DNA constructs for improved T cell immunotherapy

Abstract

Provided herein are methods and compositions for modifying the genome of human T cells.Furthermore, the compositions and methods described herein can be used to generate human T cells with altered specificity and functionality while limiting the side effects associated with T cell therapy.Provided herein are human T cells that heterologously express one or more polypeptides.In some embodiments, one or more polypeptides, for example, two or more polypeptides, are encoded by a nucleic acid construct that is inserted into the TCR locus of the cell. TIFF2025506185000058.tif91170

Description

[Technical field]

[0001] Prior Related Applications This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 309,938, filed February 14, 2022, which is incorporated by reference in its entirety. [Background technology]

[0002] 2. Background of the Invention Current technologies for the modification of ex vivo or in vivo gene-edited cells for therapeutic use focus on correcting existing mutations, limiting therapeutic applicability to conditions caused by single mutations that result in dysfunctional genes, or incorporating completely new synthetic genes, requiring extensive research and development leading to the creation of new therapeutically useful synthetic DNA sequences.Therefore, genome modification options are limited.Given the importance of T cells in adoptive cell therapy, the ability to obtain human T cells and modify them to produce edited T cells with desired functions can be beneficial in the development and application of adoptive T cell therapy. Summary of the Invention

[0003] BRIEF SUMMARY OF THE INVETION The present disclosure relates to compositions and methods for modifying the genome of T cells. The inventors have discovered that human T cells can be modified to change the specificity and function of T cells. By inserting nucleic acids encoding one or more polypeptides and a heterologous T cell receptor (TCR) or a synthetic antigen receptor (e.g., a chimeric antigen receptor (CAR)) into a specific endogenous site (e.g., a TCR locus) in the genome of a T cell, human T cells can be generated with the desired antigen specificity of the TCR or CAR and the function of the polypeptide. Furthermore, the compositions and methods described herein can be used to generate human T cells with altered specificity and functionality while limiting the side effects associated with T cell therapy.

[0004] Provided herein is a human T cell that heterologously expresses one or more polypeptides. In some embodiments, the one or more polypeptides, for example, two or more polypeptides, are encoded by a nucleic acid construct inserted into the TCR locus of the cell.

[0005] In some embodiments, the human T cell is characterized in that it is capable of 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 H Heterologously expressing a polypeptide comprising an ES2 protein; a polypeptide comprising an ATF2 protein and a polypeptide comprising a BATF 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 CCR4 protein; a polypeptide comprising a BATF3 protein and a polypeptide comprising an 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, a polypeptide comprising a TP73 protein; a polypeptide comprising a SMAD3 protein, or a polypeptide comprising a BATF3 protein.

[0006] In some embodiments, the human T cells express a polypeptide comprising a human TFAP4 protein and a polypeptide comprising a human TIM3 extracellular domain, or a portion thereof, linked via a transmembrane domain to a human 4-1BB intracellular domain (and optionally about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain).

[0007] In some embodiments, the human T cells express a polypeptide comprising a human SOX5 protein and a truncated TIGIT protein comprising the human TIGIT extracellular domain or a portion thereof, the TIGIT transmembrane domain, and about 1 to 10 (e.g., 7) amino acids of the human TIGIT intracellular domain.

[0008] In some embodiments, the human T cell expresses a polypeptide comprising a human MYC protein and a polypeptide comprising the human TGFbR2 extracellular domain, or a portion thereof, linked via a transmembrane domain to amino acids 41-142 of human MyD88 (and optionally, 1-10 (e.g., 7) amino acids of the TGFbR2 intracellular domain).

[0009] In some embodiments, the human T cell expresses a polypeptide comprising a BATF3 protein and a polypeptide comprising a 4-1BB protein.

[0010] In some embodiments, the human T cells express a polypeptide comprising a TFAP4 protein and a polypeptide comprising the human LTBR extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human IL-4R (and optionally 1-10 (e.g., 7) amino acids of the LTBR intracellular domain).

[0011] In some embodiments, the human T cells express a polypeptide comprising an ATF1 protein and a polypeptide comprising the extracellular domain of human IL4RA linked to the intracellular domain of human 4-1BB, or a portion thereof.

[0012] In some embodiments, the human T cell expresses a polypeptide comprising a BATF protein and a polypeptide comprising the human 2B4 extracellular domain or a portion thereof linked to the intracellular domain of human 4-1BB, and optionally, about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain.

[0013] In some embodiments, the human T cell expresses 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 human CD28 extracellular domain.

[0014] In some embodiments, the human T cell expresses a polypeptide comprising a BATF protein and a polypeptide comprising a CXCR1 protein.

[0015] In some embodiments, the human T cell expresses a polypeptide comprising a MAFF protein and a polypeptide comprising an IL2RA protein.

[0016] In some embodiments, the human T cells express a polypeptide comprising an ATF1 protein and a polypeptide comprising the human TNFRSF12 extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human OX40 (and optionally 1-10 (e.g., 7) amino acids of the TNFRSF12 intracellular domain).

[0017] In some embodiments, the human T cells express a polypeptide comprising the human TIM3 extracellular domain, or a portion thereof, linked via a transmembrane domain to the intracellular domain of human 4-1BB (and optionally about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain).

[0018] In some embodiments, the human T cell expresses a polypeptide comprising the human BATF2 extracellular domain, or a portion thereof, and a truncated PD-1 that includes the human PD-1 extracellular domain, or a portion thereof, the PD-1 transmembrane domain, and about 1-12 (e.g., 10) amino acids of the human PD-1 intracellular domain.

[0019] In some embodiments, the human T cells express a polypeptide comprising a human TCF7 protein and a polypeptide comprising a human LAT1 protein.

[0020] In some embodiments, the human T cells express a polypeptide comprising a human STAT5 protein and a human IL-2RA protein.

[0021] 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 linked via a transmembrane domain to the intracellular domain of human CD28 (and optionally 1-10 (e.g., 7) amino acids of the LTBR intracellular domain).

[0022] In some embodiments, the human T cell expresses a polypeptide comprising a human FOXJ2 protein and a polypeptide comprising the LTBR extracellular domain or a portion thereof linked via a transmembrane domain to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g., 7) amino acids of the LTBR intracellular domain).

[0023] In some embodiments, the human T cell expresses a polypeptide comprising a human HOPX protein and a polypeptide comprising an IL4RA extracellular domain or a portion thereof linked via a transmembrane domain to an intracellular domain of human 41-BB;

[0024] In some embodiments, the human T cell expresses a polypeptide comprising an ID2 protein and a polypeptide comprising the BLTA extracellular domain or a portion thereof linked to the intracellular domain of human 4-1BB, and optionally, about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain.

[0025] In some embodiments, the human T cells express a polypeptide comprising a human SMAD1 protein and a polypeptide comprising a MCT4 protein.

[0026] In some embodiments, the human T cells express a polypeptide comprising a human SMAD1 protein and a polypeptide comprising the LTBR extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human IL-4R (and optionally 1-10 (e.g., 7) amino acids of the LTBR intracellular domain).

[0027] In some embodiments, the human T cell expresses a polypeptide comprising the DR5 extracellular domain or a portion thereof linked via 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.

[0028] In some embodiments, the human T cells express a polypeptide comprising the TIGIT extracellular domain, or a portion thereof, linked via a transmembrane domain to the intracellular domain of human ICOS (and optionally, about 1-15 (e.g., 12) amino acids of the human ICOS extracellular domain).

[0029] In some embodiments, the human T cells express a polypeptide comprising the LTBR extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human 4-1BB (and optionally about 1-15 (e.g., 7) amino acids of the human LTBR intracellular domain). In some embodiments, the polypeptide further comprises about 1-12 amino acids (e.g., 7 amino acids) of the intracellular domain of 4-1BB linked to the intracellular domain of 4-1BB.

[0030] In some embodiments, the human T cells express a polypeptide that comprises CCR4.

[0031] 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.

[0032] In some embodiments, the T cell heterologously expresses two or more polypeptides, each polypeptide comprising 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).

[0033] In some embodiments, the heterologous nucleic acid construct comprises two or more nucleic acid sequences, each of which is at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-29 and 59-68.

[0034] In some embodiments, the T cells express an antigen-specific T cell receptor (TCR) or a synthetic antigen receptor that recognizes a target antigen. 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+ T cells or CD4+ T cells. In some embodiments, the effector T cells are CD8+CD4+ T cells. In some embodiments, the T cells are primary cells.

[0035] In some embodiments, the target insertion site is in exon 1 of the TCR-alpha subunit constant gene (TRAC). In some embodiments, the target insertion site is in exon 1 of the TCR-beta subunit constant gene (TRBC).

[0036] In some embodiments, the heterologous nucleic acid inserted into the human T cell contains, in the following order: (i) a first self-cleaving peptide sequence; (ii) a first heterologous TCR subunit chain comprising a variable region and a constant region of the TCR subunit; (iii) a second self-cleaving peptide sequence; (iv) one or more polypeptides described herein; (v) a third self-cleaving peptide sequence; (vi) a variable region of the second heterologous TCR subunit chain; and (vii) a portion of the N-terminus of an endogenous TCR subunit. and, 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, 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. Optionally, in any of the constructs described herein, the construct encodes two, three, four or more heterologous polypeptides.

[0037] In some embodiments, the heterologous nucleic acid inserted into a human T cell comprises, 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 first heterologous TCR subunit chain comprising 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. and 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 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.

[0038] 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 polyA sequence.

[0039] 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 polyA sequence.

[0040] In some embodiments, the heterologous nucleic acid construct comprises two or more nucleic acid sequences, each nucleic acid being at least 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-29 and 59-68.

[0041] (a) administering to a human T cell (i) a targeted nuclease that cleaves a target region within the TCR locus of the human T cell to create a targeted insertion site within the genome of the cell; and (ii) 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 peptide; 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 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) a polypeptide comprising a human TIM3 extracellular domain or a portion thereof linked via a transmembrane domain to a human 4-1BB intracellular domain (and optionally about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain); a polypeptide comprising (a) a human SOX5 protein and (b) a truncated TIGIT protein comprising a human TIGIT extracellular domain or a portion thereof, a TIGIT transmembrane domain, and about 1-10 (e.g., 7) amino acids of the human TIGIT intracellular domain;A polypeptide comprising (a) a human MYC protein and (b) a polypeptide comprising a human TGFbR2 extracellular domain or a portion thereof linked via a transmembrane domain to amino acids 41 to 142 of human MyD88 (and optionally, 1 to 10 (e.g., 7) amino acids of the TGFbR2 intracellular domain); a polypeptide comprising a BATF3 protein and a 4-1BB protein; a polypeptide comprising (a) a TFAP4 protein and (b) a polypeptide comprising a human LTBR extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human IL-4R (and optionally, 1 to 10 (e.g., 7) amino acids of the LTBR intracellular domain); (a) an ATF1 protein and (b) a polypeptide comprising (a) a BATF protein and (b) a human 2B4 extracellular domain or a portion thereof linked to the intracellular domain of human 4-1BB, and optionally a polypeptide comprising about 1 to 15 (e.g., 12) amino acids of the human 4-1BB extracellular domain; a polypeptide comprising (a) a RORC protein and (b) a VISTA extracellular domain or a portion thereof linked to the intracellular domain of human CD28, and optionally a polypeptide comprising about 1 to 15 (e.g., 12) amino acids of the human CD28 extracellular domain; a polypeptide comprising a BATF protein and a CXCR a polypeptide comprising an 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 linked via a transmembrane domain to a human OX40 intracellular domain (and optionally, about 1 to 10 (e.g., 7) amino acids of the TNFRSF12 intracellular domain); a polypeptide comprising a human TIM3 extracellular domain or a portion thereof linked via a transmembrane domain to a human 4-1BB intracellular domain (and optionally, about 1 to 15 (e.g., 12) amino acids of the human 4-1BB extracellular domain);a polypeptide comprising a human BATF2 extracellular domain or a portion thereof linked to a truncated PD-1 comprising a human PD-1 extracellular domain or a portion thereof, a PD-1 transmembrane domain, and about 1-12 (e.g., 10) amino acids of 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) a polypeptide comprising a LTBR extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human CD28 (and optionally, 1-10 (e.g., 7) amino acids of the LTBR intracellular domain); a polypeptide comprising (a) a human FOXJ2 protein and (b) 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) a human HOPX protein and (b) a polypeptide comprising an IL4RA extracellular domain or a portion thereof linked to an intracellular domain of human 41-BB via a transmembrane domain; a polypeptide comprising (a) an ID2 protein and (b) a BLTA extracellular domain or a portion thereof linked to an intracellular domain of human 4-1BB, and optionally a polypeptide comprising about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain; a polypeptide comprising a human SMAD1 protein and an MCT4 protein; and introducing a nucleic acid construct encoding a polypeptide comprising (a) a human SMAD1 protein and (b) a LTBR extracellular domain or a portion thereof linked to an intracellular domain of human IL-4R via a transmembrane domain (and optionally 1-10 (e.g., 7) amino acids of the LTBR intracellular domain);and (b) effecting recombination, thereby inserting the nucleic acid construct into the target insertion site to generate a modified human T cell. In any of the compositions and methods provided herein, the nucleic acid construct can include a nucleic acid sequence encoding two or more polypeptides, where the two or more polypeptides are the same polypeptide, for example, a nucleic acid construct encoding two or more transcription factors, where both transcription factors are the same (e.g., an IDE polypeptide and an ID3 polypeptide; an EOMES polypeptide and an EOMES polypeptide; a TFAP4 polypeptide and a TFAP 4 polypeptide, or a BATF polypeptide and a BATF polypeptide);

[0042] In some methods, 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-SEQ ID NO:58, SEQ ID NO:69-85, SEQ ID NO:160, SEQ ID NO:161 and SEQ ID NO:162.

[0043] In some methods, the targeted insertion site is in exon 1 of the TCR-alpha subunit constant gene (TRAC) or exon 1 of the TCR-beta subunit constant gene (TRBC).

[0044] In some methods, the nucleic acid construct is inserted by introducing a viral vector containing the nucleic acid construct into the cell. In some embodiments, 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.

[0045] In some methods, the targeting nuclease, the guide RNA and the DNA template are introduced into the cell as a ribonucleoprotein complex (RNP)-DNA template complex, the RNP-DNA template complex comprising (i) an RNP comprising the targeting nuclease and the guide RNA, and (ii) a nucleic acid construct.

[0046] In some methods, the T cells express an antigen-specific T cell receptor (TCR) or a synthetic antigen receptor that recognizes a target antigen. 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+ T cells or CD4+ T cells. In some embodiments, the effector T cells are CD8+CD4+ T cells. In some embodiments, the T cells are primary cells.

[0047] Modified T cells produced by any of the methods described herein are also provided.

[0048] Further provided is a method of enhancing immune response in a human subject, comprising administering any of the T cells described herein. In some embodiments, the T cells express an antigen-specific TCR that recognizes a target antigen in the subject. In some embodiments, the human subject has cancer, and the target antigen is a cancer-specific antigen. In some embodiments, the human subject has an autoimmune disorder or an allergic disorder, and the antigen is an antigen associated with the autoimmune disorder or the allergic disorder. In some embodiments, the subject has an infectious disease, and the target antigen is an antigen associated with the infectious disease. In some embodiments, the T cells are autologous. In some embodiments, the T cells are allogeneic. In some embodiments, the T cells are induced pluripotent stem cell (iPSC)-derived T cells. [Brief description of the drawings]

[0049] This application includes the following figures. The figures are intended to illustrate certain aspects and / or features of the compositions and methods, and to supplement any descriptions of the compositions and methods. The figures do not limit the scope of the compositions and methods unless the written description expressly indicates such.

