+ cells having a knockout of each gene of interest after the first restimulation.

[0088] FIGs. 22A-22R depict data concerning phenotypic characteristics and edited population characteristics for KRAS TCR-T cells with various knockout genes of interest including PRDM1, TNFAIP3, and REGNASE-1. FIG. 22 A depicts data concerning knockout efficiency in a population of KRAS TCR-T cells for various knockout genes of interest for various donors. FIG. 22B depicts data concerning cell expansion of KRAS TCR-T cells on day 9 having various knockout genes of interestforvarious donors. FIG.22C and FIG.22D depict data concerning2D repetitive killing assays for various donors over about 400 hours for various knockout genes of interest in a population of KRAS TCR-T cells. FIG. 22E and FIG. 22F depict data concerning 3D repetitive killing assays for various donors over about 400 hours for various knockout genes of interest in a population of KRAS TCR-T cells. FIGs. 22G-22L depict data concerning effector molecule and cytokine secretion in 2D cytotoxicity assays for various knockout genes of interest and various cytokines for re stimulations 1-5 in a population of KRAS TCR-T cells. FIGs. 22M-22R depict data concerning effector molecule and cytokine secretion in 3D cytotoxicity assays for various knockout genes of interest and various cytokines for restimulations 1-5 in a population of KRAS TCR-T cells.

[0089] FIG. 23A depicts data concerning metabolic fitness measured by oxygen consumption rate (OCR) on restimulation 10 forvarious knockout genes of interestover about 80 minutes in a population of KRAS TCR-T cells.

[0090] FIG. 23B depicts data concerning spare respiratory capacity in OCR and ATP production rate for various knockout genes of interest for multiple donors on restimulation 10 in a population of KRAS TCR-T cells.

[0091] FIGs. 24A-24J depict phenotypic characteristics and edited population characteristics for PRAME TCR-T cells with various knockout genes of interest including PRDM1, TNFAIP3, and REGNASE-1 . FIG. 24 A and FIG. 24B depict data concerning percentage editing knockout efficiency forvarious knockout genes of interest forvarious donors in PRAME TCR-T cell populations. FIG. 24C and FIG. 24D depict data concerning cell expansion of PRAME TCR-T cells on day 9 having various knockout genes of interest for various donors. FIG. 24E and FIG. 24F depict data concerning 2D cytotoxicity assays for various donors over about 100 hours for various knockout genes of interest in a population of PRAME TCR-T cells co-cultured with 624-Mel cells. FIG. 24G and FIG. 24H depict data concerning 3D repetitive killing assays forvarious donors over about 1000 to about 1500 hours forvarious knockout genes of interest in a population of PRAME TCR-T cells. FIG. 241 depicts data concerning percentage of CD127++ cells in a population of TCR+ (TCRVbl+) PRAME TCR-T cells for various knockout genes of interest in various donors. FIG. 24J depicts data concerning percentage of TCF1++ cells in apopulation ofTCR+PRAME TCR-T cells forvarious knockout genes of interest in various donors.

[0092] FIG. 25A depicts data concerning metabolic fitness measured by OCR on restimulation 3 for various knockout genes of interest over about 80 minutes in a population of PRAME TCR-T cells. FIG. 25B depicts data concerning spare respiratory capacity in OCR and ATP production rate for various knockout genes of interest for multiple donors on re stimulation 3 in a population of PRAME TCR-T cells.

[0093] FIGs. 26A-26J depict data concerning KRAS TCR cell population phenotypic characteristics for knockout genes of interest in KRAS TCR relevant versus irrelevant KRAS TCR cell populations. FIG. 26 A depicts PRDM1 knockout editing efficiency for relevant versus irrelevant KRAS TCR cell populations for various donors with PRDM1 knockout. FIG. 26B depicts data concerning editing efficiency percentage for relevant versus irrelevant KRAS TCR cell populations forvarious donors with TNFAIP3 knockout. FIG. 26C and FIG. 26D depict data concerning repetitive killing assay datafor relevant versus irrelevant KRAS TCR cell populations for various donors having PRDM1 knockout. FIG. 26E and FIG. 26F depict data concerning repetitive killing assay data for relevant versus irrelevant KRAS TCR cell populations for various donors having TNFAIP3 knockout. FIG. 26G depicts data concerning total CD4+ TCR+ cell count in a population of CD4+ TCR+ cells for relevant versus irrelevant KRAS TCR cell populations for various donors having PRDM1 knockout for re stimulation 1 and restimulation 4. FIG.26H depicts data concerningtotalCD8+TCR+ cell count in a population of CD8+ TCR+ cells for relevant versus irrelevant KRAS TCR cell populations for various donors having PRDM1 knockout for restimulation 1 and restimulation 4 having PRDM1 knockout. FIG. 261 depicts data concerning total CD4+ TCR+ cell count in a population of CD4+ TCR+ cells for relevant versus irrelevant KRAS TCR cell populations for various donors having TNFAIP3 gene knockout for restimulation 1 and restimulation 4. FIG. 26 J depicts data concerning total CD8+ TCR+ cell count in a population of CD8+ TCR+ cells for relevant versus irrelevant KRAS TCR cell populations for various donors for restimulation 1 and restimulation 4 having TNFAIP3 gene knockout.

[0094] FIGs. 27A-27F depict data concerning phenotypic assays and gene knockout data for double knockout cells. FIG. 27A depicts data concerning editing efficiency of double knockout cells with PRDM1 and TNFAIP3 knockouts for various donors. FIG. 27B depicts data concerning PRAME TCR-T cell population cell expansion on day 9 having double knockout PRDM1 and TNFAIP3 knockout, and cell expansion on day 9 in a single knockoutPRAME TCR-T cell population. FIG.27C depicts data concerning the percent population of TCRVB1+ cells having each subpopulation of T cells for double knockout PRDM1 and TNFAIP3 knockout and single knockout cells. FIG. 27D depicts percentage of population of cells that are CD127+ or TCF1+ within TCRVB1 + cells for single and double knockouts PRDM1 and TNFAIP3. FIG. 27E depicts cytotoxic assay results for double PRDM1 and TNFAIP3 knockout cells co-cultured with SKMEL5 cells for various donors. FIG. 27F depicts cytotoxic assay results for double PRDM1 and TNFAIP3 knockout cells co-cultured with 624- MEL cells for various donors.

[0095] FIGs. 28A-28B depict examples of in vivo experimental methods in mouse models. FIG. 28A depicts a KRAS-TCR T xenograft mouse model and a B16 / Ova OT-I syngeneic mouse model. FIG. 28B depicts a method of in vivo mouse model tumor challenge with gene knockouts of interest.

[0096] FIG. 29A depicts data concerning tumor volume over about 0 to 12 days post tumor rechallenge for TCR-T cells without knockout and with PRDM1 knockout.

[0097] FIG. 29B depicts data concerning TCR counts, percent central memory T cells, and percent CD39- and PD-1- cells in a population of TCR+ cells for PRDM1 knockout cells.

[0098] FIG. 30A depicts an in vivo mouse model method for unedited OT-I cell dosing.

[0099] FIG. 30B depicts the absolute tumor volume in vivo over approximately 30 days for various concentrations of B16 / Ova OT-I CD8+ T cells.

[0100] FIG. 31A depicts an in vivo mouse model method for generating Cas9 RNP-mediated gene edited OT-I cells.

[0101] FIG. 31B depicts an in vivo mouse model method for evaluating anti-tumor efficacy of CRISPR knockout edited OT-I cells.

[0102] FIG. 31C depicts data concerning knockout efficiency for various knockout genes of interest including PRDM1, TNFAIP3, and REGNASE-1 in OT-I cells tested in B16 / Ova mouse model.

[0103] FIG. 31D depicts data concerning 2D cytotoxicity assays for various knockout genes of interest.

[0104] FIG.31E depicts data concemingtumor volume over from about 0 to about40 days for various knockout genes of interest versus control cell populations.

[0105] FIG. 32 depicts a schematic illustration of a method for a single restimulation assay using CD3 / CD28 -activated T cells.

[0106] FIG. 33A depicts data concerning flow cytometry validation assays for CD8+ cells with various knockout genes of interest.

[0107] FIG. 33B depicts data concerning flow cytometry validation assays for CD4+ cells with various knockout genes of interest.

[0108] FIG. 34 depicts a schematic illustration of a method for a repeated stimulation assay.

[0109] FIG. 35A depicts percentage of each T cell subtype, including naive T cells, central memory T cells, effector memory T cells, and effector T cells for a population of CD8+ cells with various knockout genes of interest.

[0110] FIG. 35B depicts percentage of each T cell subtype, including naive T cells, central memory T cells, effector memory T cells, and effector T cells for a population of CD4+ cells with various knockout genes of interest.

[0111] FIG. 35C depicts data concerning cell fold expansions for stimulations 1 to 5 for cells with various knockout genes of interest.

[0112] FIG. 35D depicts data concerning cytokine secretion for stimulations 1 to 5 for cells with various knockout genes of interest.

[0113] FIG. 35E depicts data concerning frequency of CD8+ central memory T cells for each knockout gene of interest for cells obtained from various donors.

[0114] FIG. 35F depicts data concerning normalized cytokine concentration for various cytokines for PRDM1 knockout cells engineered from cells obtained from various donors.

[0115] FIG. 35G depicts data concemingnormalized cytokine concentration for various cytokinesfor TNFAIP3 knockout cells engineered from cells obtained from various donors.

[0116] FIG. 35H depicts data concemingnormalized cytokine concentration for various cytokinesfor REGNASE-1 knockout cells engineered from cells obtained from various donors.

[0117] FIG. 351 depicts data concerning change in frequency of CD8+ cells in a population of PD-1+ and CD8+ cells for various knockout genes of interest for cells obtained from various donors.

[0118] FIG. 35 J depicts data concerning change in frequency of CD4+ cells in a population of PD- 1+ and CD4+ cells for various knockout genes of interest for cells obtained from various donors.

[0119] FIG. 36A depicts data concerning total fold expansion of various immune cell populations with various knockout or double knockout genes of interest including PRDM1 / TNFAIP3, PRDM1 / REGNASE-1 (REG-1), TNFAIP3 / REG-1, PRDM1, TNFAIP3, and REG-1.

[0120] FIG. 36B depicts data relating to CD127+ expression in a population of CD8+ cells with various knockout or double knockout genes of interest including PRDM1 / TNFAIP3, PRDM1 / REGNASE-1 (REG-1), TNFAIP3 / REG-1, PRDM1, TNFAIP3, and REG-1.

[0121] FIG. 36C depicts data concerning differentiation percent frequency for various cell types in a population of CD8+ cells including naive T cells, central memory T cells, effector memory T cells, and effector T cells for a population of CD8+ cells with various knockout genes of interest including PRDM1 / TNFAIP3, PRDM1 / REGNASE-1 (REG-1), TNFAIP3 / REG-1, PRDM1, TNFAIP3, and REG-1.

[0122] FIG. 36D depicts data concerning proportion of Ly 108+ cells within CD8+ antigen-specific NEOSTIM cells with various knockout genes of interest.

[0123] FIG. 37 depicts a non-limiting exemplary method of evaluating single and dual genetic knockout combinations in an OT-I B16.OVA syngeneic model (OVA3).

[0124] FIG. 38A depicts data concerning absolute tumor volume over a time period of about 0 to 45 days for a genetic knockout condition of PRDM1.

[0125] FIG. 38B depicts data concerning absolute tumor volume over a time period of about 0 to 55 days for a genetic knockout condition of REGNASE-1 (REG-1).

[0126] FIG. 38C depicts data concerning absolute tumor volume over a time period of about 0 to 45 days for a genetic knockout condition of TNFAIP3.

[0127] FIG. 38D depicts data concerning absolute tumor volume over a time period of about 0 to 45 days for a genetic double knockout condition of PRDM1 / TNFAIP3.

[0128] FIG. 38E depicts data concerning absolute tumor volume over a time period of about 0 to 55 days for a genetic double knockout condition of PRDM1 / REGNASE-1 (REG-1).

[0129] FIG. 39 depicts data concerning CD45.2+ OT-I cell frequencies in the periphery for a population of CD3+ cells with various knockout genes of interest including PRDM1 / TNFAIP3, PRDM1 / REGNASE-1 (REG-1), TNFAIP3 / REG-1, PRDM1, TNFAIP3, and REG-1.

[0130] FIG. 40 depicts a non-limiting exemplary method for evaluating double genetic knockout effects in tumor challenge and re-challenge over about 80 days in a xenograft mouse model.

[0131] FIG. 41 A depicts data concerning tumor volume at a primary challenge site (right flank tumor) over about 45 days for various knockout and double-knockout gene features of interest including PRDM1, PRDM1 / TNFAIP3, and PRDM1 / REGNASE-1 (REG-1).

[0132] FIG. 4 IB depicts data concerning tumor volume at a rechallenge site (left flank tumor) over about 80 days for various knockout and double-knockout gene features of interest including PRDM1, PRDM1 / TNFAIP3, and PRDM1 / REGNASE-1 (REG-1).

[0133] FIG. 41C depicts data relating to cell expansion post adoptive cell transfer over about 21 days in the periphery for various knockout and double-knockout gene features of interest including PRDM1, PRDM1 / TNFAIP3, and PRDM1 / REGNASE-1 (REG-1).

[0134] FIGs. 42A-42E depict data concerning 3D serial cytotoxicity assays over about 600 hours for various knockout genes of interest in a population of PRAME TCR-T cells co-cultured with SKMEL-5 cells at E:T ratio of 1 :1. FIG. 42A depicts data concerning 3D serial cytotoxicity assays for PRDM1 knockout in PRAME TCR-T cells co-cultured with SKMEL-5 cells. FIG. 42B depicts data concerning 3D serial cytotoxicity assays for REG-1 knockout in PRAME TCR-T cells co- cultured with SKMEL-5 cells. FIG. 42C depicts data concerning 3D serial cytotoxicity assays for TNFAIP3 knockout in PRAME TCR-T cells co-cultured with SKMEL-5 cells. FIG. 42D depicts data concerning 3D serial cytotoxicity assays for CISH knockout in PRAME TCR-T cells co- cultured with SKMEL-5 cells. FIG. 42E depicts data concerning 3D serial cytotoxicity assays for PTPN2 knockout in PRAME TCR-T cells co-cultured with SKMEL-5 cells.

[0135] FIGs. 43A-43E depict data concerning 3D serial cytotoxicity assays over about 600 hours for various knockout genes of interest in a population of PRAME TCR-T cells co-cultured with 624MEL cells at E:T ratio of 1 : 1. FIG. 43A depicts data concerning 3D serial cytotoxicity assays for PRDM1 knockout in PRAME TCR-T cells co-cultured with 624MEL cells. FIG. 43B depicts data concerning 3D serial cytotoxicity assays for REG-1 knockout in PRAME TCR-T cells co-cultured with 624MEL cells. FIG. 43C depicts data concerning 3D serial cytotoxicity assays for TNFAIP3 knockout in PRAME TCR-T cells co-cultured with 624MEL cells. FIG. 43D depicts data concerning 3D serial cytotoxicity assays for CISH knockout in PRAME TCR-T cells co-cultured with 624MEL cells. FIG. 43E depicts data concerning 3D serial cytotoxicity assays for PTPN2 knockout in PRAME TCR-T cells co-cultured with 624MEL cells.

[0136] FIGs. 44A-44G depict data concerning 3D serial cytotoxicity assays over about 600 hours for various double knockout genes of interest in a population of PRAME TCR-T cells co-cultured with SKMEL-5 cells at E:T ratio of 1 :1. FIG. 44A depicts data concerning 3D serial cytotoxicity assays for CISH / PRDM1 double knockouts in PRAME TCR-T cells co-cultured with SKMEL cells. FIG. 44B depicts data concerning 3D serial cytotoxicity assays for CISH / PTPN2 double knockouts in PRAME TCR-T cells co-cultured with SKMEL-5 cells. FIG. 44C depicts data concerning 3D serial cytotoxicity assays for PRDM1 / TNFAIP3 double knockouts in PRAME TCR-T cells co- cultured with SKMEL-5 cells. FIG. 44D depicts data concerning 3D serial cytotoxicity assays for PRDM1 / PTPN2 double knockouts in PRAME TCR-T cells co-cultured with SKMEL-5 cells. FIG. 44E depicts data concerning 3D serial cytotoxicity assays for PRDMl / REG-1 double knockouts in PRAME TCR-T cells co-cultured with SKMEL-5 cells. FIG. 44F depicts data concerning 3D serial cytotoxicity assays for CISH / REG-1 double knockouts in PRAME TCR-T cells co-cultured with SKMEL-5 cells. FIG. 44G depicts data concerning 3D serial cytotoxicity assays for PTPN2 / REG-1 double knockouts in PRAME TCR-T cells co-cultured with SKMEL-5 cells.

[0137] FIGs. 45A-45G depict data concerning 3D serial cytotoxicity assays over about 600 hours for various double knockout genes of interest in a population of PRAME TCR-T cells co-cultured with 624MEL cells at E:T ratio of 1 :1. FIG. 45A depicts data concerning 3D serial cytotoxicity assays for CISH / PRDM1 double knockouts in PRAME TCR-T cells co-cultured with 624MEL cells. FIG. 45B depicts data concerning 3D serial cytotoxicity assays for CISH / PTPN2 double knockouts in PRAME TCR-T cells co-cultured with 624MEL cells. FIG. 45C depicts data concerning 3D serial cytotoxicity assays for PRDM1 / TNFAIP3 double knockouts in PRAME TCR-T cells co-cultured with 624MEL cells. FIG. 45D depicts data concerning 3D serial cytotoxicity assays for PRDM1 / PTPN2 double knockouts in PRAME TCR-T cells co-cultured with 624MEL cells. FIG. 45E depicts data concerning 3D serial cytotoxicity assays for PRDMl / REG-1 double knockouts in PRAME TCR-T cells co-cultured with 624MEL cells. FIG. 45F depicts data concerning 3D serial cytotoxicity assays for CISH / REG-1 double knockouts in PRAME TCR-T cells co-cultured with 624MEL cells. FIG. 45G depicts data concerning 3D serial cytotoxicity assays for PTPN2 / REG-1 double knockouts in PRAME TCR-T cells co-cultured with 624MEL cells.

[0138] FIGs. 46A-46D depict data concerning metabolic fitness measured by OCR on restimulation 1 or 5 with 624MEL cells for various knockout genes of interest over about 80 minutes in a population of PRAME TCR-T cells derived from two donors. FIG. 46A depicts data concerning metabolic fitness on restimulation 1 for various knockout genes of interest in a population of PRAME TCR-T cells derived from donor HD315. FIG. 46B depicts data concerning metabolic fitness on restimulation 5 for various knockout genes of interest in a population of PRAME TCR-Tcells derived from donor HD315. FIG. 46C depicts data concerning metabolic fitness on restimulation 1 for various knockout genes of interest in a population of PRAME TCR-T cells derived from donor HD317. FIG. 46D depicts data concerning metabolic fitness on restimulation 5 for various knockout genes of interest in a population of PRAME TCR-T cells derived from donor HD317.

[0139] FIGs. 47A-47D depict data concerning spare respiratory capacity (SRC) for various knockout genes of interest in a population of PRAME TCR-T cells on restimulation 1 or 5 with 624MEL cells for multiple donors. FIG. 47A depicts data concerning SRC for various knockout genes of interest in a population of PRAME TCR-T cells on restimulation 1 for donor HD315. FIG. 47B depicts data concerning SRC for various knockout genes of interest in a population of PRAME TCR-T cells on restimulation 1 for donor HD317. FIG. 47C depicts data concerning SRC for various knockout genes of interest in a population of PRAME TCR-T cells on restimulation 5 for donor HD315. FIG. 47D depicts data concerning SRC for various knockout genes of interest in a population of PRAME TCR-T cells on restimulation 5 for donor HD317.

[0140] FIGs. 48A-48D depict data concerning ATP production rate for various knockout genes of interest in a population of PRAME TCR-T cells on restimulation 1 or 5 with 624MEL cells for multiple donors. FIG. 48A depicts data concerning ATP production rate for various knockout genes of interest in a population of PRAME TCR-T cells on restimulation 1 for donor HD315. FIG. 48B depicts data concerning ATP production rate for various knockout genes of interest in a population of PRAME TCR-T cells on restimulation 1 for donor HD317. FIG. 48C depicts data concerning ATP production rate for various knockout genes of interest in a population of PRAME TCR-T cells on restimulation 5 for donor HD315. FIG. 48D depicts data concerning ATP production rate for various knockout genes of interest in a population of PRAME TCR-T cells on restimulation 5 for donor HD317.

[0141] FIGs. 49A-49F depict data concerning in vitro cytotoxicity for various knockout genes of interest in OT-I T cells againstB16. OVA tumor cells at a E:T ratio of 4:1 over 40 hours. FIG. 49A depicts data concerning in vitro cytotoxicity for the single knockout of PRDM1 in OT-I T cells against Bl 6. OVA tumor cells. FIG. 49B depicts data concerning in vitro cytotoxicity for the single knockout of TNFAIP3 in OT-I T cells against B16. OVA tumor cells. FIG. 49C depicts data concerning in vitro cytotoxicity for the single knockout of REG- 1 in OT-I T cells against B16. OVA tumor cells. FIG. 49D depicts data concerning in vitro cytotoxicity for the double knockouts of PRDM1 / TNFAIP3 in OT-I T cells againstB16.OVAtumor cells. FIG. 49E depicts data concerning in vitro cytotoxicity for the double knockouts of PRDM1 / REG-1 in OT-I T cells against B16. OVAtumor cells. FIG. 49F depicts data concerning in vitro cytotoxicity for the double knockouts of TNFAIP3 / REG-1 in OT-I T cells against Bl 6. OVA tumor cells.

[0142] FIG. 50 depicts data concerning absolute tumor volume over a time period of about 0 to 45 days in B16.OVA bearing mice following adoptive transfer of OT-I cells with a genetic knockout of TNF AIP3 / REG-1.

[0143] FIG. 51 depicts data concerning CD45.2+ OT-I cell frequencies at the tumor site of CD3 + cells with various knockout genes of interest including PRDM1 / TNFAIP3, PRDM1 / REGNASE-1 (REG-1), TNFAIP3 / REG-1, PRDM1, TNFAIP3, and REG-1.

[0144] FIG. 52 depicts a non-limiting exemplary method of evaluating single and dual genetic knockout combinations in an OT-I B16.OVA syngeneic model (OVA4).

[0145] FIGs. 53A-53F depict data concerning in vitro cytotoxicity for various knockout genes of interest in OT-I T cells againstB16. OVA tumor cells at a E:T ratio of 4:1 over 35 hours. FIG. 53A depicts data concerning in vitro cytotoxicity for the single knockout of PRDM1 in OT-I T cells against Bl 6. OVA tumor cells. FIG. 53B depicts data concerning in vitro cytotoxicity for the single knockout of TNFAIP3 in OT-I T cells against Bl 6. OVA tumor cells. FIG. 53C depicts data concerning in vitro cytotoxicity for the single knockout of REG- 1 in OT-I T cells against B16. OVA tumor cells. FIG. 53D depicts data concerning in vitro cytotoxicity for the single knockout of SOCS1 in OT-I T cells against B16. OVA tumor cells. FIG. 53E depicts data concerning in vitro cytotoxicity for the single knockout of CISH in OT-I T cells against B16. OVA tumor cells. FIG. 53F depicts data concerning in vitro cytotoxicity for the single knockout of PTPN2 in OT-I T cells against B16. OVA tumor cells.

[0146] FIGs. 54A-54H depict data concerning absolute tumor volume over a time period of about 0 to 45 days in B16.OVA bearing mice following adoptive transfer of OT-I cells with various knockout genes ofinterest. FIG. 54A depicts data concerning absolute tumor volume in B16.OVA bearing mice following adoptive transfer of OT-I cells with vehicle control. FIG. 54B depicts data concerning absolute tumor volume in B16.OVA bearing mice following adoptive transfer of OT-I cells with genetic knockout of CISH. FIG. 54C depicts data concerning absolute tumor volume in B16.OVAbearingmice following adoptivetransfer of OT-I cells with genetic knockout of PTPN2. FIG. 54D depicts data concerning absolute tumor volume in B16.OVA bearing mice following adoptive transfer of OT-I cells with genetic knockouts of PRDM1 / CISH. FIG. 54E depicts data concerning absolute tumor volume in B16.OVA bearing mice following adoptive transfer of OT-I cells with genetic knockouts of PRDM1 / PTPN2. FIG. 54F depicts data concerning absolute tumor volume in Bl 6. OVA bearing mice following adoptive transfer of OT-I cells with genetic knockouts of REG-l / CISH. FIG. 54G depicts data concerning absolute tumor volume in B16.OVA bearingmice following adoptive transfer of OT-I cells with genetic knockouts of REG-1 / PTPN2. FIG. 54H depicts data concerning absolute tumor volume in Bl 6. OVA bearing mice following adoptive transfer of OT-I cells with genetic knockouts of PRDM1 / REG-1.

[0147] FIG. 55 depicts a non-limiting exemplary method of generating PRAME TCR-T cells with various knockout genes of interest.

[0148] FIGs. 56A-56C depict data concerning 3D serial cytotoxicity assays over about 600 hours for various knockout genes of interest in a population of PRAME TCR-T cells co-cultured with SKMEL-5 cells at 2.5:1 E:T ratio. FIG. 56A depicts data concerning 3D serial cytotoxicity assays for PRDMl knockout in PRAME TCR-T cells co-cultured with SKMEL-5 cells. FIG. 56B depicts data concerning 3D serial cytotoxicity assays for PRDM1 / REG-1 double knockouts in PRAME TCR-T cells co-cultured with SKMEL-5 cells. FIG. 56C depicts data concerning 3D serial cytotoxicity assays for PRDM1 / TNFAIP3 double knockouts in PRAME TCR-T cells co-cultured with SKMEL-5 cells.

[0149] FIGs. 57A-57C depict data concerning characterization of phenotype of PRAME TCR-T cells with various knockout genes of interest. FIG. 57A depicts data concerning differentiation frequency for various subsets in a population of CD3+ T cells including stem cell memory T cells, central memory T cells, effector memory T cells, and effector T cells for a population of CD8+ cells with various knockout genes of interest including PRDM1, PRDMl / REG-1, and PRDM1 / TNFAIP3. FIG. 57B depicts data concerning frequency of TCR expression in PRAME TCR-T cells with PRDM1, PRDMl / REG-1, or PRDM1 / TNFAIP3 knockout genes of interest. FIG. 57C depicts data concerning mean fluorescence intensity (MFI) of TCR expression in PRAME TCR-T cells with PRDM1, PRDMl / REG-1, or PRDM1 / TNFAIP3 knockout genes of interest.

[0150] FIG. 58 depicts a non-limiting exemplary method of generating KRAS G12V / A68 TCR-T cells with knockout genes of PRDMl / REG-1.

