Compositions and methods for enhancing adoptive t cell therapeutics

Abstract

The present disclosure relates generally to compositions and methods for improving T cell therapy. In particular, the disclosure provides polypeptides and recombinant nucleic acid constructs encoding recombinant polypeptides comprising a caspase-associated recruitment domain (CARD) containing protein or a functional fragment thereof. The disclosure also provides vectors and cells including the polypeptides and / or recombinant nucleic acid constructs of the disclosure as well as methods of preparing a T cell for use in cell therapy.

Description

COMPOSITIONS AND METHODS FOR ENHANCING ADOPTIVE T CELL THERAPEUTICSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 550,520, filed on February 6, 2024, the contents of which are herein incorporated by reference in its entirety.STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided in XML file format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing XML is MNBI_002_01WO_SeqList_ST26.xml. The XML file is 538,224 bytes, and created on February 5, 2025, and is being submitted electronically via USPTO Patent Center.FIELD

[0003] The present disclosure relates generally to compositions and methods for enhancing T cell therapeutics. The disclosure provides recombinant nucleic acid constructs encoding polypeptides that promote T cell signaling, efficacy and / or in vivo persistence, cells that comprise such recombinant nucleic acids, methods for preparing T cells for use in cell therapies, as well as methods for identifying mutations useful for improving T cell therapy.BACKGROUND

[0004] Adoptive T cell therapies, including chimeric antigen receptor (CAR) T cells and engineered T cell receptor (TCR) T cells, have revolutionized cancer therapy. To date, five CD 19 targeted CAR-T cell therapies have been approved by the FDA for use against hematological B cell cancers. Despite this success, meaningful responses have not been achieved for patients with solid tumors, which represent -90% of adult cancers. In solid tumor cancers, the effectiveness of T cell therapies is limited by a complex combination of factors including: fitness of engineered T cells in tumors, T cell exhaustion, poor T cell persistence in vivo, and immunosuppressive environmental factors. Despite recent advances, there is still a need to develop alternative approaches to effectively treat solid tumor cancers.

[0005] One approach that has been explored for improving CAR T cells is to genetically modify the cells to improve their functionality in treating solid tumors. However, while there are examples of CAR T cells with improved functionality there remains a large unmet need.As such, there remains a need in the art for alternative solutions to address the significant unmet need for effective adoptive T cell therapies and for enhancing engineered T cell fitness.SUMMARY

[0006] This section provides a general summary of the disclosure, and is not comprehensive of its full scope or all of its features. The disclosure provides recombinant nucleic acid constructs and methods for enhancing adoptive T cell therapies. The recombinant nucleic acid constructs of the disclosure encode polypeptides with mutations wherein the mutations enhance the therapeutic efficacy of T cells by altering T cell signaling, decreasing T cell exhaustion and / or by enhancing in vivo persistence and fitness of engineered T cells.

[0007] In one aspect, the disclosure provides recombinant polypeptides comprising a caspase-associated recruitment domain (CARD) containing protein or a functional fragment thereof, wherein the recombinant polypeptide comprises no more than 724 amino acids.

[0008] In one aspect, the disclosure provides recombinant polypeptides comprising: (a) a CARD domain, and (b) a recruitment domain or a functional fragment thereof, wherein the recruitment domain is capable of binding to (i) a substrate localized to the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine, wherein the recombinant polypeptide comprises no more than 724 amino acids.

[0009] In one aspect, the disclosure provides recombinant polypeptides comprising: (a) a first polypeptide capable of binding to a CARD domain on BCL10, and (b) a recruitment domain or a functional fragment thereof, wherein the recruitment domain is capable of binding to (i) a substrate localized to the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine, wherein the recombinant polypeptide comprises no more than 724 amino acids.

[0010] In one aspect, the disclosure provides a recombinant polypeptide described herein, wherein when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits decreased Interferon gamma (IFNg) and / or Interleukin-2 (IL-2) production compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1. In some embodiments, the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits equivalent or enhanced: (a) BCL10 binding, (b) BCL10 and MALT1 complex formation, (c) NF-KB and / or AP-1 signaling, (d) MALT1 paracaspase activity, (e)cytokine production, (f) peak tumor expansion, (g) peak peripheral blood expansion, (h) contraction following peak tumor expansion, and / or (i) contraction following peak peripheral blood expansion, compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1. In some embodiments, the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits equivalent or higher IFNg and / or IL-2 production compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises no more than 700, no more than 650, no more than 600, no more than 550, no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, or no more than 150 amino acids.

[0011] In some embodiments, the CARD domain-containing protein or the functional fragment thereof, the CARD domain, or the recombinant polypeptide, is capable of binding to a CARD domain on BCL10. In some embodiments, the CARD domain-containing protein or the functional fragment thereof, the CARD domain, or the recombinant polypeptide, is capable of forming a complex with BCL10 and MALT1.

[0012] In some embodiments, the CARD domain-containing protein or the functional fragment thereof, or the CARD domain, comprises or consists of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 293-323. In some embodiments, the CARD domain-containing protein is selected from, or wherein the CARD domain is derived from a protein selected from, CARD9, CARDIO, CARD11, and CARD14. In some embodiments, the CARD domain-containing protein is, or wherein the CARD domain is derived from, CARD11.

[0013] In some embodiments, the recombinant polypeptide comprises a full coiled-coil domain or a truncated portion thereof, wherein the start of the full coiled-coil domain corresponds to amino acid position 123 of SEQ ID NO: 1, and wherein the end of the full coiled-coil domain corresponds to amino acid position 442 of SEQ ID NO: 1; optionally, wherein the full coiled-coil domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 123-442 of SEQ ID NO: 1. In some embodiments, the full coiled-coil domain comprises at least 310 amino acids. In some embodiments, the recombinant polypeptide comprises the full coiled-coil domain. In some embodiments, the recombinant polypeptide comprises a truncated portion of the coiled-coil domain having no more than 300, no more than 290, no more than 280, no more than 270, no more than 260, no more than 250, no more than 240, no more than 230, nomore than 220, no more than 210, no more than 200, no more than 190, no more than 180, no more than 170, no more than 160, no more than 150, no more than 140, no more than 130, no mor than 120, no more than 110, no more than 100, no more than 95, no more than 90, no more than 85, no more than 80, no more than 75, no more than 70, no more than 60, no more than 50, or no more than 40 amino acids; preferably, wherein the truncated portion of the coiled-coil domain has no more than 260 amino acids. In some embodiments, the coiled-coil domain corresponding to amino acids 253-324 of SEQ ID NO: 1 is removed or replaced by a polypeptide comprising no more than 20, 15, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids. In some embodiments, the region within the coiled-coil domain is replaced by a polypeptide comprising a sequence of SGGGGSGGGGS (SEQ ID NO:431) or QAGKRSLPD (SEQ ID NO: 432), or a sequence having at most 1, 2, or 3 mutations thereto.

[0014] In some embodiments, the recombinant polypeptide comprises an inhibitory domain or a truncated portion thereof, wherein the start of the inhibitory domain corresponds to amino acid position 443 of SEQ ID NO: 1, and wherein the end of the inhibitory domain corresponds to amino acid position 577 of SEQ ID NO: 1; optionally, wherein the inhibitory domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 443-577 of SEQ ID NO: 1. In some embodiments, the recombinant polypeptide comprises an inhibitory domain or a truncated portion thereof, wherein the inhibitory domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 441. In some embodiments, the inhibitory domain comprises at least 130, or at least 125, amino acids. In some embodiments, the recombinant polypeptide comprises the full inhibitory domain. In some embodiments, the recombinant polypeptide comprises a truncated portion of the inhibitory domain having no more than 120, no more than 110, no more than 100, no more than 95, no more than 90, no more than 85, no more than 80, no more than 75, no more than 70, no more than 60, no more than 50, or no more than 40 amino acids. In some embodiments, the inhibitory domain comprises N-terminal and / or C-terminal truncation(s). In some embodiments, the inhibitory domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 amino acids at the N-terminus. In some embodiments, the inhibitory domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 amino acids at the C-terminus. In some embodiments, the inhibitory domain comprises a truncation of at least 5, at least 10, atleast 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 amino acids in between the N-terminus and the C-terminus.

[0015] In some embodiments, the inhibitory domain or the truncated portion thereof comprises at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 serines that are capable of being phosphorylated; optionally wherein the serines are capable of being phosphorylated by a protein kinase C (PKC); optionally, wherein the PKC comprises PKCP and / or PKC9. In some embodiments, the inhibitory domain or the truncated portion thereof comprises a sequence of DASPRT (SEQ ID NO: 425), RAKSPI (SEQ ID NO: 426), DASPSS (SEQ ID NO: 427), SRSSIMS (SEQ ID NO: 428), RKFSLER (SEQ ID NO: 429), and / or FRPSVTS (SEQ ID NO: 430), or a sequence having at most 1, 2, or 3 mutations thereto; optionally, where the inhibitory domain or the truncated portion thereof comprises the sequence of RAKSPI (SEQ ID NO: 426), DASPSS (SEQ ID NO: 427), and / or SRSSIMS (SEQ ID NO: 428).

[0016] In some embodiments, the recombinant polypeptide comprises, from N-term to C- term: the coiled-coil domain or the truncated portion thereof, and the inhibitory domain or the truncated portion thereof, without any additional amino acid in between.