[0050] [Figure 1] 1A-C are diagrams outlining an exemplary pooled assembly approach described herein. (A) Schematic showing the pooled assembly approach used to generate a combinatorial library of tonic signaling GD2 CAR+100 transcription factors (TFs)×100 transcription factors, resulting in a >10,000 member library. Constructs (and order of integration) were identified by amplicon sequencing of the fusion region between two transcription factors (3' barcode of TF1, and 5' barcode of TF2). (B) Schematic of the amplicon sequencing strategy used to detect barcodes. (C) Analysis of the presentation of combinatorial constructs on plasmid and cell pools 4 days after electroporation showed >99% presentation of >10,000 constructs. [Diagram 2] Figure 2A-B shows that the TFxTF combinatorial library was knocked into primary human T cells. Because the GD2 CAR provides tonic signaling / chronic stimulation, T cells were cultured without adding target cells. Cells were sorted on days 16 and 4 post-electroporation and the log2 fold change (log2FC) was calculated (day 16 / day 4). Log2FC is shown, highlighting that the combination of TFAP4 and BATF performed best. [Diagram 3]Figure 3A-B shows the construction of the second combinatorial library. (A) A second combinatorial library was generated using the tonic signaling GD2 CAR + 100 TFs x 129 therapeutic constructs (TCs) (synthetic receptors, chemokine receptors, etc.), resulting in a 13,362 member library. Again, constructs were identified by amplicon sequencing of the fusion region. (B) Analysis of the presentation of combinatorial constructs on plasmid and cell pools 4 days after electroporation showed >99% presentation of >10,000 constructs. [Figure 4] Figure 4 shows the hits from the TFxTC screen. Screening was performed as described in Figure 2. The top hits TFAP4_TIM-3 / 4-1BB and SOX5_TIGIT_trunc are highlighted. Many of the combinatorial constructs with the highest Log2FC have either TFAP4 or BATF / BATF3 as the first insert in their combinatorial construct. Looking at the fusion receptors in the TC portion of the constructs, the 4-1BB intracellular domain was over-represented compared to the CD28 intracellular domain in the top hits. [Diagram 5] Figure 5A-D provide validation analyses. (A) For validation analyses, single knock-ins of combinatorial constructs (including GD2 CAR) were performed. 50% / 50% co-culture analyses of the various combinatorial constructs are shown, highlighting that adding TFAP4 to the constructs seems to have a stronger effect than adding BATF. (B) Activation marker expression was analyzed on CAR T cells 8 days after electroporation, showing increased CD25 levels in TFAP4-overexpressing CAR T cells. (C) Exemplary flow cytometry plots are shown for phenotypic marker analysis 14 days after electroporation. Addition of BATF, especially TFAP4, increases the percentage of CD62L-positive T cells. (D) Phenotypic analysis of the various combinatorial constructs 14 days after electroporation. [Figure 6]Figure 6A-D shows differential expression of genes. Differentially expressed genes in the BATF-TFAP4 construct compared to the mCherry-NGFR control construct were analyzed by RNA-seq 14 days after electroporation. Volcano plots show differentially expressed genes. The most differentially expressed gene was TFAP4. Colors indicate whether the respective gene was found to be less than the most differentially expressed gene when comparing TFAP4-mCherry to control, BATF-mCherry to control, or both of these conditions. Highlighted in yellow are genes that only appear as differentially expressed when comparing BATF-TFAP4 to control. (B) Log2 fold change between test conditions and control (mCherry-NGFR) correlated between BATF-TFAP4 and other constructs. Correlation analysis showed that BATF-TFAP4 shared most changes in gene expression with TFAP4-mCherry and BATF-mCherry, but the correlation between BATF-TFAP4 and mCherry-JUN was lower. (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 was increased in BATF-TFAP4 cells, whereas expression of genes involved in apoptosis was decreased. [Figure 7] 7A-B show the killing ability of combinatorial constructs. (A) Combinatorial constructs were co-cultured with Nalm-6 / GD2 target cells and target cell killing was analyzed. The BATF-TFAP4 combinatorial construct competes with other constructs for killing ability. (B) This finding was confirmed across multiple effector:target (E:T) ratios. [Figure 8]Figure 8A-B shows the in vivo results. To analyze the in vivo functionality, a xenograft model of adoptive T cell transfer was used. NSG mice were injected IV with 0.5M Nalm-6 / GFP / Luc / GD2 cells on day 0, followed by IV injection of 1M GD2 CAR T cells 3 days later. Leukemic load was determined by bioluminescence imaging. (B) Leukemic growth analysis after injection of combinatorial T cells from two different T cell donors. [Figure 9] FIG. 9 is a schematic diagram of an exemplary GD2 CAR combinatorial construct. [Figure 10]Figure 10A-D shows that pooled combinatorial knock-in screens revealed efficient transcription factor combinations. (A) The ModPoKI platform was further improved to screen pairwise combinations of transcription factors and GD2 CAR. In the combinatorial functional module, 100 TFs were combined with 100 TFs (in addition to controls) to obtain ~10,000 different TF combinations. They were combined with GD2 CAR to obtain knock-in size ranges of ~3.3 to ~8.2 kb. (B) Barcode sequencing of the TFxTF combinatorial plasmid library showed size-dependent presentation, but confirmed that >99% of the constructs were presented after pooled assembly. Statistics were performed using linear regression (lm function in R studio). (C) Combinatorial construct knock-in rates were analyzed in cell pools by amplicon sequencing 4 days after electroporation, showing >99% presentation of ~10,000 constructs. N=2 individual donors. Statistics were performed using linear regression (lm function in R studio). (D) TFxTF combinatorial libraries were knocked into primary human T cells. T cells were cultured without adding target cells because the GD2 CAR provides a tonic stimulus. Cells were sorted on days 16 and 4 post-electroporation and log2 fold change (log2FC) was calculated (day 16 / day 4). Log2FC for combinatorial TFxTF constructs is shown, highlighting that the combination of TFAP4 and BATF performed best. N=2 individual donors. Statistics were calculated using DESeq2. Two possible construct orientations (e.g., BATF-TFAP4, and TFAP4-BATF) were combined into one data set to create a volcano plot. The right panel shows the barcode representation of the two construct orientations x two donors. [Figure 11]Figure 11A-E shows the combinatorial knock-in strategy and plasmid display of TFxTF libraries, related to Figure 10. (A-B) Schematic showing the pooled assembly approach used to generate the tonic signaling GD2 CAR+100 transcription factors (TFs) x 100 TFs combinatorial library, resulting in a ~10,000 member library. Inserts at TF positions 1 and 2 were generated separately by pooled PCR from the existing TF library. The scaffold (consisting of GD2 CAR+homology arms) and the two inserts were assembled in a pooled Hifi DNA assembly reaction to obtain the combinatorial ModPoKI plasmid library. Double-stranded HDR templates were generated by pooled PCR, followed by pooled non-viral knock-in into primary human T cells. (C) The resulting fusion region between the two transcription factors (combinatorial barcoded multicistronic adapter) consisted of both the barcode of the insert at position 1 and the barcode of the insert at position 2. To read out the barcode region, mRNA was isolated, reverse transcribed to cDNA, and amplified using 5' and 3' sequencing adapters to append Illumina Read 1 and Read 2. A second PCR step appended index sequences. Amplicon sequencing was performed and mRNA / cDNA combinatorial barcode counts were calculated. (D) Exemplary knock-in of a control construct containing GD2 CAR with tNGFR and RFP containing a new combinatorial barcoded multicistronic adapter sequence into primary human T cells. (E) TFxTF combinatorial libraries were knocked-in into primary human T cells. T cells were cultured without adding target cells since GD2 CAR provides tonic signaling. Cells were sorted on days 16 and 4 after electroporation and log2 fold change (log2FC) was calculated (day 16 / day 4). Log2FC of the top 10 combinatorial TFxTF constructs is shown and compared to control. N=2 individual donors. The orientation of the two constructs x barcode representation of the two donors are shown. [Figure 12]Figure 12A-G shows that combinatorial knock-in of TFAP4 and BATF induces favorable transcriptional programs in therapeutic T cells. (A) Competitive fitness assays with specific combinatorial knock-in constructs (including GD2 CAR) were performed. Approximately 50% / 50% co-culture analysis of various combinatorial constructs is shown. N=2 individual donors in technical triplicates. Mean+SEM is shown. Unpaired t-test was performed on day 4 to determine statistical significance. (B) Activation marker expression was analyzed on CAR T cells 8 days after electroporation, showing increased CD25 levels in TFAP4 KI CAR T cells and BATF-TFAP dual KI CAR T cells compared to BATF KI CAR T cells. N=2 individual donors in technical duplicates. Mean+SEM is shown. 2-way ANOVA with Holm-Sidak multiple comparisons correction was performed to determine statistical significance. (C) Exemplary flow cytometry plots are shown for phenotypic markers 14 days after electroporation. (D) Phenotypic analysis of various combinatorial KI CAR T cells 14 days after electroporation. N=2 individual donors in technical duplicates. Mean+SEM is shown. 2-way ANOVA with Holm-Sidak multiple comparison correction was performed to determine statistical significance. (E) Differentially expressed genes in BATF-TFAP4 KI CAR T cells compared to RFP-tNGFR control KI CAR T cells were analyzed by RNA-seq 14 days after electroporation, as shown in the Volcano plot. The most differentially expressed gene was TFAP4 (not shown, log2FC=5.0, padj=6.03E-77). Color indicates whether the respective gene was also found among the most differentially expressed genes when comparing TFAP4-RFP KI CAR T cells to control KI CAR T cells, BATF-RFP KI CAR T cells to control KI CAR T cells, or both of these comparisons. Highlighted in yellow are genes selectively differentially expressed in BATF-TFAP4 versus RFP-tNGFR KI CAR T cells. N=2 individual donors.(F) Combinatorial KI CAR T cells were co-cultured with Nalm-6 / GFP / Luc / GD2 target cells and target cell killing was analyzed. N=2 individual donors in technical triplicates. Mean+SEM is shown. Two-way ANOVA with Holm-Sidak multiple comparison correction was performed to determine statistical significance. (G) To assess in vivo function, NSG mice were injected IV with 0.5e6 Nalm-6 / GFP / Luc / GD2 cells on day 0 followed by IV injection of 1e6 GD2 CAR KI T cells 3 days later. Leukemic load was determined by bioluminescence imaging. N=2 individual T cell donors, 2-5 mice / donor / group. Two different donors are shown separately in Figure 14B. Mean+SEM is shown. Two-way ANOVA with Holm-Sidak multiple comparison test was performed to compare all constructs to the control (RFP-tNGFR) (both donors combined). [Figure 13]Figure 13A-E shows the transcriptome changes driven by combinatorial BATF-TFAP4 knock-in, related to Figure 12. (A) Various combinatorial validation constructs were knocked-in into primary human T cells as described in Figure 6. Bulk RNA-seq was performed 14 days after electroporation. Differentially expressed genes are plotted between BATF-TFAP4 KI GD2 CAR T cells and RFP-tNGFR KI GD2 CAR T cells. N=2 individual donors. (B) Log2 fold change in gene expression between the tested KI conditions and control KI (RFP-tNGFR) correlated between BATF-TFAP4 and other constructs. Correlation analysis showed that BATF-TFAP4 KI is most similar in gene expression to RFP-TFAP4 and BATF-RFP, while correlation between BATF-TFAP4 KI cells and RFP-JUN KI cells is lower. N=2 individual donors. Statistics were performed using linear regression (lm function in R studio). (C-E) Gene set enrichment analysis of BATF-TFAP4 combinatorial constructs compared to RFP-tNGFR (C), RFP-TFAP4 (D) or BATF-RFP (E) without or in addition to GD2+ target cells over a 24-hour period (day 14 vs. day 15 post-electroporation, respectively). Notably, 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 following stimulation with target cells. N=2 individual donors. [Figure 14]Figure 14A-B shows the functional capacity of combinatorial BATF-TFAP4 knock-in in vitro and in vivo, related to Figure 12. Combinatorial KI GD2 CAR T cells were co-cultured with Nalm-6 / GFP / Luc / GD2 target cells and target cell killing was analyzed by Incucyte. BATF-TFAP4 combinatorial KI GD2 CAR T cells tended to outperform other combinatorial KI GD2 CAR T cells in terms of in vitro killing capacity. N=2 individual donors in technical triplicates. Two-way ANOVA with Holm Sidak's multiple comparison test was performed to compare all constructs to the control (RFP-tNGFR). Mean + SEM is shown. (B) Data from Figure 12 split by donor. Mean + SEM is shown. [Figure 15] Figure 15 shows the results of CD25 sorting screening. GD2 CAR single insert TF libraries were knocked into three individual primary human T cell donors. Cells were sorted for CD25 high vs. low expression 8 days after electroporation. Log2FC of insert presentation in CD25 high vs. low bins is shown. Data was normalized to control (GFP / RFP) and fitted to the -1 / 1 axis. For some constructs, donor-to-donor variability was observed (mean+SEM shown). [Figure 16] Figure 16 shows the results of single stimulation screen (A375 / CD19 addition vs. non-stimulation conditions) and repeated stimulation screen (5 additions of A375 / CD19) in the complementation model using CD19 / 28z CAR in combination with the complete TF and SR single insert library described in Example III. Single knock-in TF and SR libraries in combination with CD19 / 28z CAR were knocked-in into 2-3 individual primary human T cell donors. Input populations as well as single stimulation populations (non-stimulation vs. A375 / CD19 stimulation for 4 days) and repeated stimulation populations (continuous stimulation with A375 / CD19 for 2 days x 5 times) were harvested and changes in construct abundance were analyzed using Log2FC on the input population. n=3 individual donors for repeated stimulation and n=2 individual donors for single stimulation. Data were normalized to control (GFP / RFP) and fitted to the -1 / 1 axis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] definition Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, patent applications and publications mentioned throughout this disclosure are incorporated by reference in their entirety.

[0052] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, a reference to "a transcript" or "the transcript" may include a plurality of transcripts.

[0053] The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better illustrate the invention, and does not pose a limitation on the scope of the invention unless otherwise asserted.

[0054] The terms "may," "may be," "can," and "can be," and related terms, unless the context clearly indicates otherwise, are intended to convey that the subject matter involved is optional (i.e., the subject matter is present in some instances and absent in other instances), and are not a reference to the ability or probability of the subject matter.

[0055] The terms "optional" and "optionally" mean that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material does occur or is present, as well as instances where it does not occur or is not present.

[0056] Use of the terms "including," "comprising," or "having" and variations thereof herein is meant to encompass the elements listed thereafter, and equivalents thereof, as well as additional elements. Embodiments recited as "including," "comprising," or "having" particular elements are also contemplated as "consisting essentially of" and "consisting of" those particular elements. As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations interpreted in the alternative ("or").

[0057] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be construed to encompass the recited materials or steps "and which do not materially affect the basic and novel characteristics" of the claimed invention. See In re Herz, 537 F.2d 549,551-52,190 USPQ461,463 (CCPA 1976) (emphasis in original). See also MPEP §2111.03. Thus, the term "consisting essentially of" as used herein should not be construed as equivalent to "comprising."

[0058] The term "nucleic acid" or "nucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers in single-stranded or double-stranded form.Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties as reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides.Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as the sequence explicitly shown. Specifically, degenerate codon substitutions can be achieved by generating sequences 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)).

