Compositions and methods for t cell manufacturing and uses thereof

Abstract

Provided herein are methods of producing a plurality of T cells which has been ex vivo stimulated and expanded to enrich for antigen-specific T cells. This plurality of T cells can be used to provide highly specific and efficacious T cell therapies for treatment of cancers and other conditions.

Description

WSGR Docket No.: 50401-791.602 COMPOSITIONS AND METHODS FOR T CELL MANUFACTURING AND USES THEREOF CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 651,534 filed May 24, 2024 and U.S. Provisional Application No. 63 / 798,408 filed May 01, 2025, each of which is incorporated by reference herein in its entirety. BACKGROUND

[0002] T cell therapies for the treatment of cancers have improved the clinical landscape and opened new horizons in the field of oncology. Endogenous T cell responses in the context of cancer are complex and often result in anergy, exhaustion, and other states associated with immunosuppression. T cell therapies attempt to enhance the anti-cancer immune response by providing a population of T cells that have been “armed” in some manner to target and kill cancer cells more effectively. One approach utilizes identification of antigens presented by cancer cells in a particular tumor and aims to generate expanded populations of T cells that are specific for the cancer antigens which are then administered into the patient in an adoptive cell therapy process.

[0003] Most, if not all T cell-based therapeutic strategies 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, remain limited by cumbersome T cell manufacturing processes. Existing T cell manufacturing processes are 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 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

[0004] Recognized herein is a need for an improved T cell activation and expansion process to produce antigen-specific T cells (e.g., tumor antigen-specific T cells). The present disclosure provides compositions and methods of producing T cell therapies for clinical development and use. Although autologous T cell therapies are currently in use, there are a number of difficulties to achieve a high level of efficacy. The compositions and methods disclosed herein, in someWSGR Docket No.: 50401-791.602 cases, separation of certain immune cell populations and alteration of cell culture conditions (including, but not limited to, presence of certain stimulatory agents (e.g., cytokines) and gene delivery mechanisms) can, in a scalable manner, generate an expanded and activated population of T cells suitable for use in treatment of cancer and other conditions. In other embodiments there is no separation of immune cell populations alteration of cell culture conditions which, in a scalable manner, generate an expanded and activated population of T cells suitable for use in treatment of cancer and other conditions.

[0005] Provided herein is a method for producing a plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells, the method comprising: (a) providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a single biological sample from a subject; (b) separating the APCs from the T cells in the population of immune cells, thereby forming a population of APCs separated from a population of T cells; (c) culturing the population of APCs in a first vessel in the presence of a first stimulatory agent and culturing the population of T cells in a second vessel in the presence of a second stimulatory agent, wherein the first vessel and the second vessel are different vessels; (d) co-culturing APCs of the population of APCs from the first vessel and T cells of the population of T cells from the second vessel, thereby forming a population of cells comprising stimulated T cells; and (e) expanding the stimulated T cells, thereby forming the plurality of T cells that comprises antigen-specific T cells.

[0006] In some embodiments, the method further comprises, prior to (b), depleting CD25+ cells and / or CD56+ cells from the population of immune cells.

[0007] In some embodiments, the method further comprises, prior to (b), depleting CD11b+ cells.

[0008] In some embodiments, separating the APCs and the T cells in the population of immune cells in (b) comprises enriching CD14+ cells and / or CD19+ cells from the population of immune cells, thereby forming the population of APCs separated from the population of T cells.

[0009] In some embodiments, the first stimulatory agent comprises one or more agents selected from the group consisting of FLT3L, IL-4, GM-CSF, and any combination thereof.

[0010] In some embodiments, the second stimulatory agent comprises one or more agents selected from the group consisting of IL-2, IL-7, IL-15, IL-21, and any combination thereof.

[0011] In some embodiments, co-culturing in (d) comprises co-culturing the APCs of the population of APCs from the first vessel and the T cells of the population of T cells from the second vessel in the presence of IL-21.WSGR Docket No.: 50401-791.602

[0012] In some embodiments, expanding the stimulated T cells in (e) comprises expanding the stimulated T cells in the presence of one or more agents selected from the group consisting of IL-7, IL-15, IL-2 and an IL-2 variant.

[0013] In some embodiments, expanding the stimulated T cells in (e) comprises expanding the stimulated T cells in the absence of IL-21.

[0014] In some embodiments, the method further comprises, prior to co-culturing in (d), delivering (A) at least one 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 at least one polypeptide into the population of APCs.

[0015] In some embodiments, culturing in (c) further comprises delivering (A) at least one 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 at least one polypeptide into the population of APCs.

[0016] In some embodiments, delivering comprises electroporating the polynucleotide encoding the polypeptide into the population of APCs.

[0017] In some embodiments, delivering comprises contacting the population of APCs with the polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer.

[0018] In some embodiments, culturing in (c) comprises maturing the population of APCs in the first vessel.

[0019] In some embodiments, maturing comprises maturing the population of APCs in the firstvessel in the presence of IFN and / or a toll like receptor agonist lipopolysaccharide (LPS).

[0020] In some embodiments, the method further comprises, prior to co-culturing in (d), genetically editing the population of T cells.

[0021] In some embodiments, genetically editing the population of T cells comprises inhibiting expression of an endogenous gene in the population of T cells.

[0022] 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.

[0023] 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 an miRNA or siRNA that targets an RNA transcript encoded by the endogenous gene.WSGR Docket No.: 50401-791.602

[0024] 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.

[0025] In some embodiments, silencing the 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 site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator into the population of T cells via electroporation, a lipid nanoparticle, a lipoplex, a liposome, nucleofection, or a viral vector.

[0026] In some embodiments, genetically editing comprises delivering an exogenous gene into the population of T cells via transposon.

[0027] In some embodiments, the exogenous gene encodes a cell surface receptor that binds to a cytokine, or wherein the exogenous gene encodes a switch receptor or a safety switch for controlling activities of T cells.

[0028] In some embodiments, the cytokine is an IL-2 variant and the cell surface receptor is an IL-2 receptor, or wherein the cytokine is IL-18 and the cell surface receptor is IL-18 receptor.

[0029] In some embodiments, the IL-2 receptor comprises one or more mutations relative to a wildtype IL-2 receptor.

[0030] In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 5%, 10%, 20%, 40%, 60%, 80% or more.

[0031] In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 80%, 60%, 40%, 20%, 10%, 5% or less.

[0032] In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 5%, 10%, 20%, 40%, 60%, 80% or more.

[0033] Also provided herein is a method for producing a plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells, the method comprising: (a) providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a biological sample from a subject; (b) co-culturing APCs from the population of immune cells and T cells from the population of immune cells in the presence of IL-21, thereby forming a population of cells comprising stimulated T cells; and (c) expanding the stimulated T cells in the absence of IL-21, thereby forming the plurality of T cells that comprises antigen-specific T cells.

[0034] In some embodiments, expanding the stimulated T cells in (c) comprises expanding the stimulated T cells in the presence of one or more stimulatory agents selected from the group consisting of IL-7, IL-15, IL-2 and an IL-2 variant.

[0035] In some embodiments, the biological sample is a single biological sample.WSGR Docket No.: 50401-791.602

[0036] In some embodiments, the method further comprises, prior to co-culturing in (b): (i) separating the APCs and the T cells in the population of immune cells, thereby forming a population of APCs separated from a population of T cells; and (ii) culturing the population of APCs in a first vessel the presence of a first stimulatory agent and culturing the population of T cells in a second vessel in the presence of a second stimulatory agent, wherein the first vessel and the second vessel are different vessels.

[0037] In some embodiments, the first stimulatory agent comprises one or more agents selected from the group consisting of FLT3L, IL-4 GM-CSF, and any combination thereof.

[0038] In some embodiments, the second stimulatory agent comprises one or more agents selected from the group consisting of IL-2, IL-7, IL-15, IL-21, and any combination thereof.

[0039] In some embodiments, the method further comprises, prior to co-culturing in (b), delivering (A) at least one 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 at least one polypeptide into the population of APCs.

[0040] In some embodiments, delivering comprises electroporating the polynucleotide encoding the polypeptide into the population of APCs.

[0041] In some embodiments, delivering comprises contacting the population of APCs with the polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer.

[0042] In some embodiments, delivering comprises contacting a lipid nanoparticle or a lipoplex the polynucleotide encoding the polypeptide to the population of APCs.

[0043] In some embodiments, the method further comprises, prior to co-culturing in (b), maturing the population of APCs in the first vessel.

[0044] In some embodiments, maturing comprises maturing the population of APCs in the firstvessel in the presence of IFN and / or a toll like receptor agonist lipopolysaccharide (LPS).

[0045] In some embodiments, the method further comprises, prior to co-culturing in (b), genetically editing the population of T cells.

[0046] In some embodiments, genetically editing the population of T cells comprises inhibiting expression of an endogenous gene in the population of T cells.

[0047] 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.WSGR Docket No.: 50401-791.602

[0048] 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 an miRNA or siRNA that targets an RNA transcript encoded by the endogenous gene.

[0049] 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.

[0050] In some embodiments, silencing the 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 site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator into the population of T cells via electroporation, a lipid nanoparticle, a lipoplex, a liposome, nucleofection, or a viral vector.

[0051] In some embodiments, genetically editing comprises delivering an exogenous gene into the population of T cells via transposon.

[0052] In some embodiments, the exogenous gene encodes a cell surface receptor that binds to a cytokine, or wherein the exogenous gene encodes a switch receptor or a safety switch for controlling activities of T cells.

[0053] In some embodiments, the cytokine is an IL-2 variant and the cell surface receptor is an IL-2 receptor, or wherein the cytokine is IL-18 and the cell surface receptor is IL-18 receptor.

[0054] In some embodiments, the IL-2 receptor comprises one or more mutations relative to a wild type IL-2 receptor.

[0055] In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 5%, 10%, 20%, 40%, 60%, 80% or more.

[0056] In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 80%, 60%, 40%, 20%, 10%, 5% or less.

[0057] In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 5%, 10%, 20%, 40%, 60%, 80% or more.

[0058] In some embodiments, the method further comprises, prior to co-culturing in (b): depleting one or more cells selected from the group consisting of CD14+ cells, CD25+ cells and CD56+ cells from the population of immune cells.

[0059] In some embodiments, the method further comprises, prior to co-culturing in (b): (i) depleting one or more cells selected from the group consisting of CD14+ cells, CD11b+ cells, CD25+ cells and CD56+ cells from the population of immune cells, thereby forming a depleted population of immune cells comprising a first population of APCs and T cells; (ii) incubating the first population of APCs and T cells for a first time period in the presence of: FMS-like tyrosineWSGR Docket No.: 50401-791.602 kinase 3 receptor ligand (FLT3L), and (A) at least one 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 at least one polypeptide; thereby forming the population of cells comprising the stimulated T cells.

[0060] In some embodiments, the method further comprises enriching CD25+ cells, CD39+cells, 4-1BB+ cells, CD69+ cells, HLA-DR+ cells, LFA-1+ cells, IFN-g+ cells, TNF + cells,ITGB7+ cells or any combination thereof from the population of cells comprising the stimulated T cells.

[0061] In some embodiments, the method further comprises enriching CD25+ cells, 4-1BB+ cells, CD69+ cells, IFN-g+ cells, or any combination thereof from the population of cells comprising the stimulated T cells.

[0062] In some embodiments, a higher number of antigen-specific T cells is produced by the method compared to a corresponding method in which the T cells are expanded in the presence of IL-21.

[0063] In some embodiments, a higher number of antigen-specific T cells is produced by the method compared to a corresponding method in which the co-culturing is performed in the absence of IL-21.

[0064] Also provided herein is a method for producing a plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells, the method comprising: (a) depleting one or more cells selected from the group consisting of CD14+ cells, CD11b+ cells, CD25+ cells and CD56+ cells from the population of immune cells, thereby forming a depleted population of immune cells comprising a first population of APCs and T cells; (b) incubating the first population of APCs and T cells from step (a) for a first time period in the presence of: (A) at least one 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 at least one polypeptide; thereby forming a population of cells comprising stimulated T cells.

[0065] In some embodiments, the method comprises enriching CD25+ cells, CD39+ cells, 4-1BB+ cells, CD69+ cells, HLA-DR+ cells, ITGB7+ cells, LFA-1+ cells, IFN-g+ cells, TNF +cells, or any combination thereof from the population of cells comprising the stimulated T cells.

[0066] In some embodiments, depleting in (a) comprises depleting CD25+ cells from the population of immune cells.

[0067] In some embodiments, depleting in (a) comprises depleting CD25+ cells from the population of immune cells, and wherein enriching in (c) comprises enriching CD25+ cells from the population of cells comprising the stimulated T cells.WSGR Docket No.: 50401-791.602

[0068] In some embodiments, incubating in (b) is performed in the presence of FMS-like tyrosine kinase 3 receptor ligand (FLT3L).

[0069] In some embodiments, the method further comprises expanding the population of cells comprising the stimulated T cells.

[0070] In some embodiments, expanding comprises expanding the population of cells comprising the stimulated T cells in the presence of one or more stimulatory agents selected from the group consisting of IL-7, IL-15, IL-2, an IL-2 variant, and any combination thereof.

[0071] In some embodiments, incubating the first population of APCs and T cells from step (a) comprises delivering (A) the polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) the polynucleotide encoding the polypeptide into the APCs of the first population of APCs and T cells.

[0072] In some embodiments, delivering comprises contacting a first lipid delivery vehicle comprising the polynucleotide encoding the polypeptide to the APCs of the first population of APCs and T cells.

[0073] In some embodiments, the first lipid delivery vehicle is APC specific.

[0074] In some embodiments, the first lipid delivery vehicle delivers the polypeptide or the polynucleotide into APC cells and does not deliver the polypeptide or the polynucleotide into the T cells.

[0075] In some embodiments, incubating the first population of APCs and T cells from step (a) further comprises genetically editing the T cells of the first population of APCs and T cells.

[0076] In some embodiments, genetically editing the population of T cells comprises inhibiting expression of an endogenous gene in the population of T cells.

[0077] 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.

[0078] 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 an miRNA or siRNA that targets an RNA transcript encoded by the endogenous gene.

[0079] 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.

[0080] In some embodiments, silencing the 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 site-specific nuclease, the site-specific nickase, or the site-specific epigeneticWSGR Docket No.: 50401-791.602 regulator into the population of T cells via electroporation, a lipid nanoparticle, a lipoplex, a liposome, nucleofection, or a viral vector.

[0081] In some embodiments, genetically editing comprises delivering an exogenous gene into the population of T cells via transposon and the percent of T cells in the population of T cells expressing the exogenous gene is at least 5%, 10%, 20%, 40%, 60%, 80% or more.

[0082] In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 80%, 60%, 40%, 20%, 10%, 5% or less.

[0083] In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 5%, 10%, 20%, 40%, 60%, 80% or more.

[0084] In some embodiments, the method further comprises administering the plurality of T cells that comprises antigen-specific T cells into a subject in need thereof.

[0085] In some embodiments, the time from (a) to obtaining a therapeutically effective amount of antigen-specific T cells is less than 20 days, less than 15 days, or less than 10 days.

[0086] Also provided herein is a method for producing a plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells, the method comprising: (a) providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a biological sample from a subject; (b) co-culturing APCs from the population of immune cells and T cells from the population of immune cells, thereby forming a population of cells comprising stimulated T cells; and (c) expanding the stimulated T cells, thereby forming the plurality of T cells that comprise antigen-specific T cells comprising at least 106, at least 107, at least 108, or at least 109 antigen-specific T cells, wherein the time from (a) to obtaining the plurality of T cells that comprise antigen-specific cells is less than 20 days, less than 19 days, less than 18 days, less than 17 days, less than 16 days, less than 15 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, or less than 10 days.

[0087] Also provided herein is a method for producing a plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells, the method comprising: (a) incubating APCs and T cells (i) in the presence of (A) at least one 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 at least one polypeptide, or (ii) wherein the APCs comprise (A) at least one 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 at least one polypeptide, thereby forming a population of cells comprising stimulated T cells, wherein the stimulated T cells or a subset thereof express an IL-2 receptor variant, and wherein expression of the IL-2 receptor variant is induced upon T cell simulation; and (b) culturing the population ofWSGR Docket No.: 50401-791.602 cells comprising stimulated T cells in the presence of IL-2 variant, wherein the stimulated T cells or a subset thereof expressing the IL-2 receptor variant expand, thereby obtaining the plurality of T cells comprises antigen-specific T cells.

[0088] In some embodiments, the stimulated T cells or a subset thereof expressing the IL-2 receptor variant comprises a nucleic acid sequence comprising a regulatory sequence and a sequence encoding the IL-2 receptor variant.

[0089] In some embodiments, the regulatory sequence comprises one or more Nuclear Factor of Activated T cells (NFAT) response elements (REs).

[0090] In some embodiments, the regulatory sequence comprises a promoter.

[0091] In some embodiments, the promotor is a minimal promoter.

[0092] In some embodiments, the minimal promoter comprises a MinP promoter, a yb TATA promoter, or a combination thereof.

[0093] In some embodiments, the method further comprises, prior to (a), delivering a nucleic acid sequence comprising a regulatory sequence and a sequence encoding the IL-2 receptor variant into the T cells or a subset thereof.

[0094] In some embodiments, delivering comprises electroporating the nucleic acid sequence into the T cells or a subset thereof.

[0095] In some embodiments, delivering comprises delivering a transposon comprising the nucleic acid sequence into the T cells or a subset thereof.

[0096] In some embodiments, delivering comprises electroporating a transposon comprising the nucleic acid sequence into the T cells or a subset thereof.

[0097] In some embodiments, delivering comprises using lipopolysaccharide (LPS), liposome, lipid nanoparticle, or lipoplex to deliver the nucleic acid sequence into the T cells or a subset thereof.

[0098] In some embodiments, the T cells express endogenous TCRs.

[0099] In some embodiments, the T cells do not express exogenous TCRs.

[0100] In some embodiments, the stimulated T cells or the subset thereof express an activation marker.

[0101] In some embodiments, the activation marker is CD69.

[0102] In some embodiments, the frequency of T cells expressing the IL-2 receptor variant is about 5-fold, about 10-fold, about 15-fold, or about 20-fold higher among the stimulated T cells or the subset thereof compared to a corresponding population of non-stimulated T cells.WSGR Docket No.: 50401-791.602

[0103] In some embodiments, the stimulated T cell or the subset thereof have a fold expansion of 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold relative to a corresponding population of non-stimulated T cells.

[0104] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD8+ T cell epitope.

[0105] In some embodiments, the CD8+ T cell epitope comprises a sequence of about 7 to about 12 consecutive amino acids from a cancer protein.

[0106] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least two different CD8+ T cell epitope sequences.

[0107] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD4+ T cell epitope.

[0108] In some embodiments, the CD4+ T cell epitope comprises a sequence of about 13 to about 25 consecutive amino acids from a cancer protein.

[0109] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least two different CD4+ T cell epitope sequences.

[0110] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a linker between the at least two different CD8+ T cell epitope sequences and the at least two different CD4+ T cell epitope sequences.

[0111] In some embodiments, the polynucleotide encoding the polypeptide encodes a linker.

[0112] In some embodiments, the linker comprises a cleavable linker and / or a linker that is degradable by a protease.

[0113] In some embodiments, the least two different CD8+ T cell epitope sequences are separated by one or more linkers.

[0114] In some embodiments, the least two different CD4+ T cell epitope sequences are separated by one or more linkers.

[0115] In some embodiments, the one or more linkers are one or more flexible linkers.

[0116] In some embodiments, the one or more linkers are one or more cleavable linkers.

[0117] In some embodiments, the polypeptide further comprises a Sec domain sequence at its N-terminus.

[0118] In some embodiments, the polypeptide further comprises a MITD domain sequence at its C-terminus. INCORPORATION BY REFERENCEWSGR Docket No.: 50401-791.602

[0119] 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

[0120] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0121] FIG. 1 depicts an experimental outline to test efficiency of integration of transposon constructs during a process of cell coculture and enrichment of antigen specific cells.

[0122] FIG. 2 depicts an experimental outline with three arms to test the efficacy of removing certain cell populations before coculturing to enhance efficacy of transposon integration.

[0123] FIGs. 3A-3L illustrate results from the experiments outlined in FIG. 2. FIG. 3A depicts expansion of total cell numbers from human donor ID: HD278. FIG. 3C depicts expansion of total cell numbers from human donor ID: HD282. FIG. 3E depicts expansion of total cell numbers from human donor ID: HD280. FIG. 3B depicts viability of cells from human donor ID:HD278. FIG. 3D depicts viability of cells from human donor ID: HD282. FIG. 3F depicts viability of cells from human donor ID: HD280. FIG. 3G depicts frequency of antigen specific cells from human donor ID: HD278. FIG. 3I depicts frequency of antigen specific cells from human donor ID: HD282. FIG. 3K depicts frequency of antigen specific cells from human donor ID: HD280. FIG. 3H depicts number of antigen specific cells from human donor ID: 278. FIG. 3J depicts number of antigen specific cells from human donor ID: 280. FIG. 3L depicts number of antigen specific cells from human donor ID: 282.

[0124] FIGs. 4A-4F illustrate results from the experiments outlined in FIG. 2. FIG. 4A depicts Frequency of claudin 6 (CLDN6) chimeric antigen receptor (CAR) positive CD8 T cells in human donor ID: 278. FIG. 4C depicts frequency of claudin 6 (CLDN6) chimeric antigen receptor (CAR) positive CD8 T cells in human donor ID: 280. FIG. 4E depicts frequency of claudin 6 (CLDN6) chimeric antigen receptor (CAR) positive CD8 T cells in human donor ID: 282. FIG. 4B depicts frequency of CLDN6 CAR positive antigen specific cells in human donor ID: 278. FIG. 4D depicts frequency of CLDN6 CAR positive antigen specific cells in human donor ID: 280. FIG. 4F depicts frequency of CLDN6 CAR positive antigen specific cells in human donor ID: 282. All results were quantified via flow cytometry.WSGR Docket No.: 50401-791.602

[0125] FIG. 5 depicts an experimental outline to test efficiency of integration of transposon construction with a process of cell coculture and enrichment of antigen specific cells.

[0126] FIG. 6 depicts an experimental outline with three arms to test the efficacy of removing certain cell populations before coculturing and addition of TransAct reagent to enhance efficacy of transposon integration.

[0127] FIGs. 7A-7H depict results from the experiment outlined in FIG. 6. X axis depicts donor from which cells were obtained. Each bar represents a different electroporation (EP) program. Within each donor bars, from left to right, correspond to the EP programs EA100, EO115, DS137, FT150, and negative mock control. FIG. 7A depicts cell viability with no TransAct treatment. FIG. 7B depicts cell viability with TransAct treatment. FIG. 7C depicts cell numbers with no TransAct treatment. FIG. 7D depicts cell number with TransAct treatment. FIG. 7E depicts frequency of Venus positive T cells with no TransAct treatment. FIG. 7F depicts frequency of Venus positive T cells with TransAct treatment. FIG. 7G depicts mean fluorescence intensity of Venus with no TransAct treatment. FIG. 7H depicts mean fluorescence intensity of Venus with TransAct treatment.

[0128] FIG. 8 depicts an experimental outline to test the effects of altering cell type separation and coculture parameters to enhance efficiency of transposon integration.

[0129] FIGs. 9A-9C depict results from the experiment outlined in FIG. 8. FIG. 9A depicts number of total cells (upper panel) and frequency of CD8 T cells (lower panel) in human donor ID: 227. FIG. 9B depicts number of total cells (upper panel) and frequency of CD8 T cells (lower panel) in human donor ID: 278. FIG. 9C depicts number of total cells (upper panel) and frequency of CD8 T cells (lower panel) in human donor ID: 280. All data compared cell types which were separated at day 0 or day 3 of culture.

