DNA constructs for improved t cell immunotherapy

Abstract

Provided herein are compositions and methods for modifying the human T cell genome. Further, the compositions and methods described herein can be used to produce human T cells whose specificity and function are altered to limit their side effects associated with T cell therapy. Provided herein are human T cells that heterologously express one or more polypeptides. In some embodiments, the one or more polypeptides, e.g., two or more polypeptides, are encoded by a nucleic acid construct inserted into the cell TCR locus.

Description

[0001] Prior related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 309,938, filed on February 14, 2022, the entirety of which is incorporated herein by reference. Background of the Invention

[0004] Current technologies for modifying ex vivo or living gene-edited cells for therapeutic use focus on correcting existing mutations, limiting therapeutic applicability to conditions caused by single mutations that result in dysregulated gene function, or integrating completely new synthetic genes, which require extensive research and development to create new synthetic DNA sequences that can be used therapeutically. As a result, the options for genome modification are limited. Given the importance of T cells in adoptive cell therapy, the ability to obtain human T cells and modify them to generate edited T cells with a desired function or functions may facilitate the development and application of adoptive T cell therapy. Summary of the invention

[0005] The present disclosure relates to compositions and methods for modifying T cell genomes. The inventors have found that human T cells can be modified to change T cell specificity and function. By inserting nucleic acids encoding one or more polypeptides and heterologous T cell receptors (TCR) or synthetic antigen receptors (e.g., chimeric antigen receptors (CAR)) into specific endogenous sites (e.g., TCR loci) in the T cell genome, human T cells with desired TCR or CAR antigen specificity and polypeptide functions can be prepared. In addition, the compositions and methods described herein can be used to produce human T cells with specificity and functional changes while limiting the side effects associated with T cell therapy.

[0006] Provided herein are human T cells that heterologously express one or more polypeptides. In some embodiments, the one or more polypeptides, such as two or more polypeptides, are encoded by a nucleic acid construct inserted into the TCR locus of the cell.

[0007] In some embodiments, human T cells heterologously express: a polypeptide comprising a TFAP4 protein, and a polypeptide comprising a BATF protein; a polypeptide comprising a BATF3 protein, and a polypeptide comprising a TFAP4 protein; a polypeptide comprising a FOXJ2 protein, and a polypeptide comprising a RARA protein; a polypeptide comprising an ID3 protein, and a polypeptide comprising a TFAP4 protein; a polypeptide comprising a BATF protein, and a polypeptide comprising an IRF2 protein; a polypeptide comprising a NANOG protein, and a polypeptide comprising a TFAP4 protein; a polypeptide comprising a MAFF protein, and a polypeptide comprising a SATB1 protein; a polypeptide comprising a BATF protein, and a polypeptide comprising an IRF1 protein; a polypeptide comprising a TFAP4 protein, and a polypeptide comprising a HOPX protein; a polypeptide comprising a BATF protein; white polypeptide, and a polypeptide comprising HES2 protein; a polypeptide comprising ATF2 protein, and a polypeptide comprising BATF protein; a polypeptide comprising TIGIT protein, and a polypeptide comprising ICOS protein; a polypeptide comprising LTBR protein, and a polypeptide comprising 4-1BB protein; a polypeptide comprising DR5 protein, and a polypeptide comprising ICOS protein; a polypeptide comprising CCR4 protein; a polypeptide comprising BATF3 protein, and a polypeptide comprising SMAD4 protein; a polypeptide comprising FOXP3 protein, and a polypeptide comprising RELA protein; a polypeptide comprising ID3 protein, and a polypeptide comprising BATF3 protein; a polypeptide comprising ID2 protein, a polypeptide comprising TP73 protein; a polypeptide comprising SMAD3 protein, or a polypeptide comprising BATF3 protein.

[0008] In some embodiments, human T cells express a polypeptide comprising a human TFAP4 protein, and a polypeptide comprising a human TIM3 extracellular domain or a portion thereof connected to a human 4-1BB intracellular domain (and optionally about 1-15 (e.g., 12) amino acids of a human 4-1BB extracellular domain) via a transmembrane domain.

[0009] In some embodiments, human T cells express a polypeptide comprising a human SOX5 protein, and a truncated TIGIT protein comprising about 1-10 (e.g., 7) amino acids of the human TIGIT extracellular domain or a portion thereof, the TIGIT transmembrane domain, and the human TIGIT intracellular domain.

[0010] In some embodiments, human T cells express a polypeptide comprising a human MYC protein and a polypeptide comprising the extracellular domain of human TGFbR2 or a portion thereof connected to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g., 7) amino acids of the intracellular domain of TGFbR2) via a transmembrane domain.

[0011] In some embodiments, the human T cells express a polypeptide comprising a BATF3 protein and a polypeptide comprising a 4-1BB protein.

[0012] In some embodiments, the human T cells express a polypeptide comprising a TFAP4 protein and a polypeptide comprising the extracellular domain of human LTBR or a portion thereof connected to the intracellular domain of human IL-4R (and optionally, 1-10 (e.g., 7) amino acids of the intracellular domain of LTBR) via a transmembrane domain.

[0013] In some embodiments, the human T cells express a polypeptide comprising an ATF1 protein and a polypeptide comprising the extracellular domain of human IL4RA or a portion thereof linked to the intracellular domain of human 4-1BB.

[0014] In some embodiments, human T cells express a polypeptide comprising a BATF protein and a polypeptide comprising a human 2B4 extracellular domain or a portion thereof linked to a human 4-1BB intracellular domain, and optionally about 1-15 (e.g., 12) amino acids of a human 4-1BB extracellular domain.

[0015] In some embodiments, human T cells express a polypeptide comprising a RORC protein and a polypeptide comprising the VISTA extracellular domain or a portion thereof linked to the intracellular domain of human CD28, and optionally about 1-15 (e.g., 12) amino acids of the extracellular domain of human CD28.

[0016] In some embodiments, the human T cells express a polypeptide comprising a BATF protein and a polypeptide comprising a CXCR 1 protein.

[0017] In some embodiments, the human T cells express a polypeptide comprising a MAFF protein and a polypeptide comprising an IL2RA protein.

[0018] In some embodiments, human T cells express a polypeptide comprising an ATF1 protein and a polypeptide comprising the extracellular domain of human TNFRSF12 or a portion thereof connected to the intracellular domain of human OX40 (and optionally, 1-10 (e.g., 7) amino acids of the intracellular domain of TNFRSF12) via a transmembrane domain.

[0019] In some embodiments, human T cells express a polypeptide comprising an extracellular domain of human TIM3 or a portion thereof connected to the intracellular domain of human 4-1BB (and optionally about 1-15 (e.g., 12) amino acids of the extracellular domain of human 4-1BB) via a transmembrane domain.

[0020] In some embodiments, the human T cells express a polypeptide comprising the human BATF2 extracellular domain or a portion thereof and a truncated PD-1 comprising about 1-12 (e.g., 10) amino acids of the human PD-1 extracellular domain or a portion thereof, the PD-1 transmembrane domain, and the human PD-1 intracellular domain.

[0021] In some embodiments, the human T cells express a polypeptide comprising a human TCF7 protein and a polypeptide comprising a human LAT1 protein.

[0022] In some embodiments, the human T cells express a polypeptide comprising a human STAT5 protein and a human IL-2RA protein.

[0023] In some embodiments, the human T cells express a polypeptide comprising a human FOXJ2 protein and a polypeptide comprising the extracellular domain of LTBR or a portion thereof connected to the intracellular domain of human CD28 (and optionally, 1-10 (e.g., 7) amino acids of the intracellular domain of LTBR) via a transmembrane domain.

[0024] In some embodiments, the human T cells express a polypeptide comprising a human FOXJ2 protein and a polypeptide comprising the LTBR extracellular domain or a portion thereof connected to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g., 7) amino acids of the LTBR intracellular domain) via a transmembrane domain.

[0025] In some embodiments, the human T cells express a polypeptide comprising a human HOPX protein and a polypeptide comprising an extracellular domain of IL4RA or a portion thereof connected to an intracellular domain of human 41-BB via a transmembrane domain.

[0026] In some embodiments, human T cells express a polypeptide comprising an ID2 protein and a polypeptide comprising the BLTA extracellular domain or a portion thereof linked to the human 4-1BB intracellular domain, and optionally about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain.

[0027] In some embodiments, the human T cells express a polypeptide comprising a human SMAD1 protein, and a polypeptide comprising a MCT4 protein.

[0028] In some embodiments, the human T cells express a polypeptide comprising a human SMAD1 protein, and a polypeptide comprising the extracellular domain of LTBR, ​​or a portion thereof, connected to the intracellular domain of human IL-4R (and optionally, 1-10 (e.g., 7) amino acids of the intracellular domain of LTBR) via a transmembrane domain.

[0029] In some embodiments, the human T cells express a polypeptide comprising the DR5 extracellular domain or a portion thereof connected by a transmembrane domain to a polypeptide comprising about 1-12 amino acids (e.g., 7 amino acids) of the DR5 intracellular domain and a polypeptide comprising the intracellular domain of human ICOS.

[0030] In some embodiments, the human T cells express a polypeptide comprising the extracellular domain of TIGIT or a portion thereof connected to the intracellular domain of human ICOS (and optionally about 1-15 (e.g., 12) amino acids of the extracellular domain of human ICOS) via a transmembrane domain.

[0031] In some embodiments, human T cells express a polypeptide comprising an LTBR extracellular domain or a portion thereof connected to the intracellular domain of human 4-1BB (and optionally about 1-15 (e.g., 7) amino acids of the human LTBR intracellular domain) via a transmembrane domain. In some embodiments, the polypeptide further comprises about 1-12 amino acids (e.g., 7 amino acids) of the 4-1BB intracellular domain connected to the 4-1BB intracellular domain.

[0032] In some embodiments, the human T cells express a polypeptide comprising CCR4.

[0033] In some embodiments, the human T cells express a polypeptide comprising a full-length 4-1BB protein, a FOXJ2 protein, a HES2 protein, or a fragment thereof.

[0034] In some embodiments, the T cells heterologously express two or more polypeptides, wherein each polypeptide comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 160, 161, and 162 (i.e., SEQ ID NOs: 30-58, 69-85, 160, 161, and 162).

[0035] In some embodiments, the heterologous nucleic acid construct comprises two or more nucleic acid sequences, wherein each nucleic acid sequence is at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-29 and 59-68.

[0036] In some embodiments, T cells express antigen-specific T cell receptors (TCR) or synthetic antigen receptors that recognize target antigens. In some embodiments, T cells are regulatory T cells, effector T cells, memory T cells, or naive T cells. In some embodiments, effector T cells are CD8+T cells or CD4+T cells. In some embodiments, effector T cells are CD8+CD4+T cells. In some embodiments, T cells are primary cells.

[0037] In some embodiments, the target insertion site is located in exon 1 of the TCR-alpha subunit constant gene (TRAC). In some embodiments, the target insertion site is located in exon 1 of the TCR-beta subunit constant gene (TRBC).

[0038] In some embodiments, the heterologous nucleic acid inserted into the human T cell encodes in the following order: (i) a first self-cleaving peptide sequence; (ii) a first heterologous TCR subunit chain, wherein the TCR subunit chain comprises a variable region and a constant region of a TCR subunit; (iii) a second self-cleaving peptide sequence; (iv) one or more polypeptides as described herein; (v) a third self-cleaving peptide sequence; (vi) a variable region of a second heterologous TCR subunit chain; and (vii) a portion of the N-terminus of an endogenous TCR subunit, Wherein, if the endogenous TCR subunit of the cell is a TCR-alpha (TCR-α) subunit, then the first heterologous TCR subunit chain is a heterologous TCR-beta (TCR-β) subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-α subunit chain, and if the endogenous TCR subunit of the cell is a TCR-β subunit, then the first heterologous TCR subunit chain is a heterologous TCR-α subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-β subunit chain. Optionally, in any construct described herein, the construct encodes two, three, four or more heterologous polypeptides.

[0039] In some embodiments, the heterologous nucleic acid inserted into the human T cell encodes in the following order: (i) a first self-cleaving peptide sequence; (ii) one or more heterologous polypeptides as described herein; (iii) a second self-cleaving peptide sequence; (iv) a first heterologous TCR subunit chain, wherein the TCR subunit chain comprises a variable region and a constant region of a TCR subunit; (v) a third self-cleaving peptide sequence; (vi) a variable region of a second heterologous TCR subunit chain; and (vii) a portion of the N-terminus of an endogenous TCR subunit. , wherein, if the endogenous TCR subunit of the cell is a TCR-alpha (TCR-α) subunit, the first heterologous TCR subunit chain is a heterologous TCR-beta (TCR-β) subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-α subunit chain, and wherein if the endogenous TCR subunit of the cell is a TCR-β subunit, the first heterologous TCR subunit chain is a heterologous TCR-α subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-β subunit chain.

[0040] In some embodiments, the nucleic acid construct encodes in the following order: (i) a first self-cleaving peptide sequence; (ii) a synthetic antigen receptor; (iii) a second self-cleaving peptide sequence; (iv) one or more heterologous polypeptides described herein; and (v) a third self-cleaving peptide sequence or a poly A sequence.

[0041] In some embodiments, the nucleic acid construct encodes in the following order: (i) a first self-cleaving peptide sequence; (ii) one or more heterologous polypeptides described herein; (iii) a second self-cleaving peptide sequence; (iv) a synthetic antigen receptor; and (v) a third self-cleaving peptide sequence or a poly A sequence.

[0042] In some embodiments, the heterologous nucleic acid construct comprises two or more nucleic acid sequences, wherein each nucleic acid is at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-29 and 59-68.

[0043] Also provided is a method for modifying human T cells, comprising: (a) introducing into human T cells (i) a targeted nuclease that cleaves a target region in the TCR locus of the human T cells to generate a target insertion site in the cell genome; and (ii) a nucleic acid construct encoding: a polypeptide comprising a TFAP4 protein and a BATF protein; a polypeptide comprising a BATF3 protein and a TFAP4 protein; a polypeptide comprising a FOXJ2 protein and a RARA protein; a polypeptide comprising an ID3 protein and a TFAP4 protein; a polypeptide comprising a BATF protein and an IRF2 protein; a polypeptide comprising a NANOG protein and a TFAP4 protein; a polypeptide comprising a MAFF protein and a SATB1 protein; a polypeptide comprising a BATF protein and an IRF1 protein; a polypeptide comprising a T A polypeptide comprising a FAP4 protein and a HOPX protein; a polypeptide comprising a BATF protein and a HES2 protein; a polypeptide comprising an ATF2 protein and a BATF protein; a polypeptide comprising a BATF3 protein and a SMAD4 protein; a polypeptide comprising a FOXP3 protein and a RELA protein; a polypeptide comprising an ID3 protein and a BATF3 protein; a polypeptide comprising an ID2 protein and a polypeptide comprising a TP73 protein; a polypeptide comprising a SMAD3 protein, or a polypeptide comprising a BATF3 protein; a polypeptide comprising a HES2 protein; a polypeptide comprising a FOXJ2 protein; a polypeptide comprising (a) a human TFAP4 protein, and (b) comprising a polypeptide that binds to a human 4-1BB intracellular domain (and optionally a human 4-1BB extracellular domain) through a transmembrane domain A polypeptide comprising (a) a human SOX5 protein, and (b) a truncated TIGIT protein comprising about 1-10 (e.g., 7) amino acids of the human TIGIT extracellular domain or a portion thereof, a TIGIT transmembrane domain, and a human TIGIT intracellular domain; a polypeptide comprising (a) a human MYC protein, and (b) a polypeptide comprising the human TGFbR2 extracellular domain or a portion thereof connected to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g., 7) amino acids of the TGFbR2 intracellular domain) via a transmembrane domain; a polypeptide comprising a BATF3 protein and 4-1 A polypeptide comprising (a) a TFAP4 protein, and (b) a polypeptide comprising a human LTBR extracellular domain or a portion thereof connected to a human IL-4R intracellular domain (and optionally, 1-10 (e.g., 7) amino acids of the LTBR intracellular domain) via a transmembrane domain; a polypeptide comprising (a) an ATF1 protein, and (b) a polypeptide comprising a human IL4RA extracellular domain or a portion thereof connected to a human 4-1BB intracellular domain; a polypeptide comprising (a) a BATF protein, and (b) a polypeptide comprising a human 2B4 extracellular domain or a portion thereof connected to a human 4-1BB intracellular domain, and optionally, about 1-15 (e.g., 12) amino acids of a human 4-1BB extracellular domain;A polypeptide comprising (a) a RORC protein, and (b) a polypeptide comprising an extracellular domain of VISTA or a portion thereof connected to a human CD28 intracellular domain, and optionally about 1-15 (e.g., 12) amino acids of a human CD28 extracellular domain; a polypeptide comprising a BATF protein and a CXCR 1 protein; a polypeptide comprising a MAFF protein IL2RA protein; a polypeptide comprising (a) an ATF1 protein, and (b) a polypeptide comprising an extracellular domain of human TNFRSF12 or a portion thereof connected to a human OX40 intracellular domain (and optionally, 1-10 (e.g., 7) amino acids of a TNFRSF12 intracellular domain) via a transmembrane domain; a polypeptide comprising an extracellular domain of human TIM3 or a portion thereof connected to a human 4-1BB intracellular domain (and optionally about 1-15 (e.g., 12) amino acids of a human 4-1BB extracellular domain) via a transmembrane domain; A polypeptide comprising a human BATF2 extracellular domain or a portion thereof connected to a truncated PD-1, wherein the truncated PD-1 comprises about 1-12 (e.g., 10) amino acids of the human PD-1 extracellular domain or a portion thereof, the PD-1 transmembrane domain, and the human PD-1 intracellular domain; a polypeptide comprising a human TCF7 protein and a human LAT1 protein; a polypeptide comprising a human 4-1BB protein; a polypeptide comprising a human STAT5 protein and a human IL-2RA protein; a polypeptide comprising (a) a human FOXJ2 protein, and (b) comprising a human CD28 intracellular domain (and optionally, A polypeptide comprising (a) a human FOXJ2 protein, and (b) a polypeptide comprising an extracellular domain of LTBR or a portion thereof connected to amino acids 41-142 of human MyD88 (and optionally, 1-10 (e.g., 7) amino acids of the intracellular domain of LTBR) via a transmembrane domain; a polypeptide comprising (a) a human HOPX protein, and (b) an extracellular domain of IL4RA or a portion thereof connected to the intracellular domain of human 41-BB via a transmembrane domain. A polypeptide comprising (a) an ID2 protein, and (b) a polypeptide comprising an extracellular domain of BLTA or a portion thereof connected to the intracellular domain of human 4-1BB, and optionally about 1-15 (e.g., 12) amino acids of the extracellular domain of human 4-1BB; a polypeptide comprising a human SMAD1 protein and an MCT4 protein; and a polypeptide comprising (a) a human SMAD1 protein and (b) an extracellular domain of LTBR or a portion thereof connected to an amino acid of the intracellular domain of human IL-4R (and optionally, 1-10 (e.g., 7) amino acids of the intracellular domain of LTBR) via a transmembrane domain;And (b) allowing recombination to occur, thereby inserting the nucleic acid construct into the target insertion site to produce modified human T cells. In any of the compositions and methods provided herein, the nucleic acid construct may include a nucleic acid sequence encoding two or more polypeptides, wherein the two or more polypeptides are the same polypeptide, such as a nucleic acid construct encoding two or more transcription factors, wherein the two transcription factors are the same (e.g., IDE polypeptide and ID3 polypeptide; EOMES polypeptide and EOMES polypeptide; TFAP4 polypeptide and TFAP 4 polypeptide, or BATF polypeptide and BATF polypeptide). ;

[0044] In some methods, the polypeptide comprises an amino acid sequence at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:30-SEQ ID NO:58, SEQ ID NO:69-85, SEQ ID NO:160, SEQ ID NO:161, and SEQ ID NO:162.

[0045] In some methods, the target insertion site is located in exon 1 of the TCR-alpha subunit constant gene (TRAC) or in exon 1 of the TCR-beta subunit constant gene (TRBC).

[0046] In some methods, the nucleic acid construct is inserted by introducing a viral vector comprising the nucleic acid construct into the cell. In some embodiments, the targeting nuclease is selected from an RNA-guided nuclease domain, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), and a megaTAL.

[0047] In some methods, the targeting nuclease, guide RNA, and DNA template are introduced into the cell as a ribonucleoprotein complex (RNP)-DNA template complex, wherein the RNP-DNA template complex comprises: (i) RNP, wherein the RNP comprises the targeting nuclease and the guide RNA; and (ii) a nucleic acid construct.

[0048] In some methods, T cells express antigen-specific T cell receptors (TCR) or synthetic antigen receptors that recognize target antigens. In some embodiments, T cells are regulatory T cells, effector T cells, memory T cells, or naive T cells. In some embodiments, effector T cells are CD8+T cells or CD4+T cells. In some embodiments, effector T cells are CD8+CD4+T cells. In some embodiments, T cells are primary cells.

[0049] Also provided are modified T cells produced by any of the methods described herein.

[0050] Also provided is a method for enhancing the immune response of a human subject, the method comprising administering any T cell described herein. In some embodiments, the T cell expresses an antigen-specific TCR that recognizes a target antigen in the subject. In some embodiments, the human subject suffers from cancer and the target antigen is a cancer-specific antigen. In some embodiments, the human subject suffers from an autoimmune disease or an allergic disease, and the antigen is an antigen associated with the autoimmune disease or the allergic disease. In some embodiments, the subject suffers from an infection and the target antigen is an antigen associated with an infection. In some embodiments, the T cell is autologous. In some embodiments, the T cell is allogeneic. In some embodiments, the T cell is a T cell derived from an induced pluripotent stem cell (iPSC). BRIEF DESCRIPTION OF THE DRAWINGS

[0051] This application includes the following drawings. These drawings are used to illustrate certain embodiments and / or features of the compositions and methods and to supplement the description of the compositions and methods. The drawings are not intended to limit the scope of the compositions and methods unless the written description explicitly indicates otherwise.

[0052] Figure 1 AC is a schematic diagram summarizing an exemplary pooled assembly method described herein. (A) Schematic diagram showing a pooled assembly method for generating a combinatorial library of tonic signal transduction GD2 CAR plus 100 transcription factors (TF) x 100 transcription factors, thereby generating a library of>10,000 members. Constructs (and integration orders) were identified by amplicon sequencing of the fusion region between two transcription factors (3' barcode of TF1 and 5' barcode of TF2). (B) Schematic diagram of an amplicon sequencing strategy for detecting barcodes. (C) The representativeness of the combined constructs was analyzed in plasmid pools and cell pools on the 4th day after electroporation, and the representativeness of>10,000 constructs was shown to be>99%.

[0053] Figure 2 AB shows the TFxTF combinatorial library knocked into primary human T cells. Since the GD2 CAR provides tonic signaling / chronic stimulation, T cells were cultured without the addition of target cells. Cells were sorted at day 16 and day 4 after electroporation, and log2 fold change (log2FC) was calculated (day 16 / day 4). Log2FC is shown and highlights that the combination of TFAP4 and BATF performed best.