[0059] The term "gene" can refer to a segment of DNA that is involved in the production or coding of a polypeptide chain. The term "gene" can include the regions before and after the coding region (leader and trailer), as well as the intervening sequences (introns) between individual coding segments (exons). Alternatively, the term "gene" can refer to a segment of DNA that is involved in the production or coding of non-translated RNA, such as rRNA, tRNA, guide RNA (e.g., single guide RNA) or microRNA.

[0060] As used herein, the term "endogenous" in reference to a nucleic acid, e.g., a gene or protein, within a cell refers to a nucleic acid or protein that is present in that particular cell as found in nature, e.g., in its natural genomic location or locus. Moreover, a cell that "endogenously expresses" a nucleic acid or protein expresses that nucleic acid or protein as found in nature.

[0061] As used herein, the term "heterologous" refers to something that is not normally found in nature. The term "heterologous nucleotide sequence" refers to a nucleotide sequence that is not normally found in a given cell in nature. Thus, a heterologous nucleotide sequence can be (a) foreign to its host cell (i.e., exogenous to the cell), (b) naturally found in a host cell (i.e., endogenous) but present in the cell in an unnatural amount (i.e., greater or less than the amount naturally found in the host cell), or (c) naturally found in a host cell but located outside its natural locus.

[0062] "Promoter" is defined as one or more nucleic acid control sequences that guide the transcription of nucleic acid.As used herein, promoter includes the necessary nucleic acid sequence near the start site of transcription, for example, in the case of polymerase II type promoter, TATA element.Promoter also includes the distal enhancer element or repressor element, which can be located several thousand base pairs from the start site of transcription.

[0063] A 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 is positioned so as to facilitate translation.

[0064] "Polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, in which the amino acid residues are linked by covalent peptide bonds.

[0065] As used herein, the term "complementary" or "complementarity" refers to specific base pairing between nucleotides or nucleic acids. Complementary nucleotides are generally A and T (or A and U), and G and C. The guide RNA described herein can include a DNA targeting sequence that is fully complementary or substantially complementary (e.g., has 1-4 mismatches) to a sequence, e.g., a genomic sequence.

[0066] "CRISPR / Cas" system refers to a broad class of bacterial systems for defense against foreign nucleic acids. CRISPR / Cas systems are found in a wide range of eubacterial and archaeal organisms. CRISPR / Cas systems include type I, type II and type III subtypes. Wild-type type II CRISPR / Cas systems utilize an RNA-mediated nuclease, e.g., Cas9, complexed with guide RNA and activator RNA to recognize and cleave foreign nucleic acid. Guide RNAs with both guide RNA and activator RNA activity are also known in the art. In some cases, such dual activity guide RNAs are referred to as single guide RNAs (sgRNAs).

[0067] Cas9 homologs are found in a wide variety of eubacteria, including, but not limited to, bacteria from the following taxa: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydiae-Verrucomicrobia, Chloroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein. Additional Cas9 proteins and their homologues are described, for example, in 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 US A. 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. Any mutant of the Cas9 nuclease provided herein can be optimized for efficient activity or enhanced stability in host cells. Thus, 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)).

[0068] As used herein, the term "Cas9" refers to an RNA-mediated nuclease (e.g., of bacterial or archaeal origin 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, Volume 163, Issue 3, p759-771, 22 October 2015) and its homologs. As used herein, terms such as "ribonucleoprotein" complexes refer to targeted nucleases, e.g., a complex between Cas9 and a crRNA (e.g., a guide RNA or a single guide RNA), a Cas9 protein and a trans-activating crRNA (tracrRNA), a Cas9 protein and a guide RNA, or a combination thereof (e.g., a complex containing a Cas9 protein, a tracrRNA, and a crRNA guide RNA). It is understood that in any of the embodiments described herein, the Cas9 nuclease can be replaced by a Cpf1 nuclease or any other guide nuclease.

[0069] As used herein, in relation to the modification of the genome of a cell, the phrase "modifying" refers to inducing structural changes in the sequence of the genome at a target genome region.For example, modifying can take the form of inserting a nucleotide sequence into the genome of a cell.For example, a nucleotide sequence that codes for one or more polypeptides can be inserted into the genome sequence of the TCR locus of a T cell.As used throughout, "TCR locus" refers to the location in a genome where the gene that codes for the TCR alpha subunit, the TCR beta subunit, the TCR gamma subunit or the TCR delta subunit is located.

[0070] Such modification can be performed, for example, by inducing a double-stranded break in the target genomic region, or a pair of single-stranded nicks on opposite strands adjacent to the target genomic region. Methods for inducing single-stranded or double-stranded breaks at or within the target genomic region include the use of Cas9 nuclease domain or its derivatives and guide RNA or guide RNA pair directed to the target genomic region.

[0071] As used herein, in relation to the introduction of nucleic acid or a complex that contains nucleic acid, for example, RNP-DNA template complex, the phrase "introducing" refers to the translocation of 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 nucleic acid or complex from outside the cell to inside the nucleus of the cell.Various methods of such translocation are contemplated, including, but not limited to, electroporation, contact with nanowire or nanotube, receptor-mediated internalization, translocation via cell-penetrating peptide, liposome-mediated translocation, etc.

[0072] As used herein, the term "selection marker" refers to a gene that allows the selection of host cells, such as T cells, that contain the marker. Selection markers can include, but are not limited to, fluorescent markers, luminescent markers, and drug selection markers, cell surface receptors, and the like. In some embodiments, the selection can be positive selection. That is, for example, cells that express the marker are isolated from the population to create an enriched population of cells that express the selection marker. Separation can be by any convenient separation technique that is suitable for the selection marker used. For example, if a fluorescent marker is used, the cells can be separated by fluorescence-activated cell sorting, while if a cell surface marker is inserted, the cells can be separated from the heterogeneous population by affinity separation techniques, such as magnetic separation, affinity chromatography, "panning" with affinity reagents bound to solid matrices, fluorescence-activated cell sorting, or other convenient techniques.

[0073] As used herein, a "cell" may be a human T cell, or a cell that can differentiate into a T cell, such as a T cell that expresses a TCR receptor molecule. These include hematopoietic stem cells and cells derived from hematopoietic stem cells.

[0074] As used herein, the phrase "hematopoietic stem cell" refers to a type of stem cell that can give rise to blood cells. Hematopoietic stem cells can give rise to cells of myeloid or lymphoid lineages, or a combination thereof. Hematopoietic stem cells are found primarily in bone marrow, but can be isolated from peripheral blood or a portion thereof. Various cell surface markers can be used to identify, select, or purify hematopoietic stem cells. In some cases, hematopoietic stem cells are expressed by the c-kit + and 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 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 as CD34 lo / - , SCA-1 + , Thy1 + / lo , CD38 + , C-kit + , lin - In some cases, hematopoietic stem cells express CD150 + CD48- CD244 - It is.

[0075] As used herein, the phrase "hematopoietic cells" refers to cells derived from hematopoietic stem cells. Hematopoietic cells may be obtained or provided by isolation from an organism, system, organ or tissue (e.g., blood or a portion thereof). Alternatively, hematopoietic stem cells may be isolated and hematopoietic cells may be obtained or provided by differentiating the stem cells. Hematopoietic cells include cells that have limited potential to differentiate into further cell types. Such hematopoietic cells include, but are not limited to, multipotent progenitor cells, lineage-restricted progenitor cells, common myeloid progenitor cells, granulocyte-macrophage progenitor cells or megakaryocyte-erythroid progenitor cells. Hematopoietic cells include cells of lymphoid and myeloid lineages, such as lymphocytes, erythrocytes, granulocytes, monocytes and platelets. In some embodiments, the hematopoietic cell is an immune cell, such as a T cell, a B cell, a macrophage, a natural killer (NK) cell, or a dendritic cell. In some embodiments, the cell is an innate immune cell.

[0076] As used herein, the phrase "T cells" refers to lymphoid cells that express T cell receptor molecules. T cells include human alpha beta (αβ) T cells and human gamma delta (γδ) T cells. T cells include, but are not limited to, naive T cells, stimulated T cells, primary T cells (e.g., not cultured), 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 subpopulations thereof. T cells are CD4 + , CD8 + , or CD4 + and CD8 + T cells can also be CD4 - , CD8 - , or CD4 - and CD8 -The T cell can be a helper cell, e.g., a T H Type 1, T H Type 2, T H Type 3, T H Type 9, T H 17 type or T FH The T cells can be cytotoxic T cells. The regulatory T cells can be FOXP3 + or FOXP3 - The T cells may be alpha / beta T cells or gamma / delta T cells. Optionally, the T cells are CD4 + CD25 hi CD127 lo In some cases, the T cells are type 1 regulatory (Tr1) T cells, T H 3 T cells, CD8+CD28- T cells, Treg17 T cells, and Qa-1 restricted T cells, or a combination or subpopulation thereof. + T cells. In some cases, T cells are CD4 + CD25 lo CD127 hi effector T cells. In some cases, T cells are CD4 + CD25 lo CD127 hi CD45RA hi CD45RO - The T cells are naive T cells. The T cells may be genetically engineered recombinant T cells.

[0077] As used herein, the term "primary" in relation to primary cells refers to cells that are not transformed or immortalized. Such primary cells can be cultured, subcultured or passaged a limited number of times (e.g., 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 are adapted to in vitro culture conditions. In some cases, primary cells are isolated from organisms, systems, organs or tissues, and optionally selected and directly utilized without culture or subculture. In some cases, primary cells are stimulated, activated or differentiated. For example, primary T cells can be activated by contact with (e.g., cultured in the presence of) CD3, CD28 agonist, IL-2, IFN-γ or combinations thereof.

[0078] "Treating" refers to any indicia of success in treating or ameliorating or preventing a disease, condition, or disorder, e.g., any objective or subjective parameter, such as relief; remission; reducing symptoms or making the disease state more tolerable to the patient; slowing the rate of degeneration or decline; or making the end point of degeneration less debilitating.

[0079] As used herein, the term "homologous recombination repair" or HDR refers to the cellular process in which the cut or nicked end of a DNA strand is repaired by polymerization from 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, e.g., a DNA template, can be introduced to obtain a specific HDR-induced change of the sequence at the target site. In this way, a specific mutation can be introduced at the cut site, e.g., the cut site created by the target-directed nuclease. A single-stranded or double-stranded DNA template can be used by a cell as a template for editing or modifying the genome of a cell, for example, by HDR. In general, a single-stranded or double-stranded DNA template has at least one region of homology to a target site. In some cases, a single-stranded or double-stranded DNA template has two homologous regions, e.g., 5'-end and 3'-end, adjacent to the region containing the DNA template that is inserted into the target cut site or target insertion site.

[0080] The term "substantial identity" or "substantially identical," when used in reference to a polynucleotide or polypeptide sequence, refers to a sequence having at least 60% sequence identity to a reference sequence. Alternatively, the percent identity can be any integer between 60% and 100%. Exemplary embodiments include at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% as compared to a reference sequence using a program described herein, preferably BLAST using standard parameters as described below. Those skilled in the art will recognize that these values ​​can be appropriately adjusted to determine the corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame arrangement, and the like.

[0081] In sequence comparison, typically, one sequence serves as a reference sequence, and test sequence is compared to the reference sequence.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, partial sequence coordinates are designated as necessary, and sequence algorithm program parameters are designated.Default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence to reference sequence based on program parameters.

[0082] As used herein, a "comparison window" includes reference to any one segment of a number of adjacent positions selected from the group consisting of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, where a sequence can be compared to a reference sequence of the same number of adjacent positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences 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 computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0083] Suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (NCBI) website. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short strings of length W in the query sequence, which match or meet some positive threshold score T when aligned with strings of the same length in database sequences. T refers to the neighborhood string score threshold (Altschul et al, supra). These initial neighborhood string hits serve as seeds to initiate searches to find longer HSPs that contain them. The string hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score value for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the string hits in each direction is stopped when the cumulative alignment score falls by an amount X from its maximum achieved value, when the cumulative score becomes zero or below due to the accumulation of one or more negatively scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a string size (W) of 28, an expectation (E) of 10, M=1, N=-2, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a string size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0084] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & 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 provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, the smallest sum probability in a comparison between a test nucleic acid and a reference nucleic acid is less than about 0.01, more preferably less than about 10 -5 less than about 10 -20 A nucleic acid is considered to be similar to a reference sequence if it is less than 1:1.

[0085] Detailed Description of the Invention The following description lists various aspects and embodiments of the present compositions and methods. The specific embodiments are not intended to define the scope of the compositions and methods. Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least included within the scope of the disclosed compositions and methods. The description should be read from the perspective of those skilled in the art. Thus, it does not necessarily include information that is known to those skilled in the art.

[0086] The present disclosure relates to compositions and methods for modifying the genome of T cells. The inventors have discovered that human T cells can be modified to change the specificity and function of T cells. In some embodiments, human T cells are modified to heterologously express one or more transcription factors to change T cell function, e.g., to enhance 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, e.g., to enhance tumor cell killing properties of T cells. In some embodiments, human T cells are modified to heterologously express a combination of a transcription factor and a switch receptor (i.e., a chimeric molecule or fusion protein that combines a ligand binding domain with a signaling domain) to change T cell function, e.g., to enhance tumor cell killing properties of T cells.

[0087] composition Provided herein is a human T cell that heterologously expresses one or more polypeptides, wherein the one or more polypeptides are encoded by a nucleic acid construct inserted into the TCR locus of the cell.Any of the polypeptides described herein can be heterologously expressed in human T cell.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 cell.In some examples, one or more, two or more, three or more polypeptides, etc. are encoded by one or more nucleic acid constructs.

[0088] Exemplary polypeptides include, but are not limited to, the amino acid sequences set forth in Tables 1 and 2, e.g., SEQ ID NOs: 30-58, and 69-85, 160, 161, and 162. Polypeptides comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 99%, or at least 100% identical to any one 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 set forth in Table 3. Polypeptides comprising an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 99%, or at least 100% identical to any one of the amino acid sequences set forth in Table 3 can also be heterologously expressed in human T cells.

[0089] In some embodiments, the nucleic acid construct inserted into the human T cell encodes two or more polypeptides. In some embodiments, the nucleic acid construct can include two or more nucleic acid sequences in a multicistronic construct. It is understood that the two or more nucleic acid sequences encoding two or more polypeptides can be in any order within the nucleic acid construct.

[0090] 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.

[0091] 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.

[0092] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0093] 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.

[0094] 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 an IRF2 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:36.

[0095] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0096] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] 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 SMAD4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:48.

[0103] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0104] 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 BATF3 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:32.

[0105] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0106] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0107] 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 TIM-3 / 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:69.

[0108] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0109] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[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 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:55.

[0111] 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 LTBR-IL-4R polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:71.

[0112] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0113] 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 2B4 / 4-1BB polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:73.

[0114] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0115] 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 CXCR1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:57.

[0116] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0117] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0118] In some embodiments, the nucleic acid construct inserted into the human T cell encodes a TIM-3 / 4-1BB polypeptide comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:69.

[0119] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0120] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0121] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0122] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0123] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0124] 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.

[0125] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0126] In some embodiments, the nucleic acid construct inserted into the human T cell encodes a SMAD1 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:56, and an MCT4 polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO:84.

[0127] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0128] 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;

[0129] In some embodiments, the nucleic acid construct inserted into the human T cell encodes an 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;

[0130] In some embodiments, the nucleic acid construct inserted into the human T cell 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.

[0131] In some embodiments, the polypeptide comprises a full-length HES2 protein, a FOXJ2 protein, or a 41-BB protein.