[0130] FIG. 10 depicts the ratio of APCs to T cells across different donors in each experimental condition when cell types are separated at day 0 or day 3 of culture. APCs are depicted in the lower portion of each bar, T cells are depicted in the higher portion of each bar.

[0131] FIGs. 11A-11F illustrate flow cytometric analyses. FIG. 11A depicts frequency of multimer positive CD8 T cells (upper panel) and total number of multimer positive cells (lower) using cells obtained from human donor ID: 227. FIG. 11B depicts frequency of multimer positive CD8 T cells (upper panel) and total number of multimer positive cells (lower) using cells obtained from human donor ID: 278. FIG. 11C depicts frequency of multimer positive CD8 T cells (upper panel) and total number of multimer positive cells (lower) using cells obtained from human donor ID: 280. FIG. 11D depicts frequency of CLDN6 CAR positive cells (upper panel) and total number of CLDN6 CAR positive cells (lower panel) using cells obtainedWSGR Docket No.: 50401-791.602 from human donor: 227. FIG. 11E depicts frequency of CLDN6 CAR positive cells (upper panel) and total number of CLDN6 CAR positive cells (lower panel) using cells obtained from human donor: 278. FIG. 11F depicts frequency of CLDN6 CAR positive cells (upper panel) and total number of CLDN6 CAR positive cells (lower panel) using cells obtained from human donor: 280.

[0132] FIGs. 12A-12C illustrate T cell phenotype analysis via flow cytometry. FIG. 12A depicts frequency of native (Tn), stem cell memory (Tscm), central memory (Tcm), effector memory (Tem), and effector memory RA+ (TEMRA) cells in both CD8 (upper panel) and CD4 (lower panel) using cells from human donor ID: 227. FIG. 12B depicts frequency of native (Tn), stem cell memory (Tscm), central memory (Tcm), effector memory (Tem), and effector memory RA+ (TEMRA) cells in both CD8 (upper panel) and CD4 (lower panel) using cells from human donor ID: 278. FIG. 12C depicts frequency of native (Tn), stem cell memory (Tscm), central memory (Tcm), effector memory (Tem), and effector memory RA+ (TEMRA) cells in both CD8 (upper panel) and CD4 (lower panel) using cells from human donor ID: 280.

[0133] FIGs. 13A-13B depict two experimental outlines testing culture conditions. FIG. 13A depicts an experiment wherein APCs and T cells are not separated. FIG. 13B depicts an experiment wherein APCs and T cells are separated and cultured in different conditions prior to coculture.

[0134] FIG. 14 depicts six different experimental conditions used to test the ratios of T cells to APC and total number of either population during a coculture experiment.

[0135] FIGs. 15A-15L illustrate results obtained from the experimental outline presented in FIG. 14. FIG. 15A depicts fold change of cell expansion. FIG. 15B depicts CAR expression in CD8 T cells. FIG. 15C depicts CAR expression in CD4 T cells. FIG. 15D depicts frequency of antigen specific CD8 T cells. FIG. 15E depicts total number of antigen specific T cells. FIG. 15F depicts frequency of CAR expressing cells among antigen specific or non-antigen specific T cells. FIG. 15G depicts T cell subset analysis of CD8 T cells. FIG. 15H depicts expression of CD28. FIG. 15I depicts expression of CD127. FIG. 15J depicts antigen specific cell expansion measured by frequency. FIG. 15K depicts antigen specific cell expansion measured by fold change. FIG. 15L depicts cytokine production. In FIG. 15F, within each APC:T cell ratio and cell density tested, the left data points represent the antigen specific cells and the right data points represent the antigen nonspecific cells.

[0136] FIG. 16 depicts an experimental timeline wherein PBMCs are depleted of CD25 / 56 cells and CD14 / 19 expressing cells are separated. T cells remaining are cultured for 3 days and analyzed.WSGR Docket No.: 50401-791.602

[0137] FIG. 17 depicts the experimental conditions tested to determine the effects of cytokine presence on T cells after 3 days of culture.

[0138] FIGs. 18A-18G depict results obtained from the experiment outlined in FIG. 17. FIG. 18A depicts overall yield of T cells. FIG. 18B depicts viability. FIG. 18C depicts purity of T cells as measured when cultured in the indicated cytokine environments. FIG. 18D depicts expression of CD27. FIG. 18E depicts expression of CD28. FIG. 18F depicts expression of CD127. FIG. 18G depicts memory phenotype subset analysis among CD8 T cells.

[0139] FIGs. 19A-19B illustrate two experimental outlines testing the effects of enrichment methods. FIG. 19A depicts an experiment using a manual enrichment (FIG. 19A) of immune cells. FIG. 19B depicts an experiment using an intrinsic enrichment step in enhancing the generation of antigen specific T cells.

[0140] FIG. 20 illustrates exemplary data measuring expression of 4-1BB before (top) and after (bottom) enrichment steps.

[0141] FIG. 21A illustrates data measuring the frequency of antigen specific T cells before and after enrichment when stimulated with indicated antigens.

[0142] FIG. 21B depicts a cell culture set up to test the use of additional APCs for a restimulation.

[0143] FIG. 22A illustrates data measuring the expression of various activation markers by the indicated population of T cells, either antigen specific or not antigen specific and either 3hr, 7hr, or overnight (O / N) coculture.

[0144] FIG. 22B depicts exemplary flow cytometry data representing expression of activation markers in either antigen specific T cells (bottom panels) or not antigen specific (top panels).

[0145] FIGs. 23A-23B depict exemplary flow cytometry data. FIG. 23A depicts multimer expressing (antigen specific) T cells among bulk CD8, CD39, CD25, or 4-1BB. FIG. 23B depicts multimer expressing (antigen specific) T cells among CD69, HLA-DR, LFA1, IFNg, orTNF . Top panels were in the absence of a restimulation, the bottom panels were 7h followingrestimulation.

[0146] FIG. 24 depict an experimental workflow testing the effects of stimulation with viralpeptides and using a IFN / TNF secretion assay to potentiate enrichment of antigen specificcells.

[0147] FIGs. 25A-25B depict exemplary flow cytometry experiments measuring antigen specific cell frequency. FIG. 25A depicts antigen specific cell frequency among total CD8 T cells when cells are stimulated with Epstein-Barr Virus peptides (top panels) or cytomegalovirus peptides (bottom panels). FIG. 25B depicts antigen specific cell frequency gated initially onWSGR Docket No.: 50401-791.602IFN +, TNF + or IFN +TNF + CD8 T cells when cells are stimulated with Epstein-Barr Viruspeptides (top panels) or cytomegalovirus peptides (bottom panels).

[0148] FIG. 25C depicts an experimental outline to test activation of CD25+ or CD25- cells.

[0149] FIG. 25D depicts results measuring frequency of antigen specific cells.

[0150] FIG. 26 depict an experimental outline measuring the enrichment efficacy across a 14 day culture experiment.

[0151] FIG. 27 depict exemplary analysis methods used to examine the properties of T cells in the experiment outlined in FIG. 26.

[0152] FIG. 28 illustrates an exemplary experimental outline measuring enrichment of antigen specific T cells.

[0153] FIG. 29 outlines an experimental procedure wherein antigen presenting cells (APCs) are generated for restimulation.

[0154] FIG. 30 illustrates the experimental outline to measure the expression of enrichment markers without restimulation.

[0155] FIG. 31A depicts results measuring frequency of antigen specific cells. FIG. 31B depicts results of assessing the phenotypes of multimer CD8 CD45RA and CCR7 triple positive cells. Results were obtained from cells cultured in six well plates.

[0156] FIG. 32A depicts results measuring cell purity (percent multimer positive in each activation marker positive CD8s). FIG. 32B depicts results measuring cell coverage (percent multimer positive activation marker positive divided by total frequency of multimer positive in total CD8 T cells).

[0157] FIG. 33 depicts effects of enriching for the indicated markers on the generation of antigen specific CD8 T cells.

[0158] FIG. 34 depicts an experimental outline to determine the effects of using different combination of enrichment markers after restimulation.

[0159] FIGs. 35A-35D depict results obtained using the indicated enrichment markers. FIG. 35A depicts purity of the antigen specific population. FIG. 35B depicts fold improvement over non-enriched cells. FIG. 35C depicts cell coverage. FIG. 35D depicts frequency of antigen specific cells among CD8 T cells.

[0160] FIG. 36 illustrates an experimental outline wherein antigen specific T cells are sorted and expanded following sort.

[0161] FIGs. 37A-37B illustrate data obtained measuring frequency of antigen specific cells. FIG. 37A depicts exemplary flow cytometry plots. FIG. 37B depicts frequency of antigen specific cells when stimulated with various antigens.WSGR Docket No.: 50401-791.602

[0162] FIG. 38 depicts T cell subtype analysis results obtained among antigen specific T cells or non antigen specific T cells before and after the sorting step.

[0163] FIG. 39 depicts data measuring IFN secretion when sorted antigen specific T cells arestimulated with peptide.

[0164] FIG. 40 illustrates an experimental outline to determine the efficacy of utilizing IFNg catch when isolating CD8 T cells prior to expansion.

[0165] FIG. 41A depicts data obtained measuring fold change of antigen specific cells enriched post isolation.

[0166] FIG. 41B depicts data obtained measuring fold change of antigen specific cells enriched after 6 days of expansion.

[0167] FIG. 42 depicts flow cytometry data measuring frequency of antigen specific T cells in the indicated condition among 2 different donor sources.

[0168] FIG. 43 depicts data measuring the frequency of antigen specific T cells with various conditions post expansion of the antigen specific T cells.

[0169] FIG. 44 depicts data measuring total number of antigen specific T cells with various conditions post expansion of the antigen specific T cells.

[0170] FIGs. 45A-45B illustrate T cell phenotypes among multimer positive or negative populations of CD8 T cells. FIG. 45A depicts phenotypes of CD45RA / CCR7 CD8 T cells. FIG. 45B depicts phenotypes of TIM3 CD39 CD8 T cells.

[0171] FIG. 46 illustrates an experimental outline using sequencing approaches to determine the expression of certain enrichment markers over time.

[0172] FIG. 47 depicts results from a T cell receptor sequencing experiment, each bar represents a single unique TCR clone specific for Gli3 antigen.

[0173] FIG. 48 depicts RNA sequencing results measuring expression of potential enrichment markers in antigen specific T cell cluster.

[0174] FIGs. 49A-49B depict results obtained from cite-SEQ and VDJ sequencing of CD8 T cells to analyze expression profiles at different time points across the experiment. FIG. 49A depicts combined analysis at days 3, 5, 7, 9, and 11 for two donor samples HD293 and HD294. FIG. 49B depicts Antigen specific cells from both donor clustered in same location and growth kinetics by frequency.

[0175] FIG. 50 depicts expression data measuring gene expression of indicated markers in various T cell populations.

[0176] FIG. 51 depicts expression of indicated markers associated with antigen specific T cells.WSGR Docket No.: 50401-791.602

[0177] FIGs. 52A-52B depict enrichment of expression of indicated markers over time among non-antigen specific T cells (lower line) or antigen specific T cells (upper line). FIG. 52A depicts expression of IL2RA, ENTPD1, and ITGB7. FIG. 52B depicts IL2RA+ITGB7+, ITGB7+ENTPD1+, IL2RA+ENTPD1+ expression.

[0178] FIG. 53 depicts an exemplary experimental outline testing the use of certain activation markers and changes in APC / T cell separation and coculture.

[0179] FIG. 54 illustrates an experimental outline wherein T cells are engineered to express a transgene under the control of a TCR signaling-induced promoter to measure T cell activation.

[0180] FIG. 55 illustrate exemplary constructs with 6 NFAT binding sites and a minimal promoter to assess expression of the indicated marker.

[0181] FIG. 56 illustrates an experimental workflow to measure the expression of CD69, the VENUS transgene, and IL-2 secretion to measure activation of T cells.

[0182] FIG. 57A illustrates the mechanism of the VENUS transgene expression when T cells bearing the transgene become activated.

[0183] FIG. 57B depicts an exemplary construct with 6 NFAT binding sites to assess expression of reporter markers upon T cell activation.

[0184] FIGs. 58-59 illustrate exemplary experimental outlines to measure the effects of enrichment through activation markers following restimulation. FIG. 58 depicts an overview of the NEO STIM process. FIG. 59 depicts a detailed experimental design to test effects of enrichment.

[0185] FIG. 60 depicts an experimental outline evaluating the enrichment efficiency of antigen specific cells from total T cells with specific activation markers.

[0186] FIGs. 61A-61M illustrate data obtained in the experiment illustrated in FIG. 60. FIG. 61A depicts yield post isolation of antigen specific T cells. FIG. 61B depicts frequency of CD8 and CD4 T cells. FIG. 61C depicts CD8 subset analysis. FIG. 61D depicts frequency of antigen specific cells. FIG. 61E depicts enrichment marker expression following expansion. FIG. 61F depicts numbers of cells expressing IFNg, 4-1BB, or CD25. FIG. 61G depicts expression of 4- 1BB, IFNg, or CD25 when cells are restimulated with either peptide pools or with specific neoantigens. FIG. 61H depicts production of IFN-g using 4-1BB enrichment. FIG. 6I depicts production of IFN-g using IFN-g enrichment. FIG. 61J depicts production of IFN-g using CD25 enrichment. FIG. 61K depicts IFN-g production when using 4-1BB enrichment and stimulating with antigens FIG. 61L depicts IFN-g production when using IFNg enrichment and stimulating with antigens. FIG. 61M depicts IFN-g production when using CD25 enrichment andWSGR Docket No.: 50401-791.602 stimulating with antigens. In FIG. 61A, within each donor noted with labels beginning with“HD”, bars depict, from left to right, 4-1BB, IFN- and CD25.

[0187] FIG. 62 illustrates an experimental outline to evaluate enrichment efficiency of neoantigen specific using bead / column isolation and using different selection markers and combinations of selection markers.

[0188] FIGs. 63A-63F illustrate data measuring frequency and total number of antigen specific cells when cells are selected using various selection markers. FIG. 63A depicts exemplary flow cytometry plots measuring frequency of antigen specific cells. FIG. 63B depicts total antigen specific cell numbers. FIG. 63C depicts frequency of antigen specific cells when using 4-1BB enrichment. FIG. 63D depicts frequency of antigen specific cells when using IFNg enrichment. FIG. 63E depicts frequency of antigen specific cells when using CD25 enrichment. FIG. 63Fdepicts frequency of antigen specific cells when using TNF +4-1BB enrichment. This was alsomeasured across time points in the experiment.

[0189] FIGs. 64A-64C depict data measuring cells selected for using different markers as indicated. FIG. 64A depicts total cell numbers post expansion. FIG. 64B depicts cell trace violet mean fluorescence intensity using cells from human donor ID: 301. FIG. 64C depicts cell trace violet mean fluorescence intensity using cells from human donor ID: 337. For FIG. 64A, within each condition, the left bar represents day 0 post isolation, and the right bar represents day 6 post isolation.

[0190] FIGs. 65A-65B depict data measuring T cell phenotypes or overall frequency of various immune cell populations in cells selected for using different markers. FIG. 65A depicts T cell phenotypes of antigen specific cells. FIG. 65B depicts overall frequency of immune cell populations.

[0191] FIGs. 66A-66B depict data measuring frequency of certain T cell populations and frequency of regulatory T cells (Treg) in cells selected for using different markers and across different donors. FIG. 66A depicts frequency of T cell subset populations. FIG. 66B depicts frequency of Tregs. In FIG. 66B, bars within each treatment group represent data from two different donor populations.

[0192] FIGs. 67A-67F illustrate data measuring frequency of antigen specific T cells and fold change of expansion of antigen specific T cells in cells selected for using different markers and across different donors. FIG. 67A depicts antigen specific cells using 4-1BB enrichment. FIG.67B depicts antigen specific cells using IFN enrichment. FIG. 67C depicts antigen specificcells using CD25 enrichment. FIG. 67D depicts fold change using 4-1BB enrichment. FIG. 67EWSGR Docket No.: 50401-791.602 depicts fold change using IFNg enrichment. FIG. 67F depicts fold change using CD25 enrichment.

[0193] FIGs. 68-69A illustrate experimental outlines to measure the effect of electroporation on T cells and subsequent enrichment of antigen specific cells. FIG. 68 depicts an experiment testing electroporation protocols. FIG. 69A depicts an overview of the NEO-STIM process.

[0194] FIG. 69B depicts exemplary construct designs utilizing nuclear factor of activated T cell (NFAT domain).

[0195] FIG. 69C illustrates an exemplary experimental design to test the efficacy of NFAT constructs.

[0196] FIG. 70 is a schematic describing a transgene that is used to allow for selective expansion of activated antigen specific cells compared to non-activated cells.

[0197] FIG. 71 shows a transgene comprising NFAT binding sites, a minimal promoter, and a IL2Ra mut3 or Venus reporter cassette. The cargo of the transgene can be IL2Ra mut3. As a proof of concept experiment, the cargo used was Venus reporter gene. Exemplary promoters include ybTATA and MinP promoters.

[0198] FIG. 72 shows an experimental workflow to measure activation via Venus expression of T cells electroporated with transgenes encoding IL2Ra mut Venus reporters.

[0199] FIGs. 73A-73B show results of the experiment described in FIG. 72. FIG. 73A shows results measuring Venus expression among Jurkat cells expressing TCR1. FIG. 73B shows results measuring Venus expression among Jurkat cells expressing TCR5.

[0200] FIG. 74 shows a transgene comprising NFAT binding sites, a minimal reporter, a IL2Ra mut3 Venus reporter cassette and a truncated CD34.

[0201] FIG. 75A depicts results measuring activation of T cells expressing various TCRs and electroporated with various transgene constructs. Activation was measured via CD69 expression across various cognate peptide concentrations.

[0202] FIGs. 75B-75D depict quantification of CD34 expression by T cells electroporated with the indicated transgene construct, and incubated with cognate peptide at various concentrations. FIG. 75B depicts results using T cells expressing TCR1. FIG. 75C depicts results using T cells expressing TCR5. FIG. 75D depicts results using T cells expressing TCR6.

[0203] FIG. 76 is a schematic of an experiment testing the effectiveness of using CRISPR-Cas9 to knockout gene targets prior to activation and expansion of T cells.

[0204] FIG. 77A shows results measuring bulk T cell expansion when the indicated genes are knocked out. FIG. 77B shows results measuring expression of CD127 among antigen specific CD8 T cells when the indicated genes are knocked out. FIG. 77C shows results measuring theWSGR Docket No.: 50401-791.602 differentiation status of antigen specific CD8 T cells when the indicated genes are knocked out. FIG. 77D shows results measuring expression of Ly108, a TCF1 surrogate marker, among antigen specific CD8 T cells when the indicated genes are knocked out.

[0205] FIGs. 78A-78L show results measuring the expansion of antigen specific CD8 T cells when the indicated genes are knocked out and where appropriate NTC (intergenic controls) were used to mimic double stranded breaks that occur in a dual knockout combination. FIG. 78A shows results using MART1 mRNA string and targeting PRDM1 and TNFAIP3. FIG. 78B shows results using MART1 mRNA string and targeting PRDM1 and REGNASE1 (REG-1). FIG. 78C shows results using MART1 mRNA string and targeting TNFAIP3 and REG-1. FIG. 78D shows results using MART1 mRNA string and targeting PRDM1. FIG. 78E shows results using MART1 mRNA string and targeting TNFAIP3. FIG. 78F shows results measuring MART1 mRNA string and targeting REG-1. FIG. 78G shows results using CSNK1 mRNA string and targeting PRDM1 and TNFAIP3. FIG. 78H shows results using CSNK1 mRNA string and targeting PRDM1 and REG-1. FIG. 78I shows results using CSNK1 mRNA string and targeting TNFAIP3 and REG-1. FIG. 78J shows results using CSNK1 mRNA string and targeting PRDM1. FIG. 78K shows results using CSNK1 mRNA string and targeting TNFAIP3. FIG. 78L shows results measuring CSNK1 mRNA string and targeting REG-1.

[0206] FIG. 79A show results measuring fold expansion of bulk T cells when the indicated genes are knocked out. MART-1 and CSNK1 decamer APCs indicate two different immunogen strings used to induce antigen-specific cells. FIG. 79B show results measuring frequency of peptide-MHC (pMHC) bound by CD8 T cells.

[0207] FIG. 80 is a schematic of an experiment testing the effectiveness of using CRISPR-Cas9 to knockout gene targets prior to activation and expansion of T cells which are obtained from human donors and stimulated using APCs loaded with patient specific mRNA strings.

[0208] FIG. 81A show results measuring editing efficiency of the indicated genes either in single targeted groups or dual gene targeted groups. Results using samples from another donor are shown in FIG. 81B.

[0209] FIG. 82A show results measuring total cell number (left) and fold expansion (right). Results using samples from another donor are shown in FIG. 82B.

[0210] FIGs. 83A-83B show representative flow cytometry plots demonstrating frequency of antigen specific cells in either a primary or memory response. FIG. 83A shows results in NTC control or PRDM1 single knock out. FIG. 83B shows results in REG1 single knock out or PRDM1 / REG1 double knock out.WSGR Docket No.: 50401-791.602

[0211] FIG. 84A shows frequency of antigen specific CD8 T cells which are specific for the indicated patient antigens. FIG. 84B shows total cell count of antigen specific cells which are specific for indicated patient antigens.

[0212] FIGs. 85A-85H shows results characterizing CD4 and CD8 T cell stemness. FIG. 85A shows results of CD4 T cell differentiation in donor 1. FIG. 85B shows results of CD8 T cell differentiation in donor 1. FIG. 85C shows results of CD127 expression among CD4 T cells in donor 1. FIG. 85D shows results of CD127 expression among CD8 T cells in donor 1. FIG. 85E shows results of CD4 T cell differentiation in donor 2. FIG. 85F shows results of CD8 T cell differentiation in donor 2. FIG. 85G shows results of CD127 expression among CD4 T cells in donor 2. FIG. 85H shows results of CD127 expression among CD8 T cells in donor 2.

[0213] FIGs. 86A-86F show results measuring antigen specific T cell profiles among different antigen specific populations. FIG. 86A shows results for all antigen-specific populations. FIG. 86B shows results for T cells specific for the indicated antigens. FIG. 86C shows results for T cells specific to PRMT3. FIG. 86D shows results for T cells specific for RNF213. FIG. 86E shows results for T cells specific for CERS2. FIG. 86F shows results for T cells specific for DHX36.

[0214] FIGs. 87A-87F show results measuring expression of CD127 among different antigen specific populations. FIG. 87A shows results for all antigen-specific populations. FIG. 87B shows results for T cells specific for the indicated antigens. FIG. 87C shows results for T cells specific to PRMT3. FIG. 87D shows results for T cells specific for RNF213. FIG. 87E shows results for T cells specific for CERS2. FIG. 87F shows results for T cells specific for DHX36. DETAILED DESCRIPTION Definitions

[0215] The methods provided herein can generate a population of expanded and activated antigen-specific T cells. The T cell populations generated by the methods disclosed herein can provide an effective and scalable therapeutic solution to the challenges posed by current T cell manufacturing methods for use in treating cancer.

[0216] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0217] An antigen is a foreign substance 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, forWSGR Docket No.: 50401-791.602 example, a substitution in a protein sequence, a frame shift mutation, a fusion polypeptide, an in- frame deletion, an insertion, and expression of an endogenous retroviral polypeptide.

[0218] 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.

[0219] 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.

[0220] “Antigen processing” or “processing” refers to the degradation of a polypeptide or antigen into procession products, which are fragments of said polypeptide or antigen (e.g., the degradation of a polypeptide into peptides) and the association of one or more of these fragments (e.g., via binding) with MHC molecules for presentation by cells, for example, antigen presenting cells, to specific T cells.

