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.: 50401-791.603 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 1, 2025, each of which is incorporated herein by reference 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 difficultiesWSGR Docket No.: 50401-791.603 to achieve a high level of efficacy. The compositions and methods disclosed herein, in some 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] 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; and (b) incubating the first population of APCs and T cells from step (a) for a first time period in the presence of: (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding the polypeptide; thereby forming a population of cells comprising stimulated T cells.

[0006] 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, ITGB7 cells, cells, cells, TNF-a+ cells, or any combination thereof from the population of cells comprising the stimulated T cells.

[0007] 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.

[0008] 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.

[0009] In some embodiments, the first lipid delivery vehicle is APC specific.

[0010] 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.

[0011] In some embodiments, the first lipid nanoparticle comprises an APC targeting agent.WSGR Docket No.: 50401-791.603

[0012] In some embodiments, the APC targeting agent is a mono / di / oligo-saccharide or a synthetic analogue.

[0013] 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).

[0014] 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.

[0015] In some embodiments, genetically editing the population of T cells comprises inhibiting expression of an endogenous gene in the population of T cells.

[0016] 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.

[0017] 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.

[0018] 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.

[0019] 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 viral vector.

[0020] In some embodiments, the method further comprises delivering a guide RNA concurrently with delivering (i) or (ii).

[0021] In some embodiments, the method further comprises delivering a guide RNA concurrently with delivering (i) or (ii) in a same lipid nanoparticle, a same lipoplex, or a same liposome.

[0022] In some embodiments, genetically editing the T cells comprises delivering a second lipid delivery vehicle comprising a guide RNA, and (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.

[0023] In some embodiments, genetically editing the T cells comprises delivering a second lipid delivery vehicle comprising an exogenous gene into the T cells.WSGR Docket No.: 50401-791.603

[0024] In some embodiments, the second lipid delivery vehicle is T cell specific.

[0025] In some embodiments, the second lipid delivery vehicle specifically targets T cells.

[0026] In some embodiments, the second lipid delivery vehicle comprises a T cell targeting agent.

[0027] In some embodiments, the T cell targeting agent is recognized by or interacts with a molecule present on a T cell surface.

[0028] In some embodiments, the T cell targeting agent is an antibody or fragment thereof.

[0029] 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, CD137, ITGB7, 4-1BB, CD45, HLA-DR, and ICOS.

[0030] In some embodiments, incubating the first population of APCs and T cells from step (a) 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] In some embodiments, the time from (a) to obtaining a therapeutically effective amount of antigen-specific T cells is less than 30 days, less than 25 days, less than 20 days, less than 15 days, or less than 10 days.

[0036] In some embodiments, the first lipid delivery vehicle and / or second lipid delivery vehicle comprises a lipid nanoparticle.

[0037] In some embodiments, first lipid delivery vehicle and / or second lipid delivery vehicle comprises a lipoplex.

[0038] In some embodiments, the lipid nanoparticle comprises (i) a cationically ionizable lipid; (ii) a phospholipid; (iii) a steroid; and / or (iv) a grafted lipid.

[0039] In some embodiments, the lipid nanoparticle comprises (i) a cationically ionizable lipid, (ii) DSPC, (iii) cholesterol, and (iv) a grafted lipid.

[0040] In some embodiments, the cationically ionizable lipid is selected from the group consisting of: 7,7’-((4-hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide)WSGR Docket No.: 50401-791.603 (BL-51); 7,7’-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylheptane-1- sulfonamide) (BL-52); 7,7’-((4-hydroxybutyl)azanediyl)bis(N,N-dioctyl heptane-1-sulfonamide (BL-207); 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA); 2,2-dilinoleyl-4- dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA); heptatriaconta-6,9,28,31-tetraen-19- yl-4-(dimethylamino)butanoate (D-Lin-MC3-DMA); heptadecan-9-yl 8-{(2-hydroxyethyl)[6- oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102); O-[N-{(9Z,12Z)-octadeca-9,12-dien-1- yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4-(dimethylamino)butanoate (HY501); ((2-(4- (dimethylamino)butanoyl)oxy)ethyl)azanediylbis(octane 8,1-diyl) bis(2-hexyldecanoate) (EA- 405); (2-(4-(dimethylamino)butanoyl)oxy)azanediylbis(octane 8,1-diyl) bis(2-hexyldecanoate) (HY-405); bis(2-octyldodecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (BODD-C2C4-PipZ); bis(2-hexyldecyl) 3,3'-((4-(4-methylpiperazin-1- yl)butyl)azanediyl)dipropionate (BHD-C2C2-PipZ); bis(2-octyldodecyl) 3,3'-((2- (dimethylamino)ethyl)azanediyl)dipropionate (BODD-C2C2-DMA); bis(2-octyldodecyl) 3,3'- ((2-(1-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-1Me-Pyr); bis(2- octyldodecyl) 3,3'-((2-(pyrrolidin-1-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-Pyr); or a mixture of any thereof.

[0041] In some embodiments, the grafted lipid is selected from the group consisting of: a poly(ethylene glycol)(PEG)-conjugated lipid and a poly(aminoethoxy ethoxy acetic acid) (pAEEA)-conjugated lipid, optionally the grafted lipid is C16Cer-PEG2K.

[0042] In some embodiments, the lipid nanoparticle comprises (i) 50-75 mol% cationically ionizable lipid, (ii) 8-26 mol% phospholipid, (iii) 15-45 mol% steroid, and (iv) 1-4 mol% grafted lipid.

[0043] In some embodiments, the lipid nanoparticle comprises R-DODMA, DOPE, Cholesterol, and / or PEG.

[0044] In some embodiments, the ratio of R-DODMA to DOPE to Cholesterol to PEG is 40:10:48:2.

[0045] In some embodiments, the lipoplex comprises DOTMA and / or DOPE.

[0046] In some embodiments, the ratio of DOTMA to DOPE is 2:1.

[0047] In some embodiments, the lipid nanoparticle comprises a T cell targeting agent.

[0048] In some embodiments, T cell targeting agent is recognized by or interacts with a molecule present on a T cell surface.

[0049] In some embodiments, the T cell targeting agent is an antibody or fragment thereof.WSGR Docket No.: 50401-791.603

[0050] In some embodiments, the lipid nanoparticle further comprises: (a) a connector compound comprising: (i) a lipid, and (ii) a moiety capable of binding to a molecule present on a T cell surface.

[0051] In some embodiments, the lipid nanoparticle further comprises: (a) a connector compound comprising: (i) a lipid, and (ii) a first interacting moiety; and (b) a docking compound comprising: (i) a second interacting moiety, and (ii) a moiety capable of binding to a molecule present on a T cell surface; wherein the first interacting moiety and the second interacting moiety are capable of binding to each other.

[0052] In some embodiments, --B3 (I) wherein: Pis absent or comprises a polymer; L comprises a moiety capable of incorporating the compound into the nanoparticle, which is attached to B1 when P is absent or to a first end of the polymer P when present; B1 comprises a moiety capable of binding to B2, the moiety B1 being attached to L when P is absent or to a second end of the polymer P when present; omprises a moiety capable of B3 comprises a moiety capable of binding to a molecule present on a T cell surface.

[0053] In some embodiments, the connector compound is DSPE-PEG2K-ALFA-tag.

[0054] In some embodiments, the lipid nanoparticle comprises (i) 50-75 mol% cationically ionizable lipid, (ii) 8-26 mol% phospholipid, (iii) 15-45 mol% steroid, (iv) 1-4 mol% grafted lipid, and (v) 0.05-0.4 mol% connector compound.

[0055] In some embodiments, the docking compound comprises (i) an ALFA-tag binding VHH, and (ii) an antibody or fragment thereof capable of binding to a molecule present on a T cell surface.

[0056] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD8+ T cell epitope.

[0057] In some embodiments, the CD8+ T cell epitope comprises a sequence of about 7 to about 12 consecutive amino acids expressed by a cancer cell. For example, the CD8+ T cell epitope is included in the polypeptide expressed by the cancer cells of the human with cancer.

[0058] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least two CD8+ T cell epitope sequences.

[0059] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD4+ T cell epitope.WSGR Docket No.: 50401-791.603

[0060] In some embodiments, the CD4+ T cell epitope comprises a sequence of about 13 to about 25 consecutive amino acids expressed by a cancer cell. For example, the CD4+ T cell epitope is included in the polypeptide expressed by the cancer cells of the human with cancer.

[0061] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least two different CD4+ T cell epitope sequences.

[0062] 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 targeting epitopes and the at least two different CD4+ T cell epitope sequences.

[0063] In some embodiments, the linker comprises a cleavable linker and / or a linker that is degradable by a protease.

[0064] In some embodiments, the least two different CD8+ T cell epitope sequences are separated by one or more linkers.

[0065] In some embodiments, the least two different CD4+ T cell epitope sequences are separated by one or more linkers.

[0066] In some embodiments, the one or more linkers are one or more flexible linkers.

[0067] In some embodiments, the one or more linkers are one or more cleavable linkers.

[0068] In some embodiments, the polypeptide further comprises a Sec domain sequence at its N-terminus.

[0069] In some embodiments, the polypeptide further comprises a MITD domain sequence at its C-terminus.

[0070] In some embodiments, the Sec domain sequence comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NOs: 35 or 36.

[0071] In some embodiments, the MITD domain sequence comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 34.

[0072] In some embodiments, the polynucleotide encoding the polypeptide comprises a sequence encoding the Sec domain at its 5’ end.

[0073] In some embodiments, the polynucleotide encoding the polypeptide comprises a sequence encoding the MITD domain at its 3’ end.

[0074] In some embodiments, the polynucleotide encoding the polypeptide comprises one or more sequences encoding one or more linkers.

[0075] In some embodiments, the sequence encoding the Sec domain comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NOs: 32 or 33.

[0076] In some embodiments, the sequence encoding the MITD domain comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 31.WSGR Docket No.: 50401-791.603

[0077] 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 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.

[0078] In some embodiments, the lipid nanoparticle comprises R-DODMA, DOPE, Cholesterol, and / or PEG.

[0079] In some embodiments, the ratio of R-DODMA to DOPE to Cholesterol to PEG is 40:10:48:2.

[0080] 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 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.

[0081] In some embodiments, the lipoplex comprises DOTMA and / or DOPE.

[0082] In some embodiments, the ratio of DOTMA to DOPE is 2:1.

[0083] 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 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; and (c) expanding the stimulated T cells.WSGR Docket No.: 50401-791.603

[0084] In some embodiments, the second lipid nanoparticle formulation comprises a T cell targeting agent.

[0085] In some embodiments, the T cell targeting agent is recognized by or interacts with a molecule present on a T cell surface.

[0086] In some embodiments, the T cell targeting agent is an antibody or fragment thereof.

[0087] 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, CD137, ITGB7, 4-1BB, CD45, HLA-DR, and ICOS.

[0088] In some embodiments, the first lipid nanoparticle formulation comprises an APC targeting agent.

[0089] In some embodiments, the APC targeting agent is a mono / di / oligo-saccharide or a synthetic analogue.

[0090] 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).

[0091] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD8+ T cell epitope.