[0017] In some embodiments, the recombinant polypeptide comprises (b) a recruitment domain or a functional fragment thereof, wherein the recruitment domain is capable of binding to (i) a substrate localized to the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine. In some embodiments, the recruitment domain is capable of binding to a substrate indirectly localized to the intracellular side of the plasma membrane, and wherein the recruitment domain binds a polypeptide or lipid that is directly localized to the intracellular side of the plasma membrane of a cell. In some embodiments, the recruitment domain is a Src Homology region 2 (SH2) domain, a Src Homology region 3 (SH3) domain, a phosphotyrosine-binding (PTB) domain, or a pleckstrin homology (PH) domain. In some embodiments, the recruitment domain is a Src Homology region 2 (SH2) domain. In some embodiments, the recruitment domain or the functional fragment thereof comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a segment of at least 30, 40, 50, 60, 70, 80, 90, or 97 amino acids within the region of amino acids 622- 716 of SEQ ID NO: 1.

[0018] In some embodiments, the recombinant polypeptide comprises, from N-term to C- term: the inhibitory domain or the truncated portion thereof, and the recruitment domain or the functional fragment thereof, with no more than 5, no more than 10, no more than 15, nomore than 20, or no more than 25, amino acids in between. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 433-438 in between the inhibitory domain or the truncated portion thereof and the recruitment domain or the functional fragment thereof. In some embodiments, the recombinant polypeptide comprises, from N-terminus to C-terminus, the CARD domain, optionally the coiled-coil domain, optionally the inhibitory domain, and the recruitment domain.

[0019] In one aspect, the disclosure provides a nucleic acid construct comprising a polynucleotide sequence encoding a recombinant polypeptide described herein. In some embodiments, the nucleic acid construct further comprises a polynucleotide sequence encoding one or more additional polypeptides and / or a non-coding RNA. In some embodiments, the one or more additional polypeptides comprise a chimeric antigen receptor (CAR), a recombinant T cell receptor (TCR), an additional potency enhancement polypeptide, a cytokine, a chemokine, a growth factor, a safety switch, or any combination thereof. In some embodiments, the one or more additional polypeptides comprise a CAR or a recombinant TCR.

[0020] In some embodiments, the nucleic acid construct comprises a first homology arm before the 5’ end of the nucleic acid sequence encoding the recombinant polypeptide and a second homology arm after the 3’ end of the nucleic acid sequence encoding the recombinant polypeptide; optionally, wherein the lengths of the two homology arms are between about 150 bp and about 1500 bp. In some embodiments, the length of at least one of the homology arms is about 200bp, 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, or about 500 bp; optionally, wherein the lengths of both of the homology arms are about 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, about 500 bp, about 525 bp, about 550 bp, about 575 bp, or about 600 bp. In some embodiments, the total length of the homology arms is between about 400 bp and about 500 bp, about 500 bp and about 600 bp, about 600 bp and about 700 bp, about 700 bp and about 800 bp, about 800 bp and about 900 bp, about 900 bp and about 1000 bp, about 1000 bp and about 1100 bp, or about 1100 bp and about 1200 bp. In some embodiments, the nucleic acid construct is less than 10 kb, less than 9 kb, less than 8 kb, less than 7 kb, less than 6 kb, less than 5 kb, less than 4 kb, or less than 3 kb in length. In some embodiments, the nucleic acid construct is less than about 5.0kb in length. In some embodiments, the nucleic acid construct is less than about 4.7 kb in length.

[0021] In one aspect, the disclosure provides a vector comprising a nucleic acid construct described herein. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from a retrovirus vector, an adenovirus vector, and an adeno- associated virus (AAV) vector; optionally, wherein the viral vector is an AAV vector.

[0022] In one aspect, the disclosure provides an engineered cell comprising a recombinant polypeptide or a nucleic acid construct described herein. In some embodiments, activating the engineered cell results in the secretion of IFNg by the cell at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, least 30-fold, or at least 100-fold higher than the IFNg secretion level of a control cell lacking the recombinant polypeptide. In some embodiments, activating the engineered cell results in the secretion of IL-2 by the cell at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, least 30-fold, or at least 100-fold higher than the IL-2 secretion level of a control cell lacking the recombinant polypeptide.

[0023] In some embodiments, activation of the engineered cell and subsequent removal of the target antigen results in CARD11-PIK3R3 signaling by the cell at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10- fold, at least 20-fold, least 30-fold, or at least 100-fold lower than the CARD11-PIK3R3 signaling of a control cell comprising a CARD11-PIK3R3 fusion protein lacking the inhibitory domain. In some embodiments, activation of the engineered cell and subsequent removal of the target antigen results in CARD11-PIK3R3 signaling by the cell at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, least 30-fold, or at least 100-fold lower than the CARD11- PIK3R3 signaling of a control cell comprising a CARD11-PIK3R3 fusion protein lacking one of more of S466, S512, S535, S559, S644, and S652 according to SEQ ID NO: 518 in the inhibitory domain.

[0024] In some embodiments, CARD11-PIK3R3 signaling in the cell in the absence of a target antigen is at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, least 30-fold, or at least 100-fold lower than the CARD11-PIK3R3 signaling of a control cell comprising a control CARD11- PIK3R3 fusion protein lacking the inhibitory domain. In some embodiments, CARD11- PIK3R3 signaling in the cell in the absence of a target antigen is at a level that is at least10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10- fold, at least 20-fold, least 30-fold, or at least 100-fold lower than the CARD11-PIK3R3 signaling of a control cell comprising a control CARD11-PIK3R3 fusion protein lacking one of more of S466, S512, S535, S559, S644, and S652 according to SEQ ID NO: 518 in the inhibitory domain. In some embodiments, the cell is selected from the group consisting of a T cell, a macrophage, a monocyte, and a natural killer (NK) cell. In some embodiments, the cell is a T cell. In some embodiments, the cell further comprises: (i) a chimeric antigen receptor (CAR) having specificity for a target antigen; or (ii) a recombinant T cell receptor (TCR) having specificity for a target antigen.

[0025] In one aspect, the disclosure provides a composition comprising an engineered cell or a plurality of an engineered cell described herein.

[0026] In one aspect, the disclosure provides a method of preparing an engineered cell, the method comprising expressing in the cell a recombinant polypeptide described herein, thereby making the engineered cell. In some embodiments, the method comprises introducing into the cell a nucleic acid construct described herein. In some embodiments, the method comprises introducing into the cell a nucleic acid construct described herein, thereby making the engineered cell. In some embodiments, the step of introducing comprises homology-directed repair (HDR)-mediated insertion using the gene editing machinery.

[0027] In some embodiments, the HDR occurs in at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, of a population of the cells; optionally, wherein the HDR occurs in between about 20%-30%, about 30%-40%, about 40%-50%, about 50%-60%, about 60%-70%, about 70%-80%, about 80%-90%, or about 90%-100%, of the cells. In some embodiments, the step of introducing comprises HDR-mediated insertion in the absence of an HDR enhancer; optionally, wherein the HDR enhancer is M3814 or AZD7648.

[0028] In some embodiments, the nucleic acid construct comprises a first homology arm before the 5’ end of the nucleic acid sequence encoding the recombinant polypeptide and a second homology arm after the 3’ end of the nucleic acid sequence encoding the recombinant polypeptide; optionally, wherein the lengths of the two homology arms are between about 150 bp and about 1500 bp. In some embodiments, the length of each of the homology arms is at least 200 bp. In some embodiments, the length of at least one of the homology arms is about 200bp, 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, or about 500 bp; optionally, wherein the lengths of both of the homology arms are about225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, about 500 bp, about 525 bp, about 550 bp, about 575 bp, or about 600 bp. In some embodiments, the total length of the homology arms is between about 400 bp and about 500 bp, about 500 bp and about 600 bp, about 600 bp and about 700 bp, about 700 bp and about 800 bp, about 800 bp and about 900 bp, about 900 bp and about 1000 bp, about 1000 bp and about 1100 bp, or about 1100 bp and about 1200 bp.

[0029] In some embodiments of the method, the nucleic acid construct comprises, from 5’ to 3’, the first homology arm, a promoter, a CAR encoding sequence, a polynucleotide sequence encoding the recombinant polypeptide, and the second homology arm. In some embodiments of the method, the nucleic acid construct is introduced via AAV. In some embodiments, the method thereby produces an engineered cell comprising: (i) the recombinant polypeptide; (ii) the immune receptor (e.g., CAR); and (iii) the modified TRAC gene locus. In some embodiments, the method thereby produces an engineered cell comprising: (i) the recombinant polypeptide; (ii) the immune receptor (e.g., CAR); and (iii) the modified TRAJ intron splice acceptor locus.

[0030] In one aspect, the disclosure provides a method of treating a subject, comprising administering to the subject an engineered cell described herein or an engineered cell prepared by a method described herein. In some embodiments, the subject has a cancer or an autoimmune disease.

[0031] In one aspect, the disclosure provides recombinant polypeptides comprising a caspase-associated recruitment domain (CARD) containing protein or a functional fragment thereof, wherein the recombinant polypeptide comprises no more than 724 amino acids.

[0032] In one aspect, the disclosure provides recombinant polypeptides comprising a caspase-associated recruitment domain (CARD) containing protein or a functional fragment thereof, wherein the recombinant polypeptide does not comprise a full length CARD containing protein.

[0033] In one aspect, the disclosure provides recombinant polypeptides comprising a recruitment domain or a functional fragment thereof, wherein the recruitment domain is capable of binding to (i) a substrate localized to the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine, wherein the recombinant polypeptide comprises no more than 724 amino acids.

[0034] In one aspect, the disclosure provides recombinant polypeptides comprising a recruitment domain or a functional fragment thereof, wherein the recruitment domain iscapable of binding to (i) a substrate localized to the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine, wherein the recombinant polypeptide does not comprise a full length sequence of an endogenous protein comprising the recruitment domain.