[0054] Figure 3AB shows the construction of the second combinatorial library. (A) The second combinatorial library was generated using the tonic signal GD2 CAR plus 100 TF x129 therapeutic constructs (TC) (synthetic receptors, chemokine receptors, etc.), resulting in a library of 13,362 members. Similarly, the constructs were identified by amplicon sequencing of the fusion region. (B) The representative of the combined constructs was analyzed in the plasmid pool and the cell pool on the 4th day after electroporation, and the representativeness of >10,000 constructs was shown to be >99%.

[0055] Figure 4 Hits from the TF x TC screen are shown. Figure 2 Screening was performed as described. The highest hits TFAP4_TIM-3 / 4-1BB and SOX5_TIGIT_trunc were highlighted. Many of the combinatorial constructs with the highest Log2FC had TFAP4 or BATF / BATF3 as the first insertion position in their combinatorial constructs. Looking at the fusion receptors in the TC portion of the construct, the 4-1BB intracellular domain was overrepresented compared to the CD28 intracellular domain in the highest hit.

[0056] Figure 5 AD provides validation analysis. (A) For validation analysis, a single knock-in of a combination construct (including GD2CAR) was performed. A 50% / 50% co-culture analysis of different combination constructs is shown, and it is highlighted that adding TFAP4 to the construct appears to have a stronger effect than adding BATF. (B) The expression of activation markers of CAR T cells was analyzed 8 days after electroporation, and CD25 levels were increased in CAR T cells overexpressing TFAP4. (C) An exemplary flow cytometry diagram of phenotypic marker analysis 14 days after electroporation is shown. Adding BATF, especially TFAP4, increases the percentage of CD62L-positive T cells. (D) Phenotypic analysis of different combination constructs 14 days after electroporation.

[0057] Figure 6AD shows differential expression of genes. 14 days after electroporation, differentially expressed genes of the BATF-TFAP4 construct compared to the mCherry-NGFR control construct were analyzed by RNA-seq. Volcano plots show differentially expressed genes. The gene with the greatest differential expression was TFAP4. The color indicates whether the corresponding gene was also found under the most differentially expressed genes when comparing TFAP4-mCherry to control, BATF-mCherry to control, or all of these conditions. Genes highlighted in yellow are differentially expressed only when comparing BATF-TFAP4 to control. (B) Log2 fold changes between the test conditions and the control (mCherry-NGFR) are associated between BATF-TFAP4 and other constructs. Correlation analysis showed that BATF-TFAP4 shared the most changes in gene expression with TFAP4-mCherry and BATF-mCherry, while BATF-TFAP4 had a lower correlation with mCherry-JUN. (C) Gene set enrichment analysis of BATF-TFAP4 vs mCherry-NGFR cells at day 14 (baseline) highlights increased expression of genes involved in cell cycle regulation. (D) After stimulation with target cells, expression of genes involved in glycolysis, oxidative phosphorylation, and fatty acid metabolism increased in BATF-TFAP4 cells, while expression of genes involved in apoptosis decreased.

[0058] Figure 7 AB shows the killing ability of the combination constructs. (A) The combination constructs were co-cultured with Nalm-6 / GD2 target cells and analyzed for target cell killing. The BATF-TFAP4 combination construct outperformed the other constructs in terms of killing ability. (B) This finding was confirmed at multiple effector:target (E:T) ratios.

[0059] Figure 8 AB shows in vivo results. To analyze in vivo function, a xenograft model of adoptive T cell transfer was used. NSG mice were injected intravenously with 0.5M Nalm-6 / GFP / Luc / GD2 cells on day 0 and 1MGD2 CAR T cells three days later. Leukemic burden was determined by bioluminescent imaging. (B) shows analysis of leukemic growth after injection of combined T cells from two different T cell donors.

[0060] Fig. 9 is a schematic diagram of an exemplary GD2 CAR combination construct.

[0061] Fig.10AD shows that the combined knock-in screening revealed an efficient combination of transcription factors. (A) The ModPoKI platform was further developed to screen paired combinations of transcription factors and GD2 CAR. In the combined functional module, 100 TFs were combined with 100 TFs (except for the control), resulting in about 10,000 different TF combinations. They were combined with GD2 CAR, resulting in a knock-in size range of about 3.3 to about 8.2 kb. (B) Barcode sequencing of the TFxTF combinatorial plasmid library showed size-dependent representation, but confirmed that >99% of the constructs were represented after pooled assembly. Linear regression (lm function in R studio) was used for statistics. (C) The knock-in percentage of the combined constructs was analyzed in the cell pool on the 4th day after electroporation by amplicon sequencing, and the representativeness of about 10,000 constructs was shown to be >99%. N = 2 individual donors. Linear regression (lm function in R studio) was used for statistics. (D) The TFxTF combinatorial library was knocked into primary human T cells. Since GD2CAR provides tonic stimulation, T cells are cultured without adding target cells. Cells were sorted on the 16th and 4th days after electroporation, and log2 fold changes (log2FC) were calculated (16th day / 4th day). The Log2FC of the combined TFxTF constructs is shown, and the combination of TFAP4 and BATF with the best performance is highlighted. N = 2 individual donors. Statistics are calculated using DESeq2. In order to create a volcano map, two possible construct orientations (such as BATF-TFAP4 and TFAP4-BATF) are combined into one data set. The right sub-figure shows the barcode representation of two construct orientations x two donors.

[0062] Fig.11 AE shows the Fig.10Plasmid representation of the relevant combinatorial knock-in strategy and TFxTF library. (AB) Schematic diagram shows the pooled assembly method for generating a combinatorial library of tonic signal transduction GD2 CAR plus 100 transcription factors (TF) x100 TFs, thereby generating a library of approximately 10,000 members. The inserts at TF positions 1 and 2 are generated separately by PCR pooled from existing TF libraries. The backbone (consisting of GD2 CAR and homology arms) and the two inserts are assembled in a pooled Hifi DNA assembly reaction to form a combined ModPoKI plasmid library. The double-stranded HDR template is generated by pooled PCR and then knocked into primary human T cells by non-viral pooling. (C) The fusion region between the two transcription factors (combined barcoded polycistronic adapter) is composed of the barcode of the position 1 insert and the barcode of the position 2 insert. In order to read out the barcode region, mRNA is isolated, reverse transcribed into cDNA, and amplified using 5' and 3' sequencing adapters to add Illumina read 1 and read 2. Index sequences were added in the second PCR step. Amplicon sequencing was performed and mRNA / cDNA combined barcode counts were calculated. (D) An example of knocking into primary human T cells a control construct containing GD2 CAR and tNGFR and RFP containing a new combined barcoded polycistronic aptamer sequence. (D) TFxTF combinatorial library was knocked into primary human T cells. Since GD2 CAR provides tonic signal transduction, T cells were cultured without adding target cells. Cells were sorted on days 16 and 4 after electroporation, and log2 fold changes (log2FC) were calculated (day 16 / day 4). The log2FC of the first 10 combined TFxTF constructs is shown and compared with the control group. N = 2 individual donors. Shown is the barcode representation of two construct orientations x two donors.

[0063] Fig.12AG shows that the combination knock-in of TFAP4 and BATF induces a favorable transcriptional program in therapeutic T cells. (A) Competitive fitness assays were performed with specific combination knock-in constructs (including GD2 CAR). The figure shows an approximately 50% / 50% co-culture analysis of different combination constructs. N = 2 individual donors, in triplicate. Mean values ​​+ SEM are shown. An unpaired t-test was performed on day 4 to determine statistical significance. (B) Analysis of activation marker expression of CAR T cells 8 days after electroporation showed that TFAP4 KI and BATF-TFAP double KI CAR T cells had increased CD25 levels compared to BATF KI CAR T cells. N = 2 individual donors, in duplicate. Mean values ​​+ SEM are shown. Two-way ANOVA was performed using Holm-Sidak's multiple comparison correction to determine statistical significance. (C) An exemplary flow cytometry graph of phenotypic marker analysis 14 days after electroporation is shown. (D) Phenotypic analysis of KI CAR T cells in different combinations 14 days after electroporation. N = 2 individual donors, in duplicate. Mean + SEM is shown. Two-way ANOVA with Holm-Sidak's multiple comparison correction was performed to determine statistical significance. (E) 14 days after electroporation, differentially expressed genes in BATF-TFAP4 KI CAR T cells compared with RFP-tNGFR control KI CAR T cells were analyzed by RNA-seq, as shown in the volcano plot. The gene with the greatest differential expression was TFAP4 (not shown, log2FC = 5.0, padj = 6.03E-77). The color indicates whether the corresponding gene was also found under the gene with the greatest differential expression when comparing TFAP4-RFP KI CAR T cells to control KI CAR T cells, BATF-RFP KI CAR T cells to control KICAR T cells, or in all these comparisons. Genes that were selectively differentially expressed in BATF-TFAP4 vs. RFP-tNGFR KI CAR T cells are highlighted in yellow. N = 2 individual donors. (F) Combination KI CAR T cells were co-cultured with Nalm-6 / GFP / Luc / GD2 target cells and analyzed for target cell killing. N = 2 individual donors in triplicate. Mean + SEM are shown. Two-way ANOVA with Holm-Sidak's multiple comparison correction was performed to determine statistical significance. (G) To assess in vivo function, NSG mice were injected intravenously with 0.5e6 Nalm-6 / GFP / Luc / GD2 cells on day 0, followed by 1e6 GD2 CAR KI T cells three days later. Leukemic burden was determined by bioluminescent imaging. N = 2 individual T cell donors, 2-5 mice per donor per group. Fig.14Two different donors are shown separately in B. Mean + SEM are shown. Two-way ANOVA was performed using the Holm-Sedak multiple comparison test to compare all constructs with the control (RFP-tNGFR) (both donors combined).

[0064] Fig.13 AE shows the Fig.12 Transcriptome changes driven by combined BATF-TFAP4 knock-in. (A) Figure 6 As described above, different combination validation constructs were knocked into primary human T cells. Overall RNA-seq was performed 14 days after electroporation. The figure shows the differentially expressed genes of BATF-TFAP4 KI GD2 CAR T cells and RFP-tNGFR KI GD2 CAR T cells. N = 2 individual donors. (B) Log2 fold changes in gene expression between BATF-TFAP4 and other constructs, test KI conditions and control KI (RFP-tNGFR) were associated. Association analysis showed that BATF-TFAP4 KI had the most similar gene expression with RFP-TFAP4 and BATF-RFP, while BATF-TFAP4 KI cells had a lower correlation with RFP-JUN KI cells. N = 2 individual donors. Statistics were performed using linear regression (lm function in R studio). (CE) shows gene set enrichment analysis of BATF-TFAP4 combinatorial constructs without and 24 hours of GD2+ target cells added (days 14 and 15 after electroporation, respectively), compared to RFP-tNGFR (C), RFP-TFAP4 (D), or BATF-RFP (E). Notably, after target cell stimulation, expression of genes involved in fatty acid metabolism, glycolysis, oxidative phosphorylation, and IL2 / STAT5 signaling was increased in BATF-TFAP4 cells compared to RFP-tNGFR cells. N = 2 individual donors.

[0065] Fig.14 AB shows the Fig.12 Functional capacity of the associated in vitro and in vivo combinatorial BATF-TFAP4 knock-in. Combination KI GD2 CAR T cells were co-cultured with Nalm-6 / GFP / Luc / GD2 target cells and target cell killing was analyzed by Incucyte. In terms of in vitro killing capacity, BATF-TFAP4 combination KI GD2 CAR T cells trended outperforming other combination KI GD2 CAR T cells. N = 2 individual donors, triplicates. Two-way ANOVA was performed using the Holm-Sedak multiple comparison test to compare all constructs to the control (RFP-tNGFR). Means + SEM are shown. (B) Fig.12Data in are divided by donor. Shown are mean + SEM.

[0066] Fig.15 The results of CD25 sorting screening are shown. The GD2 CAR single insertion TF library was knocked into three separate primary human T cell donors. On the 8th day after electroporation, the cells were sorted for high and low expression of CD25. The figure shows the representative Log2FC of CD25 high and low box inserts. The data are normalized to the control (GFP / RFP) and fitted on the -1 / 1 axis. Donor-to-donor variability was observed for some constructs (mean + SEM shown).

[0067] Fig.16 The results of single stimulation screening (addition of A375 / CD19 compared to non-stimulated conditions) and repeated stimulation screening (addition of 5 A375 / CD19) in the complementary model using CD19 / 28z CAR combined with the full TF and SR single insertion library described in Example III are shown. The single knock-in TF and SR library was knocked into two-three separate primary human T cell donors in combination with CD19 / 28z CAR. The input group as well as the single stimulation group (unstimulated compared to A375 / CD19 stimulation for 4 days) and the repeated stimulation group (5 A375 / CD19 continuous stimulations for 2 days) were harvested, and the changes in the abundance of the constructs were analyzed using Log2FC relative to the input group. The number of individual donors for repeated stimulation = 3, and the number of individual donors for single stimulation = 2. The data were normalized to the control (GFP / RFP) and fitted on the -1 / 1 axis.

[0068] definition

[0069] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this document belongs. All patents, patent applications and publications cited throughout this disclosure are incorporated herein by reference in their entirety.

[0070] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a transcript" or "the transcript" may include plural transcripts.

[0071] Any and all embodiments / examples, or exemplary language (e.g., "for example / such as / such as") referred to herein are merely used to better illustrate the present invention and are not intended to limit the scope of protection claimed by the present invention, unless otherwise specified.

[0072] The terms "may," "might be," "can," and "could be," and related terms are intended to convey that the subject matter involved is optional (i.e., that the subject matter is present in some instances and not in other instances), rather than to indicate the likelihood or probability of the subject matter, unless the context clearly indicates otherwise.

[0073] As used herein, the terms "optional" and "optionally" mean that the subsequently described event, circumstance, or material may or may not occur, exist or not exist, and that the description includes instances where said event, circumstance, or material occurs or exists and instances where it does not or does not exist.

[0074] As used herein, the terms "include," "comprising," or "having" and variations thereof are intended to encompass the elements listed thereafter and their equivalents as well as other elements. Embodiments described as "include," "including," or "having" certain elements are also contemplated as "consisting essentially of (these specified elements)" and "consisting of (these specified elements)." As used herein, "and / or" means encompassing any and all possible combinations of one or more of the associated listed items, as well as non-combinations when interpreted in an optional context ("or").

[0075] As used herein, "consisting essentially of" (and grammatical variations) will be understood to cover the substances or steps and "those that do not materially affect the basic and novel characteristics" of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP §2111.03. Therefore, as used herein, the term "consisting essentially of" should be understood to be equivalent to "including / comprising".

[0076] The term "nucleic acid" or "nucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences as well as sequences explicitly indicated. Specifically, a degenerate codon substitution pattern can be obtained by generating a sequence in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0077] The term "gene" may refer to a DNA segment involved in producing or encoding a polypeptide chain. It may include regions before and after the coding region (leader and trailer) and intervening sequences (introns) between individual coding segments (exons). Alternatively, the term "gene" may refer to a DNA segment involved in producing or encoding non-translated RNA, such as rRNA, tRNA, guide RNA (e.g., single guide RNA) or microRNA.

[0078] As used herein, the term "endogenous" with respect to a nucleic acid (e.g., a gene) or a protein in a cell is a nucleic acid or protein that occurs in a particular cell found in nature, e.g., in its natural genomic location or locus. Additionally, a cell that "endogenously expresses" a nucleic acid or protein means a nucleic acid or protein found in nature.

[0079] As used herein, the phrase "heterologous" refers to a substance that does not normally occur naturally. The term "heterologous nucleotide sequence" refers to a nucleotide sequence that does not normally occur naturally in a given cell. Thus, a heterologous nucleotide sequence can be: (a) foreign to its host cell (i.e., exogenous to the cell); (b) naturally present in the host cell (i.e., endogenous), but present in a non-natural amount in the cell (i.e., greater or less than the amount naturally present in the host cell); or (c) naturally present in the host cell but located outside its natural locus.

[0080] "Promoter" is defined as one or more nucleic acid control sequences that direct the transcription of a nucleic acid. As used herein, a promoter includes essential nucleic acid sequences near the transcription start site, such as the TATA element of a polymerase II type promoter. A promoter also optionally includes a distal enhancer or repressor element, which may be located up to several thousand base pairs from the transcription start site.

[0081] Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it positions the site to facilitate translation.

[0082] "Polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, these terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0083] As used herein, the term "complementary" or "complementarity" refers to specific base pairing between nucleotides or between nucleic acids. Complementary nucleotides are typically A and T (or A and U) and G and C. The guide RNA described herein can comprise a sequence, for example, a DNA targeting sequence that is perfectly complementary or substantially complementary (e.g., with 1-4 mismatches) to a genomic sequence.

[0084] The "CRISPR / Cas" system refers to a broad class of bacterial systems for defending against foreign nucleic acids. The CRISPR / Cas system is present in a wide range of eubacteria and archaeal organisms. The CRISPR / Cas system includes subtypes I, II, and III. The wild-type II type CRISPR / Cas system uses RNA-mediated nucleases (e.g., Cas9,) to form a complex with guide and activation RNA to recognize and cleave foreign nucleic acids. Guide RNAs with guide RNA and activation RNA activity are also known in the art. In some cases, this type of dual-activity guide RNA is referred to as a single guide RNA (sgRNA).

[0085] Cas9 homologs exist in a variety of true bacteria, including, but not limited to, bacteria from the following taxonomic groups: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydiae-Verrucomicrobia, Chlroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 protein is Streptococcus pyogenes Cas9 protein. Other Cas9 proteins and their homologs are described in, for example, Chylinksi, et al., RNA Biol. 2013 May 1; 10(5):726–737; Nat. Rev. Microbiol. 2011 June; 9(6):467-477; Hou, et al., Proc Natl Acad Sci USA. 2013 Sep 24; 110(39):15644-9; Sampson et al., Nature. 2013 May 9; 497(7448):254-7; and Jinek, et al., Science. 2012 Aug 17; 337(6096):816-21. Variants of any Cas9 nuclease provided herein can be optimized to provide efficient activity or enhanced stability in host cells. Therefore, engineered Cas9 nucleases are also contemplated. See, e.g., “Slaymaker et al., “Rationally engineered Cas9 nucleases with improved specificity,” Science 351(6268):84-88 (2016)).

[0086] The term "Cas9" as used herein refers to an RNA-mediated nuclease (e.g., from a bacterial or archaeal source, or derived therefrom). Exemplary RNA-mediated nucleases include the aforementioned Cas9 protein and its homologs. Other RNA-mediated nucleases include Cpf1 (see, e.g., Zetsche et al., Cell, Vol. 163, No. 3, p759-771, October 22, 2015) and its homologs. As used herein, terms such as "ribonucleoprotein" complexes refer to a complex between a targeting nuclease (e.g., Cas9) and crRNA (e.g., a guide RNA or a single guide RNA), a complex between a Cas9 protein and a trans-activated crRNA (tracrRNA), a complex between a Cas9 protein and a guide RNA, or a combination thereof (e.g., a complex comprising a Cas9 protein, a tracrRNA, or a crRNA guide RNA). It should be understood that in any embodiment described herein, the Cas9 nuclease can be replaced with a Cpf1 nuclease or any other guide nuclease.

[0087] As used herein, the phrase "modification" refers to inducing structural changes in the genomic sequence at the target genomic region in the context of modifying the genome of the cell. For example, the modification can take the form of inserting a nucleotide sequence into the genome of the cell. For example, a nucleotide sequence encoding one or more polypeptides can be inserted into the genomic sequence of the T cell TCR locus. As used throughout, a "TCR locus" is the location in the genome where the gene encoding the TCR alpha subunit, TCR beta subunit, TCR gamma subunit or TCR delta subunit is located.

[0088] Such modifications can be performed, for example, by inducing double-strand breaks within the target genomic region, or pairs of single-stranded nicks located on opposite strands and flanking the target genomic region. Methods for inducing single-strand or double-strand breaks at or in the target genomic region include the use of a Cas9 nuclease domain or derivative thereof and a guide RNA or guide RNA pair directed to the target genomic region.

[0089] As used herein, in the context of introducing a nucleic acid or a complex comprising a nucleic acid, such as an RNP-DNA template complex, the phrase "introducing" refers to the translocation of a nucleic acid sequence or RNP-DNA template complex from outside the cell to inside the cell. In some cases, introducing refers to the translocation of a nucleic acid or complex from outside the cell to the nucleus. The various methods of this type of translocation are contemplated, including, but not limited to, electroporation, contact with nanowires or nanotubes, receptor-mediated internalization, translocation via cell-penetrating peptides, liposome-mediated translocation, etc.

[0090] As used herein, the term "selective marker" refers to a gene that allows selection of host cells, such as T cells, that contain the marker. Selective markers include, but are not limited to, fluorescent markers, luminescent markers, and drug-selective markers, cell surface receptors, and the like. In some embodiments, the selection can be a positive selection; that is, cells expressing the marker are separated from the population, for example, to produce an enriched cell population expressing the selective marker. Separation can be performed by any conventional separation technique suitable for the selective marker used. For example, if a fluorescent marker is used, cells can be separated by fluorescence-activated cell sorting, whereas if a cell surface marker has been inserted, cells can be separated from a heterogeneous population by affinity separation techniques, for example, magnetic separation, affinity chromatography, panning using an affinity reagent attached to a solid matrix, fluorescence-activated cell sorting, or other conventional techniques.

[0091] As used herein, "cell" may be a human T cell or a cell capable of differentiating into a T cell, for example, a T cell expressing a TCR receptor molecule. These include hematopoietic stem cells and cells derived from hematopoietic stem cells.

[0092] As used herein, the phrase "hematopoietic stem cell" refers to a type of stem cell that can produce blood cells. Hematopoietic stem cells can produce cells of the myeloid or lymphoid lineages, or a combination thereof. Hematopoietic stem cells are primarily found in the bone marrow, although they can be isolated from peripheral blood or a portion thereof. Various cell surface markers can be used to identify / recognize, sort, or purify hematopoietic stem cells. In some cases, hematopoietic stem cells are identified / recognized as c-kit + Helin - In some cases, human hematopoietic stem cells are identified as CD34 + , CD59 + , Thy1 / CD90 + , CD38 lo / - , C-kit / CD117 + ,lin - In some cases, human hematopoietic stem cells are identified as CD34 - , CD59 + , Thy1 / CD90 + , CD38 lo / - , C-kit / CD117 + ,lin - In some cases, human hematopoietic stem cells are identified as CD133 + , CD59 + , Thy1 / CD90 + , CD38 lo / - , C-kit / CD117 + ,lin- In some cases, mouse hematopoietic stem cells are identified / recognized as CD34 lo / - , SCA-1 + , Thy1 + / lo , CD38 + , C-kit + ,lin - In some cases, the hematopoietic stem cell is CD150 + CD48 - CD244 - .