[0092] In some embodiments, the CD8+ T cell epitope comprises a sequence of about 7 to about 12 consecutive amino acids expressed by a cancer cell.

[0093] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least two different CD8+ T cell epitope sequences.

[0094] In some embodiments, polypeptide comprising at least one tumor antigen epitope sequence comprises a CD4+ T cell epitope.

[0095] In some embodiments, the CD4+ T cell epitope comprises a sequence of about 13 to about 25 consecutive amino acids expressed by a cancer cell.

[0096] In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence comprises at least two different CD4+ T cell targeting epitope sequences.

[0097] 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.

[0098] In some embodiments, the polynucleotide encoding the polypeptide comprises a linker.

[0099] In some embodiments, the linker comprises a cleavable linker and / or a linker that is degradable by a protease.WSGR Docket No.: 50401-791.603

[0100] In some embodiments, the least two different CD8+ T cell epitope sequences are separated by one or more linkers.

[0101] In some embodiments, the least two different CD4+ T cell epitope sequences are separated by one or more linkers.

[0102] In some embodiments, the one or more linkers are one or more flexible linkers.

[0103] In some embodiments, the one or more linkers are one or more cleavable linkers.

[0104] In some embodiments, the polypeptide further comprises a Sec domain sequence at its N-terminus.

[0105] In some embodiments, the polypeptide further comprises a MITD domain sequence at its C-terminus.

[0106] In some embodiments, the Sec domain sequence comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NOs: 35 or 36.

[0107] In some embodiments, the MITD domain sequence comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 34.

[0108] In some embodiments, the polynucleotide encoding the polypeptide comprises a sequence encoding the Sec domain at its 5’ end.

[0109] In some embodiments, the polynucleotide encoding the polypeptide comprises a sequence encoding the MITD domain at its 3’ end.

[0110] In some embodiments, the polynucleotide encoding the polypeptide comprises one or more sequences encoding one or more linkers.

[0111] In some embodiments, the sequence encoding the Sec domain comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NOs: 32 or 33.

[0112] In some embodiments, the sequence encoding the MITD domain comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 31.

[0113] In some embodiments, the polynucleotide of the second lipid nanoparticle formulation comprises or encodes an agent for genetically editing T cells.

[0114] In some embodiments, the polynucleotide encodes a guide RNA, the site-specific nuclease, the site-specific nickase, and / or the site-specific epigenetic regulator.

[0115] In some embodiments, the polynucleotide comprises an RNA.

[0116] In some embodiments, the RNA is a guide RNA.

[0117] In some embodiments, the T cell targeting agent is an anti-CD3 antibody, an anti-CD7 antibody, or a mixture of the anti-CD3 antibody and the anti-CD7 antibody.

[0118] In some embodiments, the second lipid nanoparticle formulation further comprises another polynucleotide encoding an endonuclease.WSGR Docket No.: 50401-791.603

[0119] In some embodiments, the endonuclease is a Cas9.

[0120] In some embodiments, a total amount of the guide RNA and the polynucleotide encoding the endonuclease is at least about 1 μg.

[0121] In some embodiments, a total amount of the guide RNA and the polynucleotide encoding the endonuclease is at least about 4 μg.

[0122] In some embodiments, the ratio of the guide RNA to the polynucleotide encoding the endonuclease is 1:1.

[0123] In some embodiments, an endogenous gene is knocked out.

[0124] In some embodiments, knockout efficiency using the T cell targeting agent is at least 50 fold more than without using the T cell targeting agent.

[0125] In some embodiments, the endogenous gene is CD47.

[0126] In some embodiments, knockout efficiency of the CD47 is at least about 70%.

[0127] In some embodiments, the endogenous gene is REGNASE-1.

[0128] In some embodiments, knockout efficiency of the REGNASE-1 is at least about 60%.

[0129] In some embodiments, the at least one tumor antigen epitope sequence comprises a mutation.

[0130] Also provided herein is an ex vivo antigen presenting cell comprising a polynucleotide encoding the 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 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, and 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.

[0131] In some embodiments, the one or more CD4+ 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, and wherein the third amino acid sequence is operably linked to the first amino acid sequence.

[0132] In some embodiments, herein the one or more CD4+ T cell epitopes comprises at least 3 amino acid sequences each having a different epitope sequence.

[0133] In some embodiments, the one or more CD8+ T cell epitopes comprising a fourth amino acid sequence having a fourth epitope sequence consisting of 13 to 25 consecutive amino acids from a cancer protein, and wherein the fourth amino acid sequence is operably linked to the second amino acid sequence.WSGR Docket No.: 50401-791.603

[0134] In some embodiments, the one or more CD8+ T cell epitopes comprises at least 3 amino acid sequences each having a different epitope sequence.

[0135] In some embodiments, the one or more linkers comprise a cleavable linker and / or a linker that is degradable by a protease.

[0136] In some embodiments, the one or more linkers are one or more flexible linkers.

[0137] In some embodiments, the one or more linkers are one or more cleavable linkers.

[0138] In some embodiments, the polypeptide further comprises a Sec domain sequence at its N-terminus.

[0139] In some embodiments, polypeptide further comprises a MITD domain sequence at its C- terminus.

[0140] In some embodiments, the Sec domain sequence comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NOs: 35 or 36.

[0141] In some embodiments, the MITD domain sequence comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 34.

[0142] In some embodiments, the polynucleotide encoding the polypeptide comprises a sequence encoding the Sec domain at its 5’ end.

[0143] In some embodiments, the polynucleotide encoding the polypeptide comprises a sequence encoding the MITD domain at its 3’ end.

[0144] In some embodiments, the polynucleotide encoding the polypeptide comprises one or more sequences encoding one or more linkers.

[0145] In some embodiments, the sequence encoding the Sec domain comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NOs: 32 or 33.

[0146] In some embodiments, the sequence encoding the MITD domain comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 31. INCORPORATION BY REFERENCE

[0147] 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

[0148] 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 illustrativeWSGR Docket No.: 50401-791.603 embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0149] 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.

[0150] FIGs. 2A-2B depict two experimental outlines testing culture conditions. FIG. 2A depicts an experiment wherein APCs and T cells are not separated. FIG. 2B depicts an experiment wherein APCs and T cells are separated and cultured in different conditions prior to coculture.

[0151] FIGs. 3A-3B are schematics of exemplary RNA strings that encode for CD4 and CD8 T cell-targeting antigens. FIG. 3A depicts two separate constructs, one with CD8 T cell targeting antigens and another with CD4. FIG. 3B depicts constructs combining CD4 and CD8 T cell targeting antigens on one construct.

[0152] FIGs. 4A-4B depict results measuring expansion of antigen specific CD8 T cells when stimulated using the indicated RNA strings. FIG. 4A depicts frequency of antigen specific cells. FIG. 4B depicts number of antigen specific cells.

[0153] FIGs. 5A-5B depict results measuring expansion of antigen specific cells when cells are incubated at 2.5pM per one million cells, 12.5pM per one million cells, or 25pM per one million cells with one stimulation. FIG. 5A depicts frequency of antigen specific cells. FIG. 5B depicts number of antigen specific cells.

[0154] FIGs. 6A-6B depict results measuring expansion of antigen specific cells when cells are incubated at 2.5pM per one million cells, 12.5pM per one million cells, or 25pM per one million cells with two stimulations. FIG. 6A depicts frequency of antigen specific cells. FIG. 6B depicts number of antigen specific cells.

[0155] FIG. 7A depicts an experimental outline to test administering three different RNA strings in various combinations and concentrations.

[0156] FIG. 7B depicts an exemplary nanoplasmid system encoding up to 5 transgenes.

[0157] FIG. 7C depicts an exemplary modular nanoplasmid construct.

[0158] FIGs. 8A-8G depict results from the experiment outlined in FIG. 7A detecting expansion of cells specific for each of the indicated antigens encoded by the RNA strings. FIG. 8A depicts number of antigen specific cells using cells from human donor ID: 300. FIG. 8B depicts number of antigen specific cells using cells from human donor ID: 302. FIG. 8C depicts number of antigen specific cells using cells from human donor ID: 304. FIG. 8D depicts number of antigen specific cells using cells from human donor ID: 300. FIG. 8E depicts number of antigen specific cells using cells from human donor ID: 302. FIG. 8F depicts number of antigenWSGR Docket No.: 50401-791.603 specific cells using cells from human donor ID: 304. FIG. 8G depicts number of antigen specific cells using cells from human donor ID: 300.

[0159] FIG. 9A is a schematic of an RNA string encoding four long epitopes and 20 short epitopes.

[0160] FIG. 9B is a comparison of two different RNA string design strategies.

[0161] FIG. 10A depicts results measuring overall expansion of antigen specific CD8 T cellswhen using an RNA with a modified m1 backbone or an unmodified backbone.

[0162] FIG. 10B depicts frequency of CD8 T cells with each antigen specificity indicated whenusing an RNA with a modified m1 backbone or an unmodified backbone.

[0163] FIG. 10C depicts results measuring overall expansion of antigen specific CD8 T cells when using an RNA encoding 14mer epitope lengths, 25mer epitope lengths, or minimal epitopes.

[0164] FIG. 10D depicts results measuring frequency of CD8 T cells with each antigen specificity indicated when using an RNA encoding 14mer epitope lengths, 25mer epitope lengths, or minimal epitopes.

[0165] FIG. 11A depicts an experimental outline to test RNA strings with varying epitope composition, length, and overall design.

[0166] FIG. 11B depicts the binding sites of primers used to detect mRNA strings.

[0167] FIG. 11C illustrates a qPCR process to generate cDNA which is then detected using specific primers.

[0168] FIG. 11D is a quantification of the Cq values measured in a qPCR experiment as concentration of mRNA standard is increased.

[0169] FIG. 11E is a quantification of mRNA levels inside cells post electroporation in the NEO-STIM process.

[0170] FIG. 11F is a quantification of mRNA levels in the supernatant post electroporation in the NEO-STIM process.

[0171] FIG. 12 is an outline of a T cell culture and expansion process.

[0172] FIG. 13 is an experimental outline testing the efficacy of lipid-based delivery systems.

[0173] FIG. 14A depicts results measuring efficacy of luciferase mRNA delivery when lipid nanoparticle delivery is used with varying mRNA amounts.

[0174] FIG. 14B depicts results measuring viability when lipid nanoparticle delivery is used with varying mRNA amounts.

[0175] FIG. 15A depicts results measuring efficacy of luciferase mRNA delivery when lipoplex delivery is used with varying mRNA amounts.WSGR Docket No.: 50401-791.603

[0176] FIG. 15B depicts results measuring viability when lipoplex delivery is used with varying mRNA amounts.

[0177] FIG. 16A depicts a cell culture set up used to test the efficacy of lipid-based delivery systems.

[0178] FIG. 16B depicts an experimental outline for testing the efficacy of lipid-based delivery systems.

[0179] FIG. 17A depicts quantification of fold expansion (FIG. 17A) of cells which were delivered the indicated amount of mRNA in a lipid nanoparticle (LNP), a lipoplex (LP electroporation (EP).