[0035] In one aspect, the disclosure provides recombinant polypeptides comprising: (a) a caspase-associated recruitment domain (CARD), and (b) a recruitment domain or a functional fragment thereof, wherein the recruitment domain is capable of binding to (i) a substrate localized to the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine.

[0036] In one aspect, the disclosure provides recombinant polypeptides comprising: (a) a caspase-associated recruitment domain (CARD), and (b) a recruitment domain or a functional fragment thereof, wherein the recruitment domain is capable of binding to (i) a substrate localized to the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine, wherein the recombinant polypeptide comprises no more than 724 amino acids.

[0037] In one aspect, the disclosure provides recombinant polypeptides comprising: (a) a caspase-associated recruitment domain (CARD), and (b) a recruitment domain or a functional fragment thereof, wherein the recruitment domain is capable of binding to (i) a substrate localized to the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine, wherein the recombinant polypeptide does not comprise a full length sequence of an endogenous protein comprising the CARD and / or a full length sequence of an endogenous protein comprising the recruitment domain.

[0038] In one aspect, the disclosure provides recombinant polypeptides comprising: (a) a first polypeptide capable of binding to a caspase-associated recruitment domain (CARD) on BCL10, and (b) a recruitment domain or a functional fragment thereof, wherein the recruitment domain is capable of binding to (i) a substrate localized to the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine, wherein the recombinant polypeptide comprises no more than 724 amino acids.

[0039] In one aspect, the disclosure provides recombinant polypeptides comprising: (a) a first polypeptide capable of binding to a caspase-associated recruitment domain (CARD) on BCL10, and (b) a recruitment domain or a functional fragment thereof, wherein the recruitment domain is capable of binding to (i) a substrate localized to the intracellular sideof the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine, wherein the recombinant polypeptide does not comprise a full length sequence of an endogenous protein capable of binding the CARD recruitment domain ofBCLIO.

[0040] In some embodiments, when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits decreased: (a) BCL10 binding, (b) BCL10 and MALT1 complex formation, (c) NF AT, NF-KB and / or AP-1 signaling, (d) cytokine production, (e) JAK / STAT signaling, (f) co-stimulatory molecule signaling, (g) phospholipase gamma signaling, and / or (h) transcription factor activity, compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

[0041] In some embodiments, when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits decreased Interferon gamma (IFNg) and / or Interleukin-2 (IL-2) production compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

[0042] In some embodiments, when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits equivalent or enhanced: (a) BCL10 binding, (b) BCL10 and MALT1 complex formation, (c) NF-KB and / or AP-1 signaling, (d) MALT1 paracaspase activity, (e) cytokine production, (f) peak tumor expansion, (g) peak peripheral blood expansion, (h) contraction following peak tumor expansion, and / or (i) contraction following peak peripheral blood expansion, compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

[0043] In some embodiments, when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits equivalent or higher IFNg and / or IL-2 production compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

[0044] In some embodiments, the recombinant polypeptide comprises no more than 700, no more than 650, no more than 600, no more than 550, no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, or no more than 150 amino acids.

[0045] In some embodiments, the CARD containing protein or the functional fragment thereof, the CARD, or the recombinant polypeptide, is capable of binding to a CARD domain on BCL10.

[0046] In some embodiments, the CARD containing protein or the functional fragment thereof, the CARD, or the recombinant polypeptide, is capable of forming a complex with BCL10 and MALT 1.

[0047] In some embodiments, the CARD containing protein or the functional fragment thereof, or the CARD, comprises no more than 40, no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, no more than 100, no more than 150, no more than 200, no more than 300, no more than 400, or no more than 500 amino acids.

[0048] In some embodiments, the CARD containing protein or the functional fragment thereof, or the CARD, comprises or consists of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 293-323.

[0049] In some embodiments, the CARD containing protein is selected from, or wherein the CARD is derived from a protein selected from,CARD9, CARDIO, CARD11, and CARD14. In some embodiments, the CARD containing protein is, or wherein the CARD is derived from, CARD11.

[0050] In some embodiments, the recombinant polypeptide comprises a full CARD domain or a truncated portion thereof, wherein the start of the full CARD domain corresponds to amino acid position 11 of SEQ ID NO: 1, and wherein the end of the full CARD domain corresponds to amino acid position 103 of SEQ ID NO: 1. In some embodiments, the full CARD domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to one of SEQ ID NO: 293-323. In some embodiments, the full CARD domain comprises at least 90 amino acids. In some embodiments, the recombinant polypeptide comprises the full CARD domain.

[0051] In some embodiments, the CARD domain comprises N-terminal and / or C-terminal truncation(s). In some embodiments, the CARD domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 amino acids at the N- terminus. In some embodiments, the CARD domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 amino acids at the C- terminus. In some embodiments, the CARD domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 amino acids in between the N-terminus and the C-terminus.

[0052] In some embodiments, the recombinant polypeptide does not comprise any full or truncated portion of CARD domain having at least 30 amino acids in length.

[0053] In some embodiments, the recombinant polypeptide comprises or consists of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 225-245.

[0054] In some embodiments, the recombinant polypeptide does not comprise the polypeptide sequence “MDDY” within 10 amino acids before the N-term of the start of the CARD domain.

[0055] In some embodiments, when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits decreased: (a) BCL10 binding, (b) BCL10 and MALT1 complex formation, (c) NF AT, NF-KB and / or AP-1 signaling, (d) cytokine production, (e) JAK / STAT signaling, (f) co-stimulatory molecule signaling, (g) phospholipase gamma signaling, and / or (h) transcription factor activity, compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

[0056] In some embodiments, when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits decreased IFNg and / or IL-2 production compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

[0057] In some embodiments, when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits equivalent or enhanced: (a) BCL10 binding, (b) BCL10 and MALT1 complex formation, (c) NF-KB and / or AP-1 signaling, (d) MALT1 paracaspase activity, (e) cytokine production, (f) peak tumor expansion, (g) peak peripheral blood expansion, (h) contraction following peak tumor expansion, and / or (i) contraction following peak peripheral blood expansion, compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

[0058] In some embodiments, when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits equivalent or higher IFNg and / or IL-2 production compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

[0059] In some embodiments, the recombinant polypeptide comprises a full coiled-coil domain or a truncated portion thereof, wherein the start of the full coiled-coil domain corresponds to amino acid position 123 of SEQ ID NO: 1, and wherein the end of the full coiled-coil domain corresponds to amino acid position 442 of SEQ ID NO: 1; optionally, wherein the full coiled-coil domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 123-442 of SEQ ID NO:1. In some embodiments, the full coiled-coil domain comprises at least 310 amino acids. In some embodiments, the recombinant polypeptide comprises the full coiled-coil domain.

[0060] In some embodiments, the recombinant polypeptide comprises a truncated portion of the coiled-coil domain having no more than 300, no more than 290, no more than 280, no more than 270, no more than 260, no more than 250, no more than 240, no more than 230, no more than 220, no more than 210, no more than 200, no more than 190, no more than 180, no more than 170, no more than 160, no more than 150, no more than 140, no more than 130, no mor than 120, no more than 110, no more than 100, no more than 95, no more than 90, no more than 85, no more than 80, no more than 75, no more than 70, no more than 60, no more than 50, or no more than 40 amino acids; preferably, wherein the truncated portion of the coiled-coil domain has no more than 260 amino acids.

[0061] In some embodiments, the coiled-coil domain comprises N-terminal and / or C- terminal truncation(s). In some embodiments, the coiled-coil domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, or at least 270 amino acids at the N-terminus. In some embodiments, the coiled-coil domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, or at least 270 amino acids at the C-terminus.

[0062] In some embodiments, the coiled-coil domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, or at least 270 amino acids in between the N-terminus and the C-terminus; optionally, the coiled-coil domain comprises a truncation of about 60 to about 80 amino acids in between the N-terminus and the C-terminus.

[0063] In some embodiments, a region within the coiled-coil domain corresponding to amino acids 253-324 of SEQ ID NO: 1 is removed or replaced by a polypeptide comprising no more than 20, 15, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids.

[0064] In some embodiments, the region within the coiled-coil domain is replaced by a polypeptide comprising a sequence of SGGGGSGGGGS (SEQ ID NO: 431) or QAGKRSLPD (SEQ ID NO: 432), or a sequence having at most 1, 2, or 3 mutations thereto.

[0065] In some embodiments, the recombinant polypeptide does not comprise any full or truncated portion of coiled-coil domain having at least 30 amino acids in length.

[0066] In some embodiments, the recombinant polypeptide comprises or consists of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 193-224 and 246-276.

[0067] In some embodiments, when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits decreased: (a) BCL10 binding, (b) BCL10 and MALT1 complex formation, (c) NF AT, NF-KB and / or AP-1 signaling, (d) cytokine production, (e) JAK / STAT signaling, (f) co-stimulatory molecule signaling, (g) phospholipase gamma signaling, and / or (h) transcription factor activity, compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

[0068] In some embodiments, when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits decreased IFNg and / or IL-2 production compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

[0069] In some embodiments, when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits equivalent or enhanced: (a) BCL10 binding, (b) BCL10 and MALT1 complex formation, (c) NF-KB and / or AP-1 signaling, (d) MALT1 paracaspase activity, (e) cytokine production, (f) peak tumor expansion, (g) peak peripheral blood expansion, (h) contraction following peak tumor expansion, and / or (i) contraction following peak peripheral blood expansion, compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

[0070] In some embodiments, when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits equivalent or enhanced IFNg and / or IL-2 production compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

[0071] In some embodiments, the recombinant polypeptide comprises an inhibitory domain or a truncated portion thereof, wherein the start of the inhibitory domain corresponds to amino acid position 443 of SEQ ID NO: 1, and wherein the end of the inhibitory domain corresponds to amino acid position 577 of SEQ ID NO: 1; optionally, wherein the inhibitorydomain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 443-577 of SEQ ID NO: 1.