[0093] As used herein, the phrase "hematopoietic cell" refers to a cell derived from a hematopoietic stem cell. Hematopoietic cells can be obtained or provided by separation from an organism, system, organ or tissue (e.g., blood or a portion thereof). Alternatively, hematopoietic stem cells can be separated, and hematopoietic cells can be obtained or provided by differentiation of stem cells. Hematopoietic cells include cells that are differentiated into other cell types with limited potential. Such hematopoietic cells include, but are not limited to, multipotent progenitor cells, lineage-restricted progenitor cells, conventional myeloid progenitor cells, granulocyte-macrophage progenitor cells or megakaryocyte-erythrocyte progenitor cells. Hematopoietic cells include cells of lymphatic and myeloid lineages, such as lymphocytes, erythrocytes, granulocytes, monocytes and platelets. In some embodiments, hematopoietic cells are immune cells, such as T cells, B cells, macrophages, natural killer (NK) cells or dendritic cells. In some embodiments, cells are innate immune cells.

[0094] As used herein, the phrase "T cell" refers to a lymphocyte that expresses a T cell receptor molecule. T cells include human αβT cells and human γδT cells. T cells include, but are not limited to, naive T cells, stimulatory T cells, primary T cells (e.g., uncultured T cells), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or subsets thereof. T cells may be CD4 + , CD8 + or CD4 + and CD8 + . T cells can also be CD4 - , CD8 - or CD4 - and CD8 -. The T cells may be helper cells, e.g., T H 1. T H 2. T H 3. T H 9. T H 17 or T FH The T cell may be a cytotoxic T cell. The regulatory T cell may be a FOXP3 +or FOXP3 - The T cell can be an α / β T cell or a γ / δ T cell. In some cases, the T cell is a CD4 + CD25 高 CD127 低 In some cases, the T cell is a regulatory T cell selected from the group consisting of type 1 regulatory (Tr1), T H 3. CD8+CD28-, Treg17 and Qa-1 restricted T cells or a combination or subset thereof. In some cases, the T cells are FOXP3 + In some cases, the T cell is a CD4 + CD25 低 CD127 高 Effector T cells. In some cases, the T cells are CD4 + CD25 低 CD127 高 CD45RA 高 CD45RO - Naive T cells. T cells may be genetically manipulated recombinant T cells.

[0095] As used herein, the phrase "primary" refers to cells that have not yet been transformed or immortalized in the context of primary cells. Such primary cells can be cultured, subcultured or passed down for a limited number of times (e.g., cultured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 times). In some cases, primary cells can be adapted to in vitro culture conditions. In some cases, primary cells are separated from organisms, systems, organs or tissues, optionally, sorted, and used directly in the absence of culture or subculture. In some cases, primary cells can be stimulated, activated or differentiated. For example, primary T cells can be activated by contacting with CD3, CD28 agonists, IL-2, IFN-γ or a combination thereof (e.g., cultured in the presence thereof).

[0096] "Treatment" refers to various manifestations of successful treatment or alleviation or prevention of a disease, condition or disorder, including various objective or subjective parameters, such as reduction, regression, elimination of symptoms or making the condition more tolerable to the patient, slowing the rate of regression or decline, or making the regression endpoint less debilitating.

[0097] The term "homologous directed repair" or HDR as used herein refers to a cellular process in which the cut or nicked end of a DNA strand is repaired by polymerization of a homologous template nucleic acid. Thus, the original sequence is replaced by the sequence of the template. In some cases, an exogenous template nucleic acid, such as a DNA template, can be introduced to obtain a specific HDR-induced sequence change at the target site. In this way, a specific mutation can be introduced at the cleavage site, for example, a cleavage site produced by a targeted nuclease. The cell can use a single-stranded DNA template or a double-stranded DNA template as a template for editing or modifying the genome of the cell, for example, by HDR. Typically, the single-stranded DNA template or the double-stranded DNA template has at least one region homologous to the target site. In some cases, the single-stranded DNA template or the double-stranded DNA template has two homologous regions, such as a 5' end and a 3' end, which flanks the region of the DNA template containing the target cleavage or insertion site to be inserted.

[0098] The term "substantial identity" or "substantially identical" in the context of polynucleotide or polypeptide sequences refers to a sequence having at least 60% sequence identity to a reference sequence. Additionally, the percent identity can be any integer between 60% and 100%. Exemplary embodiments include at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% relative to a reference sequence using the programs described herein, preferably a reference sequence using BLAST with standard parameters as described below. It will be appreciated by those skilled in the art that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, reading frame positioning, etc.

[0099] For sequence comparison, generally a sequence is used as a reference sequence, and the test sequence is compared thereto. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, and if necessary, subsequence coordinates are specified, and sequence algorithm program parameters are specified. Default program parameters can be used, or other parameters can be specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters.

[0100] As used herein, a "comparison window" includes a reference to a segment of any one of a plurality of consecutive positions selected from 20 to 600, typically about 50 to about 200, more typically about 100 to about 150, wherein a sequence can be compared to a reference sequence of the same number of consecutive positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison are well known in the art. Optimal sequence alignment for comparison can be performed by the local homology algorithm of Smith and Waterman Add. APL. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48: 443 (1970), by the similarity search method of Pearson and Lipman Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988), by computer implementation of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0101] Suitable algorithms for determining percent sequence identity / identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, respectively, described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI) website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is called the neighboring word score threshold (Altschul et al., supra). These initial neighboring word hits are used as seeds to initiate searches in order to find longer HSPs containing them. The word hits are then extended in both directions along each sequence until the cumulative alignment score is improved. For nucleotide sequences, cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, cumulative scores are calculated using a scoring matrix. Extension of word hits in each direction is terminated when: the cumulative alignment score falls by X from its maximum achieved value; the cumulative score becomes zero or below due to the accumulation of one or more negative scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The defaults used by the BLASTN program (for nucleotide sequences) are as follows: word length (W) 28, expectation (E) 10, M=1, N=-2, and comparison of both chains. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0102] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which indicates the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, if the smallest sum probability when comparing a test nucleic acid to a reference nucleic acid is less than about 0.01, more preferably less than about 10 -5 , most preferably less than about 10 -20, then the nucleic acid is considered similar to the reference sequence. DETAILED DESCRIPTION

[0103] The following description lists various aspects and embodiments of the compositions and methods of the present invention. Any particular embodiment is not intended to limit the scope of the compositions and methods. Instead, the embodiments merely provide non-limiting examples of various compositions and methods at least included within the scope of the disclosed compositions and methods. This description should be read from the perspective of one of ordinary skill in the art; therefore, it does not necessarily include information well known to the skilled person.

[0104] The present disclosure relates to compositions and methods for modifying T cell genomes. The inventors have found that human T cells can be modified to change T cell specificity and function. In some embodiments, human T cells are modified to heterologously express one or more transcription factors to change T cell function, for example, to increase the tumor cell killing properties of T cells. In some embodiments, human T cells are modified to heterologously express two or more transcription factors (e.g., a combination of two or more transcription factors) to change T cell function, for example, to increase the tumor cell killing properties of T cells. In some embodiments, human T cells are modified to heterologously express a combination of transcription factors and switch receptors (i.e., a fusion protein or chimeric molecule that binds a ligand binding domain and a signal transduction domain) to change T cell function, for example, to increase the tumor cell killing properties of T cells.

[0105] Composition

[0106] Provided herein are human T cells heterologously expressing one or more polypeptides, wherein the one or more polypeptides are encoded by a nucleic acid construct inserted into a cell TCR locus. Any polypeptide described herein can be heterologously expressed in human T cells. In some examples, two or more, three or more, four or more, or five or more polypeptides described herein are heterologously expressed in human T cells. In some examples, one or more nucleic acid constructs encode one or more, two or more, three or more polypeptides, etc.

[0107] Exemplary polypeptides include, but are not limited to, the amino acid sequences shown in Tables 1 and 2, such as SEQ ID NOs: 30-58 and 69-85, 160, 161, and 162. Polypeptides comprising an amino acid sequence at least 80%, 85%, 90%, 99%, or 100% identical to any of SEQ ID NOs: 30-58 and 69-85, 160, 161, and 162 can also be expressed in human T cells. Other polypeptides that can be heterologously expressed include polypeptides comprising an amino acid sequence as shown in Table 3. Polypeptides comprising an amino acid sequence at least 80%, 85%, 90%, 99%, or 100% identical to any of the amino acid sequences shown in Table 3 can also be heterologously expressed in human T cells.

[0108] In some embodiments, the nucleic acid construct encoding two or more polypeptides is inserted into a human T cell. In some embodiments, the nucleic acid construct can include two or more nucleic acid sequences in a polycistronic construct. It should be understood that the two or more nucleic acid sequences encoding two or more polypeptides can be arranged in any order in the nucleic acid construct.

[0109] In some embodiments, the nucleic acid construct inserted into the human T cell encodes a TFAP polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:30, and a BATF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:31.

[0110] In some embodiments, the nucleic acid construct inserted into the human T cell encodes a BATF3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:32, and a TFAP4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:30.

[0111] In some embodiments, the nucleic acid construct inserted into the human T cells encodes a FOXJ2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:33, and a RARA polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:34.

[0112] In some embodiments, the nucleic acid construct inserted into the human T cell encodes an ID3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:35, and a TFAP4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:30.

[0113] In some embodiments, the nucleic acid construct inserted into the human T cell encodes a BATF3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:31, and an IRF2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:36.

[0114] In some embodiments, the nucleic acid construct inserted into the human T cells encodes a NANOG polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:37, and a TFAP4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:30.

[0115] In some embodiments, the nucleic acid construct inserted into the human T cells encodes a MAFF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:38, and a SATB1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:39.

[0116] In some embodiments, the nucleic acid construct inserted into the human T cell encodes a BATF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:32, and an IRF1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:36.

[0117] In some embodiments, the nucleic acid construct inserted into the human T cell encodes a TFAP4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:39, and a HOPX polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:51.

[0118] In some embodiments, the nucleic acid construct inserted into the human T cell encodes a TFAP4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:30, and a HOPX polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:42.

[0119] In some embodiments, the nucleic acid construct inserted into the human T cell encodes a BATF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:31, and a HES2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:43.

[0120] In some embodiments, the nucleic acid construct inserted into the human T cell encodes an ATF2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:47, and a BATF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:31.

[0121] In some embodiments, the nucleic acid construct inserted into human T cells encodes a BATF3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:32, and a SMAD4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:48.

[0122] In some embodiments, the nucleic acid construct inserted into the human T cells encodes a FOXP3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:49, and a RELA polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:50.

[0123] In some embodiments, the nucleic acid construct inserted into human T cells encodes an ID3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:35, and a BATF3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:32.

[0124] In some embodiments, the nucleic acid construct inserted into human T cells encodes a SMAD3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:46, and a BATF3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:32.

[0125] In some embodiments, the nucleic acid construct inserted into human T cells encodes an ID2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:44, and a TP73 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:45.

[0126] In some embodiments, the nucleic acid construct inserted into human T cells encodes a TFAP4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:30, and a TIM-3 / 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:69.

[0127] In some embodiments, the nucleic acid construct inserted into human T cells encodes a SOX5 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:51, and a TIM-3 / 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:69.

[0128] In some embodiments, the nucleic acid construct inserted into human T cells encodes a MYC polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:53, and a TGFbR2 / MyD88 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:70.

[0129] In some embodiments, the nucleic acid construct inserted into the human T cell encodes a BATF3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:32, and a 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:55.

[0130] In some embodiments, the nucleic acid construct inserted into human T cells encodes a TFAP4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:30, and a LTBR-IL-4R polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:71.

[0131] In some embodiments, the nucleic acid construct inserted into human T cells encodes an ATF1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:58, and an IL4RA / 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:72.

[0132] In some embodiments, the nucleic acid construct inserted into human T cells encodes a BATF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:31, and a 2B4 / 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:73.

[0133] In some embodiments, the nucleic acid construct inserted into human T cells encodes a RORC polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:79, and a VISTA / CD28 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:74.

[0134] In some embodiments, the nucleic acid construct inserted into the human T cells encodes a BATF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:31, and a CXCR1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:57.

[0135] In some embodiments, the nucleic acid construct inserted into human T cells encodes a MAFF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:38, and an IL-2RA polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:54.

[0136] In some embodiments, the nucleic acid construct inserted into human T cells encodes an ATF1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:58, and a TNFRSF12-OX40 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:75.

[0137] In some embodiments, the nucleic acid construct inserted into human T cells encodes a TIM-3 / 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:69.

[0138] In some embodiments, the nucleic acid construct inserted into human T cells encodes a BATF2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:80, and a truncated PD1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:81.

[0139] In some embodiments, the nucleic acid construct inserted into human T cells encodes a TCF7 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:82, and a LAT polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:83.

[0140] In some embodiments, the nucleic acid construct inserted into the human T cells encodes a STAT5 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:83, and an IL-2RA polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:54.

[0141] In some embodiments, the nucleic acid construct inserted into human T cells encodes a FoxJ2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:33, and a LTBR-CD28 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:76.

[0142] In some embodiments, the nucleic acid construct inserted into the human T cells encodes a FoxJ2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:33, and a LTBR-MyD88 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:77.

[0143] In some embodiments, the nucleic acid construct inserted into the human T cell encodes a HOPX polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:42, and an IL4RA / 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:72.

[0144] In some embodiments, the nucleic acid construct inserted into human T cells encodes an ID2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:44, and a BTLA / 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:85.

[0145] In some embodiments, the nucleic acid construct inserted into the human T cells encodes a SMAD1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:56, and a MCT4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:84.

[0146] In some embodiments, the nucleic acid construct inserted into the human T cells encodes a SMAD1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:56, and a LTBR-IL-4R polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:71.

[0147] In some embodiments, the nucleic acid construct inserted into the human T cell encodes a TIGIT polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 121, and an ICOS polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 159;

[0148] In some embodiments, the nucleic acid construct inserted into the human T cell encodes a LTBR polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 123, and a 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 146;

[0149] In some embodiments, the nucleic acid construct inserted into the human T cells encodes a DR5 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:133, and an ICOS polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:159.

[0150] In some embodiments, the polypeptide includes a full-length HES2 protein, a FOXJ2 protein, or a 41-BB protein.

[0151] One or more nucleic acid sequences described herein, such as one or more of SEQ ID NOs: 1-29 and 59-68, can be inserted into the TCR locus of T cells. Nucleic acid sequences encoding two or more polypeptides described herein can also be inserted into the TCR locus of T cells. In some embodiments, a nucleic acid sequence encoding one or more sequences selected from the group consisting of SEQ ID NOs: 30-SEQ ID NOs: 58, SEQ ID NOs: 69-85, SEQ ID NOs: 160, SEQ ID NOs: 161, and SEQ ID NOs: 162 is inserted into the TCR locus of T cells. In some embodiments, one or more nucleic acid sequences that are at least 80%, 85%, 90%, 99% or 100% identical to any one of the nucleic acid sequences shown in SEQ ID NOs: 1-29 and 59-68, or a nucleic acid sequence encoding two or more sequences selected from SEQ ID NO: 30-SEQ ID NO: 58, SEQ ID NOs: 69-85, SEQ ID NO: 160, SEQ ID NO: 161 and SEQ ID NO: 162 is inserted into the TCR locus of a T cell.

[0152] Any polypeptide sequence or combination of polypeptide sequences; nucleic acid sequence or combination of nucleic acid sequences; T cells comprising one or more polypeptides; T cells comprising one or more nucleic acid sequences; or methods of using the T cells, polypeptides, or nucleic acid sequences described herein may be claimed.

[0153] Inserting a heterologous coding sequence into the TCR locus means that the expression of one or more heterologous proteins will be controlled by an endogenous TCR promoter, and in some embodiments, will be expressed as part of a larger fusion protein with a TCR polypeptide, which is then cut to form a separate TCR and heterologous polypeptide. The TCR polypeptide can be endogenous, or it can be added to the TCR locus to provide a novel TCR affinity for T cells (for example, but not limited to cancer antigens). In some embodiments, the nucleic acid construct is inserted into the target insertion site in exon 1 of the TCR-α subunit constant gene (TRAC). In some embodiments, the nucleic acid construct is inserted into the target insertion site in exon 1 of the TCR-β subunit constant gene (TRBC), such as TRBC1 gene exon 1 or TRBC2 gene exon 1. After the nucleic acid construct is inserted into the TCR locus of the cell, the construct is controlled by an endogenous TCR promoter, such as a TRAC1 promoter or a TRBC promoter. As described below, the nucleic acid construct provided herein encodes a TCR or synthetic antigen receptor co-expressed with a polypeptide. Once the construct is incorporated into the genome of the T cell through HDR and is controlled by an endogenous promoter, the T cell can be cultured under conditions that allow the inserted construct to be transcribed into a single mRNA sequence encoding a fusion polypeptide, which is subsequently processed into a separate heterologous polypeptide (e.g., by cutting the peptide sequence of the connecting polypeptide). Insertion of any nucleic acid construct encoding a heterologous T cell receptor and one or more heterologous polypeptide components described herein will produce T cells with heterologous TCR receptor specificity and one or more heterologous polypeptide functions. In some embodiments, the T cell expresses an antigen-specific TCR that recognizes the target antigen. Similarly, insertion of any nucleic acid construct encoding a synthetic antigen receptor and one or more heterologous polypeptides described herein will produce T cells with synthetic antigen receptor specificity and heterologous polypeptide functions. In some embodiments, the T cell expresses a synthetic antigen receptor that recognizes the target antigen.

[0154] In some embodiments, the heterologous nucleic acid inserted into the human T cell encodes in the following order: (i) a first self-cleaving peptide sequence; (ii) a first heterologous TCR subunit chain, wherein the TCR subunit chain comprises a variable region and a constant region of a TCR subunit; (iii) a second self-cleaving peptide sequence; (iv) one or more heterologous polypeptides as described herein; (v) a third self-cleaving peptide sequence; (vi) a variable region of a second heterologous TCR subunit chain; and (vii) a portion of the N-terminus of an endogenous TCR subunit. , wherein, if the endogenous TCR subunit of the cell is a TCR-alpha (TCR-α) subunit, the first heterologous TCR subunit chain is a heterologous TCR-beta (TCR-β) subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-α subunit chain, and wherein if the endogenous TCR subunit of the cell is a TCR-β subunit, the first heterologous TCR subunit chain is a heterologous TCR-α subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-β subunit chain.

[0155] In some embodiments, the heterologous nucleic acid inserted into the human T cell encodes in the following order: (i) a first self-cleaving peptide sequence; (ii) one or more heterologous polypeptides as described herein; (iii) a second self-cleaving peptide sequence; (iv) a first heterologous TCR subunit chain, wherein the TCR subunit chain comprises a variable region and a constant region of a TCR subunit; (v) a third self-cleaving peptide sequence; (vi) a variable region of a second heterologous TCR subunit chain; and (vii) a portion of the N-terminus of an endogenous TCR subunit. , wherein, if the endogenous TCR subunit of the cell is a TCR-alpha (TCR-α) subunit, the first heterologous TCR subunit chain is a heterologous TCR-beta (TCR-β) subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-α subunit chain, and wherein if the endogenous TCR subunit of the cell is a TCR-β subunit, the first heterologous TCR subunit chain is a heterologous TCR-α subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-β subunit chain.

[0156] In the compositions and methods described herein, if the endogenous TCR subunit is a TCR-alpha (TCR-α) subunit, the first heterologous TCR subunit chain is a heterologous TCR-beta (TCR-β) subunit chain, and the second heterologous TCR subunit chain is a heterologous TCR-α subunit chain. In some methods, if the endogenous TCR subunit is a TCR-β subunit, the first heterologous TCR subunit chain is a heterologous TCR-α subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-β subunit chain.

[0157] As used throughout, the term "endogenous TCR subunit" is a TCR subunit that is endogenously expressed by the cell into which the nucleic acid construct is introduced, such as TCR-α or TCR-β. As described above, the nucleic acid constructs described herein encode multiple amino acid sequences that are expressed as polycistronic sequences that are processed (i.e., self-cleaved) to produce two or more amino acid sequences, such as TCR-α subunits, TCR-β subunits, and polypeptides encoded by the construct, or synthetic antigen receptors (e.g., CAR (see, e.g., Guedan et al. "Engineering and Design of Chimeric Antigen Receptors," Mol. Ther. Methods & Clinical Development 12: 145-156 (2019)) or SynNotch receptors (see, e.g., Cho et al. "Engineering Axl specific CAR and SynNotch receptor for cancer therapy," Nature Scientific Reports 8, Article No: 3846 (2018)) and polypeptides encoded by the construct.

[0158] In some nucleic acid constructs, the size of the nucleic acid encoding the N-terminal portion of the endogenous TCR subunit will depend on the number of nucleotides in the endogenous TRAC or TRBC nucleic acid sequence between the start of TRAC exon 1 or TRBC exon 1 and the targeted insertion site. For example, if the number of nucleotides between the start of TRAC exon 1 and the insertion site is less than or greater than 25 nucleotides, then the construct may have less than or greater than 25 nucleotides of nucleic acid encoding the N-terminal portion of the endogenous TCR-α subunit.

[0159] In the above example, translation of the mRNA sequence transcribed from the construct results in the expression of a protein that is self-cleaved into four or more independent polypeptide sequences, i.e., an inactive endogenous variable region peptide lacking a transmembrane domain, (e.g., it can be degraded or secreted in the endoplasmic reticulum after translation), a full-length heterologous antigen-specific TCR-β chain or TCR-α chain, one or more polypeptide sequences described herein, and a full-length heterologous antigen-specific TCR-α chain or TCR-β chain. The full-length antigen-specific TCR-β chain and the full-length antigen-specific TCR-α chain form a TCR with the desired antigen specificity. In some embodiments, the polypeptide enhances or confers one or more functions required by T cells. The mRNA transcribed from any other nucleic acid construct described herein is similarly processed in T cells.

[0160] In some embodiments, the nucleic acid construct encodes in the following order: (i) a first self-cleaving peptide sequence; (ii) a first heterologous TCR subunit chain, wherein the TCR subunit chain comprises a variable region and a constant region of a TCR subunit; (iii) a second self-cleaving peptide sequence; (iv) a second heterologous TCR subunit chain, wherein the TCR subunit chain comprises a variable region and a constant region of a TCR subunit; (v) a third self-cleaving peptide sequence; (vi) one or more heterologous polypeptides described herein; and (vii) a fourth self-cleaving peptide sequence. A peptide sequence or a poly A sequence, wherein if the endogenous TCR subunit is a TCR-alpha (TCR-α) subunit, the first heterologous TCR subunit chain is a heterologous TCR-beta (TCR-β) subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-α subunit chain, and wherein if the endogenous TCR subunit is a TCR-β subunit, the first heterologous TCR subunit chain is a heterologous TCR-α subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-β subunit chain.

[0161] In some embodiments, the nucleic acid construct encodes in the following order: (i) a first self-cleaving peptide sequence; (ii) a synthetic antigen receptor; (iii) a second self-cleaving peptide sequence; (iv) one or more heterologous polypeptides described herein; and (v) a third self-cleaving peptide sequence or a poly A sequence.