[0221] An “antigen presenting cell” (APC) refers to a cell which presents 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, dendritic cells, Langerhans cells) as well as other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, oligodendrocytes). The term includes professional APCs or non- professional APCs. The term also includes dendritic cell precursors (e.g., monocytes). The monocytes can be CD14+ monocytes that can differentiate into monocyte derived dendritic cells. The term also includes a cell that is engineered to express an MHC molecule or a cell that expresses an endogenous MHC molecule.

[0222] 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 dissociationWSGR Docket No.: 50401-791.602 constant between two members of a binding pair and has units of molarity. KA refers to the affinity constant between two members of a binding pair is the inverse of the dissociation constant. Affinity may be determined experimentally, for example by surface plasmon resonance (SPR) using commercially available Biacore SPR units. Koffrefers 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).

[0223] Throughout this disclosure, “binding data” results may be expressed in terms of an “IC50.” Affinity may 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, ln(IC50) refers to the natural log of the IC50. For example, an IC50may 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 et al., 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)).

[0224] 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 residuesWSGR Docket No.: 50401-791.602 “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 may include naturally occurring, recombinant, unpurified, purified or differentiated molecules or cells. For example, an expanded or induced antigen specific T cell may be derived from a T cell. For example, an expanded or induced antigen specific T cell may be derived from an antigen specific T cell in a biological sample. For example, a matured APC (e.g., a professional APC) may be derived from a non-matured APC (e.g., an immature APC). For example, an APC may be derived from a monocyte (e.g., a CD14+monocyte). For example, a dendritic cell may be derived from a monocyte (e.g., a CD14+monocyte). For example, an APC may be derived from a bone marrow cell.

[0225] 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 may 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, or less than or equal to 50 amino acidWSGR Docket No.: 50401-791.602 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.

[0226] 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).

[0227] 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 may 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 for a specific therapy such as a cancer, e.g., melanoma. When drug candidate cells pass specific qualitative and quantitative criteria for fitness for a clinical application, the drug candidate may be designated a drug product. In some cases, a drug product is selected from a number of drug candidates. In the context of this application, a drug product is 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 may 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.

[0228] 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.

[0229] 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.

[0230] A “protective immune response” or “therapeutic immune response” refers to a CTL and / or an HTL response to an antigen derived from a pathogenic antigen (e.g., a tumor antigen), which in some way prevents or at least partially arrests disease symptoms, side effects orWSGR Docket No.: 50401-791.602 progression. The immune response can also include an antibody response which has been facilitated by the stimulation of helper T cells.

[0231] 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) or infectious organisms isconferred by its TCR, which is made up of both an alpha ( chain and a beta ( chain or agamma ( and a delta ( 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.

[0232] 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 that includes a polypeptide comprising animmunoglobulin heavy chain variable domain linked to a TCR constant domain. In someembodiments, the CAR is a dimer that includes a first polypeptide comprising animmunoglobulin heavy or light chain variable domain linked to a TCR or TCR constantdomain and a second polypeptide comprising an immunoglobulin heavy or light chain variabledomain (e.g., a or variable domain) linked to a TCR or TCR constant domain.

[0233] “Major Histocompatibility Complex” or “MHC” is a cluster of genes 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).

[0234] 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 endogenousWSGR Docket No.: 50401-791.602 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 encoded by 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 -chain and -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 - and -chains and they can present antigen fragments to T-helper cells. MHC class III region can encode for other immune components, such as complement components 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).

[0235] A “receptor” refers to a biological molecule or a molecule grouping capable of binding a ligand. A receptor may 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 may consist of a protein molecule. A receptor has a structure which complements that of a ligand and may complex the ligand as a binding partner. The information is transmitted in particular by conformational changes of the receptor following complexation 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” refers 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 be 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.WSGR Docket No.: 50401-791.602

[0236] A “native” or a “wild type” sequence refers to a sequence found in nature. The term “naturally occurring” as used herein refers to the fact that 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.

[0237] 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 -amino and carboxyl groups of 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.”

[0238] 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 anchor residues. In some embodiments, an MHC class I motif identifies a peptide of 7, 89, 10, 11, 12 or 13 amino acid residues in 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).

[0239] 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 atWSGR Docket No.: 50401-791.602 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 for those amino acid residues having D-forms is represented by a lower case single letter or a lower case three letter symbol. However, when three 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.)

[0240] 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.

[0241] “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.

[0242] According to the present disclosure, the term “vaccine” relates to a pharmaceutical preparation (pharmaceutical composition) or product that upon administration induces an immune response, for example, a cellular or humoral immune response, which recognizes and attacks a pathogen or a diseased cell such as a cancer cell. A vaccine may be used for the prevention or treatment of a disease. The term “individualized cancer vaccine” or “personalized cancer vaccine” “personal cancer vaccine” concerns a particular cancer patient and means that a cancer vaccine is adapted to the needs or special circumstances of an individual cancer patient.WSGR Docket No.: 50401-791.602

[0243] 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.

[0244] 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 could be part of a vector, and / or such a polynucleotide or peptide could 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), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0245] The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences 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 substitutions as part of the sequence identity. The percent identity can be 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, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variations thereof. In some embodiments, two nucleic acids or polypeptides described herein are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments atWSGR Docket No.: 50401-791.602 least 95%, 96%, 97%, 98%, or 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.

[0246] The term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.

[0247] 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.

[0248] The terms “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to therapeutic measures that cure, slow down, lessen symptoms of, and / or halt progression of a diagnosed pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder. In some cases, treating may refer to reducing, or ameliorating a disorder and / or symptoms associated therewith (e.g., a neoplasia or tumor or infectious agent or an autoimmune disease). “Treating” can refer to administration of the therapy to a subject after the onset, or suspected onset, of a disease (e.g., cancer or infection by an infectious agent or an autoimmune disease). “Treating” includes the concepts of “alleviating”, which refers to lessening the frequency of occurrence or recurrence, or the severity, of any symptoms or other ill effects related to the disease and / or the side effects associated with therapy. The term “treating” may also encompass the concept of “managing” which refers to reducing the severity of a disease or disorder in a patient, e.g., extending the life or prolonging the survivability of a patient with the disease, or delaying its recurrence, e.g., lengthening the period of remission in a patient who had suffered from the disease. It is appreciated that, although not precluded, treatingWSGR Docket No.: 50401-791.602 a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.

[0249] The terms “prevent” or “prevention” refer to prophylactic or preventative measures that slow down the development of a targeted pathologic condition or disorder. Thus, those in need of prevention include those prone to have the disorder or those in whom the disorder is to be prevented.

[0250] 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. The cell sample can include tumor infiltrating lymphocytes (TILs). 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, CD14+cells can be depleted or removed from a PBMC sample, such as by using an antibody that binds to CD14.

[0251] 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 within protocol 1 or protocol 2 in which PBMCs are cultured together with peptide loaded APCs.

[0252] 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.

[0253] 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.

[0254] As used herein, a tumor unless otherwise mentioned, is a cancerous tumor, and the terms cancer and tumor are used interchangeably throughout the document. While a tumor is a cancerWSGR Docket No.: 50401-791.602 of solid tissue, several of the compositions and methods described herein are in principle applicable to cancers of the blood, leukemia. Overview

[0255] The present disclosure provides compositions and methods for manufacturing of expanded populations of cells for improved T cell therapies. Current T cell therapies can be susceptible upon repeated stimulation to dysfunction, lack of repeated activation, ineffectiveness, and lack of persistence. Therapeutic potential of T cells can be improved by promoting persistence, including promoting homeostatic cytokine production, enhancing stemness, enhancing memory, enhancing proliferation, and / or enhancing metabolism function. Improvement 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.

[0256] In one example, the methods provided herein can improve activation and function of stimulated T cells by separating APCs and T cells from a biological sample and culture the APCs and T cells separately in different conditions. In another example, the methods provided herein can improve activation and function of stimulated T cells by enriching for T cells expressing markers associated with activation or costimulation.

[0257] The methods provided herein can produce an expanded population of T cells with improved functionality suitable for use in a T cell therapy for treatment of cancer. The therapies provided by the methods herein address a need for improved T cell therapies for treatment of cancer. Methods of Producing T Cells

[0258] Provided herein is a method of producing a plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells. In some embodiments, the method comprises providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a single biological sample from a subject. In some embodiments, the method comprises separating the APCs from the T cells in the population of immune cells, thereby forming a population of APCs separated from a population of T cells. In some embodiments, the method comprises culturing the population of APCs in a first vessel in the presence of a first stimulatory agent and the population of T cells in a second vessel in the presence of a second stimulatory agentWSGR Docket No.: 50401-791.602 wherein the first vessel and the second vessel are different vessels. In some embodiments, the method comprises co-culturing the population of APCs and the population of T cells, thereby forming a population of cells comprising stimulated T cells. In some embodiments, the method comprises expanding the stimulated T cells, thereby forming the plurality of T cells that comprises antigen-specific T cells. In some embodiments, the method comprises (a) providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a single biological sample from a subject; (b) separating the APCs from the T cells in the population of immune cells, thereby forming a population of APCs separated from a population of T cells; (c) culturing the population of APCs in a first vessel in the presence of a first stimulatory agent and the population of T cells in a second vessel in the presence of a second stimulatory agent wherein the first vessel and the second vessel are different vessels; (d) co-culturing the population of APCs and the population of T cells, thereby forming a population of cells comprising stimulated T cells; and (e) expanding the stimulated T cells, thereby forming the plurality of T cells that comprises antigen-specific T cells.

[0259] In some embodiments, the number of T cells in a separate culture or a coculture with APCs is about 5 X 105, about 1 X 106, about 2 X 106, about 3 X 106, about 4 X 106, about 5 X 106, about 6 X 106, about 7 X 106, about 8 X 106, about 9 X 106, about 10 X 106, about 11 X 106, about 12 X 106, about 13 X 106, about 14 X 106, about 15 X 106, or about 20 X 106. In some embodiments, the number of APCs in a separate culture or a coculture with T cells is about 1 X 105, about 2 X 105, about 3 X 105, about 4 X 105, about 5 X 105, about 6 X 105, about 7 X 105, about 8 X 105, about 9 X 105, about 1 X 106, about 1.5 X 106, about 2 X 106, about 2.5 X 106, about 3 X 106, about 3.5 X 106, about 4 X 106, about 4.5 X 106, about 5 X 106, about 5.5 X 106, about 6 X 106, about 6.5 X 106, about 7 X 106, about 7.5 X 106, about 8 X 106, about 8.5 X 106, about 9 X 106, about 9.5 X 106, or about 10 X 106. In some embodiments, the total cells in a coculture of T cells and APCs is about 5 X 105, about 1 X 106, about 2 X 106, about 3 X 106, about 4 X 106, about 5 X 106, about 6 X 106, about 7 X 106, about 8 X 106, about 9 X 106, about 10 X 106, about 11 X 106, about 12 X 106, about 13 X 106, about 14 X 106, about 15 X 106, or about 20 X 106. In some embodiments, the ratio of T cells to APCs in coculture is about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In some embodiments the cell density of the coculture is about 0.5 X 106 / cm2, about 1.0 X 106 / cm2, about 1.5 X 106 / cm2, about 2.0 X 106 / cm2, about 2.5 X 106 / cm2, about 3.0 X 106 / cm2, about 3.5 X 106 / cm2, about 4.0 X 106 / cm2, about 4.5 X 106 / cm2, about 5 X 106 / cm2, about 5.5 X 106 / cm2, about 6.0 X 106 / cm2, about 6.5 X 106 / cm2, about 7.0 X 106 / cm2, about 7.5 X 106 / cm2, about 8.0 X 106 / cm2, about 8.5 X 106 / cm2, about 9 X 106 / cm2, about 10 X 106 / cm2orWSGR Docket No.: 50401-791.602 more. The total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can affect T cell priming, expansion, and / or transposon integration. In some cases, the total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can improve T cell priming, expansion, and / or transposon integration. The total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can affect LNP / LPX-mediated cargo delivery, for example, RNA delivery. In some cases, total cell numbers can affect expansion of T cells. In some cases, lower total cell numbers can increase expansion of T cells. In some cases, T cell to APC ratios can affect expansion of T cells. In some cases, lower T cell to APC ratios can increase expansion of T cells. In some cases, cell density can affect expansion of T cells. In some cases, lower cell density can increase expansion of T cells.

[0260] In some embodiments, the method further comprises, prior to separating, depleting CD25+ cells and / or CD56+ cells from the population of immune cells. In some embodiments, the method further comprises, prior to separating, depleting CD25+ cells from the population of immune cells. In some embodiments, the method further comprises, prior to separating, depleting CD56+ cells from the population of immune cells. In some embodiments, the method further comprises, prior to separating, depleting CD25+ cells and CD56+ cells from the population of immune cells. In some embodiments, the method further comprises, prior to separating, depleting CD11b+ cells.

[0261] In some embodiments, separating the APCs and T cells comprises enriching CD14+ cells and / or CD19+ cells from the population of immune cells, thereby forming the population of APCs separated from the population of T cells. In some embodiments, separating the APCs and T cells comprises enriching CD14+ cells from the population of immune cells, thereby forming the population of APCs separated from the population of T cells. In some embodiments, separating the APCs and T cells comprises enriching CD19+ cells from the population of immune cells, thereby forming the population of APCs separated from the population of T cells. In some embodiments, separating the APCs and T cells comprises enriching CD14+ cells and CD19+ cells from the population of immune cells, thereby forming the population of APCs separated from the population of T cells.

[0262] In some embodiments, the first stimulatory agent comprises one or more agents selected from the group consisting of FLT3L, IL-4, GM-CSF, and any combination thereof. In some embodiments, the first stimulatory agent comprises FLT3L. In some embodiments, the first stimulatory agent comprises IL-4. In some embodiments, the first stimulatory agent comprises GM-CSF. In some embodiments, the first stimulatory agent comprises FLT3L and IL-4. In some embodiments, the first stimulatory agent comprises FLT3L and GM-CSF. In some embodiments,WSGR Docket No.: 50401-791.602 the first stimulatory agent comprises IL-4 and GM-CSF. In some embodiments, the first stimulatory agent comprises FLT3L, GM-CSF, and IL-4. In some embodiments, the second stimulatory agent comprises one or more agents selected from the group consisting of IL-2, IL- 7, IL-15, IL-21, and any combination thereof. In some embodiments, the second stimulatory agent comprises IL-2. In some embodiments, the second stimulatory agent comprises IL-7. In some embodiments, the second stimulatory agent comprises IL-15. In some embodiments, the second stimulatory agent comprises IL-21. In some embodiments, the second stimulatory agent comprises IL-7 and IL-15. In some embodiments, the second stimulatory agent comprises IL-7 and IL-21. In some embodiments, the second stimulatory agent comprises IL-15 and IL-21. In some embodiments, the second stimulatory agent comprises IL-7, IL-15, and IL-21.

[0263] In some embodiments, co-culturing comprises co-culturing the APCs of the population of APCs from the first vessel and the T cells of the population of T cells from the second vessel in the presence of IL-21. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of one or more stimulatory agents selected from the group consisting of IL-7, IL-15, IL-2 and an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-7. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-15. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-2. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-7 and IL-15. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-7 and IL-2. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-7 and an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-15 and IL-2. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-15 and mutIL-2. In some embodiments, expanding the stimulated T cellsWSGR Docket No.: 50401-791.602 comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-2 and an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-7, IL-15 and IL-2. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-7, IL-15, and an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-7, IL-2, and an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-15, IL-2 and an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-7, IL-15, IL-2, and an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the absence of IL-21.

[0264] In some embodiments, the method further comprises, prior to co-culturing, delivering (A) at least one 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 at least one polypeptide into the population of APCs. In some embodiments, the method further comprises, prior to co-culturing, delivering a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer. In some embodiments, the peptide comprising at least one tumor antigen epitope sequence comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more tumor antigen epitope sequences. In some embodiments, the method further comprises, prior to co-culturing, delivering a polynucleotide encoding the polypeptide into the population of APCs. In some embodiments, delivering comprises electroporating a lipid nanoparticle comprising (A) the polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) the polynucleotide encoding the polypeptide into the population ofWSGR Docket No.: 50401-791.602 APCs. In some embodiments, delivering comprises electroporating a lipid nanoparticle comprising the polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer. In some embodiments, delivering comprises electroporating a lipid nanoparticle comprising the polynucleotide encoding the polypeptide into the population of APCs.

[0265] In some embodiments, culturing in (c) (e.g., culturing the population of APCs in a first vessel in the presence of a first stimulatory agent and the population of T cells in a second vessel in the presence of a second stimulatory agent wherein the first vessel and the second vessel are different vessels) comprises delivering (A) at least one 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 at least one polypeptide into the population of APCs. In some embodiments, culturing in (c) comprises delivering (A) at least one polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer into the population of APCs. In some embodiments, culturing in (c) comprises delivering a polynucleotide encoding at least one polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer into the population of APCs. In some embodiments, delivery comprises electroporating the polynucleotide encoding the polypeptide into the population of APCs. In some embodiments, delivering comprises contacting the population of APCs with the at least one polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer. In some embodiments, delivering comprises contacting a lipid nanoparticle or a lipoplex comprising the polynucleotide encoding the at least one polypeptide to the population of APCs.

[0266] In some embodiments, the method further comprises, prior to (d), maturing the population of APCs in the first vessel. In some embodiments, culturing in (c) comprises maturing the population of APCs in the first vessel. In some embodiments, maturing comprisesmaturing the population of APCs in the first vessel in the presence of IFN and / or a toll likereceptor agonist lipopolysaccharide (LPS). In some embodiments, maturing comprises maturingthe population of APCs in the first vessel in the presence of IFN . In some embodiments,maturing comprises maturing the population of APCs in the first vessel in the presence of a toll like receptor agonist. In some embodiments, maturing comprises maturing the population of APCs in the first vessel in the presence of LPS.

[0267] In some embodiments, the method further comprises, prior to co-culturing, genetically editing the population of T cells. In some embodiments, genetically editing the population of T cells comprises inhibiting expression of an endogenous gene in the population of T cells. InWSGR Docket No.: 50401-791.602 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. In some embodiments, the endogenous gene comprises TNFAIP3. In some embodiments, the endogenous gene comprises REGNASE-1. In some embodiments, the endogenous gene comprises SOCS1. In some embodiments, the endogenous gene comprises PTPN2. In some embodiments, the endogenous gene comprises CISH. In some embodiments, the endogenous gene comprises a combination of the foregoing genes. In some embodiments, the endogenous gene can comprise both PRDM1 and REGNASE-1. In some embodiments, the endogenous gene can comprise both TNFAIP3 and REGNASE-1. In some embodiments, the endogenous gene can comprise both PRDM1 and TNFAIP3. In some embodiments, the endogenous gene can comprise both PRDM1 and CISH. In some embodiments, the endogenous gene can comprise both PRDM1 and PTPN2. In some embodiments, the endogenous gene can comprise both PRDM1 and SOCS1. In some embodiments, the endogenous gene can comprise both CISH and REGNASE-1. In some embodiments, the endogenous gene can comprise both REGNASE-1 and SOCS1.

[0268] 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 an miRNA or siRNA that targets an RNA transcript encoded by the endogenous gene. 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 an miRNA that targets an RNA transcript encoded by the endogenous gene. 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 an siRNA that targets an RNA transcript encoded by 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 gene knockout using a site- specific nuclease. In some embodiments, silencing the gene locus comprises gene knockout using a site-specific nickase. In some embodiments, silencing the gene locus comprises gene knockout using a site-specific transcriptional regulator. In some embodiments, silencing the gene locus comprises gene knockout using a site-specific epigenetic regulator. In some embodiments, silencing the gene locus comprises delivering the site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator into the population of T cells via electroporation, a lipid nanoparticle, a lipoplex, a liposome, nucleofection, or a viral vector. In some embodiments, silencing the gene locus comprises delivering a nucleic acid encoding the site-specific nuclease,WSGR Docket No.: 50401-791.602 the site-specific nickase, or the site-specific epigenetic regulator into the population of T cells via electroporation, a lipid nanoparticle, a lipoplex, a liposome, nucleofection, or a viral vector.

[0269] Various methods can be used to inhibit the expression of an endogenous gene to produce the population of engineered immune cells. For example, inhibiting the expression of the endogenous gene can comprise editing a locus in the endogenous gene. Editing the 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 the endogenous gene by binding gRNA to the target endogenous gene sequence, inhibiting its transcription and activation. Other gene editing methods can be used. For another example, inhibiting the expression of the endogenous gene can comprise contacting to the cells or expressing in the cells a miRNA or siRNA that targets an RNA transcript from the endogenous gene. Other gene inhibiting methods can include, for example, inhibiting the expression of the endogenous gene via contact with the FokI cleavage domain recognized and targeted by transcription activator- like effector nucleases (TALENS). Another method of inhibiting the expression of the endogenous gene 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 the endogenous gene.

[0270] In some embodiments, inhibiting the expression of PRDM1 and REGNASE-1 can comprise silencing PRDM1 and REGNASE-1 gene loci. In some embodiments, inhibiting the expression of PRDM1 and REGNASE-1 can comprise contacting to the cells or expressing in the cells a miRNA or siRNA that targets RNA transcripts encoding PRDM1 and REGNASE-1. In some embodiments, inhibiting the expression of PRDM1 and REGNASE-1 can comprise expressing in the cells miRNAs. In some embodiments, inhibiting the expression of PRDM1 and REGNASE-1 can comprise expressing in the cells siRNAs. In some embodiments, inhibiting the expression of PRDM1 and REGNASE-1 can comprise expressing in the cells miRNAs that target RNA transcripts encoding PRDM1 and REGNASE-1. In some embodiments, inhibiting the expression of PRDM1 and REGNASE-1 can comprise expressing in the cells siRNAs that target RNA transcripts encoding PRDM1 and REGNASE-1.

[0271] In some embodiments, the population of T cells comprise tumor infiltrating lymphocytes (TILs). In some embodiments, the method comprises, prior to co-culturing, genetically editing the TILs. In some embodiments, genetically editing the TILs comprises inhibiting expression of two or more endogenous genes (e.g., 2, 3, 4, 5, 6, or more endogenous genes) in the TILs. 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 combinationWSGR Docket No.: 50401-791.602 thereof. In some embodiments, the endogenous gene comprises PRDM1. In some embodiments, the endogenous gene comprises TNFAIP3. In some embodiments, the endogenous gene comprises REGNASE-1. In some embodiments, the endogenous gene comprises SOCS1. In some embodiments, the endogenous gene comprises PTPN2. In some embodiments, the endogenous gene comprises CISH. In some embodiments, the endogenous gene comprises a combination of the foregoing genes. In some embodiments, the endogenous gene can comprise both PRDM1 and REGNASE-1. In some embodiments, the endogenous gene can comprise both TNFAIP3 and REGNASE-1. In some embodiments, the endogenous gene can comprise both PRDM1 and TNFAIP3. In some embodiments, the endogenous gene can comprise both PRDM1 and CISH. In some embodiments, the endogenous gene can comprise both PRDM1 and PTPN2. In some embodiments, the endogenous gene can comprise both PRDM1 and SOCS1. In some embodiments, the endogenous gene can comprise both CISH and REGNASE-1. In some embodiments, the endogenous gene can comprise both REGNASE-1 and SOCS1.

[0272] In some embodiments, inhibiting expression of the two or more endogenous genes results in an increase in stemness of the population of T cells compared to an otherwise identical population of T cells which have not been genetically edited. In some embodiments, stemness can be measured by CD127 expression. In some embodiments, inhibiting expression of the two or more endogenous genes results in at least at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 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% or more of the population of T cells to express CD127. In some embodiments, stemness is measured by quantifying the frequency of T cells which present a T central memory phenotype. In some embodiments, inhibiting expression of the two or more endogenous genes results in at least at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 15%, least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 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% or more to differentiate into a T central memory phenotype.