[0072] In some embodiments, the recombinant polypeptide comprises an inhibitory domain or a truncated portion thereof, wherein the inhibitory domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 441.

[0073] In some embodiments, the inhibitory domain comprises at least 130, or at least 125, amino acids. In some embodiments, the recombinant polypeptide comprises the full inhibitory domain.

[0074] In some embodiments, the recombinant polypeptide comprises a truncated portion of the inhibitory domain having no more than 120, no more than 110, no more than 100, no more than 95, no more than 90, no more than 85, no more than 80, no more than 75, no more than 70, no more than 60, no more than 50, or no more than 40 amino acids.

[0075] In some embodiments, the inhibitory domain comprises N-terminal and / or C- terminal truncation(s). In some embodiments, the inhibitory domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 amino acids at the N-terminus. In some embodiments, the inhibitory domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 amino acids at the C-terminus.

[0076] In some embodiments, the inhibitory domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 amino acids in between the N-terminus and the C-terminus.

[0077] In some embodiments, the recombinant polypeptide does not comprise any full or truncated portion of inhibitory domain having at least 30 amino acids in length.

[0078] In some embodiments, the inhibitory domain or the truncated portion thereof comprises at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 serines that are capable of being phosphorylated; optionally wherein the series are capable of being phosphorylated by a protein kinase C (PKC); optionally, wherein the PKC comprises PKCP and / or PKC 9.

[0079] In some embodiments, the inhibitory domain or the truncated portion thereof comprises a sequence of DASPRT (SEQ ID NO:425), RAKSPI (SEQ ID NO:426), DASPSS (SEQ ID NO:427), SRSSIMS (SEQ ID NO: 428), RKFSLER (SEQ ID NO: 429), and / or FRPSVTS (SEQ ID NO: 430), or a sequence having at most 1, 2, or 3 mutations thereto;optionally, where the inhibitory domain or the truncated portion thereof comprises the sequence of RAKSPI (SEQ ID NO:426), DASPSS (SEQ ID NO:427), and / or SRSSIMS (SEQ ID NO: 428).

[0080] In some embodiments, the recombinant polypeptide comprises, from N-term to C- term: the coiled-coil domain or the truncated portion thereof, and the inhibitory domain or the truncated portion thereof, without any additional amino acid in between.

[0081] In some embodiments, the recombinant polypeptide comprises or consists of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 179-192, 277-288, and 289-292.

[0082] In some embodiments, when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits equivalent or enhanced: (a) BCL10 binding, (b) BCL10 and MALT1 complex formation, (c) NF-KB and / or AP-1 signaling, and / or (d) MALT1 paracaspase activity, (e) cytokine production, (f) peak tumor expansion, (g) peak peripheral blood expansion, (h) contraction following peak tumor expansion, and / or (i) contraction following peak peripheral blood expansion, compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

[0083] In some embodiments, when the recombinant polypeptide is expressed in an immune cell, the immune cell exhibits equivalent or enhanced IFNg and / or IL-2 production compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

[0084] In some embodiments, the recombinant polypeptide comprises (b) a recruitment domain or a functional fragment thereof, wherein the recruitment domain is capable of binding to (i) a substrate localized to the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine.

[0085] In some embodiments, the substrate localized to the intracellular side of the plasma membrane of a cell is a phosphoinositide; optionally, wherein the phosphoinositide is selected from phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and phosphatidylinositol 4,5- bisphosphate (PI(4,5)P2).

[0086] In some embodiments, the recombinant polypeptide binds to the phosphoinositide with a Kd of less than 50 pM, 10 pM, 5 pM, IpM, 0.5 pM, 0.1 pM, 0.05 pM, or 0.01 pM, and wherein the Kd is analyzed using SPR.

[0087] In some embodiments, the target polypeptide is derived from IGF-1R, CTLA-4, or CD28.

[0088] In some embodiments, the phosphorylated tyrosine is located at the position corresponding to pY1221 of SEQ ID NO: 324, or to pY1346 of SEQ ID NO: 325.

[0089] In some embodiments, the polypeptide binds to the target polypeptide with a Kd of less than 10 pM, 5 pM, IpM, 0.5 pM, 0.1 pM, 0.05 pM, or 0.01 pM, and wherein the Kd is analyzed by fluorescence polarization assay.

[0090] In some embodiments, the recruitment domain is capable of binding to a substrate indirectly localized to the intracellular side of the plasma membrane, and wherein the recruitment domain binds a polypeptide or lipid that is directly localized to the intracellular side of the plasma membrane of a cell.

[0091] In some embodiments, the recruitment domain is a Src Homology region 2 (SH2) domain, a Src Homology region 3 (SH3) domain, a phosphotyrosine-binding (PTB) domain, or a pleckstrin homology (PH) domain. In some embodiments, the recruitment domain is a Src Homology region 2 (SH2) domain. In some embodiments, the start of the recruitment domain corresponds to amino acid position 622 of SEQ ID NO: 1 and wherein the end of the recruitment domain corresponds to amino acid position 716 of SEQ ID NO: 1.

[0092] In some embodiments, the recruitment domain comprises at least 90 amino acids.

[0093] In some embodiments, the recombinant polypeptide comprises the functional fragment of the recruitment domain.

[0094] In some embodiments, the recruitment domain comprises N-terminal and / or C- terminal truncation(s). In some embodiments, the recruitment domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 amino acids at the N-terminus. In some embodiments, the recruitment domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 amino acids at the C-terminus.

[0095] In some embodiments, the recruitment domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 amino acids in between the N-terminus and the C-terminus.

[0096] In some embodiments, the recruitment domain or the functional fragment thereof comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a segment of at least 30, 40, 50, 60, 70, 80, 90, or 97 amino acids within the region of amino acids 622-716 of SEQ ID NO: 1.

[0097] In some embodiments, the recombinant polypeptide does not comprise any recruitment domain having at least 30 amino acids in length.

[0098] In some embodiments, the recombinant polypeptide comprises, from N-term to C- term: the inhibitory domain or the truncated portion thereof, and the recruitment domain orthe functional fragment thereof, with no more than 5, no more than 10, no more than 15, no more than 20, or no more than 25, amino acids in between.

[0099] In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 433-438in between the inhibitory domain or the truncated portion thereof and the recruitment domain or the functional fragment thereof.

[0100] In some embodiments, the recombinant polypeptide comprises, from N-terminus to C-terminus, the CARD domain, optionally the coiled-coil domain, optionally the inhibitory domain, and the recruitment domain.

[0101] In some embodiments, the recombinant polypeptide comprises no more than 40, no more than 35, no more than 30, no more than 25, no more than 20, no more than 15, no more than 10, or no more than 5, amino acids, in between the CARD containing protein or functional fragment thereof and the recruitment domain.

[0102] In some embodiments, the recombinant polypeptide comprises or consists of a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 3-150.

[0103] In some embodiments, the recombinant polypeptide comprises or consists of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 151-178.

[0104] In some embodiments, the recombinant polypeptide comprises an aspartatic acid (D) at the position corresponding to amino acid 602 of SEQ ID NO: 1.

[0105] In one aspect, the disclosure provides nucleic acid constructs comprising a polynucleotide sequence encoding the recombinant polypeptide of the disclosure.

[0106] In some embodiments, the polynucleotide sequence encoding the recombinant polypeptide is operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter or an inducible promoter. In some embodiments, the promoter is or comprises a minimal TATA promoter, pGK promoter, actin promoter, CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, CARD9 promoter, CARDIO promoter, CARD 11 promoter, CARD 14 promoter, PIK3R3 promoter, CD25 promoter, IL2 promoter, IL7 promoter, IL 15 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CDl la promoter, CD58 promoter, CD99 promoter, CD62L promoter, CD 103 promoter, CCR4 promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter, CXCR3 promoter, CXCR4 promoter, CL A promoter,Granzyme A promoter, Granzyme B promoter, Perforin promoter, CD57 promoter, CD161 promoter, IL-18Ra promoter, CD69 promoter, GzmB promoter, T-bet promoter, IFNgamma promoter, TIM3 promoter, IL4 promoter, GAT A3 promoter, IL1 promoter, IL5 promoter, IL6 promoter, IL 13 promoter, IL 10 promoter, IL17A promoter, IL6 promoter, IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25 promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter, CD95 promoter, CD28 promoter, CD27 promoter, CD 127 promoter, PD-1 promoter, CD 122 promoter, CD 132 promoter, c-Kit promoter, nuclear factor of activated T cells (NF AT) promoter, programmed death 1 (PD-1) promoter, T cell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, lymphocyte-activation protein 3 (LAG-3) promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, TGF-beta promoter, T-bet promoter, Eomes promoter, GATA3 promoter, CD45RA promoter, 2B4 promoter, Type I interferon (IFN) alpha, Type I IFN beta promoter, IFN gamma promoter, IRF3 promoter, IRF7 promoter, NFkB promoter, AP-1 promoter, TNF-alpha promoter, CD 130 promoter, NR4A1 promoter, NR4A2, NR4A3 promoter, MND promoter, EF-1 alpha promoter, short EF-1 alpha promoter, CAG promoter, ubiquitin / S27a promoter, SV40 promoter, SV40 early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, Moloney murine leukemia virus (MMLV) long terminal repeat (LTR) region, CMV promoter, immunoglobulin promoter, heat shock promoter, polyoma virus promoter, fowlpox virus promoter, bovine papilloma virus promoter, avian sarcoma virus promoter, retrovirus promoter, hepatitis-B virus promoter, PGK promoter, vaccinia virus 7.5K promoter, TK promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of moloney virus, Epstein Barr virus (EBV) promoter, Rous sarcoma virus (RSV) promoter, U6 promoter, or UBC promoter.