[0162] In some embodiments, the nucleic acid construct encodes in the following order: (i) a first self-cleaving peptide sequence; (ii) one or more heterologous polypeptides; (iii) a second self-cleaving peptide sequence; (iv) a synthetic antigen receptor; and (v) a third self-cleaving peptide sequence or a poly A sequence. It should be understood that in any nucleic acid construct described herein, one or more heterologous polypeptides can be separated by a self-cleaving peptide, so that the one or more polypeptides are self-cleaved into one or more separate polypeptides in human T cells, as described above.

[0163] Examples of self-cleaving peptides include, but are not limited to, self-cleaving viral 2A peptides, such as porcine Teschovirus-1 (P2A) peptides, T. punctatus virus (T2A) peptides, equine rhinitis virus A (E2A) peptides, or foot-and-mouth disease virus (F2A) peptides. Self-cleaving 2A peptides enable the expression of multiple gene products from a single construct. (See, e.g., Chng et al., "Cleavage efficient 2A peptides for high level monoclonal antibody expression in CHO cells," MAbs 7(2): 403-412 (2015)). In some embodiments, the nucleic acid construct comprises two or more self-cleaving peptides. In some embodiments, the two or more self-cleaving peptides are the same. In other embodiments, at least one of the two or more self-cleaving peptides is different.

[0164] In some embodiments, one or more linker sequences separate components of the nucleic acid construct. The length of the linker sequence can be 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids or longer.

[0165] In some embodiments, nucleic acid construct comprises a side joint homology arm sequence with homology to human TCR locus. In compositions and methods as described herein, the length of one or two homology arm sequences is at least about 50, 100, 150, 200, 250, 300, 350, 400 or 450 nucleotides. In some cases, the nucleotide sequence homologous to the genomic sequence is at least 80%, 90%, 95%, 99% or 100% complementary to the genomic sequence. In some embodiments, one or two homology arm sequences optionally comprise a nucleotide sequence compared to the homologous sequence mismatch in the genomic sequence in the TCR locus of the flank insertion site in the TCR locus.

[0166] In some embodiments, the nucleic acid construct optionally encodes a selectable marker that can be used to separate or isolate a modified T cell subpopulation. In some embodiments, the nucleic acid construct optionally comprises one or more barcode sequences that indicate the identity of one or more polypeptides encoded by the construct.

[0167] Any polypeptide described herein can be encoded by any nucleic acid construct described herein. In some embodiments, the polypeptide sequence encoded by the heterologous nucleic acid construct is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 30-58 and 69-85. In some embodiments, the heterologous nucleic acid construct encodes two or more polypeptides, wherein each polypeptide is at least 95% identical to an amino acid sequence selected from SEQ ID NOs: 30-58 and 69-85.

[0168] Also provided are polypeptides that are at least 95% identical to SEQ ID NOs: 30-58 and 69-85. Also provided herein are nucleic acids encoding these polypeptides.

[0169] Also provided are human T cells comprising any of the nucleic acid sequences described herein. Also provided are a population (eg, a plurality) of human T cells comprising any of the nucleic acid sequences described herein.

[0170] Any nucleic acid construct encoding any polypeptide described herein can be used to prepare a modified T cell. In some embodiments, the method comprises: (a) introducing into a human T cell (i) a targeted nuclease that cuts a target region in a human T cell TCR locus to generate a target insertion site in the genome of the cell; and (ii) a nucleic acid construct encoding one or more polypeptides described herein, for example, a polypeptide comprising TFAP4 protein and BATF protein; a polypeptide comprising BATF3 protein and TFAP4 protein; a polypeptide comprising FOXJ2 protein and RARA protein; a polypeptide comprising ID3 protein and TFAP4 protein; a polypeptide comprising BATF protein and IRF2 protein; a polypeptide comprising NANOG protein and TFAP4 protein; a polypeptide comprising MAFF protein and SATB1 protein; a polypeptide comprising A polypeptide comprising a BATF protein and an IRF1 protein; a polypeptide comprising a TFAP4 protein and a HOPX protein; a polypeptide comprising a BATF protein and a HES2 protein; a polypeptide comprising an ATF2 protein and a BATF protein; a polypeptide comprising a BATF3 protein and a SMAD4 protein; a polypeptide comprising a FOXP3 protein and a RELA protein; a polypeptide comprising an ID3 protein and a BATF3 protein; a polypeptide comprising an EOMES protein; a polypeptide comprising a HES2 protein; a polypeptide comprising a FOXJ2 protein; a polypeptide comprising (a) a human TFAP4 protein, and (b) comprising about 1 to 15 ( A polypeptide comprising (a) a human SOX5 protein, and (b) a truncated TIGIT protein of about 1-10 (e.g., 7) amino acids comprising the human TIGIT extracellular domain or a portion thereof, the TIGIT transmembrane domain, and the human TIGIT intracellular domain; a polypeptide comprising (a) a human MYC protein, and (b) a polypeptide comprising the human TGFbR2 extracellular domain or a portion thereof connected to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g., 7) amino acids of the TGFbR2 intracellular domain) via a transmembrane domain; a polypeptide comprising a BATF3 protein and a 4-1BB protein Peptide; a polypeptide comprising (a) TFAP4 protein, and (b) a polypeptide comprising a human LTBR extracellular domain or a portion thereof connected to a human IL-4R intracellular domain (and optionally, 1-10 (e.g., 7) amino acids of the LTBR intracellular domain) via a transmembrane domain; a polypeptide comprising (a) ATF1 protein, and (b) a polypeptide comprising a human IL4RA extracellular domain or a portion thereof connected to a human 4-1BB intracellular domain; a polypeptide comprising (a) BATF protein, and (b) a polypeptide comprising a human 2B4 extracellular domain or a portion thereof connected to a human 4-1BB intracellular domain, and optionally, about 1-15 (e.g., 12) amino acids of a human 4-1BB extracellular domain;A polypeptide comprising (a) a RORC protein, and (b) a polypeptide comprising an extracellular domain of VISTA or a portion thereof connected to a human CD28 intracellular domain, and optionally about 1-15 (e.g., 12) amino acids of a human CD28 extracellular domain; a polypeptide comprising a BATF protein and a CXCR 1 protein; a polypeptide comprising a MAFF protein and an IL2RA protein; a polypeptide comprising (a) an ATF1 protein, and (b) a polypeptide comprising an extracellular domain of human TNFRSF12 or a portion thereof connected to a human OX40 intracellular domain (and optionally, 1-10 (e.g., 7) amino acids of a TNFRSF12 intracellular domain) through a transmembrane domain; a polypeptide comprising an extracellular domain of human TIM3 or a portion thereof connected to a human 4-1BB intracellular domain (and optionally about 1-15 (e.g., 12) amino acids of a human 4-1BB extracellular domain) through a transmembrane domain; a polypeptide comprising a truncated PD- 1 connected to the human BATF2 extracellular domain or a portion thereof, the truncated PD-1 comprises about 1-12 (e.g., 10) amino acids of the human PD-1 extracellular domain or a portion thereof, the PD-1 transmembrane domain and the human PD-1 intracellular domain; a polypeptide comprising a human TCF7 protein and a human LAT1 protein; a polypeptide comprising a human 4-1BB protein; a polypeptide comprising a human STAT5 protein and a human IL-2RA protein; a polypeptide comprising (a) a human FOXJ2 protein, and (b) comprising 1-10 (e.g., 7) amino acids of a human CD28 intracellular domain (and optionally, a LTBR intracellular domain) connected to the human FOXJ2 protein via a transmembrane domain. ) amino acids) linked to the extracellular domain of LTBR or a portion thereof; a polypeptide comprising (a) human FOXJ2 protein, and (b) a polypeptide comprising an extracellular domain of LTBR or a portion thereof linked to amino acids 41-142 of human MyD88 (and optionally, 1-10 (e.g., 7) amino acids of the intracellular domain of LTBR) via a transmembrane domain; a polypeptide comprising (a) human HOPX protein, and (b) a polypeptide comprising an extracellular domain of IL4RA or a portion thereof linked to the intracellular domain of human 41-BB via a transmembrane domain; a polypeptide comprising (a) ID2 protein, and (b) a polypeptide comprising an extracellular domain of IL4RA or a portion thereof linked to the intracellular domain of human 41-BB via a transmembrane domain. 1BB intracellular domain, and optionally about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain connected to the BLTA extracellular domain or a portion thereof; a polypeptide comprising a human SMAD1 protein and an MCT4 protein; or a polypeptide comprising (a) a human SMAD1 protein and (b) an LTBR extracellular domain or a portion thereof connected to the amino acids of the human IL-4R intracellular domain (and optionally, 1-10 (e.g., 7) amino acids of the LTBR intracellular domain) via a transmembrane domain; and (b) allowing recombination to occur, thereby inserting the nucleic acid construct into a target insertion site to produce modified human T cells. ;

[0171] In some embodiments, the nucleic acid is inserted into the T cell by introducing into the T cell: (a) a targeted nuclease that cuts a target region in exon 1 of a TCR-α subunit constant gene (TRAC) to generate an insertion site in the genome of the T cell; and (b) a nucleic acid construct, wherein the nucleic acid construct is incorporated into the insertion site by homology directed repair (HDR). In some embodiments, the nucleic acid construct is inserted into the T cell by introducing into the T cell: (a) a targeted nuclease that cuts a target region in exon 1 of a TCR-β subunit constant gene (TRAC) (e.g., TRBC1 or TRBC 2) to generate an insertion site in the T cell genome; and (b) a nucleic acid construct, wherein the nucleic acid construct is incorporated into the insertion site by homology directed repair (HDR).

[0172] In some embodiments, the nucleic acid construct is inserted by introducing a viral vector comprising the nucleic acid construct into the cell. Examples of viral vectors include, but are not limited to, adeno-associated virus (AAV) vectors, retroviral vectors, or lentiviral vectors. In some embodiments, the lentiviral vector is an integrase-deficient lentiviral vector.

[0173] In some embodiments, the nucleic acid construct is inserted by introducing a non-viral vector comprising the nucleic acid construct into a cell. In non-viral delivery methods, the nucleic acid can be naked DNA, or in a non-viral plasmid or vector. For non-viral delivery methods, a non-viral genome targeting scheme based on a Cas9 shuttle system and anionic polymers can be used to insert a DNA template. Transposon-based gene transfer can also be used. See, for example, Tipanee et al. "Preclinical and clinical advances in transposon-based gene therapy," Biosci Rep. 37(6): BSR20160614 (2017).

[0174] In some cases, the nucleic acid sequence is introduced into the cell as a linear DNA template. In some cases, the nucleic acid sequence is introduced into the cell as a double-stranded DNA template. In some cases, the DNA template is a single-stranded DNA template. In some cases, the single-stranded DNA template is a pure single-stranded DNA template. As used herein, reference to "pure single-stranded DNA" means that there is substantially no single-stranded DNA of other or opposite strands of DNA. Reference to "substantially no" means that one DNA strand of pure single-stranded DNA is at least 100 times less than another DNA strand. In some cases, the DNA template is a double-stranded or single-stranded plasmid or a mini-circle.

[0175] In some embodiments, the targeting nuclease is selected from an RNA-guided nuclease domain, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), and a megaTAL (see, e.g., Merkert and Martin, "Site-Specific Genome Engineering in Human Pluripotent Stem Cells," Int. J. Mol. Sci. 18(7): 1000 (2016)). In some embodiments, the RNA-guided nuclease is a Cas9 nuclease, and the method further comprises introducing into the cell a guide RNA that specifically hybridizes to a target region in the genome of the cell (e.g., a target region in exon 1 of a TRAC gene in a T cell). In other embodiments, the RNA-guided nuclease is a Cas9 nuclease, and the method further comprises introducing into the cell a guide RNA that specifically hybridizes to a target region in exon 1 of a TRBC gene.

[0176] As used throughout, a guide RNA (gRNA) sequence is a sequence that interacts with a site-specific or targeted nuclease and specifically binds or hybridizes with a target nucleic acid in a cell genome, so that the gRNA and the targeted nuclease are co-localized in the target nucleic acid in the cell genome. Each gRNA comprises a DNA targeting sequence or a prototype spacer sequence having a length of about 10 to 50 nucleotides, which specifically binds or hybridizes with a target DNA sequence in the genome. For example, the length of the DNA targeting sequence is about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. In some embodiments, the gRNA comprises a crRNA sequence and a trans-activating crRNA (tracrRNA) sequence. In some embodiments, the gRNA does not comprise a tracrRNA sequence.

[0177] Typically, the DNA targeting sequence is designed to be complementary (e.g., perfectly complementary) or substantially complementary to the target DNA sequence. In some cases, the DNA targeting sequence can incorporate wobble or degenerate bases to bind multiple genetic elements. In some cases, the 19 nucleotides at the 3' or 5' end of the binding region are perfectly complementary to one or more target genetic elements. In some cases, the binding region can be changed to increase stability. For example, non-natural nucleotides can be incorporated to increase the resistance of RNA to degradation. In some cases, the binding region can be changed or designed to avoid or reduce the formation of secondary structures in the binding region. In some cases, the binding region can be designed to optimize the GC content. In some cases, the GC content is preferably about 40% to about 60% (e.g., 40%, 45%, 50%, 55%, 60%).

[0178] In some embodiments, the Cas9 protein can be in an activated endonuclease form, so when combined with a target nucleic acid as part of a complex with a guide RNA or part of a complex with a DNA template, a double-strand break is introduced into the target nucleic acid. In the methods provided herein, a Cas9 polypeptide or a nucleic acid encoding a Cas9 polypeptide can be introduced into a cell. Double-strand breaks can be repaired with HDR to insert a DNA template into the genome of a cell. The methods described herein can utilize various Cas9 nucleases. For example, such a Cas9 nuclease can be utilized, and the Cas9 nuclease requires an NGG protospacer adjacent motif (PAM) immediately adjacent to the region 3' targeted by the guide RNA. Such Cas9 nucleases can target, for example, a region in TRAB exon 1 or TRAC exon 1 containing an NGG sequence. For another example, a Cas9 protein with an orthogonal PAM motif requirement can be used to target a sequence that does not have an adjacent NGG PAM sequence. Exemplary Cas9 proteins with orthogonal PAM sequence specificity include, but are not limited to, those described in Esvelt et al., Nature Methods 10: 1116–1121 (2013).

[0179] In some cases, Cas9 protein is a nickase, so when combined with the target nucleic acid as part of the complex with the guide RNA, a single-strand break or nick is introduced into the target nucleic acid. A pair of Cas9 nickases each combining different guide RNAs can target 2 proximal sites in the target genome region, and therefore a pair of proximal single-strand breaks are introduced into the target genome region, for example, TRAC gene exon 1 or TRBC gene exon 1. The nickase pair can provide enhanced specificity because off-target effects are likely to cause a single nick, which is usually repaired without damage by a base excision repair mechanism. Exemplary Cas9 nickases include Cas9 nucleases with D10A or H840A mutations (see, for example, Ran et al. "Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity," Cell 154 (6): 1380-1389 (2013)).

[0180] In some embodiments, the Cas9 nuclease, guide RNA, and nucleic acid sequence are introduced into the cell as a ribonucleoprotein complex (RNP)-nucleic acid sequence (e.g., DNA template) complex, wherein the RNP-nucleic acid sequence complex comprises: (i) RNP, wherein the RNP comprises the Cas9 nuclease and the guide RNA; and (ii) the nucleic acid sequence or construct.

[0181] In some embodiments, the molar ratio of RNP to DNA template can be about 3: 1 to about 100: 1. For example, the molar ratio can be about 5: 1 to 10: 1, about 5: 1 to about 15: 1, 5: 1 to about 20: 1; 5: 1 to about 25: 1; about 8: 1 to about 12: 1; about 8: 1 to about 15: 1, about 8: 1 to about 20: 1, or about 8: 1 to about 25: 1.

[0182] In some embodiments, the concentration of the DNA template in the RNP-DNA template complex is about 2.5 pM to about 25 pM. In some embodiments, the amount of the DNA template is about 1 μg to about 10 μg.

[0183] In some embodiments, the RNP-DNA template complex is formed by incubating the DNA template with the RNP for less than about 1 minute to 30 minutes at a temperature of about 20° C. to about 25° C. In some embodiments, the RNP-DNA template complex and the cell are mixed before introducing the RNP-DNA template complex into the cell.

[0184] In some embodiments, nucleic acid sequence or RNP-DNA template complex is introduced into cell by electroporation. Methods, compositions and devices for electroporating cells to import RNP-DNA template complex can include those described in the embodiments herein. Other or additional methods, compositions and devices for electroporating cells to import RNP-DNA template complex can include those described in WO / 2006 / 001614 or Kim, JA et al. Biosens.Bioelectron.23, 1353–1360 (2008). Other or additional methods, compositions and devices for electroporating cells to import RNP-DNA template complex can include those described in U.S. Patent Application Publication No. 2006 / 0094095; 2005 / 0064596; or 2006 / 0087522. Other or additional methods, compositions and devices for electroporating cells to introduce RNP-DNA template complexes may include those described in Li, LH et al. Cancer Res. Treat. 1, 341-350 (2002); U.S. Pat. Nos. 6,773,669; 7,186,559; 7,771,984; 7,991,559; 6485961; 7029916; and U.S. Patent Application Publication Nos. 2014 / 0017213 and 2012 / 0088842. Other or additional methods, compositions and devices for electroporating cells to introduce RNP-DNA template complexes may include those described in Geng, T. et al. J. Control Release 144, 91-100 (2010); and Wang, J., et al. Lab. Chip 10, 2057-2061 (2010).

[0185] In some embodiments, RNP is delivered to the cell in the presence of anionic polymer. In some embodiments, anionic polymer is anionic polypeptide or anionic polysaccharide. In some embodiments, anionic polymer is anionic polypeptide (such as polyglutamic acid (PGA), polyaspartic acid or polycarboxyglutamic acid). In some embodiments, anionic polymer is anionic polysaccharide (such as hyaluronic acid (HA), heparin, heparin sulfate or glycosaminoglycan). In some embodiments, anionic polymer is poly (acrylic acid) (PAA), poly (methacrylic acid) (PMAA), poly (styrene sulfonic acid) or polyphosphate. In some embodiments, the molecular weight of anionic polymer is at least 15kDa (such as between 15kDa and 50kDa). In some embodiments, the molar ratio of anionic polymer to Cas protein is between 10:1 and 120:1 (e.g., 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 110:1, or 120:1), respectively. In some embodiments of this aspect, the molar ratio of sgRNA:Cas protein is between 0.25:1 and 4:1 (e.g., 0.25:1, 0.5:1, 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1, 2.2:1, 2.4:1, 2.6:1, 2.8:1, 3:1, 3.2:1, 3.4:1, 3.6:1, 3.8:1, or 4:1).

[0186] In some embodiments, the donor template comprises a homology-directed repair (HDR) template and one or more DNA-binding protein target sequences. In some embodiments, the donor template has a DNA-binding protein target sequence and one or more protospacer adjacent motifs (PAMs). A complex containing a DNA-binding protein (e.g., an RNA-guided nuclease), a donor gRNA, and a donor template can shuttle the donor template to a desired intracellular location (e.g., a nucleus) without cutting the DNA-binding protein target sequence, so that the HDR template can be integrated into the cut target nucleic acid. In some embodiments, the DNA-binding protein target sequence and the PAM are located at the 5' end of the HDR template. In particular, in some embodiments, the PAM can be located at the 5' end of the DNA-binding protein target sequence. In other embodiments, the PAM can be located at the 3' end of the DNA-binding protein target sequence. In some embodiments, the DNA-binding protein target sequence and the PAM are located at the 3' end of the HDR template. In particular, in some embodiments, the PAM can be located at the 5' end of the DNA-binding protein target sequence. In other embodiments, the PAM is located at the 3' end of the DNA-binding protein target sequence. In some embodiments, the donor template has two DNA-binding protein target sequences and two PAMs. In particular, in some embodiments, the first DNA-binding protein target sequence and the first PAM are located at the 5' end of the HDR template, and the second DNA-binding protein target sequence and the second PAM are located at the 3' end of the HDR template. In some embodiments, the first PAM is located at the 5' end of the first DNA-binding protein target sequence, and the second PAM is located at the 5' end of the second DNA-binding protein target sequence. In other embodiments, the first PAM is located at the 5' end of the first DNA-binding protein target sequence, and the second PAM is located at the 3' end of the second DNA-binding protein target sequence. In some other embodiments, the first PAM is located at the 3' end of the first DNA-binding protein target sequence, and the second PAM is located at the 5' end of the second DNA-binding protein target sequence. In some other embodiments, the first PAM is located at the 3' end of the first DNA-binding protein target sequence, and the second PAM is located at the 3' end of the second DNA-binding protein target sequence.

[0187] In some embodiments, the nucleic acid sequence or RNP-DNA template complex is introduced into about 1×10 5 About 2×10 6 For example, a nucleic acid sequence or RNP-DNA template complex can be introduced into about 1×10 5 cells to about 5×10 5 cells, about 1×10 5 cells to about 1×10 6 cells, 1×10 5cells to about 1.5×10 6 cells, 1×10 5 cells to about 2×10 6 cells, about 1×10 6 cells to about 1.5×10 6 cells or about 1×10 6 cells to about 2×10 6 cells.

[0188] In the methods and compositions provided herein, the human T cells can be primary T cells. In some embodiments, the T cells are regulatory T cells, effector T cells, memory T cells, or naive T cells. In some embodiments, the effector T cells are CD8 + In some embodiments, the T cells are CD4+ cells. In some embodiments, the T cells are CD4 + CD8 + In some embodiments, the T cells are CD4 - CD8 - T cell. In some embodiments, the T cell is a T cell expressing a TCR receptor or differentiated into a T cell expressing a TCR receptor.

[0189] Treatment

[0190] Any of the methods and compositions described herein can be used to modify T cells obtained from a human subject. Any of the methods and compositions described herein can be used to modify T cells obtained from a human subject to enhance the immune response of the subject. Any of the methods and compositions described herein can be used to modify T cells obtained from a human subject to treat or prevent a disease (e.g., cancer, infectious disease, autoimmune disease, transplant rejection, graft-versus-host disease, or other inflammatory disorders in a subject).

[0191] As used herein, subject means an individual. A subject can be an adult subject or a pediatric subject. Pediatric subjects include subjects ranging in age from birth to eighteen years old.