[0151] FIG. 59 depicts an experimental design of evaluating anti-tumor efficacy of TCR-T with PRDMl / REG-1 double knockout in xenograft model.

[0152] FIGs. 60A-60D depict data concerning transduction efficacy and TCR expression in TCR-T cells with knockout genes of PRDMl / REG-1. FIG. 60A depicts data concerning the frequency of mTCR+ cells. FIG. 60B depicts data concerning the MFI of mTCR expression. FIG. 60C depicts representative FACS data showing the TCR gating strategy. FIG. 60D depicts data concerning frequency of mTCR+ cells within alive cells.

[0153] FIGs. 61A-61B depict data concerning tumor volume over a time period of about 0 to 45 days in tumor bearing mice following adoptive transfer of TCR-T cells with PRDMl / REG-1 double knockout. FIG. 61A depict data concerning tumor volume in tumor bearing mice following adoptivetransfer of 10e6 TCR-T cells with PRDM1 / REG-1 double knockout. FIG. 61B depict data concerning tumor volume in tumor bearing mice following adoptive transfer of 5e6 TCR-T cells with PRDM1 / REG-1 double knockout.

[0154] FIGs. 62A-62E depict data concerning donor T cell expansion of adoptive transferred TCR- T cells with PRDM1 / REG-1 double knockouts in the periphery. FIG. 62A depicts grouped data concerning cell counts in the periphery after adoptive transfer of 10e6 TCR-T cells with PRDM1 / REG-1 double knockouts or 10e6 control TCR-T cells. FIG. 62B depicts individual data concerning cell counts in the periphery after adoptive transfer of 10e6 TCR-T cells with PRDM1 / REG-1 double knockouts or 10e6 control TCR-T cells. FIG. 62C depicts grouped data concerning cell counts in the periphery after adoptive transfer of 5e6 TCR-T cells with PRDM1 / REG-1 double knockouts or 5e6 control TCR-T cells. FIG. 62D depicts individual data concerning cell counts in the periphery after adoptive transfer of 5e6 TCR-T cells with PRDM1 / REG-1 double knockouts or 5e6 control TCR-T cells. FIG. 62E data concerning cell counts in the periphery after adoptive transfer of 5e6 TCR-T cells with PRDM1 / REG-1 double knockouts or 10e6 control TCR-T cells.

[0155] FIG. 63 data concerning tumor volume over a time period of about 0 to 45 days in tumor bearing mice following adoptive transfer of 5e6 TCR-T cells with PRDM1 / REG-1 double knockout or 10e6 control TCR-T cells.

[0156] FIGs. 64A-64B depict data concerning percentage of multimer+CD8+ NEOSTIM cells from three donors after various genetic knockouts. FIG. 64A depicts data concerning percentage of multimer+CD8+ NEOSTIM cells from three donors after various genetic knockouts, generated with MART-1 decamer string. FIG. 64B depicts data concerning percentage of multimer+CD8+ NEOSTIM cells from three donors after various genetic knockouts, generated with CSNK1 decamer string.

[0157] FIG. 65 depicts data concerning fold expansion of antigen-specific CD8+ NEOSTIM cells with various genetic knockouts, stimulated with APC expressing MART-1 decamer or CSNK1 decamer.

[0158] FIG. 66 data concerning frequency of antigen-specific CD8+ NEOSTIM cells with various genetic knockouts, stimulated with APC expressing MART-1 decamer.

[0159] FIG. 67 depicts an exemplary method for identification of gen es / path ways that are affected by various gene knockouts upon antigen stimulation.

[0160] FIG. 68 depicts RNAseq data concerning differential gene expression in T cells with PRDM1, REG-1, or PRDMl / REG-1 knockouts upon antigen stimulation.DETAILED DESCRIPTIONIntroduction

[0161] The present disclosure provides compositions and methods for genetic enhancement of cells for improved cell therapies. Current ? cell therapies are susceptible upon repeated stimulation to dysfunction, lack of repeated activation, ineffectiveness, and lack of persistence. Genetic screening can enable identification of genes that are associated with beneficial or detrimental T cell phenotypes at scale, and enhance therapeutic potential of T cell populations. Therapeutic potential of T cells can be enhanced by promoting persistence, including promoting homeostatic cytokine production, enhancing sternness, enhancing memory, enhancing proliferation, and / or enhancing metabolism function. Genetic enhancement of T cells can also include promoting effector function, including promoting tumor infiltration, enhancing metabolic changes, enhancing proliferation, enhancing bystander cell activation, enhancing cytokine secretion, and enhancing cytotoxicity to cancerous cells. T cells can also be enhanced by boosting their activation, through improved T cell priming, improved antigen recognition, enhanced cytokine sensing, and enhanced co-stimulation. Additionally, beneficial T cell phenotypes can be genetically enhanced to avoid dysfunction, such dysfunction including overactivation, AICD, exhaustion, apoptosis, toxicity, antigen escape, immunosuppression, and lack of homeostasis.

[0162] Gene knockdown or knockout screening (e.g., CRISPR screening) can be used to increase the likelihood of success for genetic enhancement for T cell therapies. Knockdown or knockout of one or more screened genes in genetically engineered T cells alone or in combination with the NEOSTIM personalized cell therapy process can produce enhanced T cells with improved functionality in treatment for cancers and other diseases.

[0163] In one example, the methods provided herein can comprise producing a population of engineered immune cells by depleting one or more cells selected from the group consisting of CD14+ cells, CD25+ cells, and CD56+ cells from a population of immune cells comprising antigen presenting cells (APCs) and T cells, thereby forming a depleted population of immune cells; and inhibiting expression of an endogenous gene in the depleted population of immune cells, thereby producing the population of engineered immune cells.

[0164] In another example, the methods provided herein can comprise depleting one or more cells selected from the group consisting of CD14+ cells, CD25+ cells, and CD56+ cells from a population of immune cells comprising antigen presenting cells (APCs) and T cells, thereby forming a depleted population of immune cells; and inhibiting expression of one or more endogenous genes in the depleted population of immune cells, thereby producing the population of engineered immune cells.

[0165] To illustrate, the methods provided herein can comprise inhibiting expression of endogenous PRDM1, TNFAIP3, REGNASE-1, SOCS1, PTPN2, CISH, or any combination thereof in a population of immune cells.

[0166] For another illustration, the methods provided herein can comprise inhibiting expression of one or more endogenous genes in a population of immune cells, thereby producing the population of engineered immune cells, wherein the population of immune cells comprises T cells; and depleting one or more cells selected from the group consisting of CD 14+ cells, CD25+ cells, and CD56+ cells in the population of immune cells to obtain a depleted population of immune cells. In some cases, the method can further comprise inhibiting one or more endogenous genes selected from the group comprising PRDM1, TNFAIP3, REGNASE-1, SOCS1, PTPN2, CISH, or any combination thereof.

[0167] Engineered immune cells produced by the methods described herein, pharmaceutical compositions of the engineered immune cells, methods of producing the engineered immune cells, and use of the engineered immune cells are also provided.Definitions

[0168] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0169] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about’ can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about’ can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0170] The term “and / or” used herein to link one or more species means one of the species or any combinations of the one or more species. For example, depleting CD14+ cells and / or CD25+ cells and / or CD56+ cells encompass depleting CD 14+ cells only, CD25+ cells only, or CD56+ cells only, or depleting any combinations of two of the CD14+ cells, CD25+ cells and CD56+ cells, or depleting CD14+ cells, CD25+ cells and CD56+ cells.

[0171] An antigen is a foreign sub stance to the body that induces an immune response. A “neoantigen” refers to a class of tumor antigens which arise from tumor-specific changes in proteins. Neoantigens encompass, but are not limited to, tumor antigens which arise from, for example, a substitution in aprotein sequence, a frame shift mutation, a fusion polypeptide, an in-frame deletion, an insertion, and expression of an endogenous retroviral polypeptide.

[0172] A “neoepitope” refers to an epitope that is not present in a reference, such as a non-diseased cell, e.g., a non-cancerous cell or a germline cell, but is found in a diseased cell, e.g., a cancer cell. This includes situations where a corresponding epitope is found in a normal non-diseased cell or a germline cell but, due to one or more mutations in a diseased cell, e.g., a cancer cell, the sequence of the epitope is changed so as to result in the neoepitope.

[0173] A “mutation” refers to a change of or a difference in a nucleic acid sequence (e.g., a nucleotide substitution, addition or deletion) compared to a reference nucleic acid. A “somatic mutation” can occur in any of the cells of the body except the germ cells (sperm and egg) and are not passed on to children. These alterations can (but do not always) cause cancer or other diseases. In some embodiments, a mutation is a non-synonymous mutation. A “non-synonymous mutation” refers to a mutation, for (e.g., a nucleotide substitution), which does result in an amino acid change such as an amino acid substitution in the translation product. A “frameshift” occurs when a mutation disrupts the normal phase of a gene’ s codon periodicity (also known as “reading frame”), resulting in translation of a non-native protein sequence. It is possible for different mutations in a gene to achieve the same altered reading frame.

[0174] An “antigen-presenting cell” (APC) refers to a cell that expresses an MHC molecule and can present an epitope in complex with the MHC molecule. The cell can present peptide fragments of protein antigens in association with MHC molecules on its cell surface. The term includes professional antigen-presenting cells (e.g., B lymphocytes, monocytes, Langerhans cells) as well as any other cells that express an MHC and can present an epitope in complex with the MHC (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes). The APC can be a cell that is engineered to express an MHC molecule or a cell that expresses an endogenous MHC molecule.

[0175] The term “affinity” refers to a measure of the strength of binding between two members of a binding pair (e.g., a human leukocyte antigen (HLA)-binding peptide and a class I or II HLA, or a peptide-HLA complex and a T-cell receptor (TCR)). KDrefers to the dissociation constant between two members of a binding pair and has units of molarity. KArefers to the affinity constant between two members of a binding pair is the inverse of the dissociation constant. Affinity can be determined experimentally, for example by surface plasmon resonance (SPR) using commercially available Biacore SPR units. Koff refers to the off-rate constant of two members of a binding pair, (e.g. , the off- rate constant of an HLA-binding peptide and a class I or II HLA, or a peptide-HLA complex and a TCR). Konrefers to the on-rate constant of two members of a binding pair, (e.g. , the on-rate constant of an HLA-binding peptide and a class I or II HLA, or a peptide-HLA complex and a TCR).

[0176] Throughout this disclosure, “binding data” results can be expressed in terms of an “IC50.” Affinity can also be expressed as the inhibitory concentration 50 (IC50), or the concentration at which 50% of a first member of a binding pair (e.g., a peptide) is displaced. Likewise, In (IC50) refers to the natural log of the IC50. For example, an IC50can be the concentration of a tested peptide in a binding assay at which 50% inhibition of binding of a labeled reference peptide is observed. Given the conditions in which the assays are run (e.g., limiting HLA protein concentrations and / or labeled reference peptide concentrations), these values can approximate KDvalues. Assays for determining binding are well known in the art and are described in detail, for example, in PCT publications WO 94 / 20127 and WO 94 / 03205, and other publications such Sidney et al., Current Protocols in Immunology 18.3.1 (1998); Sidney, et al., J. Immunol. 154:247 (1995); and Sette, et al., Mol. Immunol. 31 :813 (1994). Alternatively, binding can be expressed relative to binding by a reference standard peptide. Binding can also be determined using other assay systems including those using live cells (e.g., Ceppellini et al., Nature 339:392 (1989); Christnick et al., Nature 352:67 (1991); Busch et al., Int. Immunol. 2:443 (1990); Hill et al., J. Immunol. 147:189(1991); del Guercio etal., J. Immunol. 154:685 (1995)), cell free systems using detergent lysates (e.g., Cerundolo et al., J. Immunol. 21 :2069 (1991)), immobilized purified MHC (e.g., Hill et al., J. Immunol. 152, 2890 (1994); Marshall et al., J. Immunol. 152:4946 (1994)), ELISA systems (e.g., Reay et al., EMBO J. 11 :2829 (1992)), surface plasmon resonance (e.g., Khilko et al., J. Biol. Chem. 268:15425 (1993)); high flux soluble phase assays (Hammer et al., J. Exp. Med. 180:2353 (1994)), and measurement of class I MHC stabilization or assembly (e.g., Ljunggren et al., Nature 346:476 (1990); Schumacher et al., Cell 62:563 (1990); Townsend et al., Cell 62:285 (1990); Parker et al., J. Immunol. 149:1896 (1992)).

[0177] The term “derived” when used to discuss an epitope is a synonym for “prepared.” A derived epitope can be isolated from a natural source, or it can be synthesized according to standard protocols in the art. Synthetic epitopes can comprise artificial amino acid residues “amino acid mimetics,” such as D isomers of natural occurring L amino acid residues or non-natural amino acid residues such as cyclohexylalanine. A derived or prepared epitope can be an analog of a native epitope. The term “derived from” refers to the origin or source, and can include naturally occurring, recombinant, unpurified, purified or differentiated molecules or cells. For example, an expanded or induced antigenspecific T cell can be derived from a T cell. For example, an expanded or induced antigen-specific T cell can be derived from an antigen-specific T cell in a biological sample. For example, a matured APC (e.g., a professional APC) can be derived from a non-matured APC (e.g., an immature APC). For example, an APC can be derived from a monocyte (e.g., a CD 14+ monocyte). For example, an APC can be derived from a bone marrow cell.

[0178] An “epitope” is the collective features of a molecule (e.g., a peptide’s charge and primary, secondary and tertiary structure) that together form a site recognized by another molecule (e.g., an immunoglobulin, T-cell receptor, HLA molecule, or chimeric antigen receptor). For example, an epitope can be a set of amino acid residues involved in recognition by a particular immunoglobulin; a Major Histocompatibility Complex (MHC) receptor; or in the context of T cells, those residues recognized by a T-cell receptor protein and / or a chimeric antigen receptor. Epitopes can be prepared by isolation from a natural source, or they can be synthesized according to standard protocols in the art. Synthetic epitopes can comprise artificial amino acid residues, amino acid mimetics, (such as D isomers of naturally-occurring L amino acid residues or non-naturally-occurring amino acid residues). Throughout this disclosure, epitopes can be referred to in some cases as peptides or peptide epitopes. In certain embodiments, there is a limitation on the length of a peptide of the present disclosure. The embodiment that is length-limited occurs when the protein or peptide comprising an epitope described herein comprises a region (i.e., a contiguous series of amino acid residues) having 100% identity with a native sequence. In order to avoid the definition of epitope from reading, e.g., on whole natural molecules, there is a limitation on the length of any region that has 100% identity with a native peptide sequence. Thus, for a peptide comprising an epitope described herein and a region with 100% identity with a native peptide sequence, the region with 100% identity to a native sequence generally has a length of: less than or equal to 600 amino acid residues, less than or equal to 500 amino acid residues, less than or equal to 400 amino acid residues, less than or equal to 250 amino acid residues, less than or equal to 100 amino acid residues, less than or equal to 85 amino acid residues, less than or equal to 75 amino acid residues, less than or equal to 65 amino acid residues, and less than or equal to 50 amino acid residues. In certain embodiments, an “epitope” described herein is comprised by a peptide having a region with less than 51 amino acid residues that has 100% identity to a native peptide sequence, in any increment down to 5 amino acid residues; for example 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues.

[0179] A “T cell epitope” refers to a peptide sequence bound by an MHC molecule in the form of a peptide-MHC (pMHC) complex. A peptide-MHC complex can be recognized and bound by a TCR of a T cell (e.g., a cytotoxic T-lymphocyte or a T-helper cell).

[0180] A “T cell” includes CD4+ T cells and CD8+ T cells. The term T cell also includes both T helper 1 type T cells and T helper 2 type T cells. T cells can be generated by the method described in the application, for a clinical application. T cells or adoptive T cells referred to here, such as for a clinical application are cells isolated from a biological source, manipulated and cultured ex vivo and prepared into a drug candidate fora specific therapy such as a cancer therapy. When candidate cells pass specificqualitative and quantitative criteria for fitness for a clinical application, the drug candidate can be designated a drug product. In some cases, a drug productis selected from a number of drug candidates. When candidate vaccines comprising candidate antigens pass specific qualitative and quantitative criteria for fitness for a clinical application, such candidate vaccines can be designated a drug product. In the context of this application, a drug product can be a vaccine, such as an mRNA-based vaccine, a T cell, more specifically, a population of T cells, or more specifically a population of T cells with heterogeneous characteristics and subtypes. For example, a drug product, as disclosed herein can have a population of T cells comprising CD8+ T cells, CD4+ T cells, with cells at least above a certain exhibiting antigen-specificity, a certain percentage of each exhibiting a memory phenotype, among others.

[0181] An “immune cell” refers to a cell that plays a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0182] An “immunogenic” peptide or an “immunogenic” epitope or an “immunogenic” peptide epitope is a peptide that binds to an HLA molecule and induces a cell-mediated or humoral response, for example, a cytotoxic T lymphocyte (CTL) response, a helper T lymphocyte (HTL) response and / or a B lymphocyte response. Immunogenic peptides described herein are capable of binding to an HLA molecule and thereafter induce a cell-mediated or humoral response (e.g. , a CTL (cytotoxic) response, or a HTL response) to the peptide.

[0183] A “protective immune response” or “therapeutic immune response” refers to a CTL and / or an HTL response to an antigen, which in some way prevents or at least partially arrests disease symptoms, side effects or progression. The immune response can also include an antibody response which has been facilitated by the stimulation of helper T cells.

[0184] A “T-cell receptor” (“TCR”) refers to a molecule, whether natural or partly or wholly synthetically produced, found on the surface of T lymphocytes (T cells) that recognizes an antigen bound to a major histocompatibility complex (MHC) molecule. The ability of a T cells to recognize an antigen associated with various diseases e.g., cancers) is conferred by its TCR, which is made up of both an alpha (a) chain and a beta (p) chain or a gamma (y) and a delta (8) chain. The proteins which make up these chains are encoded by DNA, which employs a unique mechanism for generating the tremendous diversity of the TCR. This multi-subunit immune recognition receptor associates with the CD3 complex and binds peptides presented by the MHC class I and II proteins on the surface of antigen-presenting cells (APCs). Binding of a TCR to a peptide on an APC is a central event in T cell activation.

[0185] As used herein, a “chimeric antigen receptor” or “CAR” refers to an antigen binding protein in that includes an immunoglobulin antigen binding domain (e.g., an immunoglobulin variable domain) and a T cell receptor (TCR) constant domain. As used herein, a “constant domain” of a TCR polypeptide includes a membrane-proximal TCR constant domain, a TCR transmembrane domain and / or a TCR cytoplasmic domain, or fragments thereof. For example, in some embodiments, a CAR is a monomer thatincludes a polypeptide comprising an immunoglobulin heavy chain variable domain linked to a TCR0 constant domain. In some embodiments, the CAR is a dimer that includes a first polypeptide comprising an immunoglobulin heavy or light chain variable domain linked to a TCRa or TCR0constant domain and a second polypeptide comprising an immunoglobulin heavy or light chain variable domain (e.g., a K or X variable domain) linked to a TCR0 or TCRa constant domain.

[0186] “Major Histocompatibility Complex” or “MHC” is a cluster of genes or the protein products thereof that plays a role in control of the cellular interactions responsible for physiologic immune responses. The terms “major histocompatibility complex” and the abbreviation “MHC” can include any class of MHC molecule, such as MHC class I and MHC class II molecules, and relate to a complex of genes which occurs in all vertebrates. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. Thus, a “Human Leukocyte Antigen” or “HLA” refers to a human Major Histocompatibility Complex (MHC) protein (see, e.g., Stites, et al., Immunology, 8THEd., Lange Publishing, Los Altos, Calif. (1994). For a detailed description of the MHC and HLA complexes, see, Paul, Fundamental Immunology, 3rdEd., Raven Press, New York (1993).

[0187] The major histocompatibility complex in the genome comprises the genetic region whose gene products expressed on the cell surface are important for binding and presenting endogenous and / or foreign antigens and thus for regulating immunological processes. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting cells or diseased cells in immune reactions. MHC proteins or molecules bind peptides and present them for recognition by T-cell receptors. The proteins encodedby the MHC can be expressed on the surface of cells, and display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g. , fragments of invading microorganisms) to a T-cell. MHC binding peptides can result from the proteolytic cleavage of protein antigens and represent potential lymphocyte epitopes, (e.g., T cell epitope and B cell epitope). MHCs can transport the peptides to the cell surface and present them there to specific cells, such as cytotoxic T-lymphocytes, T-helper cells, or B cells. The MHC region can be divided into three subgroups, class I, class II, and class III. MHC class I proteins can contain an a-chain and 02-microglobulin (not part of the MHC encoded by chromosome 15). They can present antigen fragments to cytotoxic T-cells. MHC class II proteins can contain a- and 0-chains and they can present antigen fragments to T-helper cells. MHC class III region can encode for other immune components, such as complementcomponents and cytokines. The MHC can be both polygenic (there are several MHC class I and MHC class II genes) and polymorphic (there are multiple alleles of each gene).

[0188] A “receptor” refers to abiological molecule or a molecule grouping capable of binding a ligand. A receptor can serve, to transmit information in a cell, a cell formation or an organism. A receptor comprises at least one receptor unit, for example, where each receptor unit can consist of a protein molecule. A receptor has a structure which complements that of a ligand and can complex the ligand as a binding partner. The information is transmitted in particular by conformational changes of the receptor following complexati on of the ligand on the surface of a cell. In some embodiments, a receptor is to be understood as meaning in particular proteins of MHC classes I and II capable of forming a receptor / ligand complex with a ligand, in particular a peptide or peptide fragment of suitable length. A “ligand” ref ers to a molecule which has a structure complementary to that of a receptor and is capable of forming a complex with this receptor. In some embodiments, a ligand is to b e understood as meaning a peptide or peptide fragment which has a suitable length and suitable binding motifs in its amino acid sequence, so that the peptide or peptide fragment is capable of forming a complex with MHC proteins such as MHC class I or MHC class II proteins. In some embodiments, a “receptor / ligand complex” is also to be understood as meaning a “receptor / peptide complex” or “receptor / peptide fragment complex”, including a peptide- or peptide fragment-presenting MHC molecule such as MHC class I or MHC class II molecules.

[0189] A “native” or a “wild type” sequence refers to a sequence found in nature. The term “naturally occurring” as used herein refers to the factthat an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally occurring The term “naturally processed” as used herein in the context of antigen processing or presentation, refers to the fact that the antigen is not pulsed or overexpressed in a cell by man in the laboratory but is presented by the cell as a product of endogenous pathways of antigen processing and presentation (e.g., via the transporter associated with antigen processing (TAP) pathway to present intracellular antigen on MHC I).

[0190] The term “motif’ refers to a pattern of residues in an amino acid sequence of defined length, for example, a peptide of less than about 15 amino acid residues in length, or less than about 13 amino acid residues in length, for example, from about 8 to about 13 amino acid residues (e.g., 8, 9, 10, 11, 12, or 13) for a class I HLA motif and from about 6 to about 25 amino acid residues (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) for a class II HLA motif, which is recognized by a particular HLA molecule. Motifs are typically different for each HLA protein encoded by a given human HLA allele. These motifs differ in their pattern of the primary and secondary anchorresidues. In some embodiments, an MHC class I motif identifies a peptide of 7, 8 9, 10, 11, 12 or 13 amino acid residuesin length. In some embodiments, an MHC class II motif identifies a peptide of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 amino acid residues in length. A “cross-reactive binding” peptide refers to a peptide that binds to more than one member of a class of a binding pair members (e.g., a peptide bound by both a class I HLA molecule and a class II HLA molecule).

[0191] The term “residue” refers to an amino acid residue or amino acid mimetic residue incorporated into a peptide or protein by an amide bond or amide bond mimetic, or that is encoded by a nucleic acid (DNA or RNA). The nomenclature used to describe peptides or proteins follows the conventional practice. The amino group is presented to the left (the amino- or N-terminus) and the carboxyl group to the right (the carboxy- or C-terminus) of each amino acid residue. When amino acid residue positions are referred to in a peptide epitope, they are numbered in an amino to carboxyl direction with the first position being the residue located at the amino terminal end of the epitope, or the peptide or protein of which it can be a part. In the formulae representing selected specific embodiments of the present invention, the amino- and carboxyl-terminal groups, although not specifically shown, are in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by standard three letter or single letter designations. The L-form of an amino acid residue is represented by a capital single letter or a capital first letter of a three-letter symbol, and the D-form forthose amino acid residues having D-forms is represented by a lower case single letter ora lower case three letter symbol. However, whenthree letter symbols or full names are used without capitals, they can refer to L amino acid residues. Glycine has no asymmetric carbon atom and is simply referred to as “Gly” or“G”. The amino acid sequences of peptides set forth herein are generally designated using the standard single letter symbol. (A, Alanine; C, Cysteine; D, Aspartic Acid; E, Glutamic Acid; F, Phenylalanine; G, Glycine; H, Histidine; I, Isoleucine; K, Lysine; L, Leucine; M, Methionine; N, Asparagine; P, Proline; Q, Glutamine; R, Arginine; S, Serine; T, Threonine; V, Valine; W, Tryptophan; and Y, Tyrosine.)

[0192] The terms “peptide” and “peptide epitope” are used interchangeably with “oligopeptide” in the present specification to designate a series of residues connected one to the other, typically by peptide bonds between the a-amino andcarboxylgroupsof adjacent amino acid residues. A“synthetic peptide” refers to a peptide that is obtained from a non-natural source, e.g., is man-made. Such peptides can be produced using such methods as chemical synthesis or recombinant DNA technology. “Synthetic peptides” include “fusion proteins.”

[0193] A “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine,histidine), acidic side chains e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate peptide function are well-known in the art.

[0194] “Pharmaceutically acceptable” refers to a generally non-toxic, inert, and / or physiologically compatible composition or component of a composition. A “pharmaceutical excipient” or “excipient’ comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservatives, and the like. A “pharmaceutical excipient” is an excipient which is pharmaceutically acceptable.

[0195] The terms “polynucleotide” and “nucleic acid” are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA, for example, mRNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. In some embodiments, the polynucleotide and nucleic acid can be in vitro transcribed mRNA. In some embodiments, the polynucleotide that is administered using the methods of the invention is mRNA.

[0196] The terms “isolated” or “biologically pure” refer to material which is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides described herein do not contain some or all of the materials normally associated with the peptides in their in situ environment. For example, an “isolated” epitope can be an epitope that does not include the whole sequence of the protein from which the epitope was derived. For example, a naturally -occurring polynucleotide or peptide present in a living animal is not isolated, but the same polynucleotide or peptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such a polynucleotide can be part of a vector, and / or such a polynucleotide or peptide can be part of a composition, and still be “isolated” in that such vector or composition is not part of its natural environment. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules described herein, and further include such molecules produced synthetically. In some embodiments, a polypeptide, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure. The term “substantially pure” as used herein refers to material which is at least 50% pure (i.e., free from contaminants), atleast90% pure, atleast95% pure, at least98% pure, or at least 99% pure.