[0107] In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is or comprises a CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, CARD9 promoter, CARDIO promoter, CARD 11 promoter, CARD 14 promoter, or PIK3R3 promoter.

[0108] In some embodiments, the promoter is or comprises an MND promoter or a short EFla promoter.

[0109] In some embodiments, the polynucleotide sequence encoding the recombinant polypeptide is not operably linked to a promoter.

[0110] In some embodiments, the one or more cleavable linkers comprise a P2A, E2A, F2A, or T2A self-cleaving peptide.[OHl] In some embodiments, the nucleic acid construct further comprises one or more nucleic acid coding or regulatory sequence. In some embodiments, the one or more nucleic acid coding or regulatory sequence is or comprises ribosomal binding sites, enhancer elements, activator elements, translational start sequences, translational termination sequences, transcription start sequences, transcription termination sequences, polyadenylation signal sequences, a 70 bp poly(A) tract, a 100 bp poly(A) tract, a 172 bp poly(A) tract, a 200 bp poly(A) tract, a 300 bp poly(A) tract, a 325 bp poly(A) tract, replication elements, RNA processing and export elements, transposon sequences, transposase sequences, insulator sequences, internal ribosome entry sites (IRES), 5’UTRs, 3’UTRs, mRNA 3’ end processing sequences, boundary elements, locus control regions (LCR), matrix attachment regions (MAR), recombination or cassette exchange sequences, linker sequences, cleavable linker sequences, secretion signals, resistance markers, anchoring peptides, localization signals, fusion tags, affinity tags, chaperonins, proteases, or any combination thereof.

[0112] In some embodiments, the nucleic acid construct further comprises a polynucleotide sequence encoding one or more additional polypeptides and / or a non-coding RNA. In some embodiments, the non-coding RNA comprises a shRNA or a microRNA. In some embodiments, the one or more additional polypeptides comprise a chimeric antigen receptor (CAR), a recombinant T cell receptor (TCR), an additional potency enhancement polypeptide, a cytokine, a chemokine, a growth factor, a safety switch, or any combination thereof. In some embodiments, the one or more additional polypeptides comprise a CAR or a recombinant TCR.

[0113] In some embodiments, the CAR or recombinant TCR has specificity for a target antigen selected from the group consisting of CD1, CD la, CD lb, CDlc, CD Id, CDle, CD2, CD3d, CD3e, CD3g, CD3s, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44v6, CD45, CD46, CD47 CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD213A2, CD246, CD252, CD253, CD261, CD262,CD272, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), CDH17, CEA, CLECL1, CLL-1, CLDN6, CLDN18.2, CS1, DLL3, LY6G6D, GCC, p53R175H, PRAME, EGFR, EGFRvIII, FGFR2, AFP, CA125, MUC-1, MAGE, ALPI, alkaline phosphatase placental-like 2 (ALPPL2), B-cell maturation antigen (BCM A), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), KLK2, KLK3, Mesothelin, IL13Ra2, ggcc, signal regulatory protein a (SIRPa), TCRalpha, TCRbeta, TSHR, GD2, GD3, Tn Ag, cMET, Axl, R0R1, R0R2, GPC1, GPC2, GPC3, FLT3, TAG72, CEA, EPC AM, KIT (CD117), IL-13Ra2, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, PDGFRp, SSEA-4, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, STEAP1, STEAP2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, R0PN1, GPRC5D, GPA33, CX0RF61, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE- Al, legumain, HPV E6,E7, MAGE-A4, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, p53R175H, KRAS, mutant KRAS, KRAS G12D, prostein, surviving, telomerase, PCTA-l / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70- 2, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, FCRL5, IGLL1, PSMA, TROP2, citrullinated vimentin, the extracellular portion of the APRIL protein, and any combinations thereof.

[0114] In some embodiments, the CAR or recombinant TCR has specificity for a target antigen selected from the group consisting of B7H3, BCMA, CD19, CD20, CD22, CD70, CD79a, CD79b, CDH17, CLDN6, CLDN18.2, DLL3, GCC, GD2, GD3, GPC3, GPRC5D, GPA33, KLK3, LY6G6D, p53R175H, PRAME, ROPN1, STEAP1, and STEAP2.

[0115] In some embodiments, the CAR comprises one or more costimulatory domains. In some embodiments, the one or more costimulatory domains are derived from a protein selected from the group comprising CD3zeta, CD28, 4-1BB, 0X40, IL2Rb turbodomains, MYD88 / CD40, MYD88 / CD40 inducible constimulatory domain, CD2, B7-1 / CD80; B7- 2 / CD86; B7-H1 / PD-L1; B7-H2; B7-H3; B7-H4; B7-H6; B7-H7; BTLA / CD272; CD28; CTLA-4; Gi24 / VISTA / B7-H5; ICOS / CD278; PD-1; PD-L2 / B7-DC; PDCD6); 4-1BB / TNFSF9 / CD137; 4-1BB Ligand / TNFSF9; BAFF / BLyS / TNFSF13B; BAFF R / TNFRSF13C; CD27 / TNFRSF7; CD27 Ligand / TNFSF7; CD30 / TNFRSF8; CD30 Ligand / TNFSF8; CD40 / TNFRSF5; CD40 / TNFSF5; CD40 Ligand / TNFSF5;DR3 / TNFRSF25; GITR / TNFRSF18; GITR Ligand / TNFSF18; HVEM / TNFRSF14;LIGHT / TNFSF14; Lymphotoxin-alpha / TNF-beta; OX40 / TNFRSF4; 0X40 Ligand / TNFSF4; RELT / TNFRSF19L; TACI / TNFRSF13B; TL1A / TNFSF15; TNF-alpha; TNF RII / TNFRSF1B); 2B4 / CD244 / SLAMF4; BLAME / SLAMF8; CD2; CD2F-10 / SLAMF9; CD48 / SLAMF2; CD58 / LFA-3; CD84 / SLAMF5; CD229 / SLAMF3; CRACC / SLAMF7;NTB- A / SLAMF6; SLAM / CD150); CD2; CD7; CD53; CD82 / Kai-1; CD90 / Thyl; CD96; CD160; CD200; CD300a / LMIRl; HLA Class I; HLA-DR; Ikaros; Integrin alpha 4 / CD49d; Integrin alpha 4 beta 1; Integrin alpha 4 beta 7 / LPAM-l; LAG-3; TCL1A; TCL1B; CRTAM; DAP12; Dectin- 1 / CLEC7A; DPPIV / CD26; EphB6; TIM-l / KIM-l / HAVCR; TIM-4; TSLP; TSLP R; lymphocyte function associated antigen-1 (LFA-1); NKG2C, an immunoreceptor tyrosinebased activation motif (ITAM), CD27, CD134 / OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD7, LIGHT, NKG2C, and any combinations thereof.

[0116] In some embodiments, the additional potency enhancement polypeptide is or comprises a dominant negative form of an inhibitor of a cell-mediated immune response of the immune cell (e.g., TGFPR2 DNR), c-Jun, CCL19, CCL21, IL2R, IL7, IL7Ralpha, IL15, IL15RA, IL18, decoy-resistant IL18 (DR-18), MyD88 / CD40, PD1-CD28 switch receptor, PD1-41BB switch receptor, CD40L-CD28 switch receptor, CTBR12 switch receptor, or CD8alpha / beta.

[0117] In some embodiments, the polynucleotide sequence encoding the recombinant polypeptide and the polynucleotide sequence encoding the one or more additional polypeptides are separated by a polynucleotide sequence encoding one or more cleavable linkers, and wherein all the polynucleotide sequences form a continuous open reading frame.

[0118] In some embodiments, the polynucleotide sequence encoding the recombinant polypeptide and the polynucleotide sequence encoding the one or more additional polypeptides are separated by an internal ribozyme entry site (IRES).

[0119] In some embodiments, the nucleic acid construct is a DNA construct. In some embodiments, the nucleic acid construct is an RNA construct. In some embodiments, the RNA construct is an mRNA construct.

[0120] In some embodiments, the nucleic acid construct comprises one or more modified nucleotides. In some embodiments, the one or more modified nucleotides are or comprisepseudouridine, N1 -methylpseudouridine, 4’-thiouridine, 5 -methylcytosine, 2-thio-l-methyl- 1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl- pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5- methoxyuridine, 2’-O-methyl uridine, or any combination thereof. In some embodiments, one or more modified nucleotides are selected from the group consisting of pseudouridine, N1 -methylpseudouridine, 5-methylcytosine, 5- methoxyuridine, and a combination thereof. In some embodiments, one or more modified nucleotides are Nl- methylpseudouridines.

[0121] In some embodiments, the nucleic acid construct comprises a first homology arm before the 5’ end of the nucleic acid sequence encoding the recombinant polypeptide and a second homology arm after the 3’ end of the nucleic acid sequence encoding the recombinant polypeptide; optionally, wherein the lengths of the two homology arms are between about 150 bp and about 1500 bp. In some embodiments, the length of at least one of the homology arms is about 200bp, 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, or about 500 bp; optionally, wherein the lengths of both of the homology arms are about 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, about 500 bp, about 525 bp, about 550 bp, about 575 bp, or about 600 bp. In some embodiments, the total length of the homology arms is between about 400 bp and about 500 bp, about 500 bp and about 600 bp, about 600 bp and about 700 bp, about 700 bp and about 800 bp, about 800 bp and about 900 bp, about 900 bp and about 1000 bp, about 1000 bp and about 1100 bp, or about 1100 bp and about 1200 bp.