[0132] One or more of the nucleic acid sequences described herein, for example, one or more of SEQ ID NOs:1-29 and 59-68, can be inserted into the TCR locus of a T cell. A nucleic acid sequence encoding two or more of the polypeptides described herein can also be inserted into the TCR locus of a T cell. In some embodiments, a nucleic acid sequence encoding one or more sequences selected from the group consisting of SEQ ID NOs:30-58, 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 a T cell. In some embodiments, one or more nucleic acid sequences that are at least 80%, at least 85%, at least 90%, at least 99% or at least 100% identical to any one of the nucleic acid sequences set forth as SEQ ID NOs:1-29 and 59-68, or a nucleic acid sequence encoding two or more sequences 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, is inserted into the TCR locus of a T cell.

[0133] Any polypeptide sequence, or combination of polypeptide sequences; a nucleic acid sequence, or combination of nucleic acid sequences; a T cell comprising one or more polypeptides; a T cell comprising one or more nucleic acid sequences; or a method of using a T cell, polypeptide or nucleic acid sequence described herein may be claimed.

[0134] Insertion of a heterologous coding sequence into a TCR locus means that expression of one or more heterologous proteins is controlled by an endogenous TCR promoter and, in some embodiments, is expressed as part of a larger fusion protein with a TCR polypeptide that is then cleaved to form separate TCR and heterologous polypeptides. The TCR polypeptide can be endogenous or can be added to the TCR locus to provide T cells with new TCR affinity (for example, but not limited to, for cancer antigens). In some embodiments, the nucleic acid construct is inserted into a target insertion site within exon 1 of the TCR-alpha subunit constant gene (TRAC). In some embodiments, the nucleic acid construct is inserted into a target insertion site within exon 1 of the TCR-beta subunit constant gene (TRBC), for example, exon 1 of the TRBC1 gene, or exon 1 of the TRBC2 gene. When the nucleic acid construct is inserted into the TCR locus of a cell, the construct is under the control of an endogenous TCR promoter, for example, the TRAC1 promoter or the TRBC promoter. As described below, the nucleic acid constructs provided herein encode a TCR or a synthetic antigen receptor that is co-expressed with a polypeptide. Once the construct is integrated into the genome of the T cell by HDR, the T cell can be cultured under conditions that allow transcription of the inserted construct into a single mRNA sequence encoding the fusion polypeptide under the control of an endogenous promoter, which is then processed into separate heterologous polypeptides (e.g., by cleavage of the peptide sequence linking the polypeptides). Insertion of any of the nucleic acid constructs described herein that encode a heterologous T cell receptor and one or more heterologous polypeptide components produces T cells with the specificity of the heterologous TCR receptor and the function of the heterologous polypeptide. In some embodiments, the T cell expresses an antigen-specific TCR that recognizes the target antigen. Similarly, insertion of any of the nucleic acid constructs described herein that encode a synthetic antigen receptor and one or more heterologous polypeptides produces T cells with the specificity of the synthetic antigen receptor and the function of the heterologous polypeptide. In some embodiments, the T cell expresses a synthetic antigen receptor that recognizes the target antigen.

[0135] In some embodiments, the heterologous nucleic acid inserted into a human T cell comprises, in the following order: (i) a first self-cleaving peptide sequence; (ii) a first heterologous TCR subunit chain comprising a variable region and a constant region of a TCR subunit; (iii) a second self-cleaving peptide sequence; (iv) one or more heterologous polypeptides described herein; (v) a third self-cleaving peptide sequence; (vi) a variable region of the second heterologous TCR subunit chain; and (vii) a polypeptide encoding a portion of the N-terminus of an endogenous TCR subunit. and 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 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.

[0136] In some embodiments, the heterologous nucleic acid inserted into a human T cell comprises, 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 first heterologous TCR subunit chain comprising 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. and 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 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.

[0137] In the compositions and methods described herein, when 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, when 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.

[0138] As used throughout, the term "endogenous TCR subunit" is a TCR subunit that is endogenously expressed by a cell into which a nucleic acid construct is introduced, e.g., TCR-α or TCR-β. As described above, the nucleic acid constructs described herein encode two or more amino acid sequences that are processed, i.e., self-cleaved, to produce two or more amino acid sequences, e.g., a TCR-α subunit, a TCR-β subunit, and a polypeptide encoded by the construct, or a synthetic antigen receptor (e.g., a CAR (see, e.g., Guedan et al. "Engineering and Design of Chimeric Antigen Receptors," Mol. Ther. Methods & Clinical Development 12:145-156 (2019)) or a SynNotch receptor (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 a polypeptide encoded by the construct.

[0139] In some nucleic acid constructs, the size of the nucleic acid encoding the N-terminal portion of the endogenous TCR subunit depends on the number of nucleotides of 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 more than 25 nucleotides, there may be less than or more than 25 nucleotides of nucleic acid encoding the N-terminal portion of the endogenous TCR-α subunit in the construct.

[0140] In the above example, translation of the mRNA sequence transcribed from the construct results in the expression of four or more separate polypeptide sequences, namely, an inactive endogenous variable region peptide lacking a transmembrane domain (which may be degraded, for example, in the endoplasmic reticulum or secreted post-translationally), a full-length heterologous antigen-specific TCR-β or TCR-α chain, one or more polypeptide sequences described herein, and one protein that self-cleaves into a full-length heterologous antigen-specific TCR-α 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 a desired function in the T cell. The mRNA transcribed from any of the other nucleic acid constructs described herein is similarly processed in the T cell.

[0141] In some embodiments, the nucleic acid construct comprises, in the following order: (i) a first self-cleaving peptide sequence; (ii) a first heterologous TCR subunit chain comprising 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 comprising 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 or encodes a polyA sequence, and 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 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.

[0142] 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 polyA sequence.

[0143] 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 polyA sequence. It is understood that in any of the nucleic acid constructs described herein, the heterologous polypeptide or polypeptides can be cleaved by an auto-cleaving peptide, as described above, such that the heterologous polypeptide or polypeptides are auto-cleaved into one or more separate polypeptides in human T cells.

[0144] Examples of self-cleaving peptides include, but are not limited to, self-cleaving viral 2A peptides, such as porcine teschovirus-1 (P2A) peptide, Zosea asignavirus (T2A) peptide, equine rhinitis A virus (E2A) peptide, or foot and mouth disease virus (F2A) peptide. Self-cleaving 2A peptides allow expression of multiple gene products from a single construct. (See, for example, 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 all the same. In other embodiments, at least one of the two or more self-cleaving peptides is different.

[0145] In some embodiments, one or more linker sequences separate the components of the nucleic acid construct. The linker sequences can be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids in length.

[0146] In some embodiments, the nucleic acid construct comprises adjacent homology arm sequences having homology to the human TCR locus. In the compositions and methods described herein, the length of one or both homology arm sequences is at least about 50, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400 or at least about 450 nucleotides. In some cases, the nucleotide sequence that is homologous to the genomic sequence is at least 80%, at least 90%, at least 95%, at least 99% or at least 100% complementary to the genomic sequence. In some embodiments, one or both homology arm sequences optionally comprise mismatched nucleotide sequences compared to the homologous sequence in the genomic sequence in the TCR locus adjacent to the insertion site in the TCR locus.

[0147] In some embodiments, the nucleic acid construct optionally encodes a selectable marker that can be used to separate or isolate subpopulations of the modified T cells. 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.

[0148] Any of the polypeptides described herein can be encoded by any of the nucleic acid constructs 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 the group consisting of SEQ ID NOs: 30-58, and 69-85. In some embodiments, the heterologous nucleic acid construct encodes two or more polypeptides, each polypeptide being at least 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 30-58, and 69-85.

[0149] Also provided are polypeptides that are at least 95% identical to SEQ ID NOs: 30-58, and 69-85. Nucleic acids encoding these polypeptides are also provided herein.

[0150] Also provided is a human T cell comprising any of the nucleic acid sequences described herein. Also provided is a population of human T cells (e.g., a plurality of human T cells) comprising any of the nucleic acid sequences described herein.

[0151] Any of the nucleic acid constructs encoding any of the polypeptides described herein can be used to generate engineered T cells. In some embodiments, the method includes administering to a human T cell (i) a targeted nuclease that cleaves a target region within the TCR locus of the human T cell to create a target insertion site within the genome of the cell; and (ii) 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; an ATF2 protein; a polypeptide comprising a 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) a polypeptide comprising a human TIM3 extracellular domain or a portion thereof linked via a transmembrane domain to a human 4-1BB intracellular domain (and optionally about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain); a polypeptide comprising (a) a human SOX5 protein and (b) a truncated TIGIT protein comprising a human TIGIT extracellular domain or a portion thereof, a TIGIT transmembrane domain, and about 1-10 (e.g., 7) amino acids of the human TIGIT intracellular domain;A polypeptide comprising (a) a human MYC protein and (b) a polypeptide comprising a human TGFbR2 extracellular domain or a portion thereof linked via a transmembrane domain to amino acids 41 to 142 of human MyD88 (and optionally, 1 to 10 (e.g., 7) amino acids of the TGFbR2 intracellular domain); a polypeptide comprising a BATF3 protein and a 4-1BB protein; a polypeptide comprising (a) a TFAP4 protein and (b) a polypeptide comprising a human LTBR extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human IL-4R (and optionally, 1 to 10 (e.g., 7) amino acids of the LTBR intracellular domain); (a) an ATF1 protein and (b) a polypeptide comprising (a) a BATF protein and (b) a human 2B4 extracellular domain or a portion thereof linked to the intracellular domain of human 4-1BB, and optionally a polypeptide comprising about 1 to 15 (e.g., 12) amino acids of the human 4-1BB extracellular domain; a polypeptide comprising (a) a RORC protein and (b) a VISTA extracellular domain or a portion thereof linked to the intracellular domain of human CD28, and optionally a polypeptide comprising about 1 to 15 (e.g., 12) amino acids of the human CD28 extracellular domain; a polypeptide comprising a BATF protein and a CXCR a polypeptide comprising an 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 linked via a transmembrane domain to a human OX40 intracellular domain (and optionally, about 1 to 10 (e.g., 7) amino acids of the TNFRSF12 intracellular domain); a polypeptide comprising a human TIM3 extracellular domain or a portion thereof linked via a transmembrane domain to a human 4-1BB intracellular domain (and optionally, about 1 to 15 (e.g., 12) amino acids of the human 4-1BB extracellular domain);a polypeptide comprising a human BATF2 extracellular domain or a portion thereof linked to a truncated PD-1 comprising a human PD-1 extracellular domain or a portion thereof, a PD-1 transmembrane domain, and about 1-12 (e.g., 10) amino acids of 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) a polypeptide comprising a LTBR extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human CD28 (and optionally, 1-10 (e.g., 7) amino acids of the LTBR intracellular domain); a polypeptide comprising (a) a human FOXJ2 protein and (b) a polypeptide comprising a LTBR extracellular domain or a portion thereof linked via a transmembrane domain to amino acids 41-142 of human MyD88 (and optionally, 1-10 (e.g., 7) amino acids of the LTBR intracellular domain). introducing a nucleic acid construct encoding one or more of a peptide; a polypeptide comprising (a) a human HOPX protein and (b) a polypeptide comprising an IL4RA extracellular domain or a portion thereof linked via a transmembrane domain to an intracellular domain of human 41-BB; a polypeptide comprising (a) an ID2 protein and (b) a BLTA extracellular domain or a portion thereof linked via a transmembrane domain to an intracellular domain of human 4-1BB, and optionally a polypeptide comprising about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain; 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 linked via a transmembrane domain to an intracellular domain of human IL-4R (and optionally 1-10 (e.g., 7) amino acids of the LTBR intracellular domain); and (b) causing recombination, thereby inserting the nucleic acid construct into the target insertion site to generate an engineered human T cell;

[0152] In some embodiments, a nucleic acid is inserted into a T cell by introducing into the T cell (a) a targeted nuclease that cleaves a target region within exon 1 of a TCR-α subunit constant gene (TRAC) to create an insertion site in the genome of the T cell, and (b) a nucleic acid construct that integrates into the insertion site by homology directed repair (HDR). In some embodiments, a nucleic acid construct is inserted into a T cell by introducing into the T cell (a) a targeted nuclease that cleaves a target region within exon 1 of a TCR-β subunit constant gene (TRBC), e.g., TRBC1 or TRBC2, to create an insertion site in the genome of the T cell, and (b) a nucleic acid construct, where the nucleic acid sequence integrates into the insertion site by homology directed repair (HDR).

[0153] In some embodiments, the nucleic acid construct is inserted by introducing the viral vector that contains the nucleic acid construct into cells.Examples of viral vectors include, but are not limited to, adeno-associated virus (AAV) vector, retrovirus vector or lentivirus vector.In some embodiments, the lentivirus vector is an integrase-deficient lentivirus vector.

[0154] In some embodiments, the nucleic acid construct is inserted by introducing a non-viral vector containing the nucleic acid construct into the cell. In non-viral delivery methods, the nucleic acid can be naked DNA or can be in a non-viral plasmid or a non-viral vector. In non-viral delivery methods, the DNA template can be inserted using a non-viral genome targeting protocol based on the Cas9 shuttle system and anionic polymers. 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).

[0155] 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, "pure single-stranded DNA" refers to a single-stranded DNA that is substantially devoid of the other or opposite strand of DNA. By "substantially devoid," it is meant that the pure single-stranded DNA is devoid of at least 100 times the single strand of another strand of DNA. In some cases, the DNA template is a double-stranded or single-stranded plasmid or minicircle.

[0156] In some embodiments, 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 (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 the 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 the TRBC gene.

[0157] As used throughout, a guide RNA (gRNA) sequence is a sequence that specifically binds or hybridizes to a target nucleic acid in the genome of a cell such that it interacts with a site-specific or targeting nuclease and the gRNA and the targeting nuclease co-localize to the target nucleic acid in the genome of the cell. Each gRNA contains a DNA targeting sequence or protospacer sequence of about 10-50 nucleotides in length that specifically binds or hybridizes to a target DNA sequence in the genome. For example, the DNA targeting sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49 or about 50 nucleotides in length. In some embodiments, the gRNA comprises a crRNA sequence and a transactivating crRNA (tracrRNA) sequence. In some embodiments, the gRNA does not comprise a tracrRNA sequence.

[0158] Generally, the DNA targeting sequence is designed to be complementary (e.g., fully complementary) or substantially complementary to the target DNA sequence. In some cases, the DNA targeting sequence can incorporate wobble or degenerate bases to bind to multiple genetic elements. In some cases, the 19 nucleotides at the 3' or 5' end of the binding region are fully complementary to the target genetic element or elements. In some cases, the binding region can be altered to increase stability. For example, non-natural nucleotides can be incorporated to increase RNA resistance to degradation. In some cases, the binding region can be altered or designed to avoid or reduce the formation of secondary structures within the binding region. In some cases, the binding region can be designed to optimize GC content. In some cases, the GC content is preferably about 40% to about 60% (e.g., 40%, 45%, 50%, 55%, 60%). In some embodiments, the Cas9 protein can be in the form of an active endonuclease such that upon binding to the target nucleic acid as part of a complex with a guide RNA or as part of a complex with a DNA template, a double-stranded break is introduced into the target nucleic acid. In the methods provided herein, Cas9 polypeptide or a nucleic acid encoding Cas9 polypeptide can be introduced into a cell. Double-strand breaks can be repaired by HDR to insert DNA template into the genome of the cell. Various Cas9 nucleases can be utilized in the methods described herein. For example, Cas9 nucleases that require a NGG protospacer adjacent motif (PAM) immediately 3' to the region targeted by guide RNA can be utilized. Such Cas9 nucleases can target regions within exon 1 of TRAC or exon 1 of TRAB that contain, for example, NGG sequences. As another example, Cas9 proteins that require an orthogonal PAM motif can be used to target sequences that do not have adjacent NGG PAM sequences. 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).