[0180] FIG. 17B depicts quantification of viability of cells which were delivered the indicated

[0181] FIG. 18A depicts frequency of CD8 T cells when APCs are delivered mRNA in a lipid

[0182] FIG. 18B depicts frequency of antigen-specific CD8 T cells when APCs are deliveredmRNA in a lipid nanoparticle (LNP), a li

[0183] FIG. 19A systems.

[0184] FIG. 19B depicts quantification of total cells when APCs from two donors are delivered density indicated in parentheses.

[0185] FIG. 19C depicts cell fold expansion when APCs from two donors are delivered mRNA in indicated in parentheses.

[0186] FIG. 19D depicts viability when APCs from two donors are delivered mRNA in a lipid in parentheses.

[0187] FIG. 20A depicts quantification of total cells when APCs from two donors are delivered density indicated in parentheses.

[0188] FIG. 20B depicts cell fold expansion when APCs from two donors are delivered mRNA in a lipid nanoparticle (LNP), a lipoplex indicated in parentheses.WSGR Docket No.: 50401-791.603

[0189] FIG. 20C depicts viability when APCs from two donors are delivered mRNA in a lipid dicated in parentheses.

[0190] FIG. 21 depicts frequency of CD8 T cells in the samples from FIGs. 19A-19D and FIGs. 20A-20C.

[0191] FIGs. 22A-22B depicts a cell culture set up to determine the efficacy of delivery of mRNA to APCs by either electroporation or LNP in combination with electroporation of T cells. FIG. 22A is an overview of the culture system. FIG. 22B is an illustration of treatment groups for the experiment shown in FIG. 22A.

[0192] FIG. 23 is an experimental overview illustrating the use of T cell- and APC-targeting LNPs.

[0193] FIG. 24A depicts results quantifying luciferase activity from an experiment delivering a T cell targeting LNP carrying luciferase into T cells.

[0194] FIG. 24B depicts results quantifying GFP expression) from an experiment delivering a T cell targeting LNP carrying GFP into T cells.

[0195] FIG. 24C depicts overall cell count from an experiment delivery a T cell targeting LNP carrying GFP into T cells.

[0196] FIG. 25A is a schematic diagram of a nanoplasmid construct encoding NFAT binding sites.

[0197] FIG. 25B depicts a gel image illustrating levels of supercoiledness of nanoplasmid constructs.

[0198] FIG. 25C is a quantification of the yield of different nanoplasmid constructs.

[0199] FIG. 26 is a schematic of an in-vitro assay using LNPs carrying Cas9, and gRNA directed to CD47 to assess gene editing efficiency.

[0200] FIG. 27 depicts results measuring CD47 knockout efficiency in activated T cells.

[0201] FIG. 28A depicts results measuring expression of Thy1.1 as a measure of cargo uptake.

[0202] FIG. 28B depicts results measuring REGNASE-1 (REG-1) knockout efficiency in activated T cells.

[0203] FIG. 29 depicts the effect of different string designs (model antigen string, split string A, split string B, concatenated AB (S1-10), concatenated AB (S11-S20)) on inducing antigen specific CD8+ T cells across donors.

[0204] FIG. 30A depicts the effect of split A pool and concatenated AB on inducing hit rate and antigen specific CD8+ T cells across donors.WSGR Docket No.: 50401-791.603

[0205] FIG. 30B depicts the effect of split B pool and concatenated AB on inducing hit rate and antigen specific CD8+ T cells across donors. DETAILED DESCRIPTION Definitions

[0206] 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.

[0207] To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below.

[0208] 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, for 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.

[0209] 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.

[0210] 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.

[0211] “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 theseWSGR Docket No.: 50401-791.603 fragments (e.g., via binding) with MHC molecules for presentation by cells, for example, antigen presenting cells, to specific T cells.

[0212] 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.

[0213] The term “affinity” refers to a measure of the strength of binding between two members of a binding pair (e.g., a human leukocyte antigen (HLA)-binding peptide and a class I or II HLA, or a peptide-HLA complex and a T cell receptor (TCR)). KDrefers to the dissociation constant between two members of a binding pair and has units of molarity. KArefers to the affinity constant between two members of a binding pair is the inverse of the dissociation constant. Affinity 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).

[0214] 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 alsoWSGR Docket No.: 50401-791.603 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)).

[0215] The term “derived” when used to discuss an epitope is a synonym for “prepared.” A derived epitope can be isolated from a natural source, or it can be synthesized according to standard protocols in the art. Synthetic epitopes can comprise artificial amino acid residues “amino acid mimetics,” such as D isomers of natural occurring L amino acid residues or non- natural amino acid residues such as cyclohexylalanine. A derived or prepared epitope can be an analog of a native epitope. The term “derived from” refers to the origin or source, and 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.

[0216] 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 aminoWSGR Docket No.: 50401-791.603 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 acid residues. In certain embodiments, an “epitope” described herein is comprised by a peptide having a region with less than 51 amino acid residues that has 100% identity to a native peptide sequence, in any increment down to 5 amino acid residues; for example 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues.

[0217] 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).

[0218] 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+WSGR Docket No.: 50401-791.603 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.

[0219] 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.

[0220] 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.

[0221] 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 or progression. The immune response can also include an antibody response which has been facilitated by the stimulation of helper T cells.

[0222] 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.

[0223] 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 someWSGR Docket No.: 50401-791.603 embodiments, 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.

[0224] “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).

[0225] The major histocompatibility complex in the genome comprises the genetic region whose gene products expressed on the cell surface are important for binding and presenting endogenous and / or foreign antigens and thus for regulating immunological processes. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune reactions. MHC proteins or molecules bind peptides and present them for recognition by T-cell receptors. The proteins 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).

[0226] 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 mayWSGR Docket No.: 50401-791.603 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.

[0227] 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.

[0228] 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.”

[0229] 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.WSGR Docket No.: 50401-791.603 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).

[0230] The term “residue” refers to an amino acid residue or amino acid mimetic residue incorporated into a peptide or protein by an amide bond or amide bond mimetic, or that is encoded by a nucleic acid (DNA or RNA). The nomenclature used to describe peptides or proteins follows the conventional practice. The amino group is presented to the left (the amino- or N-terminus) and the carboxyl group to the right (the carboxy- or C-terminus) of each amino acid residue. When amino acid residue positions are referred to in a peptide epitope, they are numbered in an amino to carboxyl direction with the first position being the residue located at the amino terminal end of the epitope, or the peptide or protein of which it can be a part. In the formulae representing selected specific embodiments of the present invention, the amino- and carboxyl-terminal groups, although not specifically shown, are in the form they would assume at physiologic pH values, unless otherwise specified. In the amino acid structure formulae, each residue is generally represented by standard three letter or single letter designations. The L-form of an amino acid residue is represented by a capital single letter or a capital first letter of a three- letter symbol, and the D-form 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.)

[0231] 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 nucleotideWSGR Docket No.: 50401-791.603 and amino acid conservative substitutions which do not eliminate peptide function are well- known in the art.

[0232] “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.

[0233] 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.

[0234] 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.

[0235] 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. TheWSGR Docket No.: 50401-791.603 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.

[0236] 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 at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the sequences that is at least about 10, at least about 20, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as an amino acid sequence of a peptide or a coding region of a nucleotide sequence.

[0237] 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.

[0238] 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.WSGR Docket No.: 50401-791.603

[0239] 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, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.

[0240] 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.

[0241] 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.

[0242] 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.WSGR Docket No.: 50401-791.603

[0243] 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.

[0244] 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.

[0245] 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 cancer of solid tissue, several of the compositions and methods described herein are in principle applicable to cancers of the blood, leukemia.

[0246] CDR numbering systems are known in the art. The Kabat numbering system is based on sequence variability and is the most commonly used (see http: / / www.bioinf.org.uk / abs / ). The ImMunoGeneTics information system (IMGT) (see http: / / www.imgt.org) can also be used. According to this numbering system, a complementarity determining region (CDR-IMGT) is a loop region of a variable domain, delimited according to the IMGT unique numbering for V domain. There are three CDR-IMGT in a variable domain: CDR1-IMGT (loop BC), CDR2- -IMGT (loop FG). Other CDR numbering systems have also been developed, such as the Chothia, the AbM and the contact definitions (see http: / / www.imgt.org). The CDRs of the sdAbs according to the present invention may be defined using any suitable system, such as any suitable system known in the art.

[0247] The “Chothia-Nanobody” system is a Chothia-based numbering, with modifications to account for the structure of nanobodies / VHH. In the Chothia-Nanobody system, VHH sequences are numbered according to the Chothia numbering scheme, with CDR1 defined as H26-H35, CDR2 defined as H51-H60 and CDR3 as H93-102.

[0248] The IMGT and Kabat numbering systems are described above.

[0249] “Humanized antibody” may refer to a genetically engineered non-human antibody, which contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains.WSGR Docket No.: 50401-791.603

[0250] “Humanized antibody” may also refer to a sdAb which has been modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the three (or six) non-human antibody complementarity-determining regions (CDRs), which together form the antigen binding site, onto a homologous human acceptor framework region (FR). Non-limiting examples of antibody humanization methods include CDR grafting, and resurfacing (i.e. replacing surface residues to obtain a “more human” surface), and germline- based humanization. For example, in order to fully reconstitute the binding affinity and specificity of the parental antibody, the substitution of framework residues from the parental antibody (i.e. the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modelling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and, optionally, fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back-mutations, may be introduced to obtain a humanized antibody with preferred characteristics, such as affinity and biochemical properties. Humanization of non-human therapeutic antibodies is performed to minimize its immunogenicity in man while such humanized antibodies at the same time maintain the specificity and binding affinity of the antibody of non-human origin. Exemplary methods for humanization of VHHs are described in Vincke et al. (Journal of Biological Chemistry; 2009; 284(5); 3273-3284) and Rossotti et al. (FEBS; 2021; doi:10.1111 / febs / 15809). Overview

[0251] 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.WSGR Docket No.: 50401-791.603

[0252] 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.

[0253] 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

[0254] 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 agent 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.WSGR Docket No.: 50401-791.603

[0255] The population of immune cells can be from a biological sample. The biological sample can comprise blood, serum, and / or plasma. The biological sample can be a peripheral blood mononuclear cell (PBMC) sample or a leukapheresis sample. In some embodiments, the PBMC sample is freshly isolated PBMC sample. In some embodiments the PBMC sample is a frozen PBMC sample. In some embodiments, the PBMC sample is an autologous PBMC sample isolated from the subject or the patient. In some embodiments the biological sample is from a liquid or solid biopsy of a tissue. In some embodiments, the liquid or solid biopsy is taken from a site in or near a tumor. In some embodiments, the population of immune cells comprise tumor infiltrating lymphocytes (TILs).

[0256] The T cells described herein can be TILs. In some embodiments, the T cells are from a sample of blood, serum, and / or plasma. In some embodiments, the T cells are from a PBMC sample or a leukapheresis sample. In some embodiments, the PBMC sample is freshly isolated PBMC sample. In some embodiments the PBMC sample is a frozen PBMC sample. In some embodiments, the PBMC sample is an autologous PBMC sample isolated from the subject or the patient. In some embodiments the T cells are from a liquid or solid biopsy of a tissue. In some embodiments, the liquid or solid biopsy is taken from a site in or near a tumor.