[0122] In some embodiments, the nucleic acid construct is less than 10 kb, less than 9 kb, less than 8 kb, less than 7 kb, less than 6 kb, less than 5 kb, less than 4 kb, or less than 3 kb in length. In some embodiments, the nucleic acid construct is less than about 5.0 kb in length. In some embodiments, the nucleic acid construct is less than about 4.7 kb in length.

[0123] In some embodiments, the length of the polynucleotide sequence encoding the one or more additional polypeptides is less than about 2100 bp, less than about 2150 bp, less than about 2200 bp, less than about 2250 bp, less than about 2300 bp, less than about 2350 bp, or less than about 2400 bp.

[0124] In some embodiments, the length of the one or more additional polypeptides is less than about 700 amino acids, less than about 725 amino acids, less than about 750 aminoacids, less than about 775 amino acids, less than about 800 amino acids, less than about 825 amino acids, less than about 850 amino acids, less than about 875 amino acids, or less than about 900 amino acids.

[0125] In one aspect, the disclosure provides nucleic acid constructs comprising, from 5’ to 3’ : (1) a promoter, (2) a CAR encoding sequence, (3) a cleavable linker encoding sequence, and (4) a polynucleotide encoding the recombinant polypeptide of the disclosure.

[0126] In one aspect, the disclosure provides nucleic acid constructs comprising, from 5’ to 3’ : (1) a promoter, (2) a polynucleotide encoding the recombinant polypeptide of the disclosure, (3) a cleavable linker encoding sequence, and (4) a CAR encoding sequence.

[0127] In one aspect, the disclosure provides nucleic acid constructs comprising, from 5’ to 3’ : (1) a promoter, (2) a CAR encoding sequence, (3) a cleavable linker encoding sequence, (4) a polynucleotide encoding the recombinant polypeptide of the disclosure, (5) a cleavable linker encoding sequence, and (6) a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor.

[0128] In one aspect, the disclosure provides nucleic acid constructs comprising, from 5’ to 3’ : (1) a promoter, (2) a polynucleotide sequence encoding the recombinant polypeptide of the disclosure, (3) a cleavable linker encoding sequence, (4) a CAR encoding sequence, (5) a cleavable linker encoding sequence, and (6) a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor.

[0129] In one aspect, the disclosure provides nucleic acid constructs comprising, from 5’ to 3’ : (1) a promoter, (2) a polynucleotide sequence encoding the recombinant polypeptide of the disclosure, (3) a cleavable linker encoding sequence, (4) a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor (5) a cleavable linker encoding sequence, and (6) a CAR encoding sequence.

[0130] In one aspect, the disclosure provides nucleic acid constructs comprising, from 5’ to 3’ : (1) a promoter, (2) a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor, (3) a cleavable linker encoding sequence, (4) a polynucleotide sequence encoding the recombinant polypeptide of the disclosure, (5) a cleavable linker encoding sequence, and (6) a CAR encoding sequence.

[0131] In one aspect, the disclosure provides nucleic acid constructs comprising, from 5’ to 3’ : (1) a promoter, (2) a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor, (3) a cleavable linker encoding sequence, (4) a CAR encoding sequence, (5) a cleavable linker encoding sequence, and (6) a polynucleotide sequence encoding the recombinant polypeptide of the disclosure.

[0132] In one aspect, the disclosure provides nucleic acid constructs comprising, from 5’ to 3’ : (1) a cleavable linker encoding sequence, (2) a CAR encoding sequence, (3) a cleavable linker encoding sequence, (4) a polynucleotide sequence encoding the recombinant polypeptide of the disclosure, and (5) a cleavable linker encoding sequence.

[0133] In one aspect, the disclosure provides nucleic acid constructs comprising, from 5’ to 3’ : (1) a cleavable linker encoding sequence, (2) a polynucleotide sequence encoding the recombinant polypeptide of the disclosure, (3) a cleavable linker encoding sequence, (4) a CAR encoding sequence, and (5) a cleavable linker encoding sequence.

[0134] In one aspect, the disclosure provides nucleic acid constructs comprising, from 5’ to 3’ : (1) a cleavable linker encoding sequence, (2) a CAR encoding sequence, (3) a cleavable linker encoding sequence, (4) a polynucleotide sequence encoding the recombinant polypeptide of the disclosure, (5) a cleavable linker encoding sequence, (6) a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor, (7) a cleavable linker encoding sequence.

[0135] In one aspect, the disclosure provides nucleic acid constructs comprising, from 5’ to 3’ : (1) a cleavable linker encoding sequence, (2) a CAR encoding sequence, (3) a cleavable linker encoding sequence, (4) a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor, (5) a cleavable linker encoding sequence, (6) a polynucleotide sequence encoding the recombinant polypeptide of the disclosure, (7) a cleavable linker encoding sequence.

[0136] In one aspect, the disclosure provides nucleic acid constructs comprising, from 5’ to 3’ : (1) a cleavable linker encoding sequence, (2) a polynucleotide sequence encoding the recombinant polypeptide of the disclosure, (3) a cleavable linker encoding sequence, (4) a CAR encoding sequence, (5) a cleavable linker encoding sequence, (6) a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor, (7) a cleavable linker encoding sequence.

[0137] In one aspect, the disclosure provides nucleic acid constructs comprising, from 5’ to 3’ : (1) a cleavable linker encoding sequence, (2) a polynucleotide sequence encoding the recombinant polypeptide of the disclosure, (3) a cleavable linker encoding sequence, (4) a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor, (5) a cleavable linker encoding sequence, (6) a CAR encoding sequence, (7) a cleavable linker encoding sequence.

[0138] In one aspect, the disclosure provides nucleic acid constructs comprising, from 5’ to 3’ : (1) a cleavable linker encoding sequence, (2) a polynucleotide encoding an additionalpotency enhancement polypeptide, a cytokine, a chemokine, or a growth factor, (3) a cleavable linker encoding sequence, (4) a CAR encoding sequence, (5) a cleavable linker encoding sequence, (6) a polynucleotide sequence encoding the recombinant polypeptide of the disclosure, (7) a cleavable linker encoding sequence.

[0139] In one aspect, the disclosure provides nucleic acid constructs comprising, from 5’ to 3’ : (1) a cleavable linker encoding sequence, (2) a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor, (3) a cleavable linker encoding sequence, (4) a polynucleotide sequence encoding the recombinant polypeptide of the disclosure, (5) a cleavable linker encoding sequence, (6) a CAR encoding sequence, (7) a cleavable linker encoding sequence.

[0140] In one aspect, the disclosure provides nucleic acid constructs comprising: (1) a CAR encoding sequence, and (2) a polynucleotide sequence encoding the recombinant polypeptide of the disclosure, wherein the nucleic acid construct is an RNA construct.

[0141] In one aspect, the disclosure provides compositions comprising: (1) a first nucleic acid construct encoding a CAR, and (2) a second nucleic acid construct encoding the recombinant polypeptide of the disclosure, wherein the first and the second nucleic acid constructs are both RNA constructs.

[0142] In one aspect, the disclosure provides compositions comprising: (1) a CAR encoding sequence, (2) a polynucleotide sequence encoding the recombinant polypeptide of the disclosure, and (3) a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor, wherein the nucleic acid construct is an RNA construct.

[0143] In one aspect, the disclosure provides compositions comprising: (1) a first nucleic acid construct encoding a CAR, (2) a second nucleic acid construct encoding the recombinant polypeptide of the disclosure, and (3) a third nucleic acid construct encoding a polynucleotide encoding an additional potency enhancement polypeptide, a cytokine, a chemokine, or a growth factor, and wherein the first, second, and third nucleic acid constructs are each RNA constructs.

[0144] In one aspect, the disclosure provides vectors comprising the nucleic acid construct of the disclosure. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a synthetic DNA vector. In some embodiments, the vector is a linear DNA vector. In some embodiments, the vector is a closed linear DNA vector. In some embodiments, the vector is a synthetic RNA vector. In some embodiments, the vector is a phagemid vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector isselected from a retrovirus vector, an adenovirus vector, and an adeno-associated virus (AAV) vector; optionally, wherein the viral vector is an AAV vector. In some embodiments, the AAV vector is AAV6 vector. In some embodiments, the AAV vector is AAV9 vector. In some embodiments, the AAV vector is a split-intein dual AAV vector. In some embodiments, the retrovirus is a lentivirus. In some embodiments, the lentivirus is a VSV-G pseudotyped lentivirus. In some embodiments, the vector is a redirected lentiviral vector. In some embodiments, the vector is a fusosome. In some embodiments, the vector is a lentiviral particle engineered with the anti-CD3 Cocal glycoprotein. In some embodiments, the vector is an enveloped delivery vehicle. In some embodiments, the vector is self-replicating RNA virus. In some embodiments, the vector is mRNA-packaging virus-like particle. In some embodiments, the vector is RNP-packaging virus-like particle.

[0145] In some embodiments, activating the engineered cell results in the secretion of IL-2 by the cell at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2- fold, at least 5-fold, at least 10-fold, at least 20-fold, least 30-fold, or at least 100-fold higher than the IL-2 secretion level of a control cell lacking the recombinant polypeptide.

[0146] In some embodiments, activation of the engineered cell and subsequent removal of the target antigen results in CARD11-PIK3R3 signaling by the cell at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10- fold, at least 20-fold, least 30-fold, or at least 100-fold lower than the CARD11-PIK3R3 signaling of a control cell comprising a CARD11-PIK3R3 fusion protein lacking the inhibitory domain.