[0159] In some cases, the Cas9 protein is a nickase, so that when it binds to a target nucleic acid as part of a complex with a guide RNA, a single-strand break or nick is introduced into the target nucleic acid. A pair of Cas9 nickases, each bound to a structurally distinct guide RNA, can target two proximal sites in the target genomic region, so that a pair of proximal single-strand breaks can be introduced into the target genomic region, for example, exon 1 of the TRAC gene or exon 1 of the TRBC gene. The nickase pair can provide enhanced specificity, since off-target effects are more likely to result in a single nick, which is generally repaired without damage by base excision repair mechanisms. Exemplary Cas9 nickases include Cas9 nucleases with D10A or H840A mutations (see, e.g., Ran et al. “Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity,” Cell 154(6):1380-1389 (2013)).

[0160] 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, the RNP-nucleic acid sequence complex comprising (i) an RNP comprising the Cas9 nuclease and the guide RNA, and (ii) the nucleic acid sequence or nucleic acid construct.

[0161] In some embodiments, the molar ratio of RNP to DNA template can be from about 3:1 to about 100:1. For example, the molar ratio can be from about 5:1 to about 10:1, from about 5:1 to about 15:1, from 5:1 to about 20:1, from 5:1 to about 25:1, from about 8:1 to about 12:1, from about 8:1 to about 15:1, from about 8:1 to about 20:1, or from about 8:1 to about 25:1.

[0162] In some embodiments, the DNA template in the RNP-DNA template complex is at a concentration of about 2.5 pM to about 25 pM. In some embodiments, the amount of DNA template is about 1 μg to about 10 μg.

[0163] In some cases, the RNP-DNA template complex is formed by incubating the RNP with the DNA template for less than 1 minute to about 30 minutes at a temperature of about 20° C. to about 25° C. In some embodiments, the RNP-DNA template complex is mixed with the cells prior to introducing the RNP-DNA template complex into the cells.

[0164] In some embodiments, the nucleic acid sequence or the RNP-DNA template complex is introduced into the cell by electroporation. Methods, compositions and devices for electroporating cells to introduce the RNP-DNA template complex may include those described in the Examples herein. Additional or alternative methods, compositions and devices for electroporating cells to introduce the RNP-DNA template complex may include those described in WO / 2006 / 001614 or Kim, JA et al. Biosens. Bioelectron. 23, 1353-1360 (2008). Additional or alternative methods, compositions and devices for electroporating cells to introduce the RNP-DNA template complex may include those described in U.S. Patent Application Publication No. 2006 / 0094095, U.S. Patent Application Publication No. 2005 / 0064596 or U.S. Patent Application Publication No. 2006 / 0087522. Additional or alternative methods, compositions and devices for electroporating cells to introduce RNP-DNA template complexes may include those described in Li, L Het al. Cancer Res. Treat. 1, 341-350 (2002), U.S. Patent No. 6,773,669, U.S. Patent No. 7,186,559, U.S. Patent No. 7,771,984, U.S. Patent No. 7,991,559, U.S. Patent No. 6,485,961, U.S. Patent No. 7,029,916, and U.S. Patent Application Publication No. 2014 / 0017213 and U.S. Patent Application Publication No. 2012 / 0088842. Additional or alternative 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).

[0165] In some embodiments, the RNP is delivered to the cell in the presence of an anionic polymer. In some embodiments, the anionic polymer is an anionic polypeptide or an anionic polysaccharide. In some embodiments, the anionic polymer is an anionic polypeptide (e.g., polyglutamic acid (PGA), polyaspartic acid, or polycarboxyglutamic acid). In some embodiments, the anionic polymer is an anionic polysaccharide (e.g., hyaluronic acid (HA), heparan, heparan sulfate, or glycosaminoglycan). In some embodiments, the anionic polymer is poly(acrylic acid) (PAA), poly(methacrylic acid) (PMAA), poly(styrene sulfonate), or polyphosphate. In some embodiments, the anionic polymer has a molecular weight of at least 15 kDa (e.g., 15 kDa to 50 kDa). In some embodiments, the anionic polymer and the Cas protein are in a molar ratio of 10:1 to 120:1, respectively (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). In some embodiments of this aspect, the molar ratio of sgRNA:Cas protein is 0.25:1 to 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).

[0166] 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 one 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 subcellular location (e.g., the nucleus) without cleaving the DNA binding protein target sequence so that the HDR template can be integrated into the cleaved 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 may 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 yet 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 yet 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.

[0167] In some embodiments, the nucleic acid sequence or RNP-DNA template complex is about 1×10 5 ~Approx. 2×10 6 For example, the nucleic acid sequence or RNP-DNA template complex is introduced into about 1×10 5 ~ approx. 5 x 10 cells 5 cells, approximately 1 x 10 5 ~ approx. 1 x 10 cells 6 cells, 1 x 10 5 ~ approx. 1.5 x 10 cells 6 cells, 1 x 10 5 ~ approx. 2 x 10 cells 6 cells, approximately 1 x 10 6 ~ approx. 1.5 x 10 cells 6 cells, or approximately 1 x 10 6 ~ approx. 2 x 10 cells 6 The antibody can be introduced into a cell.

[0168] 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 - A T cell. In some embodiments, the T cell is a T cell that expresses a TCR receptor or that differentiates into a T cell that expresses a TCR receptor.

[0169] Treatment method Any of the methods and compositions described herein can be used to modify T cells obtained from human subjects. Any of the methods and compositions described herein can be used to modify T cells obtained from human subjects to enhance immune response in subjects. Any of the methods and compositions described herein can be used to modify T cells obtained from human subjects to treat or prevent disease (e.g., cancer, infectious disease, autoimmune disease, transplant rejection, graft-versus-host disease or other inflammatory disorder in subjects).

[0170] As used herein, subject means an individual. A subject may be an adult subject or a pediatric subject. Pediatric subjects include subjects ranging in age from birth to 18 years of age.

[0171] The modified T cells described herein, i.e., polypeptides 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; (a) a polypeptide comprising a human TFAP4 protein; and (b) a polypeptide comprising a human TIM3 extracellular domain or a portion thereof linked via a transmembrane domain to a human 4-1BB intracellular domain (and optionally about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain); (a) a polypeptide comprising a human SOX5 protein; and (b) a truncated TIGIT protein comprising a human TIGIT extracellular domain or a portion thereof, a TIGIT transmembrane domain, and about 1 to 10 (e.g., 7) amino acids of the human TIGIT intracellular domain; (a) a polypeptide comprising a human MYC protein; and (b) a polypeptide comprising the human TGFbR2 extracellular domain or a portion thereof linked via a transmembrane domain to amino acids 41-142 of human MyD88 (and optionally, 1-10 (e.g., 7) amino acids of the TGFbR2 intracellular domain); A polypeptide comprising a BATF3 protein and a 4-1BB protein; (a) a polypeptide comprising a TFAP4 protein; and (b) a polypeptide comprising the human LTBR extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human IL-4R (and optionally 1 to 10 (e.g., 7) amino acids of the LTBR intracellular domain); (a) a polypeptide comprising an ATF1 protein and (b) a polypeptide comprising the extracellular domain of human IL4RA linked to the intracellular domain of human 4-1BB or a portion thereof; (a) a BATF protein; and (b) a polypeptide comprising a human 2B4 extracellular domain or a portion thereof linked to an intracellular domain of human 4-1BB, and optionally a polypeptide comprising about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain; (a) a RORC protein; and (b) a polypeptide comprising a VISTA extracellular domain or a portion thereof linked to the intracellular domain of human CD28, and optionally a polypeptide comprising about 1-15 (e.g., 12) amino acids of the human CD28 extracellular domain; a polypeptide comprising a BATF protein and a CXCR1 protein; a polypeptide comprising a MAFF protein and an IL2RA protein; (a) a polypeptide comprising an ATF1 protein; and (b) a polypeptide comprising the human TNFRSF12 extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human OX40 (and optionally 1 to 10 (e.g., 7) amino acids of the TNFRSF12 intracellular domain); a polypeptide comprising a human TIM3 extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human 4-1BB (and optionally about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain); a polypeptide comprising the human BATF2 extracellular domain or a portion thereof linked to a truncated PD-1 comprising the human PD-1 extracellular domain or a portion thereof, the PD-1 transmembrane domain, and about 1-12 (e.g., 10) amino acids of the human PD-1 intracellular domain; A polypeptide comprising a human TCF7 protein and a human LAT1 protein; A polypeptide comprising the human 4-1BB protein; A polypeptide comprising a human STAT5 protein and a human IL-2RA protein; (a) a polypeptide comprising a human FOXJ2 protein; and (b) a polypeptide comprising the LTBR extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human CD28 (and optionally 1 to 10 (e.g., 7) amino acids of the LTBR intracellular domain); (a) a polypeptide comprising a human FOXJ2 protein; and (b) a polypeptide comprising the LTBR extracellular domain or a portion thereof linked via a transmembrane domain to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g., 7) amino acids of the LTBR intracellular domain); (a) a polypeptide comprising a human HOPX protein and (b) a polypeptide comprising the 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) an ID2 protein; and (b) a polypeptide comprising a BLTA extracellular domain or a portion thereof linked to an intracellular domain of human 4-1BB, and optionally a polypeptide comprising about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain; A polypeptide comprising a human SMAD1 protein and a MCT4 protein; or Provided herein are methods of enhancing an immune response in a human subject, comprising administering either (a) a human SMAD1 protein; and (b) a T cell that heterologously expresses a polypeptide comprising the LTBR extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human IL-4R (and optionally, 1 to 10 (e.g., 7) amino acids of the LTBR intracellular domain).

[0172] In some embodiments, T cells are obtained from a subject and modified to express antigen-specific TCR or synthetic antigen receptor using any of the methods provided herein before administering modified T cells to the subject.In some embodiments, the subject has cancer and the target antigen is a cancer-specific antigen.In some embodiments, the subject has an autoimmune disorder and the antigen is an antigen associated with the autoimmune disorder.In some embodiments, the subject has an infectious disease and the target antigen is an antigen associated with the infectious disease.

[0173] Also provided is a method for treating cancer in a human subject, comprising the steps of: a) obtaining T cells from a subject; b) modifying T cells in the subject to express an antigen-specific TCR or synthetic antigen receptor that recognizes a target antigen using any of the methods provided herein; and c) administering the modified T cells to the subject, wherein the human subject has cancer and the target antigen is a cancer-specific antigen.As used throughout, the phrase "cancer-specific antigen" refers to an antigen that is unique to cancer cells or that is more abundantly expressed in cancer cells than in non-cancerous cells.In some embodiments, the cancer-specific antigen is a tumor-specific antigen.

[0174] As used herein, cancer is a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a blood cancer or hematological 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, renal cancer, liver cancer, brain cancer, lymphoma, leukemia (e.g., acute myeloid leukemia), myeloma, lung cancer, and the like. It is understood that the methods provided herein can also be used to target circulating cancer cells, for example, cells shed by solid tumors into the bloodstream of a subject.

[0175] In some embodiments, a heterogeneous and cancer-specific T cell population, tumor infiltrating lymphocytes, is obtained from a cancer subject and expanded ex vivo. The characteristics of the patient's cancer determine a set of tailored cell modifications, which are applied to tumor infiltrating lymphocytes using any of the methods described herein.

[0176] Also provided herein are methods of treating an autoimmune disease, an allergic disorder, or transplant rejection in a human subject, comprising the steps of: a) obtaining T cells from a subject, b) modifying the T cells in the subject to express an antigen-specific TCR or a synthetic antigen receptor that recognizes a target antigen using any of the methods provided herein, and c) administering the modified T cells to the subject, wherein the human subject has 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.

[0177] As used herein, autoimmune disease refers to the disease in which immune system cannot distinguish between subject's own cells and foreign cells, and thus immune system attacks healthy cells in the body by mistake.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, Sjogren's syndrome, autoimmune thyroid disease and celiac disease.

[0178] Also provided is a method of treating an infectious disease in a human subject, comprising the steps of: a) obtaining T cells from a subject; b) modifying the T cells in the subject to express an antigen-specific TCR or a synthetic antigen receptor that recognizes a target antigen using any of the methods provided herein; and c) administering the modified T cells to the subject, wherein the subject has an infectious disease and the target antigen is an antigen associated with the infectious disease in the subject.

[0179] In some embodiments, the T cells are autologous (i.e., from the same subject receiving the modified cells) or allogeneic (i.e., from a subject other than the subject receiving the modified cells). In some examples, the T cells are iPSC-derived T cells. See, e.g., Nagano et al. Mol. Therapy Methods & Clinical Development 16:126-135 (2020). Any of the therapeutic methods provided herein may further include expanding the population of T cells before the T cells are modified. Any of the therapeutic methods provided herein may further include expanding the population of T cells after the T cells are modified and before administration to the subject.

[0180] Screening Methods Also provided are methods for identifying combinations of heterologous polypeptides, e.g., combinations of two or more transcription factors, or combinations of transcription factor switch receptors, that can alter one or more properties of a human T cell, e.g., that can enhance the cell killing properties of a human T cell or a population of human T cells. Provided herein is a method for identifying targeted insertion in the genome of a cell, comprising the steps of: (a) (i) introducing into a population of cells a targeted nuclease that cuts a target region in the genome of the cell to create a target insertion site, and (ii) a plurality of DNA templates that are different from each other in sequence, each DNA template comprising: i. two or more heterologous coding nucleic acid sequences or heterologous non-coding nucleic acid sequences, and ii. a unique barcode nucleotide sequence that indicates the identity of each heterologous coding nucleic acid sequence or each heterologous non-coding nucleic acid sequence; (b) allowing recombination to occur, thereby creating a population of modified cells; (c) (i) isolating genomic DNA, or (ii) isolating mRNA from the cell and generating cDNA from the mRNA; and (d) sequencing genomic DNA or cDNA to identify the DNA template that is inserted into the target insertion site of the cell.In some methods, isolating mRNA further comprises digesting the DNA from the cell. In some methods, a barcode sequence within the genomic DNA or cDNA is sequenced to identify two or more heterologous coding or non-coding nucleic acid sequences.

[0181] 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 skilled in the art. Any of the screening methods described herein can further include, for example, amplifying genomic DNA or cDNA before sequencing genomic DNA or cDNA. In some methods, two or more heterologous coding nucleic acid sequences or heterologous non-coding nucleic acid sequences are separated by a linker, and each heterologous coding nucleic acid sequence or heterologous non-coding nucleic acid sequence comprises a unique barcode sequence. In some methods, the barcode that identifies each heterologous coding nucleic acid sequence or heterologous non-coding nucleic acid sequence is placed between the heterologous coding nucleic acid sequence or heterologous non-coding nucleic acid sequence, for example, between the first heterologous coding sequence and the second heterologous coding sequence. See exemplary FIG. 1A, in which a nucleic acid sequence comprising a nucleic acid sequence encoding transcription factor 1 (TF1) and a first barcode identifying TF1 (3'BC TF1) is linked 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).

[0182] In some methods, the DNA template comprises 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 comprises a heterologous nucleic acid sequence encoding a transcription factor and a heterologous nucleic acid sequence encoding a switch receptor.