[0257] The APCs described herein can be from a biological sample. In some embodiments, the APCs are from a sample of blood, serum, and / or plasma. In some embodiments, the APCs are from a PBMC sample or a leukapheresis sample. In some embodiments, the PBMC sample is freshly isolated PBMC sample. In some embodiments the PBMC sample is a frozen PBMC sample. In some embodiments, the PBMC sample is an autologous PBMC sample isolated from the subject or the patient. In some embodiments the APCs are from a liquid or solid biopsy. In some embodiments, the liquid or solid biopsy is taken from a site in or near a tumor.

[0258] In some embodiments, the number of T cells in a separate culture or a coculture with APCs is5 6 6 6 6106 6 6 6 6 6 6, 66 6,6 6. In some embodiments, the number of APCs in a separate culture or a coculture with T cells 105 5 5 5 5 5 5, 5, about 95 6 6 6 6, 66 6 6 6 6, 66 6 6 6, about6, 66 6. In some embodiments, the total cells in a 56 6 6,WSGR Docket No.: 50401-791.603 66 6 6,6 6, about 66 6 6 6 6, or 6. 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, 6 / cm2, 6 / cm2 6 / cm2 6 / cm2 6 / cm2, about 3.0 06 / cm2 6 / cm2 6 / cm2 6 / cm2 6 / cm2, 6 / cm2 6 / cm2 6 / cm2 6 / cm2, about 7.5 6 / cm2 6 / cm2 6 / cm2 6 / cm2,6 / 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 -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.

[0259] 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.

[0260] 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.WSGR Docket No.: 50401-791.603 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.

[0261] 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, 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.

[0262] 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 cellsWSGR Docket No.: 50401-791.603 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 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. In some embodiments, expanding the stimulated T cells comprises expanding the stimulated T cells in the absence of IL-21.

[0263] In some embodiments, the method further comprises, prior to co-culturing, delivering (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding the polypeptide 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 atWSGR Docket No.: 50401-791.603 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.

[0264] 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) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding the polypeptide into the population of APCs. In some embodiments, culturing in (c) comprises delivering (A) a 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 a 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 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 particle (e.g., lipid nanoparticle or a lipoplex) comprising the polynucleotide encoding the polypeptide to the population of APCs.

[0265] 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 maturing comprises maturingthe 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.

[0266] 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 TWSGR Docket No.: 50401-791.603 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, CD47, 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 CD47. 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 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 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 viral vector.

[0267] 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 someWSGR Docket No.: 50401-791.603 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 described herein.

[0268] 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 someWSGR Docket No.: 50401-791.603 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.

[0269] 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 cellsWSGR Docket No.: 50401-791.603 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%.

[0270] 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 TWSGR Docket No.: 50401-791.603 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.

[0271] In some embodiments, the polypeptide or the polynucleotide encoding the peptide is delivered in a lipid particle. 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, 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,WSGR Docket No.: 50401-791.603 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. In some embodiments, the lipid nanoparticle comprise DODMA / Chol / DOPE / C16Cer-PEG2k / DSPE-PEG2K-ALFA with molar ratios 40.0 / 48.0 / 10.0 / 1.8 / 0.2. In some embodiments, the molar ratio of DODMA / Chol / DOPE / C16Cer- PEG2k / DSPE-PEG2K-ALFA is at least about 10-25, at least about 25-40, at least about 40-55, at least about 55-70, at least about 70-85, or at least about 85-100 DODMA to at least about 10- 25, at least about 25-40, at least about 40-55, at least about 55-70, at least about 70-85, or at least about 85-100 cholesterol to at least about 1-5, at least about 5-10, at least about 10-15, at least about 15-20, at least about 20-25, at least about 25-30, at least about 30-35, at least about 35-40, at least about 40-45, or at least about 45-50 DOPE to at least about 1-5, at least about 5- 10, at least about 10-15, at least about 15-20 at least about 20-25, at least about 25-30, at least about 30-35, at least about 35-40, at least about 40-45, or at least about 45-50 C16Cer-PEG2k disclosed herein to at least about 0.05-0.1, at least about 0.1-0.4, or at least about 0.1-0.4 DSPE- PEG2K-ALFA disclosed herein. In some embodiments, the molar ratio of DODMA / Chol / DOPE / C16Cer-PEG2k / DSPE-PEG2K-ALFA is about 50-75 / 15-45 / 8-26 / 1- 4 / 0.05-0.4.

[0272] In some embodiments, the molar ratio of an ionizable lipid / Chol / DSPC / C16Cer- PEG2k / DSPE-PEG2K-ALFA is at least about 10-25, at least about 25-40, at least about 40-55, at least about 55-70, at least about 70-85, or at least about 85-100 ionizable lipid disclosed herein to at least about 10-25, at least about 25-40, at least about 40-55, at least about 55-70, at least about 70-85, or at least about 85-100 cholesterol to at least about 1-5, at least about 5-10, at least about 10-15, at least about 15-20, at least about 20-25, at least about 25-30, at least about 30-35, at least about 35-40, at least about 40-45, or at least about 45-50 DSPC to at least about 1-5, at least about 5-10, at least about 10-15, at least about 15-20, at least about 20-25, at least about 25-30, at least about 30-35, at least about 35-40, at least about 40-45, or at least about 45- 50 C16Cer-PEG2k to at least about 0.05-0.1, at least about 0.1-0.4, or at least about 0.1-0.4 DSPE-PEG2K-ALFA. In some embodiments, the molar ratio of an ionizableWSGR Docket No.: 50401-791.603 lipid / Chol / DSPC / C16Cer-PEG2k / DSPE-PEG2K-ALFA is at least about 50-75 / 15-45 / 8-26 / 1- 4 / 0.05-0.4.

[0273] 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. In some embodiments, the lipoplex comprises DOTMA and / or DOPE. In some embodiments, the lipoplex comprises DOTMA. In some embodiments, the lipoplex comprises DOPE. In some embodiments, the lipoplex comprises DOTMA and DOPE. In some embodiments, the ratio of DOTMA 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 DOTMA 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 DOTMA to DOPE is 2:1.

[0274] In some embodiments, the lipid particle comprises a T cell targeting agent. 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 cases, the T cell targeting agent is an anti-CD3 antibody, an anti-CD7 antibody, or a mixture of the anti-CD3-antibody and the anti-CD7 antibody. For example, a single lipid particle (e.g., lipid nanoparticle or lipoplex) can comprise both anti-CD3-antibody and the anti-CD7 antibody. For another example, a plurality of lipid particles (e.g., lipid nanoparticles or lipoplexes) can comprise a mixture of two types of lipid particles, each type comprising a single type ofWSGR Docket No.: 50401-791.603 antibody (e.g., an anti-CD3-antibody or an anti-CD7 antibody). 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, CD69, CD137, ITGB7, 4-1BB, CD45, HLA-DR, and ICOS. 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 CD137.

[0275] In some embodiments, the lipid particle comprises an APC targeting agent. 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.

[0276] 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 epitopeWSGR Docket No.: 50401-791.603 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.

[0277] 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.

[0278] 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 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: 35 or 36. 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: 34.WSGR Docket No.: 50401-791.603

[0279] 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: 32 or 33. 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: 31.

[0280] 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.

[0281] In some embodiments, the number of T cells in a separate culture or a coculture with 56 6 6 6106 6 6 6 6 6 6, 66 6,6 6. In some 105 5 5 5 5 5 5, 5, about 95 6 6 6 6, 66 6 6 6 6, 66 6 6 6, about 8.56, 66 6. In some embodiments, the total cells in a 56 6 6,WSGR Docket No.: 50401-791.603 66 6 6, ab 6 6, about66 6 6 6 6, or 6. 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, 6 / cm2, 6 / cm2 6 / cm2 6 / cm2 6 / cm2, about 3.0 06 / cm2 6 / cm2 6 / cm2 6 / cm2 6 / cm2, 6 / cm2 6 / cm2 6 / cm2 6 / cm2, about 7.5 6 / cm2 6 / cm2 6 / cm2 6 / cm2,6 / 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, orratio of APCs and T cells (in a cocultur -mediated cargo delivery, forexample 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.

[0282] 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 inWSGR Docket No.: 50401-791.603 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.

[0283] 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. In some embodiments the method further 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 agent wherein the first vessel and the second vessel are different vessels. In some embodiments the method further comprises (i) 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; and (ii) culturingWSGR Docket No.: 50401-791.603 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.

[0284] 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, 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 IL-7. In some embodiments, the second stimulatory agent comprises IL-15. In some embodiments, the second stimulatory agent comprises one or more agents is 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-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.

[0285] In some embodiments, the method further comprises, prior to co-culturing, delivering (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding the polypeptide 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 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. 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 the polynucleotide encoding the polypeptide into the population of APCs. In some embodiments, delivering comprises contacting the population of APCs with theWSGR Docket No.: 50401-791.603 polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer.

[0286] In some embodiments, delivering comprises contacting a lipid particle (e.g., lipid nanoparticle or lipoplex) 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 of APCs. In some embodiments, delivering comprises contacting a lipid particle (e.g., 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 contacting a lipid particle (e.g., lipid nanoparticle) comprising the polynucleotide encoding the polypeptide into the population of APCs. In some embodiments, delivering comprises contacting a lipoplex 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 contacting a lipoplex comprising the polynucleotide encoding the polypeptide into the population of APCs.

[0287] In some embodiments, the method further comprises, prior to co-culturing in (b), 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 maturing comprises maturingthe 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.

[0288] 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, genetically editing the population of T cells comprises inhibiting expression of two or more endogenous genes (e.g., 2, 3, 4, 5, 6, or more endogenous genes) 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, CD47, 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 CD47. In some embodiments, the endogenous gene comprisesWSGR Docket No.: 50401-791.603 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.

[0289] 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 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 viral vector.

[0290] 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 canWSGR Docket No.: 50401-791.603 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.

[0291] 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.

[0292] 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, CD47 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 CD47. In some embodiments, the endogenous gene comprises SOCS1. In some embodiments, the endogenous gene comprises PTPN2. In some embodiments, the endogenousWSGR Docket No.: 50401-791.603 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.

[0293] 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 comprises one or more mutations relative to a wildtype IL-2 receptor.

[0294] 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 theWSGR Docket No.: 50401-791.603 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, the percent of T cells in the population of T cells expressing the exogenous gene is at least 99% or more.

[0295] 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 ofWSGR Docket No.: 50401-791.603 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 most 95%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 99%.

[0296] 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 TWSGR Docket No.: 50401-791.603 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 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.

[0297] In some embodiments, the polypeptide or the polynucleotide encoding the peptide is delivered in a lipid particle. 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 someWSGR Docket No.: 50401-791.603 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, 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. In some embodiments, the lipoplex comprises DOTMA and / or DOPE. In some embodiments, the lipoplex comprises DOTMA. In some embodiments, the lipoplex comprises DOPE. In some embodiments, the lipoplex comprises DOTMA and DOPE. In some embodiments, the ratio of DOTMA 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, aboutWSGR Docket No.: 50401-791.603 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 DOTMA 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 DOTMA to DOPE is 2:1.