[0197] The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or sub sequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid sub stitutions as part of the sequence identity. The percent identity canbe measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that can be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, for example, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof. In some embodiments, two nucleic acids or polypeptides described herein are substantially identical, meaningthey have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the sequences that is at least about 10, at least about 20, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as an amino acid sequence of a peptide or a coding region of a nucleotide sequence.

[0198] The term “subject” refers to any animal (e.g., a mammal), including, for example, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. In some embodiments, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.

[0199] The terms “effective amount” or “therapeutically effective amount” or “therapeutic effect’ refer to an amount of a therapeutic effective to “treat” a disease or disorder in a subject or mammal. The therapeutically effective amount of a drug has a therapeutic effect and as such can prevent the development of a disease or disorder; slow down the development of a disease or disorder; slow down the progression of a disease or disorder; relieve to some extent one or more of the symptoms associated with a disease or disorder; reduce morbidity and mortality; improve quality of life; or a combination of such effects.

[0200] The terms “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatmentinclude those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.

[0201] The term “depleted” when used to describe a cell sample (e.g., a peripheral blood mononuclear cell (PBMC) sample) refers to a cell sample in which a subpopulation of cells has been removed or depleted. For example, an immune cell sample depleted of CD25 expressing cells refers to an immune cell sample in which CD25 expressing cells have been removed or depleted. For example, one or more binding agents can be used to remove or deplete one or more cells or cell types from a sample. For example, CD 14+ cells can be depleted or removed from a PBMC sample, such as by using an antibody that binds to CD14.

[0202] The “stimulation” refers to a response induced by binding of a stimulatory molecule with its cognate ligand thereby mediating a signal transduction event. For example, stimulation of a T cell can refer to binding of a TCR of a T cell to a peptide-MHC complex. For example, stimulation of a T cell can refer to a step in which PBMCs are cultured together with peptide loaded APCs.

[0203] The term “enriched” refers to a composition or fraction wherein an object species has been partially purified such that the concentration of the object species is substantially higher than the naturally occurring level of the species in a finished product without enrichment. The term “induced cell” refers to a cell that has been treated with an inducing compound, cell, or population of cells that affects the cell’s protein expression, gene expression, differentiation status, shape, morphology, viability, and the like.

[0204] The term “silencing” in the context of gene silencing as used herein encompass various ways of inhibiting the gene or genes. In some cases, silencing the gene(s) can comprise inhibiting, downregulating, editing, knocking down, knocking out, cutting or using other methods to render the gene(s) not expressed or with reduced expression, or render the gene product(s) not functional.

[0205] A “reference” can be used to correlate and / or compare the results obtained in the methods of the present disclosure from a diseased specimen. Typically, a “reference” may be obtained on the basis of one or more normal specimens, in particular specimens which are not affected by a disease, either obtained from an individual or one or more different individuals (e.g., healthy individuals), such as individuals of the same species. A “reference” can be determined empirically by testing a sufficiently large number of normal specimens.

[0206] As used herein, a tumor unless otherwise mentioned, is a cancerous tumor, andthe terms cancer and tumor are used interchangeably through outthe document. While a tumor is a cancer of solid tissue, several of the compositions and methods described herein are in principle applicable to cancers of the blood, such as leukemia.Methods for Producing Engineered Cells

[0207] The present disclosure provides a method of producing a population of engineered immune cells. The method can comprise depleting CD 14+ cells, CD25+ cells, CD56+ cells or a combination of one or more of CD14+ and / or CD25+ cells and / or CD56+ cells, from a population of immune cells. In some cases, the method can comprise depleting only one type of cells selected from the group consisting of CD14+ cells, CD25+ cells and CD56+ cells from a population of immune cells. In some cases, the method can comprise depletingtwo types of cells comprising CD 14+ cells and CD25+ cells, CD25+ cells and CD56+ cells, or CD 14+ and CD56+ cells from a population from immune cells. In some cases, the method can comprise depleting CD14+ cells, CD25+ cells and CD56+ cells from a population from immune cells. The population ofimmune cells canbe isolatedfrom a subject in need of treatment. The population of immune cells can be from a PBMC sample from the subject. The population of immune cells can comprise antigen presenting cells (APCs) and T cells. By deleting CD14+ and / or CD25 and / or CD56+ cells from the population ofimmune cells, a CD14 and / or CD25 and / or CD56 depleted population of immune cells can be produced. The method can further comprise inhibiting expression of one or more endogenous genes in the depleted population of immune cells, thereby producing the population of engineered immune cells. In some cases, the method can further comprise inhibiting one or more endogenous genes selected from the group comprising PRDM1 , TNFAIP3, REGNASE-1, SOCS1, PTPN2, CISH, or any combination thereof (e.g., two or more, three or more, four or more, five or more, or all). In some cases, inhibiting the expression of the endogenous gene comprises silencing a gene locus (e.g., editing a gene locus or rendering the gene locus not being transcribed). In some cases, inhibiting the expression of the endogenous gene comprises contacting to the cells a miRNA. In some cases, inhibiting the expression of the endogenous gene comprises expressing in the cells a miRNA. In some cases, inhibiting the expression of the endogenous gene comprises contacting to the cells a siRNA. In some cases, inhibiting the expression of the endogenous gene comprises expressing in the cells a siRNA. In some cases, the miRNA targets an RNA transcript encoding the endogenous gene. In some cases, the siRNA targets an RNA transcript encoding the endogenous gene.

[0208] The method can further comprise silencing the gene locus by delivering (i) the site specific nuclease, the site specific nickase, or the site-specific epigenetic regulator, or (ii) a nucleic acid encoding the site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator into the depleted population of immune cells via electroporation, lipid nanoparticles, liposomes, nucleof ection, or viral vector. In some cases, silencing the gene locus comprises gene knockout using a site-specific nuclease. In some cases, silencing the gene locus comprises gene knockout using a sitespecific nickase. In other cases, silencing the gene locus comprises gene knockout using a site-specificepigenetic regulator. In some cases, silencing the gene locus can comprise delivering the site-specific nuclease. In other cases, silencing the gene locus can comprise delivering the site-specific nickase. In some cases, silencing the gene locus can comprise delivering the site-specific epigenetic regulator. In some cases, silencing the gene locus can comprise delivering a nucleic acid encoding the site-specific nuclease. In other cases, silencing the gene locus can comprise delivering a nucleic acid encoding the site-specific nickase. In some cases, silencing the gene locus can comprise delivering a nucleic acid encoding the site-specific epigenetic regulator. In some embodiments, the delivery can be into the depleted population of immune cells. In some embodiments, the delivery can take place into the depleted population of immune cells via electroporation. In some cases, the delivery can take place into the depleted population of immune cells via lipid nanoparticles. In some cases, the delivery can take place into the depleted population ofimmune cells via liposomes. In some cases, the delivery can take place into the depleted population ofimmune cells via nucleofection. In some cases, the delivery can take place into the depleted population of immune cells via viral vector.

[0209] The method can comprise depleting CD14+ cells, CD25+ cells, CD56+ cells or a combination of one or more of CD14+ and / or CD25+ cells and / or CD56+ cells, from a population ofimmune cells. In some cases, the method can comprise depleting only one type of cells selected from the group consisting of CD14+ cells, CD25+ cells and CD56+ cells from a population ofimmune cells. In some cases, the method can comprise depletingtwo types of cells comprising CD 14+ cells and CD25+ cells, CD25+ cells and CD56+ cells, or CD 14+ and CD56+ cells from a population from immune cells. In some cases, the method can comprise depleting CD 14+ cells, CD25+ cells and CD56+ cells from a population from immune cells. The population ofimmune cells canbe isolatedfrom a subject in need of treatment. The population of immune cells can be from a PBMC sample from the subject. The population of immune cells can comprise antigen presenting cells (APCs) and T cells. By deleting CD14+ and / or CD25 and / or CD56+ cells from the population of immune cells, a CD14 and / or CD25 and / or CD56 depleted population ofimmune cells can be produced. The method can further comprise inhibiting expression of one or more endogenous genes in the CD14 and / or CD25 and / or CD56 depleted population of immune cells, thereby producing the population of engineered immune cells. In some cases, PRDM1 can be inhibited in a CD 14 depleted population of immune cells, thereby producing the population of engineered immune cells. In some cases, PRDM1 can be inhibited in a CD25 depleted population ofimmune cells, producing the population of engineered immune cells. In some cases, PRDM1 can be inhibited in a CD56 depleted population of immune cells, producing the population of engineered immune cells. In some cases, TNFAIP3 can be inhibited in a CD14 depleted population of immune cells, thereby producing the population of engineered immune cells. In some cases, TNFAIP3 can be inhibited in a CD25 depleted populations of immune cells, producing thepopulation of engineered immune cells. In some cases, both PRDM1 and TNFAIP3 can be inhibited in a CD 14 depleted population of immune cells, producingthe population of engineered immune cells. In some cases, both PRDM1 and TNFAIP3 can be inhibited in a CD25 depleted population of immune cells, producing the population of engineered immune cells. In some cases, both PRDM1 and TNFAIP3 can be inhibited in a CD56 depleted population of immune cells, producing the population of engineered immune cells.

[0210] Various methods can be used to inhibit the expression of an endogenous gene to produce the population of engineered immune cells. For example, inhibitingthe expression of endogenous PRDM1 can comprise editing a PRDM1 gene locus. Editing the PRDM1 gene locus can comprise delivering CRISPR-associated machinery (e.g., Cas9 and gRNA) into the immune cells for gene editing CRISPR / Cas9 editing technology can be used to inhibit PRDM1 by binding gRNA to the target PRDM1 sequence, inhibiting its transcription and activation. Other gene editing methods can be used. For another example, inhibiting the expression of endogenous PRDM1 can comprise contacting to the cells or expressing in the cells a miRNA or siRNA that targets an RNA transcript encoding PRDM1. Other gene inhibiting methods can include, for example, inhibiting the expression of endogenous PRDM1 via contact with the FokI cleavage domain recognized and targeted by transcription activatorlike effector nucleases (TALENS). Another method of inhibiting the expression of endogenous PRDM1 can include contacting with FokI bound zinc-finger DNA binding domain nucleases (SFNs). Homing endonucleases or meganucleases can also be used to inhibit expression of endogenous PRDM1. Inhibitingthe expression of endogenous TNFAIP3 can comprise editing a TNFAIP3 gene locus. Inhibiting the expression of endogenous TNFAIP3 can comprise contacting to the cells or expressing in the cells a miRNA or siRNA that targets an RNA transcript encoding TNFAIP3. Editing the TNFAIP3 gene locus can comprise delivering CRISPR-associated machinery (e.g., Cas9 and gRNA) into the immune cells for gene editing. CRISPR / Cas9 editing technology can be used to inhibit TNFAIP3 by binding gRNA to the target TNFAIP3 sequence, inhibiting its transcription and activation. Other gene editing methods can be used. Other gene inhibiting methods can include, for example, inhibitingthe expression of endogenous TNFAIP3 via contact with the FokI cleavage domain recognized and targeted by transcription activator-like effector nucleases (TALENS). Another method of inhibiting the expression of endogenous TNFAIP3 can include contacting with FokI bound zinc- finger DNA binding domain nucleases (SFNs). Homing endonucleases or meganucleases can also be used to inhibit expression of endogenous TNFAIP3. Inhibiting the expression of PRDM1 and TNFAIP3 can comprise editingPRDMl and TNFAIP3 gene loci. Inhibitingthe expression ofPRDMl and TNFAIP3 can comprise contacting to the cells or expressing in the cells miRNAs or exogenous siRNAs that target RNA transcripts encoding PRDM1 and TNFAIP3. Editing the PRDM1 andTNFAIP3 gene locus can comprise delivering CRISPR-associated machinery (e.g., Cas9 and gRNA) into the immune cells for gene editing. CRISPR / Cas9 editing technology can be used to inhibit PRDM1 and TNFAIP3 by binding gRNA to the target PRDM1 and TNFAIP3 sequence, inhibiting its transcription and activation. Other gene editing methods can be used. Other gene inhibiting methods can include, for example, inhibiting the expression of endogenous PRDM1 and TNFAIP3 via contact with the FokI cleavage domain recognized and targeted by transcription activator-like effector nucleases (TALENS). Another method of inhibiting the expression of endogenous PRDM1 and TNFAIP3 can include contacting with FokI bound zinc-finger DNA binding domain nucleases (SFNs). Homing endonucleases or meganucleases can also be used to inhibit expression of endogenous PRDM1 and TNFAIP3.

[0211] In some cases, silencing the PRDM1 and / or TNFAIP3 gene locus can comprise a gene knockout using a site-specific nuclease. In other cases, silencing the PRDM1 and / or TNFAIP3 gene locus can comprise a gene knockoutusing a site-specific nickase. In some cases, silencingthe PRDM1 and / or TNFAIP3 gene locus can comprise a gene knockout using a site-specific epigenetic regulator. In some cases, silencingthe PRDM1 and / or TNFAIP3 gene locus can comprise delivering (i) the sitespecific nuclease, the site-specific nickase, or the site-specific epigenetic regulator, or (ii) a nucleic acid encoding the site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator into the depleted population of immune cells via electroporation, lipid nanoparticles, liposomes, nucleofection, or viral vector. In some cases, silencing the gene locus comprises gene knockout using a site-specific nuclease. In some cases, silencing the gene locus comprises gene knockout using a site-specific nickase. In other cases, silencing the gene locus comprises gene knockout using a site-specific epigenetic regulator. In some cases, silencing the gene locus can comprise delivering the site-specific nuclease. In other cases, silencing the gene locus can comprise delivering the site-specific nickase. In some cases, silencing the gene locus can comprise delivering the site-specific epigenetic regulator. In some cases, silencingthe gene locus can comprise delivering a nucleic acid encodingthe site-specific nuclease. In other cases, silencingthe gene locus can comprise delivering a nucleic acid encodingthe site-specific nickase. In some cases, silencing the gene locus can comprise delivering a nucleic acid encoding the site-specific epigenetic regulator. In some embodiments, the delivery can be into the depleted population of immune cells. In some embodiments, the delivery can take place into the depleted population of immune cells via electroporation. In some cases, the delivery can take place into the depleted population of immune cells via lipid nanoparticles. In some cases, the delivery can take place into the depleted population of immune cells via liposomes. In some cases, the delivery can take place into the depleted population of immune cells vianucleofection. In some cases, the delivery can take place into the depleted population of immune cells via viral vector.

[0212] The depleted population of immune cells can comprise, in some embodiments, a first population of APCs and T cells. In some embodiments, the first population of APCs and T cells can be incubated in the presence of FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, the firstpopulation of APCs and T cells can be incubated for a firsttime period. In some cases, the first time period can be 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some cases, the first time period can be 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or more than 30 days.

[0213] In some embodiments, the APCs and T cells can be incubated in the presence of FLT3L and a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, thereby forming a population of engineered immune cells comprising stimulated T cells. In some embodiments, the APCs and T cells for the first time period can be incubated in the presence of FLT3L and a polynucleotide encoding a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, thereby forming a population of engineered immune cells comprising stimulated T cells. In some cases, the APCs and T cells can be incubated in the presence of FLT3L and a polypeptide comprising at least two tumor antigen epitope sequences expressed by cancer cells of a human subject with cancer. In some cases, the APCs and T cells can be incubated in the presence of FLT3L and a polypeptide comprising at least three tumor antigen epitope sequences expressed by cancer cells of a human subject with cancer. In some cases, the APCs and T cells can be incubated in the presence of FLT3L and a polypeptide comprising at least four tumor antigen epitope sequences expressed by cancer cells of a human subject with cancer. In some cases, the APCs and T cells can be incubated in the presence of FLT3L and a polypeptide comprising at least five tumor antigen epitope sequences expressedby cancer cells of a human subject with cancer. In some cases, the APCs and T cells can be incubated in the presence of FLT3L and a polypeptide comprising at least six tumor antigen epitope sequences expressed by cancer cells of a human subject with cancer. In some cases, the APCs and T cells can be incubated in the presence of FLT3L and a polypeptide comprising at least seven tumor antigen epitope sequences expressedby cancer cells of a human subject with cancer. In some cases, the APCs and T cells can be incubated in the presence of FLT3L and a polypeptide comprising at least eight tumor antigen epitope sequences expressed by cancer cells of a human subject with cancer. In some cases,the APCs and T cells can be incubated in the presence of FLT3L and a polypeptide comprising at least nine tumor antigen epitope sequences expressed by cancer cells of a human subject with cancer. In some cases, the APCs and T cells can be incubated in the presence of FLT3L and a polypeptide comprising at least ten tumor antigen epitope sequences expressed by cancer cells of a human subject with cancer.

[0214] In some embodiments, a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer can comprise personalized neoantigens derived from the human subject.

[0215] In some embodiments, a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer can compriseaMART-1 protein ora MART- 1 protein fragment. In some cases, the polypeptide can comprise proteins involved in T cell proliferation. In some cases, the proteins can be GLI-3 or a fragment thereof.

[0216] Incubatingthe first population of APCs and T cells can be performed priorto or subsequently to inhibiting the endogenous PRDM1 or TNFAIP3, or both.

[0217] Subsequently to inhibiting the endogenous PRDM1 and / or TNFAIP3, and subsequently to incubatingthe first population of APCs and T cells in the presence of FLT3L, the stimulated T cells can be expanded, thereby forming an expanded population of cells. The expanded population of cells, in some embodiments, can comprise a population of tumor antigen-specific T cells. In some cases, the population of tumor antigen-specific T cells can comprise T cells that are specific to a complex, for example a complex that includes at least one tumor antigen epitope sequence. In some cases, the epitope can comprise a MART-1 protein or fragment thereof. In some embodiments, the epitope can be a tumor associated antigen, in some cases comprising an epitope from MART-1, or GLI-3, or a combination thereof. In some embodiments, the complex can also comprise an MHC protein expressed by the cancer cells. In other cases, the complex can also comprise APCs of the human subject. In some embodiments, the APCs can comprise APCs incubated with the T cells and a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or a polynucleotide encoding the polypeptide. In some cases, the polypeptide can comprise a protein or fragment thereof of MART-1 and / or GLI-3.

[0218] In some embodiments, inhibiting of one or more endogenous genes in the depleted population of immune cells can also include inhibiting expression of endogenous REGNASE-1 . In some cases, one or more endogenous genes and / or REGNASE-1 can be inhibited in a depleted population of CD14 and / or CD25 of immune cells. In some embodiments, inhibiting REGNASE-1 can comprise inhibiting the expression of endogenous REGNASE-1. In other embodiments, inhibiting REGNASE-1 can comprise editing the REGNASE-1 gene locus to eliminate expression of endogenous REGNASE-1.In yet other embodiments, inhibiting REGNASE-1 can comprise expression of an exogenous miRNA or an exogenous siRNA that specifically targets REGNASE-1 .

[0219] In some embodiments, inhibition of one or more endogenous genes and / or REGNASE-1 can additionally comprise inhibiting expression of one or more additional endogenous genes, for example PRDM1, TNFAIP3, SOCS1, PTPN2, CISH, or any combination thereof.

[0220] Also disclosed herein is a method of producing a population of engineered immune cells, the method can comprise: inhibiting expression of an endogenous gene in a population of immune cells, thereby producing the population of engineered immune cells, wherein the population of immune cells can comprise T cells; and depleting one or more cells selected from the group consisting of CD 14+ cells, CD25+ cells, and CD56+ cells in the population of immune cells to obtain a depleted population of immune cells. In some embodiments, the method can comprise inhibiting expression of an endogenous gene in a population of immune cells. In some cases, the inhibition can produce the population of engineered immune cells. In some cases, the population of immune cells can comprise T cells. In some cases, the method can further comprise depleting CD 14+ cells in the population of immune cells. In some cases, the population of immune cells can comprise T cells. In some cases, the method can further comprise depleting CD25+ cells in the population of immune cells. In some cases, the population of immune cells can comprise T cells. In some cases, the method can further comprise depleting CD56+ cells in the population of immune cells. In some cases, the population of immune cells can comprise T cells. In some cases, the method can further comprise depleting CD 14+ and CD25+ cells in the population of immune cells. In some cases, the method can further comprise depleting CD14+ cells and CD56+ cells in the population of immune cells. In some cases, the population of immune cells can comprise T cells. In some cases, the method can further comprise depleting CD25+ cells and CD56+ cells in the population of immune cells. In some cases, the population of immune cells can comprise T cells. In some cases, the method can further comprise depleting CD14+ cells, CD25+ cells, and CD56+ cells in the population of immune cells. In some cases, the population of immune cells can comprise T cells. In some cases, the method can further comprise obtaining a depleted population of immune cells.

[0221] In some embodiments, expression of the one or more endogenous genes and / or REGNASE-1 can be inhibited prior to depletingone or more of CD14+cells and / or CD25+cells and / or CD56+ cells in the population of immune cells to obtain a depleted population of immune cells. In some embodiments, incubatingthefirstpopulationof APCsand T cells forafirsttime period in the presence of FLTL3 can take place subsequently to depleting one or more of CD14+ cells and / or CD25+ cells and / or CD56+ cells in the population of immune cells to obtain a depleted population of immune cells. In some embodiments, a method of producing a population of engineered immune cells can beperformed. In some embodiments, the method can comprise inhibiting expression of one or more endogenous genes in the CD14 and / or CD25 and / or CD56 depleted population of immune cells, thereby producing the population of engineered immune cells. The method can further comprise depleting CD 14+ cells, CD25+ cells, CD56+ cells or a combination of one or more of CD14+ and / or CD25+ cells and / or CD56+ cells, from a population of immune cells. The population of immune cells can comprise antigen presenting cells (APCs) and / or T cells. By depleting CD 14+ and / or CD25 and / or CD56+ cells from the population of immune cells, a CD14 and / or CD25 and / or CD56 depleted population of immune cells can be produced. In some cases, the method canfurther comprise inhibiting one or more endogenous genes selected from the group comprising PRDM1, TNFAIP3, REGNASE- 1, SOCS1, PTPN2, CISH, or any combination thereof.

[0222] In some embodiments, the CD14 and / or CD25 and / or CD56+ depleted population of immune cells can comprise a first population of APCs. In some embodiments, the CD 14 and / or CD25 and / or CD56+ depleted population of immune cells can comprise a first population of APCs and / or T cells. The method can further comprise incubating the first population of APCs and T cells for a first time period in the presence of (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L). Incubating the first population of APCs and T cells for the firsttime period can further compriseincubatingin the presence of (i) FLT3L, and (ii) (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding the polypeptide, thereby forming a population of engineered immune cells comprising stimulated T cells. In some embodiments, the first time period is at least 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days,18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days. . In some embodiments, the first time period is no more than 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days,17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days. In some embodiments, the first time period is at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, or 9 days. In some embodiments, the first time period is no more than, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. In some embodiments, the first time period is at 0 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, the first time period is no more than 0 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours.

[0223] Disclosed herein is additionally a method of producing a population of engineered immune cells, the method can comprise: inhibiting expression of an endogenous gene in a population of immune cells, wherein the population of immune cells can comprise T cells; and incubating the population of immune cells for a first time period in the presence of FMS-like tyrosine kinase 3 receptor ligand (FLT3L). In some embodiments, the method can comprise inhibiting expression of an endogenous gene in a population of immune cells. In some cases, the population of immune cells can comprise T cells. In some embodiments, the method can further comprise incubating the population of immune cells for a first time period in the presence of FLT3L. In some embodiments, the first time period is at least 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days. . In some embodiments, the first time period is no more than 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days. In some embodiments, the first time period is at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, or 9 days. In some embodiments, the first time period is no more than, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. In some embodiments, the first time period is at 0 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, the first time period is no more than 0 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, the incubation of the first population of APCs and / or T cells for a firsttime period in the presence of FLT3L can be performed subsequentto depleting CD14+ cells, CD25+ cells, CD56+ cells or a combination of one or more of CD14+ and / or CD25+ cells and / or CD56+ cells, from a population of immune cells.

[0224] In some embodiments, the method can further comprise, subsequently to the depletion and incubation in the presence of FLT3L, expandingthe stimulated T cells. In some embodiments, the expanded stimulated T cells can form an expanded population of cells. In some cases, the expanded population of cells can comprise tumor-antigen-specific T cells. In some embodiments, the tumor antigen-specific T cells can comprise T cells that are specific to a complex. In some embodiments, the complex can comprise (1) at least one tumor antigen epitope sequence. In some cases, the at least one tumor antigen epitope sequence can comprise a MART-1 protein or fragment thereof. In some embodiments, the complex can further comprise (2) an MHC protein. In some embodiments, the MHCprotein can be a protein expressed by the cancer cells. In other embodiments, the MHC protein can be expressed by APCs of the human subject. In some embodiments, the APCs can comprise APCs incubated with the T cells and a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or a polynucleotide encoding the polypeptide. In some cases, the polypeptide can comprise a protein or fragment thereof of MART-1 and / or GLI-3.

[0225] In some embodiments, a population of engineered immune cells can be produced by inhibiting expression of one or more endogenous genes and / or REGNASE-1 and subsequently incubating the population of engineered immune cells for a firsttime period in the presence of FLT3L. In some cases, the method can further comprise inhibiting one or more endogenous genes selected from the group comprising PRDM1, TNFAIP3, REGNASE-1, SOCS1, PTPN2, CISH, or any combination thereof.

[0226] Incubatingthe first population of APCs and T cells forthe firsttime period can further comprise incubating in the presence of (i) FLT3L, and (ii) (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding the polypeptide, thereby forming a population of engineered immune cells comprising stimulated T cells. In some embodiments, the first time period is at least 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days. . In some embodiments, the first time period is no more than 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days. In some embodiments, the first time period is at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, or 9 days. In some embodiments, the first time period is no more than, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, or 10 days. In some embodiments, the first time period is at 0 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. In some embodiments, the first time period is no more than 0 hours, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours.

[0227] In some embodiments, CD14+ cells and / or CD25+ cells and / or CD56+ cells can subsequently be depleted from the population of immune cells. In some cases, one or more of CD 14+ cells and / or CD25+ cells and / or CD56+ and / or CD19+ cells can be depleted from the population of immune cells. In some embodiments, CD 1 lb+ cells can be depleted from the population of immune cells. In somecases, one or more of CD14+ cells and / or CD25+ cells and / or CD56+ and / or CD 19+ and / or CD llb+ cells can be depleted from the population of immune cells. In some embodiments, expression of the one or more endogenous genes (e g., PRDM1, TNFAIP3, REGNASE-1, SOCS1, PTPN2, CISH, or any combination thereof) can be inhibited prior to depleting one ormore of CD 14+ cells and / or CD25+ cells and / or CD56+ cells in the population of immune cells to obtain a depleted population of immune cells. In some embodiments, incubating the first population of APCs and T cells for a first time period in the presence of FLTL3 can take place subsequently to depleting one or more of CD 14+ cells and / or CD25+ cells and / or CD56+ cells in the population of immune cells to obtain a depleted population of immune cells. In some embodiments, a method of producing a population of engineered immune cells can be performed. In some embodiments, the method can comprise inhibiting expression of one or more endogenous genes in the CD14 and / or CD25 and / or CD56 depleted population of immune cells, thereby producing the population of engineered immune cells. The method can further comprise depleting CD 14+ cells, CD25+ cells, CD56+ cells or a combination of one or more of CD14+ and / or CD25+ cells and / or CD56+ cells, from a population ofimmune cells. The population of immune cells can comprise antigen presenting cells (APCs) and / orT cells. By depleting CD 14+ and / or CD25 and / or CD56+ cells from the population of immune cells, a CD14+ and / or CD25+ and / or CD56+ depleted population of immune cells can be produced.