[0273] In some embodiments, inhibiting expression of the two or more endogenous genes results in at least a fold expansion of about 1.2-fold, about 1.5-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30- fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 75-fold, about 100-fold, about 125-fold, about 150-fold, about 175-fold, about 200-fold, about 225-fold, about 250-fold, or about 300-fold of the population of T cells. In some embodiments, inhibiting expression of the two or more endogenous genes results in at least at least 0.1%, at least 0.5%, at least 1%, atWSGR Docket No.: 50401-791.602 least 5%, at least 10%, at least 15%, least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 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% or more frequency of antigen-specific cells in the population of T cells.

[0274] In some embodiments, genetically editing comprises delivering an exogenous gene into the population of T cells via transposon. In some embodiments, the exogenous gene encodes a cell surface receptor that binds to a cytokine. In some embodiments, the exogenous gene encodes a switch receptor or a safety switch for controlling activities of T cells. In some embodiments, the cytokine is an IL-2 variant. In some embodiments, the cell surface receptor is an IL-2 receptor. In some embodiments, the cytokine is an IL-2 variant and the cell surface receptor is an IL-2 receptor. In some embodiments, the cytokine is IL-18. In some embodiments, the cell surface receptor is an IL-18 receptor. In some embodiments, the cytokine is IL-18 and the cell surface receptor is an IL-18 receptor. In some embodiments, the IL-2 receptor (e.g., IL- 2R or variant thereof) comprises one or more mutations relative to a wildtype IL-2 receptor. The IL-2 receptor can be an IL-2R variant.

[0275] In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99% or more. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 1%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 2%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 3%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 4%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 5%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 10%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 15%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 20%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 25%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 30%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 35%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 40%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous geneWSGR Docket No.: 50401-791.602 is at least 45%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 50%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 55%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 60%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 65%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 70%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 75%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 80%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 90%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 95%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 99% or more.

[0276] In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 99%, 95%, 90%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 55, 4%, 3%, 2%, 1% or less. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 1% or less. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 2%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 3%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 4%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 5%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 10%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 15%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 20%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 25%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 30%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 35%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 40%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 45%. In some embodiments, the percent of T cells inWSGR Docket No.: 50401-791.602 the population of T cells expressing the endogenous gene is at most 50%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 55%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 60%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 65%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 70%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 75%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 80%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 90%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 95%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 99%.

[0277] In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99% or more. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 1%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 2%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 3%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 4%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 5%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 10%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 15%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 20%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 25%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 30%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 35%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 40%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 45%. In some embodiments, the percent of T cells in the population of TWSGR Docket No.: 50401-791.602 cells with the endogenous gene inhibited is at least 50%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 55%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 60%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 65%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 70%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 75%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 80%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 90%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 95%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 99% or more.

[0278] In some embodiments, the polypeptide or the polynucleotide encoding the peptide is delivered in a lipid nanoparticle. In some embodiments, the polypeptide or the polynucleotide encoding the peptide is delivered in a lipoplex. In some embodiments, the lipid nanoparticle comprises R-DODMA, DOPE, cholesterol, and / or PEG. In some embodiments, the lipid nanoparticle comprises R-DODMA. In some embodiments, the lipid nanoparticle comprises DOPE. In some embodiments, the lipid nanoparticle comprises cholesterol. In some embodiments, the lipid nanoparticle comprises PEG. In some embodiments, the lipid nanoparticle comprises R-DODMA and DOPE. In some embodiments, the lipid nanoparticle comprises R-DODMA and cholesterol. In some embodiments, the lipid nanoparticle comprises R-DODMA and PEG. In some embodiments, the lipid nanoparticle comprises DOPE and cholesterol. In some embodiments, the lipid nanoparticle comprises DOPE and PEG. In some embodiments, the lipid nanoparticle comprises cholesterol and PEG. In some embodiments, the lipid nanoparticle comprises R-DODMA, DOPE, and cholesterol. In some embodiments, the lipid nanoparticle comprises R-DODMA, DOPE, and PEG. In some embodiments, the lipid nanoparticle comprises R-DODMA, cholesterol, and PEG. In some embodiments, the lipid nanoparticle comprises DOPE, cholesterol, and PEG. In some embodiments, the lipid nanoparticle comprises R-DODMA, DOPE, cholesterol, and PEG. In some embodiments, the ratio of R-DODMA to DOPE is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1: 30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100. In some embodiments, the ratio of R-DODMA to DOPE is about 1:1, about 2:1, about 3:1, about 4:1,WSGR Docket No.: 50401-791.602 about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1. In some embodiments, the ratio of R-DODMA to cholesterol is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1: 30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100. In some embodiments, the ratio of R-DODMA to cholesterol is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1. In some embodiments, the ratio of R-DODMA to PEG is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1: 30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100. In some embodiments, the ratio of R-DODMA to PEG is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1In some embodiments, the ratio of R-DODMA to DOPE to cholesterol to PEG is 40:10:48:2. In some embodiments, the lipoplex comprises DOTAP and / or DOPE. In some embodiments, the lipoplex comprises DOTAP. In some embodiments, the lipoplex comprises DOPE. In some embodiments, the lipoplex comprises DOTAP and DOPE. In some embodiments, the ratio of DOTAP to DOPE is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1: 30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100. In some embodiments, the ratio of DOTAP to DOPE is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1. In some embodiments, the ratio of DOTAP to DOPE is 2:1.

[0279] In some embodiments the lipid nanoparticle or lipoplex comprises a T cell targeting agent. In some embodiments the lipid nanoparticle comprises a T cell targeting agent. In some embodiments, the lipoplex comprises a T cell targeting agent. In some embodiments, the T cell targeting agent is recognized by or interacts with a molecule present on a T cell surface. In some embodiments, the T cell targeting agent recognizes a molecule present on a T cell surface. In some embodiments, the T cell targeting agent interacts with a molecule present on a T cell surface. In some embodiments, the T cell targeting agent is an antibody or fragment thereof. In some embodiments, the molecule present on the T cell surface is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD56, CD39, ICAM-1, CD366, CD279,WSGR Docket No.: 50401-791.602 CD200, CD25, CD69, CD39, 4-1BB, ITGB7, ICOS and CD137. In some embodiments, the molecule present on the T cell surface is CD2. In some embodiments, the molecule present on the T cell surface is CD3. In some embodiments, the molecule present on the T cell surface is CD4. In some embodiments, the molecule present on the T cell surface is CD5. In some embodiments, the molecule present on the T cell surface is CD7. In some embodiments, the molecule present on the T cell surface is CD8. In some embodiments, the molecule present on the T cell surface is CD56. In some embodiments, the molecule present on the T cell surface is CD39. In some embodiments, the molecule present on the T cell surface is ICAM-1. In some embodiments, the molecule present on the T cell surface is CD366. In some embodiments, the molecule present on the T cell surface is CD279. In some embodiments, the molecule present on the T cell surface is CD200. In some embodiments, the molecule present on the T cell surface is CD25. In some embodiments, the molecule present on the T cell surface is CD69. In some embodiments, the molecule present on the T cell surface is CD39. In some embodiments, the molecule present on the T cell surface is 4-1BB. In some embodiments, the molecule present on the T cell surface is ITGB7. In some embodiments, the molecule present on the T cell surface is ICOS. In some embodiments, the molecule present on the T cell surface is CD137.

[0280] In some embodiments the lipid nanoparticle or lipoplex comprises an APC targeting agent. In some embodiments the lipid nanoparticle comprises an APC targeting agent. In some embodiments, the lipoplex comprises an APC targeting agent. In some embodiments, the APC targeting agent is a mono / di / oligo-saccharide or a synthetic analogue. In some embodiments, the APC targeting agent is a monosaccharide. In some embodiments, the APC targeting agent is a disaccharide. In some embodiments, the APC targeting agent is an oligosaccharide. In some embodiments, the APC targeting agent is a synthetic analogue. In some embodiments, the APC targeting agent binds to a macrophage galactose-type lectin (MGL), a mineralcorticoid receptor (MR), or a Dendritic Cell Specific Intracellular adhesion molecule 3-Grabbing Non-integrin (DC-SIGN). In some embodiments, the APC targeting agent binds to an MGL. In some embodiments, the APC targeting agent binds to an MR. In some embodiments, the APC targeting agent binds to a DC-SIGN.

[0281] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD8+ T cell epitope. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more CD8+ T cell epitope sequences. In some embodiments, each of the CD8+ T cell epitopes have a different epitope sequence. In some embodiments, CD8+ T cell epitope comprises a sequence of about 2 to 20,WSGR Docket No.: 50401-791.602 about 3 to 19, about 4 to 18, about 5 to 17, about 6 to 16, about 7 to 15, about 8 to 14, about 9 to 13, or about 10 to 12 consecutive amino acids from a cancer protein. In some embodiments, CD8+ T cell epitope comprises a sequence of about 7 to about 12 consecutive amino acids from a cancer protein.

[0282] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD4+ T cell epitope. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more CD4+ T cell epitope sequences. In some embodiments, each of the CD4+ T cell epitopes have a different epitope sequence. In some embodiments, the CD4+ T cell epitope comprises a sequence of about 1 to 40, about 2 to 39, about 3 to 38, about 4 to 37, about 5 to 36, about 6 to 35, about 7 to 34, about 8 to 33, about 9 to 32, about 10 to 31, about 11 to 30, about 12 to 29, about 13 to 28, about 14 to 27, about 15 to 26, about 16 to 25, about 17 to 24, about 18 to 23, about 17 to 22, about 18 to 21, or about 19 to 20 consecutive amino acids from a cancer protein. In some embodiments, the CD4+ T cell epitope comprises a sequence of about 13 to 25 consecutive amino acids from a cancer protein. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a linker between the at least two CD8+ T cell epitopes and the at least two CD4+ T cell epitopes.

[0283] In some embodiments, the polynucleotide encoding the polypeptide comprises a linker. In some embodiments, the linker comprises a cleavable linker and / or a linker that is degradable by a protease. In some embodiments, the linker comprises a linker that is degradable by a protease. In some embodiments, the protease is a proteasome. In some embodiments, the linker comprises a cleavable linker. In some embodiments, wherein the least two different CD8+ T cell epitope sequences are separated by one or more linkers. In some embodiments, the least two different CD4+ T cell epitope sequences are separated by one or more linkers. In some embodiments, the one or more linkers are one or more flexible linkers. In some embodiments, wherein the one or more linkers are one or more cleavable linkers. In some embodiments, the polypeptide further comprises a Sec domain sequence at its N-terminus. In some embodiments, the polypeptide further comprises a MITD domain sequence at its C-terminus. In some embodiments, the Sec domain sequence comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity to a sequence of SEQ ID NOs: 20 or 21. In some embodiments, the MITD domain sequence comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity to a sequence of SEQ ID NO: 19.WSGR Docket No.: 50401-791.602

[0284] In some embodiments, the polynucleotide encoding the polypeptide comprises a sequence encoding the Sec domain at its 5’ end. In some embodiments, the polynucleotide encoding the polypeptide comprises a sequence encoding the MITD domain at its 3’ end. In some embodiments, the polynucleotide encoding the polypeptide comprises one or more sequences encoding one or more linkers. In some embodiments, the Sec domain comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity to a sequence of SEQ ID NOs: 23 or 24. In some embodiments, the sequence encoding the MITD domain comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity to a sequence of SEQ ID NO: 22.

[0285] Also provided herein is a method for producing a plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells. In some embodiments, the method comprises providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a biological sample from a subject. In some embodiments, the method comprises co-culturing APCs from the population of immune cells and T cells from the population of immune cells in the presence of IL-21, thereby forming a population of cells comprising stimulated T cells. In some embodiments, the method comprises expanding the stimulated T cells in the absence of IL-21. In some embodiments, the method comprises (a) providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a biological sample from a subject; (b) co-culturing APCs from the population of immune cells and T cells from the population of immune cells in the presence of IL-21, thereby forming a population of cells comprising stimulated T cells; and (c) expanding the stimulated T cells in the absence of IL-21, thereby forming the plurality of T cells that comprises antigen-specific T cells.

[0286] In some embodiments, the number of T cells in a separate culture or a coculture with APCs is about 5 X 105, about 1 X 106, about 2 X 106, about 3 X 106, about 4 X 106, about 5 X 106, about 6 X 106, about 7 X 106, about 8 X 106, about 9 X 106, about 10 X 106, about 11 X 106, about 12 X 106, about 13 X 106, about 14 X 106, about 15 X 106, or about 20 X 106. In some embodiments, the number of APCs in a separate culture or a coculture with T cells is about 1 X 105, about 2 X 105, about 3 X 105, about 4 X 105, about 5 X 105, about 6 X 105, about 7 X 105, about 8 X 105, about 9 X 105, about 1 X 106, about 1.5 X 106, about 2 X 106, about 2.5 X 106, about 3 X 106, about 3.5 X 106, about 4 X 106, about 4.5 X 106, about 5 X 106, about 5.5 X 106, about 6 X 106, about 6.5 X 106, about 7 X 106, about 7.5 X 106, about 8 X 106, about 8.5 X 106, about 9 X 106, about 9.5 X 106, or about 10 X 106. In some embodiments, the total cells in a coculture of T cells and APCs is about 5 X 105, about 1 X 106, about 2 X 106, about 3 X 106,WSGR Docket No.: 50401-791.602 about 4 X 106, about 5 X 106, about 6 X 106, about 7 X 106, about 8 X 106, about 9 X 106, about 10 X 106, about 11 X 106, about 12 X 106, about 13 X 106, about 14 X 106, about 15 X 106, or about 20 X 106. In some embodiments, the ratio of T cells to APCs in coculture is about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In some embodiments the cell density of the coculture is about 0.5 X 106 / cm2, about 1.0 X 106 / cm2, about 1.5 X 106 / cm2, about 2.0 X 106 / cm2, about 2.5 X 106 / cm2, about 3.0 X 106 / cm2, about 3.5 X 106 / cm2, about 4.0 X 106 / cm2, about 4.5 X 106 / cm2, about 5 X 106 / cm2, about 5.5 X 106 / cm2, about 6.0 X 106 / cm2, about 6.5 X 106 / cm2, about 7.0 X 106 / cm2, about 7.5 X 106 / cm2, about 8.0 X 106 / cm2, about 8.5 X 106 / cm2, about 9 X 106 / cm2, about 10 X 106 / cm2or more. The total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can affect T cell priming, expansion, and / or transposon integration. In some cases, the total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can improve T cell priming, expansion, and / or transposon integration. The total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can affect LNP / LPX-mediated RNA delivery or electroporation. In some cases, total cell numbers can affect expansion of T cells. In some cases, lower total cell numbers can increase expansion of T cells. In some cases, T cell to APC ratios can affect expansion of T cells. In some cases, lower T cell to APC ratios can increase expansion of T cells. In some cases, cell density can affect expansion of T cells. In some cases, lower cell density can increase expansion of T cells.

[0287] In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of one or more stimulatory agents selected from the group consisting of IL-7, IL-15, IL-2 and an IL-2 variant (e.g., mutIL2). In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-7. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-15. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-2. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-7 and IL-15. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-7 and IL-2. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells inWSGR Docket No.: 50401-791.602 the presence of IL-7 and an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-15 and IL-2. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-15 and mutIL-2. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-2 and an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-7, IL-15 and IL-2. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-7, IL-15, and an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-7, IL-2, and an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-15, IL-2 and an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the presence of IL-7, IL-15, IL-2, and an IL-2 variant.

[0288] In some embodiments, the biological sample is a single biological sample. In some embodiments, the method further comprises, prior to co-culturing, separating the APCs from the T cells in the population of immune cells, thereby forming a population of APCs separated from a population of T cells.

[0289] In some embodiments, the method further comprises, prior to co-culturing, genetically editing the population of T cells. In some embodiments, genetically editing the population of T cells comprises inhibiting expression of an endogenous gene in the population of T 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 combinationWSGR Docket No.: 50401-791.602 thereof. In some embodiments, the endogenous gene comprises PRDM1. In some embodiments, the endogenous gene comprises TNFAIP3. In some embodiments, the endogenous gene comprises REGNASE-1. In some embodiments, the endogenous gene comprises SOCS1. In some embodiments, the endogenous gene comprises PTPN2. In some embodiments, the endogenous gene comprises CISH. In some embodiments, the endogenous gene comprises a combination of the foregoing genes. 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 an miRNA or siRNA that targets an RNA transcript encoded by the endogenous gene. 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 an miRNA that targets an RNA transcript encoded by the endogenous gene. 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 an siRNA that targets an RNA transcript encoded by 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 gene knockout using a site-specific nuclease. In some embodiments, silencing the gene locus comprises gene knockout using a site-specific nickase. In some embodiments, silencing the gene locus comprises gene knockout using a site-specific transcriptional regulator. In some embodiments, silencing the gene locus comprises gene knockout using a site-specific epigenetic regulator. In some embodiments, silencing the gene locus comprises delivering the site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator into the population of T cells via electroporation, a lipid nanoparticle, a lipoplex, a liposome, nucleofection, or a viral vector. In some embodiments, silencing the gene locus comprises delivering a nucleic acid encoding the site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator into the population of T cells via electroporation, a lipid nanoparticle, a lipoplex, a liposome, nucleofection, or a viral vector.

[0290] In some embodiments, genetically editing comprises delivering an exogenous gene into the population of T cells via transposon. In some embodiments, the exogenous gene encodes a cell surface receptor that binds to a cytokine. In some embodiments, the exogenous gene encodes a switch receptor or a safety switch for controlling activities of T cells. In some embodiments, the cytokine is an IL-2 variant. In some embodiments, the cell surface receptor is an IL-2 receptor. In some embodiments, the cytokine is an IL-2 variant and the cell surface receptor is an IL-2 receptor. In some embodiments, the cytokine is IL-18. In some embodiments,WSGR Docket No.: 50401-791.602 the cell surface receptor is an IL-18 receptor. In some embodiments, the cytokine is IL-18 and the cell surface receptor is an IL-18 receptor. In some embodiments, the IL-2 receptor comprises one or more mutations relative to a wildtype IL-2 receptor.

[0291] In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99% or more. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 1%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 2%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 3%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 4%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 5%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 10%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 15%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 20%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 25%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 30%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 35%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 40%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 45%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 50%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 55%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 60%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 65%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 70%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 75%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 80%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 90%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 95%. In some embodiments,WSGR Docket No.: 50401-791.602 the percent of T cells in the population of T cells expressing the exogenous gene is at least 99% or more.

[0292] In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 99%, 95%, 90%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 55, 4%, 3%, 2%, 1% or less. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 1% or less. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 2%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 3%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 4%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 5%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 10%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 15%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 20%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 25%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 30%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 35%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 40%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 45%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 50%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 55%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 60%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 65%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 70%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 75%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 80%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 90%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at mostWSGR Docket No.: 50401-791.602 95%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 99%.

[0293] In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99% or more. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 1%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 2%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 3%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 4%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 5%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 10%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 15%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 20%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 25%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 30%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 35%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 40%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 45%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 50%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 55%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 60%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 65%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 70%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 75%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 80%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 90%. In some embodiments, the percent of T cells in the population of T cells with the endogenous geneWSGR Docket No.: 50401-791.602 inhibited is at least 95%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 99% or more.

[0294] In some embodiments, the polypeptide or the polynucleotide encoding the peptide is delivered in a lipid nanoparticle. In some embodiments, the polypeptide or the polynucleotide encoding the peptide is delivered in a lipoplex. In some embodiments, the lipid nanoparticle comprises R-DODMA, DOPE, cholesterol, and / or PEG.

[0295] In some embodiments the lipid nanoparticle or lipoplex comprises a T cell targeting agent. In some embodiments the lipid nanoparticle comprises a T cell targeting agent. In some embodiments, the lipoplex comprises a T cell targeting agent. In some embodiments, the T cell targeting agent is recognized by or interacts with a molecule present on a T cell surface. In some embodiments, the T cell targeting agent recognizes a molecule present on a T cell surface. In some embodiments, the T cell targeting agent interacts with a molecule present on a T cell surface. In some embodiments, the T cell targeting agent is an antibody or fragment thereof. In some embodiments, the molecule present on the T cell surface is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD56, CD39, ICAM-1, CD366, CD279, CD200, CD25, CD103, HLA-DR, CD69, 4-1BB, ITGB7, ICOS, and CD137. In some embodiments, the molecule present on the T cell surface is CD2. In some embodiments, the molecule present on the T cell surface is CD3. In some embodiments, the molecule present on the T cell surface is CD4. In some embodiments, the molecule present on the T cell surface is CD5. In some embodiments, the molecule present on the T cell surface is CD7. In some embodiments, the molecule present on the T cell surface is CD8. In some embodiments, the molecule present on the T cell surface is CD56. In some embodiments, the molecule present on the T cell surface is CD39. In some embodiments, the molecule present on the T cell surface is ICAM-1. In some embodiments, the molecule present on the T cell surface is CD366. In some embodiments, the molecule present on the T cell surface is CD279. In some embodiments, the molecule present on the T cell surface is CD200. In some embodiments, the molecule present on the T cell surface is CD25. In some embodiments, the molecule present on the T cell surface is CD69. In some embodiments, the molecule present on the T cell surface is CD39. In some embodiments, the molecule present on the T cell surface is 4-1BB. In some embodiments, the molecule present on the T cell surface is ITGB7. In some embodiments, the molecule present on the T cell surface is ICOS. In some embodiments, the molecule present on the T cell surface is CD137. In some embodiments the lipid nanoparticle or lipoplex comprises an APC targeting agent. In some embodiments the lipid nanoparticle comprises an APC targeting agent. In some embodiments, the lipoplex comprises an APC targeting agent. In some embodiments, the APCWSGR Docket No.: 50401-791.602 targeting agent is a mono / di / oligo-saccharide or a synthetic analogue. In some embodiments, the APC targeting agent is a monosaccharide. In some embodiments, the APC targeting agent is a disaccharide. In some embodiments, the APC targeting agent is an oligosaccharide. In some embodiments, the APC targeting agent is a synthetic analogue. In some embodiments, the APC targeting agent binds to a macrophage galactose-type lectin (MGL), a mineralcorticoid receptor (MR), or a Dendritic Cell Specific Intracellular adhesion molecule 3-Grabbing Non-integrin (DC-SIGN). In some embodiments, the APC targeting agent binds to an MGL. In some embodiments, the APC targeting agent binds to an MR. In some embodiments, the APC targeting agent binds to a DC-SIGN.

[0296] In some embodiments, the method further comprises, prior to co-culturing, depleting one or more cells selected from the group consisting of CD14+ cells, CD25+ cells and CD56+ cells from the population of immune cells. In some embodiments, the method further comprises, prior to co-culturing, depleting CD14+ cells from the population of immune cells. In some embodiments, the method further comprises, prior to co-culturing, depleting CD25+ cells from the population of immune cells. In some embodiments, the method further comprises, prior to co-culturing, depleting CD56+ cells from the population of immune cells. In some embodiments, the method further comprises, prior to co-culturing, depleting CD14+ cells and CD25+ cells from the population of immune cells. In some embodiments, the method further comprises, prior to co-culturing, depleting CD14+ cells and CD56+ cells from the population of immune cells. In some embodiments, the method further comprises, prior to co-culturing, depleting CD25+ cells and CD56+ cells from the population of immune cells. In some embodiments, the method further comprises, prior to co-culturing, depleting CD14+ cells, CD25+ cells and CD56+ cells from the population of immune cells.

[0297] In some embodiments, the method further comprises, prior to co-culturing, depleting one or more cells selected from the group consisting of CD14+ cells, CD11b+ cells, CD25+ cells and CD56+ cells from the population of immune cells, thereby forming a depleted population of immune cells comprising a first population of APCs and T cells. In some embodiments, the method further comprises, prior to co-culturing, 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), and a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer. In some embodiments, the method further comprises, prior to co-culturing, (i) depleting one or more cells selected from the group consisting of CD14+ cells, CD11b+ cells, CD25+ cells and CD56+ cells from the population of immune cells, thereby forming a depleted population of immune cells comprising a firstWSGR Docket No.: 50401-791.602 population of APCs and T cells; (ii) 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), and (A) at least one 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 at least one polypeptide; thereby forming the population of cells comprising the stimulated T cells. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more tumor antigen epitope sequences.