[0298] In some embodiments, the lipid particle comprises a T cell targeting agent. 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 cases, the T cell targeting agent is an anti-CD3 antibody, an anti-CD7 antibody, or a mixture of the anti-CD3-antibody and the anti-CD7 antibody. 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, CD137, ITGB7, 4-1BB, CD45, HLA-DR, and ICOS. 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 CD137. In some embodiments, the lipid particle comprises an APC targeting agent. 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 someWSGR Docket No.: 50401-791.603 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.

[0299] 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.

[0300] 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 first population of APCs and T cells; (ii) incubating the first population of APCs and T cells for aWSGR Docket No.: 50401-791.603 first time period in the presence of: FMS-like tyrosine kinase 3 receptor ligand (FLT3L), and (A) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer or (B) a polynucleotide encoding the polypeptide; thereby forming 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.

[0301] 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, ITGB7 cells, cells, cells, TNF-a+ 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, ITGB7 cells, CD69+ cells, IFN-g+ 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 LFA-1+ cells from the population of cells comprising the stimulated T cells. In some embodiments, the method further comprises enriching IFN-g+ cells from the population of cells comprising the stimulated T cells. In some embodiments, the method further comprises enriching TNF-a+ cells from the population of cells comprising the stimulated T cells.

[0302] 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 a 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 someWSGR Docket No.: 50401-791.603 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.

[0303] 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.

[0304] 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.

[0305] 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 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 moreWSGR Docket No.: 50401-791.603 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: 35 or 36. 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: 34.

[0306] 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: 32 or 33. 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: 31.

[0307] 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) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding the polypeptide; thereby forming a population of 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, at 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, IFN-g+ cells, ITGB7 cells, cells, cells, TNF-a+ cells, or any combination thereof from the population of cells comprising the stimulated T cells. In some embodiments,WSGR Docket No.: 50401-791.603 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) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding the polypeptide; thereby forming a population of cells comprising stimulated T cells; and (c) enriching CD25+ cells, CD39+ cells, 4-1BB+ cells, CD69+ cells, HLA-DR+ cells, LFA-1+ cells, IFN-g+ cells, ITGB7 cells, cells, cells, TNF-a+ cells, or any combination thereof from the population of cells comprising the stimulated T cells.

[0308] In some embodiments, the number of T cells in a separate culture or a coculture with APCs is5 6 6 6 6106 6 6 6 6 6 6, 66 6 6, or about 2 6. In some105 5 5 5 5 5 5, 55 6, abou 6 6 6,66 6 6 6 6, 66 6 6 6 6, 6,6 6. In some embodiments, the total cells in a 56 6 6, 66 6 6 6106, about 66 6 6 6 6, or 6. 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, 6 / cm2, 6 / cm2 6 / cm2 6 / cm2 6 / cm2, about 3.0 6 / cm2 6 / cm2 6 / cm2 6 / cm2 6 / cm2, 6 / cm2 6 / cm2 6 / cm2 6 / cm2, about 7.5 6 / cm2 6 / cm2 6 / cm2 6 / cm2 6 / 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, orWSGR Docket No.: 50401-791.603 -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.

[0309] 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).

[0310] 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 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 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,WSGR Docket No.: 50401-791.603 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.

[0311] 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 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, 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.WSGR Docket No.: 50401-791.603

[0312] 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.

[0313] 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, CD47, 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 CD47. 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 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 silencingWSGR Docket No.: 50401-791.603 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 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 viral vector.

[0314] In some embodiments, genetically editing the T cells comprises delivering a second lipid delivery vehicle comprising a guide RNA, and (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. In some embodiments, genetically editing the T cells comprises delivering a second lipid delivery vehicle comprising a guide RNA and an polynucleotide encoding an endonuclease disclosed herein (e.g., Cas9). In some embodiments, the total amount of the guide RNA and the polynucleotide encoding the endonuclease disclosed herein is at least about 1 μg, at least about 2 μg, at least about 3 μg, at least about 4 μg, at least about 5 μg, at least about 10 μg, at least about 15 μg, at least about 20 μg, at least about 25 μg, at least about 30 μg, at least about 35 μg, at least about 40 μg¸ at least about 45 μg¸ or at least about 50 μg. In some embodiments, the ratio of the guide RNA to the polynucleotide encoding the endonuclease is 1:1, 1:2, 2:1, 1:3, 3:1, 2:3, 3:2. 1:4, 4:1, 1:5, or 5:1.

[0315] 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 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 cases,WSGR Docket No.: 50401-791.603 the T cell targeting agent is an anti-CD3 antibody, an anti-CD7 antibody, or a mixture of the anti-CD3-antibody and the anti-CD7 antibody. 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, CD137, ITGB7, 4-1BB, CD45, HLA-DR, and ICOS. 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 CD137.

[0316] 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,WSGR Docket No.: 50401-791.603 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, the percent of T cells in the population of T cells expressing the exogenous gene is at least 99% or more.

[0317] 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 ofWSGR Docket No.: 50401-791.603 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 most 95%. In some embodiments, the percent of T cells in the population of T cells expressing the endogenous gene is at most 99%.

[0318] 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 TWSGR Docket No.: 50401-791.603 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 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.

[0319] 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.WSGR Docket No.: 50401-791.603

[0320] In some embodiments, the first lipid delivery vehicle comprises a lipid particle. 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.

[0321] 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, 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,WSGR Docket No.: 50401-791.603 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. 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. In some embodiments, the lipoplex comprises DOTMA and / or DOPE. In some embodiments, the lipoplex comprises DOTMA. In some embodiments, the lipoplex comprises DOPE. In some embodiments, the lipoplex comprises DOTMA and DOPE. In some embodiments, the ratio of DOTMA 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 DOTMA 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 DOTMA to DOPE is 2:1.

[0322] 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.WSGR Docket No.: 50401-791.603

[0323] 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.

[0324] 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 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 NO: 35 or 36. 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: 34.

[0325] 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:WSGR Docket No.: 50401-791.603 32 or 33. 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: 31.

[0326] 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 particle (e.g., 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 particle (e.g., 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.

[0327] In some embodiments, the number of T cells in a separate culture or a coculture with 56 6 6 6, 106 6 6 6 6 6 6, 66 6 6 6. In some embodiments, the number of APCs in a separate 105 5 5 5 5 5 5, 55 6 6 6 6, 6, abo 6 6 6 6 6,66 6 6 6 6, 66 6. In some embodiments, the total cells in a 56 6 6, 66 6 6 6 6, about 66 6 6 6 6, or 6. In some embodiments, the ratio of T cells to APCs in coculture is about 1:2,WSGR Docket No.: 50401-791.603 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 some6 / cm2, 6 / cm2 6 / cm2 6 / cm2 6 / cm2, about 3.0 6 / cm2 6 / cm2 6 / cm2 6 / cm2 6 / cm2, 6 / cm2 6 / cm2 6 / cm2 6 / cm2, about 7.5 6 / cm2 6 / cm2 6 / cm2 6 / cm2 6 / 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 -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.

[0328] 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 isWSGR Docket No.: 50401-791.603 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.

[0329] 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 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.

[0330] In some embodiments, the number of T cells in a separate culture or a coculture with 56 6 6 6WSGR Docket No.: 50401-791.603 106 6 6 6 6 6 6, 66 6 6 6. In some 105 5 5 5 5 5 5, 5, about5 6 6 6 6, 66 6 6 6 6, 66 6 6 6, about 8. 6,66 6. In some embodiments, the total cells in a 56 6 6, 66 6 6, a 6 6, about66 6 6 6 6, or 6. 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, 6 / cm2, 6 / cm2 6 / cm2 6 / cm2 6 / cm2, about 3.0 6 / cm2 6 / cm2 6 / cm2 6 / cm2 6 / cm2, 6 / cm2 6 / cm2 6 / cm2 6 / cm2, about 7.5 6 / cm2 6 / cm2 6 / cm2 6 / cm2 6 / 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, orratio of APCs and T cells (in a cocultur -mediated cargo delivery, forexample 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.

[0331] 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 someWSGR Docket No.: 50401-791.603 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.

[0332] In some embodiments, the lipoplex comprises DOTMA and / or DOPE. In some embodiments, the lipoplex comprises DOTMA. In some embodiments, the lipoplex comprises DOPE. In some embodiments, the lipoplex comprises DOTMA and DOPE. In some embodiments, the ratio of DOTMA 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 DOTMA 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 DOTMA to DOPE is 2:1.

[0333] 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 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; and (c) expanding the stimulated T cells.WSGR Docket No.: 50401-791.603

[0334] In some embodiments, the number of T cells in a separate culture or a coculture with 56 6 6 6106 6 6 6 6 6 6, 6, abo 6 6 6 6. In some105 5 5 5 5 5,5, 55 6 6 6 6, 66 6 6 6 6, 66 6, abou 6 6 6,66 6. In some embodiments, the total cells in a 56 6 6, 6 106 6 6 6 6, about 66 6 6 6 6, or 6. 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, 6 / cm2, 6 / cm2 6 / cm2 6 / cm2,6 / cm2, about 3.0 6 / cm2 6 / cm2 6 / cm2 6 / cm2 6 / cm2, 6 / cm2 6 / cm2 6 / cm2 6 / cm2, about 7.5 6 / cm2 6 / cm2, about6 / cm2 6 / cm2 6 / 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 -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.

[0335] 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 recognizes a molecule present on a T cell surface. In some embodiments, the T cell targetingWSGR Docket No.: 50401-791.603 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 cases, the T cell targeting agent is an anti-CD3 antibody, an anti-CD7 antibody, or a mixture of the anti-CD3-antibody and the anti- CD7 antibody. 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, CD137, ITGB7, 4-1BB, CD45, HLA-DR, and ICOS. 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 CD137.

[0336] 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, the 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.WSGR Docket No.: 50401-791.603

[0337] 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.

[0338] 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.

[0339] 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.

[0340] 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 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, aboutWSGR Docket No.: 50401-791.603 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.

[0341] 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 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: 35 or 36. 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: 34.

[0342] 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: 32 or 33. 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: 31.

[0343] 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)WSGR Docket No.: 50401-791.603 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 8 antigen 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 9 antigen 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 11antigen specific T cells. In some embodiments, the plurality of T cells comprises at least 1012antigen specific T cells.

[0344] 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 someWSGR Docket No.: 50401-791.603 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 less 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.

[0345] In some embodiments, the number of T cells in a separate culture or a coculture with 56 6 6 6WSGR Docket No.: 50401-791.603 106 6 6 6 6 6 6, 66 6, abo 6 6. In some105 5 5 5 5 5 5, 5 05 6 6 6 6, 66 6 6 6 6, 66 6 6 606, 66 6. In some embodiments, the total cells in a 56 6 6, 66 6 6,6 6, about 66 6 6 6 6, or 6. 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, 6 / cm2, 6 / cm2 6 / cm2 6 / cm2 6 / cm2, about 3.0 6 / cm2 6 / cm2 6 / cm2 6 / cm2 6 / cm2, 6 / cm2 6 / cm2 6 / cm2 6 / cm2, about 7.5 6 / cm2 6 / cm2 6 / cm2 6 / cm2,6 / 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 -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.