[0228] In some embodiments, the method can further comprise inhibiting (or silencing) the endogenous gene from the group comprising PRDM1, TNFAIP3, REGNASE-1, SOCS1, PTPN2, CISH, and any combination thereof. In some embodiments, inhibiting the expression of the endogenous gene can comprise silencing a gene locus or contacting to the cells or expressing in the cells a miRNAor siRNA that targets anRNA transcript encodingthe endogenous gene. In some cases, inhibiting the expression of the endogenous gene can comprise silencing a gene locus. In other cases, inhibiting the expression of the endogenous gene can comprise contacting to the cells a miRNA. inhibiting the expression of the endogenous gene can comprise expressing in the cells a miRNA. inhibiting the expression of the endogenous gene can comprise expressing in the cells a miRNA. inhibiting the expression of the endogenous gene can comprise expressing in the cells a siRNA. In some cases, the miRNA and siRNA can target an RNA transcript encodingthe endogenous gene. In some embodiments, silencing the gene locus can comprise gene knockout using a site-specific nuclease, gene knockout using a site-specific nickase, or gene silencingusing a site-specific epigenetic regulator. In some cases, silencing the gene locus can comprise gene knockout using a site-specific nuclease. In some cases, silencing the gene locus can comprise gene knockout using a site-specific nickase. In some cases, silencing the gene locus can comprise gene knockout using a site-specific epigenetic regulator. In some embodiments, silencing the gene locus can comprise delivering (i) thesite-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator, or (ii) a nucleic acid encoding the site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator into the depleted population of immune cells via electroporation, lipid nanoparticles, liposomes, nucleofection, or viral vector. In some cases, silencing the gene locus comprises gene knockout using a site-specific nuclease. In some cases, silencing the gene locus comprises gene knockout using a site-specific nickase. In other cases, silencing the gene locus comprises gene knockout using a site-specific epigenetic regulator. In some cases, silencing the gene locus can comprise delivering the site-specific nuclease. In other cases, silencing the gene locus can comprise delivering the site-specific nickase. In some cases, silencing the gene locus can comprise delivering the site-specific epigenetic regulator. In some cases, silencing the gene locus can comprise delivering a nucleic acid encodingthe site-specific nuclease. In other cases, silencingthe gene locus can comprise delivering a nucleic acid encodingthe site-specific nickase. In some cases, silencing the gene locus can comprise delivering a nucleic acid encoding the site-specific epigenetic regulator. In some embodiments, the delivery can be into the depleted population of immune cells. In some embodiments, the delivery can take place into the depleted population of immune cells via electroporation. In some cases, the delivery can take place into the depleted population of immune cells via lipid nanoparticles. In some cases, the delivery can take place into the depleted population of immune cells via liposomes. In some cases, the delivery can take place into the depleted population of immune cells via nucleofection. In some cases, the delivery can take place into the depleted population of immune cells via viral vector.

[0229] In some embodiments, the CD14+ and / or CD25+ and / or CD56+ depleted population of immune cells can comprise a first population of APCs. In some embodiments, the CD 14 and / or CD25 and / or CD56+ depleted population of immune cells can comprise a first population of APCs and / or T cells. The method can further comprise incubating the first population of APCs and T cells for a first time period in the presence of (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L).

[0230] In some embodiments, the incubation of the first population of APCs and / or T cells for a first time period in the presence ofFLT3L can be performed subsequent to depleting CD 14+ cells, CD25+ cells, CD56+ cells ora combination of one ormore of CD14+and / orCD25+cells and / orCD56+cells, from a population of immune cells.

[0231] In some embodiments, endogenous REGNASE-1 expression can be inhibited in the population of immune cells. In some embodiments, endogenous REGNASE-1 expression can be inhibited prior to incubation with FLT3L and prior to depletion.

[0232] In some embodiments, inhibiting the expression of endogenous REGNASE-1 can comprise editing a REGNASE-1 gene locus. Editing the REGNASE-1 gene locus can comprise deliveringCRISPR-associated machinery (e.g., Cas9 and gRNA) into the immune cells for gene editing CRISPR / Cas9 editing technology can be used to inhibit REGNASE-1 by binding gRNA to the target REGNASE-1 sequence, inhibiting its transcription and activation. Other gene editing methods can be used. For another example, inhibiting the expression of endogenous REGNASE-1 can comprise contacting to the cells or expressing in the cells a miRNA or siRNA that targets an RNA transcript encoding REGNASE-1. Other gene inhibiting methods can include, for example, inhibiting the expression of endogenous REGNASE-1 via contact with the FokI cleavage domain recognized and targeted by transcription activator-like effector nucleases (TALENS). Another method of inhibiting the expression of endogenous REGNASE-1 can include contacting with FokI bound zinc-finger DNA binding domain nucleases (SFNs).

[0233] In some embodiments, the method can additionally comprise inhibiting expression of one or more additional endogenous genes, for example an endogenous gene selected from the group consisting of PRDM1, TNFAIP3, REGNASE-1, SOCS1, PTPN2, CISH, or any combination thereof. Methods for Screening Gene Targets in Engineered Cells

[0234] The present disclosure provides methods of producing a population of engineered immune cells. The engineered immune cells can comprise one or more reduced expression of an endogenous gene. The engineered immune cells can be used for screening for endogenous genes that can be down- regulated for genetic enhancement of T cell therapies.

[0235] The screening methods described herein can comprise obtaining a plurality of immune cells. The plurality of immune cells can be isolated immune cells. The plurality of isolated immune cells can be, for example, PBMC cells. The plurality of immune cells can be, for example, T cells and / or APC cells. In some cases, the plurality of immune cells can be, for example, TCR-T cells, CAR-T cells, or T cells activated through NEO STIM. Next, the plurality of immune cells can be activated. The plurality of immune cells can be depleted of one or more types of cells, for example, CD 14+ cells, CD25+ cells, and / or CD56+ cells. Next, the plurality of activated immune cells can undergo transduction. Transduction can be performed by, for example, a viral vector system. Transduction can, for example, comprise transducing the activated immune cells with one or more lentivirusesfrom a lentiviral pool. In some examples, the lentiviral pool can comprise a lentiviral library. Next, the plurality of immune cells can undergo transfection. Transfection can be performed by, for example, calcium phosphate precipitation, lipofection, electroporation, or viral delivery. Transfection can be performed by, for example, electroporation to produce a population of edited immune cells. In some cases, the population of immune cells can first undergo transduction. In some cases, the population of immune cells can undergo activation after undergoing transduction. In some cases, the population of immune cells can undergo electroporation after transduction and activation. In some cases, the population of immunecells can undergo electroporation first. In some cases, the population of immune cells can undergo activation and transduction after undergoing electroporation. In some cases, the population of immune cells can undergo electroporation before undergoing transduction, and next undergoing activation. Electroporation can be performed by, for example, delivering a molecule into the cells of the cell population. For example, electroporation can deliver a component for gene silencing (e.g., a Cas9 molecule or a nucleic acid encoding the Cas9) into the cells. For example, transfection can be performed to knock out one or more genes. Knocking out one or more genes in the cell population can be performed by various methods described in the present disclosure, for example, sgRNA or siRNA knockout of genes.

[0236] Next, the plurality of immune cells can undergo one or more phenotypic assays. Phenotypic assays can comprise, for example, cell sternness, proliferative capacity, resistance to repeated stimulation, resistance to repeated exhaustion, robustness, cytokine secretion, cytotoxic capability, or metabolic fitness. Next, the plurality of immune cells can undergo selective pressure screening In some examples, selective pressure screening can comprise pooled gain-of-function screening with open reading frames. Next, the plurality of immune cells can undergo flow cytometry sorting Alternatively, next the plurality of immune cells can undergo flow cytometry sorting prior to undergoing selective pressure analysis. Next, one or more genes can be identified form the screening The genes identified can comprise, for example, PRDM1, TNFAIP3, REGNASE-1, SOCS1, PTPN2, CISH, and any combination thereof. Next, the gDNA can be isolated and sequenced from the cells.

[0237] FIG. 2 provides an example of the screening method. For example, the method can comprise transactivation of isolated PBMC cells. The method can further comprise transduction of retrovirus TCR with cells, and lentivirus guide transduction from a lentiviral pool. The method can further comprise positive selection for sgRNA-transduced cells (e.g., CD19+ positive selection). The method can further comprise post-selection Cas9 electroporation to produce edited T cells, that can then be expanded. The method can further comprise incubation for approximately 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days. The method can further comprise resuspending the expanded transduced T cell population and transferring them onto fresh media approximately every 0 days, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or more than 7 days until a loss of functionality can be seen.

[0238] FIG. 4 provides an example of the phenotypic assay screening. For example, edited immune cells can rest for 7-10 days. Edited immune cells can be, for example, edited T cells. The edited immune cells can be, for example, labeled. The labeling molecule can be for example, CFSE. Next, the edited immune cell population can be restimulated. Next, the edited immune population can be screened for markers of phenotypic qualities. Markers can include cytokines, sternness markers (e.g,CCR7), proliferative markers, exhaustion markers, or cytotoxic markers. Next, the genomic DNA can be isolated. In another example, edited immune cells (e.g., T cells), can be rep-plated with an antigenexpressing cell line. The re-plating can occur 0 times, 1 time, 2 times, 3 times, or 4 times, or more than 4 times. The edited immune cells can undergo genomic DNAisolation. FIG.6 provides model systems of activated T cells that can be used for screening. Additional model systems can be used forvalidation of identified gene targets to confirm their functions of enhancing T cell therapies. In some cases, the model systems used for validation may comprise ribonucleoprotein complex (RNP) edited cells.T Cell Manufacturing

[0239] The engineered immune cells provided here in can be ex vivo induced or expanded. Generating antigen-specific T cells by controlled ex vivo induction or expansion of T cells (e.g., autologous T cells) can provide highly specific and beneficial T cell and T-cell receptors (TCRs). The present disclosure provides T cell manufacturing methods, TCRs, CARs, therapeutic T cell compositions which can be used for therapies including, for example, treatment of cancer or other illnesses. Preparing stimulated and expanded depleted populations of engineered T cells, CARs, and engineered immune cells with TCRs can provide performance advantages, for example increased specificity, increased persistence after repeated stimulation, increased proliferation, and increased cytotoxic activity. Engineered CARs can provide customized targeting of autologous cancer cells, without requiring an MHC complex, and their function can be enhanced with NEOSTIM manufacturing Engineered TCRs can reduce side effects while enhancing specificity. The manufacturing process for preparing the antigen-specific T cells using ex vivo induction or expansion can be referred to as NEOSTIM process described herein. Additional details of the NEOSTIM process are disclosed in International Application No. PCT / US2018 / 059896, PCT / US2020 / 031898, PCT / US2021 / 045800, and PCT / US2023 / 083256, the entire content of each of which is incorporated herein by reference.Preparing Activated, Antigen-Specific T Cells

[0240] Provided herein are methods for stimulating T cells. For example, the methods provided herein can be used to stimulate antigen-specific T cells. The methods provided herein can be used to induce or activate T cells. For example, the methods provided herein can be used to expand activated T cells. For example, the methods provided herein can be used to induce naive T cells. For example, the methods provided herein can be used to expand antigen-specific CD8+ T cells. For example, the methods provided herein can be used to expand antigen-specific CD4+ T cells. For example, the methods provided herein can be used to expand antigen-specific CD8+ T cells having memory phenotype. For example, the therapeutic compositions can comprise antigen-specific CD8+ T cells. For example, the therapeutic compositions can comprise antigen -specific memory T cells.

[0241] T cells can be activated ex vivo with a composition comprising antigenic peptides or polynucleotides encoding the antigenic peptides. T cells can be activated ex vivo with a composition comprising antigen loaded antigen-presenting cells. In some embodiments, the APCs and / or T cells are derived from a biological sample which is peripheral blood mononuclear cells (PBMC). In some embodiments, the subjectis administered FLT3L prior to obtainingthe biological sample for preparing the APCs and / or T cells.

[0242] In some embodiments, the population of immune cells can be from a biological sample. In some cases, the biological sample can be from a human subject. In other cases, the biological sample can be from an animal subject, for example a murine subject. In some embodiments, the population of immune cells can be from the same human or animal subject from which cancer cells can be obtained. In some embodiments, the APCs and / or T cells are derived from a biological sample, for example, from healthy human donors.

[0243] In some embodiments, the biological sample comprisesa percentage of the atleastone antigenspecific T cell in the composition is at least about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0. 1%, 0.5%. In some embodiments, the biological sample comprises less than 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%. 1%, 2%, 3%, 4%, 5%, or less than 10% antigen activated T cells of the total cell count in the biological sample that is derived from peripheral blood or leukapheresis. In some embodiments, the biological sample comprises less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30% antigen activated T cells of the total cell count in the biological sample that is derived from peripheral blood.

[0244] In some embodiments, the biological sample comprises antigen naive T cells. In some embodiments, the biological sample comprises greater than about 0.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0. 1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% antigen naive cells of the total cell count in the biological sample that is derived from peripheral blood or leukapheresis.

[0245] In some embodiments, a percentage of at least one antigen-specific CD8+ T cell in the composition is less than aboutO.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5% in the biological sample derived from peripheral blood or leukapheresis. In some embodiments, a percentage of at least one antigen -specific CD4+ T cell in the composition is at least aboutO.00001%, 0.00002%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0. 1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, of in the biological sample derived from peripheral blood or leukapheresis.

[0246] In some embodiments, a percentage of the at least one antigen-specific T cell in the biological sample is at most about O.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1% or 0.5% of the total immune cells. In some embodiments, a percentage of atleast one antigenspecific CD8+T cell in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1% or 0.5% of the total immune cells. In some embodiments, a percentage of atleast one antigen-specific CD4+ T cell in the biological sample is at most about 0.00001%, 0.00005%, 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1% or 0.5% of the total immune cells. In some embodiments, a percentage of antigen-specific T cells in the biological sample is at most about 0.5%. In some embodiments, a percentage of antigen-specific CD8+ T cells in the biological sample is at most about 0.5%. In some embodiments, a percentage of antigenspecific CD4+ T cells in the biological sample is at most about 0.5% in the biological sample.Expanding the Depleted Population of T cells

[0247] Immune cells can be characterized by cell surface molecules. In some embodiments the immune cells are preferably selected based on the cell surface markers, for example, from the biological sample, by using antibodies that can bind to the cell surface receptors. In some embodiments some cells are negatively selected to enrich one or more cell types that do not express the cell surface molecule that they are negatively selected for.

[0248] In some embodiments, the expanded population of cells can comprise at least IxlO3, 1x104, at least IxlO5, at least IxlO6, at least IxlO7, at least IxlO8, at least IxlO9, at least IxlO10, at least IxlO11, at least IxlO12, at least IxlO13, at least IxlO14, at least IxlO15or more total cells. In some embodiments, the expanded population of cells can comprise at least IxlO6total cells. In some embodiments, the expanded population of cells can comprise at least IxlO7total cells. In some embodiments, the expanded population of cells can comprise at least IxlO8total cells. In some embodiments, the expanded population of cells can comprise at least IxlO9total cells. In some embodiments, the expanded population of cells can comprise at least about IxlO10total cells. In some embodiments, the expanded population of cells can comprise at least about IxlO11total cells. In some embodiments, the expanded population of cells can comprise at least about IxlO9total cells. In some embodiments, the expanded population of cells can comprise at least about 5xl07total cells. In some embodiments, the expanded population of cells can comprise at least about 5xl08total cells. In some embodiments, the expanded population of cells can comprise at least about 5xl09total cells. In some embodiments, the expanded population of cells can comprise at least about 5xl010total cells. In some embodiments, the expanded population of cells can comprise at least about 5xl0ntotal cells. In some embodiments, the expanded population of cells can comprise from about lxl08to about IxlO11total cells. In some embodiments, the expanded population of cells can comprisebetween about 0.75 xlO8total cells to about 1.25 xlO10total cells. In some embodiments, the expanded population of cells can comprise from about 5xl08to about IxlO9total cells. In some embodiments the expanded population of cells can comprise from about 5xl08to about 2xl09total cells. In some embodiments, the expanded population of cells can comprise about 5xl08to about IxlO10total cells.

[0249] In some embodiments, the depleting can comprise depleting CD14+ cells and / or CD25+ cells and / or CD56+ cells and / or CD 19+ and / or CD 1 lb+ cells directly from a washed and / or cryopreserved peripheral blood mononuclear cell (PBMC) sample. In some cases, the PBMC sample can be from a human subject. In other cases, the PBMC sample can be from an animal subject, for example a murine subject. In some embodiments, incubating and expanding of the cell population can be performed in about less than 28 days. In some embodiments, incubating and expanding of the cell population can be performed in about less than 30 days, about less than 29 days, about less than 28 days, about less than 27 days, about less than 26 days, about less than 25 days, about less than 24 days, about less than 23 days, about less than 22 days, about less than 21 days, about less than 20 days, about less than 19 days, about less than 18 days, about less than 17 days, about less than 16 days, about less than 15 days, aboutlessthan 14 days, aboutlessthan 13 days, aboutlessthan 12 days, aboutlessthan 11 days, about less than 10 days, aboutless than 9 days, about less than 8 days, about less than 7 days, aboutless than 6 days, about less than 5 days, about less than 3 days, aboutless than 1 days, or about less than 1 day. In some embodiments, incubating and expanding of the cell population can be performed in about more than 28 days, in about more than 29 days, or in more than about 30 days.

[0250] In some embodiments, the fraction of CD8+ antigen-specific T cells of the total number of CD8+ T cells in the expanded population of cells can be atleast abouttwo-fold higher than the fraction of CD8+ antigen-specific T cells of the total number of CD8+ T cells in the depleted population of immune cells. In some cases, the fraction of CD8+ antigen-specific T cells of the total number of CD8+ T cells in the expanded population of cells can be at least about2.5-fold higher, about 3-fold higher, about 3.5-fold higher, about 4-fold higher, about 4.5 -fold higher, about 5 -fold higher, about 5.5-fold higher, about 6-fold higher, about 6.5-fold higher, about 7-fold higher, about 7.5-fold higher, about 8- fold higher, about 8.5-fold higher, about 9-fold higher, about 9.5-fold higher, about 10-fold higher, about 11-fold higher, about 12-fold higher, about 13-fold higher, about 14-fold higher, about 15-fold higher, about 16-fold higher, about 17-fold higher, about 18-fold higher, about 19-fold higher, about 20-fold higher, about 25-fold higher, about 30-fold higher, about 35-fold higher, about 40-fold higher, about45-fold higher, about 50-fold higher, about 55-fold higher, about 60 -fold higher, about 65-fold higher, about 70-fold higher, about 75-fold higher, about 80-fold higher, about 85 -fold higher, about 90-fold higher, about 95-fold higher, or more than about 100-fold higher than the fraction of CD8+antigen-specific T cells of the total number of CD8+ T cells in the depleted population of immune cells.

[0251] In some embodiments, the fraction of CD4+ antigen-specific T cells of the total number of CD4+ T cells in the expanded population of cells can be atleast abouttwo-fold higher than the fraction of CD4+ antigen-specific T cells of the total number of CD4+ T cells in the depleted population of immune cells. In some cases, the fraction of CD4+ antigen-specific T cells of the total number of CD4+ T cells in the expanded population of cells can be at least about2.5-fold higher, about 3-fold higher, about 3.5-fold higher, about 4-fold higher, about 4.5 -fold higher, about 5 -fold higher, about 5.5-fold higher, about 6-fold higher, about 6.5-fold higher, about 7-fold higher, about 7.5-fold higher, about 8- fold higher, about 8.5-fold higher, about 9-fold higher, about 9.5-fold higher, about 10-fold higher, about 11-fold higher, about 12-fold higher, about 13-fold higher, about 14-fold higher, about 15-fold higher, about 16-fold higher, about 17-fold higher, about 18-fold higher, about 19-fold higher, about 20-fold higher, about 25-fold higher, about 30-fold higher, about 35-fold higher, about 40-fold higher, about45-fold higher, about 50-fold higher, about 55-fold higher, about 60-fold higher, about 65-fold higher, about 70-fold higher, about 75-fold higher, about 80-fold higher, about 85-fold higher, about 90-fold higher, about 95-fold higher, or more than about 100-fold higher than the fraction of CD4+ antigen-specific T cells of the total number of CD4+ T cells in the depleted population of immune cells.

[0252] In some embodiments, atleast about 0.1% ofthe CD8+ T cells in the expanded population of cells can be CD8+ antigen-specific T cells. In some embodiments, the CD8+ antigen-specific T cells can be derived from naive CD8+ T cells. In some cases, at least about 0.05%, atleast about 0.15%, at least about 0.2%, atleast about 0.3%, atleast about 0.4%, atleast about 0.5%, atleast about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, atleast about 1%, at least about 2%, atleast about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, atleast about25%, atleast about 30%, atleast about 35%, atleast about40%, at least about 45%, at least about 50%, at least about 55%, atleast about 60%, atleast about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than about 95% of the CD8+ T cells in the expanded population of cell can be CD8+ antigenspecific T cells, which can be derived from naive CD8+ T cells.

[0253] In some embodiments, at least about 0.1% ofthe CD4+ T cells in the expanded population of cells can be CD4+ antigen-specific T cells. In some embodiments, the CD4+ antigen-specific T cells can be derived from naive CD4+ T cells. In some cases, atleast about 0.05%, atleast about 0.15%, at least about 0.2%, atleast about 0.3%, atleast about 0.4%, atleast about 0.5%, atleast about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, atleast about 1%, at least about 2%, atleastabout 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about25%, atleast about 30%, atleastabout35%, atleast about40%, at least about 45%, at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more than about 95% of the CD4+ T cells in the expanded population of cell can be CD4+ antigenspecific T cells, which can be derived from naive CD4+ T cells.

[0254] In some embodiments, the method can comprise introducing the polynucleotide encoding the polypeptide or the mRNA encoding the polypeptide into the APCs of the first population of APCs and T cells. In some embodiments, introducing comprises electroporating. In some embodiments, introducing comprises nucleofecting. In some embodiments, introducing can be carried out without separatingthe T cells from the APCs of the firstpopulationof APCs and T cells. In some embodiments, the method can be performedin less than 35 days. In some embodiments, the method canbe performed in less than 30 days. In some embodiments, the method can be performed in less than 28 days. In some embodiments, the method can be performed in less than 25 days. In some embodiments, the method can be performedin less than 20 days, in some embodiments, the method can be performedin less than 15 days. In some embodiments, the method can be performed in less than 10 days. In some embodiments, the method can be performed in less than 5 days. In some embodiments, CD8+ antigenspecific T cells are expanded. In some embodiments CD4+ antigen-specific T cells are expanded. In some embodiments, the fraction of CD8+ antigen-specific T cells of the total number of T cells in the expanded population of cells is at least two-fold higher than the fraction of CD8+ antigen-specific T cells of the total number of CD8+ T cells in the CD 14 and / or CD25 depleted population of immune cell. In some embodiments, the fraction of CD8+ antigen-specific T cells of the total number of T cells in the expanded population of cells is atleast 1.5-fold, at least 2-fold, at least 2.5 -fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10- fold higher than the fraction of CD8+ antigen-specific T cells of the total number of CD8+ T cells in the CD14 and / or CD25 depleted population of immune cell. In some embodiments, the fraction of CD4+ antigen-specific T cells of the total number of T cells in the expanded population of cells is at least two-fold higher than the fraction of CD4+ antigen-specific T cells of the total number of CD4+ T cells in the CD14 and / or CD25 depleted population of immune cell. In some embodiments, the fraction of CD4+ antigen-specific T cells of the total number of T cells in the expanded population of cells is at least 1.5-fold, at least 2-fold, at least 2.5 -fold, at least 3 -fold, at least 4-fold, at least 5 -fold, at least 6-fold, at least 7-fold, atleast 8 -fold, at least 9-fold, atleast 10-fold higher than the fraction of CD4+ antigen-specific T cells of the total number of CD4+ T cells in the CD 14 and / or CD25 depleted population of immune cell. In some embodiments, at least 0.1% of the CD8+ T cells in the expandedpopulation of cells are CD8+ antigen-specific T cells derived from naive CD8+ T cells. In some embodiments, atleast0.5% ofthe CD8+T cells in the expanded population of cells are CD8+ antigenspecific T cells derived from naive CD8+ T cells. In some embodiments, at least 1% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from naive CD8+ T cells. In some embodiments, at least 5% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from naive CD8+ T cells. In some embodiments, at least 10% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from naive CD8+ T cells. In some embodiments, at least 20% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from naive CD8+ T cells. In some embodiments, at least 30% of the CD8+ T cells in the expanded population of cells are CD8+ antigenspecific T cells derived from naive CD8+ T cells. In some embodiments, at least 40% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from naive CD8+ T cells. In some embodiments, at least 50% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from naive CD8+ T cells. In some embodiments, at least 60% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from naive CD8+ T cells. In some embodiments, at least 70% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from naive CD8+ T cells. In some embodiments, at least 80% of the CD8+ T cells in the expanded population of cells are CD8+ antigenspecific T cells derived from naive CD8+ T cells. In some embodiments, at least 90% of the CD8+ T cells in the expanded population of cells are CD8+ antigen-specific T cells derived from naive CD8+ T cells. In some embodiments, at least 0.1% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from naive CD4+ T cells. In some embodiments, at least 0.5% of the CD4+T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from naive CD4+ T cells. In some embodiments, at least 1% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from naive CD4+ T cells. In some embodiments, at least 5% of the CD4+ T cells in the expanded population of cells are CD4+ antigenspecific T cells derived from naive CD4+ T cells. In some embodiments, at least 10% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from naive CD4+ T cells. In some embodiments, at least 20% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from naive CD4+ T cells. In some embodiments, at least 30% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from naive CD4+ T cells. In some embodiments, at least 40% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from naive CD4+ T cells. In some embodiments, at least 50% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from naive CD4+ T cells. In some embodiments, at least 60% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from naive CD4+ T cells. In some embodiments, at least 70% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from naive CD4+ T cells. In some embodiments, at least 80% of the CD4+ T cells in the expandedpopulation of cells are CD4+ antigen-specific T cells derived from naive CD4+ T cells. In some embodiments, at least 90% of the CD4+ T cells in the expanded population of cells are CD4+ antigen-specific T cells derived from naive CD4+ T cells.