[0147] In some embodiments, activation of the engineered cell and subsequent removal of the target antigen results in CARD11-PIK3R3 signaling by the cell at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10- fold, at least 20-fold, least 30-fold, or at least 100-fold lower than the CARD11-PIK3R3 signaling of a control cell comprising a CARD11-PIK3R3 fusion protein lacking one of more of S466, S512, S535, S559, S644, and S652 according to SEQ ID NO: 518 in the inhibitory domain.

[0148] In some embodiments, CARD11-PIK3R3 signaling in the cell in the absence of a target antigen is at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, least 30-fold, or at least 100-fold lower than the CARD11-PIK3R3 signaling of a control cell comprising a control CARD11- PIK3R3 fusion protein lacking the inhibitory domain.

[0149] In some embodiments, CARD11-PIK3R3 signaling in the cell in the absence of a target antigen is at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, least 30-fold, or at least 100-fold lower than the CARD11-PIK3R3 signaling of a control cell comprising a control CARD11- PIK3R3 fusion protein lacking one of more of S466, S512, S535, S559, S644, and S652 according to SEQ ID NO: 518in the inhibitory domain.

[0150] In some embodiments, the CARD11-PIK3R3 signaling is measured by: (a) BCL10 binding, (b) BCL10 and MALT1 complex formation, (c) NF AT, NF-KB and / or AP-1 signaling, (d) cytokine production, (e) JAK / STAT signaling, (f) co-stimulatory molecule signaling, (g) phospholipase gamma signaling, and / or (h) transcription factor activity.

[0151] In some embodiments, expression of the recombinant polypeptide is under the control of an endogenous promoter. In some embodiments, the endogenous promoter is a TCRa promoter, a TCRb promoter, a CD3d promoter, a CD3g promoter, a CD3e promoter, a CD3z promoter, a CARD9 promoter, a CARDIO promoter, a CARD 11 promoter, a CARD 14 promoter, or a PIK3R3 promoter.

[0152] In some embodiments, expression of the recombinant polypeptide is under the control of an exogenous promoter. In some embodiments, the exogenous promoter is or comprises a minimal TATA promoter, pGK promoter, actin promoter, CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, CARD9 promoter, CARDIO promoter, CARD 11 promoter, CARD 14 promoter, PIK3R3 promoter, CD25 promoter, IL2 promoter, IL7 promoter, IL 15 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CDl la promoter, CD58 promoter, CD99 promoter, CD62L promoter, CD 103 promoter, CCR4 promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter, CXCR3 promoter, CXCR4 promoter, CL A promoter, Granzyme A promoter, Granzyme B promoter, Perforin promoter, CD57 promoter, CD161 promoter, IL-18Ra promoter, CD69 promoter, GzmB promoter, T-bet promoter, IFNgamma promoter, TIM3 promoter, IL4 promoter, GAT A3 promoter, IL1 promoter, IL5 promoter, IL6 promoter, IL 13 promoter, IL 10 promoter, IL17A promoter, IL6 promoter, IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25 promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter, CD95 promoter, CD28 promoter, CD27 promoter, CD 127 promoter, PD-1 promoter, CD 122 promoter, CD 132 promoter, c-Kit promoter, nuclear factor of activated T cells (NF AT) promoter, programmed death 1 (PD-1) promoter, T cell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic Tlymphocyte antigen-4 (CTLA4) promoter, lymphocyte-activation protein 3 (LAG-3) promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, TGF-beta promoter, T-bet promoter, Eomes promoter, GATA3 promoter, CD45RA promoter, 2B4 promoter, Type I interferon (IFN) alpha, Type I IFN beta promoter, IFN gamma promoter, IRF3 promoter, IRF7 promoter, NFkB promoter, AP-1 promoter, TNF-alpha promoter, CD 130 promoter, NR4A1 promoter, NR4A2, NR4A3 promoter, MND promoter, EF-1 alpha promoter, short EF-1 alpha promoter, CAG promoter, ubiquitin / S27a promoter, SV40 promoter, SV40 early promoter, adenovirus major late promoter, mouse metallothionein- 1 promoter, Moloney murine leukemia virus (MMLV) long terminal repeat (LTR) region, CMV promoter, immunoglobulin promoter, heat shock promoter, polyoma virus promoter, fowlpox virus promoter, bovine papilloma virus promoter, avian sarcoma virus promoter, retrovirus promoter, hepatitis-B virus promoter, PGK promoter, vaccinia virus 7.5K promoter, TK promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of oloney virus, Epstein Barr virus (EBV) promoter, Rous sarcoma virus (RSV) promoter, U6 promoter, or UBC promoter.

[0153] In some embodiments, the nucleic acid construct is inserted in a T-cell receptor (TCR) locus, a CD3 locus, a B2 microglobulin (B2M) locus, a class II transactivator (CIITA) locus, a CARD9 locus, a CARDIO locus, a CARD11 locus, a CARD14 locus, a PIK3R3 locus, or a safe harbor locus. In some embodiments, the TCR locus is or comprises a TRAC locus, a TRBC1 locus, a TRBC2 locus, or a TRAJ locus.

[0154] In some embodiments, the cell does not express a gene product of an endogenous TRAC locus. In some embodiments, the cell does not express a gene product of an endogenous TRBC1 locus. In some embodiments, the cell does not express a gene product of an endogenous TRBC2 locus.

[0155] In some embodiments, the CD3 locus is or comprises: a CD3d locus, a CD3g locus, a CD3e locus, or CD3z locus. In some embodiments, the cell does not express a gene product of an endogenous CD3d locus. In some embodiments, the cell does not express a gene product of an endogenous CD3g locus. In some embodiments, the cell does not express a gene product of an endogenous CD3e locus. In some embodiments, the cell does not express a gene product of an endogenous CD3z locus.

[0156] In some embodiments, the safe harbor locus is or comprises an AAVS1, ABO, CCR5, CLYBL, CXCR4, F3, FUT1, HMGB1, KDM5D, LRP1, MICA, MICB, RHD, ROSA26, or SHS231 locus.

[0157] In some embodiments, the nucleic acid construct is inserted is an exon, an intron, between an intron and an exon, or a regulatory region.

[0158] In some embodiments, the cell is selected from the group of consisting of a T cell, a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), a gamma delta T cell (yST), an invariant natural killer T (iNKT) cell, a mucosal associated invariant T (MAIT) cell, a macrophage, a monocyte, a natural killer (NK) cell, a tumor infiltrating lymphocyte (TIL), a cytotoxic T cell, a T helper cell, a memory T cell, a central memory T (TCM) cell, a stem memory T (TSCM) cell, a stem-cell-like memory T cell (or stem-like memory T cells), an effector memory T (TEM) cell, a TEMRA (CD45RA+) cell, an effector T cell, a Thl cell, a Th2 cell, a Th9 cell, a Thl7 cell, a Th22 cell, a Tfh (follicular helper) cell, a natural killer T (NKT) cell, a transitional memory T (TTM) cell, a terminal effector T (TTE) cell, a naive T (TN) cell, a hematopoietic stem cell, and a progenitor cell of the lymphoid lineage. In some embodiments, the cell is selected from the group consisting of a T cell, a macrophage, a monocyte, and a natural killer (NK) cell. In some embodiments, the cell is a T cell. In some embodiments, the T cell is selected from the group consisting of a regulatory T cell (Treg), a gamma delta T cell, a CD8+ T cell, an invariant iNKT cell, a MAIT cell, a CAR T cell, a tumor-infiltrating lymphocyte, and an engineered T cell comprising a transcriptional receptor.

[0159] In some embodiments, the cell is an autologous cell. In some embodiments, the cell is an allogeneic cell. In some embodiments, the cell is a primary cell. In some embodiments, the cell is derived from a stem cell. In some embodiments, the cell is genetically modified.

[0160] In some embodiments, the cell has reduced or eliminated expression of an endogenous T cell receptor.

[0161] In some embodiments, the cell is more prone to display central memory phenotype than a control cell lacking the recombinant polypeptide.

[0162] In some embodiments, the cell further comprises: (i) a chimeric antigen receptor (CAR) having specificity for a target antigen; or (ii) a recombinant T cell receptor (TCR) having specificity for a target antigen.

[0163] In some embodiments, the engineered cell comprises a nucleic acid construct expressing the CAR or recombinant TCR; optionally, wherein the nucleic acid construct is an RNA construct.

[0164] In some embodiments, the target antigen is CD1, CD la, CD lb, CDlc, CD Id, CDle, CD2, CD3d, CD3e, CD3g, CD3s, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44v6, CD45, CD46, CD47 CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD213A2, CD246, CD252, CD253, CD261, CD262, CD272, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), CDH17, CEA, CLECL1, CLL-1, CLDN6, CLDN18.2, CS1, DLL3, LY6G6D, GCC, p53R175H, PRAME, EGFR, EGFRvIII, FGFR2, AFP, CA125, MUC-1, MAGE, ALPI, alkaline phosphatase placental-like 2 (ALPPL2), B-cell maturation antigen (BCM A), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), KLK2, KLK3, Mesothelin, IL13Ra2, ggcc, signal regulatory protein a (SIRPa), TCRalpha, TCRbeta, TSHR, GD2, GD3, Tn Ag, cMET, Axl, ROR1, ROR2, GPC1, GPC2, GPC3, FLT3, TAG72, CEA, EPC AM, KIT (CD117), IL-13Ra2, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, PDGFRp, SSEA-4, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, STEAP1, STEAP2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, ROPN1, GPRC5D, GPA33, CXORF61, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE- Al, legumain, HPV E6,E7, MAGE-A4, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, p53R175H, KRAS, mutant KRAS, KRAS G12D, prostein, surviving, telomerase, PCTA-l / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70- 2,LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, FCRL5, IGLL1, PSMA, TROP2, citrullinated vimentin, the extracellular portion of the APRIL protein, and any combinations thereof. In some embodiments, the target antigen is B7H3, BCMA, CD19, CD20, CD22, CD70, CD79a, CD79b, CDH17, CLDN6, CLDN18.2, DLL3, GCC, GD2, GD3, GPC3, GPRC5D, GPA33, KLK3, LY6G6D, p53R175H, PRAME, ROPN1, STEAP1, or STEAP2.