[0346] 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.WSGR Docket No.: 50401-791.603 In some embodiments, the method comprises expanding the stimulated T cells in a single vessel, thereby forming the plurality of T cells that comprises antigen-specific T cells, wherein the single vessel comprises a therapeutically effective amount of antigen-specific T cells or at least 106antigen-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 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 in a single vessel, thereby forming the plurality of T cells that comprises antigen-specific T cells, wherein the single vessel comprises a therapeutically effective amount of antigen-specific T cells or at least 106antigen-specific T cells. In some embodiments, the single vessel comprises at least 1,000 antigen specific T cells. In some embodiments, the single vessel comprises at least 5,000 antigen specific T cells. In some embodiments, the single vessel comprises at least 10,000 antigen specific T cells. In some embodiments, the single vessel comprises at least 50,000 antigen specific T cells. In some embodiments, the single vessel comprises at least 100,000 antigen specific T cells. In some embodiments, the single vessel comprises at least 500,000 antigen specific T cells. In some embodiments, the single vessel comprises at least 1,000,000 antigen specific T cells. In some embodiments, the single vessel comprises at least 5,000,000 antigen specific T cells. In some embodiments, the single vessel comprises at least 10,000,000 antigen specific T cells. In some embodiments, the single vessel comprises at least 50,000,000 antigen specific T cells. In some embodiments, the single vessel comprises at least 108antigen specific T cells. In some embodiments, the single vessel comprises at least8antigen specific T cells. In some embodiments, the single vessel comprises at least 109antigen specific T cells. In some 9 antigen specific T cells. In some embodiments, the single vessel comprises at least 1010antigen specific T cells. . In some 11antigen specific T cells. In some embodiments, the single vessel comprises at least 1012antigen specific T cells. In some embodiments, a volume of the single vessel is at most 200cm3, at most 400cm3, at most 600cm3, at most 800cm3, at most 1000cm3, at most 1200cm3, at most 1400cm3, at most 1600cm3, at most 1800cm3, at most 2000cm3, at most 2500cm3, at most 3000cm3, at most 3500cm3, at most 4000cm3, at most 4500cm3, at most 5000cm3, at most 5500cm3, at most 6000cm3, at most 6500cm3, at most 7000cm3or more. In some embodiments, a volume of the single vessel is at most 1000cm3. In some embodiments, a surface area of the single vessel is at most 25cm2, at most 75cm2, at most 100cm2, 200cm2, at most 400cm2, at most 600cm2, at most 800cm2, at mostWSGR Docket No.: 50401-791.603 1000cm2, at most 1200cm2, at most 1400cm2, at most 1600cm2, at most 1800cm2, at most 2000cm2, at most 2500cm2, at most 3000cm2, at most 3500cm2, at most 4000cm2, at most 4500cm2, at most 5000cm2, at most 5500cm2, at most 6000cm2, at most 6500cm2, at most 7000cm2or more. In some embodiments, a surface area of the single vessel is at least 25cm2, 75cm2, 100cm2, 200cm2, at least 400cm2, at least 600cm2, at least 800cm2, at least 1000cm2, at least 1200cm2, at least 1400cm2, at least 1600cm2, at least 1800cm2, at least 2000cm2, at least 2500cm2, at least 3000cm2, at least 3500cm2, at least 4000cm2, at least 4500cm2, at least 5000cm2, at least 5500cm2, at least 6000cm2, at least 6500cm2, at least 7000cm2or more.

[0347] In some embodiments, the number of T cells in a separate culture or a coculture with 56 6 6 6106 6 6,6 6 6 6, 66 6 6 6. In some105, abo 5 5 5 5 5 5,55 6 6 6 6, 66 6 606 6, 66 6 6 6 6, 66 6. In some embodiments, the total cells in a 56 6 6, 66 6 6 6 6, about 66 6 6 6 6, or 6. 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, 106 / cm2, 6 / cm2 6 / cm2 6 / cm2 6 / cm2, about 3.0 6 / cm2 6 / cm2 6 / cm2 6 / cm2 6 / cm2, 6 / cm2 6 / cm2 6 / cm2 6 / cm2, about 7.5 6 / cm2 6 / cm2 6 / cm2 6 / cm2 6 / 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 -mediated cargo delivery, for example RNA delivery. In some cases, total cell numbers can affect expansion of T cells. InWSGR Docket No.: 50401-791.603 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.

[0348] 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, 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.

[0349] 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, restimulating comprises culturing the population of cells in the presence of IL-4, GMCSF, and / or FLT3L. 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 stepWSGR Docket No.: 50401-791.603 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.

[0350] 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.

[0351] 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.

[0352] 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%, atWSGR Docket No.: 50401-791.603 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.

[0353] 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% ofthe 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.

[0354] 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 cellsWSGR Docket No.: 50401-791.603 in the plurality of T cells that comprises antigen-specific T cells is at least 0.5% of the antigen- specific T cells.

[0355] In some embodiments, the percentage of D107a+ 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 s in the plurality of T cells that comprises antigen-specific T cells is at least 5% of the antigen- specific T cells.

[0356] 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 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 0.1% of the antigen-specific T cells.

[0357] 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+ TWSGR Docket No.: 50401-791.603 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%.

[0358] 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%.

[0359] 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 some 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%.

[0360] 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%, atWSGR Docket No.: 50401-791.603 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%.

[0361] 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%.

[0362] 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%, 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 effector memory T cells (CD62L- and CD45RA-) is at least 60%.

[0363] 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%.

[0364] 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 leastWSGR Docket No.: 50401-791.603 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%.

[0365] 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 produce cytokines and cause degranulation upon recognition of cognate antigen.

[0366] 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 least 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.

[0367] 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 leastWSGR Docket No.: 50401-791.603 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.

[0368] 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, 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,WSGR Docket No.: 50401-791.603 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.

[0369] 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 of 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-foldWSGR Docket No.: 50401-791.603 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.

[0370] 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%, at 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.

[0371] 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%,WSGR Docket No.: 50401-791.603 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. 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 12 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 particle (e.g., 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 sequenceWSGR Docket No.: 50401-791.603 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 particle (e.g., 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 a 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 is operably linked to the first amino acid sequence. In some embodiments, the one or more CD8+ T cell epitopes comprise 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 amino acid sequences each having a different epitope sequence. In some embodiments, the one or more CD8+ T cell epitopes comprise at least 3 amino acid sequences each having a different epitope sequence.WSGR Docket No.: 50401-791.603

[0377] In some embodiments, the one or more CD4+ T cell epitopes comprise a fourth amino acid sequence having a fourth epitope sequence. In some embodiments, the fourth epitope sequence 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 fourth epitope sequence consists of 13 to 25 consecutive amino acids from a cancer protein. In some embodiments, the fourth amino acid is operably linked to the second amino acid. In some embodiments, the one or more CD4+ T cell epitopes comprise 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 amino acid sequences each having a different epitope sequence. In some embodiments, the one or more CD4+ T cell epitopes comprise at least 3 amino acid sequences each having a different epitope sequence.

[0378] In some embodiments, one or more linkers comprise a cleavable linker and / or a linker that is degradable by a protease. In some embodiments, the one or more linkers comprise a linker that is degradable by a protease. In some embodiments, the one or more linkers comprise 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: 35 or 36. 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: 34.

[0379] 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%WSGR Docket No.: 50401-791.603 sequence identity to a sequence of SEQ ID NOs: 32 or 33. 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: 31. Lipid Particles

[0380] In some embodiments, the polynucleotides and / or nucleic acids as described herein can be formulated in lipid particles. In some embodiments, the term “lipid particle” relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the terms “particle” and “lipid particle” can be used interchangeably. In some embodiments, the particle and lipid particle include nanoparticles. A lipid particle can be used to deliver DNA and / or RNA to a cell, e.g., a human immune cell such as a human T cell. In some embodiments, a lipid particle comprises DNA, RNA or a mixture thereof. For example, a cell, e.g., a human T cell, can be transfected with a lipid particle (e.g., lipid nanoparticle, lipoplex) as described herein. A lipid particle may be formed from lipids comprising at least one cationic or cationically ionizable lipid. Without wishing to be bound by theory, it is contemplated herein that the cationic or cationically ionizable lipid combines together with the DNA and / or RNA to form aggregates, and this aggregation results in colloidally stable particles.

[0381] Lipid -based and lipid nanoparticle (LNP)- thereof are described for example in WO2022 / 069632, WO2022 / 101470, WO2022 / 101486, WO2023 / 148276, and WO2023 / 148277, all of which are hereby incorporated by reference.

[0382] In some embodiments, the polynucleotide and / or nucleic acid disclosed herein is disclosed herein.

[0383] In some embodiments, the polynucleotide encoding a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer In some embodiments, the polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human The LNPs and / or

[0384] In some embodiments, the nucleic acid encoding the site-specific nuclease, the site- specific nickase, or the site- some embodiments, the polynucleotide encoding an exogenous protein is formulated in LNPs herein.WSGR Docket No.: 50401-791.603

[0385] The polynucleotide / nucleic acid disclosed herein can be referred to as “nucleic acid payload.” In some embodiments, the nucleic acid payload is the polynucleotide or nucleic acid disclosed herein.

[0386] In some embodiments, a T cell targeting agent disclosed herein is formulated in LNPs

[0387] In some embodiments, the lipid particle is a functionalized particle. In some embodiments, the polypeptide or polynucleotide / nucleic acid, as described herein, is formulated as a functionalized particle comprising: (a) one or more particle forming components; (b) the polypeptide or polynucleotide / nucleic acid as described herein; and (c) a moiety capable of binding to a molecule present on the surface of a target cell. In some embodiments the target cell is a T cell. In some embodiments, the polynucleotide / nucleic acid, as described herein, is formulated as a functionalized particle comprising: (a) one or more particle forming components; (b) the polypeptide or polynucleotide / nucleic acid as described herein; and (c) a T cell targeting agent that is recognized by or interacts with a molecule present on a T cell surface. In some embodiments, the polypeptide or polynucleotide / nucleic acid, as described herein, is formulated as a functionalized particle comprising: (a) one or more particle forming components; (b) the polypeptide or polynucleotide / nucleic acid as described herein; and (c) a moiety capable of binding to a molecule present on a T cell surface disclosed herein. The molecule present on the T cell surface may be CD2, CD3, CD4, CD5, CD7, CD8, CD56, CD39, ICAM-1, CD366, CD279, CD200, CD25, CD103, HLA-DR, CD69, CD39, 4-1BB, ITGB7, ICOS, or CD137. The molecule present on the T cell surface may be CD2, CD3, CD4, CD7, or CD8.