[0183] In some embodiments, the method further comprises determining the relative number of cells in the population with the various DNA templates inserted at the target insertion site. In some embodiments, the method further comprises applying selection pressure to the population of modified cells. In some embodiments, the method further comprises comparing the relative number of cells in the population with the various DNA templates inserted at the target insertion site 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 the cells. In some methods, the population is a population of mammalian cells. In some methods, the mammalian cells are human cells. In some methods, the human cells are T cells. In some methods, the T cells are regulatory T cells, effector T cells or naive T cells. In some methods, the effector T cells are CD8+ T cells or CD4+ T cells. In some methods, the effector T cells are CD8+CD4+ T cells. In some methods, the cells are primary cells. In some methods, the DNA template comprises nucleic acids encoding two or more heterologous polypeptides.

[0184] Disclosed are materials, compositions and components that can be used for, used in conjunction with, used in preparation for, or are products of the disclosed methods and compositions.These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that each of the various individual and collective combinations and permutations of these compounds are specifically contemplated and described herein, although specific references may not be explicitly disclosed.For example, when a method is disclosed and described, and several modifications that can be made to one or more molecules contained in the method are described, any and all combinations and permutations of the method, and possible modifications, are specifically contemplated, unless otherwise indicated.Similarly, any subset or combination of these is specifically contemplated and disclosed.This concept applies to all aspects of the present disclosure, including, without limitation, the steps in the method of using the disclosed compositions. Thus, if there are various additional steps that may be performed, it is understood that each of these additional steps may be performed by any particular method step, or combination of method steps, of the disclosed methods, and that each such combination, or subset of combinations, is to be considered as specifically contemplated and disclosed.

[0185] Publications cited herein and the material for which they are cited are specifically incorporated herein by reference in their entireties. EXAMPLES

[0186] Example I Isolation and culture of primary human T cells T cell isolation was performed as previously described (Roth et al., Cell 181:728-744 (2020)). In summary, human T cells were isolated from leukapheresis products (Stemcell, samples collected by approved Stemcell IRB) using the EasySep Human T Cell Isolation Kit (Stemcell). The use of human material was approved by the UCSF Committee on Human Research (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-cystine (VWR). Prior to electroporation, T cells were stimulated for 48 hours with 1e6 cells per ml of medium containing 500U / ml IL-2 (Peprotech), 5ng / ml IL-7 (ThermoFisher), 5ng / ml IL-15 (ThermoFisher), and CTS (Cell Therapy Systems) CD3 / CD28 Dynabeads (ThermoFisher, 1:1 bead:cell ratio). After nucleofection, T cells were cultured in X-VIVO 15 medium containing 500U / ml IL-2.

[0187] Generation of combinatorial libraries for pooled knock-in A combinatorial GD2 CAR plasmid library was generated by pooled Gibson assembly of the GD2 CAR pUC19 backbone and insert 1 and insert 2. The backbone included the published GD2 CAR sequence (Lynn et al., 2019) with CD28 costimulation and mutations that increase tonic signaling within the IgG1 CH2 region (Watanabe et al., 2016). Inserts were PCR amplified from the existing library (as described in International Patent Application Publication 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 combinatorial inserts. An exemplary plasmid map of the final construct is shown in Figure 9. For insert 1, a transcription factor library was used as described in International Patent Application Publication WO2022072932. For insert 2, a transcription factor library or a switch receptor (SR) library (as described in International Patent Application Publication WO2022072932) was used. The GD2 CAR backbone, the pool of insert 1, and the pool of insert 2 were assembled using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs (NEB)). The assembled products were SPRI-purified using Sera-Mag SpeedBeads and transformed into Endura electrocompetent cells (Lucigen) and maxiprepped (Zymo Research) for subsequent use. HDR templates (HDRT) were generated as previously described (Roth et al., 2020). In summary, combinatorial GD2 plasmid pools were used as templates for PCR amplification (KAPA HiFi HotStart ReadyMix, Roche) to generate double-stranded DNA templates. Templates were SPRI-purified and eluted in H2O. Plasmids for arrayed knock-ins of various combinations during the validation phase were generated in a similar manner.Instead of a library, a single construct served as a template for PCR to generate insert 1 and insert 2, followed by Gibson assembly with the GD2 pUC19 backbone. The GD2 CAR sequence was provided by Robbie Majzner and Crystal Mackall (Stanford University).

[0188] Cas9 RNP electroporation Electroporation was performed as previously described (Roth et al., 2020). To generate ribonucleoproteins (RNPs), crRNA and tracrRNA (stock 160uM, both Dharmacon) were mixed in a 1:1 volume ratio and annealed by incubation at 37C for 30 minutes to form an 80μM guide RNA (gRNA) solution. Poly-L-glutamic acid (PGA, stock 125mg / ml, Sigma) was mixed with gRNA in a 0.8:1 volume ratio and then complexed with Cas9-NLS (QB3 Macrolab) for a final volume ratio of 1:0.8:1 gRNA:PGA:Cas9. These were incubated at 37C for 15 minutes to form a 14.3μM RNP solution. RNP and HDRT were mixed with T cells prior to electroporation (1ug = 3.5ul of RNP with 1uL of HDRT). Bulk T cells were resuspended in electroporation buffer P3 (Lonza) at 0.75e6 cells / 20μl per well and transferred to a 96-well electroporation plate with 4.5uL of RNP / HDRT mix per well. Pulse code EH115 was used. Cells were rescued in X-VIVO 15 without cytokines for 15 minutes and then cultured in X-VIVO 15 with 500U / ml IL-2.

[0189] Pooled combinatorial knock-in screening Primary human T cells were electroporated with the combinatorial library 2 days after activation. Four days after electroporation, knock-in rates were analyzed by flow cytometry, and the input population was harvested and transferred to TRI Reagent (Sigma). Cells were cultured in X-VIVO 15 supplemented with 500 U / ml IL-2 for 12 days and split every 2–3 days. The output population was harvested 16 days after electroporation and transferred to TRI Reagent (Sigma). RNA was isolated from the input and output populations using the Direct-zol RNA kit (Zymo Research) and reverse transcribed to cDNA using the Maxima H Minus First Strand cDNA Synthesis Kit (ThermoFisher). Sequencing libraries were generated by two rounds of PCR. PCR1 was performed for 18 cycles using KAPA HiFi HotStart ReadyMix (Roche). Primers were designed to add Illumina Read1 and Read2 sequences, including a forward primer that binds to the furin / GSGS region upstream of the two barcodes and a reverse primer that binds downstream of the two barcodes in the constant E2A region (Figure 9). Amplicons from PCR1 were SPRI purified. In PCR2, P5 and P7 Illumina sequencing adapters were added using NEB Next Ultra II Q5 polymerase (NEB) for 10 cycles. PCR2 products were SPRI purified and normalized libraries were pooled across samples and sequenced using NextSeq500 (Illumina). Barcode distribution was analyzed and log2 fold change in barcode representation in the output relative to the input population was calculated to detect changes in abundance.

[0190] Competition assay After arraying the knock-ins of the different combinatorial constructs, cells were sorted on day 7 post-electroporation and competition assays were set up on day 8 post-electroporation. T cells were cultured at a 50 / 50 ratio with control T cells in X-VIVO 15 containing 50U / ml IL-2. The cell ratio was confirmed by flow analysis of the cell mixture and the exact percentage of control T cells was determined at baseline levels (NGFR expression). Every 2 days, the change in cell ratio was analyzed by flow, cells were split 1:2 and IL-2 was added to a final concentration of 50U / ml. The change in cell ratio was normalized based on the percentage on day 0 of the assay.

[0191] Activation markers and phenotypic analysis Activation marker expression (CD25, CD69) was analyzed by flow cytometry 8 days after electroporation. CD62L / CD45RA expression levels were analyzed by flow cytometry 14 days after electroporation.

[0192] Flow cytometry and fluorescence-activated cell sorting (FACS) For flow cytometry analysis, T cells (unless sorted) were first stained for CAR expression using anti-mouse IgG, F(ab')2 fragment-specific antibody (Jackson ImmunoResearch) at 1:100 dilution for 10 min at 4°C. Cells were washed and blocked with PBS / 2% mouse serum before staining with remaining extracellular antibodies in flow buffer (PBS / 2% FCS). Cells were analyzed using an Attune NxT Flow Cytometer (ThermoFisher, Waltham, Massachusetts, USA) or BD LSRFortessa (BD Biosciences, San Jose, California, USA).

[0193] For FACS, cells were stained with anti-mouse IgG, F(ab')2 fragment-specific antibody (Jackson ImmunoResearch) and anti-human TCRab (Biolegend) and sorted for CAR+ / TCR- cells using a BD FACSAria Fusion (BD Biosciences, San Jose, California, USA).

[0194] RNA sequencing (RNA-seq) On day 14, edited cells were sorted for CAR+ / TCR- expression. A portion of the sorted population was stored in Tri Reagent, and the other portion was stimulated with GD2+Nalm-6 cells at an E:T ratio of 1:1. After 24 hours, stimulated T cells were sorted again for CAR+ / TCR- cells and stored in Tri Reagent. RNA was isolated using the Direct-zol RNA kit (Zymo Research). RNA was prepared for sequencing as previously described by the Functional Genomics Laboratory at UC Berkeley (Cortez et al., 2020) and sequenced by the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley. Reads were mapped to the human reference transcriptome using Kallisto, and genes with counts of 0 in >80% of 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 hallmark gene set as the reference gene list.

[0195] In vitro killing assay For the IncuCyte assay, flat-bottom 96-well plates were coated with 50ul of 0.01% poly-L-ornithine (PLO) solution (Sigma) for 1 hour. PLO was removed and plates were allowed to dry for 30-60 minutes. 10,000 GFP+ / GD2+Nalm-6 cells per well were mixed with sorted T cells at various effector:target (E:T) ratios. Assay medium consisted of X-VIVO 15 as above supplemented with 500U / ml IL-2 and 1X glucose solution (ThermoFisher). Cell counts (GFP+) were analyzed every 6 hours using the IncuCyte Live Cell Analysis System (Essen BioScience).

[0196] In vivo mouse model NOD / SCID / IL2Rg-null (NSG) mice were purchased from Jackson Laboratory. 8-12 week old female mice were used and mouse experiments were performed in accordance with ethical regulations following an approved IACUC protocol (UCSF). On day 0, mice were IV injected with 0.5e6 Nalm-6 / luciferase / GFP / GD2 cells. Three days later, edited human T cells were IV injected (1e6 / mouse). To avoid graft-versus-host disease in mice, T cells were TCR-depleted (TCR-depleted) one day before injection using EasySep Human TCR Alpha / Beta Depletion Kit (Stemcell). Knock-in rates were adjusted between groups by adding TCR-negative T cells without CAR knock-in immediately before injection. For imaging, 200 uL (3 mg) of D-Luciferin Potassium Salt (Gold BioTechnology) was injected IP and mice were imaged once / twice weekly using an IVIS Spectrum In Vivo Imaging System (PerkinElmer).

[0197] result Analysis of single transcription factor overexpression in combination with T cell receptor (TCR) or chimeric antigen receptor (CAR) raised the question of whether a combination of two transcription factors (TF) or one transcription factor and one switch receptor / therapeutic construct (TC) could further improve T cell functionality and overcome exhaustion. A 10,404 member library of 100 transcription factors (+2 controls) in combination with GD2 CAR and 100 transcription factors (+2 controls). The GD2 CAR pUC19-based backbone and the transcription factor library were PCR amplified for insertion position 1 and insertion position 2 using different primer pairs as described in International Patent Application Publication WO2022072932 (Figure 1A). 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. Pooled Gibson assembly generated a fusion region consisting of the 3' barcode of the TF in the first position, a constant linker (linker2-linker1 junction), and the 5' barcode of the TF in the second position. Homologous recombination repair templates (HDRTs) were generated from the plasmid library by PCR and non-viral knock-in of the library into the TCR alpha constant chain (TRAC) locus of primary human T cells. The fusion region between TF1 and TF2 was used to identify the abundance and orientation (insert1 vs. insert2) of the various combinatorial constructs by amplicon sequencing (Figure 1B). Amplicon sequencing of plasmid and cell pools 4 days after electroporation showed size-dependent presentation / knock-in rates of the constructs, but confirmed >99% presentation of the constructs at both stages of the protocol (Figure 1C).

[0198] We tested which TFxTF combinations enhance T cell expansion and survival in association with chronic antigen stimulation (tonic signaling GD2 CAR). The abundance of >10,000 different TFxTF combinations was analyzed after 16 days of culture and compared to baseline abundance at day 4 post-electroporation. Statistical analysis of two independent donors showed that while most constructs were dropped over time, the combination of TFAP4 and BATF significantly increased the abundance of GD2 CAR T cells (Figure 2A). Upon further analysis of the constructs with the highest positive log2 fold change after 16 days compared to 4 days, most of the best performing constructs were found to be composed of either TFAP4 or BATF / BATF3, or a combination of the two, suggesting that in this model, BATF(3) and TFAP4 are key transcription factors driving increased fitness (Figure 2B).

[0199] Similarly, a second combinatorial library was generated with a transcription factor in the first position and a switch receptor / therapeutic construct (TC) in the second position (see International Patent Application Publication WO2022072932), resulting in a 13,362-member TFxTC library (Figure 3A). Again, amplicon sequencing showed a size-dependent presentation / knock-in rate of the inserts, but confirmed that >99% of the constructs were presented in the plasmid library (Figure 3B) and 4 days after electroporation (Figure 3C). A significant increase in abundance of the combinatorial constructs TFAP4-TIM-3 / 4-1BB and SOX5-TIGITtrunc was found when calculating the log2 fold change at day 16 versus day 4 after electroporation of the TFxTC library (Figure 4A). Again, many of the constructs with a large fold change in abundance were observed to have either TFAP4 or BATF(3) as the insert in the first position (Figure 4B). Because the GD2 CAR may be a CD28-based costimulatory CAR, a relatively higher number of 4-1BB-based switch receptors were found in the best-performing hits compared to CD28-based switch receptors.

[0200] To verify our findings that the combination of BATF and TFAP4 seems to be beneficial in the tonic signaling model, we next performed single knock-ins of GD2 CAR and BATF-TFAP4 combinations, as well as controls with overexpression of only one transcription factor (mCherry-TFAP, and BATF-mCherry) or no transcription factor (mCherry-NGFR). The combinatorial constructs were co-cultured in a 50 / 50 ratio with constructs containing only one of the transcription factors, and the abundance was monitored over time (Figure 5A). Adding TFAP4 to the constructs seemed to exert a stronger effect than adding BATF, suggesting the fact that most of the proliferation or anti-apoptotic effects are driven by TFAP4. Consistent with the observations made with the single knock-in constructs containing TFAP4, we found increased CD25 expression levels in the TFAP4-containing combinatorial constructs, while CD69 expression remained comparable (Figure 5B). When analyzing the phenotype of GD2 CAR T cells 14 days after electroporation, it was 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 markedly reduced percentage of Temra cells (Figure 5C+D).

[0201] To evaluate the transcriptome profile driven by overexpression of the BATF-TFAP4 construct, CAR+ / TCR- cells were sorted 14 days after electroporation and bulk RNA-seq was performed. 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). Furthermore, it was observed that while BATF-TFAP4 shared many differentially expressed genes such as CCR3, CCR4 and CCR8 with its single knock-in controls (BATF-mCherry and mCherry-TFAP4), the combination of BATF and TFAP4 further led to unique differential expression of various genes highlighted in yellow (Figure 6A). Correlation analysis of the log2 fold change between each test condition and the control (mCherry-NGFR) showed that BATF-TFAP4 shared the highest similarity with mCherry-TFAP4, followed by correlation with BATF-mCherry (Figure 6B). BATF-TFAP4 was poorly correlated with mCherry-JUN. Gene set enrichment analysis of BATF-TFAP4 versus mCherry-NGFR highlighted increased expression of genes involved in cell cycle regulation at 14 days post-electroporation (Figure 6C). After stimulation with target cells, BATF-TFAP4 cells showed increased expression of genes involved in glycolysis, oxidative phosphorylation and fatty acid metabolism, whereas expression of genes involved in apoptosis was decreased (Figure 6D).