[0192] Provided herein is a method for enhancing an immune response in a human subject, comprising administering any of the modified T cells described herein, i.e., T cells heterologously expressing a polypeptide described herein, e.g., a polypeptide comprising a TFAP4 protein and a BATF protein; a polypeptide comprising a BATF3 protein and a TFAP4 protein; a polypeptide comprising a FOXJ2 protein and a RARA protein; a polypeptide comprising an ID3 protein and a TFAP4 protein; a polypeptide comprising a BATF protein and an IRF2 protein; a polypeptide comprising a NANOG protein and a TFAP4 protein; a polypeptide comprising a MAFF protein and a SATB1 protein; a polypeptide comprising a BATF protein and an IRF1 protein; a polypeptide comprising a TFAP4 protein and a HOPX protein; a polypeptide comprising a BATF protein and a HES2 protein; a polypeptide comprising an ATF2 protein and a BATF protein; a polypeptide comprising a BATF3 protein and a SMAD4 protein; a polypeptide comprising a FOXP3 protein and a RELA protein; a polypeptide comprising an ID3 protein and a BATF3 protein; a polypeptide comprising an EOMES protein; a polypeptide comprising a HES2 protein; a polypeptide comprising a FOXJ2 protein;

[0193] A polypeptide comprising (a) a human TFAP4 protein, and (b) a polypeptide comprising a human TIM3 extracellular domain or a portion thereof connected to a human 4-1BB intracellular domain (and optionally about 1-15 (e.g., 12) amino acids of a human 4-1BB extracellular domain) via a transmembrane domain;

[0194] A polypeptide comprising (a) a human SOX5 protein, and (b) a truncated TIGIT protein comprising about 1-10 (e.g., 7) amino acids of a human TIGIT extracellular domain or a portion thereof, a TIGIT transmembrane domain, and a human TIGIT intracellular domain;

[0195] A polypeptide comprising (a) a human MYC protein, and (b) a polypeptide comprising the extracellular domain of human TGFbR2 or a portion thereof, connected to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g., 7) amino acids of the intracellular domain of TGFbR2) via a transmembrane domain;

[0196] A polypeptide comprising BATF3 protein and 4-1BB protein;

[0197] A polypeptide comprising (a) a TFAP4 protein, and (b) a polypeptide comprising a human LTBR extracellular domain or a portion thereof connected to a human IL-4R intracellular domain (and optionally, 1-10 (e.g., 7) amino acids of the LTBR intracellular domain) via a transmembrane domain;

[0198] A polypeptide comprising (a) an ATF1 protein, and (b) a polypeptide comprising an extracellular domain of human IL4RA or a portion thereof connected to an intracellular domain of human 4-1BB;

[0199] A polypeptide comprising (a) a BATF protein, and (b) a polypeptide comprising a human 2B4 extracellular domain or a portion thereof connected to a human 4-1BB intracellular domain, and optionally about 1-15 (e.g., 12) amino acids of a human 4-1BB extracellular domain;

[0200] A polypeptide comprising (a) a RORC protein, and (b) a polypeptide comprising a VISTA extracellular domain or a portion thereof connected to a human CD28 intracellular domain, and optionally about 1-15 (e.g., 12) amino acids of a human CD28 extracellular domain;

[0201] A polypeptide comprising BATF protein and CXCR 1 protein;

[0202] A polypeptide comprising a MAFF protein and an IL2RA protein;

[0203] A polypeptide comprising (a) an ATF1 protein, and (b) a polypeptide comprising a human TNFRSF12 extracellular domain or a portion thereof connected to a human OX40 intracellular domain (and optionally, 1-10 (e.g., 7) amino acids of a TNFRSF12 intracellular domain) via a transmembrane domain;

[0204] A polypeptide comprising a human TIM3 extracellular domain or a portion thereof connected to the intracellular domain of human 4-1BB (and optionally about 1-15 (e.g., 12) amino acids of the extracellular domain of human 4-1BB) via a transmembrane domain;

[0205] A polypeptide comprising a human BATF2 extracellular domain or a portion thereof linked to a truncated PD-1, wherein the truncated PD-1 comprises about 1-12 (e.g., 10) amino acids of the human PD-1 extracellular domain or a portion thereof, a PD-1 transmembrane domain, and a human PD-1 intracellular domain;

[0206] A polypeptide comprising human TCF7 protein and human LAT1 protein;

[0207] A polypeptide comprising a human 4-1BB protein;

[0208] A polypeptide comprising human STAT5 protein and human IL-2RA protein;

[0209] A polypeptide comprising (a) a human FOXJ2 protein, and (b) a polypeptide comprising an extracellular domain of LTBR or a portion thereof connected to a human CD28 intracellular domain (and optionally, 1-10 (e.g., 7) amino acids of the LTBR intracellular domain) via a transmembrane domain;

[0210] A polypeptide comprising (a) a human FOXJ2 protein, and (b) a polypeptide comprising an extracellular domain of LTBR or a portion thereof connected to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g., 7) amino acids of an intracellular domain of LTBR) via a transmembrane domain;

[0211] A polypeptide comprising (a) a human HOPX protein, and (b) a polypeptide comprising an IL4RA extracellular domain or a portion thereof connected to an intracellular domain of human 41-BB via a transmembrane domain;

[0212] A polypeptide comprising (a) an ID2 protein, and (b) a polypeptide comprising a BLTA extracellular domain or a portion thereof connected to the intracellular domain of human 4-1BB, and optionally about 1-15 (e.g., 12) amino acids of the extracellular domain of human 4-1BB;

[0213] A polypeptide comprising human SMAD1 protein and MCT4 protein; or

[0214] A polypeptide comprising (a) a human SMAD1 protein and (b) an extracellular domain of LTBR or a portion thereof connected to amino acids of the intracellular domain of human IL-4R (and optionally, 1-10 (e.g., 7) amino acids of the intracellular domain of LTBR) via a transmembrane domain.

[0215] In some embodiments, T cells are obtained from a subject and modified using any of the methods provided herein to express an antigen-specific TCR or synthetic antigen receptor, and the modified T cells are then administered to the subject. In some embodiments, the subject suffers from cancer and the target antigen is a cancer-specific antigen. In some embodiments, the subject suffers from an autoimmune disorder and the antigen is an antigen associated with an autoimmune disorder. In some embodiments, the subject suffers from an infection and the target antigen is an antigen associated with an infection.

[0216] Also provided is a method for treating cancer in a human subject, comprising: a) obtaining T cells from a subject; b) modifying the T cells using any of the methods provided herein to express an antigen-specific TCR or a synthetic antigen receptor that recognizes a target antigen in a subject; and c) administering the modified T cells to a subject, wherein the human subject suffers from cancer and the target antigen is a cancer-specific antigen. As used throughout, the phrase "cancer-specific antigen" means an antigen that is unique to a cancer cell or an antigen that is more abundantly expressed in a cancer cell than in a non-cancerous cell. In some embodiments, the cancer-specific antigen is a tumor-specific antigen.

[0217] As used herein, cancer is a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or spread to other parts of the body through the bloodstream and lymphatic system. In some embodiments, cancer is a solid tumor. In some embodiments, cancer is a blood or blood cancer. Exemplary cancers include, but are not limited to: breast cancer, prostate cancer, ovarian cancer, glioblastoma, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, bladder cancer, endometrial cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia (e.g., acute myeloid leukemia), myeloma, lung cancer, etc. It should be understood that the methods provided herein can also be used to target circulating cancer cells, such as cells that solid tumors shed into the bloodstream of a subject.

[0218] In some embodiments, tumor infiltrating lymphocytes are a heterogeneous and cancer-specific population of T cells obtained from a subject with cancer and expanded ex vivo. The characteristics of the patient's cancer determine the customized set of cell modifications, and these modifications are applied to tumor infiltrating lymphocytes using any of the methods described herein.

[0219] Also provided herein is a method for treating an autoimmune disease, an allergic disease, or a transplant rejection in a human subject, comprising: a) obtaining T cells from a subject; b) modifying the T cells using any of the methods provided herein to express an antigen-specific TCR or a synthetic antigen receptor that recognizes a target antigen in a subject; and c) administering the modified T cells to a subject, wherein the human subject suffers from an autoimmune disorder and the target antigen is an antigen associated with the autoimmune disorder. In some embodiments, the T cells are regulatory T cells.

[0220] As used herein, an autoimmune disease is a disease in which the immune system cannot distinguish between the subject's own cells and foreign cells, resulting in the immune system mistakenly attacking healthy cells in the body. Examples of autoimmune disorders include, but are not limited to, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, systemic lupus erythematosus, Graves' disease, type 1 diabetes, Sjögren's syndrome, autoimmune thyroid disease, and celiac disease.

[0221] Also provided herein are methods of treating an infection in a human subject comprising: a) obtaining T cells from the subject; b) modifying the T cells using any of the methods provided herein to express an antigen-specific TCR or a synthetic antigen receptor that recognizes a target antigen in the subject; and c) administering the modified T cells to the subject, wherein the subject has an infection and the target antigen is an antigen associated with the infection in the subject.

[0222] In some embodiments, the T cells are autologous (ie, from the same subject that will receive the modified cells) or allogeneic (ie, from a subject different from the subject that will receive the modified cells). In some embodiments, the T cells are iPSC-derived T cells. See, for example, Nagano et al. Mol. Therapy Methods & Clinical Development 16: 126-135 (2020). Any treatment methods provided herein may also include expanding the T cell population before modifying the T cells. Any treatment methods provided herein may also include expanding the T cell population after the T cells are modified and before being administered to the subject.

[0223] Screening methods

[0224] Also provided are methods for identifying heterologous polypeptide combinations, for example, a combination of two or more transcription factors, or a combination of transcription factors and switch receptors, which polypeptides can change one or more properties of human T cells, such as enhancing the cell killing properties of human T cells or human T cell populations. A method for identifying targeted insertion in a cell genome is provided herein, the method comprising: (a) introducing into a cell population (i) a targeted nuclease that cuts a target region in the cell genome to produce a target insertion site; and (ii) a plurality of DNA templates having sequences that are different from each other, wherein each DNA template comprises: i. two or more heterologous coding or non-coding nucleic acid sequences; and ii. a unique barcode nucleotide sequence that identifies the identity of each heterologous coding or non-coding nucleic acid sequence; (b) allowing recombination to occur, thereby producing a modified cell population; (c) (i) isolating genomic DNA; or (ii) isolating mRNA from the cell and generating cDNA from the mRNA; and (d) sequencing the genomic DNA or cDNA to identify the DNA template inserted into the cell target insertion site. In some methods, the step of isolating mRNA further comprises digesting DNA from the cell. In some methods, a barcode sequence in genomic DNA or cDNA is sequenced to identify two or more heterologous coding or non-coding nucleic acid sequences.

[0225] In any of the screening methods described herein, cDNA can be generated from mRNA by reverse transcribing mRNA from cells using techniques known to those of skill in the art. Any of the screening methods described herein can also include amplifying the genomic DNA or cDNA, for example, prior to sequencing the genomic DNA or cDNA. In some methods, two or more heterologous coding or non-coding nucleic acid sequences are separated by a linker, and each heterologous coding or non-coding nucleic acid sequence contains a unique barcode sequence. In some methods, the barcode that identifies each heterologous coding or non-coding nucleic acid sequence is located between the heterologous coding or non-coding nucleic acid sequences, for example, between a first heterologous coding sequence and a second heterologous coding sequence. See, Examples Figure 1 A, wherein a nucleic acid sequence comprising a nucleic acid sequence encoding transcription factor 1 (TF1) and a first barcode identifying TF1 (3' BC TF1) is sequentially connected to a nucleic acid sequence comprising a second barcode identifying a transcription factor (5' BC TF2) and a nucleic acid sequence encoding transcription factor 2 (TF2).

[0226] In some methods, the DNA template includes two heterologous nucleic acid sequences encoding two transcription factors. In some methods, the transcription factors are the same. In some methods, the transcription factors are different. In some methods, the DNA template includes a heterologous nucleic acid sequence encoding a transcription factor and a heterologous nucleic acid sequence encoding a switch receptor.

[0227] In some embodiments, the method further comprises determining the relative number of cells having different DNA templates inserted at the target insertion site in the population. In some embodiments, the method further comprises applying selection pressure to the modified cell population. In some embodiments, the method further comprises comparing the relative number of cells having different DNA templates inserted at the target insertion site in the population before and after applying selection pressure to the cells. In some methods, the DNA template is inserted by introducing a viral vector comprising the DNA template into a T cell. In some methods, the population is a mammalian cell population. In some methods, the mammalian cell is a human cell. In some embodiments, the human cell is a human T cell. In some methods, the T cell is a regulatory T cell, an effector T cell, or a primary T cell. In some methods, the effector T cell is a CD8+T cell or a CD4+T cell. In some methods, the effector T cell is a CD8+CD4+T cell. In some methods, the cell is a primary cell. In some methods, the DNA template comprises a nucleic acid encoding two or more heterologous polypeptides.

[0228] The materials, compositions, and components disclosed herein can be used in the methods and compositions disclosed herein, can be used in conjunction with the methods and compositions disclosed herein, can be used to prepare the methods and compositions disclosed herein, or are products of the methods and compositions disclosed herein. The above and other materials are disclosed herein, and it should be understood that when disclosing combinations, subsets, interactions, groups, etc. of these materials, even if there is no explicit specific reference to each of the multiple individual or individual combinations and collective combinations and permutations of these compounds, each of these is specifically covered and specifically described herein. For example, if a method is disclosed and discussed, and multiple changes that can be implemented on one or more molecules in the method are discussed, then each and all possible combinations and permutations of the changes and the method are specifically covered, unless otherwise specified. Similarly, any subset or combination of these is also specifically considered and disclosed. This concept applies to all aspects of the present disclosure, including but not limited to the steps of the methods of the disclosed compositions. Thus, if there are multiple other steps that can be performed, it should be considered that each of these other steps can be performed in conjunction with any specific method step or combination of method steps of the disclosed method, and each of such combinations or combination subsets is specifically covered and considered disclosed. All publications cited herein and the relevant materials in which they are cited are specifically incorporated herein by reference in their entirety.

[0229] Embodiment 1

[0230] Isolation and culture of primary human T cells

[0231] T cell isolation was performed as previously described (Roth et al., Cell 181:728-744 (2020)). In brief, human T cells were isolated from leukocyte removal products (Stemcell, using samples collected by approved Stemcell IRB) using the EasySep Human T Cell Isolation Kit (Stemcell). The use of human materials was approved by the UCSF Human Research Committee (CHR#13-11950). T cells were cultured in X-VIVO 15 medium (Lonza) supplemented with 5% fetal bovine serum (FBS), 50 μM 2-mercaptoethanol (ThermoFisher) and 10 mM N-acetyl L-cysteine ​​(VWR). Prior to electroporation, T cells were stimulated at 1e6 cells / ml in medium containing 500U / ml IL-2 (Peprotech), 5ng / ml IL-7 (Thermo Fisher), 5ng / ml IL-15 (Thermo Fisher), and CTS (Cell Therapy Systems) CD3 / CD28 Dynabeads (Thermo Fisher, bead: cell ratio 1:1) for 48 hours. After nucleofection, T cells were cultured in X-VIVO 15 medium containing 500U / ml IL-2.

[0232] Generation of combinatorial libraries for pooled knock-ins

[0233] A combinatorial GD2CAR plasmid library was generated by pooled Gibson assembly of the GD2 CAR pUC19 backbone and inserts 1 and 2. The backbone contained the published GD2 CAR sequence (Lynn et al., 2019) with CD28 co-stimulation and mutations to increase tonic signaling in the IgG1 CH2 region (Watanabe et al., 2016). Inserts were PCR amplified from a pre-existing library (as described in International Patent Application Publication No. WO2022072932) using primers that removed the 5' barcode of the first insert and the 3' barcode of the second insert, and added a constant linker between the two combined inserts. Fig. 9The example plasmid map of the final construct is shown.For insert 1, the transcription factor library described in International Patent Application Publication No. WO2022072932 is used.For insert 2, a transcription factor library or a switch receptor (SR) library (as described in International Patent Application Publication No. WO2022072932) is used. The GD2 CAR skeleton, insert 1 pool, and insert 2 pool are assembled using NEBuilder HiFi DNA assembly master mix (New England Biolabs (NEB)). The assembled product is subjected to SPRI purification using Sera-MagSpeedBead, transformed into Endura electrocompetent cells (Lucigen) and prepared in large quantities (Zymo Research Company (ZymoResearch)) for further use. HDR template (HDRT) is produced as previously described (Roth et al., 2020). Briefly, the combined GD2 plasmid pool was used as a template for PCR amplification (KAPA HiFiHotStart Ready mixture, Roche) to generate double-stranded DNA templates. The template was purified by SPRI and eluted into H2O. In the validation phase, plasmids for arrayed knock-ins of different combinations were generated in a similar manner. A single construct (rather than a library) was used as a template for PCR to generate insert 1 and insert 2, which were then Gibson assembled with the GD2 pUC19 backbone. The GD2 CAR sequence was provided by Robbie Majzner and Crystal Mackall (Stanford University).

[0234] Cas9 RNP electroporation

[0235] Electroporation was performed as previously described (Roth et al., 2020). To produce ribonucleoproteins (RNPs), crRNA and tracrRNA (stock solution 160uM, both Dharmacon) were mixed by volume 1:1 and incubated at 37C for 30 minutes to anneal to form an 80μM guide RNA (gRNA) solution. Poly-L-glutamic acid (PGA, stock solution 125mg / ml, Sigma) was mixed with gRNA at a volume ratio of 0.8:1 and then complexed with Cas9-NLS (QB3Macrolab), with a final volume ratio of gRNA:PGA:Cas9 of 1:0.8:1. It was incubated at 37°C for 15 minutes to form a 14.3μM RNP solution. Prior to electroporation, RNP and HDRT were mixed with T cells (3.5ul RNP and 1ug=1uL of HDRT). The subject T cells were resuspended in electroporation buffer P3 (Lonsard) at 0.75e6 cells per 20 μl (per well) and transferred to a 96-well electroporation plate with 4.5 uL RNP / HDRT mixture per well. Pulse code EH115 was used. The cells were rescued in X-VIVO 15 without cytokines for 15 minutes and then cultured in X-VIVO 15 containing 500 U / ml IL-2.

[0236] Combinatorial pooled knock-in screening

[0237] On the second day after activation, primary human T cells were electroporated with the combinatorial library. On the fourth day after electroporation, the knock-in rate was analyzed by flow cytometry, and the input group was harvested and transferred to TRI reagent (Sigma). The cells were cultured in X-VIVO 15 with 500U / ml IL-2 for 12 days and split every 2-3 days. The output group was harvested on the 16th day after electroporation and transferred to TRI reagent (Sigma). RNA was isolated from input and output populations using Direct-zol RNA kit (Zymo Research Company) and reverse transcribed into cDNA using Maxima H Minus first-strand cDNA synthesis kit (Thermo Fisher Scientific). Sequencing libraries were generated by two PCRs. PCR1, 18 cycles were performed using KAPA HiFi HotStart Ready mixture (Roche). Primers were designed to add Illumina read 1 and read 2 sequences and included a forward primer that binds to the furin / GSGS region upstream of the two barcodes and a reverse primer that binds to the two barcodes downstream of the constant E2A region ( Fig. 9). Amplicons from PCR1 were purified by SPRI. For PCR2, 10 cycles were performed using NEB Next UltraIIQ5 polymerase (NEB) to attach P5 and P7 Illumina sequencing adapters. PCR2 products were purified by SPRI, and the normalized libraries of each sample were pooled and sequenced on NextSeq500 (Illumina). Barcode distribution was analyzed and the log2 fold change of barcode presentation in the output and input populations was calculated to detect changes in abundance.

[0238] Competition test

[0239] After array knock-in of different combination constructs, cells were sorted on the 7th day after electroporation, and competition assays were established on the 8th day after electroporation. T cells were cultured with control T cells at a 50 / 50 ratio in X-VIVO 15 containing 50U / ml IL-2. The cell ratio was confirmed by flow cytometry of the cell mixture, and the exact percentage of control T cells was determined at the baseline level (NGFR expression). Every two days, the changes in cell ratios were analyzed by flow cytometry, the cells were separated at a 1:2 ratio, and IL-2 was added to a final concentration of 50U / ml. The changes in cell ratios were standardized based on the percentage of the test day 0.

[0240] Activation markers and phenotypic analysis

[0241] On day 8 after electroporation, the expression of activation markers (CD25, CD69) was analyzed by flow cytometry. On day 14 after electroporation, the expression levels of CD62L / CD45RA were analyzed by flow cytometry.

[0242] Flow cytometry and fluorescence activated cell sorting (FACS)

[0243] For flow cytometry analysis, T cells (unless sorted) were first stained for CAR expression using a 1:100 dilution of anti-mouse IgG, F(ab')2 fragment-specific antibody (Jackson ImmunoResearch) at 4C for 10 minutes. The cells were washed and blocked with PBS / 2% mouse serum, and then stained with the remaining extracellular antibodies in flow buffer (PBS / 2% FCS). Cells were analyzed on an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA) or a BD LSRFortessa (BD Biosciences, San Jose, California, USA).

[0244] For FACS, cells were stained with anti-mouse IgG, F(ab')2 fragment-specific antibodies (Jackson ImmunoResearch) and anti-human TCRab (Biolegend), and CAR+ / TCR- cells were sorted on a BD FACSAria Fusion (BD Biosciences, San Jose, CA, USA).

[0245] RNA sequencing (RNA-seq)

[0246] Edited cells were sorted for CAR+ / TCR- expression on day 14. Part of the sorted population was stored in Tri reagent, and the other part was stimulated with GD2+Nalm-6 cells at an E:T ratio of 1:1. After 24 hours, the stimulated T cells were sorted for CAR+ / TCR- cells again and stored in Tri reagent. RNA was isolated using the Direct-zol RNA kit (Zymo Research). RNA was prepared for sequencing by the Functional Genomics Laboratory at the University of California, Berkeley as previously described (Cortez et al., 2020) and sequenced by the Vincent J. Coates Genomics Sequencing Laboratory at the University of California, Berkeley. Reads were mapped to the human reference transcriptome using Kallisto, and genes with zero counts in more than 80% of the samples were removed from the analysis. The DESeq2 R package was used for differential gene expression, and the fgsea package was used for gene set enrichment analysis (GSEA), with the MSigDB v7.2 feature gene set as the reference gene list.

[0247] In vitro killing assay

[0248] For IncuCyte assay, flat-bottomed 96-well plates were coated with 50ul of 0.01% poly-L-ornithine (PLO) solution (Sigma) for one hour. PLO was removed and the plates were dried for 30-60 minutes. 10,000 GFP+ / GD2+Nalm-6 per well were mixed with sorted T cells at various effector: target (E:T) ratios. The assay medium consisted of X-VIVO 15 as described above, supplemented with 500U / ml IL-2 and 1X glucose solution (Thermo Fisher Scientific). Cell counts (GFP+) were analyzed every six hours using the IncuCyte live cell analysis system (Essen BioScience, Ann Arbor, Michigan, USA).

[0249] In vivo mouse model

[0250] NOD / SCID / IL2Rg-null (NSG) mice were purchased from Jackson Laboratory. Female mice aged 8-12 weeks were used and mouse experiments were performed according to the ethical regulations of the approved IACUC protocol (UCSF). On day 0, mice were injected intravenously with 0.5e6Nalm-6 / Luciferase / GFP / GD2 cells. Three days later, edited human T cells (1e6 per mouse) were injected intravenously. One day before injection, the T cell TCR was depleted using the EasySep Human TCRα / β Depletion Kit (Stemcell) to avoid graft-versus-host disease in mice. The knock-in rate between groups was adjusted by adding TCR-negative T cells without CAR knock-in immediately before injection. For imaging, 200 uL (3 mg) D-luciferin potassium salt (Gold BioTechnology) was injected intraperitoneally and mice were imaged once / twice weekly using the IVIS Spectrum In Vivo Imaging System (PerkinElmer).