[0298] In some embodiments, the method further comprises enriching CD25+ cells, CD39+ cells, 4-1BB+ cells, CD69+ cells, HLA-DR+ cells, LFA-1+ cells, ITGB7+ cells, cells, cells, cells, or any combination thereof from the population of cells comprising the stimulated T cells. In some embodiments, the method further comprises enriching CD25+ cells, 4-1BB+ cells, CD69+ cells, + cells, or any combination thereof from the population of cells comprising the stimulated T cells. In some embodiments, the method further comprises enriching CD25+ cells from the population of cells comprising the stimulated T cells. In some embodiments, the method further comprises enriching CD39+ cells from the population of cells comprising the stimulated T cells. In some embodiments, the method further comprises enriching 4-1BB+ cells from the population of cells comprising the stimulated T cells. In some embodiments, the method further comprises enriching CD69+ cells from the population of cells comprising the stimulated T cells. In some embodiments, the method further comprises enriching HLA-DR+ cells from the population of cells comprising the stimulated T cells. In some embodiments, the method further comprises enriching ITGB7+ cells from the population of cells comprising the stimulated T cells. In some embodiments, the method further comprises enriching LFA-1+ cells from the population of cells comprising the stimulated T cells. In some embodiments, the method further comprises enriching + cells from the population of cells comprising the stimulated T cells. In some embodiments, the method further comprises enriching + cells from the population of cells comprising the stimulated T cells.

[0299] In some embodiments, a higher number of antigen-specific T cells is produced by the method compared to a corresponding method in which the T cells are expanded in the presence of IL-21. In some embodiments, the number of antigen-specific T cells is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or more compared to aWSGR Docket No.: 50401-791.602 corresponding method in which the T cells are expanded in the presence of IL-21. In some embodiments, a higher number of antigen-specific T cells is produced by the method compared to a corresponding method in which the T cells are expanded in the absence of IL-21. In some embodiments, the number of antigen-specific T cells is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or more compared to a corresponding method in which the T cells are expanded in the absence of IL-21.

[0300] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD8+ T cell epitope. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more CD8+ T cell epitopes. In some embodiments, each of the CD8+ T cell epitopes have a different epitope sequence. In some embodiments, CD8+ T cell epitope comprises a sequence of about 2 to 20, about 3 to 19, about 4 to 18, about 5 to 17, about 6 to 16, about 7 to 15, about 8 to 14, about 9 to 13, or about 10 to 12 consecutive amino acids from a cancer protein. In some embodiments, CD8+ T cell epitope comprises a sequence of about 7 to about 12 consecutive amino acids from a cancer protein.

[0301] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD4+ T cell epitope. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more CD4+ T cell epitopes. In some embodiments, each of the CD4+ T cell epitopes have a different epitope sequence. In some embodiments, the CD4+ T cell epitope comprises a sequence of about 1 to 40, about 2 to 39, about 3 to 38, about 4 to 37, about 5 to 36, about 6 to 35, about 7 to 34, about 8 to 33, about 9 to 32, about 10 to 31, about 11 to 30, about 12 to 29, about 13 to 28, about 14 to 27, about 15 to 26, about 16 to 25, about 17 to 24, about 18 to 23, about 17 to 22, about 18 to 21, or about 19 to 20 consecutive amino acids from a cancer protein. In some embodiments, the CD4+ T cell epitope comprises a sequence of about 13 to 25 consecutive amino acids from a cancer protein. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a cleavable linker between the at least two CD8+ T cell epitopes and the at least two CD4+ T cell epitopes.

[0302] In some embodiments, the polynucleotide encoding the polypeptide comprises a linker. In some embodiments, the linker comprises a cleavable linker and / or a linker that is degradable by a protease. In some embodiments, the linker comprises a linker that is degradable by a protease. In some embodiments, the protease is a proteasome. In some embodiments, the linkerWSGR Docket No.: 50401-791.602 comprises a cleavable linker. In some embodiments, wherein the least two different CD8+ T cell epitope sequences are separated by one or more linkers. In some embodiments, the least two different CD4+ T cell epitope sequences are separated by one or more linkers. In some embodiments, the one or more linkers are one or more flexible linkers. In some embodiments, wherein the one or more linkers are one or more cleavable linkers. In some embodiments, the polypeptide further comprises a Sec domain sequence at its N-terminus. In some embodiments, the polypeptide further comprises a MITD domain sequence at its C-terminus. In some embodiments, the Sec domain sequence comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity to a sequence of SEQ ID NO: 20 or 21. In some embodiments, the MITD domain sequence comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity to a sequence of SEQ ID NO: 19.

[0303] In some embodiments, the polynucleotide encoding the polypeptide comprises a sequence encoding the Sec domain at its 5’ end. In some embodiments, the polynucleotide encoding the polypeptide comprises a sequence encoding the MITD domain at its 3’ end. In some embodiments, the polynucleotide encoding the polypeptide comprises one or more sequences encoding one or more linkers. In some embodiments, the Sec domain comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity to a sequence of SEQ ID NOs: 23 or 24. In some embodiments, the sequence encoding the MITD domain comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity to a sequence of SEQ ID NO: 22.

[0304] Also provided herein is a method for producing a plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells. In some embodiments, the method comprises depleting one or more cells selected from the group consisting of CD14+ cells, CD11b+ cells, CD25+ cells and CD56+ cells from the population of immune cells, thereby forming a depleted population of immune cells comprising a first population of APCs and T cells. In some embodiments, the method comprises incubating the first population of APCs and T cells for a first time period in the presence of (A) at least one 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 at least one polypeptide; thereby forming a population of cells comprising stimulated T cells. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, atWSGR Docket No.: 50401-791.602 least 24, at least 25 or more tumor antigen epitope sequences. In some embodiments, the method comprises enriching CD25+ cells, CD39+ cells, 4-1BB+ cells, CD69+ cells, HLA-DR+ cells, LFA-1+ cells, ITGB7+ cells, cells, cells, or any combination thereof from the population of cells comprising the stimulated T cells. In some embodiments, the method comprises (a) depleting one or more cells selected from the group consisting of CD14+ cells, CD11b+ cells, CD25+ cells and CD56+ cells from the population of immune cells, thereby forming a depleted population of immune cells comprising a first population of APCs and T cells; (b) incubating the first population of APCs and T cells for a first time period in the presence of (A) at least one 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 at least one polypeptide; thereby forming a population of cells comprising stimulated T cells; and (c) enriching CD25+ cells, CD39+ cells, 4-1BB+ cells, CD69+ cells, HLA-DR+ cells, LFA- 1+ cells, ITGB7+ cells, cells, cells, or any combination thereof from the population of cells comprising the stimulated T cells.

[0305] In some embodiments, the number of T cells in a separate culture or a coculture with APCs is about 5 X 105, about 1 X 106, about 2 X 106, about 3 X 106, about 4 X 106, about 5 X 106, about 6 X 106, about 7 X 106, about 8 X 106, about 9 X 106, about 10 X 106, about 11 X 106, about 12 X 106, about 13 X 106, about 14 X 106, about 15 X 106, or about 20 X 106. In some embodiments, the number of APCs in a separate culture or a coculture with T cells is about 1 X 105, about 2 X 105, about 3 X 105, about 4 X 105, about 5 X 105, about 6 X 105, about 7 X 105, about 8 X 105, about 9 X 105, about 1 X 106, about 1.5 X 106, about 2 X 106, about 2.5 X 106, about 3 X 106, about 3.5 X 106, about 4 X 106, about 4.5 X 106, about 5 X 106, about 5.5 X 106, about 6 X 106, about 6.5 X 106, about 7 X 106, about 7.5 X 106, about 8 X 106, about 8.5 X 106, about 9 X 106, about 9.5 X 106, or about 10 X 106. In some embodiments, the total cells in a coculture of T cells and APCs is about 5 X 105, about 1 X 106, about 2 X 106, about 3 X 106, about 4 X 106, about 5 X 106, about 6 X 106, about 7 X 106, about 8 X 106, about 9 X 106, about 10 X 106, about 11 X 106, about 12 X 106, about 13 X 106, about 14 X 106, about 15 X 106, or about 20 X 106. In some embodiments, the ratio of T cells to APCs in coculture is about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In some embodiments the cell density of the coculture is about 0.5 X 106 / cm2, about 1.0 X 106 / cm2, about 1.5 X 106 / cm2, about 2.0 X 106 / cm2, about 2.5 X 106 / cm2, about 3.0 X 106 / cm2, about 3.5 X 106 / cm2, about 4.0 X 106 / cm2, about 4.5 X 106 / cm2, about 5 X 106 / cm2, about 5.5 X 106 / cm2, about 6.0 X 106 / cm2, about 6.5 X 106 / cm2, about 7.0 X 106 / cm2, about 7.5 X 106 / cm2, about 8.0 X 106 / cm2, about 8.5 X 106 / cm2, about 9 X 106 / cm2, about 10 X 106 / cm2orWSGR Docket No.: 50401-791.602 more. The total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can affect T cell priming, expansion, and / or transposon integration. In some cases, the total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can improve T cell priming, expansion, and / or transposon integration. The total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can affect LNP / LPX-mediated RNA delivery or electroporation. In some cases, total cell numbers can affect expansion of T cells. In some cases, lower total cell numbers can increase expansion of T cells. In some cases, T cell to APC ratios can affect expansion of T cells. In some cases, lower T cell to APC ratios can increase expansion of T cells. In some cases, cell density can affect expansion of T cells. In some cases, lower cell density can increase expansion of T cells.

[0306] In some embodiments, depleting comprises depleting CD25+ cells from the population of immune cells. In some embodiments, depleting comprises depleting CD25+ cells from the population of immune cells and enriching comprises enriching CD25+ cells from the population of cells comprising the stimulated T cells. In some embodiments, incubating is performed in the presence of FMS-like tyrosine kinase 3 receptor ligand (FLT3L).

[0307] In some embodiments, the method further comprises expanding the population of cells comprising the stimulated T cells. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of one or more stimulatory agents selected from the group consisting of IL-7, IL-15, IL-2, an IL-2 variant, and any combination thereof. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of IL-7. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of IL- 15. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of IL-2. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of IL-7 and IL-15. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of mutIL-2. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of IL-7 and IL-2. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of IL-7 and an IL-2 variant. In some embodiments, expanding comprises expanding the population of cells comprisingWSGR Docket No.: 50401-791.602 stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of IL- 15 and IL-2. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of IL-15 and mutIL-2. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of IL-2 and an IL-2 variant. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of IL-7, IL-15 and IL-2. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of IL-7, IL-15, and an IL-2 variant. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of IL-7, IL-2, and an IL-2 variant. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of IL-15, IL-2 and an IL-2 variant. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of an IL-2 variant. In some embodiments, expanding comprises expanding the population of cells comprising stimulated T cells in the presence of IL-7, IL-15, IL-2, and an IL-2 variant. Examples of an IL-2 variant and the IL-2R variants are described herein, see e.g., Table 9.

[0308] In some embodiments, the method comprises incubating the first population of APCs and T cells comprises delivering the polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer into the APCs of the first population of APCs and T cells. In some embodiments, the method comprises incubating the first population of APCs and T cells comprises delivering the polynucleotide encoding the polypeptide into the APCs of the first population of APCs and T cells. In some embodiments, delivering comprises contacting a first lipid delivery vehicle comprising the polypeptideWSGR Docket No.: 50401-791.602 comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer into the APCs of the first population of APCs and T cells. In some embodiments, delivering comprises contacting a first lipid delivery vehicle comprising the polynucleotide encoding the polypeptide into the APCs of the first population of APCs and T cells.

[0309] In some embodiments, the first lipid delivery vehicle is APC specific. In some embodiments, the first lipid delivery vehicle delivers the polypeptide or the polynucleotide into APC cells and does not deliver the polypeptide or the polynucleotide into the T cells. In some embodiments, the first lipid delivery vehicle comprises an APC targeting agent. In some embodiments, the APC targeting agent is a mono / di / oligo-saccharide or a synthetic analogue. In some embodiments, the APC targeting agent is a monosaccharide. In some embodiments, the APC targeting agent is a disaccharide. In some embodiments, the APC targeting agent is an oligosaccharide. In some embodiments, the APC targeting agent is a synthetic analogue. In some embodiments, the APC targeting agent binds to a macrophage galactose-type lectin (MGL), a mineralcorticoid receptor (MR), or a Dendritic Cell Specific Intracellular adhesion molecule 3- Grabbing Non-integrin (DC-SIGN). In some embodiments, the APC targeting agent binds to an MGL. In some embodiments, the APC targeting agent binds to an MR. In some embodiments, the APC targeting agent binds to a DC-SIGN.

[0310] In some embodiments, incubating the first population of APCs and T cells further comprises genetically editing the T cells of the first population of APCs and T cells. In some embodiments, genetically editing the population of T cells comprises inhibiting expression of an endogenous gene in the population of T 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, the endogenous gene comprises PRDM1. In some embodiments, the endogenous gene comprises TNFAIP3. In some embodiments, the endogenous gene comprises REGNASE-1. In some embodiments, the endogenous gene comprises SOCS1. In some embodiments, the endogenous gene comprises PTPN2. In some embodiments, the endogenous gene comprises CISH. In some embodiments, the endogenous gene comprises a combination of the foregoing genes. 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 an miRNA or siRNA that targets an RNA transcript encoded by the endogenous gene. 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 an miRNA that targets an RNA transcript encoded by the endogenousWSGR Docket No.: 50401-791.602 gene. 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 an siRNA that targets an RNA transcript encoded by 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 gene knockout using a site-specific nuclease. In some embodiments, silencing the gene locus comprises gene knockout using a site- specific nickase. In some embodiments, silencing the gene locus comprises gene knockout using a site-specific transcriptional regulator. In some embodiments, silencing the gene locus comprises gene knockout using a site-specific epigenetic regulator. In some embodiments, silencing the gene locus comprises delivering the site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator into the population of T cells via electroporation, a lipid nanoparticle, a lipoplex, a liposome, nucleofection, or a viral vector. In some embodiments, silencing the gene locus comprises delivering a nucleic acid encoding the site-specific nuclease, the site-specific nickase, or the site-specific epigenetic regulator into the population of T cells via electroporation, a lipid nanoparticle, a lipoplex, a liposome, nucleofection, or a viral vector.

[0311] In some embodiments, genetically editing the T cells comprises delivering a second lipid delivery vehicle comprising an exogenous gene into the T cells. In some embodiments, the second lipid delivery vehicle is T cell specific. In some embodiments, the second lipid delivery vehicle specifically targets T cells. In some embodiments, the second lipid delivery vehicle comprises a T cell targeting agent. In some embodiments, the T cell targeting agent is recognized by or interacts with a molecule present on a T cell surface. In some embodiments, the T cell targeting agent is recognized by a molecule present on a T cell surface. In some embodiments, the T cell targeting agent interacts with a molecule present on a T cell surface. In some embodiments, the T cell targeting agent is an antibody or fragment thereof. In some embodiments, the molecule present on the T cell surface is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD56, CD39, ICAM-1, CD366, CD279, CD200, CD25, CD103, HLA-DR, CD69, 4-1BB, ITGB7, ICOS, and CD137. In some embodiments, the molecule present on the T cell surface is CD2. In some embodiments, the molecule present on the T cell surface is CD3. In some embodiments, the molecule present on the T cell surface is CD4. In some embodiments, the molecule present on the T cell surface is CD5. In some embodiments, the molecule present on the T cell surface is CD7. In some embodiments, the molecule present on the T cell surface is CD8. In some embodiments, the molecule present on the T cell surface is CD56. In some embodiments, the molecule present on the T cell surface isWSGR Docket No.: 50401-791.602 CD39. In some embodiments, the molecule present on the T cell surface is ICAM-1. In some embodiments, the molecule present on the T cell surface is CD366. In some embodiments, the molecule present on the T cell surface is CD279. In some embodiments, the molecule present on the T cell surface is CD200. In some embodiments, the molecule present on the T cell surface is CD25. In some embodiments, the molecule present on the T cell surface is CD69. In some embodiments, the molecule present on the T cell surface is CD39. In some embodiments, the molecule present on the T cell surface is 4-1BB. In some embodiments, the molecule present on the T cell surface is ITGB7. In some embodiments, the molecule present on the T cell surface is ICOS. In some embodiments, the molecule present on the T cell surface is CD137.

[0312] In some embodiments, incubating the first population of APCs and T cells comprises delivering the first lipid delivery vehicle and the second lipid delivery vehicle into the first population of APCs and T cells concurrently in the same mixture. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99% or more. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 1%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 2%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 3%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 4%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 5%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 10%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 15%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 20%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 25%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 30%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 35%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 40%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 45%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 50%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 55%. In some embodiments, the percent of T cells inWSGR Docket No.: 50401-791.602 the population of T cells expressing the exogenous gene is at least 60%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 65%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 70%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 75%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 80%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 90%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 95%. In some embodiments, the percent of T cells in the population of T cells expressing the exogenous gene is at least 99% or more.

[0313] In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 99%, 95%, 90%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 55, 4%, 3%, 2%, 1% or less. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 1% or less. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 2%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 3%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 4%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 5%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 10%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 15%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 20%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 25%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 30%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 35%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 40%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 45%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 50%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 55%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 60%. In some embodiments, the percent of T cells in the populationWSGR Docket No.: 50401-791.602 of T cells expressing the endogenous gene is at most 65%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 70%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 75%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 80%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 90%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 95%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 99%.

[0314] In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 99% or more. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 1%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 2%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 3%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 4%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 5%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 10%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 15%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 20%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 25%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 30%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 35%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 40%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 45%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 50%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 55%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 60%. In some embodiments, the percent of T cells in the population of TWSGR Docket No.: 50401-791.602 cells with the endogenous gene inhibited is at least 65%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 70%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 75%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 80%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 90%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 95%. In some embodiments, the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 99% or more.

[0315] In some embodiments, the method further comprises administering the plurality of T cells that comprises antigen-specific T cells into a subject in need thereof. In some embodiments, the time from depleting to obtaining a therapeutically effective amount of antigen-specific cells is less than 30 days, less than 25 days, less than 20 days, less than 15 days, less than 10 days, less than 5 days, or less than 2 days. In some embodiments, the time from depleting to obtaining a therapeutically effective amount of antigen-specific cells is less than 30 days. In some embodiments, the time from depleting to obtaining a therapeutically effective amount of antigen specific cells is less than 26 days. In some embodiments, the time from depleting to obtaining a therapeutically effective amount of antigen specific cells is less than 20 days. In some embodiments, the time from depleting to obtaining a therapeutically effective amount of antigen specific cells is less than 15 days. In some embodiments, the time from depleting to obtaining a therapeutically effective amount of antigen specific cells is less than 10 days. In some embodiments, the time from depleting to obtaining a therapeutically effective amount of antigen specific cells is less than 5 days. In some embodiments, the time from depleting to obtaining a therapeutically effective amount of antigen specific cells is less than 2 days.

[0316] In some embodiments, the first lipid delivery vehicle comprises a lipid nanoparticle. In some embodiments, the first delivery vehicle comprises a lipoplex. In some embodiments, the second lipid delivery vehicle comprises a lipid nanoparticle. In some embodiments, the second delivery vehicle comprises a lipoplex.

[0317] In some embodiments, the lipid nanoparticle comprises R-DODMA, DOPE, cholesterol, and / or PEG. In some embodiments, the lipid nanoparticle comprises R-DODMA. In some embodiments, the lipid nanoparticle comprises DOPE. In some embodiments, the lipid nanoparticle comprises cholesterol. In some embodiments, the lipid nanoparticle comprises PEG. In some embodiments, the lipid nanoparticle comprises R-DODMA and DOPE. In someWSGR Docket No.: 50401-791.602 embodiments, the lipid nanoparticle comprises R-DODMA and cholesterol. In some embodiments, the lipid nanoparticle comprises R-DODMA and PEG. In some embodiments, the lipid nanoparticle comprises DOPE and cholesterol. In some embodiments, the lipid nanoparticle comprises DOPE and PEG. In some embodiments, the lipid nanoparticle comprises cholesterol and PEG. In some embodiments, the lipid nanoparticle comprises R-DODMA, DOPE, and cholesterol. In some embodiments, the lipid nanoparticle comprises R-DODMA, DOPE, and PEG. In some embodiments, the lipid nanoparticle comprises R-DODMA, cholesterol, and PEG. In some embodiments, the lipid nanoparticle comprises DOPE, cholesterol, and PEG. In some embodiments, the lipid nanoparticle comprises R-DODMA, DOPE, cholesterol, and PEG. In some embodiments, the ratio of R-DODMA to DOPE is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1: 30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100. In some embodiments, the ratio of R-DODMA to DOPE is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1. In some embodiments, the ratio of R-DODMA to cholesterol is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1: 30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100. In some embodiments, the ratio of R- DODMA to cholesterol is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1. In some embodiments, the ratio of R-DODMA to PEG is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1: 30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100. In some embodiments, the ratio of R-DODMA to PEG is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1In some embodiments, the ratio of R-DODMA to DOPE to cholesterol to PEG is 40:10:48:2. In some embodiments, the lipoplex comprises DOTAP and / or DOPE. In some embodiments, the lipoplex comprises DOTAP. In some embodiments, the lipoplex comprises DOPE. In some embodiments, the lipoplex comprises DOTAP and DOPE. In some embodiments, the ratio of DOTAP to DOPE is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1: 30, about 1:40, about 1:50, about 1:60, about 1:70, aboutWSGR Docket No.: 50401-791.602 1:80, about 1:90, or about 1:100. In some embodiments, the ratio of DOTAP to DOPE is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1. In some embodiments, the ratio of DOTAP to DOPE is 2:1.

[0318] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD8+ T cell epitope. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more CD8+ T cell epitopes. In some embodiments, each of the CD8+ T cell epitopes have a different epitope sequence. In some embodiments, CD8+ T cell epitope comprises a sequence of about 2 to 20, about 3 to 19, about 4 to 18, about 5 to 17, about 6 to 16, about 7 to 15, about 8 to 14, about 9 to 13, or about 10 to 12 consecutive amino acids from a cancer protein. In some embodiments, CD8+ T cell epitope comprises a sequence of about 7 to about 12 consecutive amino acids from a cancer protein.

[0319] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD4+ T cell epitope. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more CD4+ T cell epitopes. In some embodiments, each of the CD4+ T cell epitopes have a different epitope sequence In some embodiments, the CD4+ T cell epitope comprises a sequence of about 1 to 40, about 2 to 39, about 3 to 38, about 4 to 37, about 5 to 36, about 6 to 35, about 7 to 34, about 8 to 33, about 9 to 32, about 10 to 31, about 11 to 30, about 12 to 29, about 13 to 28, about 14 to 27, about 15 to 26, about 16 to 25, about 17 to 24, about 18 to 23, about 17 to 22, about 18 to 21, or about 19 to 20 consecutive amino acids from a cancer protein. In some embodiments, the CD4+ T cell epitope comprises a sequence of about 13 to 25 consecutive amino acids from a cancer protein. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a linker between the at least two different CD8+ T cell epitopes and the at least two different CD4+ T cell epitopes.