[0202] We next analyzed whether the addition of TFAP4 and BATF could increase the functionality of GD2 CAR T cells in terms of cancer cell killing. Indeed, when co-cultured with GD2-positive target cells, the combination of BATF and TFAP4 performed best across multiple E:T ratios in in vitro killing assays (Figure 7A+B). Finally, when the combinatorial cells were included in the NOD / SCID / IL2Rg-null (NSG) xenograft mouse model of adoptive T cell transfer (Figure 8A), we observed that the combination of TFAP4 and BATF showed the greatest potential to control leukemia growth, while TFAP4 overexpression alone seemed insufficient in one of the donors (Figure 8B).

[0203] Example II We investigated whether specific combinations of TFs work in concert to enhance T cell fitness in the context of tonic CAR signaling. Analyzing 100 different transcription factor combinations would require I) a knock-in library size (~10,000 members) that has not been previously tested in this context, and II) successful knock-in of very large constructs, especially when performed in combination with CAR (average construct size ~5.5 kb + homology arms), which cannot be easily performed with AAV (adeno-associated virus) HDR templates due to viral packaging size limitations.

[0204] Pooled modular knock-in screening (ModPoKI) molecular biology was used to overcome these challenges. The ModPoKI screening platform was adapted for large-scale pooled combinatorial knock-in screening (Figure 10A and Figure 11A). A ~10,000 member library (100 transcription factors + 100 transcription factors combined with 2 controls + 2 controls) was generated and cloned into a construct containing the GD2 CAR. In summary, to achieve this, a GD2 CAR pUC19-based scaffold (specificity module) was first PCR amplified, containing homology arms targeting the human TRAC locus. Then, two transcription factor inserts were generated by PCR amplification from an existing TF library using different primer pairs for position 1 vs. position 2 within the functional module (Figure 11B). 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. Pooled Gibson assembly generated a DNA site consisting of the 3' barcode of the TF at the first position, a constant linker (linker2-linker1 junction), and the 5' barcode of the TF at the second position, generating a unique combinatorial barcode for each TFxTF combination. HDR templates were generated from the plasmid library by PCR and non-viral knock-in of the library into the TRAC locus of primary human T cells. Remarkably, the predicted 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 served as a barcode combination to identify the abundance and orientation (TF1 vs. TF2) of different combinatorial constructs by amplicon sequencing (Figure 11C). Amplicon sequencing of plasmid pools and ModPoKI T cell pools 4 days after electroporation confirmed presentation of >99% constructs at both steps of the protocol, despite the expected construct size-dependent effect on library presentation / knock-in rates (Figure 10B-C).Thus, we were able to generate pooled libraries with thousands of different members and successfully achieved diverse knock-ins containing constructs as large as ∼7.6 kb based on barcode sequencing.

[0205] We used combinatorial ModPoKI to test which TFxTF constructs enhance T cell fitness in the context of tonic CAR signaling. ModPoKI cells expanded in culture due to GD2 CAR tonic signaling. We compared the abundance of each TFxTF combination construct after 16 days of culture with its baseline abundance in the ModPoKI T cell population at day 4 post-electroporation. Most TFxTF combinations were depleted from the pool over time (Figure 12A-B), consistent with our previous evidence that major transcriptional changes can be detrimental to fitness. Analysis of constructs that increased relative abundance the most (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 can cooperatively promote increased T cell fitness during iterative simulations (Figure 10D and Figure 11E). Analysis of screens performed on cells from two human donors identified TFAP4 and BATF combination constructs as having the most significant increase in abundance across two different barcode combinations / knock-in orientations (TFAP4-BATF, and BATF-TFAP4) (Figure 10D). In summary, these data indicate that large-scale combinatorial knock-in screening of approximately 10,000 different constructs with an average knock-in size of approximately 5.5 kb is feasible using the ModPoKI screening platform and can help generate a map of combinatorial KI constructs with the potential to enhance therapeutic T cells.

[0206] Combined TFAP4 and BATF KI induces favorable conditions in therapeutic T cells To verify and characterize the advantages of KI constructs that combine BATF and TFAP4, we next generated specific knock-in constructs with GD2 CAR and 1) BATF+TFAP4 combination, 2) single TF+control (RFP-TFAP and BATF-RFP), or 3) control+control (RFP-tNGFR). Competitive fitness assays were performed to evaluate whether combination KIs outperform individual TF KIs. KI cells with BATF+TFAP4 combinatorial constructs were co-cultured with KI cells with constructs that contain only single TFs (along with control genes) at a ratio of about 50 / 50, and relative abundance was monitored over time (Figure 12A). KI cells with BATF+TFAP4 combinatorial constructs outperformed both TFAP4+control KI cells and BATF+control KI cells. The relative advantage of the BATF+TFAP4 combination was stronger compared to BATF than to TFAP4 alone, suggesting that most (if not all) of the fitness advantage was conferred by the TFAP4 KI. Consistent with the effect of single TFAP4 KI constructs, we found increased CD25 expression levels in TFAP4-containing combinatorial constructs, whereas CD69 levels were not significantly affected (Figure 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 to a stem cell-like and central memory state with a notable decrease in the percentage of Temra cells (Figure 12C-D).

[0207] We next assessed the transcriptional effects of the BATF-TFAP4 combination constructs compared to single TF and control constructs. GD2 CAR+ / TCR- cells were sorted from each KI population 14 days after electroporation and RNA-seq was performed without the addition of target cells (tonic signaling only). Correlation analysis of the log2 fold change between each test 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 (Figure 13A-B). BATF-TFAP4 KI cells correlated even less well with RFP-JUN KI cells in this context, suggesting that the transcriptional program promoted by these TFs is distinct from the previously reported program promoted by JUN overexpression. Several genes, including CCR3, CCR4, and CCR8, were induced by BATF+TFAP4 KI, BATF-RFP KI, and RFP-TFAP4 KI (compared to control KI cells). However, the combined KI of BATF and TFAP4 also promoted differential expression of various genes highlighted in yellow, e.g., TP63 (tumor protein 63), GNLY (granulysin), TNFRSF11A (tumor necrosis factor receptor superfamily member 11A) and CLECL1 (encoding T cell costimulatory protein C-type lectin-like domain family 1), that were not differentially affected by either BATF or TFAP4 KI alone (Figure 12E). Gene set enrichment analysis of BATF-TFAP4 versus RFP-tNGFR highlighted increased expression of genes involved in cell cycle control (such as E2F targets and G2M checkpoint genes) at day 14 post-electroporation, whereas, interestingly, genes involved in the P53 pathway and IFN-g response appeared to be decreased (Figure 13C, left panel). After stimulation with target cells, BATF-TFAP4 cells showed increased expression of genes involved in glycolysis, oxidative phosphorylation, and fatty acid metabolism (FIG. 13C, center and right panels, and FIG. 13D-E (compared to single KI)).Taken together, these results suggest that combinatorial knock-in of BATF and TFAP4 can promote overlapping but distinct transcriptional changes to promote a fitness advantage compared with single BATF or TFAP4 KIs in the GD2 CAR model of tonic signaling.

[0208] Polycistronic TFAP4 single KI construct improved the cancer killing ability of GD2 TRAC CAR T cells in vitro and in vivo. We wanted to evaluate whether the combinatorial KI construct of TFAP4 and BATF could further enhance the anti-cancer function of GD2 TRAC CAR T cells. This is because this combination conferred additional T cell fitness advantage. 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 across multiple E:T ratios (Figure 12F and Figure 14A). Finally, when combinatorial KI cells were tested in an in vivo NSG xenograft mouse model of adoptive T cell transfer (Figure 12G and Figure 14B), it was observed that KI CAR T cells harboring a TFAP4+BATF combinatorial construct exhibited the best ability to control leukemia growth compared to CAR T cells harboring either single TF KI constructs or control KI constructs. In summary, pooled combinatorial modular knock-in screening of thousands of different synthetic gene constructs can inform the design of combinatorial genetic programs that promote enhanced persistence and function to improve adoptive T cell therapy.

[0209] Example III Creation of plasmid libraries for pooled single construct knock-in The 231 constructs included in the pooled knock-in library (see, e.g., Blaeschke et al., “Modular Pooled Discovery of Synthetic Knockin Sequences to Program Durable Cell Therapies,” bioRxiv 2022.07.27.501186; doi:https: / / doi.org / 10.1101 / 2022.07.27.501186) were designed using Twist Bioscience codon optimization tools, commercially synthesized, and cloned into a custom pUC19 plasmid containing the NY-ESO-1 TCR sequence (Twist Bioscience) (with the exception of 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 single construct plasmids into each library (transcription factors, 100 members; surface receptor constructs, 129 members; controls, 2) or into one complete pool. CD19 / 28z CAR plasmid pools were created in a pooled assembly format by amplifying constructs from the TCR plasmid pool as DNA templates. A pooled library of amplicons with small overhangs homologous to the pUC19 plasmid containing the CD19 / 28z CAR HDR sequence was created by PCR amplification (Kapa Hot Start polymerase, Roche). The amplicon pool was treated with DpnI restriction enzyme (New England Biolabs, NEB) to remove residual circular TCR plasmid, bead purified (Sera-Mag SpeedBeads), and eluted in H2O. Gibson Assembly (NEB) was then used to construct a plasmid pool containing all 231 library members and knock-in controls, as well as the new CAR sequence.CD19 / 28z CAR plasmid pools were bead-purified, transformed into Endura electrocompetent cells (Lucigen) and maxiprepped (Plasmid Plus Midi or Maxi Kit, Qiagen) for subsequent use. The GD2 CAR single knock-in library was generated in a similar manner. The NY-ESO-1 TCR library was pooled at the plasmid stage (plasmids were synthesized individually), whereas all other plasmid libraries in this project (anti-CD19 / 28z CAR, anti-GD2 CAR, combinatorial library) were generated by pooled Gibson assembly of plasmids. The CD19 CAR sequence used in this study was kindly provided by Tobias Feuchtinger, Dr. von Hauner Children's Hospital, University Hospital, LMU Munich. The GD2 CAR sequence was kindly provided by Crystal Mackall and Robbie Majzner, Stanford (Lynn et al., 2019). The assembled products were SPRI-purified using Sera-Mag SpeedBeads and transformed into Endura electrocompetent cells (Lucigen) and maxiprepped (Zymo Research) for subsequent use. HDR templates (HDRT) were generated as previously described (Roth et al., 2020). In summary, single knock-in plasmid pools were used as templates for PCR amplification (KAPA HiFi HotStart ReadyMix, Roche) to generate double-stranded DNA templates. Templates were SPRI-purified and eluted in H2O. The GD2 CAR sequence was provided by Robbie Majzner and Crystal Mackall (Stanford University).

[0210] Repeated stimulation screening One day before the start of the repeated stimulation screen, A375 cells were counted and transferred to 24-well plates (25,000 cells / well in 1 ml of complete RPMI medium), assuming that they would double within 24 hours. One day later, edited T cell pools were counted and diluted with 10 eV in TRI Reagent (Sigma-Aldrich) for amplicon sequencing. 6 Cells were frozen (input population). The 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 2 ml of X-VIVO 15 containing supplements + 50 U / ml IL-2. After 24 hours, fresh A375 cells were seeded as above. One day later, the medium of the new A375 plate was removed and replaced with 1 ml of fresh X-VIVO 15 + 1 ml of T cell suspension from the first plate, containing 50 U / ml IL-2 calculated in total volume per well. The remaining T cells were counted and aliquoted in TRI Reagent (Sigma-Aldrich) for amplicon sequencing. e 6 cells were transferred. This procedure was repeated every other day for a total of 5 stimulations with target cells. Multiple wells of a 24-well plate were used per screen to reach cell coverage. When working with CD19 CAR in combination with A375 cells, A375 cells overexpressing CD19 (SFFV promoter knocked in upstream of endogenous CD19) were used.

[0211] Single Stimulus Screening One day before setting up the screen, add 2.5 mL of complete RPMI medium (RPMI + NEAA, glutamine, Hepes, Pen / Strep, sodium pyruvate (all from Thermo Fisher Scientific) and 10% FCS (Sigma-Aldrich, St. Louis, Missouri, USA)) per T75 flask, assuming a doubling time within 24 hours. e6 A375 were seeded. One day later (=7 days after electroporation), the edited T cell pool was counted and washed once. 10 e 6 T cells were transferred into TRI Reagent (Sigma-Aldrich). For each screening condition, 10e6 T cells were transferred into one T75 flask in 20ml of X-VIVO 15 (Lonza Bioscience) supplemented with 5% FCS, 2-mercaptoethanol (Thermo Fisher Scientific), N-acetyl-L-cystine (VWR), and 50U / ml IL-2 (Proleukin). cRPMI was removed and the flask was filled with 20ml of X-VIVO 15+supplement and 10e6 T cells. After 2 days, 10ml of X-VIVO 15 was added to all conditions with supplements and 50U / ml IL-2. After another 2 days, cells were counted and 10e6 cells were transferred into TRI Reagent (Sigma-Aldrich) for RNA isolation and amplicon sequencing.

[0212] CD25 sorting screening For sorting screening for CD25 expression, T cells were electroporated with the GD2 CAR TF library and sorted for CD25 expression (pre-gated for CAR+ / TCR-) 8 days after electroporation (~20% CD25 expression up and down, 0.5M cells / donor / bin). RNA was isolated from CD25 high and CD25 low populations using the Direct-zol RNA kit (Zymo Research) and reverse transcribed to cDNA using the Maxima H Minus First Strand cDNA Synthesis Kit (ThermoFisher). Sequencing libraries were generated by two rounds of PCR. PCR1 was performed for 18 cycles using KAPA HiFi HotStart ReadyMix (Roche). Primers were designed to add Illumina Read1 and Read2 sequences upstream and downstream of the 3' barcode region. Amplicons from PCR1 were SPRI purified. In PCR2, P5 and P7 Illumina sequencing adapters were added using NEB Next Ultra II Q5 polymerase (NEB) for 10 cycles. PCR2 products were SPRI purified and normalized libraries were pooled across samples and sequenced using NextSeq500 (Illumina). Barcode distribution was analyzed and the log2 fold change of barcode representation in CD25 high versus CD25 low bins was calculated.

[0213] result Because increased CD25 expression was observed on GD2 CAR T cells with TFAP4 knock-in, a selective screen for CD25 high vs. CD25 low expression was performed 8 days after electroporation with the GD2 CAR single insert TF library. Data show normalized log2FC of insert presentation of CD25 high vs. CD25 low bins in three individual donors (mean + SEM). Screening revealed a trend toward increased CD25 expression on T cells with TFAP4 or BATF knock-in (Figure 15).

[0214] To complement the previous dataset, we performed single stimulation screens (A375 / CD19 addition vs. non-stimulated conditions) and repeated stimulation screens (five additions of A375 / CD19) in a complementary model using CD19 / 28z CAR in combination with the full TF and SR single insert libraries. Normalized Log2FC of stimulated / non-stimulated / repeated stimulation conditions relative to input is shown to assess the changes in abundance. Indeed, a trend towards increased abundance of hits was observed in the model complementary to the one used for combinatorial screening (GD2 CAR) (e.g., BATF2, IL2RA, TFAP4, BATF, LAT1, BATF3, CXCR1, MYC, ID3, ID2, IRF1, TIGIT / ICOS, LTBR / 4-1BB, DR5 / ICOS, CCR4) (Figure 16).