[0251] result

[0252] Analysis of single transcription factor overexpression in combination with T cell receptors (TCRs) or chimeric antigen receptors (CARs) raises the question of whether a combination of two transcription factors (TFs) or a transcription factor with a switch receptor / therapeutic construct (TC) can further improve T cell function and overcome exhaustion. A 10,404-member library of 100 transcription factors (plus two controls) was combined with 100 transcription factors (plus two controls) and combined with GD2 CAR. The GD2CAR pUC19-based backbone as well as the transcription factor library were PCR amplified using different primer pairs for insertion position 1 and insertion position 2 as described in International Patent Application Publication No. WO2022072932 ( Figure 1 A). The PCR of the two inserts was designed to remove the 5' barcode and constant 5' linker of the first construct and the 3' barcode and constant 3' linker of the second construct. By pooled Gibson assembly, a fusion region was created, which consisted of the 3' barcode of TF at position 1, the constant linker (Linker 2-Linker 1 junction) and the 5' barcode of TF at position 2. Homology-mediated repair templates (HDRTs) were generated from the plasmid library by PCR, and the library was non-virally knocked into the TCRα constant chain (TRAC) locus of primary human T cells. The fusion region between TF1 and TF2 was used to identify the abundance of different combination constructs as well as the orientation (Insert 1 vs. Insert 2) by amplicon sequencing ( Figure 1B). Amplicon sequencing of plasmid and cell pools 4 days after electroporation showed construct size-dependent representation / knock-in rates, but constructs with >99% representation were confirmed at both steps of the protocol ( Figure 1 C).

[0253] We tested which TFxTF combinations would enhance T cell expansion and survival in the context of chronic antigen stimulation (tonic signaling GD2 CAR). The abundance of >10,000 different TFxTF combinations was analyzed after 16 days in culture and compared to baseline abundance at day 4 post-electroporation. Statistical analysis of two independent donors showed that while most constructs were shed over time, the combination of TFAP4 and BATF significantly increased abundance in GD2 CAR T cells ( Figure 2 A). When we further analyzed constructs with the highest positive log2 fold change after 16 days compared to 4 days, we found that most of the best performing constructs consisted of either TFAP4 or BATF / BATF3 or a combination of both, suggesting that BATF(3) and TFAP4 are key transcription factors driving the improved fit of the model ( Figure 2 B).

[0254] Similarly, a second combinatorial library was generated in which the transcription factor was located at position 1 and the switch receptor / therapeutic construct (TC) (see International Patent Application Publication No. WO2022072932) was located at position 2, resulting in a 13,362-member TFxTC library ( Figure 3 A). Similarly, amplicon sequencing showed insert size-dependent representation / knock-in rates, but confirmed that the plasmid library ( Figure 3 B) and 4 days after electroporation ( Figure 3 C) More than 99% of the constructs are represented. When calculating the log2 fold change at day 16 versus day 4 after electroporation of the TFxTC library, we found a significant increase in abundance of the combined constructs TFAP4-TIM-3 / 4-1BB and SOX5-TIGITtrunc ( Figure 4 A). Similarly, we observed that many constructs with large fold changes in abundance had TFAP4 or BATF (3) as the first insertion position ( Figure 4 B). Likely because the GD2 CAR is a CD28-based co-stimulatory CAR, we found that the top performing hits had relatively more 4-1BB-based switch receptors than CD28-based switch receptors.

[0255] To validate our finding that the combination of BATF and TFAP4 appears to be beneficial in a tonic signaling model, we next performed single knock-ins of the GD2 CAR and BATF-TFAP4 combination as well as controls that overexpressed either one transcription factor alone (mCherry-TFAP and BATF-mCherry) or none (mCherry-NGFR). Combination constructs were co-cultured at a 50 / 50 ratio with constructs containing only one transcription factor and the abundance ( Figure 5 A). Adding TFAP4 to the construct appears to have a stronger effect than adding BATF, suggesting that most of the proliferative or anti-apoptotic effects are driven by TFAP4. Consistent with our observations with single knock-in constructs containing TFAP4, we found that CD25 expression levels were increased in the combination constructs containing TFAP4, while CD69 expression remained unchanged ( Figure 5 B). When analyzing the phenotype of GD2 CAR T cells 14 days after electroporation, we observed that control (mCherry-NGFR) and BATF-overexpressing (BATF-mCherry) T cells had a high percentage of terminally differentiated Temra cells, whereas the phenotype of TFAP4-overexpressing cells shifted to a stem cell-like memory stage with a significantly reduced percentage of Temra cells ( Figure 5 C+D).

[0256] To assess the transcriptome profile driven by overexpression of the BATF-TFAP4 construct, we sorted CAR+ / TCR- cells at day 14 post-electroporation and performed bulk RNA sequencing. Differential gene expression analysis showed that the most differentially expressed gene between BATF-TFAP4 and mCherry-NGFR conditions was TFAP4 (not shown, log2 FC 5.0, padj 6.03e-77). In addition, we observed that while BATF-TFAP4 shared many differentially expressed genes with the single knock-in controls (BATF-mCherry and mCherry-TFAP4), such as CCR3, CCR4, and CCR8, the combination of BATF and TFAP4 also produced unique differential expression of multiple genes highlighted in yellow ( Figure 6 A). Correlation analysis of log2 fold changes between each test condition and the control (mCherry-NGFR) showed that BATF-TFAP4 had the highest similarity to mCherry-TFAP4, followed by BATF-mCherry ( Figure 6B). BATF-TFAP4 has only a minor association with mCherry-JUN. Gene set enrichment analysis of BATF-TFAP4 and mCherry-NGFR highlights increased expression of genes involved in cell cycle regulation at day 14 after electroporation ( Figure 6 C). After stimulation with target cells, the expression of genes involved in glycolysis, oxidative phosphorylation, and fatty acid metabolism increased in BATF-TFAP4 cells, while the expression of genes involved in apoptosis decreased ( Figure 6 D).

[0257] We next analyzed whether the addition of TFAP4 and BATF could render GD2 CAR T cells more functional in cancer cell killing. Indeed, when co-cultured with GD2-positive target cells, the combination of BATF and TFAP4 performed best at multiple E:T ratios in an in vitro killing assay ( Figure 7 A+B). Finally, we incorporated the combined cells into the NOD / SCID / IL2Rg-null (NSG) xenograft mouse adoptive T cell transfer model ( Figure 8 A), and observed that while overexpression of TFAP4 alone appeared to be insufficient in one of the donors, the combination of TFAP4 and BATF showed the greatest potential to control leukemia growth ( Figure 8 B).

[0258] Example II

[0259] We examined whether specific combinations of TFs synergize to enhance T cell fitness in the context of tonic CAR signaling. Analysis of combinations of 100 different transcription factors requires I) knock-in library sizes (~10,000 members) that have not been tested in this setting, and II) very large constructs to successfully knock-in, especially when used in combination with CAR (average construct size ~5.5 kb plus homology arms), and therefore cannot be easily implemented using AAV (adeno-associated virus) HDR templates due to viral packaging size limitations.

[0260] Modular Pooled Knock-in Screening (ModPoKI) molecular biology was used to overcome these challenges: the ModPoKI screening platform was adapted for large-scale combinatorial pooled knock-in screening ( Fig.10A and 11A). A library of approximately 10,000 members (100 transcription factors plus two controls combined with 100 transcription factors plus two controls) was created and cloned into the construct with the GD2 CAR. Briefly, to achieve this, we first PCR amplified the GD2 CAR pUC19-based backbone (specificity module) containing homology arms to target the human TRAC locus. Next, we created two transcription factor inserts from the existing TF library by PCR amplification using different primer pairs at position 1 than at position 2 within the functional module ( Fig.11 B). PCR of the two library inserts was designed to remove the 5' barcode and constant 5' linker of the first construct and the 3' barcode and constant 3' linker of the second construct. By pooled Gibson assembly, a DNA locus was created consisting of the 3' barcode of the TF at position 1, the constant linker (Linker 2-Linker 1 junction), and the 5' barcode of the TF at position 2, thereby creating a unique combinatorial barcode for each TFxTF combination. HDR templates were generated by PCR from the plasmid library and the library was non-virally knocked into the TRAC locus of primary human T cells. Notably, the expected knock-in templates spanned a large size range from ~3.3 to ~8.2 kb (without homology arms). The fusion region between TF1 and TF2 was used as a barcode combination to identify the abundance of different combinatorial constructs as well as the orientation (TF1 vs. TF2) by amplicon sequencing ( Fig.11 C). Amplicon sequencing of plasmid pools and ModPoKIT cell pools 4 days after electroporation confirmed >99% construct representation at both steps of the protocol, despite the expected effect of construct size on library representation / knock-in rate ( Fig.10 BC). We were thus able to generate pooled libraries with thousands of different members and successfully achieved barcode sequencing-based knock-ins of various types, including constructs as large as 7.6 kb.

[0261] We used combinatorial ModPoKI to test which TFxTF constructs would enhance T cell fitness in the context of tonic CAR signaling. ModPoKI cells expanded in culture due to tonic signaling with the GD2 CAR. We compared the abundance of each TFxTF combinatorial construct after 16 days in culture to the baseline abundance in the ModPoKI T cell population at day 4 post-electroporation. Most TFxTF combinations were depleted from the pool over time, consistent with our previous evidence that major transcriptional changes can be detrimental to fitness ( Fig.12AB). Analysis of constructs with the greatest increase in relative abundance (log2 fold change) highlighted that several of the best performing constructs included combinations of TFAP4 and BATF (or BATF3), suggesting that TFAP4 and BATF (3) are key transcription factors that synergistically drive increased T cell fitness during repeated simulations ( Fig.10 D and 11E). Screening analysis of cells from two human donors found that the abundance of the TFAP4 and BATF combination construct was increased most significantly in two different barcode combinations / knock-in orientations (TFAP4-BATF and BATF-TFAP4). Fig.10 D). Taken together, these data demonstrate that the use of the ModPoKI screening platform enables large-scale combinatorial knock-in screening of ~10,000 different constructs (average knock-in size ~5.5 kb) and facilitates the creation of a map of combinatorial KI constructs with enhanced therapeutic T cell potential.

[0262] Combination TFAP4 and BATF KI induces a favorable state in therapeutic T cells

[0263] To validate and characterize the benefits of KI constructs with a combination of BATF and TFAP4, we next generated specific knock-in constructs with GD2CAR, along with: 1) BATF+TFAP4 combination, 2) single TF+control (RFP-TFAP and BATF-RFP), or 3) control+control (RFP-tNGFR). We performed a competitive fitness assay to assess whether combination KIs outperform single TF KIs. KI cells with BATF+TFAP4 combination constructs were co-cultured with KI cells with constructs containing only a single TF (along with a control gene) at a ratio of approximately 50 / 50, and the relative abundance ( Fig.12 A). KI cells with BATF+TFAP4 combination constructs outcompeted TFAP4+control KI cells and BATF+control KI cells. The relative benefit of the BATF+TFAP4 combination was stronger than that of BATF alone and TFAP4 alone, suggesting that most, though not all, of the fitness benefit was conferred by TFAP4 KI. Consistent with the effect of a single TFAP4 KI construct, we found increased CD25 expression levels in the combination construct containing TFAP4, while CD69 levels were not significantly affected ( Fig.12B). When analyzing the phenotype of GD2 CAR T cells 14 days after electroporation, we observed that control (RFP-tNGFR) and BATF KI (BATF-RFP) T cells had a high percentage of terminally differentiated Temra cells (CD62L- / CD45RA+), whereas the phenotype of TFAP4 KI cells (both TFAP4+ control and TFAP4+BATF KI) shifted toward a stem cell-like and central memory state, with a significant decrease in the percentage of Temra cells ( Fig.12 CD).

[0264] We next assessed the transcriptional effects of the BATF-TFAP4 combo construct compared to individual TF constructs and control constructs. We sorted GD2CAR+ / TCR- cells from each KI population at day 14 post-electroporation and performed RNA sequencing without the addition of target cells (tonic signaling only). Correlation analysis of the log2 fold change between each tested condition and the control (RFP-tNGFR) showed that BATF-TFAP4 KI cells were more similar to RFP-TFAP4 KI cells than to BATF-RFP KI cells ( Fig.13 AB). In this case, BATF-TFAP4 KI cells were less related to RFP-JUN KI cells, suggesting that these TFs promote a transcriptional program that is distinct from that promoted by JUN overexpression as previously reported. Several genes, including CCR3, CCR4, and CCR8, were induced by BATF+TFAP4 KI, BATF-RFP KI, and RFP-TFAP4 KI relative to control KI cells. However, the combined BATF and TFAP4 KI also promoted differential expression of multiple genes highlighted in yellow that were not differentially affected by BATF or TFAP4 KI alone ( Fig.12 E), such as TP63 (tumor protein 63), GNLY (granulysin), TNFRSF11A (tumor necrosis factor receptor superfamily member 11A), and CLECL1 (encoding T-cell co-stimulatory protein C-type lectin-like domain family 1). Gene set enrichment analysis of BATF-TFAP4 versus RFP-tNGFR highlighted an increase in the expression of genes involved in cell cycle control (e.g., E2F targets and G2M checkpoint genes) at day 14 post-electroporation, while interestingly, genes involved in the P53 pathway and IFN-g response appeared to be decreased ( Fig.13 C, left panel). After stimulation with target cells, the expression of genes involved in glycolysis, oxidative phosphorylation, and fatty acid metabolism increased in BATF-TFAP4 cells ( Fig.13 C, middle and right images and Fig.13DE (compared with single KI). Together, these results suggest that combined knock-in of BATF and TFAP4 can drive overlapping but distinct transcriptional changes compared with single BATF or TFAP4 KIs, thereby promoting a fitness advantage in the GD2 CAR tonic signaling model.

[0265] The polycistronic TFAP4 single KI construct enhanced the cancer killing capacity of GD2 TRAC CAR T cells in vitro and in vivo. We now wanted to evaluate whether the combined TFAP4 and BATF KI constructs could further enhance the anticancer function of GD2 TRAC CAR T cells, as this combination confers additional T cell fitness benefits. Indeed, when co-cultured with GD2-positive cancer target cells, BATF+TFAP4 KI GD2 TRAC CAR T cells performed best in in vitro killing assays at multiple E:T ratios ( Fig.12 F and 14A). Finally, we tested the combination KI cells in an in vivo NSG xenograft mouse adoptive T cell transfer model ( Fig.12 G and Fig.14 B), and observed that KI CAR T cells with the TFAP4+BATF combo construct exhibited the best ability to control leukemia growth compared with CAR T cells with either a single TF KI construct or a control KI construct. In conclusion, combinatorial modular pooled knock-in screening of thousands of different synthetic gene constructs can inform the design of combinatorial genetic programs to enhance persistence and function to improve adoptive T cell therapies.

[0266] Example III

[0267] Generate a plasmid library for pooling single construct knock-ins

[0268] The 231 constructs included in the pooled knock-in library (see, e.g., Blaeschke et al., “Modular pooled discovery of synthetic knock-in sequences to program durable cell therapies,” bioRxiv 2022.07.27.501186; doi: https: / / doi.org / 10.1101 / 2022.07.27.501186) were designed using the TwistBioscience codon optimization tool and commercially synthesized and cloned (TwistBioscience) into custom pUC19 plasmids containing the NY-ESO-1 TCR sequence (except for the HIF1A, JUN, and TCF7 constructs, which were cloned individually using gBlocks gene fragments (Integrated DNA Technologies)). Individual pooled plasmid libraries were created by pooling individual construct plasmids into the corresponding library (transcription factors, 100 members; surface receptor constructs, 129 members; controls, 2) or into a complete pool. The CD19 / 28z CAR plasmid pool was created by amplifying constructs from the TCR plasmid pool as DNA templates in a pooled assembly manner. PCR amplification (Kapa hot start polymerase, Roche) produced a pooled amplicon library with small overhangs homologous to the pUC19 plasmid containing the CD19 / 28z CAR HDR sequence. The amplicon pool was treated with DpnI restriction enzyme (New England Biolabs, NEB) to remove residual circular TCR plasmids, purified with beads (Sera-MagSpeedBead), and eluted into H2O. We then constructed a plasmid pool containing all 231 library members and knock-in controls, as well as new CAR sequences, using Gibson assembly (NEB). The CD19 / 28z CAR plasmid pool was bead purified, transformed into Endura electrocompetent cells (Lucigen), and prepared in large quantities (PlasmidPlus Midi or Maxi kit, Qiagen) for further use. The GD2 CAR single knock-in library was generated in a similar manner. While the NY-ESO-1 TCR library was pooled at the plasmid stage (plasmids were synthesized individually), all other plasmid libraries in this project (anti-CD19 / 28z CAR, anti-GD2 CAR, combinatorial library) were generated by Gibson assembly of pooled plasmids. The CD19 CAR sequence used in this study was kindly provided by Tobias Feuchtinger at von Hauner Children's Hospital, University Hospital Munich. The GD2 CAR sequence was kindly provided by Crystal Mackall and Robbie Majzner at Stanford University (Lynn et al., 2019).The assembled products were SPRI purified using Sera-Mag SpeedBead, transformed into Endura electrocompetent cells (Lucigen) and prepared in large quantities (Zymo Research) for further use. HDR templates (HDRT) were generated as previously described (Roth et al., 2020). In brief, single knock-in plasmid pools were used as templates for PCR amplification (KAPA HiFi HotStart Ready mixture, Roche) to generate double-stranded DNA templates. The templates were SPRI purified and eluted into H2O. The GD2 CAR sequence was provided by Robbie Majzner and Crystal Mackall (Stanford University).

[0269] Repeated stimulus screening

[0270] One day before the start of the repeated stimulation screening, A375 cells were counted and transferred to 24-well plates (25,000 cells / well in 1ml complete RPMI medium), assuming that they doubled within 24 hours. One day later, the edited T cell library was counted and 10e6 cells were frozen in TRI reagent (Sigma-Aldrich) for amplicon sequencing (input population). The culture medium of A375 cells was removed. 50,000 edited T cells (CAR positive, effector: target ratio of approximately 1: 1) were transferred to each well of a 24-well plate and co-cultured with A375 cells in 2ml of X-VIVO 15 containing supplement + 50U / ml IL-2. After 24 hours, fresh A375 cells were plated as described above. One day later, the medium of the new A375 plate was removed and replaced with 1 ml of fresh X-VIVO 15 plus 1 ml of T cell suspension from the first plate, including 50 U / ml IL-2 calculated based on the total volume of each well. The remaining T cells were counted and 10 e 6 cells were transferred to TRI reagent (Sigma Aldrich) for amplicon sequencing. This process was repeated every other day for a total of 5 target cell stimulations. Multiple wells of a 24-well plate were used for each screening to achieve cell coverage. When CD19 CAR was used in combination with A375 cells, A375 cells overexpressing CD19 were used (SFFV promoter knocked in endogenous CD19 upstream).

[0271] Single stimulus screening

[0272] The day before setting up the screening, add 2.5 eSix A375 were inoculated into each T75 flask in complete RPMI medium (RPMI+NEAA, glutamine, Hepes, Pen / Strep, sodium pyruvate (all from Thermo Fisher Scientific) and 10% FCS (Sigma-Aldrich, St. Louis, MO, USA)) assuming that they doubled within 24 hours. One day later (= 7 days after electroporation), the edited T cell pool was counted and washed once. 10 e 6 T cells were transferred to TRI reagent (Sigma Aldrich), representing the input population for amplicon sequencing. 10e6 T cells from each screening condition were transferred to a T75 flask containing 20ml X-VIVO 15 (Lonza Bioscience), supplemented with 5% FCS, 2-mercaptoethanol (Thermo Fisher Scientific), N-acetyl-L-cysteine ​​(VWR) and 50U / ml IL-2 (Proleukin). The cRPMI was removed and the flask was filled with 20ml X-VIVO 15 plus additives and 10e6 T cells. Two days later, 10ml of X-VIVO 15 was added to all conditions, including supplements and 50U / ml IL-2. After another two days, the cells were counted and 10e6 cells were transferred to TRI reagent (Sigma Aldrich) for RNA isolation and amplicon sequencing.

[0273] CD25 sorting and screening

[0274] For sorting screening of CD25 expression, T cells were electroporated with the GD2 CAR TF library and sorted for CD25 expression (pre-gated on CAR+ / TCR-) on day 8 after electroporation (approximately 20% CD25 expression at the top and bottom, 0.5M cells per box per donor). RNA was isolated from high CD25 and low CD25 populations using the Direct-zol RNA kit (Zymo Research) and reverse transcribed into cDNA using the Maxima H Minus first-strand cDNA synthesis kit (Thermo Fisher Scientific). Sequencing libraries were generated by two PCRs. PCR1 was performed using KAPA HiFi HotStart Ready mixture (Roche) for 18 cycles. Primers were designed to add Illumina read 1 and read 2 sequences upstream and downstream of the 3' barcode region. The amplicons from PCR1 were purified by SPRI. For PCR2, 10 cycles were performed using NEB Next Ultra IIQ5 polymerase (NEB) to attach P5 and P7 Illumina sequencing adapters. PCR2 products were purified by SPRI, and normalized libraries were pooled for each sample and sequenced on NextSeq500 (Illumina). The barcode distribution was analyzed and the log2 fold change of barcode representation in the high CD25 and low CD25 bins was calculated.

[0275] Since increased CD25 expression was observed on GD2 CAR T cells with TFAP4 knock-in, a screening for CD25 high vs. CD25 low expression was performed using the GD2 CAR single insert TF library on day 8 post electroporation. Data show representative normalized log2FC (mean + SEM) of inserts in high CD25 vs. low CD25 bins from 3 individual donors. Screening showed a trend toward increased CD25 expression on T cells with either TFAP4 or BATF knock-in ( Fig.15 ).

[0276] To complement the previous dataset, a single stimulation screen (addition of A375 / CD19 compared to non-stimulated conditions) and a repeated stimulation screen (addition of A375 / CD19 5 times) were performed in the complementation model using the CD19 / 28z CAR in combination with the full TF and SR single-insert libraries. The normalized Log2FC of the stimulated / unstimulated / re-stimulated conditions relative to the input is shown to assess changes in abundance. Indeed, a trend towards increased abundance of hits was observed in the model complemented with the model used for the combinatorial screen (GD2 CAR) (e.g., BATF2, IL2RA, TFAP4, BATF, LAT1, BATF3, CXCR1, MYC, ID3, ID2, IRF1, TIGIT / ICOS, LTBR / 4-1BB, DR5 / ICOS, CCR4) ( Fig.16 ).

[0277] All patents, patent applications, and other published references cited in this specification are hereby incorporated by reference in their entirety.