[0320] In some embodiments, the polynucleotide encoding the polypeptide comprises a linker. In some embodiments, the linker comprises a cleavable linker and / or a linker that is degradable by a protease. In some embodiments, the linker comprises a linker that is degradable by a protease. In some embodiments, the protease is a proteasome. In some embodiments, the linker comprises a cleavable linker. In some embodiments, wherein the least two different CD8+ T cell epitope sequences are separated by one or more linkers. In some embodiments, the least two different CD4+ T cell epitope sequences are separated by one or more linkers. In someWSGR Docket No.: 50401-791.602 embodiments, the one or more linkers are one or more flexible linkers. In some embodiments, wherein the one or more linkers are one or more cleavable linkers. In some embodiments, the polypeptide further comprises a Sec domain sequence at its N-terminus. In some embodiments, the polypeptide further comprises a MITD domain sequence at its C-terminus. In some embodiments, the Sec domain sequence comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity to a sequence of SEQ ID NO: 20 or 21. In some embodiments, the MITD domain sequence comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity to a sequence of SEQ ID NO: 19.

[0321] In some embodiments, the polynucleotide encoding the polypeptide comprises a sequence encoding the Sec domain at its 5’ end. In some embodiments, the polynucleotide encoding the polypeptide comprises a sequence encoding the MITD domain at its 3’ end. In some embodiments, the Sec domain comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity to a sequence of SEQ ID NOs: 23 or 24. In some embodiments, the sequence encoding the MITD domain comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity to a sequence of SEQ ID NO: 22.

[0322] Also provided herein is a method for producing a plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells. In some embodiments, the method comprises providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a biological sample from a subject. In some embodiments, the method comprises co-culturing APCs from the population of immune cells and T cells from the population of immune cells in the presence of a lipid nanoparticle formulation comprising 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 cells comprising stimulated T cells. In some embodiments, the method comprises expanding the stimulated T cells. In some embodiments, the method comprises (a) providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a biological sample from a subject; (b) co-culturing APCs from the population of immune cells and T cells from the population of immune cells in the presence of a lipid nanoparticle formulation comprising 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 cells comprising stimulated T cells; and (c) expanding the stimulated T cells.WSGR Docket No.: 50401-791.602

[0323] In some embodiments, the number of T cells in a separate culture or a coculture with APCs is about 5 X 105, about 1 X 106, about 2 X 106, about 3 X 106, about 4 X 106, about 5 X 106, about 6 X 106, about 7 X 106, about 8 X 106, about 9 X 106, about 10 X 106, about 11 X 106, about 12 X 106, about 13 X 106, about 14 X 106, about 15 X 106, or about 20 X 106. In some embodiments, the number of APCs in a separate culture or a coculture with T cells is about 1 X 105, about 2 X 105, about 3 X 105, about 4 X 105, about 5 X 105, about 6 X 105, about 7 X 105, about 8 X 105, about 9 X 105, about 1 X 106, about 1.5 X 106, about 2 X 106, about 2.5 X 106, about 3 X 106, about 3.5 X 106, about 4 X 106, about 4.5 X 106, about 5 X 106, about 5.5 X 106, about 6 X 106, about 6.5 X 106, about 7 X 106, about 7.5 X 106, about 8 X 106, about 8.5 X 106, about 9 X 106, about 9.5 X 106, or about 10 X 106. In some embodiments, the total cells in a coculture of T cells and APCs is about 5 X 105, about 1 X 106, about 2 X 106, about 3 X 106, about 4 X 106, about 5 X 106, about 6 X 106, about 7 X 106, about 8 X 106, about 9 X 106, about 10 X 106, about 11 X 106, about 12 X 106, about 13 X 106, about 14 X 106, about 15 X 106, or about 20 X 106. In some embodiments, the ratio of T cells to APCs in coculture is about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In some embodiments the cell density of the coculture is about 0.5 X 106 / cm2, about 1.0 X 106 / cm2, about 1.5 X 106 / cm2, about 2.0 X 106 / cm2, about 2.5 X 106 / cm2, about 3.0 X 106 / cm2, about 3.5 X 106 / cm2, about 4.0 X 106 / cm2, about 4.5 X 106 / cm2, about 5 X 106 / cm2, about 5.5 X 106 / cm2, about 6.0 X 106 / cm2, about 6.5 X 106 / cm2, about 7.0 X 106 / cm2, about 7.5 X 106 / cm2, about 8.0 X 106 / cm2, about 8.5 X 106 / cm2, about 9 X 106 / cm2, about 10 X 106 / cm2or more. The total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can affect T cell priming, expansion, and / or transposon integration. In some cases, the total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can improve T cell priming, expansion, and / or transposon integration. The total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can affect LNP / LPX-mediated RNA delivery. In some cases, total cell numbers can affect expansion of T cells. In some cases, lower total cell numbers can increase expansion of T cells. In some cases, T cell to APC ratios can affect expansion of T cells. In some cases, lower T cell to APC ratios can increase expansion of T cells. In some cases, cell density can affect expansion of T cells. In some cases, lower cell density can increase expansion of T cells.

[0324] In some embodiments, the lipid nanoparticle comprises R-DODMA, DOPE, cholesterol, and / or PEG. In some embodiments, the lipid nanoparticle comprises R-DODMA. In some embodiments, the lipid nanoparticle comprises DOPE. In some embodiments, the lipid nanoparticle comprises cholesterol. In some embodiments, the lipid nanoparticle comprisesWSGR Docket No.: 50401-791.602 PEG. In some embodiments, the lipid nanoparticle comprises R-DODMA and DOPE. In some embodiments, the lipid nanoparticle comprises R-DODMA and cholesterol. In some embodiments, the lipid nanoparticle comprises R-DODMA and PEG. In some embodiments, the lipid nanoparticle comprises DOPE and cholesterol. In some embodiments, the lipid nanoparticle comprises DOPE and PEG. In some embodiments, the lipid nanoparticle comprises cholesterol and PEG. In some embodiments, the lipid nanoparticle comprises R-DODMA, DOPE, and cholesterol. In some embodiments, the lipid nanoparticle comprises R-DODMA, DOPE, and PEG. In some embodiments, the lipid nanoparticle comprises R-DODMA, cholesterol, and PEG. In some embodiments, the lipid nanoparticle comprises DOPE, cholesterol, and PEG. In some embodiments, the lipid nanoparticle comprises R-DODMA, DOPE, cholesterol, and PEG. In some embodiments, the ratio of R-DODMA to DOPE is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1: 30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100. In some embodiments, the ratio of R-DODMA to DOPE is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1. In some embodiments, the ratio of R-DODMA to cholesterol is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1: 30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100. In some embodiments, the ratio of R- DODMA to cholesterol is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1. In some embodiments, the ratio of R-DODMA to PEG is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1: 30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100. In some embodiments, the ratio of R-DODMA to PEG is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1. In some embodiments, the ratio of R-DODMA to DOPE to cholesterol to PEG is 40:10:48:2.

[0325] Also provided herein is a method for producing a plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells. In some embodiments, the method comprises providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a biological sample from a subject. In some embodiments, the methodWSGR Docket No.: 50401-791.602 comprises co-culturing APCs from the population of immune cells and T cells from the population of immune cells in the presence of a lipoplex formulation comprising 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 cells comprising stimulated T cells. In some embodiments, the method comprises expanding the stimulated T cells. In some embodiments, the method comprises (a) providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a biological sample from a subject; (b) co-culturing APCs from the population of immune cells and T cells from the population of immune cells in the presence of a lipoplex formulation comprising 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 cells comprising stimulated T cells; and (c) expanding the stimulated T cells.

[0326] In some embodiments, the number of T cells in a separate culture or a coculture with APCs is about 5 X 105, about 1 X 106, about 2 X 106, about 3 X 106, about 4 X 106, about 5 X 106, about 6 X 106, about 7 X 106, about 8 X 106, about 9 X 106, about 10 X 106, about 11 X 106, about 12 X 106, about 13 X 106, about 14 X 106, about 15 X 106, or about 20 X 106. In some embodiments, the number of APCs in a separate culture or a coculture with T cells is about 1 X 105, about 2 X 105, about 3 X 105, about 4 X 105, about 5 X 105, about 6 X 105, about 7 X 105, about 8 X 105, about 9 X 105, about 1 X 106, about 1.5 X 106, about 2 X 106, about 2.5 X 106, about 3 X 106, about 3.5 X 106, about 4 X 106, about 4.5 X 106, about 5 X 106, about 5.5 X 106, about 6 X 106, about 6.5 X 106, about 7 X 106, about 7.5 X 106, about 8 X 106, about 8.5 X 106, about 9 X 106, about 9.5 X 106, or about 10 X 106. In some embodiments, the total cells in a coculture of T cells and APCs is about 5 X 105, about 1 X 106, about 2 X 106, about 3 X 106, about 4 X 106, about 5 X 106, about 6 X 106, about 7 X 106, about 8 X 106, about 9 X 106, about 10 X 106, about 11 X 106, about 12 X 106, about 13 X 106, about 14 X 106, about 15 X 106, or about 20 X 106. In some embodiments, the ratio of T cells to APCs in coculture is about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In some embodiments the cell density of the coculture is about 0.5 X 106 / cm2, about 1.0 X 106 / cm2, about 1.5 X 106 / cm2, about 2.0 X 106 / cm2, about 2.5 X 106 / cm2, about 3.0 X 106 / cm2, about 3.5 X 106 / cm2, about 4.0 X 106 / cm2, about 4.5 X 106 / cm2, about 5 X 106 / cm2, about 5.5 X 106 / cm2, about 6.0 X 106 / cm2, about 6.5 X 106 / cm2, about 7.0 X 106 / cm2, about 7.5 X 106 / cm2, about 8.0 X 106 / cm2, about 8.5 X 106 / cm2, about 9 X 106 / cm2, about 10 X 106 / cm2or more. The total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can affect T cell priming, expansion, and / or transposon integration. In some cases, the total cellWSGR Docket No.: 50401-791.602 numbers, seeding density, or ratio of APCs and T cells (in a coculture) can improve T cell priming, expansion, and / or transposon integration. The total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can affect LNP / LPX-mediated RNA delivery. In some cases, total cell numbers can affect expansion of T cells. In some cases, lower total cell numbers can increase expansion of T cells. In some cases, T cell to APC ratios can affect expansion of T cells. In some cases, lower T cell to APC ratios can increase expansion of T cells. In some cases, cell density can affect expansion of T cells. In some cases, lower cell density can increase expansion of T cells.

[0327] In some embodiments, the lipoplex comprises DOTAP and / or DOPE. In some embodiments, the lipoplex comprises DOTAP. In some embodiments, the lipoplex comprises DOPE. In some embodiments, the lipoplex comprises DOTAP and DOPE. In some embodiments, the ratio of DOTAP to DOPE is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:15, about 1:20, about 1: 30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, or about 1:100. In some embodiments, the ratio of DOTAP to DOPE is about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, about 20:1, about 30:1, about 40:1, about 50:1, about 60:1, about 70:1, about 80:1, about 90:1, or about 100:1. In some embodiments, the ratio of DOTAP to DOPE is 2:1.

[0328] Also provided herein is a method for producing plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells. In some embodiments, the method comprises providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a biological sample from a subject. In some embodiments, the method comprises co- culturing APCs from the population of immune cells and T cells from the population of immune cells in the presence of a first lipid nanoparticle formulation comprising a polynucleotide encoding a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer and a second lipid nanoparticle formulation comprising a polynucleotide, wherein the second lipid nanoparticle formulation is different than the first lipid nanoparticle formulation, thereby forming a population of cells comprising stimulated T cells. In some embodiments, the method comprises expanding the stimulated T cells. In some embodiments, the method comprises (a) providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a biological sample from a subject; (b) co-culturing APCs from the population of immune cells and T cells from the population of immune cells in the presence of a first lipid nanoparticle formulation comprising a polynucleotide encoding a polypeptide comprising at least one tumor antigen epitope sequenceWSGR Docket No.: 50401-791.602 expressed by cancer cells of a human subject with cancer and a second lipid nanoparticle formulation comprising a polynucleotide, wherein the second lipid nanoparticle formulation is different than the first lipid nanoparticle formulation, thereby forming a population of cells comprising stimulated T cells; and (c) expanding the stimulated T cells.

[0329] In some embodiments, the number of T cells in a separate culture or a coculture with APCs is about 5 X 105, about 1 X 106, about 2 X 106, about 3 X 106, about 4 X 106, about 5 X 106, about 6 X 106, about 7 X 106, about 8 X 106, about 9 X 106, about 10 X 106, about 11 X 106, about 12 X 106, about 13 X 106, about 14 X 106, about 15 X 106, or about 20 X 106. In some embodiments, the number of APCs in a separate culture or a coculture with T cells is about 1 X 105, about 2 X 105, about 3 X 105, about 4 X 105, about 5 X 105, about 6 X 105, about 7 X 105, about 8 X 105, about 9 X 105, about 1 X 106, about 1.5 X 106, about 2 X 106, about 2.5 X 106, about 3 X 106, about 3.5 X 106, about 4 X 106, about 4.5 X 106, about 5 X 106, about 5.5 X 106, about 6 X 106, about 6.5 X 106, about 7 X 106, about 7.5 X 106, about 8 X 106, about 8.5 X 106, about 9 X 106, about 9.5 X 106, or about 10 X 106. In some embodiments, the total cells in a coculture of T cells and APCs is about 5 X 105, about 1 X 106, about 2 X 106, about 3 X 106, about 4 X 106, about 5 X 106, about 6 X 106, about 7 X 106, about 8 X 106, about 9 X 106, about 10 X 106, about 11 X 106, about 12 X 106, about 13 X 106, about 14 X 106, about 15 X 106, or about 20 X 106. In some embodiments, the ratio of T cells to APCs in coculture is about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In some embodiments the cell density of the coculture is about 0.5 X 106 / cm2, about 1.0 X 106 / cm2, about 1.5 X 106 / cm2, about 2.0 X 106 / cm2, about 2.5 X 106 / cm2, about 3.0 X 106 / cm2, about 3.5 X 106 / cm2, about 4.0 X 106 / cm2, about 4.5 X 106 / cm2, about 5 X 106 / cm2, about 5.5 X 106 / cm2, about 6.0 X 106 / cm2, about 6.5 X 106 / cm2, about 7.0 X 106 / cm2, about 7.5 X 106 / cm2, about 8.0 X 106 / cm2, about 8.5 X 106 / cm2, about 9 X 106 / cm2, about 10 X 106 / cm2or more. The total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can affect T cell priming, expansion, and / or transposon integration. In some cases, the total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can improve T cell priming, expansion, and / or transposon integration. The total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can affect LNP / LPX-mediated RNA delivery. In some cases, total cell numbers can affect expansion of T cells. In some cases, lower total cell numbers can increase expansion of T cells. In some cases, T cell to APC ratios can affect expansion of T cells. In some cases, lower T cell to APC ratios can increase expansion of T cells. In some cases, cell density can affect expansion of T cells. In some cases, lower cell density can increase expansion of T cells.WSGR Docket No.: 50401-791.602

[0330] In some embodiments the second lipid nanoparticle formulation comprises a T cell targeting agent. In some embodiments, the T cell targeting agent is recognized by or interacts with a molecule present on a T cell surface. In some embodiments, the T cell targeting agent is recognized by a molecule present on a T cell surface. In some embodiments, the T cell targeting agent interacts with a molecule present on a T cell surface. In some embodiments, the T cell targeting agent is an antibody or fragment thereof. In some embodiments, the molecule present on the T cell surface is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD8, CD56, CD39, ICAM-1, CD366, CD279, CD200, CD25, CD103, HLA-DR, CD69, 4-1BB, ITGB7, ICOS, and CD137. In some embodiments, the molecule present on the T cell surface is CD2. In some embodiments, the molecule present on the T cell surface is CD3. In some embodiments, the molecule present on the T cell surface is CD4. In some embodiments, the molecule present on the T cell surface is CD5. In some embodiments, the molecule present on the T cell surface is CD7. In some embodiments, the molecule present on the T cell surface is CD8. In some embodiments, the molecule present on the T cell surface is CD56. In some embodiments, the molecule present on the T cell surface is CD39. In some embodiments, the molecule present on the T cell surface is ICAM-1. In some embodiments, the molecule present on the T cell surface is CD366. In some embodiments, the molecule present on the T cell surface is CD279. In some embodiments, the molecule present on the T cell surface is CD200. In some embodiments, the molecule present on the T cell surface is CD25. In some embodiments, the molecule present on the T cell surface is CD69. In some embodiments, the molecule present on the T cell surface is 4-1BB. In some embodiments, the molecule present on the T cell surface is ITGB7. In some embodiments, the molecule present on the T cell surface is ICOS. In some embodiments, the molecule present on the T cell surface is CD137.

[0331] In some embodiments, the polynucleotide of the second lipid nanoparticle formulation comprises or encodes an agent for genetically editing T cells. In some embodiments, the polynucleotide of the second lipid nanoparticle formulation comprises an agent for genetically editing T cells. In some embodiments, the polynucleotide of the second lipid nanoparticle formulation encodes an agent for genetically editing T cells. In some embodiments, the polynucleotide encodes a guide RNA, a site-specific nuclease, a site-specific nickase, and / or a site-specific-epigenetic regulator. In some embodiments, the polynucleotide encodes a guide RNA. In some embodiments, the polynucleotide encodes a site-specific nuclease. In some embodiments, the polynucleotide encodes a site-specific nickase. In some embodiments, the polynucleotide encodes a site-specific-epigenetic regulator. In some embodiments, the polynucleotide encodes a guide RNA and a site-specific nuclease. In some embodiments, theWSGR Docket No.: 50401-791.602 polynucleotide encodes a guide RNA and a site-specific nickase. In some embodiments, the polynucleotide encodes a guide RNA and a site-specific epigenetic regulator. In some embodiments, the polynucleotide comprises an RNA. In some embodiments, the RNA is a guide RNA.

[0332] In some embodiments, the first lipid nanoparticle formulation comprises an APC targeting agent. In some embodiments, the APC targeting agent is a mono / di / oligo-saccharide or a synthetic analogue. In some embodiments, the APC targeting agent is a monosaccharide. In some embodiments, the APC targeting agent is a disaccharide. In some embodiments, the APC targeting agent is an oligosaccharide. In some embodiments, the APC targeting agent is a synthetic analogue. In some embodiments, the APC targeting agent binds to a macrophage galactose-type lectin (MGL), a mineralcorticoid receptor (MR), or a Dendritic Cell Specific Intracellular adhesion molecule 3-Grabbing Non-integrin (DC-SIGN). In some embodiments, the APC targeting agent binds to an MGL. In some embodiments, the APC targeting agent binds to an MR. In some embodiments, the APC targeting agent binds to a DC-SIGN.

[0333] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more tumor antigen epitope sequences.

[0334] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD8+ T cell epitope. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more CD8+ T cell epitopes. In some embodiments, the polypeptide comprises at least one tumor antigen epitope sequence comprises at least two different CD8+ T cell epitope sequences. In some embodiments, each of the CD8+ T cell epitopes have a different epitope sequence. In some embodiments, CD8+ T cell epitope comprises a sequence of about 2 to 20, about 3 to 19, about 4 to 18, about 5 to 17, about 6 to 16, about 7 to 15, about 8 to 14, about 9 to 13, or about 10 to 12 consecutive amino acids from a cancer protein. In some embodiments, CD8+ T cell epitope comprises a sequence of about 7 to about 12 consecutive amino acids from a cancer protein.

[0335] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD4+ T cell epitope. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more CD4+ T cell epitopes. InWSGR Docket No.: 50401-791.602 some embodiments, each of the CD4+ T cell epitopes have a different epitope sequence. In some embodiments, the polypeptide comprises at least one tumor antigen epitope sequence comprises at least two different CD4+ T cell epitope sequences. In some embodiments, the CD4+ T cell epitope comprises a sequence of about 1 to 40, about 2 to 39, about 3 to 38, about 4 to 37, about 5 to 36, about 6 to 35, about 7 to 34, about 8 to 33, about 9 to 32, about 10 to 31, about 11 to 30, about 12 to 29, about 13 to 28, about 14 to 27, about 15 to 26, about 16 to 25, about 17 to 24, about 18 to 23, about 17 to 22, about 18 to 21, or about 19 to 20 consecutive amino acids from a cancer protein. In some embodiments, the CD4+ T cell epitope comprises a sequence of about 13 to 25 consecutive amino acids from a cancer protein. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a linker between the at least two CD8+ T cell epitopes and the at least two CD4+ T cell epitopes.

[0336] In some embodiments, the polynucleotide encoding the polypeptide comprises a linker. In some embodiments, the linker comprises a cleavable linker and / or a linker that is degradable by a protease. In some embodiments, the linker comprises a linker that is degradable by a protease. In some embodiments, the protease is a proteasome. In some embodiments, the linker comprises a cleavable linker. In some embodiments, wherein the least two different CD8+ T cell epitope sequences are separated by one or more linkers. In some embodiments, the least two different CD4+ T cell epitope sequences are separated by one or more linkers. In some embodiments, the one or more linkers are one or more flexible linkers. In some embodiments, wherein the one or more linkers are one or more cleavable linkers. In some embodiments, the polypeptide further comprises a Sec domain sequence at its N-terminus. In some embodiments, the polypeptide further comprises a MITD domain sequence at its C-terminus. In some embodiments, the Sec domain sequence comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity to a sequence of SEQ ID NOs: 20 or 21. In some embodiments, the MITD domain sequence comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity to a sequence of SEQ ID NO: 19.

[0337] In some embodiments, the polynucleotide encoding the polypeptide comprises a sequence encoding the Sec domain at its 5’ end. In some embodiments, the polynucleotide encoding the polypeptide comprises a sequence encoding the MITD domain at its 3’ end. In some embodiments, the polynucleotide encoding the polypeptide comprises one or more sequences encoding one or more linkers. In some embodiments, the Sec domain comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity to a sequence of SEQ ID NOs: 23 or 24. In some embodiments, the sequenceWSGR Docket No.: 50401-791.602 encoding the MITD domain comprises a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% sequence identity to a sequence of SEQ ID NO: 22.

[0338] Also provided herein is a method for producing a plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells. In some embodiments, the method comprises providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a biological sample from a subject. In some embodiments, the method comprises co-culturing APCs from the population of immune cells and T cells from the population of immune cells, thereby forming a population of cells comprising stimulated T cells. In some embodiments, the method comprises (a) providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a biological sample from a subject; (b) co-culturing APCs from the population of immune cells and T cells from the population of immune cells, thereby forming a population of cells comprising stimulated T cells; and (c) expanding the stimulated T cells, thereby forming the plurality of T cells that comprise antigen- specific T cells comprising a therapeutically effective amount of antigen-specific T cells or at least 106, at least 107, at least 108, or at least 109antigen-specific T antigen-specific T cells. In some embodiments, the plurality of T cells comprises at least 1,000 antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 5,000 antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 10,000 antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 50,000 antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 100,000 antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 500,000 antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 1,000,000 antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 5,000,000 antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 10,000,000 antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 50,000,000 antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 108antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 5 X 108antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 109antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 5 X 109antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 1010antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 5 X 1011antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 1012antigen specific T cells.WSGR Docket No.: 50401-791.602

[0339] In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 30 days, less than 25 days, less than 20 days, less than 19 days, less than 18 days, less than 17 days, less than 16 days, less than 15 days, less than 14 days, less than 13 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, less than 10 days, less than 9 days, less than 8 days, less than 7 days, less than 6 days, less than 5 days, less than 4 days, less than 3 days, less than 2 days or less than 1 day. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 30 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 25 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 20 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 19 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 18 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 17 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 16 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 15 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 14 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 13 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 12 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 11 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 10 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 9 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 8 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 7 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 6 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 5 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is lessWSGR Docket No.: 50401-791.602 than 4 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 3 days. In some embodiments, the time from providing to obtaining the plurality of T cells that comprise antigen specific cells is less than 2 days.