[0255] In some embodiments, a composition comprises a population of immune cells that have been incubated with one or more APCs or APC preparations. For example, a composition can comprise a population of immune cells that has been incubated with one or more cytokine, growth factor and / or ligand stimulated APCs or cytokine, growth factor and / or ligand stimulated APC preparations. For example, a composition can comprise a population of immune cells that has been incubated with one or more cytokine stimulated APCs or cytokine stimulated APC preparations. For example, a composition can comprise a population of immune cells that have been incubated with one or more growth factor stimulated APCs or growth factor stimulated APC preparations. For example, a composition can comprise a population of immune cells that has been incubated with one or more ligand stimulated APCs or ligand stimulated APC preparations.

[0256] In some embodiments, antigen-presenting cells (APCs) are prepared from the biological sample by selectingfrom APCs or precursor cells that can be cultured in presence of antigenic peptides to generate antigen-loaded APCs, which are used for activating T cells. Some of the related cell surface markers for selecting and / or enriching for a set of cells is described below.

[0257] In some embodiments, the APC is derived from a CD14+ monocyte. In some embodiments, the APCs can be obtained from skin, spleen, bone marrow, thymus, lymph nodes, peripheral blood, or cord blood. In some embodiments, the CD 14+ monocyte is from a biological sample from a subject comprising PBMCs. For example, a CD 14+ monocyte can be isolated from, enriched from, or purified from a biological sample from a subject comprising PBMCs. In some embodiments, the CD14+ monocyte is stimulated with one or more cytokines or growth factors. In some embodiments, the one or more cytokines or growth factors comprise GM-CSF, IL-4, FLT3L, TNF-a, IL-1 , PGE1 , IL-6, IL- 7, IL-15, IFN-y, IFN-a, R848, LPS, ss-rna40, poly I:C, or a combination thereof. In some embodiments, the CD14+ monocyte is from a second biological sample comprising PBMCs.

[0258] In some embodiments, the expansion can comprise contacting the population of cell comprising stimulated T cells with a second population of mature APCs. In some cases, the second population of mature APCs can be incubated with FLT3L prior to contacting the stimulated T cells. In some embodiments, the second population of mature APCs can present the peptide consisting of the epitopesequence from the protein expressed by the cancer cells of the human or animal subj ect. In some cases, the peptide can comprise a protein or fragment thereof of MART-1 and / or GLI-3. In some embodiments, the population of cells comprising stimulated T cells can be expandedfor a second time period, thereby forming an expanded population of T cells. In some embodiments, the second population of mature APCs can be incubated with FLT3L for at least 1 day prior to contacting the population of cells comprising stimulated T cells with the second population of mature APCs. In some cases, the second population of mature APCs can be incubated with FLT3L for at least about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or more than about 30 days prior to contacting the population of cells comprising stimulated T cells with the second population of mature APCs. In some embodiments, the subjectcanbeincubatedwithFLT3L at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, or 12 hours before contacting the population of cells comprising stimulated T cells with the second population of mature APCs. In some embodiments, the APCs can be incubated with FLT3L at least about 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks before contacting the population of cells comprising stimulated T cells with the second population of mature APCs. In some embodiments, the APC can be an autologous APC, an allogenic APC, or an artificial APC. In some embodiments, the APC can be an autologous APC. In some embodiments, an isolated population of APCs can be enriched or substantially enriched. In some embodiments, the isolated population of APCs is at least 30%, at least 50%, at least 75%, or at least 90% homogeneous. In some embodiments, the isolated population of APCs is at least 60%, at least 75%, or at least 90% homogeneous. In some embodiments, the isolated population of APCs is at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, atleast 6%, at least 7%, atleast 8%, atleast 9%, at least 10%, atleast 11%, atleast 12%, atleast 13%, atleast 14%, atleast 15%, at least 16%, at least 17%, atleast 18%, atleast 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 least 31%, at least 32%, atleast 33%, atleast 34%, atleast 35%, atleast 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, atleast48%, atleast49%, atleast 50%, atleast 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, atleast 78%, atleast 79%, atleast 80%, atleast 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% enriched. APCs, such as APCs can include, for example, APCs derived in culture from monocytic dendritic precursors as well as endogenously-derived APCs present in tissues such as, for example, peripheral blood, cord blood, skin, spleen, bone marrow, thymus, and lymph nodes.

[0259] In some embodiments, the method can further comprise reducing or depleting CD14+ cells from the cell population for preparing and enriching antigen activated T cells. In some embodiments, the method can further comprise reducing or depleting CD25+ cells from the cell population for preparing and enriching antigen activated T cells. In some embodiments, the method can further comprise reducing or depleting one or more of CD19+, CD14+, CD25+, CD56+, or CD1 lb+ cells from the cell population for activating or enriching antigen activated T cells. In some embodiments, depleting comprises depleting CD 14+ cells directly from a washed peripheral blood mononuclear cell (PBMC) sample from a human subject. In some embodiments, depleting comprises depleting CD25+ cells directly from a washed peripheral blood mononuclear cell (PBMC) sample from a human subject. In some embodiments, depleting comprises depleting CD 14+ cells and CD25+ cells directly from a washed peripheral blood mononuclear cell (PBMC) sample from a human subject. In some embodiments, depleting comprises depleting CD 14+ cells directly from a cryopreserved peripheral blood mononuclear cell (PBMC) sample from a human subject. In some embodiments, depleting comprises depleting CD25+ cells directly from a cryopreserved peripheral blood mononuclear cell (PBMC) sample from a human subject. In some embodiments, depleting comprises depleting CD14+ cells and CD25+ cells directly from a cryopreserved peripheral blood mononuclear cell (PBMC) sample from a human subject. In some embodiments, the depleted population of cells is incubated for a firsttime period in the presence of FLT3L and a polypeptide comprising atleasttwo different epitope sequences. In some embodiments, the depleted population of cells is incubated for a first time period in the presence of FLT3L and a polynucleic acid encoding a polypeptide comprising at least two different epitope sequences.

[0260] In some embodiments, depleting one or more cells selected from the group consisting of CD14+ cells, CD25+ cells, CD56+ cells, CD19+ cells, and CDl lb+ cells from the population of immune cells can comprise contacting the population of immune cells with a CD 14 binding agent, a CD25 binding agent, a CD56 binding agent, a CD 19 binding agent, and / or a CD 1 lb binding agent. In some cases, the CD 14 binding agent can comprise, for example, components of the gram-positive cell wall and / or soluble lipopolysaccharides. In some cases, the CD25 binding agent can comprise, for example, functionally inactive TGF- 1. In some cases, the CD56 binding agent can comprise, for example, FGFR1 .

[0261] In some embodiments, the percentage of CD3+ cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least 40% of the total cell population. In some embodiments, the percentage of CD3+ cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least 50% of the total cell population. In some embodiments, the percentage of CD3+ cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least 60% of the total cell population. In some embodiments, the percentage of CD3+ cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least 1% of the total cell population, at least 2% of the total cell population, at least 5% of the total cell population, at least 10% of the total cell population, at least 15% of the total cell population, at least 20% of the total cell population, at least 25% of the total cell population, at least 30% of the total cell population, at least 40% of the total cell population, at least 50% of the total cell population, at least 60% of the total cell population, at least 70% of the total cell population, at least 80% of the total cell population, at least 90% of the total cell population, or at least more than 90% of the total cell population. In some embodiments, the percentage of CD3+ cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least 1% of the total cell population, at least 2% of the total cell population, at least 3% of the total cell population, at least 4% of the total cell population, at least 5% of the total cell population, at least 6% of the total cell population, at least 7% of the total cell population, at least 8% of the total cell population, at least 9% of the total cell population, at least 10% of the total cell population, at least 11% of the total cell population, at least 12% of the total cell population, at least 13% of the total cell population, at least 14% of the total cell population, at least 15% of the total cell population, at least 16% of the total cell population, at least 17% of the total cell population, at least 18% of the total cell population, at least 19% of the total cell population, at least 20% of the total cell population, at least 21% of the total cell population, at least 22% of the total cell population, at least 23% of the total cell population, at least 24% of the total cell population, at least 25% of the total cell population, at least 26% of the total cell population, at least 27% of the total cell population, at least 28% of the total cell population, at least 29% of the total cell population, at least 30% of the total cell population, at least 31% of the total cell population, at least 32% of the total cell population, at least 33% of the total cell population, at least 34% of the total cell population, at least 35% of the total cell population, at least 36% of the total cell population, at least 37% of the total cell population, at least 38% of the total cell population, at least 39% of the total cell population, at least 40% of the total cell population, at least 41% of the total cell population, at least 42% of the total cell population, at least 43% of the total cell population, at least 44% of the total cell population, at least 45% of the total cell population, at least 46% of the total cell population, at least 47% of the total cell population, at least 48% of the total cell population, at least 49% of the total cell population, at least50% of the total cell population, at least 51% of the total cell population, at least 52% of the total cell population, at least 53% of the total cell population, at least 54% of the total cell population, at least 55% of the total cell population, at least 56% of the total cell population, at least 57% of the total cell population, at least 58% of the total cell population, at least 59% of the total cell population, at least 60% of the total cell population, at least 61% of the total cell population, at least 62% of the total cell population, at least 63% of the total cell population, at least 64% of the total cell population, at least 65% of the total cell population, at least 66% of the total cell population, at least 67% of the total cell population, at least 68% of the total cell population, at least 69% of the total cell population, at least 70% of the total cell population, at least 71% of the total cell population, at least 72% of the total cell population, at least 73% of the total cell population, at least 74% of the total cell population, at least 75% of the total cell population, at least 76% of the total cell population, at least 77% of the total cell population, at least 78% of the total cell population, at least 79% of the total cell population, at least 80% of the total cell population, at least 81% of the total cell population, at least 82% of the total cell population, at least 83% of the total cell population, at least 84% of the total cell population, at least 85% of the total cell population, at least 86% of the total cell population, at least 87% of the total cell population, at least 88% of the total cell population, at least 89% of the total cell population, at least 90% of the total cell population, at least 91% of the total cell population, at least 92% of the total cell population, at least 93% of the total cell population, at least 94% of the total cell population, at least 95% of the total cell population, at least 96% of the total cell population, at least 97% of the total cell population, at least 98% of the total cell population, at least 99% of the total cell population, or 100% of the total cell population.

[0262] In some embodiments, the percentage of CD107a+ cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least 10% of the total cell population. In some embodiments, the percentage of CD 107a+ cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least 1% of the total cell population, at least 2% of the total cell population, at least 3% of the total cell population, at least 4% of the total cell population at least 5% of the total cell population, at least 6% of the total cell population, at least 7% of the total cell population, at least 8% of the total cell population, at least 9% of the total cell population, at least 10% of the total cell population, at least 15% of the total cell population, at least 20% of the total cell population, at least 25% of the total cell population, at least 30% of the total cell population, at least 35% of the total cell population, at least 40% of the total cell population, at least 45% of the total cell population, at least 50% of the total cell population, at least 60% of the total cell population, at least 70% of the total cell population, at least 80% of the total cell population, at least 90% of the total cell population, or at least more than 90% of the total cell population. In some embodiments, the percentageof CD107a+ cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least 1% of the total cell population, at least 2% of the total cell population, atleast 3% of the total cell population, at least 4% of the total cell population, at least 5% of the total cell population, at least 6% of the total cell population, at least 7% of the total cell population, at least 8% of the total cell population, atleast 9% of the total cell population, at least 10% of the total cell population, at least 11% of the total cell population, at least 12% of the total cell population, atleast 13% of the total cell population, atleast 14% of the total cell population, at least 15% of the total cell population, atleast 16% of the total cell population, at least 17% of the total cell population, at least 18% of the total cell population, at least 19% of the total cell population, at least 20% of the total cell population, at least 21% of the total cell population, at least 22% of the total cell population, at least 23% of the total cell population, at least 24% of the total cell population, at least 25% of the total cell population, at least 26% of the total cell population, at least 27% of the total cell population, at least 28% of the total cell population, at least 29% of the total cell population, atleast 30% of the total cell population, at least 31% of the total cell population, atleast 32% of the total cell population, atleast 33% of the total cell population, at least 34% of the total cell population, atleast 35% of the total cell population, at least 36% of the total cell population, at least 37% of the total cell population, at least 38% of the total cell population, atleast 39% of the total cell population, at least 40% of the total cell population, at least 41% of the total cell population, at least 42% of the total cell population, at least 43% of the total cell population, at least 44% of the total cell population, at least 45% of the total cell population, at least 46% of the total cell population, at least 47% of the total cell population, at least 48% of the total cell population, at least 49% of the total cell population, atleast 50% of the total cell population, at least 51% of the total cell population, at least 52% of the total cell population, atleast 53% of the total cell population, at least 54% of the total cell population, at least 55% of the total cell population, at least 56% of the total cell population, at least 57% of the total cell population, atleast 58% of the total cell population, at least 59% of the total cell population, at least 60% of the total cell population, at least 61 % of the total cell population, at least 62% of the total cell population, at least 63% of the total cell population, at least 64% of the total cell population, at least 65% of the total cell population, at least 66% of the total cell population, at least 67% of the total cell population, at least 68% of the total cell population, at least 69% of the total cell population, at least 70% of the total cell population, at least 71% of the total cell population, at least 72% of the total cell population, at least 73% of the total cell population, at least 74% of the total cell population, at least 75% of the total cell population, at least 76% of the total cell population, at least 77% of the total cell population, at least 78% of the total cell population, at least 79% of the total cell population, at least 80% of the total cell population, atleast 81% of the total cell population, at least 82% of the total cell population, atleast 83% of the total cellpopulation, at least 84% of the total cell population, at least 85% of the total cell population, at least 86% of the total cell population, at least 87% of the total cell population, at least 88% of the total cell population, at least 89% of the total cell population, at least 90% of the total cell population, at least 91 % of the total cell population, at least 92% of the total cell population, at least 93% of the total cell population, at least 94% of the total cell population, at least 95% of the total cell population, at least 96% of the total cell population, at least 97% of the total cell population, at least 98% of the total cell population, at least 99% of the total cell population, or 100% of the total cell population.

[0263] In some embodiments, the percentage of TNFa+ cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least 5% of the total cell population. In some embodiments, the percentage of TNFa+ cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least 1% of the total cell population, at least 2% of the total cell population, at least 3% of the total cell population, at least 4% of the total cell population at least 5% of the total cell population, at least 6% of the total cell population, at least 7% of the total cell population, at least 8% of the total cell population, at least 9% of the total cell population, at least 10% of the total cell population, at least 15% of the total cell population, at least 20% of the total cell population, at least 25% of the total cell population, at least 30% of the total cell population, at least 35% of the total cell population, at least 40% of the total cell population, at least 45% of the total cell population, at least 50% of the total cell population, at least 60% of the total cell population, at least 70% of the total cell population, at least 80% of the total cell population, at least 90% of the total cell population, or more than 90% of the total cell population. In some embodiments, the percentage of TNFa+ cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least 1 % of the total cell population, at least 2% of the total cell population, at least 3 % of the total cell population, at least 4% of the total cell population, at least 5% of the total cell population, at least 6% of the total cell population, at least 7% of the total cell population, at least 8% of the total cell population, at least 9% of the total cell population, at least 10% of the total cell population, at least 11% of the total cell population, at least 12% of the total cell population, at least 13% of the total cell population, at least 14% of the total cell population, at least 15% of the total cell population, at least 16% of the total cell population, at least 17% of the total cell population, at least 18% of the total cell population, at least 19% of the total cell population, at least 20% of the total cell population, at least 21% of the total cell population, at least 22% of the total cell population, at least 23% of the total cell population, at least 24% of the total cell population, at least 25% of the total cell population, at least 26% of the total cell population, at least 27% of the total cell population, at least 28% of the total cell population, at least 29% of the total cell population, at least 30% of the total cell population, at least 31% of the total cell population, at least 32% of the total cell population, at least 33% of the total cellpopulation, at least 34% of the total cell population, atleast 35% of the total cell population, at least 36% of the total cell population, at least 37% of the total cell population, at least 38% of the total cell population, at least 39% of the total cell population, at least 40% of the total cell population, at least 41% of the total cell population, at least 42% of the total cell population, at least 43% of the total cell population, at least 44% of the total cell population, at least 45% of the total cell population, at least 46% of the total cell population, at least 47% of the total cell population, at least 48% of the total cell population, at least 49% of the total cell population, atleast 50% of the total cell population, at least 51% of the total cell population, at least 52% of the total cell population, atleast 53% of the total cell population, at least 54% of the total cell population, at least 55% of the total cell population, at least 56% of the total cell population, at least 57% of the total cell population, atleast 58% of the total cell population, atleast 59% of the total cell population, atleast 60% of the total cell population, atleast 61% of the total cell population, at least 62% of the total cell population, at least 63% of the total cell population, at least 64% of the total cell population, at least 65% of the total cell population, at least 66% of the total cell population, at least 67% of the total cell population, at least 68% of the total cell population, at least 69% of the total cell population, at least 70% of the total cell population, at least 71 % of the total cell population, at least 72% of the total cell population, at least 73% of the total cell population, at least 74% of the total cell population, at least 75% of the total cell population, at least 76% of the total cell population, at least 77% of the total cell population, at least 78% of the total cell population, at least 79% of the total cell population, at least 80% of the total cell population, at least 81% of the total cell population, at least 82% of the total cell population, atleast 83% of the total cell population, at least 84% of the total cell population, at least 85% of the total cell population, at least 86% of the total cell population, at least 87% of the total cell population, at least 88% of the total cell population, at least 89% of the total cell population, at least 90% of the total cell population, at least 91% of the total cell population, at least 92% of the total cell population, at least 93% of the total cell population, at least 94% of the total cell population, at least 95% of the total cell population, atleast 96% of the total cell population, at least 97% of the total cell population, at least 98% of the total cell population, at least 99% of the total cell population, or 100% of the total cell population.

[0264] In some embodiments, the percentage of IFNy+ cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least 15% of the total cell population. In some embodiments, the percentage of IFNy+ cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least 1% of the total cell population, at least 2% of the total cell population, at least 3% of the total cell population, at least 4% of the total cell population, at least 5% of the total cell population, at least 6% of the total cell population, at least 7% of the total cell population, at least 8% of the total cell population, at least 9% of the total cell population, at least 10%of the total cell population, at least 15% of the total cell population, at least 20% of the total cell population, at least 25% of the total cell population, at least 30% of the total cell population, at least 35% of the total cell population, at least 40% of the total cell population, at least 45% of the total cell population, at least 50% of the total cell population, at least 60% of the total cell population, at least 70% of the total cell population, at least 80% of the total cell population, at least 90% of the total cell population, or atleastmore than 90% of the total cell population. In some embodiments, the percentage of IFNy+ cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least 1 % of the total cell population, at least 2% of the total cell population, at least 3 % of the total cell population, at least 4% of the total cell population, at least 5% of the total cell population, at least 6% of the total cell population, at least 7% of the total cell population, at least 8% of the total cell population, at least 9% of the total cell population, at least 10% of the total cell population, at least 11% of the total cell population, at least 12% of the total cell population, at least 13% of the total cell population, at least 14% of the total cell population, at least 15% of the total cell population, at least 16% of the total cell population, at least 17% of the total cell population, at least 18% of the total cell population, at least 19% of the total cell population, at least 20% of the total cell population, at least 21 % of the total cell population, at least 22% of the total cell population, at least 23% of the total cell population, at least 24% of the total cell population, at least 25% of the total cell population, at least 26% of the total cell population, at least 27% of the total cell population, at least 28% of the total cell population, at least 29% of the total cell population, at least 30% of the total cell population, at least 31% of the total cell population, at least 32% of the total cell population, at least 33% of the total cell population, at least 34% of the total cell population, atleast 35% of the total cell population, at least 36% of the total cell population, at least 37% of the total cell population, at least 38% of the total cell population, atleast 39% of the total cell population, at least 40% of the total cell population, at least 41% of the total cell population, at least 42% of the total cell population, at least 43% of the total cell population, at least 44% of the total cell population, at least 45% of the total cell population, atleast 46% of the total cell population, at least 47% of the total cell population, at least 48% of the total cell population, at least 49% of the total cell population, atleast 50% of the total cell population, at least 51% of the total cell population, atleast 52% of the total cell population, atleast 53% of the total cell population, at least 54% of the total cell population, at least 55% of the total cell population, at least 56% of the total cell population, atleast 57% of the total cell population, at least 58% of the total cell population, at least 59% of the total cell population, at least 60% of the total cell population, at least 61% of the total cell population, at least 62% of the total cell population, at least 63% of the total cell population, at least 64% of the total cell population, at least 65% of the total cell population, at least 66% of the total cell population, at least 67% of the total cell population, at least 68% of the total cellpopulation, at least 69% of the total cell population, at least 70% of the total cell population, at least 71% of the total cell population, at least 72% of the total cell population, at least 73% of the total cell population, at least 74% of the total cell population, at least 75% of the total cell population, at least 76% of the total cell population, at least 77% of the total cell population, at least 78% of the total cell population, at least 79% of the total cell population, at least 80% of the total cell population, at least 81% of the total cell population, at least 82% of the total cell population, at least 83% of the total cell population, at least 84% of the total cell population, at least 85% of the total cell population, at least 86% of the total cell population, at least 87% of the total cell population, at least 88% of the total cell population, at least 89% of the total cell population, at least 90% of the total cell population, at least 91 % of the total cell population, at least 92% of the total cell population, at least 93% of the total cell population, at least 94% of the total cell population, at least 95% of the total cell population, at least 96% of the total cell population, at least 97% of the total cell population, at least 98% of the total cell population, at least 99% of the total cell population, or 100% of the total cell population.

[0265] In some embodiments, the percentage of TNFa+ and IFNy+ cells in the expanded population of cells comprising tumor antigen-specific T cells together can be at least 2% of the tumor antigenspecific T cell population. In some embodiments, the percentage of TNFa+ and IFNy+ cells in the expanded population of cells comprising tumor antigen-specific T cells can together be at least 1% of the total cell population, at least 2% of the total cell population, at least 3% of the total cell population, at least 4% of the total cell population at least 5% of the total cell population, at least 6% of the total cell population, at least 7% of the total cell population, at least 8% of the total cell population, at least 9% of the total cell population, at least 10% of the total cell population, at least 15% of the total cell population, at least 20% of the total cell population, at least 25% of the total cell population, at least 30% of the total cell population, at least 35% of the total cell population, at least 40% of the total cell population, at least 45% of the total cell population, at least 50% of the total cell population, at least 60% of the total cell population, at least 70% of the total cell population, at least 80% of the total cell population, at least 90% of the total cell population, or at least more than 90% of the total cell population. In some embodiments, the percentage of TNFa+ cells and IFNy+ cells together in the expanded population of cells comprising tumor antigen-specific T cells can be at least 1% of the total cell population, at least 2% of the total cell population, at least 3% of the total cell population, at least 4% of the total cell population, at least 5% of the total cell population, at least 6% of the total cell population, at least 7% of the total cell population, at least 8% of the total cell population, at least 9% of the total cell population, at least 10% of the total cell population, at least 11% of the total cell population, at least 12% of the total cell population, at least 13% of the total cell population, at least 14% of the total cell population, at least 15% of the total cell population, at least 16% of the total cellpopulation, at least 17% of the total cell population, at least 18% of the total cell population, at least 19% of the total cell population, at least 20% of the total cell population, at least 21 % of the total cell population, at least 22% of the total cell population, at least 23% of the total cell population, at least 24% of the total cell population, at least 25% of the total cell population, at least 26% of the total cell population, at least 27% of the total cell population, at least 28% of the total cell population, at least 29% of the total cell population, at least 30% of the total cell population, at least 31 % of the total cell population, at least 32% of the total cell population, at least 33% of the total cell population, at least 34% of the total cell population, at least 35% of the total cell population, at least 36% of the total cell population, at least 37% of the total cell population, at least 38% of the total cell population, at least 39% of the total cell population, at least 40% of the total cell population, at least 41 % of the total cell population, at least 42% of the total cell population, at least 43% of the total cell population, at least 44% of the total cell population, at least 45% of the total cell population, at least 46% of the total cell population, at least 47% of the total cell population, at least 48% of the total cell population, at least 49% of the total cell population, at least 50% of the total cell population, at least 51% of the total cell population, at least 52% of the total cell population, at least 53% of the total cell population, at least 54% of the total cell population, at least 55% of the total cell population, at least 56% of the total cell population, at least 57% of the total cell population, at least 58% of the total cell population, at least 59% of the total cell population, at least 60% of the total cell population, at least 61% of the total cell population, at least 62% of the total cell population, at least 63% of the total cell population, at least 64% of the total cell population, at least 65% of the total cell population, at least 66% of the total cell population, at least 67% of the total cell population, at least 68% of the total cell population, at least 69% of the total cell population, at least 70% of the total cell population, at least 71 % of the total cell population, at least 72% of the total cell population, at least 73% of the total cell population, at least 74% of the total cell population, at least 75% of the total cell population, at least 76% of the total cell population, at least 77% of the total cell population, at least 78% of the total cell population, at least 79% of the total cell population, at least 80% of the total cell population, at least 81% of the total cell population, at least 82% of the total cell population, at least 83% of the total cell population, at least 84% of the total cell population, at least 85% of the total cell population, at least 86% of the total cell population, at least 87% of the total cell population, at least 88% of the total cell population, at least 89% of the total cell population, at least 90% of the total cell population, at least 91 % of the total cell population, at least 92% of the total cell population, at least 93% of the total cell population, at least 94% of the total cell population, at least 95% of the total cell population, at least 96% of the total cell population, at least 97% of the total cell population, at least 98% of the total cell population, at least 99% of the total cell population, or 100% of the total cell population.