[0388] In some embodiments, the lipid particle (e.g., lipid nanoparticle) comprises (i) a cationically ionizable lipid; (ii) a phospholipid; (iii) a steroid; and / or (iv) a grafted lipid. In some embodiments, the lipid particle (e.g., lipid nanoparticle) comprises (i) a cationically ionizable lipid, (ii) DSPC, (iii) cholesterol, and (iv) a grafted lipid. In some embodiments, the lipid particle (e.g., lipid nanoparticle) comprises (i) 50-75 mol% cationically ionizable lipid, (ii) 8-26 mol% phospholipid, (iii) 15-45 mol% steroid, and (iv) 1-4 mol% grafted lipid. In some embodiments, the lipid particle (e.g., lipid nanoparticle) comprises at least about 10-25 mol%, at least about 25-40 mol%, at least about 40-55 mol%, at least about 55-70 mol%, at least about 70-85 mol%, or at least about 85-100 mol% cationically ionizable lipid disclosed herein. In some embodiments, the lipid particle (e.g., lipid nanoparticle) comprises at least about 50-75 mol% cationically ionizable lipid disclosed herein. In some embodiments, the lipid particle (e.g., lipid nanoparticle) comprises at least about 1-5 mol%, at least about 5-10 mol%, at least aboutWSGR Docket No.: 50401-791.603 10-15 mol%, at least about 15-20 mol%, at least about 20-25 mol%, at least about 25-30 mol%, at least about 30-35 mol%, at least about 35-40 mol%, at least about 40-45mol%, or at least about 45-50 mol%, phospholipid disclosed herein. In some embodiments, the lipid particle (e.g., lipid nanoparticle) comprises at least about 8-26 mol% phospholipid. In some embodiments, the lipid particle (e.g., lipid nanoparticle) comprises at least about 10-25 mol%, at least about 25-40 mol%, at least about 40-55 mol%, at least about 55-70 mol%, at least about 70-85 mol%, or at least about 85-100 mol% steroid disclosed herein. In some embodiments, the lipid particle (e.g., lipid nanoparticle) comprises at least about 15-45 mol% steroid disclosed herein. In some embodiments, the lipid particle (e.g., lipid nanoparticle) comprises at least about 1-5 mol%, at least about 5-10 mol%, at least about 10-15 mol%, at least about 15-20 mol%, at least about 20- 25 mol%, at least about 25-30 mol%, at least about 30-35 mol%, at least about 35-40 mol%, at least about 40-45 mol%, or at least about 45-50 mol% grafted lipid disclosed herein. In some embodiments, the lipid particle (e.g., lipid nanoparticle) comprises at least about 1-4 mol% grafted lipid disclosed herein.

[0389] In some embodiments, the one or more particle forming components comprise a cationic or cationically ionizable lipid, as described herein. In some embodiments, the one or more particle forming components comprise a cationic lipid (e.g., DOTMA) and a phospholipid (e.g., DOPE), as described herein. In some preferred embodiments, the one or more particle forming components comprise (i) a cationically ionizable lipid; (ii) a phospholipid; (iii) a steroid; and optionally (iv) a grafted lipid. In some embodiments, the functionalized particle comprises, (a) one or more particle forming components comprising: (i) a cationically ionizable lipid; (ii) a phospholipid; (iii) a steroid; and optionally (iv) a grafted lipid; and (b) a connector compound (e.g., a compound of formula (A)), and optionally (c) a docking compound (e.g., a compound of formula (I)). Cationic or cationically ionizable lipid

[0390] In some embodiments, the cationic or cationically ionizable lipid of the nucleic acid particle is selected from the group consisting of: 7,7’-((4-hydroxybutyl)azanediyl)bis(N-hexyl- N-octylheptane-1-sulfonamide); (BL-51); 7,7’-((4-(3,3- dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N-octylheptane-1-sulfonamide) (BL-52); 7,7’- ((4-hydroxybutyl)azanediyl)bis(N,N-dioctyl heptane-1-sulfonamide (BL-207); [(4- hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315); ((3- hydroxypropyl)azanediyl)-bis(nonane-9,1-diyl) bis(2-butyloctanoate) (ALC-0366); 1,2- dioleoyloxy-3-dimethylaminopropane (DODMA); heptatriaconta-6,9,28,31-tetraen-19-yl-4- (dimethylamino)butanoate (D-Lin-MC3-DMA); heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-WSGR Docket No.: 50401-791.603 (undecyloxy)hexyl]amino}-octanoate) (SM-102); O-[N-{(9Z,12Z)-octadeca-9,12-dien-1-yl)}-N- {7-pentadecylcarbonyloxyoctyl}-amino]4-(dimethylamino)butanoate (HY501); ((2-(4- (dimethylamino)butanoyl)oxy)ethyl)azanediylbis(octane 8,1-diyl) bis(2-hexyldecanoate) (EA- 405); (2-(4-(dimethylamino)butanoyl)oxy)azanediylbis(octane 8,1-diyl) bis(2-hexyldecanoate) (HY-405); 1,2-dioleoyl-3 trimethylammonium propane (DOTAP); 1,2-dioleoyl-3- dimethylammomium propane (DODAP); and 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); bis(2-octyldodecyl) 3,3'-((4-(4-methylpiperazin-1- yl)butyl)azanediyl)dipropionate (BODD-C2C4-PipZ); bis(2-hexyldecyl) 3,3'-((4-(4- methylpiperazin-1-yl)butyl)azanediyl)dipropionate (BHD-C2C2-PipZ); bis(2-octyldodecyl) 3,3'- ((2-(dimethylamino)ethyl)azanediyl)dipropionate (BODD-C2C2-DMA); bis(2-octyldodecyl) 3,3'-((2-(1-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-1Me-Pyr); bis(2- octyldodecyl) 3,3'-((2-(pyrrolidin-1-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-Pyr); or a mixture of any thereof. Lipids BL-51, BL-52 and BL-207 are described in WO2025 / 027089, incorporated by reference herein. Lipids HY501, EA-405, HY-405 are described in WO2018 / 087753, incorporated by reference herein. Lipids BODD-C2C4-PipZ, BHD-C2C2- PipZ, BODD-C2C2-DMA, BODD-C2C2-1Me-Pyr, and BODD-C2C2-Pyr are described in WO2022 / 081750, incorporated by reference herein.

[0391] In some embodiments, the cationic or cationically ionizable lipid is selected from the group consisting of: 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA); heptatriaconta- 6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (D-Lin-MC3-DMA); heptadecan-9-yl 8- {(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102); O-[N-{(9Z,12Z)- octadeca-9,12-dien-1-yl)}-N-{7-pentadecylcarbonyloxyoctyl}-amino]4- (dimethylamino)butanoate (HY501); ((2-(4- (dimethylamino)butanoyl)oxy)ethyl)azanediylbis(octane 8,1-diyl) bis(2-hexyldecanoate) (EA- 405); (2-(4-(dimethylamino)butanoyl)oxy)azanediylbis(octane 8,1-diyl) bis(2-hexyldecanoate) (HY-405); 1,2-dioleoyl-3 trimethylammonium propane (DOTAP); 1,2-dioleoyl-3- dimethylammomium propane (DODAP); 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA); bis(2-octyldodecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (BODD-C2C4-PipZ); bis(2-hexyldecyl) 3,3'-((4-(4-methylpiperazin-1- yl)butyl)azanediyl)dipropionate (BHD-C2C2-PipZ); bis(2-octyldodecyl) 3,3'-((2- (dimethylamino)ethyl)azanediyl)dipropionate (BODD-C2C2-DMA); bis(2-octyldodecyl) 3,3'- ((2-(1-methylpyrrolidin-2-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-1Me-Pyr); bis(2- octyldodecyl) 3,3'-((2-(pyrrolidin-1-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-Pyr); 7,7’-WSGR Docket No.: 50401-791.603 ((4-hydroxybutyl)azanediyl)bis(N,N-dioctyl heptane-1-sulfonamide (BL-207); or a mixture of any thereof.

[0392] In some embodiments, the cationic or cationically ionizable lipid is selected from the group consisting of: 7,7’-((4-hydroxybutyl)azanediyl)bis(N-hexyl-N-octylheptane-1- sulfonamide) (BNT-51); 7,7’-((4-(3,3-dimethylthioureido)butyl)azanediyl)bis(N-hexyl-N- octylheptane-1-sulfonamide) (BNT-52); [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2- hexyldecanoate) (ALC-0315); ((3-hydroxypropyl)azanediyl)-bis(nonane-9,1-diyl) bis(2- butyloctanoate) (ALC-0366); 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA); heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (D-Lin-MC3-DMA); heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}-octanoate) (SM-102); bis(2-octyldodecyl)3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (BODD- C2C4-PipZ); bis(2-hexyldecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (BHD-C2C2-PipZ); bis(2-octyldodecyl) 3,3'-((2-(1-methylpyrrolidin-2- yl)ethyl)azanediyl)dipropionate (BODD-C2C2-1Me-Pyr); bis(2-octyldodecyl) 3,3'-((2- (pyrrolidin-1-yl)ethyl)azanediyl)dipropionate (BODD-C2C2-Pyr); 7,7’-((4- hydroxybutyl)azanediyl)bis(N,N-dioctyl heptane-1-sulfonamide (BL-207); or a mixture of any thereof.

[0393] In some embodiments, the cationic or cationically ionizable lipid is [(4- hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315). In some embodiments, the cationic or cationically ionizable lipid is ((3-hydroxypropyl)azanediyl)- bis(nonane-9,1-diyl) bis(2-butyloctanoate) (ALC-0366). In some embodiments, the cationic or cationically ionizable lipid is 1,2-dioleoyloxy-3-dimethylaminopropane (DODMA). In some embodiments, the cationic or cationically ionizable lipid is heptadecan-9-yl 8-{(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]...

Claims

WSGR Docket No.: 50401-791.603 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) 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) a polypeptide comprising at least one tumor antigen epitope sequence expressed by cancer cells of a human subject with cancer, or (B) a polynucleotide encoding the polypeptide; thereby forming a population of cells comprising stimulated T cells.

2. The method of claim 1, further comprising enriching CD25+ cells, CD39+ cells, 4-1BB+ cells, CD69+ cells, HLA-DR+ cells, LFA-1+ cells, IFN-g+ cells, ITGB7 cells, cells, cells, TNF-a+ cells, or any combination thereof from the population of cells comprising the stimulated T cells.

3. The method of claim 1 or 2, wherein 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.

4. The method of claim 3, 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.

5. The method of claim 4, wherein the first lipid delivery vehicle is APC specific.

6. The method of claims 4 or 5, 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.

7. The method of any one of claims 4-6, wherein the first lipid nanoparticle comprises an APC targeting agent.WSGR Docket No.: 50401-791.603 8. The method of any one of claims 1-7, 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.

9. The method of claim 8, wherein genetically editing the population of T cells comprises inhibiting expression of an endogenous gene in the population of T cells.

10. The method of claim 9, 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.

11. The method of claim 9 or 10, 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.

12. The method of claim 11, 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.

13. The method of claim 11, 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 viral vector.

14. The method of claim 8, wherein genetically editing the T cells comprises delivering a second lipid delivery vehicle comprising an exogenous gene into the T cells.

15. The method of claim 14, wherein the second lipid delivery vehicle is T cell specific.

16. The method of claims 14 or 15, wherein the second lipid delivery vehicle specifically targets T cells.

17. The method of claim 16, wherein the second lipid delivery vehicle comprises a T cell targeting agent.

18. The method of claim 17, wherein the T cell targeting agent is recognized by or interacts with a molecule present on a T cell surface.