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371] Additional Sequence

[0372] RORC (SEQ ID NO:79)

[0373] MDRAPQRQHRASRELLAAKKTHTSQIEVIPCKICGDKSSGIHYGVITCEGCKGFFRRSQRCNAAYSCTRQQNCPIDRTSRNRCQHCRLQKCLALGMSRDAVKFGRMSKKQRDSLHAEVQKQLQQRQQQQQEPVVKTPPAGAQGADTLTYTLGLPDGQLPLGSSPDLPEASACPPGLLKASGSGPSYSNNLAKAGLNGASCHLEYSPERGKAEGRESFYSTGSQLTPDRCGLRFEEHRHPGLGELGQGPDSYGSPSFRSTPEAPYASLTEIEHLVQSVCKSYRETCQLRLEDLLRQRSNIFSREEVTGYQRKSMWEMWERCAHHLTEAIQYVVEFAKRLSGFMELCQNDQIVLLKAGAMEVVLVRMCRAYNADNRTVFFEGKYGGMELFRALGCSELISSIFDFSHSLSALHFSEDEIALYTALVLINAHRPGLQEKRKVEQLQYNLELAFHHHLCKTHRQSILAKLPPKGKLRSLCSQHVERLQIFQHLHPIVVQAAFPPLYKELFSTETESPVGLSK

[0374] BATF2 (SEQ ID NO:80)

[0375] MHLCGGNGLLTQTDPKEQQRQLKKQKNRAAAQRSRQKHTDKADALHQQHESLEKDNLALRKEIQSLQAELAWWSRTLHVHERLCPMDCASCSAPGLLGCWDQAEGLLGPGPQGQHGCREQLELFQTPGSCYPAQPLSPGPQPHDSPSLLQCPLPSLSLGPAVVAEPPVQLSPSPLLFASHTGSSLQGSSSKLSALQPSLTAQTAPPQPLELEHPTRGKLGSSPDNPSSALGLARLQSREHKPALSAATWQGLVVDPSPHPLLAFPLLSSAQVHF

[0376] PD_1 truncated (SEQ ID NO:81)

[0377] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIG

[0378] TCF7(SEQ ID NO:82)

[0379] MPQLDSGGGGAGGGDDLGAPDELLAFQDEGEEQDDKSRDSAAGPERDLAELKSSLVNESEGAAGGAGIPGVPGAGAGARGEAEALGREHAAQRLFPDKLPEPLEDGLKAPECTSGMYKETVYSAFNLLMHYPPPSGAGQHPQPQPPLHKANQPPHGVPQLSLYEHFNSPHPTPAPADISQKQVHRPLQTPDLSGFYSLTSGSMGQLPHTVSWFTHPSLMLGSGVPGHPAAIPHPAIVPPSGKQELQPFDRNLKTQAESKAEKEAKKPTIKKPLNAFMLYMKEMRAKVIAECTLKESAAINQILGRRWHALSREEQAKYYELARKERQLHMQLYPGWSARDNYGKKKRRSREKHQESTTGGKRNAFGTYPEKAAAPAPFLPMTVL

[0380] LAT1(SEQ ID NO:83)

[0381] MAGAGPKRRALAAPAAEEKEEAREKMLAAKSADGSAPAGEGEGVTLQRNITLLNGVAIIVGTIIGSGIFVTPTGVLKEAGSPGLALVVWAACGVFSIVGALCYAELGTTISKSGGDYAYMLEVYGSLPAFLKLWIELLIIRPSSQYIVALVFATYLLKPLFPTCPVPEEAAKLVCLCVLLLTAVNCYSVKAATRVQDAFAAAKLLALALIILLGFVQIGKGDVSNDPNFSFEGTKLDVGNIVLALYSGLFAYGGWNYLNFVTEEMINPYRNLPLAIIISLPIVTLVYVLTNLAYFTTLSTEQMLSSEAVAVDFGNYHLGVMSWIIPVFVGLSCFGSVNGSLFTSSRLFFVGSREGHLPSILSMIHPQLLTPVPPSLVFTCVMTLLYAFSKDIFSVINFFSFFNWLCVALAIIGMIWLRHRKPELERPIKVNLALPVFFILACLFLIAVSFWKTPVECGIGFTIILSGLPVYFFGVWWKNKPKWLLQGIFSTTVLCQKLMQVVPQET

[0382] STAT5(SEQ ID NO:84)

[0383] MAGWIQAQQLQGDALRQMQVLYGQHFPIEVRHYLAQWIESQPWDAIDLDNPQDRAQATQLLEGLVQELQKKAEHQVGEDGFLLKIKLGHYATQLQKTYDRCPLELVRCIRHILYNEQRLVREANNCSSPAGILVDAMSQKHLQINQTFEELRLVTQDTENELKKLQQTQEYFIIQYQESLRIQAQFAQLAQLSPQERLSRETALQQKQVSLEAWLQREAQTLQQYRVELAEKHQKTLQLLRKQQTIILDDELIQWKRRQQLAGNGGPPEGSLDVLQSWCEKLAEIIWQNRQQIRRAEHLCQQLPIPGPVEEMLAEVNATITDIISALVTSTFIIEKQPPQVLKTQTKFAATVRLLVGGKLNVHMNPPQVKATIISEQQAKSLLKNENTRNECSGEILNNCCVMEYHQATGTLSAHFRNMSLKRIKRADRRGAESVTEEKFTVLFESQFSVGSNELVFQVKTLSLPVVVIVHGSQDHNATATVLWDNAFAEPGRVPFAVPDKVLWPQLCEALNMKFKAEVQSNRGLTKENLVFLAQKLFNNSSSHLEDYSGLSVSWSQFNRENLPGWNYTFWQWFDGVMEVLKKHHKPHWNDGAILGFVNKQQAHDLLINKPDGTFLLRFSDSEIGGITIAWKFDSPERNLWNLKPFTTRDFSIRSLADRLGDLSYLIYVFPDRPKDEVFSKYYTPVLAKAVDGYVKPQIKQVVPEFVNASADAGGSSATYMDQAPSPAVCPQAPYNMYPQNPDHVLDQDGEFDLDETMDVARHVEELLRRPMDSLDSRLSPPAGLFTSARGSLS

[0384] MCT4(SLC16A3)(SEQ ID NO:85)

[0385] MGGAVVDEGPTGVKAPDGGWGWAVLFGCFVITGFSYAFPKAVSVFFKELIQEFGIGYSDTAWISSILLAMLYGTGPLCSVCVNRFGCRPVMLVGGLFASLGMVAASFCRSIIQVYLTTGVITGLGLALNFQPSLIMLNRYFSKRRPMANGLAAAGSPVFLCALSPLGQLLQDRYGWRGGFLILGGLLLNCCVCAALMRPLVVTAQPGSGPPRPSRRLLDLSVFRDRGFVLYAVAASVMVLGLFVPPVFVVSYAKDLGVPDTKAAFLLTILGFIDIFARPAAGFVAGLGKVRPYSVYLFSFSMFFNGLADLAGSTAGDYGGLVVFCIFFGISYGMVGALQFEVLMAIVGTHKFSSAIGLVLLMEAVAVLVGPPSGGKLLDATHVYMYVFILAGAEVLTSSLILLLGNFFCIRKKPKEPQPEVAAAEEEKLHKPPADSGVDLREVEHFLKAEPEKNGEVVHTPETSV

[0386] Truncated PD_1 (SEQ ID NO:86)

[0387] ATGCAAATTCCCCAAGCCCCATGGCCAGTTGTATGGGCAGTTCTCCAGCTCGGGTGGCGGCCCGGCTGGTTTCTCGATAGCCCGGATCGGCCTTGGAATCCACCAACATTTAGTCCTGCACTTCTGGTCGTTACAGAGGGTGATAATGCAACATTTACATGTTCCTTTAGCAATACCTCAGAATCCTTTGTTTTGAATTGGTATCGAATGTCTCCAAGTAATCAAACCGATAAACTCGCGGCATTTCCAGAAGATAGGTCCCAACCGGGTCAAGATTGTAGGTTTAGAGTGACTCAGCTTCCAAATGGTCGGGATTTTCATATGTCTGTCGTGCGCGCTCGCAGAAACGATTCAGGTACATATCTTTGCGGCGCTATAAGTCTCGCGCCGAAAGCCCAAATAAAGGAATCTCTTAGGGCCGAACTGCGAGTTACGGAAAGGCGGGCCGAGGTTCCTACTGCTCATCCTTCTCCTAGCCCTCGCCCTGCAGGGCAATTTCAGACTCTTGTCGTGGGCGTAGTAGGCGGTTTGCTTGGGTCACTTGTATTGCTCGTTTGGGTGCTCGCTGTGATTTGTTCTAGGGCAGCCAGAGGAACTATCGGC

[0388] BATF2(SEQ ID NO:87)

[0389] ATGCACCTCTGTGGGGGCAATGGGCTGCTGACCCAGACAGACCCCAAGGAGCAACAAAGGCAGCTGAAGAAGCAGAAGAACCGGGCAGCCGCCCAGCGAAGCCGGCAGAAGCACACAGACAAGGCAGACGCCCTGCACCAGCAGCACGAGTCTCTGGAAAAAGACAACCTCGCCCTGCGGAAGGAGATCCAGTCCCTGCAGGCCGAGCTGGCGTGGTGGAGCCGGACCCTGCACGTGCATGAGCGCCTGTGCCCCATGGATTGTGCCTCCTGCTCAGCTCCAGGGCTCCTGGGCTGCTGGGACCAGGCTGAGGGGCTCCTGGGCCCTGGCCCACAGGGACAACATGGCTGCCGGGAGCAGCTGGAGCTGTTCCAGACCCCGGGTTCCTGTTACCCAGCTCAGCCGCTCTCTCCAGGTCCACAGCCTCATGATTCTCCCAGCCTCCTCCAGTGCCCCCTGCCCTCACTGTCCCTTGGCCCCGCTGTGGTTGCTGAACCTC CTGTCCAGCTGTCCCCCAGCCCTCTCCTGTTTGCCTCGCACACTGGTTCCAGCCTGCAGGGGTCTTCCTCTAAGCTCAGTGCCCTCCAGCCCAGCCTCACGGCCCAAACTGCCCCTCCACAGCCCCTCGAGCTGGAGCATCCCACCAGAGGGAAGCTGGGGTCCTCTCCCGACAACCCTTCCTCTGCCCTGGGGCTTGCACGTCTGCAGAGCAGGGAGCACAAACCTGCTCTCTCAGCAGCCACTTGGCAAGGGCTGGTTGTGGATCCCAGCCCTCACCCTCTCCTGGCCTTTCCTCTGCTCTCCTCTGCTCAAGTCCACTTC

[0390] RORC(SEQ ID NO:88)

[0391]

[0392] TCF7(SEQ ID NO:89)

[0393] ATGCCGCAGCTGGATTCTggcGGAGGAGGAgcgggcGGAGGAGATGATctcggcgcgccgGATGAGCTGCTGGCCTTCCAGGATGAAGGCGAGGAGCAGGATGACAAGAGCCGAGATAGCGCCGCCGGTCCTGAGCGCGACCTGGCCGAGCTCAAGTCGTCGCTTGTGAACGAATCCGAGGGAGCAGCCGGAGGAGCAGGAATCCCGGGAGTTCCGGGAGCCGGCGCCGGAGCCCGAGGCGAGGCCGAAGCTCTTGGACGAGAACACGCTGCGCAGAGACTCTTCCCGGACAAACTTCCAGAGCCCCTGGAGGACGGCCTGAAGGCCCCGGAGTGCACCAGCGGCATGTACAAAGAGACCGTCTACTCCGCCTTCAATCTGCTCATGCATTACCCAC

[0394] CTCCTTCGGGAGCAGGACAGCACCCTCAGCCGCAGCCTCCGCTGCACAAGGCCAATCAGCCTCCTCACGGTGTCCCTCAACTCTCTCTCTACGAACATTTCAACAGCCCACATCCCACCCCTGCACCTGCGGACATCAGCCAGAAGCAAGTTCACAGGCCTCTGCAGACCCCTGACCTCTCTGGCTTCTACTCCCTGAC CTCAGGCAGCATGGGGCAGCTCCCCCACACTGTGAGCTGGTTCACCCACCCATCCTTGATGCTAGGTTCTGGTGTACCTGGTCACCCAGCAGCCATCCCTCACCCGGCCATTGTGCCTCCTTCAGGGAAGCAGGAGCTGCAGCCTTTCGACCGCAACCTGAAGACACAAGCAGAGTCCAAGGCAGAGAAGGAGGCCAAGAAGCCAACCATCAAGAAGCCCCTCAATGCCTTCATGCTGTACATGAAGGAGATGAGAGCCAAGGTCATTGCAGAGTGCACACTTAAGGAGAGCGCTGCCATCAACCAGATCCTGGGCCGCAGGTGGCACGCGCTGTCGCGAGAAGAGCAGGCCAAGTACTATGAGCTGGCCCGCAAGGAGAGGCAGCTGCACATGCAGCTATACCCAGGCTGGTCAGCGCGGGACAACTACGGGAAGAAGAAGAGGCGGTCGAGGGAAAAGCACCAAGAATCCACCACAGGAGGAAAAAGAAATGCATTCGGTACTTACCCGGAGAAGGCCGCTGCCCCAGCCCCGTTCCTTCCGATGACAGTGCTC

[0395] STAT5(SEQ ID NO:90)

[0396]

[0397] MCT4(SLC16A3)(SEQ ID NO:91)

[0398] ATGGGAGGGGCCGTGGTGGACGAGGGCCCCACAGGCGTCAAGGCCCCTGACGGCGGCTGGGGCTGGGCCGTGCTCTTCGGCTGTTTCGTCATCACTGGCTTCTCCTACGCCTTCCCCAAGGCCGTCAGTGTCTTCTTCAAGGAGCTCATACAGGAGTTTGGGATCGGCTACAGCGACACAGCCTGGATCTCCTCCATCCTGCTGGCCATGCTCTACGGGACAGGTCCGCTCTGCAGTGTGTGCGTGAACCGCTTTGGCTGCCGGCCCGTCATGCTTGTGGGGGGTCTCTTTGCGTCGCTGGGCATGGTGGCTGCGTCCTTTTGCCGGAGCATCATCCAGGTCTACCTCACCACTGGGGTCATCACGGGGTTGGGTTTGGCACTCAACTTCCAGCCCTCGCTCATCATGCTGAACCGCTACTTCAGCAAGCGGCGCCCCATGGCCAACGGGCTGGCGGCAGCAGGTAGCCCTGTCTTCCTGTGTGCCCTGAGCCCGCTGGGGCAGCTGCTGCAGGACCGCTACGGCTGGCGGGGCGGCTTCCTCATCCTGGGCGGCCTGCTGCTCAACTGCTGCGTGTGTGCCGCACTCATGAGGCCCCTGGTGGTCACGGCCCAGCCGGGCTCGGGGCCGCCGCGACCCTCCCGGCGCCTGCTAGACCTGAGCGTCTTCCGGGACCGCGGCTTTGTGCTTTACGCCGTGGCCGCCTCGGTCATGGTGCTGGGGCTCTTCGTCCCGCCCGTGTTCGTGGTGAGCTACGCCAAGGACCTGGGCGTGCCCGACACCAAGGCCGCCTTCCTGCTCACCATCCTGGGCTTCATTGACATCTTCGCGCGGCCGGCCGCGGGCTTCGTGGCGGGGCTTGGGAAGGTGCGGCCCTACTCCGTCTACCTCTTCAGCTTCTCCATGTTCTTCAACGGCCTCGCGGACCTGGCGGGTTCTACGGCGGGCGACTACGGCGGCCTCGTGGTCTTCTGCATCTTCTTTGGCATCTCCTACGGCATGGTGGGGGCCCTGCAGTTCGAGGTGCTCATGGCCATCGTGGGCACCCACAAGTTCTCCAGTGCCATTGGCCTGGTGCTGCTGATGGAGGCGGTGGCCGTGCTCGTCGGGCCCCCTTCGGGAGGCAAACTCCTGGATGCGACCCACGTCTACATGTACGTGTTCATCCTGGCGGGGGCCGAGGTGCTCACCTCCTCCCTGATTTTGCTGCTGGGCAACTTCTTCTGCATTAGGAAGAAGCCCAAAGAGCCACAGCCTGAGGTGGCGGCCGCGGAGGAGGAGAAGCTCCACAAGCCTCCTGCAGACTCGGGGGTGGACTTGCGGGAGGTGGAGCATTTCCTGAAGGCTGAGCCTGAGAAAAACGGGGAGGTGGTTCACACCCCGGAAACAAGTGTC

[0399] LAT1(SEQ ID NO:92)

[0400] ATGGCGGGTGCGGGCCCGAAGCGGCGCGCGCTAGCGGCGCCGGCGGCCGAGGAGAAGGAAGAGGCGCGGGAGAAGATGCTGGCCGCCAAGAGCGCGGACGGCTCGGCGCCGGCAGGCGAGGGCGAGGGCGTGACCCTGCAGCGGAACATCACGCTGCTCAACGGCGTGGCCATCATCGTGGGGACCATTATCGGCTCGGGCATCTTCGTGACGCCCACGGGCGTGCTCAAGGAGGCAGGCTCGCCGGGGCTGGCGCTGGTGGTGTGGGCCGCGTGCGGCGTCTTCTCCATCGTGGGCGCGCTCTGCTACGCGGAGCTCGGCACCACCATCTCCAAATCGGGCGGCGACTACGCCTACATGCTGGAGGTCTACGGCTCGCTGCCCGCCTTCCTCAAGCTCTGGATCGAGCTGCTCATCATCCGGCCTTCATCGCAGTACATCGTGGCCCTGGTCTTCGCCACCTACCTGCTCAAGCCGCTCTTCCCCACCTGCCCGGTGCCCGAGGAGGCAGCCAAGCTCGTGGCCTGCCTCTGCGTGCTGCTGCTCACGGCCGTGAACTGCTACAGCGTGAAGGCCGCCACCCGGGTCCAGGATGCCTTTGCCGCCGCCAAGCTCCTGGCCCTGGCCCTGATCATCCTGCTGGGCTTCGTCCAGATCGGGAAGGGTGATGTGTCCAATCTAGATCCCAACTTCTCATTTGAAGGCACCAAACTGGATGTGGGGAACATTGTGCTGGCATTATACAGCGGCCTCTTTGCCTATGGAGGATGGAATTACTTGAATTTCGTCACAGAGGAAATGATCAACCCCTACAGAAACCTGCCCCTGGCCATCATCATCTCCCTGCCCATCGTGACGCTGGTGTACGTGCTGACCAACCTGGCCTACTTCACCACCCTGTCCACCGAGCAGATGCTGTCGTCCGAGGCCGTGGCCGTGGACTTCGGGAACTATCACCTGGGCGTCATGTCCTGGATCATCCCCGTCTTCGTGGGCCTGTCCTGCTTCGGCTCCGTCAATGGGTCCCTGTTCACATCCTCCAGGCTCTTCTTCGTGGGGTCCCGGGAAGGCCACCTGCCCTCCATCCTCTCCATGATCCACCCACAGCTCCTCACCCCCGTGCCGTCCCTCGTGTTCACGTGTGTGATGACGCTGCTCTACGCCTTCTCCAAGGACATCTTCTCCGTCATCAACTTCTTCAGCTTCTTCAACTGGCTCTGCGTGGCCCTGGCCATCATCGGCATGATCTGGCTGCGCCACAGAAAGCCTGAGCTTGAGCGGCCCATCAAGGTGAACCTGGCCCTGCCTGTGTTCTTCATCCTGGCCTGCCTCTTCCTGATCGCCGTCTCCTTCTGGAAGACACCCGTGGAGTGTGGCATCGGCTTCACCATCATCCTCAGCGGGCTGCCCGTCTACTTCTTCGGGGTCTGGTGGAAAAACAAGCCCAAGTGGCTCCTCCAGGGCATCTTCTCCACGACCGTCCTGTGTCAGAAGCTCATGCAGGTGGTCCCCCAGGAGACA

[0401] CCR4(SEQ ID NO:163)

[0402] MNPTDIADTTLDESIYSNYYLYESIPKPCTKEGIKAFGELFLPPLYSLVFVFGLLGNSVVVLVLFKYKRLRSMTDVYLLNLAISDLLFVFSLPFWGYYAADQWVFGLGLCKMISWMYLVGFYSGIFFVMLMSIDRYLAIVHAVFSLRARTLTYGVITSLATWSVAVFASLPGFLFSTCYTERNHTYCKTKYSLNSTTWKVLSSLEINILGLVIPLGIMLFCYSMIIRTLQHCKNEKKNKAVKMIFAVVVLFLGFWTPYNIVLFLETLVELEVLQDCTFERYLDYAIQATETLAFVHCCLNPIIYFFLGEKFRKYILQLFKTCRGLFVLCQYCGLLQIYSADTPSSSYTQSTMDHDLHDAL