[0340] In some embodiments, the number of T cells in a separate culture or a coculture with APCs is about 5 X 105, about 1 X 106, about 2 X 106, about 3 X 106, about 4 X 106, about 5 X 106, about 6 X 106, about 7 X 106, about 8 X 106, about 9 X 106, about 10 X 106, about 11 X 106, about 12 X 106, about 13 X 106, about 14 X 106, about 15 X 106, or about 20 X 106. In some embodiments, the number of APCs in a separate culture or a coculture with T cells is about 1 X 105, about 2 X 105, about 3 X 105, about 4 X 105, about 5 X 105, about 6 X 105, about 7 X 105, about 8 X 105, about 9 X 105, about 1 X 106, about 1.5 X 106, about 2 X 106, about 2.5 X 106, about 3 X 106, about 3.5 X 106, about 4 X 106, about 4.5 X 106, about 5 X 106, about 5.5 X 106, about 6 X 106, about 6.5 X 106, about 7 X 106, about 7.5 X 106, about 8 X 106, about 8.5 X 106, about 9 X 106, about 9.5 X 106, or about 10 X 106. In some embodiments, the total cells in a coculture of T cells and APCs is about 5 X 105, about 1 X 106, about 2 X 106, about 3 X 106, about 4 X 106, about 5 X 106, about 6 X 106, about 7 X 106, about 8 X 106, about 9 X 106, about 10 X 106, about 11 X 106, about 12 X 106, about 13 X 106, about 14 X 106, about 15 X 106, or about 20 X 106. In some embodiments, the ratio of T cells to APCs in coculture is about 1:2, about 1:1, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1. In some embodiments the cell density of the coculture is about 0.5 X 106 / cm2, about 1.0 X 106 / cm2, about 1.5 X 106 / cm2, about 2.0 X 106 / cm2, about 2.5 X 106 / cm2, about 3.0 X 106 / cm2, about 3.5 X 106 / cm2, about 4.0 X 106 / cm2, about 4.5 X 106 / cm2, about 5 X 106 / cm2, about 5.5 X 106 / cm2, about 6.0 X 106 / cm2, about 6.5 X 106 / cm2, about 7.0 X 106 / cm2, about 7.5 X 106 / cm2, about 8.0 X 106 / cm2, about 8.5 X 106 / cm2, about 9 X 106 / cm2, about 10 X 106 / cm2or more. The total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can affect T cell priming, expansion, and / or transposon integration. In some cases, the total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can improve T cell priming, expansion, and / or transposon integration. The total cell numbers, seeding density, or ratio of APCs and T cells (in a coculture) can affect LNP / LPX-mediated RNA delivery. In some cases, total cell numbers can affect expansion of T cells. In some cases, lower total cell numbers can increase expansion of T cells. In some cases, T cell to APC ratios can affect expansion of T cells. In some cases, lower T cell to APC ratios can increase expansion of T cells. In some cases, cell density can affect expansion of T cells. In some cases, lower cell density can increase expansion of T cells.WSGR Docket No.: 50401-791.602

[0341] In some embodiments, the method comprises, prior to expanding the stimulated T cells, restimulating the stimulated T cells with one or more additional preparations of APCs. In some embodiments, restimulating comprises obtaining a population of cells comprising APCs from a biological sample of a subject. In some embodiments, the restimulation step comprises depleting the population of cells of CD56+ cells, CD25+ cells, and CD3+ cells. In some embodiments, the restimulation step comprises positively selecting CD14+ cells and CD19+ cells from the population of cells. In some embodiments, restimulating comprises culturing the population of cells in the presence of IL-4, GMCSF, and / or FLT3L. In some embodiments, the restimulating comprises contacting the population of cells with (A) the polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, (B) the polynucleotide encoding the polypeptide into the population of cells, or (C) electroporating the polynucleotide encoding the polypeptide to the population of cells. In some embodiments, the restimulation step comprises co-culturing the population of cells with the stimulated T cells. In some embodiments, restimulating comprises (i) comprises obtaining a population of cells comprising APCs from a biological sample of a subject, (ii) depleting the population of cells of CD56+ cells, CD25+ cells, and CD3+ cells, (iii) culturing the population of cells in the presence of IL-4, GMCSF, and / or FLT3L, (iv) contacting the population of cells with (A) the polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, (B) the polynucleotide encoding the polypeptide into the population of cells, or (C) electroporating the polynucleotide encoding the polypeptide to the population of cells, and (v) co-culturing the population of cells with the stimulated T cells. In some embodiments, restimulating comprises (i) comprises obtaining a population of cells comprising APCs from a biological sample of a subject, (ii) positively selecting CD14+ cells and CD19+ cells from the population of cells, (iii) culturing the population of cells in the presence of IL-4, GMCSF, and / or FLT3L, (iv) contacting the population of cells with (A) the polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, (B) the polynucleotide encoding the at least one polypeptide into the population of cells, or (C) electroporating the polynucleotide encoding the at least one polypeptide to the population of cells, and (v) co-culturing the population of cells with the stimulated T cells.

[0342] In some embodiments, the method comprises, prior to expanding the stimulated T cells, restimulating the stimulated T cells with one or more additional preparations of APCs. In some embodiments, restimulating comprises obtaining a population of cells comprising APCs from a biological sample of a subject. In some embodiments, the restimulation step comprises depleting the population of cells of CD56+ cells, CD25+ cells, and CD3+ cells. In some embodiments,WSGR Docket No.: 50401-791.602 restimulating comprises culturing the population of cells in the presence of IL-4, GMCSF, and / or FLT3L. In some embodiments, the restimulation step comprises positively selecting CD14+ cells and CD19+ cells from the population of cells. In some embodiments, the restimulating comprises electroporating the population of cells with the polynucleotide encoding the polypeptide, the polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer into the population of cells. In some embodiments, the restimulation step comprises co-culturing the population of cells with the stimulated T cells. In some embodiments, restimulating comprises (i) comprises obtaining a population of cells comprising APCs from a biological sample of a subject, (ii) depleting the population of cells of CD56+ cells, CD25+ cells, and CD3+ cells, (iii) culturing the population of cells in the presence of IL-4, GMCSF, and / or FLT3L, (iv) electroporating the population of cells with the polynucleotide encoding the polypeptide, the polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer into the population of cells, and (v) co-culturing the population of cells with the stimulated T cells. In some embodiments, restimulating comprises (i) comprises obtaining a population of cells comprising APCs from a biological sample of a subject, (ii) positively selecting CD14+ cells and CD19+ cells from the population of cells, (iii) culturing the population of cells in the presence of IL-4, GMCSF, and / or FLT3L, (iv) electroporating the population of cells with the polynucleotide encoding the polypeptide, the polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer into the population of cells, and (v) co-culturing the population of cells with the stimulated T cells.

[0343] In some embodiments, the percentage of CD107a+ cells in the plurality of T cells that comprises antigen-specific T cells is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of the antigen-specific T cells. In some embodiments, the percentage of CD107a+ cells in the plurality of T cells that comprises antigen-specific T cells is at least 10% of the antigen-specific T cells.WSGR Docket No.: 50401-791.602

[0344] In some embodiments, the percentage of TNF + cells in the plurality of T cells thatcomprises antigen-specific T cells is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of theantigen-specific T cells. In some embodiments, the percentage of TNF + cells in the plurality ofT cells that comprises antigen-specific T cells is at least 5% of the antigen-specific T cells.

[0345] In some embodiments, the percentage of IFN + cells in the plurality of T cells thatcomprises antigen-specific T cells is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of theantigen-specific T cells. In some embodiments, the percentage of IFN + cells in the plurality ofT cells that comprises antigen-specific T cells is at least 15% of the antigen-specific T cells.

[0346] In some embodiments, the percentage of IFN + and TNF + cells in the plurality of Tcells that comprises antigen-specific T cells is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% ofWSGR Docket No.: 50401-791.602the antigen-specific T cells. In some embodiments, the percentage of IFN + and TNF + cells inthe plurality of T cells that comprises antigen-specific T cells is at least 2% of the antigen- specific T cells.

[0347] In some embodiments, the percentage of in the plurality of T cells that comprises antigen-specific T cells is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of the antigen-specific T cells. In some embodiments, the percentage of cells in the plurality of T cells that comprises antigen-specific T cells is at least 0.5% of the antigen- specific T cells.

[0348] In some embodiments, the percentage of in the plurality of T cells that comprises antigen-specific T cells is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of the antigen-specific T cells. In some embodiments, the percentage of in the plurality of T cells that comprises antigen-specific T cells is at least 5% of the antigen- specific T cells.

[0349] In some embodiments, the percentage of in the plurality of T cells that comprises antigen-specific T cells is at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 4%, at least 6%, at leastWSGR Docket No.: 50401-791.602 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99% of the antigen-specific T cells. In some embodiments, the percentage of in the plurality of T cells that comprises antigen-specific T cells is at least 0.1% of the antigen-specific T cells.

[0350] In some embodiments, the percentage of CD4+ T cells in the plurality of T cells that comprises antigen-specific T cells that are naive T cells (CD62L+ and CD45RA+) is at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, at most 5%, at most 1%. In some embodiments, the percentage of CD4+ T cells in the plurality of T cells that comprises antigen-specific T cells that are naive T cells (CD62L+ and CD45RA+) is at most 15%.

[0351] In some embodiments, the percentage of CD4+ T cells in the plurality of T cells that comprises antigen-specific T cells that are effector memory T cells (CD62L- and CD45RA-) is at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%. In some embodiments, the percentage of CD4+ T cells in the plurality of T cells that comprises antigen-specific T cells that are effector memory T cells (CD62L- and CD45RA-) is at least 60%.

[0352] In some embodiments, percentage of CD4+ T cells in the plurality of T cells that comprises antigen-specific T cells that are effector T cells (CD62L- and CD45RA+) is at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, at most 5%, at most 4%, at most 3%, at most 2%, or at most 1%. In someWSGR Docket No.: 50401-791.602 embodiments, the percentage of CD4+ T cells in the plurality of T cells that comprises antigen- specific T cells that are effector T cells (CD62L- and CD45RA+) is at most 5%.

[0353] In some embodiments, the percentage of CD4+ T cells in the plurality of T cells that comprises antigen-specific T cells that are central memory T cells (CD62L+ and CD45RA-) is at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%. In some embodiments, the percentage of CD4+ T cells in the plurality of T cells that comprises antigen-specific T cells that are central memory T cells (CD62L+ and CD45RA-) is at least 10%.

[0354] In some embodiments, the percentage of CD8+ T cells in the plurality of T cells that comprises antigen-specific T cells that are naive T cells (CD62L+ and CD45RA+) is at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, at most 5%, at most 1%. In some embodiments, the percentage of CD8+ T cells in the plurality of T cells that comprises antigen-specific T cells that are naive T cells (CD62L+ and CD45RA+) is at most 25%.

[0355] In some embodiments, the percentage of CD8+ T cells in the plurality of T cells that comprises antigen-specific T cells that are effector memory T cells (CD62L- and CD45RA-) is at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, atWSGR Docket No.: 50401-791.602 least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%. In some embodiments, the percentage of CD8+ T cells in the plurality of T cells that comprises antigen-specific T cells that are effector memory T cells (CD62L- and CD45RA-) is at least 60%.

[0356] In some embodiments the percentage of CD8+ T cells in the plurality of T cells that comprises antigen-specific T cells that are effector T cells (CD62L- and CD45RA+) is at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%, at most 5%, at most 1%. In some embodiments, the percentage of CD8+ T cells in the plurality of T cells that comprises antigen-specific T cells that are effector T cells (CD62L- and CD45RA+) is at most 10%.

[0357] In some embodiments, the percentage of CD8+ T cells in the plurality of T cells that comprises antigen-specific T cells that are central memory T cells (CD62L+ and CD45RA-)is at least 0.01%, at least 0.02%, at least 0.03%, at least 0.04%, at least 0.05%, at least 0.06%, at least 0.07%, at least 0.08%, at least 0.09%, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 4%, at least 6%, at least 8%, at least 10%, at least 12%, at least 14%, at least 16%, at least 18%, at least 20%, at least 22%, at least 24%, at least 26%, at least 28%, at least 30%, at least 32%, at least 34%, at least 36%, at least 38%, at least 40%, at least 42%, at least 44%, at least 46%, at least 48%, at least 50%, at least 52%, at least 54%, at least 56%, at least 58%, at least 60%, at least 62%, at least 64%, at least 66%, at least 68%, at least 70%, at least 72%, at least 74%, at least 76%, at least 78%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%. In some embodiments, the percentage of CD8+ T cells in the plurality of T cells that comprises antigen-specific T cells that are central memory T cells (CD62L+ and CD45RA-) is at least 15%.

[0358] In some embodiments, the plurality of T cells that comprises antigen-specific T cells produces cytokines upon recognition of target cells. In some embodiments, the plurality of T cells that comprises antigen-specific T cells cause degranulation upon recognition of target cells. In some embodiments, the plurality of T cells that comprises antigen-specific T cells produces cytokines and cause degranulation upon recognition of target cells. For example, the antigen- specific T cells can produce cytokines and cause degranulation upon recognition of cognate antigen.

[0359] In some embodiments, the number of T cells in the plurality of T cells that comprises antigen-specific T cells is at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at leastWSGR Docket No.: 50401-791.602 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90-fold, at least 95-fold, at least 100-fold, at least 110-fold, at least 120-fold, at least 130-fold, at least 140-fold, at least 160-fold, at least 180-fold, at least 200-fold, at least 225-fold, at least 250-fold, at least 275-fold, at least 300-fold, at least 325-fold, at least 350-fold, at least 375-fold, at least 400-fold, at least 425-fold, at least 450-fold, at least 475-fold, at least 500-fold, at least 525-fold, at least 550-fold, at least 575-fold, at least 600-fold, at least 625-fold, at least 650-fold, at least 675-fold, at least 700-fold, at least 725-fold, at least 750-fold, at least 775-fold, at least 800-fold, at least 825-fold, at least 850-fold, at least 875-fold, at least 900-fold, at least 925-fold, at least 950-fold, at least 975-fold, at least 1000-fold or more higher than the number of T cells in the population of immune cells.

[0360] In some embodiments, the percentage of antigen-specific T cells in the plurality of T cells produced by the methods described herein are at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, 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%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, 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%, at least 98.5%, at least 99% or more.

[0361] 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 comprising tumor antigen specific T cells can be at least two-fold higher than the fraction of CD8+ antigen-specific T cells of the total number of CD8+ T cells in the biological sample. 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 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+ antigen-specific T cells of the total number of CD8+ T cells in the biological sample. 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 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-fold higher, about 6-fold higher,WSGR Docket No.: 50401-791.602 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, about 20- 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-fold higher than the fraction of CD8+ antigen- specific T cells of the total number of CD8+ T cells in the biological sample.

[0362] In some embodiments, the fraction of CD4+ 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+ antigen-specific T cells of the total number of CD8+ T cells in the biological sample. 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 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+ antigen-specific T cells ofWSGR Docket No.: 50401-791.602 the total number of CD4+ T cells in the biological sample. 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 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-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, about 20- 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-fold higher than the fraction of CD4+ antigen- specific T cells of the total number of CD4+ T cells in the biological sample.

[0363] In some embodiments, at least 0.1% of the CD8+ T cells in the expanded population of cells comprising antigen-specific T cells can be CD8+ antigen-specific T cells derived from naïve CD8+ 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%, at least 0.35%, at least 0.4%, at least 0.45%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 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%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, atWSGR Docket No.: 50401-791.602 least 85%, at least 90%, at least 95%, or at least more than 95% of the CD8+ T cells in the expanded population of cells comprising antigen-specific T cells can be CD8+ antigen-specific T cells derived from naïve CD8+ T cells.

[0364] In some embodiments, at least 0.1% of the CD4+ T cells in the expanded population of cells comprising antigen-specific T cells can be CD4+ antigen-specific T cells derived from naïve 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%, at least 0.35%, at least 0.4%, at least 0.45%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 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%, 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% of the CD4+ T cells in the expanded population of cells comprising antigen-specific T cells can be CD4+ antigen-specific T cells derived from naïve CD4+ T cells. In some embodiments, at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, 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%, 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%, 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%, 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% of the CD4+ T cells in the expanded population of cells comprising antigen-specific T cells can be CD4+ antigen-specific T cells derived from naïve CD4+ T cells.

[0365] Also provided herein is a method for producing a plurality of T cells. In some embodiments, the plurality of T cells comprises antigen-specific T cells. In some embodiments, the method comprises incubating APCs and T cells in the presence of (A) at least one polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells ofWSGR Docket No.: 50401-791.602 a human subject with cancer, or (B) a polynucleotide encoding the at least one polypeptide thereby forming a population of cells comprising stimulated T cells. In some embodiments, the method comprises incubating APCs and T cells, wherein the APCs comprise (A) at least one 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 at least one polypeptide thereby forming a population of cells comprising stimulated T cells. In some embodiments, the stimulated T cells or a subset thereof express an IL-2 receptor variant. In some embodiments, expression of the IL-2 receptor variant is induced upon T cell simulation. In some embodiments, the method comprises culturing the population of cells comprising stimulated T cells in the presence of IL-2 variant. In some embodiments, the stimulated T cells or a subset thereof expressing the IL-2 receptor variant expand, thereby obtaining the plurality of T cells comprises antigen-specific T cells. In some embodiments, the method comprises (a) incubating APCs and T cells (i) in the presence of (A) at least one 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 at least one polypeptide, or (ii) wherein the APCs comprise (A) at least one 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 at least one polypeptide, thereby forming a population of cells comprising stimulated T cells, wherein the stimulated T cells or a subset thereof express an IL-2 receptor variant, and wherein expression of the IL-2 receptor variant is induced upon T cell simulation and (b) culturing the population of cells comprising stimulated T cells in the presence of IL-2 variant, wherein the stimulated T cells or a subset thereof expressing the IL-2 receptor variant expand, thereby obtaining the plurality of T cells comprises antigen-specific T cells.

[0366] In some embodiments, the stimulated T cells or a subset thereof expressing the IL-2 receptor variant comprises a nucleic acid sequence comprising a regulatory sequence and a sequence encoding the IL-2 receptor variant. In some embodiments, the regulatory sequence comprises one or more Nuclear Factor of Activated T cells (NFAT) response elements (REs) or repeats. For example, the regulatory sequence comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more NFAT response elements or repeats. In some embodiments, the regulatory sequence comprises a promoter. In some embodiments, the promoter is a minimal promoter. In some embodiments, the minimal promoter comprises a MinP promoter, a ybTATA promoter, or a combination thereof. In some embodiments, the minimal promoter comprises a MinP promoter. In some embodiments, the minimal promoter comprises a ybTATA promoter. In some embodiments, the minimal promoter comprises a MinP promoter and a ybTATA promoter. InWSGR Docket No.: 50401-791.602 some embodiments, the nucleic acid sequence comprises, from 5’ to 3’, the regulatory sequence operably linked to a sequence encoding a cargo such as IL-2 receptor variant. In some embodiments, the nucleic acid sequence comprises, from 5’ to 3’, NFAT response elements (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more) operably linked to a MinP promoter operably linked to the sequence encoding the cargo. In some embodiments, the nucleic acid sequence comprises, from 5’ to 3’, NFAT response elements (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more) operably linked to a ybTATA promoter operably linked to the sequence encoding the cargo. In some embodiments, the nucleic acid sequence further comprises a sequence encoding a truncated CD34. The truncated CD34 can function as a distinct marker for inducible system. The sequence encoding the truncated CD34 can be located at the 3’ end of the nucleic acid sequence.

[0367] In some embodiments, the method further comprises, prior to (a), delivering a nucleic acid sequence comprising a regulatory sequence and a sequence encoding the IL-2 receptor variant into the T cells or a subset thereof. In some embodiments, delivering comprises electroporating the nucleic acid sequence into the T cells or a subset thereof. In some embodiments, delivering comprises delivering a transposon comprising the nucleic acid sequence into the T cells or a subset thereof. In some embodiments, delivering comprises electroporating a transposon comprising the nucleic acid sequence into the T cells or a subset thereof. In some embodiments, delivering comprises using lipopolysaccharide (LPS), liposome, lipid nanoparticle, or lipoplex to deliver the nucleic acid sequence into the T cells or a subset thereof. In some embodiments, delivering comprises using lipopolysaccharide (LPS) to deliver the nucleic acid sequence into the T cells or a subset thereof. In some embodiments, delivering comprises using liposome to deliver the nucleic acid sequence into the T cells or a subset thereof. In some embodiments, delivering comprises using lipid nanoparticle to deliver the nucleic acid sequence into the T cells or a subset thereof. In some embodiments, delivering comprises using lipoplex to deliver the nucleic acid sequence into the T cells or a subset thereof.

[0368] In some embodiments, the T cells express endogenous TCRs. In some embodiments, the T cells express endogenous TCRs. In some embodiments, the stimulated T cells or the subset thereof express an activation marker. In some embodiments, the activation marker is CD69. In some embodiments, the frequency of T cells expressing the IL-2 receptor variant is about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25- fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, or about 50-fold or more higher among the stimulated T cells or the subset thereof compared to a corresponding population of non-stimulated T cells. In some embodiments, the stimulated T cell or the subset thereof have a fold expansion of about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-WSGR Docket No.: 50401-791.602 fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 75-fold, about 100-fold, about 125-fold, about 150-fold, about 175-fold, about 200-fold, about 225-fold, about 250-fold, about 300-fold or more relative to a corresponding population of non-stimulated T cells.

[0369] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD8+ T cell epitope. In some embodiments, the CD8+ T cell epitope comprises a sequence of about 7 to about 12 consecutive amino acids from a cancer protein. In some embodiments, the CD8+ T cell epitope comprises a sequence of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 consecutive amino acids from a cancer protein. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least two different CD8+ T cell epitopes. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least three different CD8+ T cell epitopes. In some embodiments, the polypeptide comprising at two or more tumor antigen epitope sequences comprises at least four different CD8+ T cell epitopes. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least five different CD8+ T cell epitopes. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises no more than 40, no more than 30, no more than 20, no more than 10 or less different CD8+ T cell epitopes. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises no more than 60, no more than 50, no more than 40, no more than 30, no more than 20, no more than 10, no more than 5 or less different CD8+ T cell epitopes.

[0370] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD4+ T cell epitope. In some embodiments, the CD4+ T cell epitope comprises a sequence of about 13 to about 25 consecutive amino acids from a cancer protein. In some embodiments, the CD4+ T cell epitope comprises a sequence of about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, or about 29 consecutive amino acids from a cancer protein. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least two different CD4+ T cell epitope sequences. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least three different CD4+ T cell epitopes. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least four different CD4+ T cell epitopes. In some embodiments, the polypeptideWSGR Docket No.: 50401-791.602 comprising at least one tumor antigen epitope sequence comprises at least five different CD4+ T cell epitopes. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises no more than 20, no more than 15, no more than 10, no more than 6 or less different CD4+ T cell epitope sequences. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises no more than 30, no more than 40, no more than 30, no more than 20, no more than 10, no more than 5, or less different CD4+ T cell epitopes.

[0371] In some embodiments, the least two different CD8+ T cell epitope sequences are separated by one or more linkers. In some embodiments, the at least two different CD8+ T cell epitope sequences are separated by one, two, three, four, five, six, seven, eight, nine, ten or more linkers. In some embodiments, the least two different CD4+ T cell epitope sequences are separated by one or more linkers. In some embodiments, the at least two different CD4+ T cell epitope sequences are separated by one, two, three, four, five, six, seven, eight, nine, ten or more linkers. In some embodiments, the one or more linkers are one or more flexible linkers. In some embodiments, the one or more linkers are one or more cleavable linkers. In some embodiments, the polypeptide comprises at least one CD8+ T cell epitope and at least one CD4+ T cell epitope. In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a linker between the at least two different CD8+ T cell epitope sequences and the at least two different CD4+ T cell epitope sequences. In some embodiments, the polynucleotide encoding the polypeptide encodes a linker. In some embodiments, the linker comprises a cleavable linker and a linker that is degradable by a protease. In some embodiments, the linker comprises a cleavable linker. In some embodiments, the linker comprises a linker that is degradable by a protease. In some embodiments, the protease is a proteasome. In some embodiments, the polypeptide further comprises a Sec domain sequence at its N-terminus. In some embodiments, the polypeptide further comprises a MITD domain sequence at its C- terminus. Antigen Presenting Cells

[0372] Ex vivo antigen presenting cells (APCs) are also provided herein. These cells may comprise polynucleotides comprising CD4+ T cell epitopes derived from cancer antigens and CD8+ T cell epitopes derived from cancer antigens and may be able to activate T cells to enhance their anti-cancer activity.