[0266] In some embodiments, the percentage of TNFa+ and CD107a+ cells together in the expanded population of cells comprisingtumor antigen-specific T cells together can be at least 0.5% of the tumor antigen-specific T cell population. In some embodiments, the percentage of TNFa+and CD107a+ceDs together in the expanded population of cells comprising tumor antigen-specific T cells can together be at least 0.1% of the total cell population, at least 0.2% of the total cell population, at least 0.3% of the total cell population, at least 0.4% of the total cell population, at least 0.5% of the total cell population, at least 0.6% of the total cell population, at least 0.7% of the total cell population, at least 0.8% of the total cell population, at least 0.9% of the total cell population, at least 1% of the total cell population, at least 2% of the total cell population, at least 3% of the total cell population, at least 4% of the total cell population, at least 5% of the total cell population, at least 6% of the total cell population, at least 7% of the total cell population, at least 8% of the total cell population, at least 9% of the total cell population, at least 10% of the total cell population, at least 15% of the total cell population, at least 20% of the total cell population, at least 25% of the total cell population, at least 30% of the total cell population, at least 35% of the total cell population, at least 40% of the total cell population, at least 45% of the total cell population, at least 50% of the total cell population, at least 60% of the total cell population, at least 70% of the total cell population, at least 80% of the total cell population, at least 90% of the total cell population, or at least more than 90% of the total cell population. In some embodiments, the percentage of TNFa+ and CD107a+ cells together in the expanded population of cells comprising tumor antigen-specific T cells can be at least 1% of the total cell population, at least 2% of the total cell population, at least 3% of the total cell population, at least 4% of the total cell population, at least 5% of the total cell population, at least 6% of the total cell population, at least 7% of the total cell population, at least 8% of the total cell population, at least 9% of the total cell population, at least 10% of the total cell population, at least 11% of the total cell population, at least 12% of the total cell population, at least 13% of the total cell population, at least 14% of the total cell population, at least 15% of the total cell population, at least 16% of the total cell population, at least 17% of the total cell population, at least 18% of the total cell population, at least 19% of the total cell population, at least 20% of the total cell population, at least 21% of the total cell population, at least 22% of the total cell population, at least 23% of the total cell population, at least 24% of the total cell population, at least 25% of the total cell population, at least 26% of the total cell population, at least 27% of the total cell population, at least 28% of the total cell population, at least 29% of the total cell population, at least 30% of the total cell population, at least 31% of the total cell population, at least 32% of the total cell population, at least 33% of the total cell population, at least 34% of the total cell population, at least 35% of the total cell population, at least 36% of the total cell population, at least 37% of the total cell population, at least 38% of the total cell population, at least 39% of the total cellpopulation, at least 40% of the total cell population, at least 41% of the total cell population, at least 42% of the total cell population, at least 43% of the total cell population, at least 44% of the total cell population, at least 45% of the total cell population, at least 46% of the total cell population, at least 47% of the total cell population, at least 48% of the total cell population, at least 49% of the total cell population, at least 50% of the total cell population, at least 51% of the total cell population, at least 52% of the total cell population, at least 53% of the total cell population, at least 54% of the total cell population, at least 55% of the total cell population, at least 56% of the total cell population, at least 57% of the total cell population, at least 58% of the total cell population, at least 59% of the total cell population, at least 60% of the total cell population, at least 61% of the total cell population, at least 62% of the total cell population, at least 63% of the total cell population, at least 64% of the total cell population, at least 65% of the total cell population, at least 66% of the total cell population, at least 67% of the total cell population, at least 68% of the total cell population, at least 69% of the total cell population, at least 70% of the total cell population, at least 71% of the total cell population, at least 72% of the total cell population, at least 73% of the total cell population, at least 74% of the total cell population, at least 75% of the total cell population, at least 76% of the total cell population, at least 77% of the total cell population, at least 78% of the total cell population, at least 79% of the total cell population, at least 80% of the total cell population, at least 81% of the total cell population, at least 82% of the total cell population, at least 83% of the total cell population, at least 84% of the total cell population, at least 85% of the total cell population, at least 86% of the total cell population, at least 87% of the total cell population, at least 88% of the total cell population, at least 89% of the total cell population, at least 90% of the total cell population, at least 91% of the total cell population, at least 92% of the total cell population, at least 93% of the total cell population, at least 94% of the total cell population, at least 95% of the total cell population, at least 96% of the total cell population, at least 97% of the total cell population, at least 98% of the total cell population, at least 99% of the total cell population, or 100% of the total cell population.

[0267] In some embodiments, the percentage of IFNy+ and CD107a+ cells together in the expanded population of cells comprising tumor antigen-specific T cells together can be at least 5% of the tumor antigen-specific T cell population. In some embodiments, the percentage of IFNy+ and CD107a+ cells together in the expanded population of cells comprising tumor antigen-specific T cells can together be at least 1% of the total cell population, at least 2% of the total cell population, at least 3% of the total cell population, at least 4% of the total cell population, at least 5% of the total cell population, at least 6% of the total cell population, at least 7% of the total cell population, at least 8% of the total cell population, at least 9% of the total cell population, at least 10% of the total cell population, at least 15% of the total cell population, at least 20% of the total cell population, at least 25% of the total cellpopulation, at least 30% of the total cell population, at least 35% of the total cell population, at least 40% of the total cell population, at least 45% of the total cell population, at least 50% of the total cell population, at least 60% of the total cell population, at least 70% of the total cell population, at least 80% of the total cell population, at least 90% of the total cell population, or at least more than 90% of the total cell population. In some embodiments, the percentage of IFNy+ cells and CD107a+ cells together in the expanded population of cells comprising tumor antigen-specific T cells can be at least 1% of the total cell population, at least 2% of the total cell population, at least 3% of the total cell population, at least 4% of the total cell population, at least 5% of the total cell population, at least 6% of the total cell population, at least 7% of the total cell population, at least 8% of the total cell population, at least 9% of the total cell population, at least 10% of the total cell population, at least 11% of the total cell population, at least 12% of the total cell population, at least 13% of the total cell population, at least 14% of the total cell population, at least 15% of the total cell population, at least 16% of the total cell population, at least 17% of the total cell population, at least 18% of the total cell population, at least 19% of the total cell population, at least 20% of the total cell population, at least 21% of the total cell population, at least 22% of the total cell population, at least 23% of the total cell population, at least 24% of the total cell population, at least 25% of the total cell population, at least 26% of the total cell population, at least 27% of the total cell population, at least 28% of the total cell population, at least 29% of the total cell population, at least 30% of the total cell population, at least 31% of the total cell population, atleast 32% of the total cell population, atleast 33% of the total cell population, at least 34% of the total cell population, atleast 35% of the total cell population, at least 36% of the total cell population, at least 37% of the total cell population, at least 38% of the total cell population, atleast 39% of the total cell population, at least 40% of the total cell population, at least 41% of the total cell population, at least 42% of the total cell population, at least 43% of the total cell population, at least 44% of the total cell population, at least 45% of the total cell population, at least 46% of the total cell population, at least 47% of the total cell population, at least 48% of the total cell population, at least 49% of the total cell population, atleast 50% of the total cell population, at least 51% of the total cell population, atleast 52% of the total cell population, atleast 53% of the total cell population, at least 54% of the total cell population, at least 55% of the total cell population, at least 56% of the total cell population, at least 57% of the total cell population, atleast 58% of the total cell population, at least 59% of the total cell population, at least 60% of the total cell population, at least 61 % of the total cell population, at least 62% of the total cell population, at least 63% of the total cell population, at least 64% of the total cell population, at least 65% of the total cell population, at least 66% of the total cell population, at least 67% of the total cell population, at least 68% of the total cell population, at least 69% of the total cell population, at least 70% of the total cell population, atleast71% of the total cell population, at least 72% of the total cell population, at least 73% of the total cell population, at least 74% of the total cell population, at least 75% of the total cell population, at least 76% of the total cell population, at least 77% of the total cell population, at least 78% of the total cell population, at least 79% of the total cell population, at least 80% of the total cell population, at least 81% of the total cell population, at least 82% of the total cell population, at least 83% of the total cell population, at least 84% of the total cell population, at least 85% of the total cell population, at least 86% of the total cell population, at least 87% of the total cell population, at least 88% of the total cell population, at least 89% of the total cell population, at least 90% of the total cell population, at least 91% of the total cell population, at least 92% of the total cell population, at least 93% of the total cell population, at least 94% of the total cell population, at least 95% of the total cell population, at least 96% of the total cell population, at least 97% of the total cell population, at least 98% of the total cell population, at least 99% of the total cell population, or 100% of the total cell population.

[0268] In some embodiments, the percentage of TNFa+ and IFNy+ cells together in the expanded population of cells comprisingtumor antigen-specific T cells together can be at least 0. 1% of the tumor antigen-specific T cell population. In some embodiments, the percentage of TNFa+ and IFNy+ cells together in the expanded population of cells comprising tumor antigen-specific T cells can together be at least 0.05% of the total cell population, at least 0.1% of the total cell population, at least 0.2% of the total cell population, at least 0.3% of the total cell population, at least 0.4% of the total cell population, at least 0.5% of the total cell population, at least 0.6% of the total cell population, at least 0.7% of the total cell population, at least 0.8% of the total cell population, at least 0.9% of the total cell population, at least 1% of the total cell population, at least 2% of the total cell population, at least 3% of the total cell population, at least 4% of the total cell population, at least 5% of the total cell population, at least 6% of the total cell population, at least 7% of the total cell population, at least 8% of the total cell population, at least 9% of the total cell population, at least 10% of the total cell population, at least 15% of the total cell population, at least 20% of the total cell population, at least 25% of the total cell population, at least 30% of the total cell population, at least 35% of the total cell population, at least 40% of the total cell population, at least 45% of the total cell population, at least 50% of the total cell population, at least 60% of the total cell population, at least 70% of the total cell population, at least 80% of the total cell population, at least 90% of the total cell population, or at least more than 90% of the total cell population. In some embodiments, the percentage of TNFa+ cells and IFNy+ cells together in the expanded population of cells comprising tumor antigen-specific T cells can be at least 1% of the total cell population, at least 2% of the total cell population, at least 3% of the total cell population, at least 4% of the total cell population, at least 5% of the total cell population, at least 6% of the total cell population, at least 7% of the total cell population, at least 8% of the total cellpopulation, at least 9% of the total cell population, at least 10% of the total cell population, at least 11% of the total cell population, at least 12% of the total cell population, at least 13% of the total cell population, at least 14% of the total cell population, at least 15% of the total cell population, at least 16% of the total cell population, at least 17% of the total cell population, at least 18% of the total cell population, at least 19% of the total cell population, at least 20% of the total cell population, at least 21% of the total cell population, at least 22% of the total cell population, at least 23% of the total cell population, at least 24% of the total cell population, at least 25% of the total cell population, at least 26% of the total cell population, at least 27% of the total cell population, at least 28% of the total cell population, at least 29% of the total cell population, at least 30% of the total cell population, at least 31% of the total cell population, at least 32% of the total cell population, at least 33% of the total cell population, at least 34% of the total cell population, atleast 35% of the total cell population, at least 36% of the total cell population, at least 37% of the total cell population, at least 38% of the total cell population, at least 39% of the total cell population, at least 40% of the total cell population, at least 41% of the total cell population, at least 42% of the total cell population, at least 43% of the total cell population, at least 44% of the total cell population, at least 45% of the total cell population, at least 46% of the total cell population, at least 47% of the total cell population, at least 48% of the total cell population, at least 49% of the total cell population, atleast 50% of the total cell population, at least 51% of the total cell population, at least 52% of the total cell population, atleast 53% of the total cell population, at least 54% of the total cell population, atleast 55% of the total cell population, atleast 56% of the total cell population, at least 57% of the total cell population, atleast 58% of the total cell population, at least 59% of the total cell population, at least 60% of the total cell population, at least 61% of the total cell population, at least 62% of the total cell population, at least 63% of the total cell population, at least 64% of the total cell population, at least 65% of the total cell population, at least 66% of the total cell population, at least 67% of the total cell population, at least 68% of the total cell population, at least 69% of the total cell population, at least 70% of the total cell population, at least 71% of the total cell population, at least 72% of the total cell population, at least 73% of the total cell population, at least 74% of the total cell population, at least 75% of the total cell population, at least 76% of the total cell population, at least 77% of the total cell population, at least 78% of the total cell population, at least 79% of the total cell population, at least 80% of the total cell population, at least 81% of the total cell population, at least 82% of the total cell population, atleast 83% of the total cell population, atleast 84% of the total cell population, atleast 85% of the total cell population, atleast 86% of the total cell population, at least 87% of the total cell population, at least 88% of the total cell population, at least 89% of the total cell population, at least 90% of the total cell population, atleast 91% of the total cell population, at least 92% of the total cell population, at least 93% of the total cellpopulation, at least 94% of the total cell population, at least 95% of the total cell population, at least 96% of the total cell population, at least 97% of the total cell population, at least 98% of the total cell population, at least 99% of the total cell population, or 100% of the total cell population.

[0269] In some embodiments, the percentage of CD4+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are naive T cells can be at most 15%. In some embodiments, the percentage of CD4+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are naive T cells can be at most less than 0.5%, at most 0.5%, at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most 9%, at most 10%, at most 11%, at most 12%, at most 13%, at most 14%, or at most 15%. In some embodiments, the naive T cells can be CD62L+ cells and / or CD45RA+ cells. In some embodiments, the percentage of CD4+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are naive T cells can be at most 0.1%, at most 0.2%, at most 0.3%, at most 0.4%, at most 0.5%, atmost O.6%, atmost O.7%, atmost 0.8%, atmost O.9%, atmost 1%, atmost2%, atmost 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most 9%, 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%, atmost20%, atmost21%, atmost22%, at most 23%, atmost24%, atmost25%, at most 26%, atmost27%, atmost28%, atmost29%, at most 30%, at most 31%, atmost 32%, atmost 33%, at most 34%, at most 35%, at most 36%, at most 37%, at most 38%, at most 39%, at most 40%, at most 41%, atmost 42%, atmost43%, atmost 44%, at most 45%, atmost46%, atmost47%, at most 48%, atmost49%, atmost 50%, atmost 51%, atmost 52%, atmost 53%, atmost 54%, at most 55%, at most 56%, at most 57%, at most 58%, at most 59%, at most 60%, at most 61%, at most 62%, at most 63%, at most 64%, at most 65%, at most 66%, at most 67%, at most 68%, at most 69%, at most 70%, atmost 71%, atmost 72%, atmost 73%, at most 74%, atmost 75%, at most 76%, atmost 77%, at most 78%, at most 79%, at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most 85%, atmost 86%, atmost 87%, atmost 88%, at most 89%, atmost 90%, atmost 91%, atmost 92%, at most 93%, at most 94%, at most 95%, at most 96%, at most 97%, at most 98%, or at most 99%.

[0270] In some embodiments, the percentage of CD4+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector memory T cells can be at least 60%. In some embodiments, the percentage of CD4+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector memory T cells can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least more than 90%. In some embodiments, the effector memory T cells can be CD62L- cells and / or CD45RA- cells. In some embodiments, the percentage of CD4+ cells in the expanded population of cells comprising tumorantigen-specific T cells that are effector memory T cells can be at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, atleast 78%, atleast 79%, atleast 80%, atleast 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0271] In some embodiments, the percentage of CD4+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector T cells can be at most 5%. In some embodiments, the percentage of CD4+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector T cells can be at most less than 0.5%, atmost 0.5%, atmost 1%, at most 1.5%, at most 2%, at most 2.5%, at most 3%, at most 3.5%, atmost 4%, atmost4.5%, or at most 5%. In some embodiments, the percentage of CD4+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector T cells can be at most 0.1%, at most 0.2%, at most 0.3%, at most O.4%, atmost 0.5%, atmost O.6%, at most O.7%, atmost 0.8%, at most O.9%, at most 1%, at most 1.1%, atmost 1.2%, atmost 1.3%, at most 1.4%, at most 1.5%, at most 1.6%, at most 1.7%, at most 1.8%, at most 1.9%, at most 2%, at most 2.1%, at most 2.2%, at most 2.3%, at most 2.4%, at most 2.5%, at most 2.6%, at most 2.7%, at most 2.8%, at most 2.9%, at most 3%, at most 3.1%, at most 3.2%, atmost 3.3%, atmost 3.4%, at most 3.5%, atmost 3.6%, at most 3.7%, at most 3.8%, at most 3.9%, at most 4%, at most 4.1%, at most 4.2%, at most 4.3%, at most 4.4%, at most 4.5%, at most 4.6%, at most 4.7%, at most 4.8%, at most 4.9%, or at most 5%. In some embodiments, the effector T cells can be CD62L- cells and / or CD45RA+ cells.

[0272] In some embodiments, the percentage of CD4+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are central memory T cells can be at least 10%. In some embodiments, the percentage of CD4+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are central memory T cells can be at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least40%, atleast 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least more than 95%. In some embodiments, the percentage of CD4+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are central memory T cells can be at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, atleast 18%, atleast 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 least 31%, at least 32%, atleast 33%, atleast 34%, atleast 35%, atleast 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least47%, atleast48%, atleast49%, atleast 50%, atleast 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, atleast 78%, atleast 79%, atleast 80%, atleast 81%, atleast 82%, atleast 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In some embodiments, the central memory T cells can be CD62L+ cells and / or CD45RA- cells.

[0273] In some embodiments, the percentage of CD8+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are naive T cells can be at most 25%. In some embodiments, the percentage of CD8+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are naive T cells can be at most less than 0.5%, atmost 0.5%, atmost 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most 9%, at most 10%, at most 11%, at most 12%, at most 13%, at most 14%, at most 15%, most 16%, at most 17%, at most 18%, at most 19%, at most 20%, or at most 25%. In some embodiments, the percentage of CD8+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are naive T cells can be at most 0.1%, at most O.2%, atmost 0.3%, at most 0.4%, atmost 0.5%, atmost0.6%, at most 0.7%, at most 0.8%, at most 0.9%, at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most 9%, 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%, or at most 25%. In some embodiments, the naive T cells can be CD62L+ cells and / or CD45RA+ cells.

[0274] In some embodiments, the percentage of CD8+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector memory T cells can be at least 60%. In some embodiments, the percentage of CD8+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector memory T cells can be at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least more than 90%. In some embodiments, the percentage of CD8+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector memory T cells can be at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, atleast 78%, atleast 79%, atleast 80%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% In some embodiments, the effector memory T cells can be CD62L- cells and / or CD45RA- cells.

[0275] In some embodiments, the percentage of CD8+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector T cells can be at most 10%. In some embodiments, the percentage of CD8+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector T cells can be at most less than 0.5%, atmost 0.5%, at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most 8%, at most9%, or at most 10%. In some embodiments, the percentage of CD8+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector T cells canbe at most 0.1%, at most 0.2%, atmost 0.3%, atmost O.4%, at most 0.5%, at most 0.6%, atmost O.7%, atmost 0.8%, atmost0.9%, atmost 1%, atmost2%, atmost 3%, atmost 4%, atmost 5%, at most 6%, atmost 7%, atmost8%, at most 9%, or at most 10%. In some embodiments, the effector T cells can be CD62L- cells and / orCD45RA+ cells.

[0276] In some embodiments, the percentage of CD8+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are central memory T cells canbe at least 15%. In some embodiments, the percentage of CD8+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are central memory T cells can be at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, atleast 40%, atleast 45%, atleast 50%, at least 55%, at least 60%, at least 65%, atleast 70%, atleast 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least more than 95%. In some embodiments, the percentage of CD8+ cells in the expanded population of cells comprising tumor antigen-specific T cells that are central memory T cells can be atleast 15%, atleast 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 least 31%, atleast 32%, atleast 33%, atleast 34%, atleast 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, atleast 62%, atleast 63%, atleast 64%, atleast 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, atleast 77%, atleast 78%, atleast 79%, atleast 80%, atleast 81%, atleast 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, atleast 93%, atleast 94%, at least 95%, at least 96%, atleast 97%, atleast 98%, or at least 99%. In some embodiments, the central memory T cells canbe CD62L+ cells and / or CD45RA- cells.

[0277] In some embodiments, the expanded population of cells comprising tumor antigen-specific T cells can produce cytokines. In some embodiments, the expanded population of cells comprisingtumor antigen-specific T cells can produce cytokines and cause degranulation. In some embodiments, the degranulation can be caused upon recognition of target cells.

[0278] In some embodiments, the depletion can comprise depleting CD14+ cells and / or CD25+ cells and / or CD56+ cells and / or CD19+ cells and / or CD1 lb+ cells from a peripheral blood mononuclear cell (PBMC) sample. In some embodiments, the depletion can comprise depleting CD 14+ cells from a peripheral blood mononuclear cell (PBMC) sample. In some embodiments, the PBMC sample can be from a human subject. In some embodiments, the PBMC sample can be from an animal subject, for example a murine subject. In some embodiments, the PBMC sample could not have been subject to a step of monocyte maturation into mature dendritic cells (DCs).

[0279] In some embodiments, the depletion can comprise depleting CD 14+ cells and / or CD25+ cells and / or CD56+ cells and / or CD19+ cells and / or CDl lb+ cells from a peripheral blood mononuclear cell (PBMC) sample. In some embodiments, the depletion can comprise depleting CD1 lb+ cells from a peripheral blood mononuclear cell (PBMC) sample. In some embodiments, the PBMC sample can be from a human subject. In some embodiments, the PBMC sample can be from an animal subject, for example a murine subject. In some embodiments, the PBMC sample could not have been subject to a step of monocyte maturation into mature dendritic cells (DCs).

[0280] In some embodiments, the fraction of CD8+ tumor antigen-specific T cells of the total number of CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least two-fold higher than the fraction of CD8+ tumor antigen -specific T cells of the total number of CD8+ T cells in the biological sample. In some cases, the fraction of CD8+ tumor antigen-specific T cells of the total number of CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least about more than 2-fold higher, about 2.5-fold higher, about 3- fold higher, about 3.5-fold higher, about 4-fold higher, about 4.5-fold higher, about 5-fold higher, about 5.5-fold higher, about 6-fold higher, about 6.5 -fold higher, about 7-fold higher, about 7.5-fold higher, about 8-fold higher, about 8.5-fold higher, about 9-fold higher, about 9.5-fold higher, about 10-fold higher, about 15-fold higher, about 20-fold higher, or more than about 20-fold higher than the fraction of CD8+ tumor antigen-specific T cells of the total number of CD8+ T cells in the biological sample. In some embodiments, the fraction of CD8+tumor antigen-specific T cells of the total number of CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells can be atleast about 1-fold higher, about 2-fold higher, about 3-fold higher, about 4-fold higher, about 5-fold higher, about 6-fold higher, about 7-fold higher, about 8-fold higher, about 9-fold higher, about 10- fold higher, about 11-fold higher, about 12-fold higher, about 13-fold higher, about 14-fold higher,about 15-fold higher, about 16-fold higher, about 17-fold higher, about 18-fold higher, about 19-fold higher, about20-fold higher, about 21 -fold higher, about 22-fold higher, about 23 -fold higher, about 24-fold higher, about 25-fold higher, about 26-fold higher, about 27-fold higher, about 28-fold higher, about 29-fold higher, about 30-fold higher, about 31-fold higher, about 32-fold higher, about 33-fold higher, about 34-fold higher, about 35 -fold higher, about 36-fold higher, about 37-fold higher, about 38-fold higher, about 39-fold higher, about 40-fold higher, about 41 -fold higher, about 42-fold higher, about 43-fold higher, about 44-fold higher, about 45-fold higher, about 46-fold higher, about 47-fold higher, about 48-fold higher, about 49-fold higher, about 50-fold higher, about 51 -fold higher, about 52-fold higher, about 53-fold higher, about 54-fold higher, about 55-fold higher, about 56-fold higher, about 57-fold higher, about 58-fold higher, about 59-fold higher, about 60 -fold higher, about 61-fold higher, about 62-fold higher, about 63 -fold higher, about 64-fold higher, about 65-fold higher, about 66-fold higher, about 67-fold higher, about 68-fold higher, about 69-fold higher, about 70-fold higher, about 71 -fold higher, about 72-fold higher, about 73-fold higher, about 74-fold higher, about 75-fold higher, about 76-fold higher, about 77-fold higher, about 78-fold higher, about 79-fold higher, about 80-fold higher, about 81 -fold higher, about 82-fold higher, about 83-fold higher, about 84-fold higher, about 85-fold higher, about 86-fold higher, about 87-fold higher, about 88-fold higher, about 89-fold higher, about 90-fold higher, about 91 -fold higher, about 92-fold higher, about 93 -fold higher, about 94-fold higher, about 95-fold higher, about 96-fold higher, about 97-fold higher, about 98-fold higher, about 99-fold higher, or about 100-foldhigherthan the fraction of CD8+tumor antigen-specific T cells of the total number of CD8+ T cells in the biological sample.

[0281] In some embodiments, the fraction of CD4+ tumor antigen-specific T cells of the total number of CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least two-fold higher than the fraction of CD4+ tumor antigen-specific T cells of the total number of CD8+ T cells in the biological sample. In some cases, the fraction of CD4+ tumor antigen-specific T cells of the total number of CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least about more than 2-fold higher, about 2.5-fold higher, about 3- fold higher, about 3.5-fold higher, about 4-fold higher, about 4.5-fold higher, about 5-fold higher, about 5.5-fold higher, about 6-fold higher, about 6.5-fold higher, about 7-fold higher, about 7.5-fold higher, about 8-fold higher, about 8.5-fold higher, about 9-fold higher, about 9.5-fold higher, about 10-fold higher, about 15-fold higher, about 20-fold higher, or more than about 20-fold higher than the fraction of CD4+ tumor antigen-specific T cells of the total number of CD4+ T cells in the biological sample. In some embodiments, the fraction of CD4+ tumor antigen-specific T cells of the total number of CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells can be at least about 1-fold higher, about 2-fold higher, about 3 -fold higher, about 4-fold higher, about 5-foldhigher, about 6-fold higher, about ? -fold higher, about 8 -fold higher, about 9-fold higher, about 10- fold higher, about 11-fold higher, about 12-fold higher, about 13-fold higher, about 14-fold higher, about 15-fold higher, about 16-fold higher, about 17-fold higher, about 18-fold higher, about 19-fold higher, about20-fold higher, about 21 -fold higher, about22-fold higher, about 23 -fold higher, about 24-fold higher, about 25-fold higher, about 26-fold higher, about 27-fold higher, about 28-fold higher, about 29-fold higher, about 30-fold higher, about 31-fold higher, about 32-fold higher, about 33-fold higher, about 34-fold higher, about 35 -fold higher, about 36-fold higher, about 37-fold higher, about 38-fold higher, about 39-fold higher, about 40-fold higher, about 41 -fold higher, about 42-fold higher, about 43-fold higher, about 44-fold higher, about 45-fold higher, about 46 -fold higher, about 47-fold higher, about 48-fold higher, about 49-fold higher, about 50-fold higher, about 51 -fold higher, about 52-fold higher, about 53-fold higher, about 54-fold higher, about 55-fold higher, about 56-fold higher, about 57-fold higher, about 58-fold higher, about 59-fold higher, about 60-fold higher, about 61-fold higher, about 62-fold higher, about 63 -fold higher, about 64-fold higher, about 65-fold higher, about 66-fold higher, about 67-fold higher, about 68-fold higher, about 69-fold higher, about 70-fold higher, about 71 -fold higher, about 72-fold higher, about 73-fold higher, about 74-fold higher, about 75-fold higher, about 76-fold higher, about 77-fold higher, about 78-fold higher, about 79-fold higher, about 80-fold higher, about 81 -fold higher, about 82-fold higher, about 83 -fold higher, about 84-fold higher, about 85-fold higher, about 86-fold higher, about 87-fold higher, about 88-fold higher, about 89-fold higher, about 90-fold higher, about 91 -fold higher, about 92-fold higher, about 93 -fold higher, about 94-fold higher, about 95-fold higher, about 96-fold higher, about 97-fold higher, about 98-fold higher, about 99-fold higher, or about 1 OO-foldhigherthan the fraction of CD4+tumor antigen-specific T cells of the total number of CD4+ T cells in the biological sample.