Claims

1. A human T cell heterologously expressing: A polypeptide comprising a TFAP4 protein and a polypeptide comprising a BATF protein; A polypeptide comprising a BATF3 protein and a polypeptide comprising a TFAP4 protein; A polypeptide comprising a FOXJ2 protein and a polypeptide comprising a RARA protein; A polypeptide comprising an ID3 protein and a polypeptide comprising a TFAP4 protein; A polypeptide comprising a BATF protein and a polypeptide comprising an IRF2 protein; A polypeptide comprising a NANOG protein and a polypeptide comprising a TFAP4 protein; A polypeptide comprising a MAFF protein and a polypeptide comprising a SATB1 protein; A polypeptide comprising a BATF protein and a polypeptide comprising an IRF1 protein; A polypeptide comprising a TFAP4 protein and a polypeptide comprising a HOPX protein; A polypeptide comprising a BATF protein and a polypeptide comprising a HES2 protein; A polypeptide comprising an ATF2 protein and a polypeptide comprising a BATF protein; A polypeptide comprising a BATF3 protein and a polypeptide comprising a SMAD4 protein; A polypeptide comprising a FOXP3 protein and a polypeptide comprising a RELA protein; A polypeptide comprising an ID3 protein and a polypeptide comprising a BATF3 protein; A polypeptide comprising an ID2 protein and a polypeptide comprising a TP73 protein; A polypeptide comprising a TIGIT protein and a polypeptide comprising an ICOS protein; A polypeptide comprising an LTBR protein and a polypeptide comprising a 4-1BB protein; A polypeptide comprising a DR5 protein and a polypeptide comprising an ICOS protein; A polypeptide comprising a SMAD3 protein and a polypeptide comprising a BATF3 protein; A polypeptide comprising a HES2 protein; A polypeptide comprising a FOXJ2 protein; A polypeptide comprising a CCR4 protein; A polypeptide comprising a human TFAP4 protein, and a polypeptide comprising a human TIM3 extracellular domain or a portion thereof connected to a human 4-1BB intracellular domain (and optionally about 1-15 (e.g., 12) amino acids of a human 4-1BB extracellular domain) via a transmembrane domain; A polypeptide comprising a human SOX5 protein, and a truncated TIGIT protein comprising about 1-10 (e.g., 7) amino acids of a human TIGIT extracellular domain or a portion thereof, a TIGIT transmembrane domain, and a human TIGIT intracellular domain; A polypeptide comprising a human MYC protein, and a polypeptide comprising the extracellular domain of human TGFbR2 or a portion thereof connected to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g. 7) amino acids of the intracellular domain of TGFbR2) via a transmembrane domain; A polypeptide comprising a BATF3 protein and a polypeptide comprising a 4-1BB protein; A polypeptide comprising a TFAP4 protein, and a polypeptide comprising a human LTBR extracellular domain or a portion thereof connected to a human IL-4R intracellular domain (and optionally, 1-10 (e.g., 7) amino acids of the LTBR intracellular domain) via a transmembrane domain; A polypeptide comprising an ATF1 protein, and a polypeptide comprising the extracellular domain of human IL4RA or a portion thereof connected to the intracellular domain of human 4-1BB; A polypeptide comprising a BATF protein, and a polypeptide comprising a human 2B4 extracellular domain or a portion thereof connected to a human 4-1BB intracellular domain, and optionally about 1-15 (e.g., 12) amino acids of a human 4-1BB extracellular domain; A polypeptide comprising a RORC protein, and a polypeptide comprising the VISTA extracellular domain or a portion thereof connected to a human CD28 intracellular domain, and optionally about 1-15 (e.g., 12) amino acids of a human CD28 extracellular domain; A polypeptide comprising a BATF protein and a polypeptide comprising a CXCR 1 protein; A polypeptide comprising a MAFF protein and a polypeptide comprising an IL2RA protein; A polypeptide comprising an ATF1 protein, and a polypeptide comprising the extracellular domain of human TNFRSF12 or a portion thereof connected to the intracellular domain of human OX40 (and optionally, 1-10 (e.g., 7) amino acids of the intracellular domain of TNFRSF12) via a transmembrane domain; A polypeptide comprising a human TIM3 extracellular domain or a portion thereof connected to the intracellular domain of human 4-1BB (and optionally about 1-15 (e.g., 12) amino acids of the extracellular domain of human 4-1BB) via a transmembrane domain; A polypeptide comprising a human BATF2 extracellular domain or a portion thereof and a truncated PD-1, wherein the truncated PD-1 includes about 1-12 (e.g., 10) amino acids of the human PD-1 extracellular domain or a portion thereof, the PD-1 transmembrane domain, and the human PD-1 intracellular domain; A polypeptide comprising a human TCF7 protein and a polypeptide comprising a human LAT1 protein; A polypeptide comprising a human 4-1BB protein; A polypeptide comprising human STAT5 protein and human IL-2RA protein; A polypeptide comprising a human FOXJ2 protein, and a polypeptide comprising an LTBR extracellular domain or a portion thereof connected to a human CD28 intracellular domain (and optionally, 1-10 (e.g., 7) amino acids of the LTBR intracellular domain) via a transmembrane domain; A polypeptide comprising a human FOXJ2 protein, and a polypeptide comprising an extracellular domain of LTBR or a portion thereof connected to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g. 7) amino acids of the LTBR intracellular domain) via a transmembrane domain; A polypeptide comprising a human HOPX protein, and a polypeptide comprising an IL4RA extracellular domain or a portion thereof connected to an intracellular domain of human 41-BB via a transmembrane domain; A polypeptide comprising an ID2 protein, and a polypeptide comprising a BLTA extracellular domain or a portion thereof connected to about 1-15 (e.g., 12) amino acids of a human 4-1BB intracellular domain and optionally a human 4-1BB extracellular domain; A polypeptide comprising a human SMAD1 protein and a polypeptide comprising a MCT4 protein; A polypeptide comprising a SMAD1 protein, and a polypeptide comprising an extracellular domain of LTBR or a portion thereof connected to a human IL-4R intracellular domain (and optionally, 1-10 (e.g., 7) amino acids of the LTBR intracellular domain) via a transmembrane domain; A polypeptide comprising about 1-12 amino acids (e.g., 7 amino acids) of the DR5 intracellular domain, and a polypeptide comprising the human ICOS intracellular domain via a transmembrane domain; A polypeptide comprising the extracellular domain of human TIGIT or a portion thereof connected to the intracellular domain of human ICOS (and optionally about 1-15 (e.g., 12) amino acids of the extracellular domain of human ICOS) via a transmembrane domain; A polypeptide comprising the extracellular domain of human LTBR or a portion thereof connected to the intracellular domain of human 4-1BB (and optionally about 1-15 (e.g., 7) amino acids of the intracellular domain of human LTBR) via a transmembrane domain, wherein the one or more polypeptides are encoded by a heterologous nucleic acid construct inserted into a target genomic locus of the cell, optionally wherein the target genomic locus is a T cell receptor (TCR) locus of the cell, optionally wherein the heterologous nucleic acid construct is inserted in a non-viral manner.

2. The human T cell according to claim 1, wherein The T cells heterologously express one or more polypeptides, wherein each polypeptide comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:30-SEQ ID NO:58, SEQ ID NO:69-85, SEQ ID NO:160, SEQ ID NO:161, and SEQ ID NO:

162.

3. The human T cell according to claim 1 or 2, wherein The target insertion site is located in exon 1 of the TCR-α subunit constant gene (TRAC).

4. The human T cell according to claim 1 or 2, wherein The target insertion site is located in exon 1 of the TCR-β subunit constant gene (TRBC).

5. The human T cell according to claim 4, wherein The TRBC is TRBC1 or TRBC2.

6. The human T cell according to any one of claims 1 to 5, wherein The heterologous nucleic acid construct comprises one or more nucleic acid sequences, wherein each nucleic acid is at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-29 and 59-68.

7. The human T cell according to any one of claims 1 to 6, wherein The T cells express an antigen-specific T cell receptor (TCR) or a synthetic antigen receptor that recognizes a target antigen.

8. The human T cell according to claim 7, wherein The synthetic antigen receptor is a CAR or a SynNotch receptor.

9. The human T cell according to any one of claims 1 to 8, wherein The T cells are regulatory T cells, effector T cells, memory T cells or naive T cells.

10. The human T cell according to claim 9, wherein The effector T cells are CD8+ T cells or CD4+ T cells.

11. The human T cell according to claim 10, wherein The effector T cells are CD8+CD4+ T cells.

12. The human T cell according to any one of claims 1 to 11, wherein The T cells are primary cells.

13. The human T cell according to any one of claims 1 to 12, wherein The nucleic acid construct encodes: (i) a first self-cleaving peptide sequence; (ii) a first heterologous TCR subunit chain, wherein the TCR subunit chain comprises a variable region and a constant region of a TCR subunit; (iii) a second self-cleaving peptide sequence; (iv) one or more polypeptides, wherein each polypeptide comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:30-58, SEQ ID NOs:69-85, SEQ ID NO:160, SEQ ID NO:161, and SEQ ID NO:162; (v) a third self-cleaving peptide sequence; (vi) a variable region of a second heterologous TCR subunit chain; and (vii) an N-terminal portion of an endogenous TCR subunit, wherein, if the endogenous TCR subunit of the cell is a TCR-alpha (TCR-α) subunit, the first heterologous TCR subunit chain is a heterologous TCR-beta (TCR-β) subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-α subunit chain, and, wherein, if the endogenous TCR subunit of the cell is a TCR-β subunit, the first heterologous TCR subunit chain is a heterologous TCR-α subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-β subunit chain.

14. The human T cell according to any one of claims 1 to 12, wherein The heterologous nucleic acid construct encodes: (i) a first self-cleaving peptide sequence; (ii) one or more polypeptides, wherein each polypeptide comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:30-58, SEQ ID NOs:69-85, SEQ ID NO:160, SEQ ID NO:161, and SEQ ID NO:162; (iii) a second self-cleaving peptide sequence; (iv) a first heterologous TCR subunit chain, wherein the TCR subunit chain comprises a variable region and a constant region of a TCR subunit; (v) a third self-cleaving peptide sequence; (vi) a variable region of a second heterologous TCR subunit chain; and (vii) an N-terminal portion of an endogenous TCR subunit, wherein, if the endogenous TCR subunit of the cell is a TCR-alpha (TCR-α) subunit, the first heterologous TCR subunit chain is a heterologous TCR-beta (TCR-β) subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-α subunit chain, and, wherein, if the endogenous TCR subunit of the cell is a TCR-β subunit, the first heterologous TCR subunit chain is a heterologous TCR-α subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-β subunit chain.

15. The human T cell according to any one of claims 1 to 12, wherein The nucleic acid construct encodes in the following order: (i) a first self-cleaving peptide sequence; (iv) one or more polypeptides, wherein each polypeptide comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:30-58, SEQ ID NOs:69-85, SEQ ID NO:160, SEQ ID NO:161, and SEQ ID NO:162; (iii) a second self-cleaving peptide sequence; (iv) synthetic antigen receptors; and (v) a third self-cleaving peptide sequence or a poly A sequence.

16. The human T cell according to any one of claims 1 to 12, wherein The nucleic acid construct encodes in the following order: (i) a first self-cleaving peptide sequence; (ii) synthetic antigen receptors; (iii) a second self-cleaving peptide sequence; (iv) one or more polypeptides, wherein each polypeptide comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:30-58, SEQ ID NOs:69-85, SEQ ID NO:160, SEQ ID NO:161, and SEQ ID NO:162; and (v) a third self-cleaving peptide sequence or a poly A sequence.

17. The human T cell according to claim 15 or 16, wherein The synthetic antigen receptor is a CAR or a SynNotch receptor.

18. A nucleic acid sequence encoding: a TFAP polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:30, and a BATF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:31; a BATF3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:32, and a TFAP4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:30; a FOXJ2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:33, and a RARA polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:34; an ID3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:35, and a TFAP4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:30; a BATF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:31, and an IRF2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:36; a NANOG polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:37, and a TFAP4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:30; a MAFF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:38, and a SATB1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:39; a BATF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:32, and an IRF1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:36; a TFAP4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:39, and a HOPX polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:51; a TFAP4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:30, and a HOPX polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:42; a BATF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:31, and a HES2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:43; an ATF2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:47, and a BATF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:31; a BATF3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:32, and a SMAD4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:48; a FOXP3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:49, and a RELA polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:50; an ID3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:35, and a BATF3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:32; a TFAP4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:30, and a TIM-3 / 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:69; a SOX5 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:51, and a TIM-3 / 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:69; a MYC polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:53, and a TGFbR2 / MyD88 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:70; a BATF3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:32, and a 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:55; a TFAP4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:30, and a LTBR-IL-4R polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:71; an ATF1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:58, and an IL4RA / 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:72; a BATF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:31, and a 2B4 / 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:73; a RORC polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 79, and a VISTA / CD28 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 74; a BATF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:31, and a CXCR1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:57; a MAFF polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:38, and an IL-2RA polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:54; an ATF1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:58, and a TNFRSF12-OX40 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:75; A TIM-3 / 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 69, a BATF2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:80, and a truncated PD1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:81; a TCF7 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:82, and a LAT polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:83; a STAT5 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:84, and an IL-2RA polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:54; a FOXJ2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:33, and a LTBR-CD28 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:76; a FOXJ2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:33, and a LTBR-MyD88 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:77; a HOPX polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:42, and an IL4RA / 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:72; an ID2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:44, and a BTLA / 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:78; a SMAD1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:56, and a MCT4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:85; a SMAD1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:56, and a LTBR-IL-4R polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:71; a TIGIT polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 121, and an ICOS polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 159; an LTBR polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 97, and a 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 146; A DR5 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:133, and an ICOS polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:

159.

19. The nucleic acid according to claim 18, wherein The nucleic acid comprises flanking homology arm sequences that are homologous to the human TCR locus.

20. A human T cell comprising the nucleic acid of claim 18 or claim 19.

21. A nucleic acid construct encoding in the following sequence: (i) a first self-cleaving peptide sequence; (ii) a first heterologous TCR subunit chain, wherein the TCR subunit chain comprises a variable region and a constant region of a TCR subunit; (iii) a second self-cleaving peptide sequence; (iv) one or more polypeptides, wherein each polypeptide comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:30-58, SEQ ID NOs:69-85, SEQ ID NO:160, SEQ ID NO:161, and SEQ ID NO:162; (v) a third self-cleaving peptide sequence; (vi) a variable region of a second heterologous TCR subunit chain; and (vii) an N-terminal portion of an endogenous T cell TCR subunit, wherein If the endogenous TCR subunit is a TCR-alpha (TCR-α) subunit, the first heterologous TCR subunit chain is a heterologous TCR-beta (TCR-β) subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-α subunit chain, and, wherein, if the endogenous TCR subunit is a TCR-β subunit, the first heterologous TCR subunit chain is a heterologous TCR-α subunit chain and the second heterologous TCR subunit chain is a heterologous TCR-β subunit chain.

22. The nucleic acid construct of claim 21, wherein The nucleic acid construct comprises two or more nucleic acid sequences, wherein each nucleic acid is at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-29 and 59-68.

23. A method for modifying human T cells, comprising: (a) introducing into the human T cells: (i) a targeted nuclease that cleaves a target region in the human T cell TCR locus to generate a target insertion site in the cell genome; and (ii) a nucleic acid construct encoding: a polypeptide comprising TFAP4 protein and BATF protein; a polypeptide comprising BATF3 protein and TFAP4 protein; a polypeptide comprising FOXJ2 protein and RARA protein; a polypeptide comprising ID3 protein and TFAP4 protein; a polypeptide comprising BATF protein and IRF2 protein; a polypeptide comprising NANOG protein and TFAP4 protein; a polypeptide comprising MAFF protein and SATB1 protein; a polypeptide comprising BATF protein and IRF1 protein; a polypeptide comprising TFAP4 protein and HOPX protein; a polypeptide comprising BATF protein and HES2 protein; a polypeptide comprising ATF2 protein and BATF protein; a polypeptide comprising BATF3 protein and SMAD4 protein; a polypeptide comprising FOXP3 protein and RELA protein; a polypeptide comprising ID3 protein and BATF3 protein; a polypeptide comprising EOMES protein; a polypeptide comprising HES2 protein; a polypeptide comprising FOXJ2 protein; A polypeptide comprising (a) a human TFAP4 protein, and (b) a polypeptide comprising a human TIM3 extracellular domain or a portion thereof connected to a human 4-1BB intracellular domain (and optionally about 1-15 (e.g., 12) amino acids of a human 4-1BB extracellular domain) via a transmembrane domain; A polypeptide comprising (a) a human SOX5 protein, and (b) a truncated TIGIT protein comprising about 1-10 (e.g., 7) amino acids of a human TIGIT extracellular domain or a portion thereof, a TIGIT transmembrane domain, and a human TIGIT intracellular domain; A polypeptide comprising (a) a human MYC protein, and (b) a polypeptide comprising the extracellular domain of human TGFbR2 or a portion thereof, connected to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g., 7) amino acids of the intracellular domain of TGFbR2) via a transmembrane domain; A polypeptide comprising BATF3 protein and 4-1BB protein; A polypeptide comprising (a) a TFAP4 protein, and (b) a polypeptide comprising a human LTBR extracellular domain or a portion thereof connected to a human IL-4R intracellular domain (and optionally, 1-10 (e.g., 7) amino acids of the LTBR intracellular domain) via a transmembrane domain; A polypeptide comprising (a) an ATF1 protein, and (b) a polypeptide comprising an extracellular domain of human IL4RA or a portion thereof connected to an intracellular domain of human 4-1BB; A polypeptide comprising (a) a BATF protein, and (b) a polypeptide comprising a human 2B4 extracellular domain or a portion thereof connected to a human 4-1BB intracellular domain, and optionally about 1-15 (e.g., 12) amino acids of a human 4-1BB extracellular domain; A polypeptide comprising (a) a RORC protein, and (b) a polypeptide comprising a VISTA extracellular domain or a portion thereof connected to a human CD28 intracellular domain, and optionally about 1-15 (e.g., 12) amino acids of a human CD28 extracellular domain; A polypeptide comprising BATF protein and CXCR 1 protein; A polypeptide comprising a MAFF protein and an IL2RA protein; A polypeptide comprising (a) an ATF1 protein, and (b) a polypeptide comprising a human TNFRSF12 extracellular domain or a portion thereof connected to a human OX40 intracellular domain (and optionally, 1-10 (e.g., 7) amino acids of a TNFRSF12 intracellular domain) via a transmembrane domain; A polypeptide comprising a human TIM3 extracellular domain or a portion thereof connected to the intracellular domain of human 4-1BB (and optionally about 1-15 (e.g., 12) amino acids of the extracellular domain of human 4-1BB) via a transmembrane domain; A polypeptide comprising a human BATF2 extracellular domain or a portion thereof linked to a truncated PD-1, wherein the truncated PD-1 comprises about 1-12 (e.g., 10) amino acids of the human PD-1 extracellular domain or a portion thereof, a PD-1 transmembrane domain, and a human PD-1 intracellular domain; A polypeptide comprising human TCF7 protein and human LAT1 protein; A polypeptide comprising a human 4-1BB protein; A polypeptide comprising human STAT5 protein and human IL-2RA protein; A polypeptide comprising (a) a human FOXJ2 protein, and (b) a polypeptide comprising an extracellular domain of LTBR or a portion thereof connected to a human CD28 intracellular domain (and optionally, 1-10 (e.g., 7) amino acids of the LTBR intracellular domain) via a transmembrane domain; A polypeptide comprising (a) a human FOXJ2 protein, and (b) a polypeptide comprising an extracellular domain of LTBR or a portion thereof connected to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g., 7) amino acids of an intracellular domain of LTBR) via a transmembrane domain; A polypeptide comprising (a) a human HOPX protein, and (b) a polypeptide comprising an IL4RA extracellular domain or a portion thereof connected to an intracellular domain of human 41-BB via a transmembrane domain; A polypeptide comprising (a) an ID2 protein, and (b) a polypeptide comprising a BLTA extracellular domain or a portion thereof connected to a human 4-1BB intracellular domain, and optionally about 1 to 15 (e.g., 12) amino acids of a human 4-1BB extracellular domain; A polypeptide comprising human SMAD1 protein and MCT4 protein; and A polypeptide comprising (a) a human SMAD1 protein and (b) an extracellular domain of LTBR or a portion thereof connected to amino acids of the intracellular domain of human IL-4R (and optionally, 1-10 (e.g., 7) amino acids of the intracellular domain of LTBR) via a transmembrane domain; (b) allowing recombination to occur, thereby inserting the nucleic acid construct into the target insertion site to generate modified human T cells.

24. The method of claim 23, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:30 to SEQ ID NO:58, and SEQ ID NO:69-85, 160, 161 and 162.

25. The method of claim 24, wherein: The nucleic acid construct is the nucleic acid construct according to claim 22.

26. The method of any one of claims 23 to 25, wherein: The target insertion site is located in exon 1 of the TCR-α subunit constant gene (TRAC) or in exon 1 of the TCR-β subunit constant gene (TRBC).

27. The method of any one of claims 23 to 26, wherein: The nucleic acid construct is inserted into cells by introducing a viral vector comprising the nucleic acid construct.

28. The method of any one of claims 23-27, wherein the targeting nuclease is selected from the group consisting of an RNA-guided nuclease domain, a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease (ZFN), and a megaTAL.

29. The method of claim 28, wherein: The targeted nuclease, guide RNA and DNA template are introduced into the cell as a ribonucleoprotein complex (RNP)-DNA template complex, wherein the RNP-DNA template complex comprises: (i) the RNP, wherein the RNP comprises the targeting nuclease and the guide RNA; and (ii) the nucleic acid construct.

30. The method of any one of claims 22 to 29, wherein: The T cells are regulatory T cells, effector T cells, memory T cells or naive T cells.

31. The method of claim 30, wherein: The effector T cells are CD8+ T cells or CD4+ T cells.

32. The method of claim 31, wherein: The effector T cells are CD8+CD4+ T cells.

33. The method of any one of claims 22 to 32, wherein: The cells are primary cells.

34. A modified T cell produced by the method of any one of claims 22-33.

35. A method of enhancing an immune response in a human subject, comprising administering to the subject the T cell of any one of claims 1-16, 20 or 34.

36. The method of claim 35, wherein: The T cells express an antigen-specific TCR or synthetic antigen receptor that recognizes a target antigen in the subject.

37. The method of claim 35 or 36, wherein: The human subject has cancer and the target antigen is a cancer-specific antigen.

38. The method of claim 37, wherein: The human subject has a solid tumor.

39. The method of claim 35 or 36, wherein: The human subject has an infection.

40. The method of claim 35 or 36, wherein: The human subject suffers from an autoimmune disease and the antigen is an antigen associated with the autoimmune disease, allergic disease or transplant rejection.

41. The method of any one of claims 35 to 40, wherein: The T cells are autologous.

42. The method of any one of claims 35 to 40, wherein: The T cells are allogeneic.

43. The method of any one of claims 35 to 40, wherein: The T cells are iPSC-derived T cells.

44. A method for identifying a targeted insertion in a cell genome, the method comprising: (a) Introducing into cell populations (i) a targeted nuclease that cleaves a target region in the cellular genome to generate a target insertion site; and (ii) a plurality of DNA templates having different sequences from each other, wherein each DNA template comprises: i. two or more heterologous coding or non-coding nucleic acid sequences; and ii. a unique barcode nucleotide sequence that identifies the identity of each heterologous coding or non-coding nucleic acid sequence; (b) allowing recombination to occur, thereby producing a population of modified cells; (c)(i) isolating genomic DNA; or (ii) isolating mRNA from the cell and generating cDNA from the mRNA; (d) Sequencing the genomic DNA or cDNA to identify the DNA template that is inserted into the target insertion site in the cell.

45. The method of claim 44, wherein the step of isolating mRNA further comprises digesting DNA from the cells.

46. ​​The method of claim 44 or 45, wherein the barcode sequence in the genomic DNA or cDNA is sequenced to identify two or more heterologous coding or non-coding nucleic acid sequences.

47. The method of any one of claims 44-46, wherein the DNA template comprises two heterologous nucleic acid sequences encoding two transcription factors.

48. The method of any one of claims 44-47, wherein the DNA template comprises two heterologous nucleic acid sequences, wherein each heterologous nucleic acid sequence encodes a transcription factor.

49. The method of claim 48, wherein the transcription factors are the same or the transcription factors are different.

50. The method of any one of claims 44-49, wherein the DNA template comprises a heterologous nucleic acid sequence encoding a transcription factor and a heterologous nucleic acid sequence encoding a switch receptor.

51. The method of any one of claims 44-50, further comprising determining the relative number of cells in the population having a different DNA template inserted at the target insertion site.

52. The method of any one of claims 44-51, further comprising applying selective pressure to the modified cell population.

53. The method of claim 52, further comprising comparing the relative number of cells in the population having a different DNA template inserted at the target insertion site before and after applying selective pressure to the cells.

54. The method of any one of claims 44-53, wherein the DNA template is inserted by introducing a viral vector comprising the DNA template into the cell.

55. The method of any one of claims 44 to 54, wherein: The population is a population of mammalian cells.

56. The method of claim 55, wherein: The mammalian cell is a human cell.

57. The method of claim 56, wherein: The human cell is a T cell.

58. The method of claim 57, wherein: The T cells are regulatory T cells, effector T cells or naive T cells.

59. The method of claim 57 or 58, wherein: The effector T cells are CD8+ T cells or CD4+ T cells.

60. The method of claim 59, wherein: The effector T cells are CD8+CD4+ T cells.

61. The method of any one of claims 44 to 60, wherein: The cells are primary cells.

62. The method of any one of claims 44-61, wherein the DNA template comprises nucleic acids encoding two or more heterologous polypeptides.

Images