[0215] All patents, patent applications and other published references cited herein are hereby incorporated by reference in their entirety.

[0216] (Table 1) TIFF2025506185000002.tif228144TIFF2025506185000003.tif228159TIFF2025506185000004.tif228159TIFF2025506185000005.tif228159TIFF2025506185000006.tif228159TIFF2025506185000007.tif228155TIFF2025506185000008.tif228159TIFF2025506185000009.tif228159TIFF2025506185000010.tif228159TIFF2025506185000011.tif228159TIFF2025506185000012.tif228160TIFF2025506185000013.tif228159TIFF2025506185000014.tif228159TIFF2025506185000015.tif228159TIFF2025506185000016.tif228159TIFF2025506185000017.tif228159TIFF2025506185000018.tif228159TIFF2025506185000019.tif228159TIFF2025506185000020.tif228159TIFF2025506185000021.tif228159TIFF2025506185000022.tif228159TIFF2025506185000023.tif228160TIFF2025506185000024.tif228161TIFF2025506185000025.tif228157TIFF2025506185000026.tif228161TIFF2025506185000027.tif22881

[0217] (Table 2) TIFF2025506185000028.tif226142TIFF2025506185000029.tif226159TIFF2025506185000030.tif226160TIFF2025506185 000031.tif226160TIFF2025506185000032.tif226160TIFF2025506185000033.tif226160TIFF2025506185000034.tif22616 0TIFF2025506185000035.tif226160TIFF2025506185000036.tif226160TIFF2025506185000037.tif226148TIFF2025506185 000038.tif226160TIFF2025506185000039.tif226160TIFF2025506185000040.tif226160TIFF2025506185000041.tif22626

[0218] (Table 3) TIFF2025506185000042.tif232151TIFF2025506185000043.tif232159TIFF2025506185 000044.tif232159TIFF2025506185000045.tif232159TIFF2025506185000046.tif23215 9TIFF2025506185000047.tif232159TIFF2025506185000048.tif232159TIFF2025506185 000049.tif232159TIFF2025506185000050.tif232159TIFF2025506185000051.tif23280

[0219] Additional Arrays TIFF2025506185000052.tif187161TIFF2025506185000053.tif217161TIFF2025506185000054.tif220161TIFF2025506185000055.tif221161TIFF2025506185000056.tif219161TIFF2025506185000057.tif9159

Claims

1. 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 linked via a transmembrane domain to a human 4-1BB intracellular domain (and optionally about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain); a polypeptide comprising a human SOX5 protein and a truncated TIGIT protein comprising the human TIGIT extracellular domain or a portion thereof, the TIGIT transmembrane domain, and about 1-10 (e.g., 7) amino acids of the human TIGIT intracellular domain; a polypeptide comprising the human MYC protein, and a polypeptide comprising the human TGFbR2 extracellular domain or a portion thereof linked via a transmembrane domain to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g., 7) amino acids of the TGFbR2 intracellular domain); a polypeptide comprising a BATF3 protein, and a polypeptide comprising a 4-1BB protein; a polypeptide comprising the TFAP4 protein, and a polypeptide comprising the human LTBR extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human IL-4R (and optionally 1-10 (e.g., 7) amino acids of the LTBR intracellular domain); a polypeptide comprising an ATF1 protein, and a polypeptide comprising the extracellular domain of human IL4RA linked to the intracellular domain of human 4-1BB, or a portion thereof; a polypeptide comprising a BATF protein and a polypeptide comprising the human 2B4 extracellular domain linked to the intracellular domain of human 4-1BB or a portion thereof, and optionally about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain; a polypeptide comprising a RORC protein and a polypeptide comprising a 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 human CD28 extracellular domain; a polypeptide comprising a BATF protein, and a polypeptide comprising a CXCR1 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 human TNFRSF12 extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human OX40 (and optionally 1-10 (e.g., 7) amino acids of the TNFRSF12 intracellular domain); a polypeptide comprising the human TIM3 extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human 4-1BB (and optionally from about 1 to 15 (e.g., 12) amino acids of the human 4-1BB extracellular domain); a polypeptide comprising the human BATF2 extracellular domain or a portion thereof, and a truncated PD-1 comprising the human PD-1 extracellular domain or a portion thereof, the PD-1 transmembrane domain, and about 1 to 12 (e.g., 10) amino acids of the human PD-1 intracellular domain; a polypeptide comprising a human TCF7 protein, and a polypeptide comprising a human LAT1 protein; A polypeptide comprising the human 4-1BB protein; A polypeptide comprising a human STAT5 protein and a human IL-2RA protein; a polypeptide comprising the human FOXJ2 protein, and a polypeptide comprising the LTBR extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human CD28 (and optionally 1-10 (e.g., 7) amino acids of the LTBR intracellular domain); a polypeptide comprising the human FOXJ2 protein, and a polypeptide comprising the LTBR extracellular domain or a portion thereof linked via a transmembrane domain to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g., 7) amino acids of the LTBR intracellular domain); a polypeptide comprising the human HOPX protein, and a polypeptide comprising the IL4RA extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human 41-BB; a polypeptide comprising an ID2 protein and a polypeptide comprising a BLTA extracellular domain or a portion thereof linked to the intracellular domain of human 4-1BB, and optionally about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain; a polypeptide comprising a human SMAD1 protein, and a polypeptide comprising a MCT4 protein; a polypeptide comprising a human SMAD1 protein, and a polypeptide comprising an LTBR extracellular domain or a portion thereof linked via a transmembrane domain to an intracellular domain of human IL-4R (and optionally 1-10 (e.g., 7) amino acids of the LTBR intracellular domain); a polypeptide comprising about 1-12 amino acids (e.g., 7 amino acids) of the DR5 intracellular domain, via a transmembrane domain, and a polypeptide comprising the intracellular domain of human ICOS; a polypeptide comprising the TIGIT extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human ICOS (and optionally about 1-15 (e.g., 12) amino acids of the human ICOS extracellular domain); A polypeptide comprising the LTBR extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human 4-1BB (and optionally from about 1 to 15 (e.g., 7) amino acids of the human LTBR intracellular domain). A human T cell heterologously expressing the one or more polypeptides are encoded by a heterologous nucleic acid construct inserted into a target genomic locus of the cell, optionally the target genomic locus is a T cell receptor (TCR) locus of the cell, and optionally the heterologous nucleic acid construct is inserted non-virally. The human T cell.

2. 2. The human T cell of claim 1, wherein the T cell heterologously expresses one or more polypeptides, each polypeptide comprising 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.

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

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

5. The human T cell of claim 4, wherein the TRBC is TRBC1 or TRBC2.

6. 6. The human T cell of any one of claims 1 to 5, wherein the heterologous nucleic acid construct comprises one or more nucleic acid sequences, each nucleic acid being 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 of any one of claims 1 to 6, which expresses an antigen-specific T cell receptor (TCR) or a synthetic antigen receptor that recognizes a target antigen.

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

9. The human T cell of any one of claims 1 to 8, which is a regulatory T cell, an effector T cell, a memory T cell or a naive T cell.

10. The human T cell of claim 9, wherein the effector T cell is a CD8+ T cell or a CD4+ T cell.

11. The human T cell of claim 10, wherein the effector T cell is a CD8+CD4+ T cell.

12. 12. The human T cell of any one of claims 1 to 11, which is a primary cell.

13. The nucleic acid construct comprises: (i) a first self-cleaving peptide sequence; (ii) a first heterologous TCR subunit chain comprising a variable region and a constant region of a TCR subunit; (iii) a second self-cleaving peptide sequence; (iv) one or more polypeptides, each polypeptide comprising an amino acid sequence 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) a portion of the N-terminus of an endogenous TCR subunit Code the following: when 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; when 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; A human T cell according to any one of claims 1 to 12.

14. The heterologous nucleic acid construct comprises: (i) a first self-cleaving peptide sequence; (ii) one or more polypeptides, each polypeptide comprising an amino acid sequence 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 comprising 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 Code the following: when 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; when 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; A human T cell according to any one of claims 1 to 12.

15. The nucleic acid construct comprises, in the following order: (i) a first self-cleaving peptide sequence; (ii) one or more polypeptides, each polypeptide comprising an amino acid sequence 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 synthetic antigen receptor; and (v) a third self-cleaving peptide sequence or a polyA sequence. The human T cell of any one of claims 1 to 12, wherein the human T cell encodes:

16. The nucleic acid construct comprises, 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, each polypeptide comprising an amino acid sequence 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 polyA sequence. The human T cell of any one of claims 1 to 12, wherein the human T cell encodes:

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

18. 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. A nucleic acid sequence encoding

19. 20. The nucleic acid of claim 18, comprising adjacent homology arm sequences having homology to a human TCR locus.

20. 20. A human T cell comprising a nucleic acid according to claim 18 or claim 19.

21. In the following order: (i) a first self-cleaving peptide sequence; (ii) a first heterologous TCR subunit chain comprising a variable region and a constant region of a TCR subunit; (iii) a second self-cleaving peptide sequence; (iv) one or more polypeptides, each polypeptide comprising an amino acid sequence 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) a portion of the N-terminus of an endogenous T cell TCR subunit Code the following: when 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; When 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. Nucleic acid constructs.

22. 22. The nucleic acid construct of claim 21, wherein the nucleic acid construct comprises two or more nucleic acid sequences, each nucleic acid being 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) Human T cells, (i) a targeted nuclease that cleaves a target region within the TCR locus of a human T cell to create a targeted insertion site within the genome of said cell; and (ii) 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; (a) a polypeptide comprising a human TFAP4 protein; and (b) a polypeptide comprising a human TIM3 extracellular domain or a portion thereof linked via a transmembrane domain to a human 4-1BB intracellular domain (and optionally about 1 to 15 (e.g., 12) amino acids of the human 4-1BB extracellular domain); (a) a polypeptide comprising a human SOX5 protein; and (b) a truncated TIGIT protein comprising the human TIGIT extracellular domain or a portion thereof, the TIGIT transmembrane domain, and about 1-10 (e.g., 7) amino acids of the human TIGIT intracellular domain; (a) a polypeptide comprising a human MYC protein; and (b) a polypeptide comprising the human TGFbR2 extracellular domain or a portion thereof linked via a transmembrane domain to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g., 7) amino acids of the TGFbR2 intracellular domain); A polypeptide comprising a BATF3 protein and a 4-1BB protein; (a) a polypeptide comprising a TFAP4 protein; and (b) a polypeptide comprising the human LTBR extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human IL-4R (and optionally 1 to 10 (e.g., 7) amino acids of the LTBR intracellular domain); (a) a polypeptide comprising an ATF1 protein and (b) a polypeptide comprising the extracellular domain of human IL4RA linked to the intracellular domain of human 4-1BB or a portion thereof; (a) a BATF protein; and (b) a polypeptide comprising the human 2B4 extracellular domain linked to the intracellular domain of human 4-1BB, or a portion thereof, and optionally a polypeptide comprising about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain; (a) a RORC protein; and (b) a polypeptide comprising a VISTA extracellular domain or a portion thereof linked to the intracellular domain of human CD28, and optionally a polypeptide comprising about 1-15 (e.g., 12) amino acids of the human CD28 extracellular domain; a polypeptide comprising a BATF protein and a CXCR1 protein; a polypeptide comprising a MAFF protein and an IL2RA protein; (a) a polypeptide comprising an ATF1 protein; and (b) a polypeptide comprising the human TNFRSF12 extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human OX40 (and optionally 1 to 10 (e.g., 7) amino acids of the TNFRSF12 intracellular domain); a polypeptide comprising the human TIM3 extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human 4-1BB (and optionally from about 1 to 15 (e.g., 12) amino acids of the human 4-1BB extracellular domain); a polypeptide comprising the human BATF2 extracellular domain or a portion thereof linked to a truncated PD-1 comprising the human PD-1 extracellular domain or a portion thereof, the PD-1 transmembrane domain, and about 1-12 (e.g., 10) amino acids of the human PD-1 intracellular domain; A polypeptide comprising a human TCF7 protein and a human LAT1 protein; A polypeptide comprising the human 4-1BB protein; A polypeptide comprising a human STAT5 protein and a human IL-2RA protein; (a) a polypeptide comprising a human FOXJ2 protein; and (b) a polypeptide comprising the LTBR extracellular domain or a portion thereof linked via a transmembrane domain to the intracellular domain of human CD28 (and optionally 1 to 10 (e.g., 7) amino acids of the LTBR intracellular domain); (a) a polypeptide comprising a human FOXJ2 protein; and (b) a polypeptide comprising the LTBR extracellular domain or a portion thereof linked via a transmembrane domain to amino acids 41-142 of human MyD88 (and optionally 1-10 (e.g., 7) amino acids of the LTBR intracellular domain); (a) a polypeptide comprising a human HOPX protein; and (b) a polypeptide comprising the IL4RA extracellular domain or a portion thereof linked to the intracellular domain of human 41-BB via a transmembrane domain; (a) a polypeptide comprising an ID2 protein; and (b) a BLTA extracellular domain or a portion thereof linked to the intracellular domain of human 4-1BB, and optionally a polypeptide comprising about 1-15 (e.g., 12) amino acids of the human 4-1BB extracellular domain; A polypeptide comprising a human SMAD1 protein and a MCT4 protein; and (a) a human SMAD1 protein; and (b) a polypeptide comprising an LTBR extracellular domain or a portion thereof linked via a transmembrane domain to an intracellular domain of human IL-4R (and optionally 1 to 10 (e.g., 7) amino acids of the LTBR intracellular domain). A nucleic acid construct encoding introducing; (b) allowing recombination to occur, thereby inserting the nucleic acid construct into the target insertion site to generate an engineered human T cell. A method for modifying a human T cell, comprising:

24. 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 to 85, 160, 161 and 162.

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

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

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

28. 28. The method of any one of claims 23 to 27, wherein the targeted 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 targeting nuclease, guide RNA and DNA template are introduced into the cell as a ribonucleoprotein complex (RNP)-DNA template complex, and the RNP-DNA template complex is (i) an RNP comprising the targeting nuclease and the guide RNA; (ii) the nucleic acid construct; 30. The method of claim 28, comprising:

30. The method of any one of claims 22 to 29, wherein the T cell is a regulatory T cell, an effector T cell, a memory T cell or a naive T cell.

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

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

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

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

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

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

39. 37. The method of claim 35 or 36, wherein the human subject has an infectious disease.

40. 37. The method of claim 35 or 36, wherein the human subject has an autoimmune disorder and the antigen is an antigen associated with an autoimmune disorder, an allergic disorder, 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) (i) a targeted nuclease that cleaves a target region in the genome of a cell to create a targeted insertion site; (ii) Multiple DNA templates with different sequences into a population of cells, wherein each DNA template comprises: i. two or more heterologous coding or non-coding nucleic acid sequences; ii. a unique barcode nucleotide sequence indicating the identity of each heterologous coding nucleic acid sequence or each heterologous non-coding nucleic acid sequence; A process comprising: (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 producing cDNA from the mRNA; (d) sequencing the genomic DNA or the cDNA to identify the DNA template inserted into the target insertion site in the cell.

23. A method for identifying a targeted insertion in the genome of a cell, comprising:

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

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

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

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

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

50. 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 to 50, further comprising determining the relative numbers of cells within the population that have different DNA templates inserted into the target insertion site.

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

53. 53. The method of claim 52, further comprising comparing the relative numbers of cells within the population with the various DNA templates inserted at the targeted insertion site before and after applying selection pressure to the cells.

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

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

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

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

58. 58. The method of claim 57, wherein the T cell is a regulatory T cell, an effector T cell or a naive T cell.

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

60. 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.

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