[0282] In some embodiments, atleast 0.1% of the CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells can be CD8+ tumor antigen-specific T cells derived from naive CD8+ T cells. In some cases, atleast 0.05%, at least 0.1%, atleast 0.15%, at least 0.2%, atleast 0.3%, atleast 0.35%, atleast O.4%, at least O.45%, atleast 0.5%, at least 1%, atleast 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, atleast 65%, atleast 70%, atleast 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least more than 95% of the CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells can be CD8+ tumor antigen-specific T cells derived from naive CD8+ T cells.

[0283] In some embodiments, at least 0.1% of the CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells can be CD4+ tumor antigen-specific T cells derived fromnaive CD4+ T cells. In some cases, at least 0.05%, at least 0.1%, at least 0.15%, at least 0.2%, at least 0.3%, atleast O.35%, atleast O.4%, at least O.45%, atleast O.5%, at least 1%, atleast 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, atleast 65%, atleast 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least more than 95% of the CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells can be CD4+ tumor antigen-specific T cells derived from naive CD4+ T cells. In some embodiments, atleast 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, atleast 0.6%, at least O.7%, at least 0.8%, at least 0.9%, at least 1%, at least2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, atleast 12%, atleast 13%, atleast 14%, atleast 15%, at least 16%, atleast 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%, atleast27%, atleast28%, atleast29%, atleast 30%, at least 31%, atleast 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, atleast 42%, atleast43%, atleast 44%, atleast45%, at least46%, atleast47%, at least48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, atleast 72%, atleast 73%, atleast 74%, atleast 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, atleast 87%, atleast 88%, atleast 89%, atleast 90%, atleast 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, atleast 98%, or at least 99% of the CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells can be CD4+ tumor antigen-specific T cells derived from naive CD4+ T cells.

[0284] In some embodiments, expanding can comprise contacting the population of cells comprising stimulated T cells with a second population of mature APCs. In some embodiments, the second population of mature APCs can be incubated with FLT3L and / or can present the at least one tumor antigen epitope sequence. In some cases, the epitope can comprise an epitope from a MART-1 protein or fragment thereof. In some embodiments, the epitope can comprise an epitope from a GLI-3 protein or fragment thereof. In some embodiments, the expanding can comprise expanding the population of cells comprising stimulated T cells for a second time period, thereby forming an expanded population of T cells. In some cases, the second time period can comprise about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, about 24 hours, about2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or about more than 30 days.

[0285] In some embodiments, the second population of mature APCs can be incubated with FLT3L for at least 1 day prior to contacting the population of cells comprising stimulated T cells with the second population of mature APCs. In some cases, the second population of mature APCs can be incubated with FLT3L for at least about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or more than about 30 days prior to contacting the population of cells comprising stimulated T cells with the second population of mature APCs. In some embodiments, the subject can be incubated with FLT3L at least about 1 week, 2 weeks, 3 weeks, 4 weeks, or 5 weeks before contacting the population of cells comprising stimulated T cells with the second population of mature APCs.

[0286] In some embodiments, the biological sample can be a peripheral blood sample, a leukapheresis, sample, or an apheresis sample.

[0287] In some embodiments, the expanded population of cells comprising tumor antigen-specific T cells can be harvested. In some embodiments, the expanded population of cells comprising tumor antigen-specific T cells can be cryopreserved. In some embodiments, a pharmaceutical composition can be prepared containingthe expanded population of cells comprisingtumor antigen-specific T cells.

[0288] In some embodiments, the incubating can comprise incubating the depleted population of immune cells. In some embodiments, the depleted population of immune cells can comprise a first population of APCs and T cells for a first time period in the presence of FLT3L and an RNA encoding the polypeptide. In some cases, the polypeptide can comprise a MART-1 protein or fragment thereof. In some embodiments, the polypeptide can comprise a GLI3 protein or fragment thereof.

[0289] In some embodiments, the expanding comprises contacting the population of cells comprising stimulated T cells with a second population of mature APCs. In some embodiments, the second population of mature APCs have been incubated with FLT3L. In some embodiments, the second population of mature APCs have been incubated with FLT3L for at least 1 day prior to contacting the population of cells comprising stimulated T cells with the second population of mature APCs. In some embodiments, the second population of mature APCs have been incubated with FLT3L for 2 days. In some embodiments, the second population of mature APCs have been incubated with FLT3L for 3 days. In some embodiments, the second population of mature APCs have been incubated with FLT3L for 4 days. In some embodiments, the second population of mature APCs have been incubated with FLT3L for 5 days. In some embodiments, the second population of mature APCs present the peptideconsisting of the epitope. In some embodiments, the second population of mature APCs have been incubated with FLT3L and present the peptide consisting of the epitope sequence. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells for a second time period, thereby forming an expanded population of T cells. In some embodiments, depleting CD 14+ cells from the population of immune cells comprising a first population of APCs and T cells comprises contacting the population of immune cells comprising a first population of APCs and T cells with a CD14 binding agent. In some embodiments, depleting CD25+ cells from the population of immune cells comprising a first population of APCs and T cells comprises contacting the population of immune cells comprisingafirstpopulation of APCs and T cells with a CD25 binding agent. In some embodiments, depleting CD 14+ cells and / or CD25+ cells from the population of immune cells comprising a first population of APCs and T cells comprises contacting the population of immune cells comprising a first population of APCs and T cells with a CD 14 binding agent and / or a CD25 binding agent.

[0290] In some embodiments the stimulant for activating the cells comprises FL3TL. In some embodiments the agent promoting cell grow...

Claims

CLAIMSWhat is claimed is:

1. A method of producing a population of engineered immune cells, the method comprising:(a) depleting one or more cells selected from the group consisting of CD14+ cells, CD25+ cells, and CD56+ cells from a population of immune cells comprising antigen presenting cells (APCs) and T cells, thereby forming a depleted population of immune cells; and(b) inhibiting expression of an endogenous gene in the depleted population of immune cells, thereby producing the population of engineered immune cells.2 The method of claim 1, wherein the endogenous gene comprises one or more genes selected from the group consisting of PRDM1, TNFAIP3, REGNASE-1, SOCS1, PTPN2, CISH, and any combination thereof.3 The method of claim 1 or 2, wherein inhibiting the expression of the endogenous gene comprises silencing a gene locus or contacting to the cells or expressing in the cells an miRNA or siRNA that targets an RNA transcript encoding the endogenous gene.4 The method of claim 3, wherein silencing the gene locus comprises gene knockout using a site-specific nuclease, gene knockout using a site-specific nickase, or gene silencing using a site-specific transcriptional or epigenetic regulator.5 The method of claim 4, wherein silencing the gene locus comprises delivering (i) the sitespecific nuclease, the site-specific nickase, or the site-specific epigenetic regulator, or (ii) a nucleic acid encoding the site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator into the depleted population of immune cells via electroporation, lipid nanoparticles, liposomes, nucleofection, or viral vector.6 The method of any one of claims 2-5, wherein the endogenous gene comprises PRDM1 and / or TNFAIP3.7 The method of claim 6, wherein inhibiting the expression of endogenous PRDM1 comprises silencing a PRDM1 gene locus or contacting to the cells or expressing in the cells an miRNA or siRNA that targets an RNA transcript encoding PRDM1.8 The method of claim 6, wherein inhibiting the expression of endogenous TNFAIP3 comprises silencing a TNFAIP3 gene locus or contacting to the cells or expressing in the cells an miRNA or siRNA that targets an RNA transcript encoding TNFAIP3.9 The method of any one of claims 6-8, wherein inhibiting the expression of PRDM1 and TNFAIP3 comprises silencing PRDM1 and TNFAIP3 gene loci or contacting to the cells orexpressing in the cells miRNAs or siRNAs that target RNA transcripts encoding PRDM1 and TNFAIP3.

10. The method of any one of claims 7-9, wherein silencing the PRDM1 and / or TNFAIP3 gene locus comprises gene knockout using a site-specific nuclease, gene knockout using a sitespecific nickase, or gene silencing using a site-specific epigenetic regulator.

11. The method of claim 10, wherein silencing the PRDM1 and / or TNFAIP3 gene locus comprises delivering (i) the site-specific nuclease, the site-specific nickase, or the sitespecific epigenetic regulator, or (ii) a nucleic acid encoding the site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator into the depleted population of immune cells via electroporation, lipid nanoparticles, liposomes, nucleofection, or viral vector.

12. The method of any one of claims 2-5, wherein the endogenous gene comprises PRDM1 and / or REGNASE-1.

13. The method of claim 12, wherein inhibiting the expression of endogenous PRDM1 comprises silencing a PRDM1 gene locus or contacting to the cells or expressing in the cells an miRNA or siRNA that targets an RNA transcript encoding PRDM1.

14. The method of claim 12, wherein inhibiting the expression of endogenous REGNASE-1 comprises silencing a REGNASE-1 gene locus or contacting to the cells or expressing in the cells an miRNA or siRNA that targets an RNA transcript encoding REGNASE-1.

15. The method of any one of claims 12-14, wherein inhibiting the expression of PRDM1 and REGNASE-1 comprises silencing PRDM1 and REGNASE-1 gene loci or contacting to the cells or expressing in the cells miRNAs or siRNAs that target RNA transcripts encoding PRDM1 and REGNASE-1.

16. The method of any one of claims 12-15, wherein silencing the PRDM1 and / or REGNASE-1 gene locus comprises gene knockout using a site-specific nuclease, gene knockout using a site-specific nickase, or gene silencing using a site-specific epigenetic regulator.

17. The method of claim 16, wherein silencing the PRDM1 and / or REGNASE-1 gene locus comprises delivering (i) the site-specific nuclease, the site-specific nickase, or the sitespecific epigenetic regulator, or (ii) a nucleic acid encoding the site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator into the depleted population of immune cells via electroporation, lipid nanoparticles, liposomes, nucleofection, or viral vector.

18. The method of any one of claims 2-5, wherein the endogenous gene comprises TNFAIP3 and / or REGNASE-1.

19. The method of claim 18, wherein inhibiting the expression of endogenous TNFAIP3 comprises silencing a TNFAIP3 gene locus or contacting to the cells or expressing in the cells an miRNA or siRNA that targets an RNA transcript encoding TNFAIP3.

20. The method of claim 18, wherein inhibiting the expression of endogenous REGNASE-1 comprises silencing a REGNASE-1 gene locus or contacting to the cells or expressing in the cells an miRNA or siRNA that targets an RNA transcript encoding REGNASE-1.21 . The method of claims 18-20, wherein inhibitingthe expression of TNFAIP3 and REGNASE- 1 comprises silencing TNFAIP3 and REGNASE-1 gene loci or contacting to the cells or expressing in the cells miRNAs or siRNAs that target RNA transcripts encoding TNFAIP3 and REGNASE-1.

22. The method of any one of claims 18-21, wherein silencingthe TNFAIP3 and / or REGNASE-1 gene locus comprises gene knockout using a site-specific nuclease, gene knockout using a site-specific nickase, or gene silencing using a site-specific epigenetic regulator.

23. The method of claim 22, wherein silencing the TNFAIP3 and / or REGNASE-1 gene locus comprises delivering (i) the site-specific nuclease, the site-specific nickase, or the sitespecific epigenetic regulator, or (ii) a nucleic acid encoding the site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator into the depleted population of immune cells via electroporation, lipid nanoparticles, liposomes, nucleofection, or viral vector.

24. The method of any one of claims 1-23, wherein the depleted population of immune cells comprises a first population of APCs and T cells, and the method further comprises (c) incubating the first population of APCs and T cells for a first time period in the presence of (i) FMS-like tyrosine kinase 3 receptor ligand (FLT3L).

25. The method of claim 24, wherein incubating the first population of APCs and T cells for the first time period further comprises incubating in the presence of (i) FLT3L, and (ii) (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding the polypeptide, thereby forming a population of engineered immune cells comprising stimulated T cells.

26. The method of claim 25, wherein the polypeptide comprises a cancer protein or a fragment thereof comprising at least one tumor antigen epitope sequence expressed by cancer cells.

27. The method of any one of claims 24-26, wherein incubating in (c) is performed prior to or subsequent to inhibiting in (b).

28. The method of any one of claims 24-27, further comprising, subsequent to (b) and (c), expanding the stimulated T cells, thereby forming an expanded population of cellscomprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells that are specific to a complex comprising (1) the at least one tumor antigen epitope sequence from step (c)(ii), and, (2) an MHC protein expressed by the cancer cells, or APCs of the human subject of (c)(ii).

29. The method of any one of claims 1-28, wherein inhibiting in (b) comprises inhibiting expression of endogenous REGNASE-1 in the depleted population of immune cells.

30. The method of claim 29, wherein inhibiting the expression of endogenous REGNASE-1 comprises silencing REGNASE-1 gene locus or comprises expression of an exogenous miRNA or an exogenous siRNA that targets REGNASE-1 .

31. A method of producing a population of engineered immune cells, the method comprising:(a) inhibiting expression of an endogenous gene in a population of immune cells, thereby producing the population of engineered immune cells, wherein the population of immune cells comprises T cells; and(b) depleting one or more cells selected from the group consisting of CD 14+ cells, CD25+ cells, and CD56+ cells in the population of immune cells to obtain a depleted population of immune cells.

32. The method of claim 31, wherein the population of immune cells further comprises antigen presenting cells (APCs).

33. The method of claim 31 or 32, wherein the depleted population of immune cells comprises a first population of APCs and T cells, and the method further comprises (c) incubating the first population of APCs and T cells for a first time period in the presence of FMS-like tyrosine kinase 3 receptor ligand (FLT3L).

34. The method of claim 32, wherein incubatingthe first population of APCs and T cells for the first time period further comprises incubating in the presence of (i) FLT3L, and (ii) (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding the polypeptide, thereby forming a population of engineered immune cells comprising stimulated T cells.

35. The method of any one of claims 33-34, wherein (c) is performed subsequent to depleting in (b).

36. The method of any one of claims 33-35, further comprising, subsequent to (b) and (c), expanding the stimulated T cells, thereby forming an expanded population of cells comprising tumor antigen-specific T cells, wherein the tumor antigen-specific T cells comprise T cells that are specific to a complex comprising (1) the at least one tumor antigenepitope sequence from step (c)(ii), and, (2) an MHC protein expressed by the cancer cells, or APCs of the human subject of (c)(ii).

37. A method of producing a population of engineered immune cells, the method comprising:(a) inhibiting expression of an endogenous gene in a population of immune cells, wherein the population of immune cells comprises T cells; and(b) incubating the population of immune cells from (a) for a first time period in the presence of FMS-like tyrosine kinase 3 receptor ligand (FLT3L).

38. The method of claim 37, further comprising depleting one or more cells selected from the group consisting of CD14+ cells, CD25+ cells, and CD56+ cells in the population of immune cells after (a).

39. The method of claim 38, further comprising depleting one or more cells selected from the group consisting of CD14+ cells, CD25+ cells, and CD56+ cells in the population of immune cells after (a) and prior to (b).

40. The method of any one of claims 37-39, wherein the population of immune cells in (a) have been depleted of one or more cells selected from the group consisting of CD14+ cells, CD25+ cells, and CD56+ cells.

41. The method of any one of claims 1-40, wherein depleting further comprises depleting CD 19+ cells from the population of immune cells.

42. The method of any one of claims 1-41, wherein depleting further comprises depleting CD1 lb+ cells from the population of immune cells.

43. The method of any one of claims claim 31 -42, wherein the endogenous gene comprises one or more genes selected from the group consisting of PRDM1, TNFAIP3, REGNASE-1, SOCS1, PTPN2, CISH, and any combination thereof.

44. The method of claim 43 , wherein inhibiting the expression of the endogenous gene comprises silencing a gene locus or contacting to the cells or expressing in the cells an miRNA or siRNA that targets an RNA transcript encoding the endogenous gene.

45. The method of claim 44, wherein silencing the gene locus comprises gene knockout using a site-specific nuclease, gene knockout using a site-specific nickase, or gene silencing using a site-specific epigenetic regulator.

46. The method of claim 45, wherein silencing the gene locus comprises delivering (i) the sitespecific nuclease, the site-specific nickase, or the site-specific epigenetic regulator, or (ii) a nucleic acid encoding the site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator into the depleted population of immune cells via electroporation, lipid nanoparticles, liposomes, nucleofection, or viral vector.

47. The method of any one of claims 43-46, wherein the endogenous gene comprises PRDM1 and / or TNFAIP3.

48. The method of any one of clams 43-46, wherein the endogenous gene comprises PRDM1 and / or REGNASE-1.

49. The method of any one of clams 43-46, wherein the endogenous gene comprises TNFAIP3 and / or REGNASE-1.

50. The method of any one of claims 31-47, wherein inhibiting in (a) comprises inhibiting in the population of immune cells expression of endogenous REGNASE-1.

51. The method of claim 50, wherein inhibiting the expression of endogenous REGNASE-1 comprises silencing the REGNASE-1 gene locus to eliminate expression of endogenous REGNASE-1, or expressing an exogenous miRNA or an exogenous siRNA that targets REGNASE-1.

52. The method of any one of claims 1-51, wherein the population of immune cells is from a biological sample from a human subject.

53. The method of any one of claims 1-52, wherein the population of immune cells is from the same human subject from which the cancer cells are obtained.

54. The method of any one of claims 28-53, wherein the expanded population of cells comprises at least about 1x106total cells.

55. The method of any one of claims 28-54, wherein the expanded population of cells comprises at least about 1x107total cells.

56. The method of any one of claims 28-55, wherein the expanded population of cells comprises at least about 1x108total cells.

57. The method of any one of claims 28-56, wherein the expanded population of cells comprises from about 1x108to about 1x1011total cells.

58. The method of any one of claims 28-57, wherein the expanded population of cells comprises from about 0.75xl08to about 1.25xl010total cells.

59. The method of any one of claims 28-57, wherein the expanded population of cells comprises from about 5xl08to about IxlO10total cells, about 5xl08to about IxlO9total cells, or from about 5xl08to about 2xl09total cells.

60. The method of any one of claims 1-59, wherein the depleting comprises depleting CD14+ cells and / or CD25+ cells directly from a washed and / or cryopreserved peripheral blood mononuclear cell (PBMC) sample from a human subject.61 . The method of any one of claims 1-60, wherein incubating and expanding are performed in less than 28 days.

62. The method of any one of claims 28-61, wherein the fraction of CD8+ antigen-specific T cells of the total number of CD8+ T cells in the expanded population of cells is at least twofold higher than the fraction of CD8+ antigen-specific T cells of the total number of CD8+ T cells in the depleted population of immune cells.

63. The method of any one of claims 28-61, wherein the fraction of CD4+ antigen-specific T cells of the total number of CD4+ T cells in the expanded population of cells is at least twofold higher than the fraction of CD4+ antigen-specific T cells of the total number of CD4+ T cells in the depleted population of immune cells.

64. The method of any one of claims 28-63, wherein at least 0.1% of the CD8+ T cells in the expanded population of cells are CD8+ antigen -specific T cells derived from naive CD8+ T cells.

65. The method of any one of claims 28-64, wherein at least 0.1% of the CD4+ T cells in the expanded population of cells are CD4+ antigen -specific T cells derived from naive CD4+ T cells.

66. The method of any one of claims 28-65, wherein expanding comprises (A) contacting the population of cells comprising stimulated T cells with a second population of mature APCs, wherein the second population of mature APCs (i) have been incubated with FLT3L and (ii) present the peptide consisting of the epitope sequence from the protein expressed by the cancer cells of the human subject; and (B) expanding the population of cells comprising stimulated T cells for a second time period, thereby forming an expanded population of T cells.

67. The method of claim 66, wherein the second population of mature APCs have been incubated with FLT3L for at least 1 day prior to contacting the population of cells comprising stimulated T cells with the second population of mature APCs.

68. The method of any one of claims 1-67, wherein depleting one or more cells selected from the group consisting of CD14+ cells, CD25+ cells, and CD56+ cells from the population of immune cells comprises contacting the population of immune cells with a CD 14 binding agent, a CD25 binding agent, and / or a CD56 binding agent.

69. The method of any one of claims 28-68, wherein the percentage of CD3+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 40%, at least 50%, or at least 60% of the total cell population.

70. The method of any one of claims 28-69, wherein the percentage of CD107a+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 10% of the tumor antigen-specific T cell population.

71. The method of any one of claims 28-70, wherein the percentage of TNFa+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 5% of the tumor antigen-specific T cell population.

72. The method of any one of claims 28-71, wherein the percentage of IFNy+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 15% of the tumor antigen-specific T cell population.

73. The method of any one of claims 28-72, wherein the percentage of TNFa+and IFNy+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 2% of the tumor antigen-specific T cell population.

74. The method of any one of claims 28-73, wherein the percentage of TNFa+ and CD107a+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 0.5% of the tumor antigen-specific T cell population.

75. The method of any one of claims 28-74, wherein the percentage of IFNy+and CD107a+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 5% of the tumor antigen-specific T cell population.

76. The method of any one of claims 28-75, wherein the percentage of TNFa+ and IFNy+ and CD107a+ cells in the expanded population of cells comprising tumor antigen-specific T cells is at least 0.1% of the tumor antigen-specific T cell population.

77. The method of any one of claims 28-76, wherein the percentage of CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are naive T cells (CD62L+ and CD45RA+) is at most 15%.

78. The method of any one of claims 28-77, wherein the percentage of CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector memory T cells (CD62L- and CD45RA-) is at least 60%.

79. The method of any one of claims 28-78, wherein the percentage of CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector T cells (CD62L- and CD45RA+) is at most 5%.

80. The method of any one of claims 28-79, wherein the percentage of CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are central memory T cells (CD62L+ and CD45RA-) is at least 10%.

81. The method of any one of claims 28-80, wherein the percentage of CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are naive T cells (CD62L+ and CD45RA+) is at most 25%.

82. The method of any one of claims 28-81, wherein the percentage of CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector memory T cells (CD62L- and CD45RA-) is at least 60%.

83. The method of any one of claims 28-82, wherein the percentage of CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are effector T cells (CD62L- and CD45RA+) is at most 10%.

84. The method of any one of claims 28-83, wherein the percentage of CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are central memory T cells (CD62L+ and CD45RA-) is at least 15%.

85. The method of any one of claims 28-84, wherein the percentage of CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells that are CD 127+ is at least 40%.

86. The method of any one of claims 1-77, wherein the expanded population of cells comprising tumor antigen-specific T cells produce cytokines and cause degranulation upon recognition of target cells.

87. The method of any one of claims 1-86, wherein depleting comprises depleting CD14+ cells and CD25+ cells from a peripheral blood mononuclear cell (PBMC) sample from a human subject that has not been subject to a step of monocyte maturation into mature dendritic cells (DCs).

88. The method of claim 87, wherein depleting further comprises depleting CD1 lb+ cells from the peripheral blood mononuclear cell (PBMC) sample from the human subject that has not been subject to a step of monocyte maturation into mature dendritic cells (DCs).

89. The method of any one of claims 28-88, wherein the fraction of CD8+ tumor antigen-specific T cells of the total number of CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells is at least two-fold higher than the fraction of CD8+ tumor antigen-specific T cells of the total number of CD8+ T cells in the biological sample.

90. The method of any one of claims 28-89, wherein the fraction of CD4+ tumor antigen-specific T cells of the total number of CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells is at least two-fold higher than the fraction of CD4+ tumor antigen-specific T cells of the total number of CD4+ T cells in the biological sample.91 . The method of any one of claims 28-90, wherein at least 0.1% of the CD8+ T cells in the expanded population of cells comprising tumor antigen-specific T cells are CD8+ tumor antigen-specific T cells derived from naive CD8+ T cells.

92. The method of any one of claims 28-91, wherein at least 0.1% of the CD4+ T cells in the expanded population of cells comprising tumor antigen-specific T cells are CD4+ tumor antigen-specific T cells derived from naive CD4+ T cells.

93. The method of any one of claims 28-65 or 68-92, wherein expanding comprises (A) contacting the population of cells comprising stimulated T cells with a second population of mature APCs, wherein the second population of mature APCs (i) have been incubated with FLT3L and (ii) present the at least one tumor antigen epitope sequence; and (B) expanding the population of cells comprising stimulated T cells for a second time period, thereby forming an expanded population of T cells.

94. The method of claim 93, wherein the second population of mature APCs have been incubated with FLT3L for at least 1 day prior to contacting the population of cells comprising stimulated T cells with the second population of mature APCs.

95. The method of any one of claims 1-94, wherein the biological sample is a peripheral blood sample, a leukapheresis sample or an apheresis sample.

96. The method of any one of claims 28-95, wherein the method further comprises harvesting the expanded population of cells comprising tumor antigen -specific T cells, cry opreserving the expanded population of cells comprising tumor antigen-specific T cells or preparing a pharmaceutical composition containing the expanded population of cells comprising tumor antigen-specific T cells.

97. The method of any one of claims 1-96, wherein incubating comprises incubating the depleted population of immune cells comprising a first population of APCs and T cells for a first time period in the presence of FLT3L and an RNA encoding the polypeptide.

98. The method of any one of claims 1-97, wherein the population of engineered immune cells does not comprise a cell expressing a chimeric antigen receptor (CAR).

99. The method of any one of claims 1-98, wherein the population of engineered immune cells exhibit enhanced T cell activation, enhanced effector function, enhanced persistence, enhanced proliferation, or enhanced avoidance of dysfunction, or any combination thereof compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene.

100. The method of claim 99, wherein the enhanced T cell activation comprises enhanced T cell priming, enhanced antigen recognition, enhanced cytokine sensing, or enhanced costimulation properties, or any combination thereof.

101. The method of claim 99, wherein the enhanced effector function comprises enhanced tumor infiltration, enhanced metabolic changes, enhanced proliferation, enhanced bystanderactivation, enhanced cytokine secretion, or enhanced cytotoxicity, or any combination thereof.

102. The method of any one of claims 1-101, wherein the population of engineered immune cells has a decreased proportion of PD-l + T cells, TIM3+ T cells, TIGIT+ T cells, Lag-3+ T cells, CD39+ T cells, and / or TOX+ T cells compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene.

103. The method of claim 102, wherein the percentage of PD-1+ T cells, TIM3+ T cells, TIGIT+ T cells, Lag-3+ T cells, CD39+ T cells, and / or TOX+ T cells is at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% less than the percentage of PD-1+ T cells, TIM3+ T cells, TIGIT+ T cells, Lag-3 + T cells, CD39+ T cells, and / or TOX+ T cells in an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene.

104. The method of any one of claims 1-103, wherein the population of engineered immune cells has an increased cytokine release compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene.