[0373] Also provided herein is an ex vivo antigen presenting cell comprising a polynucleotide encoding a polypeptide, wherein the polypeptide comprises one or more CD8+ T cell epitopes comprising a first amino acid sequence having a first epitope sequence consisting of 7 to 12WSGR Docket No.: 50401-791.602 consecutive amino acids from a cancer protein. In some embodiments, the polypeptide comprises one or more CD4+ T cell epitopes comprising a second amino acid sequence having a second epitope sequence consisting of 13 to 25 consecutive amino acids from a cancer protein. In some embodiments, the polypeptide comprises one or more linkers between the one or more CD4+ T cell epitopes and the one or more CD8+ T cell epitopes. In some embodiments, the polynucleotide is delivered via a lipid nanoparticle. In some embodiments, the polypeptide comprises one or more CD8+ T cell epitopes comprising a first amino acid sequence having a first epitope sequence consisting of 7 to 12 consecutive amino acids from a cancer protein, one or more CD4+ T cell epitopes comprising a second amino acid sequence having a second epitope sequence consisting of 13 to 25 consecutive amino acids from a cancer protein, one or more linkers between the one or more CD4+ T cell epitopes and the one or more CD8+ T cell epitopes, wherein the polynucleotide is delivered via a lipid nanoparticle.

[0374] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more CD8+ T cell epitopes. In some embodiments, each of the CD8+ T cell epitopes have a different epitope sequence. In some embodiments, CD8+ T cell epitope comprises a sequence of about 2 to 20, about 3 to 19, about 4 to 18, about 5 to 17, about 6 to 16, about 7 to 15, about 8 to 14, about 9 to 13, or about 10 to 12 consecutive amino acids from a cancer protein. In some embodiments, CD8+ T cell epitope comprises a sequence of about 7 to about 12 consecutive amino acids from a cancer protein.

[0375] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more CD4+ T cell epitopes. In some embodiments, each of the CD4+ T cell epitopes have a different epitope sequence. In some embodiments, the CD4+ T cell epitope comprises a sequence of about 1 to 40, about 2 to 39, about 3 to 38, about 4 to 37, about 5 to 36, about 6 to 35, about 7 to 34, about 8 to 33, about 9 to 32, about 10 to 31, about 11 to 30, about 12 to 29, about 13 to 28, about 14 to 27, about 15 to 26, about 16 to 25, about 17 to 24, about 18 to 23, about 17 to 22, about 18 to 21, or about 19 to 20 consecutive amino acids from a cancer protein. In some embodiments, the CD4+ T cell epitope comprises a sequence of about 13 to 25 consecutive amino acids from a cancer protein.

[0376] In some embodiments, the one or more CD8+ T cell epitopes comprise a third amino acid sequence having a third epitope sequence. In some embodiments, the third epitope sequence comprises a sequence of about 2 to 20, about 3 to 19, about 4 to 18, about 5 to 17, about 6 to 16, about 7 to 15, about 8 to 14, about 9 to 13, or about 10 to 12 consecutive amino acids from aWSGR Docket No.: 50401-791.602 cancer protein. In some embodiments, the one or more CD8+ T cell epitopes comprise a third amino acid sequence having a third epitope sequence consisting of 7 to 12 consecutive amino acids from a cancer protein. In some embodiments, the third amino acid sequence i...

Claims

WSGR Docket No.: 50401-791.602 CLAIMS WHAT IS CLAIMED IS:

1. A method for producing a plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells, the method comprising: (a) providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a single biological sample from a subject; (b) separating the APCs from the T cells in the population of immune cells, thereby forming a population of APCs separated from a population of T cells; (c) culturing the population of APCs in a first vessel in the presence of a first stimulatory agent and culturing the population of T cells in a second vessel in the presence of a second stimulatory agent, wherein the first vessel and the second vessel are different vessels; (d) co-culturing APCs of the population of APCs from the first vessel and T cells of the population of T cells from the second vessel, thereby forming a population of cells comprising stimulated T cells; and (e) expanding the stimulated T cells, thereby forming the plurality of T cells that comprises antigen-specific T cells.

2. The method of claim 1, further comprising, prior to (b), depleting CD25+ cells and / or CD56+ cells from the population of immune cells.

3. The method of claim 1 or 2, further comprising, prior to (b), depleting CD11b+ cells.

4. The method of any one of claims 1-3, wherein separating the APCs and the T cells in the population of immune cells in (b) comprises enriching CD14+ cells and / or CD19+ cells from the population of immune cells, thereby forming the population of APCs separated from the population of T cells.

5. The method of any one of claims 1-4, wherein the first stimulatory agent comprises one or more agents selected from the group consisting of FLT3L, IL-4, GM-CSF, and any combination thereof.

6. The method of any one of claims 1-5, wherein the second stimulatory agent comprises one or more agents selected from the group consisting of IL-2, IL-7, IL-15, IL-21, and any combination thereof.

7. The method of any one of claims 1-6, wherein co-culturing in (d) comprises co-culturing the APCs of the population of APCs from the first vessel and the T cells of the population of T cells from the second vessel in the presence of IL-21.WSGR Docket No.: 50401-791.602 8. The method of any one of claims 1-7, wherein expanding the stimulated T cells in (e) comprises expanding the stimulated T cells in the presence of one or more agents selected from the group consisting of IL-7, IL-15, IL-2 and an IL-2 variant.

9. The method of any one of claims 1-8, wherein expanding the stimulated T cells in (e) comprises expanding the stimulated T cells in the absence of IL-21.

10. The method of any one of claims 1-9, wherein the method further comprises, prior to co- culturing in (d), delivering (A) at least one 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 at least one polypeptide into the population of APCs.

11. The method of claim 10, wherein culturing in (c) further comprises delivering (A) at least one 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 at least one polypeptide into the population of APCs.

12. The method of claim 10 or 11, wherein delivering comprises electroporating the polynucleotide encoding the at least one polypeptide into the population of APCs.

13. The method of any one of claims 10-12, wherein delivering comprises contacting the population of APCs with the at least one polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer.

14. The method of any one of claims 1-13, wherein culturing in (c) comprises maturing the population of APCs in the first vessel.

15. The method of claim 14, wherein maturing comprises maturing the population of APCs in the first vessel in the presence of IFN and / or a toll like receptor agonistlipopolysaccharide (LPS).

16. The method of any one of claims 1-15, wherein the method further comprises, prior to co- culturing in (d), genetically editing the population of T cells.

17. The method of claim 16, wherein genetically editing the population of T cells comprises inhibiting expression of an endogenous gene in the population of T cells.

18. The method of claim 17, 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.

19. The method of claim 17 or 18, 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 encoded by the endogenous gene.WSGR Docket No.: 50401-791.602 20. The method of claim 19, 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.

21. The method of claim 20, wherein silencing the 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 site-specific nuclease, the site-specific nickase, or the site- specific epigenetic regulator into the population of T cells via electroporation, a lipid nanoparticle, a lipoplex, a liposome, nucleofection, or a viral vector.

22. The method of any one of claims 16-21, wherein genetically editing comprises delivering an exogenous gene into the population of T cells via transposon.

23. The method of claim 22, wherein the exogenous gene encodes a cell surface receptor that binds to a cytokine, or wherein the exogenous gene encodes a switch receptor or a safety switch for controlling activities of T cells.

24. The method of claim 23, wherein the cytokine is an IL-2 variant and the cell surface receptor is an IL-2 receptor, or wherein the cytokine is IL-18 and the cell surface receptor is IL-18 receptor.

25. The method of claim 24, wherein the IL-2 receptor comprises one or more mutations relative to a wildtype IL-2 receptor.

26. The method of any one of claims 22-24, wherein the percent of T cells in the population of T cells expressing the exogenous gene is at least 5%, 10%, 20%, 40%, 60%, 80% or more.

27. The method of any one of claims 18-26, wherein the percent of T cells in the population of T cells expressing the endogenous gene is at most 80%, 60%, 40%, 20%, 10%, 5% or less.

28. The method of any one of claims 18-27, wherein the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 5%, 10%, 20%, 40%, 60%, 80% or more.

29. A method for producing a plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells, the method comprising: (a) providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a biological sample from a subject; (b) co-culturing APCs from the population of immune cells and T cells from the population of immune cells in the presence of IL-21, thereby forming a population of cells comprising stimulated T cells; andWSGR Docket No.: 50401-791.602 (c) expanding the stimulated T cells in the absence of IL-21, thereby forming the plurality of T cells that comprises antigen-specific T cells.

30. The method of claim 29, wherein expanding the stimulated T cells in (c) comprises expanding the stimulated T cells in the presence of one or more stimulatory agents selected from the group consisting of IL-7, IL-15, IL-2 and an IL-2 variant.

31. The method of claim 29 or 30, wherein the biological sample is a single biological sample.

32. The method of any one of claims 29-31, wherein the method further comprises, prior to co-culturing in (b): (i) separating the APCs and the T cells in the population of immune cells, thereby forming a population of APCs separated from a population of T cells; and (ii) culturing the population of APCs in a first vessel the presence of a first stimulatory agent and culturing the population of T cells in a second vessel in the presence of a second stimulatory agent, wherein the first vessel and the second vessel are different vessels.

33. The method of any one of claims 29-32, wherein the first stimulatory agent comprises one or more agents selected from the group consisting of FLT3L, IL-4 GM-CSF, and any combination thereof.

34. The method of any one of claims 29-33, wherein the second stimulatory agent comprises one or more agents selected from the group consisting of IL-2, IL-7, IL-15, IL-21, and any combination thereof.

35. The method of any one of claims 29-34, wherein the method further comprises, prior to co-culturing in (b), delivering (A) at least one 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 at least one polypeptide into the population of APCs.

36. The method of claim 35, wherein delivering comprises electroporating the polynucleotide encoding the polypeptide into the population of APCs.

37. The method of claim 35, wherein delivering comprises contacting the population of APCs with the at least one polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer.

38. The method of claim 35, wherein delivering comprises contacting a lipid nanoparticle or a lipoplex the polynucleotide encoding the polypeptide to the population of APCs.

39. The method of any one of claims 32-38, further comprising, prior to co-culturing in (b), maturing the population of APCs in the first vessel.WSGR Docket No.: 50401-791.602 40. The method of claim 39, wherein maturing comprises maturing the population of APCs in the first vessel in the presence of IFN and / or a toll like receptor agonistlipopolysaccharide (LPS).

41. The method of any one of claims 29-40, wherein the method further comprises, prior to co-culturing in (b), genetically editing the population of T cells.

42. The method of claim 41, wherein genetically editing the population of T cells comprises inhibiting expression of an endogenous gene in the population of T cells.

43. The method of claim 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 42 or 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 encoded by 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 transcriptional or epigenetic regulator.

46. The method of claim 45, wherein silencing the 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 site-specific nuclease, the site-specific nickase, or the site- specific epigenetic regulator into the population of T cells via electroporation, a lipid nanoparticle, a lipoplex, a liposome, nucleofection, or a viral vector.

47. The method of any one of claims 41-46, wherein genetically editing comprises delivering an exogenous gene into the population of T cells via transposon.

48. The method of claim 47, wherein the exogenous gene encodes a cell surface receptor that binds to a cytokine, or wherein the exogenous gene encodes a switch receptor or a safety switch for controlling activities of T cells.

49. The method of claim 48, wherein the cytokine is an IL-2 variant and the cell surface receptor is an IL-2 receptor, or wherein the cytokine is IL-18 and the cell surface receptor is IL-18 receptor.

50. The method of claim 49, wherein the IL-2 receptor comprises one or more mutations relative to a wild type IL-2 receptor.

51. The method of any one of claims 47-50, wherein the percent of T cells in the population of T cells expressing the exogenous gene is at least 5%, 10%, 20%, 40%, 60%, 80% or more.WSGR Docket No.: 50401-791.602 52. The method of any one of claims 43-46, wherein the percent of T cells in the population of T cells expressing the endogenous gene is at most 80%, 60%, 40%, 20%, 10%, 5% or less.

53. The method of any one of claims 43-46, wherein the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 5%, 10%, 20%, 40%, 60%, 80% or more.

54. The method of any one of claims 29-53, wherein the method further comprises, prior to co-culturing in (b): depleting one or more cells selected from the group consisting of CD14+ cells, CD25+ cells and CD56+ cells from the population of immune cells.

55. The method of any one of claims 29-54, wherein the method further comprises, prior to co-culturing in (b): (i) depleting one or more cells selected from the group consisting of CD14+ cells, CD11b+ cells, CD25+ cells and CD56+ cells from the population of immune cells, thereby forming a depleted population of immune cells comprising a first population of APCs and T cells; (ii) 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), and (A) at least one 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 at least one polypeptide; thereby forming the population of cells comprising the stimulated T cells.

56. The method of any one of claims 29-55, wherein the method further comprises enriching CD25+ cells, CD39+ cells, 4-1BB+ cells, CD69+ cells, HLA-DR+ cells, ITGB7+ cells, LFA-1+ cells, IFN-g+ cells, TNF + cells, or any combination thereof from the populationof cells comprising the stimulated T cells.

57. The method of any one of claims 29-55, wherein the method further comprises enriching CD25+ cells, 4-1BB+ cells, CD69+ cells, IFN-g+ cells, or any combination thereof from the population of cells comprising the stimulated T cells.

58. The method of any one of claims 29-57, wherein a higher number of antigen-specific T cells is produced by the method compared to a corresponding method in which the T cells are expanded in the presence of IL-21.WSGR Docket No.: 50401-791.602 59. The method of any one of claims 29-57, wherein a higher number of antigen-specific T cells is produced by the method compared to a corresponding method in which the co- culturing is performed in the absence of IL-21.

60. A method for producing a plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells, the method comprising: (a) depleting one or more cells selected from the group consisting of CD14+ cells, CD11b+ cells, CD25+ cells and CD56+ cells from the population of immune cells, thereby forming a depleted population of immune cells comprising a first population of APCs and T cells; and (b) incubating the first population of APCs and T cells from step (a) for a first time period in the presence of: (A) at least one 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 at least one polypeptide; thereby forming a population of cells comprising stimulated T cells.

61. The method of claim 60, further comprising, subsequent to (b), enriching CD25+ cells, CD39+ cells, HLA-DR+ cells, ITGB7+ cells, LFA-1+ cells, IFN-g+ cells, TNF + cells,or any combination thereof from the population of cells comprising the stimulated T cells.

62. The method of claim 60 or 61, wherein depleting in (a) comprises depleting CD25+ cells from the population of immune cells.

63. The method of any one of claims 60-62, wherein depleting in (a) comprises depleting CD25+ cells from the population of immune cells, and wherein enriching in (c) comprises enriching CD25+ cells from the population of cells comprising the stimulated T cells.

64. The method of any one of claims 60-63, wherein incubating in (b) is performed in the presence of FMS-like tyrosine kinase 3 receptor ligand (FLT3L).

65. The method of any one of claims 60-64, the method further comprising expanding the population of cells comprising the stimulated T cells.

66. The method of any one of claims 60-65, wherein expanding comprises expanding the population of cells comprising the stimulated T cells in the presence of one or more stimulatory agents selected from the group consisting of IL-7, IL-15, IL-2, an IL-2 variant, and any combination thereof.

67. The method of any one of claims 60-66, wherein incubating the first population of APCs and T cells from step (a) comprises delivering (A) the at least one polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subjectWSGR Docket No.: 50401-791.602 with cancer, or (B) the polynucleotide encoding the at least one polypeptide into the APCs of the first population of APCs and T cells.

68. The method of any one of claims 60-67, wherein delivering comprises contacting a first lipid delivery vehicle comprising the polynucleotide encoding the polypeptide to the APCs of the first population of APCs and T cells.

69. The method of claim 68, wherein the first lipid delivery vehicle is APC specific.

70. The method of claims 68 or 69, wherein the first lipid delivery vehicle delivers the polypeptide or the polynucleotide into APC cells and does not deliver the polypeptide or the polynucleotide into the T cells.

71. The method of any one of claims 60-70, wherein incubating the first population of APCs and T cells from step (a) further comprises genetically editing the T cells of the first population of APCs and T cells.

72. The method of claim 71, wherein genetically editing the population of T cells comprises inhibiting expression of an endogenous gene in the population of T cells.

73. The method of claim 72, 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.

74. The method of claim 72 or 73, 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 encoded by the endogenous gene.

75. The method of claim 74, 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.

76. The method of claim 74, wherein silencing the 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 site-specific nuclease, the site-specific nickase, or the site- specific epigenetic regulator into the population of T cells via electroporation, a lipid nanoparticle, a lipoplex, a liposome, nucleofection, or a viral vector.

77. The method of any one of claims 72-76, wherein genetically editing comprises delivering an exogenous gene into the population of T cells via transposon and the percent of T cells in the population of T cells expressing the exogenous gene is at least 5%, 10%, 20%, 40%, 60%, 80% or more.WSGR Docket No.: 50401-791.602 78. The method of any one of claims 72-76, wherein the percent of T cells in the population of T cells expressing the endogenous gene is at most 80%, 60%, 40%, 20%, 10%, 5% or less.

79. The method of any one of claims 72-76, wherein the percent of T cells in the population of T cells with the endogenous gene inhibited is at least 5%, 10%, 20%, 40%, 60%, 80% or more.

80. The method of any one of claims 60-79, further comprising administering the plurality of T cells that comprises antigen-specific T cells into a subject in need thereof.

81. The method of any one of claims 60-80, wherein the time from (a) to obtaining a therapeutically effective amount of antigen-specific T cells is less than 20 days, less than 15 days, or less than 10 days.

82. A method for producing a plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells, the method comprising: (a) providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a biological sample from a subject; (b) co-culturing APCs from the population of immune cells and T cells from the population of immune cells, thereby forming a population of cells comprising stimulated T cells; and (c) expanding the stimulated T cells, thereby forming the plurality of T cells that comprise antigen-specific T cells comprising at least 106, at least 107, at least 108, or at least 109antigen-specific T cells, wherein the time from (a) to obtaining the plurality of T cells that comprise antigen-specific cells is less than 20 days, less than 19 days, less than 18 days, less than 17 days, less than 16 days, less than 15 days, less than 14 days, less than 13 days, less than 12 days, less than 11 days, or less than 10 days.

83. A method for producing a plurality of T cells, wherein the plurality of T cells comprises antigen-specific T cells, the method comprising: (a) incubating APCs and T cells (i) in the presence of (A) at least one 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 at least one polypeptide, or (ii) wherein the APCs comprise (A) at least one 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 at least one polypeptide,WSGR Docket No.: 50401-791.602 thereby forming a population of cells comprising stimulated T cells, wherein the stimulated T cells or a subset thereof express an IL-2 receptor variant, and wherein expression of the IL-2 receptor variant is induced upon T cell simulation; and (b) culturing the population of cells comprising stimulated T cells in the presence of IL-2 variant, wherein the stimulated T cells or a subset thereof expressing the IL-2 receptor variant expand, thereby obtaining the plurality of T cells comprises antigen-specific T cells.

84. The method of claim 83, wherein the stimulated T cells or a subset thereof expressing the IL-2 receptor variant comprises a nucleic acid sequence comprising a regulatory sequence and a sequence encoding the IL-2 receptor variant.

85. The method of claim 84, wherein the regulatory sequence comprises one or more Nuclear Factor of Activated T cells (NFAT) response elements (REs).

86. The method of claim 84 or 85, wherein the regulatory sequence comprises a promoter.

87. The method of claim 86, wherein the promotor is a minimal promoter.

88. The method of claim 87, wherein the minimal promoter comprises a MinP promoter, a yb TATA promoter, or a combination thereof.

89. The method of any one of claims 83-88, further comprising, prior to (a), delivering a nucleic acid sequence comprising a regulatory sequence and a sequence encoding the IL- 2 receptor variant into the T cells or a subset thereof.

90. The method of claim 89, wherein delivering comprises electroporating the nucleic acid sequence into the T cells or a subset thereof.

91. The method of claim 89, wherein delivering comprises delivering a transposon comprising the nucleic acid sequence into the T cells or a subset thereof.

92. The method of claim 91, wherein delivering comprises electroporating a transposon comprising the nucleic acid sequence into the T cells or a subset thereof.

93. The method of any one of claims 89-92, wherein delivering comprises using lipopolysaccharide (LPS), liposome, lipid nanoparticle, or lipoplex to deliver the nucleic acid sequence into the T cells or a subset thereof.

94. The method of any one of claims 83-93, wherein the T cells express endogenous TCRs.

95. The method of any one of claims 83-93, wherein the T cells do not express exogenous TCRs.

96. The method of any one of claims 83-95, wherein the stimulated T cells or the subset thereof express an activation marker.

97. The method of claim 96, wherein the activation marker is CD69.WSGR Docket No.: 50401-791.602 98. The method of claim 96 or 97, wherein the frequency of T cells expressing the IL-2 receptor variant is about 5-fold, about 10-fold, about 15-fold, or about 20-fold higher among the stimulated T cells or the subset thereof compared to a corresponding population of non-stimulated T cells.

99. The method of any one of claims 89-98, wherein the stimulated T cell or the subset thereof have a fold expansion of 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold relative to a corresponding population of non-stimulated T cells.

100. The method of any one of claims 83-99, wherein the at least one polypeptide comprising at least one tumor antigen epitope sequence comprises a CD8+ T cell epitope.

101. The method of claim 100, wherein the CD8+ T cell epitope comprises a sequence of about 7 to about 12 consecutive amino acids from a cancer protein.

102. The method of claim 100 or 101, wherein the at least one polypeptide comprising at least one tumor antigen epitope sequence comprises at least two different CD8+ T cell epitope sequences.

103. The method of any one of claims 83-102, wherein the at least one polypeptide comprising at least one tumor antigen epitope sequence comprises a CD4+ T cell epitope.

104. The method of claim 103, wherein the CD4+ T cell epitope comprises a sequence of about 13 to about 25 consecutive amino acids from a cancer protein.

105. The method of claim 103 or 104, wherein the at least one polypeptide comprising at least one tumor antigen epitope sequence comprises at least two different CD4+ T cell epitope sequences.

106. The method of claim 102 or 105, wherein the at least one polypeptide comprising at least one tumor antigen epitope sequence comprises a linker between the at least two different CD8+ T cell epitope sequences and the at least two different CD4+ T cell epitope sequences.

107. The method of any one of claims 83-106, wherein the polynucleotide encoding the polypeptide encodes a linker.

108. The method of claim 106 or 107, wherein the linker comprises a cleavable linker and / or a linker that is degradable by a protease.

109. The method of claim 102, wherein the least two different CD8+ T cell epitope sequences are separated by one or more linkers.

110. The method of claim 105 or 106, wherein the least two different CD4+ T cell epitope sequences are separated by one or more linkers.WSGR Docket No.: 50401-791.602 111. The method of claim 109 or 110, wherein the one or more linkers are one or more flexible linkers.

112. The method of claim 109 or 110, wherein the one or more linkers are one or more cleavable linkers.

113. The method of any one of claims 83-112, wherein the polypeptide further comprises a Sec domain sequence at its N-terminus.

114. The method of any one of claims 83-113, wherein the polypeptide further comprises a MITD domain sequence at its C-terminus.

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