19. The method of claim 17, wherein the T cell targeting agent is an antibody or fragment thereof.

20. The method of claim 18 or 19, wherein 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, CD137, ITGB7, 4- 1BB, CD45, HLA-DR, and ICOS.WSGR Docket No.: 50401-791.603 21. The method of any one of claims 14-20, wherein incubating the first population of APCs and T cells from step (a) 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.

22. The method of any one of claims 14-21, 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.

23. The method of any one of claims 10-13, 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.

24. The method of any one of claims 10-13, 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.

25. The method of any one of claims 1-24, further comprising administering the plurality of T cells that comprises antigen-specific T cells into a subject in need thereof.

26. The method of any one of claims 1-25, wherein the time from (a) to obtaining a therapeutically effective amount of antigen-specific T cells is less than 30 days, less than 25 days, less than 20 days, less than 15 days, or less than 10 days.

27. The method of any one of claims 3-26, wherein the first lipid delivery vehicle and / or second lipid delivery vehicle comprises a lipid nanoparticle.

28. The method of any one of claims 3-26, wherein the first lipid delivery vehicle and / or second lipid delivery vehicle comprises a lipoplex.

29. The method of claim 27, wherein the lipid nanoparticle comprises R-DODMA, DOPE, Cholesterol, and / or PEG.

30. The method of claim 29, wherein the ratio of R-DODMA to DOPE to Cholesterol to PEG is 40:10:48:

2.

31. The method of claim 28, wherein the lipoplex comprises DOTMA and / or DOPE.

32. The method of claim 31, wherein the ratio of DOTMA to DOPE is 2:

1.

33. The method of any one of claims 1-32, wherein the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD8+ T cell epitope.

34. The method of claim 33, wherein the CD8+ T cell epitope comprises a sequence of about 7 to about 12 consecutive amino acids expressed by a cancer cell.

35. The method of claim 33 or 34, wherein the polypeptide comprising at least one tumor antigen epitope sequence comprises at least two CD8+ T cell epitope sequences.WSGR Docket No.: 50401-791.603 36. The method of any one of claims 1-35, wherein the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD4+ T cell epitope.

37. The method of claim 36, wherein the CD4+ T cell epitope comprises a sequence of about 13 to about 25 consecutive amino acids expressed by a cancer cell.

38. The method of claim 36 or 37, wherein the polypeptide comprising at least one tumor antigen epitope sequence comprises at least two different CD4+ T cell epitope sequences.

39. The method of claim 35 or 38, wherein the polypeptide comprising at least one tumor antigen epitope sequence comprises a linker between the at least two different CD8+ T cell targeting epitopes and the at least two different CD4+ T cell epitope sequences.

40. The method of claim 39, wherein the linker comprises a cleavable linker and / or a linker that is degradable by a protease.

41. The method of claim 40, wherein the least two different CD8+ T cell epitope sequences are separated by one or more linkers.

42. The method of claim 40 or 41, wherein the least two different CD4+ T cell epitope sequences are separated by one or more linkers.

43. The method of claim 41 or 42, wherein the one or more linkers are one or more flexible linkers.

44. The method of claim 41 or 42, wherein the one or more linkers are one or more cleavable linkers.

45. The method of any one of claims 33-44, wherein the polypeptide further comprises a Sec domain sequence at its N-terminus.

46. The method of any one of claims 33-45, wherein the polypeptide further comprises a MITD domain sequence at its C-terminus.

47. The method of claim 45, wherein the Sec domain sequence comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NOs: 35 or 36.

48. The method of claim 46, wherein the MITD domain sequence comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO:

34.

49. The method of any one of claims 45-48, wherein the polynucleotide encoding the polypeptide comprises a sequence encoding the Sec domain at its 5’ end.

50. The method of any one of claims 46-48, wherein the polynucleotide encoding the polypeptide comprises a sequence encoding the MITD domain at its 3’ end.

51. The method of any one of claims 41-48, wherein the polynucleotide encoding the polypeptide comprises one or more sequences encoding one or more linkers.WSGR Docket No.: 50401-791.603 52. The method of claim 49, wherein the sequence encoding the Sec domain comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NOs: 32 or 33.

53. The method of claim 50, wherein the sequence encoding the MITD domain comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO:

31.

54. 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 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.

55. The method of claim 54, wherein the lipid nanoparticle comprises R-DODMA, DOPE, Cholesterol, and / or PEG.

56. The method of claim 55, wherein the ratio of R-DODMA to DOPE to Cholesterol to PEG is 40:10:48:

2.

57. 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 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.

58. The method of claim 57, wherein the lipoplex comprises DOTMA and / or DOPE.

59. The method of claim 58, wherein the ratio of DOTMA to DOPE is 2:

1.

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) providing a population of immune cells comprising antigen presenting cells (APCs) and T cells from a biological sample from a subject;WSGR Docket No.: 50401-791.603 (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 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; and (c) expanding the stimulated T cells.

61. The method of claim 60, wherein the second lipid nanoparticle formulation comprises a T cell targeting agent.

62. The method of claim 61, wherein the T cell targeting agent is recognized by or interacts with a molecule present on a T cell surface.

63. The method of claim 62, wherein the T cell targeting agent is an antibody or fragment thereof.

64. The method of claim 62 or 63, wherein 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, CD137, ITGB7, 4- 1BB, CD45, HLA-DR, and ICOS.

65. The method of claim 60-64, wherein the first lipid nanoparticle formulation comprises an APC targeting agent.

66. The method of any one of claims 54-65, wherein the polypeptide comprising at least one tumor antigen epitope sequence comprises a CD8+ T cell epitope.

67. The method of claim 66, wherein the CD8+ T cell epitope comprises a sequence of about 7 to about 12 consecutive amino acids expressed by a cancer cell.

68. The method of claim 66 or 67, wherein the polypeptide comprising at least one tumor antigen epitope sequence comprises at least two different CD8+ T cell epitope sequences.

69. The method of any one of claims 54-68, wherein polypeptide comprising at least one tumor antigen epitope sequence comprises a CD4+ T cell epitope.

70. The method of claim 69, wherein the CD4+ T cell epitope comprises a sequence of about 13 to about 25 consecutive amino acids expressed by a cancer cell.

71. The method of claim 69 or 70, wherein the polypeptide comprising at least one tumor antigen epitope sequence comprises at least two different CD4+ T cell targeting epitope sequences.WSGR Docket No.: 50401-791.603 72. The method of claim 68 or 71, wherein 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.

73. The method of any one of claims 57-72, wherein the polynucleotide encoding the polypeptide comprises a linker.

74. The method of claim 72 or 73, wherein the linker comprises a cleavable linker and / or a linker that is degradable by a protease.

75. The method of claim 74, wherein the least two different CD8+ T cell epitope sequences are separated by one or more linkers.

76. The method of claim 74 or 75, wherein the least two different CD4+ T cell epitope sequences are separated by one or more linkers.

77. The method of claim 75 or 76, wherein the one or more linkers are one or more flexible linkers.

78. The method of claim 75 or 76, wherein the one or more linkers are one or more cleavable linkers.

79. The method of any one of claims 66-78, wherein the polypeptide further comprises a Sec domain sequence at its N-terminus.

80. The method of any one of claims 66-78, wherein the polypeptide further comprises a MITD domain sequence at its C-terminus.

81. The method of claim 79, wherein the Sec domain sequence comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NOs: 35 or 36.

82. The method of claim 80, wherein the MITD domain sequence comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO:

34.

83. The method of any one of claims 79-82, wherein the polynucleotide encoding the polypeptide comprises a sequence encoding the Sec domain at its 5’ end.

84. The method of any one of claims 80-82, wherein the polynucleotide encoding the polypeptide comprises a sequence encoding the MITD domain at its 3’ end.

85. The method of any one of claims 75-82, wherein the polynucleotide encoding the polypeptide comprises one or more sequences encoding one or more linkers.

86. The method of claim 83, wherein the sequence encoding the Sec domain comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NOs: 32 or 33.

87. The method of claim 84, wherein the sequence encoding the MITD domain comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 31.WSGR Docket No.: 50401-791.603 88. The method of any one of claims 60-87, wherein the polynucleotide of the second lipid nanoparticle formulation comprises or encodes an agent for genetically editing T cells.

89. The method of claim 88, wherein the polynucleotide encodes a guide RNA, the site- specific nuclease, the site-specific nickase, and / or the site-specific epigenetic regulator.

90. The method of claim 88, wherein the polynucleotide comprises an RNA.

91. The method of claim 90, wherein the RNA is a guide RNA.

92. The method of any one of claims 54-91, wherein the at least one tumor antigen epitope sequence comprises a mutation.

93. An ex vivo antigen presenting cell comprising a polynucleotide encoding the 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 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, and 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.

94. The ex vivo antigen presenting cell of claim 93, wherein the one or more CD4+ 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, and wherein the third amino acid sequence is operably linked to the first amino acid sequence.

95. The ex vivo antigen presenting cell of claim 93, wherein herein the one or more CD4+ T cell epitopes comprises at least 3 amino acid sequences each having a different epitope sequence.

96. The ex vivo antigen presenting cell of any one of claims 93-95, wherein the one or more CD8+ T cell epitopes comprising a fourth amino acid sequence having a fourth epitope sequence consisting of 13 to 25 consecutive amino acids from a cancer protein, and wherein the fourth amino acid sequence is operably linked to the second amino acid sequence.

97. The ex vivo antigen presenting cell of any one of claims 93-95, wherein the one or more CD8+ T cell epitopes comprises at least 3 amino acid sequences each having a different epitope sequence.WSGR Docket No.: 50401-791.603 98. The ex vivo antigen presenting cell of any one of claims 93-97, wherein the one or more linkers comprise a cleavable linker and / or a linker that is degradable by a protease.

99. The ex vivo antigen presenting cell of any one of claims 93-97, wherein the one or more linkers are one or more flexible linkers.

100. The ex vivo antigen presenting cell of claim 93-97, wherein the one or more linkers are one or more cleavable linkers.

101. The ex vivo antigen presenting cell of any one of claims 93-100, wherein the polypeptide further comprises a Sec domain sequence at its N-terminus.

102. The ex vivo antigen presenting cell of any one of claims 93-101, wherein the polypeptide further comprises a MITD domain sequence at its C-terminus.

103. The ex vivo antigen presenting cell of claim 101, wherein the Sec domain sequence comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NOs: 35 or 36.

104. The ex vivo antigen presenting cell of claim 102, wherein the MITD domain sequence comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO:

34.

105. The ex vivo antigen presenting cell of any one of claims 101-104, wherein the polynucleotide encoding the polypeptide comprises a sequence encoding the Sec domain at its 5’ end.

106. The ex vivo antigen presenting cell of any one of claims 102-104, wherein the polynucleotide encoding the polypeptide comprises a sequence encoding the MITD domain at its 3’ end.

107. The ex vivo antigen presenting cell of any one of claims 93-104, wherein the polynucleotide encoding the polypeptide comprises one or more sequences encoding one or more linkers.

108. The ex vivo antigen presenting cell of claim 105, wherein the sequence encoding the Sec domain comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NOs: 32 or 33.

109. The ex vivo antigen presenting cell of claim 106, wherein the sequence encoding the MITD domain comprises a sequence having at least 80% sequence identity to a sequence of SEQ ID NO: 31.

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