105. The method of claim 104, wherein the cytokine comprises IL-2, IFNg, TNF-a, Perforin, IL- 18, or any combination thereof.

106. The method of claim 104 or 105, wherein the increased cytokine release comprises at least about 0.5 -fold, 1 -fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 100-fold, 150-fold, or more than a cytokine release of an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene.

107. The method of any one of claims 99-106, wherein the enhanced persistence comprises enhanced homeostatic cytokine release, enhanced memory function, enhanced proliferation, enhanced sternness, or enhanced metabolism, or any combination thereof.

108. The method of any one of claims 1-107, wherein the population of engineered immune cells has an increased fold expansion compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene.

109. The method of claim 108, wherein a fold expansion is at least about 1.1-fold, 1.2-fold, 1.3- fold, 1.4-fold, 1.5 -fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3- fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 3.1-fold, 3.2-fold, 3.3- fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4-fold, 4.1-fold, 4.2-fold, 4.3- fold, 4.4-fold, 4.5-fold, 4.6-fold, 4.7-fold, 4.8-fold, 4.9-fold, 5-fold, 5.5-fold, 6-fold, 6.5-fold, 7-fold, 7.5-fold, 8-fold, 8.5-fold, 9-fold, 9.5-fold, 10-fold, 11-fold, 12-fold, 13-fold, or morethan a 13 -fold expansion of an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene after culturing for a period of time.

110. The method of claim of 109, wherein the period of time is at least about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more.

111. The method of any one of claims 1-110, wherein the population of engineered immune cells has an increased persistence or expansion in a peripheral blood sample compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene(s) after being administered into a subject.

112. The method of any one of claims 1-110, wherein the population of engineered immune cells has an increased proportion of central memory T cells compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene.

113. The method of claim 112, wherein the increased proportion of central memory T cells comprises at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% more than the proportion of central memory T cells in an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene.

114. The method of any one of claims 1-113, wherein the population of engineered immune cells with reduced expression of the endogenous gene(s) has an increased proportion of CD 127+ expressing cells compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene(s).

115. The method of any one of claims 99-113, wherein the enhanced avoidance of dysfunction comprises reduced overactivation, reduced AICD expression, reduced exhaustion, reduced apoptosis, reduced toxicity, reduced antigen escape, reduced immunosuppression, or enhanced homeostasis, or any combination thereof.

116. The method of any one of claims 1-115, wherein the population of engineered immune cells has an increased percent frequency of Texf’ros2cells or Ly 108+CD69- cells in a population of antigen-specific CD8+ cells compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene(s).

117. The method of claim 116, wherein the increased percent frequency of TexPro§2cells or Ly 108+CD69- cells in the population of engineered immune cells is at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to that of an identical population of engineered immune cells not having the reduced expression of the endogenous gene(s).

118. The method of any one of claims 1-114, wherein the population of engineered immune cells has an increased suppression of tumor growth compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene.

119. The method of claim 118, wherein a tumor volume in a subject administered with the population of engineered immune cells is at least about 1 %, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% less than that of an identical subject administered with an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene.

120. The method of any one of claims 101-119, wherein the enhanced cytotoxicity comprises enhanced suppression of tumor cell growth compared to an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene.

121. The method of claim 120, wherein a rate of tumor cell growth is reduced by a factor of 1.2- fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30- fold, 40-fold, 50-fold or more compared to a rate of tumor cell growth in the presence of an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene.

122. The method of any one of claims 1-121, further comprising preparing a pharmaceutical composition comprising the population of engineered immune cells.

123. The method of claim 122, further comprising administering the pharmaceutical composition in a subject in need thereof.

124. The method of claim 123, wherein the subject is the same subject from which the biological sample is obtained.

125. An engineered immune cell comprising: a nucleic acid molecule encoding a chimeric antigen receptor (CAR) comprising a target binding domain, a transmembrane domain and an intracellular signaling domain; and an agent for reducing expression of an endogenous gene, wherein the engineered immune cell has reduced expression of the endogenous gene compared to an otherwise identical engineered immune cell not having the agent.

126. The engineered immune cell of claim 125, wherein the endogenous gene comprises one or more genes selected from the group consisting of PRDM1, TNFAIP3, REGNASE-1, SOCS1, PTPN2, CISH, and any combination thereof.

127. The engineered immune cell of claim 125 or 126, wherein the endogenous gene comprises PRDM1 and / or TNFAIP3.

128. The engineered immune cell of claim 127, wherein the endogenous gene comprises PRDM1 and TNFAIP3.

129. The engineered immune cell of any one of claims 125-128, wherein the endogenous gene comprises REGNASE-1.

130. The engineered immune cell of claim 125 or 126, wherein the endogenous gene comprises PRDM1 and / or REGNASE-1.

131. The engineered immune cell of claim 130, wherein the endogenous gene comprises PRDM1 and REGNASE-1.

132. The engineered immune cell of claim 125 or 126, wherein the endogenous gene comprises TNFAIP3 and / or REGNASE-1.

133. The engineered immune cell of claim 132, wherein the endogenous gene comprises TNFAIP3 and REGNASE-1.

134. The engineered immune cell of any one of claims 125-129, wherein the agent is a nucleic acid encoding a component for gene silencing, or the agent is an miRNA or siRNA that targets an RNA transcript encoding the endogenous gene.

135. The engineered immune cell of claim 134, wherein the component for gene silencing comprises a guide RNA.

136. The engineered immune cell of any one of claims 125-135, wherein the target binding domain is an antigen binding domain.

137. The engineered immune cell of any one of claims 125-136, wherein the target binding domain comprises an antibody, an antibody fragment, an scFv, an Fv, a Fab, a (Fab')2, a single domain antibody (SdAb), a VH or VL domain, or a camelid VHH domain.

138. The engineered immune cell of any one of claims 125-137, wherein the transmembrane domain is fused to an extracellular domain comprising the target binding domain.

139. The engineered immune cell of any one of claims 125-138, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CD1 la, CD 18), ICOS (CD278), 4-1BB (CD 137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIId, ITGAE, CD 103, ITGAL, CDIIa, LFA-1, ITGAM, CDIIb, ITGAX, CDIIc, ITGBI, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160(BY55), PSGLI, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C, a synthetic material, or a functional variant thereof.

140. The engineered immune cell of any one of claims 125-139, wherein the intracellular signaling domain comprises a co-stimulatory domain.

141. The engineered immune cell of any oneof claims 125-140, wherein the intracellular signaling domain comprises a CD3-zeta signaling domain.

142. The engineered immune cell of any oneof claims 125-141, wherein the intracellular signaling domain comprises a CD3-zeta signaling domain and a costimulatory signaling domain.

143. The engineered immune cell of any one of claims 125-142, wherein the CAR comprises multiple costimulatory domains.

144. The engineered immune cell of claim 142 or 143, wherein the one or more costimulatory domains comprise CD28, CD137 (4-1BB), TNFR, CD134 (0X40), CD278, or any combination thereof.

145. The engineered immune cell of any one of claims 125-144, wherein the CAR further comprises a CD8a hinge domain.

146. The engineered immune cell of claim 145, wherein the hinge domain forms a link between the transmembrane domain and the extracellular domain.

147. The engineered immune cell of any one of claims 125-146, wherein the CAR further comprises a fusion protein.

148. The engineered immune cell of claim 147, wherein the fusion protein further comprises a linker, a signal peptide, or any combination thereof.

149. An engineered immune cell comprising: a nucleic acid molecule encoding a T-cell receptor (TCR), wherein the TCR binds to KRAS, PRAME, or GAT A3; and an agent for reducing expression of an endogenous gene, wherein the engineered immune cell has reduced expression of the endogenous gene compared to an otherwise identical engineered immune cell not having the agent.

150. The engineered immune cell of claim 149, wherein the endogenous gene comprises one or more genes selected from the group consisting of PRDM1, TNFAIP3, REGNASE-1, S0CS1, PTPN2, CISH, and any combination thereof.

151. The engineered immune cell of claim 149 or 150, wherein the endogenous gene comprises PRDM1 and / or TNFAIP3.

152. The engineered immune cell of claim 151, wherein the endogenous gene comprises PRDM1 and TNFAIP3.

153. The engineered immune cell of any one of claims 149-152, wherein the endogenous gene comprises REGNASE-1.

154. The engineered immune cell of claim 149 or 150, wherein the endogenous gene comprises PRDM1 and / or REGNASE-1.

155. The engineered immune cell of claim 154, wherein the endogenous gene comprises PRDM1 and REGNASE-1.

156. The engineered immune cell of claim 149 or 150, wherein the endogenous gene comprises TNFAIP3 and / or REGNASE-1.

157. The engineered immune cell of claim 156, wherein the endogenous gene comprises TNFAIP3 and REGNASE-1.

158. The engineered immune cell of any one of claims 149-153, wherein the agent is a nucleic acid encoding a component for gene silencing, or the agent is a miRNA or siRNA that targets an RNA transcript encoding the endogenous gene.

159. The engineered immune cell of claim 158, wherein the component for gene silencing comprises a guide RNA.

160. The engineered immune cell of any one of claims 149-159, wherein the TCR binds to a PRAME epitope in complex with an MHC encoded by a HLA-A02:01 allele.

161. The engineered immune cell of claim 160, wherein the PRAME epitope comprises an amino acid sequence of SEQ ID NO: 116.

162. The engineered immune cell of any one of claims 149-161, wherein the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 105.

163. The engineered immune cell of claim 162, wherein the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115.

164. The engineered immune cell of claim 162 or 163, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 103 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 104.

165. The engineered immune cell of any one of claims 162-164, wherein the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acidsequence set forth in SEQ ID NO: 100, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 101, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 102.

166. The engineered immune cell of any one of claims 162-165, wherein the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 113.

167. The engineered immune cell of any one of claims 162-166, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 124, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 124, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 123, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 123.

168. The engineered immune cell of any one of claims 162-166, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 126, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 126, and (b) an alpha chain having an amino acid sequence setforthin SEQ ID NO: 125, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 125.

169. The engineered immune cell of any one of claims 149-159, wherein the TCR binds to a complex comprising (i) an epitope from human RAS comprising a mutation G12V and (ii) an MHC protein encoded by an HLA-A11 :01 allele.

170. The engineered immune cell of any one of claims 149-159, wherein the TCR binds to the epitope comprising an amino acid sequence of SEQ ID NO: 51, 52, 53, 55 or 56 in complex with an MHC encoded by a HLA-A11 :01 allele.

171. The engineered immune cell of any one of claims 149-159 or 169-170, wherein the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 6.

172. The engineered immune cell of claim 171, wherein the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 12.

173. The engineered immune cell of claim 171 or 172, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 4 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 5.

174. The engineered immune cell of any one of claims 171-173, wherein the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acidsequence set forth in SEQ ID NO: 1, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 2, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 3.

175. The engineered immune cell of any one of claims 171-174, wherein the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 9.

176. The engineered immune cell of any oneof claims 171-175, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 13.

177. The engineered immune cell of any oneof claims 171-175, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 17, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 14.

178. The engineered immune cell of any one of claims 149-159, wherein the TCR binds to a complex comprising (i) an epitope from human RAS comprising a mutation G12V and (ii) an MHC protein encoded by an HLA-A68:01 allele.

179. The engineered immune cell of any one of claims 149-159, wherein the TCR binds to the epitope comprising an amino acid sequence of SEQ ID NO: 51 in complex with an MHC encoded by a HLA-A68:01 allele.

180. The engineered immune cell of any one of claims 149-159 or 178-179, wherein the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 132.

181. The engineered immune cell of claim 180, wherein the TCRbeta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 134.

182. The engineered immune cell of claim 180 or 181, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence setforth in SEQ IDNO: 130 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 131.

183. The engineered immune cell of any one of claims 180-182, wherein the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acidsequence set forth in SEQ ID NO: 127, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 128, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 129.

184. The engineered immune cell of any one of claims 180-183, wherein the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 133.

185. The engineered immune cell of any one of claims 125-167, wherein the engineered immune cell exhibits enhanced T cell activation, enhanced effector function, enhanced persistence, enhanced proliferation, or enhanced avoidance of dysfunction, or any combination thereof compared to an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene.

186. The engineered immune cell of claim 185, wherein the enhanced T cell activation comprises enhanced T cell priming, enhanced antigen recognition, enhanced cytokine sensing, or enhanced costimulation properties, or any combination thereof.

187. The engineered immune cell of claim 185, wherein the enhanced effector function comprises enhanced tumor infiltration, enhanced metabolic changes, enhanced proliferation, enhanced bystander activation, enhanced cytokine secretion, or enhanced cytotoxicity, or any combination thereof.

188. The engineered immune cell of any one of claims 125-187, wherein the engineered immune cell has an increased cytokine release compared to an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene.

189. The engineered immune cell of claim 188, wherein the cytokine comprises IL-2, IFNg, TNF- a, Perforin, IL-18, or any combination thereof.

190. The engineered immune cell of claim 188 or claim 189, wherein the increased cytokine release comprises at least about 0.5 -fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 100- fold, 150-fold, or more than a cytokine release of an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene.

191. The engineered immune cell of claim 185, wherein the enhanced persistence comprises enhanced homeostatic cytokine release, enhanced memory function, enhanced proliferation, enhanced sternness, or enhanced metabolism, or any combination thereof.

192. The engineered immune cell of any one of claims 125-191, wherein the engineered immune cell has an increased fold expansion compared to an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene.

193. The engineered immune cell of claim 192, wherein a fold expansion is at least about 1.1 -fold, 1 .2-fold The method of claim, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, or more than a fold expansion of an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene after culturing for a period of time.

194. The engineered immune cell of claim of 193, wherein the period of time is at least about 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more.

195. The engineered immune cell of any one of claims 125-194, wherein the engineered immune cell comprises a population of engineered immune cells, and wherein the population of engineered immune cells has an increased persistence as measured by cell count of TCR+ / CD3+ cells in a sample from a subject administered with the population of engineered immune cells compared to that of a sample from a subject administered with an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene.

196. The engineered immune cell of claim 195, wherein the TCR+ / CD3+ cell count is increased by 1-fold, 10-fold, 100-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, or more compared to the amount of TCR+ / CD3+ in the sample from the subject administered with an otherwise identical population of engineered immune cells not having a reduced expression of the endogenous gene.

197. The engineered immune cell of claim 195, wherein the TCR+ / CD3 + cell count in a peripheral blood sample from a subject administered with the population of engineered immune cells is increased by 1-fold, 10-fold, 100-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, or more compared to the amount of TCR+ / CD3+ in a peripheral blood sample from a subject administered with an otherwise identical population of engineered immune cells in the peripheral blood sample not having a reduced expression of the endogenous gene.

198. The engineered immune cell of claim 195, wherein the TCR+ / CD3 + cell count in a peripheral blood sample from a subject administered with the population of engineered immune cells is increased by 1-fold, 10-fold, 100-fold, 1000-fold, 2000-fold, 3000-fold, 4000-fold, 5000-fold, or more compared to the amount of TCR+ / CD3+ in a peripheral blood sample from a subject administered with an otherwise identical population of engineered immune cells in the peripheral blood sample not having a reduced expression of the endogenous gene, after at least 8 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days or more post adoptive cell transfer of the population of engineered immune cells.

199. The engineered immune cell of any one of claims 185-198, wherein the enhanced avoidance of dysfunction comprises reduced overactivation, reduced AICD expression, reduced exhaustion, reduced apoptosis, enhanced toxicity, reduced antigen escape, reduced immunosuppression, or enhanced homeostasis, or any combination thereof.

200. The engineered immune cell of any one of claims 125-199, wherein the engineered immune cell has an increased suppression of tumor growth compared to an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene.

201. The engineered immune cell of claim 200, wherein a tumor volume in a subject administered with the engineered immune cell is atleast about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% less than that of an identical subject administered with an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene.

202. The engineered immune cell of any one of claims 187-201, wherein the enhanced cytotoxicity comprises enhanced suppression of tumor cell growth compared to an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene.

203. The engineered immune cell of claim 202, wherein a rate of tumor cell growth is reduced by a factor of 1.2-fold, 1.5 -fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 20-fold, 30-fold, 40-fold, 50-fold or more compared to a rate of tumor cell growth in the presence of an otherwise identical engineered immune cell not having a reduced expression of the endogenous gene.

204. A population of engineered immune cells comprising the engineered immune cell of any one of claims 125-203.

205. A population of engineered immune cells, wherein expression of an endogenous gene is inhibited in the population of engineered immune cells; and wherein the population of engineered immune cells is from a biological sample from a subject and the percentage of CD14+ cells and / or CD25+ cells and / or CD56+ cells in the population of engineered immune cells is lower compared to the percentage of CD 14+ cells and / or CD25+ cells and / or CD56+ cells in the biological sample.

206. The population of engineered immune cells of claim 205, wherein the endogenous gene comprises one or more genes selected from the group consisting of PRDM1, TNFAIP3, REGNASE-1, SOCS1, PTPN2, CISH, and any combination thereof.

207. The population of engineered immune cells of claim 205 or 206, wherein the endogenous gene comprises PRDM1 and / or TNFAIP3.

208. The population of engineered immune cells of claim 207, wherein the endogenous gene comprises PRDM1 and TNFAIP3.

209. The population of engineered immune cells of any one of claims 205-208, wherein the endogenous gene comprises REGNASE-1.

210. The population of engineered immune cells of claim 205 or 206, wherein the endogenous gene comprises PRDM1 and / or REGNASE-1.

211. The population of engineered immune cells of claim 210, wherein the endogenous gene comprises PRDM1 and REGNASE-1.

212. The population of engineered immune cells of claim 205 or 206, wherein the endogenous gene comprises TNFAIP3 and / or REGNASE-1.

213. The population of engineered immune cells of claim 212, wherein the endogenous gene comprises TNFAIP3 and REGNASE-1.

214. The population of engineered immune cells of any one of claims 205-209, wherein the agent is a nucleic acid encoding a component for gene silencing, or the agent is a miRNA or siRNA that targets an RNA transcript encoding the endogenous gene.

215. The population of engineered immune cells of claim 214, wherein the component for gene silencing comprises a guide RNA.

216. The population of engineered immune cells of any one of claims 205-215, wherein the population of engineered immune cells comprises at least IxlO5cells.

217. The population of engineered immune cells of any one of claims 205-216, wherein the engineered immune cells in the population of engineered immune cells further comprise a chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

218. A method of preparing an engineered immune cell expressing a chimeric antigen receptor (CAR), the method comprising:(a) introducing into an immune cell a nucleic acid sequence encoding the CAR comprising a target binding domain, a transmembrane domain and an intracellular signaling domain; and(b) prior to, subsequent to, or concurrently to (a), inhibiting in the immune cell an expression of an endogenous gene, wherein the endogenous gene comprises one or more genes selected from the group consisting of PRDM1, TNFAIP3, REGNASE-1, SOCS1, PTPN2, CISH, and any combination thereof.

219. The method of claim 218, wherein the endogenous gene comprises PRDM1 and / or TNFAIP3.

220. The method of claim 219, wherein the endogenous gene comprises PRDM1 and TNFAIP3.

221. The method of any one of claims 218-220, wherein the endogenous gene comprises REGNASE-1.

222. The method of claim 218, wherein the endogenous gene comprises PRDM1 and / or REGNASE-1.

223. The method of claim 222, wherein the endogenous gene comprises PRDM1 and REGNASE- 1.

224. The method of claim 218, wherein the endogenous gene comprises TNFAIP3 and / or REGNASE-1.

225. The method of claim 224, wherein the endogenous gene comprises TNFAIP3 and REGNASE-1.

226. The method of any one of claims 218-221, wherein inhibiting the expression of the endogenous gene comprises silencing a gene locus or contacting to the cells or expressing in the cells a miRNA or siRNA that targets an RNA transcript encoding the endogenous gene.

227. The method of any one of claims 218-226, wherein the CAR comprises a target binding domain, a transmembrane domain, and a T cell signaling domain.

228. The method of any one of claims 218-227, wherein the target binding domain is an antigen binding domain.

229. The method of any one of claims 218-228, wherein the target binding domain comprises an antibody, an antibody fragment, an scFv, an Fv, a Fab, a (Fab')2, a single domain antibody (sdAb), a VH or VL domain, or a VHH domain.

230. The method of any one of claims 218-229, wherein the transmembrane domain is fused to an extracellular domain.

231. The method of any one of claims 218-230, wherein the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDIId, ITGAE, CD103, ITGAL, CDIIa, LFA-1, ITGAM, CDIIb, ITGAX, CDIIc, ITGBI, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGLI, CDIOO (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C, a synthetic material, or a functional variant thereof.

232. The method of any one of claims 218-231, wherein the intracellular signaling domain comprises a co-stimulatory domain.

233. The method of any one of claims 218-232, wherein the intracellular signaling domain comprises a CD3-zeta signaling domain.

234. The method of any one of claims 218-233, wherein the intracellular signaling domain comprises a CD3-zeta signaling domain and a costimulatory signaling domain.

235. The method of any one of claims 218-234, wherein the CAR comprises multiple costimulatory domains.

236. The method of claim 234 or claim 235, wherein the one or more costimulatory domains comprise CD28, CD137 (4-1BB), TNFR, CD134 (0X40), CD278, or any combination thereof.

237. The method of any one of claims 218-236, wherein the CAR further comprises a CD8a hinge domain.

238. The method of claim 237, wherein the hinge domain forms a link between the transmembrane domain and the extracellular domain.

239. The method of any one of claims 218-238, wherein the CAR further comprises a fusion protein.

240. The method of claim 239, wherein the fusion protein further comprises a linker, a signal peptide, or any combination thereof.

241. A method of preparing an engineered immune cell expressing a T-cell receptor (TCR) targeting KRAS, PRAME or GAT A3, the method comprising:(a) introducing into an immune cell a nucleic acid sequence encoding the TCR, wherein the TCR binds to KRAS, PRAME or GATA3; and(b) prior to, subsequent to, or concurrently to (a), inhibiting in the immune cell an expression of an endogenous gene, wherein the endogenous gene comprises one or more genes selected from the group consisting of PRDM1, TNFAIP3, REGNASE-1, S0CS1, PTPN2, CISH, and any combination thereof.

242. The method of claim 241, wherein the endogenous gene comprises PRDM1 and / or TNFAIP3.

243. The method of claim 242, wherein the endogenous gene comprises PRDM1 and TNFAIP3.

244. The method of any one of claims 241-243, wherein the endogenous gene comprises REGNASE-1.

245. The method of claim 241, wherein the endogenous gene comprises PRDM1 and / or REGNASE-1.

246. The method of claim 245, wherein the endogenous gene comprises PRDM1 and REGNASE- 1.

247. The method of claim 241, wherein the endogenous gene comprises TNFAIP3 and / or REGNASE-1.

248. The method of claim 247, wherein the endogenous gene comprises TNFAIP3 and REGNASE-1.

249. The method of any one of claims 241-244, wherein inhibiting the expression of the endogenous gene comprises silencing a gene locus or contacting to the cells or expressing in the cells a miRNA or siRNA that targets an RNA transcript encoding the endogenous gene.

250. The method of any one of claims 241-249, wherein the TCR binds to a PRAME epitope in complex with an MHC encoded by a HLA-A02:01 allele.

251. The method of claim 250, wherein the PRAME epitope comprises an amino acid sequence of SEQ ID NO: 116.

252. The method of any one of claims 241-251, wherein the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence of SEQ ID NO: 105.

253. The method of claim 252, wherein the TCRbeta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 115.

254. The method of claim 252 or 253, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 103 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 104.

255. The method of any one of claims 252-254, wherein the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 100, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 101, and the CDR3 has an amino acid sequence SEQ ID NO: 102.

256. The method of any one of claims 252-255, wherein the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 113.

257. The method of any one of claims 252-256, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 124, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 124, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 123, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 123.

258. The method of any one of claims 252-256, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 126, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 126, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 125, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 125.

259. The method of claim 241, wherein the TCR binds to a complex comprising (i) an epitope from human RAS comprising a mutation G12V and (ii) an MHC protein encoded by an HLA-A1 E01 allele.

260. The method of claim 241, wherein the TCR binds to a K-RAS epitope in complex with an MHC encoded by an HLA-A11 :01 allele.

261. The method of any one of claims 241-251, wherein the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence SEQ ID NO: 6.

262. The method of claim 261, wherein the TCR beta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 12.

263. The method of claim 261 or 262, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO: 4 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 5.

264. The method of any one of claims 261 -263, wherein the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 1, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 2, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 3.

265. The method of any one of claims 261-264, wherein the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 9.

266. The method of any one of claims 261-265, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 16, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 16, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 13, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 13.

267. The method of any one of claims 261-265, wherein the TCR comprises: (a) a beta chain having an amino acid sequence set forth in SEQ ID NO: 17, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 17, and (b) an alpha chain having an amino acid sequence set forth in SEQ ID NO: 14, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 14.

268. The method of claim 241, wherein the TCR binds to a K-RAS epitope in complex with an MHC encoded by an HLA-A68:01 allele.

269. The method of any one of claims 241-251, wherein the TCR comprises a TCR beta chain construct and a TCR alpha chain construct, wherein the TCR beta chain construct comprises a complementarity determining region 3 (CDR3) having an amino acid sequence SEQ ID NO: 132.

270. The method of claim 269, wherein the TCRbeta chain construct comprises a variable region having an amino acid sequence with at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO: 134.

271. The method of claim 269 or 270, wherein the TCR beta chain construct comprises a complementarity determining region 1 (CDR1) having an amino acid sequence set forth in SEQ ID NO : 130 and a complementarity determining region 2 (CDR2) having an amino acid sequence set forth in SEQ ID NO: 131.

272. The method of any one of claims 269-271, wherein the TCR alpha chain construct comprises a CDR1, a CDR2, and a CDR3, wherein the CDR1 has an amino acid sequence set forth in SEQ ID NO: 127, the CDR2 has an amino acid sequence set forth in SEQ ID NO: 128, and the CDR3 has an amino acid sequence set forth in SEQ ID NO: 129.

273. The method of any one of claims 269-272, wherein the TCR alpha chain construct comprises a variable region having an amino acid sequence having at least 80% sequence identity to an amino acid sequence set forth in SEQ ID NO: 133.

274. A pharmaceutical composition comprising the population of engineered immune cells produced according to the method of any one of claims 1-124 and 218-273, the engineered immune cell of any one of claims 125-203 or the population of engineered immune cells of any one of claims 204-217; and a pharmaceutically acceptable carrier.

275. A method of treating a disease or condition in a subject in need thereof comprising administering the pharmaceutical composition of claim 274 to the subject.

276. The method of claim 275, wherein the subject is the same subject from which the biological sample is obtained.

277. Use of the engineered immune cells of any one of claims 125-203 or the population of engineered immune cells of any one of claims 204-217 in the manufacture of a medicament for treating a cancer in a subject.

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