[0165] In some embodiments, the engineered cell has cell surface expression of the CAR.

[0166] In some embodiments, the cell further comprises a chimeric auto antigen receptor (CAAR) having specificity for a target antigen. In some embodiments, the engineered cell has cell surface expression of the CAAR.

[0167] In some embodiments, the cell further comprises a B-cell autoantibody receptor (BAR) having specificity for a target antigen. In some embodiments, the engineered cell has cell surface expression of the BAR.

[0168] In some embodiments, the cell further comprises a non-coding RNA. In some embodiments, the non-coding RNA comprises a shRNA or a microRNA.

[0169] In some embodiments, the cell further comprises an additional potency enhancement polypeptide, a cytokine, a chemokine, a growth factor, a tolerogenic factor, a safety switch, or any combination thereof. In some embodiments, the additional potency enhancement polypeptide is or comprises a dominant negative form of an inhibitor of a cell-mediated immune response of the immune cell (e.g., TGFPR2 DNR), c-Jun, CCL19, CCL21, IL2R, IL7, IL7Ralpha, IL15, IL15RA, IL18, decoy-resistant IL 18 (DR-18), MyD88 / CD40, PD1- CD28 switch receptor, PD1-41BB switch receptor, CD40L-CD28 switch receptor, CTBR12 switch receptor, or CD8alpha / beta.

[0170] In some embodiments, the safety switch is or comprises a herpesvirus thymidine kinase (HSV-tk) gene, an Escherichia coli cytosine deaminase (EC-CD) gene, or an inducible Caspase9 (iCasp9) protein.

[0171] In some embodiments, the cell further comprises one or more modifications that inactivate or disrupt one or more alleles of TGFbeta, Regnase-1, FAS, PTPN2, NR4A3, CD52, PD-1, B2M, CIITA, SOCS1, CBLB, DGKalpha, and / or DGK^.

[0172] In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is selected from the group consisting of a human cell, a mouse cell, and a canine cell.

[0173] In some embodiments, the cell further comprises a sequence specific nuclease, a nucleic acid programmable DNA binding protein, an RNA guided nuclease, an RNA-guided nuclease comprising a Cas nuclease and a guide RNA (CRISPR-Cas combination), a ribonucleoprotein (RNP) complex comprising a gRNA and a Cas nuclease, a homing endonuclease, a zinc finger nuclease (ZF) nucleic acid binding entity, a transcription activator-like effector (TALE) nucleic acid binding entity, a meganuclease, a Cas nuclease, a core Cas protein, a homing endonuclease, an endonuclease-deficient-Cas protein, an enzymatically inactive Cas protein, a CRISPR- associated transposase (CAST), a Type II or Type V Cas protein, or a functional portion thereof.

[0174] In some embodiments, the cell further comprises Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl 2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Cas 12g, Casl2h, Casl2i, Cas 12k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxl 1, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, or a functional fragment thereof.

[0175] In some embodiments, the recombinant nucleic acid sequence is integrated at exon 8 of an endogenous PIK3R3 locus. In some embodiments, the recombinant nucleic acid sequence is integrated at intron 7 of an endogenous PIK3R3 locus. In some embodiments, the recombinant nucleic acid sequence is integrated at exon 13 of an endogenous CARD 11 locus. In some embodiments, the recombinant nucleic acid sequence is integrated at intron 12 of an endogenous CARD 11 locus.

[0176] In some embodiments, the cell has reduced exhaustion, increased proliferative capacity, enhanced replicative lifespan, decreased replicative senescence, enhanced antitumor effect, reduced dysfunction, enhanced persistence, and / or increase intratumoral presence in vivo.

[0177] In some embodiments, the cell has increased or decreased signaling through the CARD1 1-BCL10-MALT1 complex, NF-KB, AP-1, NF AT, JAK / STAT, and / or MEK / ERK pathways.

[0178] In one aspect, the disclosure provides compositions comprising the engineered cell of the disclosure, or a plurality of the engineered cell of the disclosure. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

[0179] In some embodiments, at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the engineered cells, or of the total cells, in the composition express the recombinant polypeptide of the disclosure.

[0180] In some embodiments, at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the engineered cells, or of the total cells, in the composition express the CAR or recombinant TCR. In some embodiments, at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the engineered cells, or of the total cells, in the composition do not express a gene product of an endogenous TRAC locus. In some embodiments, at least at or about 70%, 75%, 80%, 85%,90%, or 95% of the engineered cells, or of the total cells, in the composition express the recombinant polypeptide of the disclosure; at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the engineered cells, or of the total cells, in the composition express the CAR or recombinant TCR; and / or at least at or about 70%, 75%, 80%, 85%, 90%, or 95% of the engineered cells, or of the total cells, in the composition do not express a gene product of an endogenous TRAC locus.

[0181] In some embodiments, the composition comprises CD4+ T cells and CD8+ T cells, and the percentage of CD4+ T cells in the composition is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the composition; and / or the percentage of CD8+ T cells in the composition is between at or about 20% and at or about 80%, or at or about 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the total cells in the composition; and / or the ratio of CD4+ T cells to CD8+ T cells is from at or about 1 :3 to at or about 3: 1, optionally at or about 1 : 1.

[0182] In one aspect, the disclosure provides methods of preparing the engineered cell the disclosure, the method comprising expressing the recombinant polypeptide in the cell, thereby making the engineered cell.

[0183] In one aspect, the disclosure provides methods of preparing an engineered cell, the method comprising expressing in the cell the recombinant polypeptide of the disclosure, thereby making the engineered cell.

[0184] In some embodiments, the method comprises introducing into the cell the nucleic acid construct of the disclosure.

[0185] In one aspect, the disclosure provides methods of preparing an engineered cell, the method comprising introducing into the cell the nucleic acid construct of the disclosure, thereby making the engineered cell.

[0186] In some embodiments, the cell is in vivo. In some embodiments, the cell is prepared in vivo. In some embodiments, the cell is prepared in the body of a subject in need of treatment.

[0187] In some embodiments, the recombinant polypeptide is expressed in the cell, and / or the nucleic acid construct is introduced into the cell, by contacting the cell with a vector or a particle.

[0188] In some embodiments, the vector is a plasmid, a synthetic DNA vector, a linear DNA vector, a closed linear DNA vector, a phagemid vector, an RNA vector, an mRNA vector, or a viral vector. In some embodiments, the viral vector is selected from a retrovirus vector, an adenovirus vector, and an adeno-associated virus (AAV) vector. In some embodiments, theretrovirus is a lentivirus, optionally a VSV-G pseudotyped lentivirus. In some embodiments, the AAV vector is an AAV6 vector or an AAV9 vector.

[0189] In some embodiments, the vector is a fusosome, a lentiviral particle engineered with the anti-CD3 Cocal glycoprotein, or an enveloped delivery vehicle. In some embodiments, the vector is a self-replicating RNA virus, an mRNA-packaging virus-like particle, or a ribonucleoprotein (RNP)-packaging virus-like particle. In some embodiments, the particle is a lipid nanoparticle (LNP), a selective organ targeting (SORT) LNP, or an antibody targeted LNP. In some embodiments, the LNP comprises: (i) an ionizable lipid (e.g., an amino lipid), (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG-lipid (e.g., a PEG-modified lipid). In some embodiments, the particle is a polymer nanoparticle or a protein nanoparticle.

[0190] In some embodiments, the cell is ex vivo. In some embodiments, the cell is prepared ex vivo. In some embodiments, the cell is prepared outside of the body of a subject in need of treatment.

[0191] In some embodiments, the nucleic acid construct is introduced into the cell by electroporation or transfection. In some embodiments, the transfection is lipofection.

[0192] In some embodiments, the nucleic acid construct is a plasmid, a synthetic DNA vector, a linear DNA vector, a closed linear DNA vector, a phagemid vector, an RNA vector, or an mRNA vector. In some embodiments, the nucleic acid construct is introduced via a viral vector. In some embodiments, the viral vector is selected from a retrovirus vector, an adenovirus vector, and an adeno-associated virus (AAV) vector. In some embodiments, the retrovirus is a lentivirus, optionally a VSV-G pseudotyped lentivirus. In some embodiments, the AAV vector is an AAV6 vector or an AAV9 vector.

[0193] In some embodiments, the method comprises genetically modifying the cell for expression of the recombinant polypeptide.

[0194] In some embodiments, the method comprises introducing into the cell one or more nucleic acids encoding a sequence specific nuclease or a nucleic acid programmable DNA binding protein.

[0195] In some embodiments, the method additionally comprises introducing into the cell one or more guide RNAs. In some embodiments, the sequence-specific nuclease is an RNA guided nuclease. In some embodiments, the sequence-specific nuclease or nucleic acid programmable DNA binding domain is a homing endonuclease, a zinc finger nuclease (ZF) nucleic acid binding entity, a transcription activator-like effector (TALE) nucleic acid binding entity, a meganuclease, a Cas nuclease, a core Cas protein, a homing endonuclease,an endonuclease-deficient-Cas protein, an enzymatically inactive Cas protein, a CRISPR- associated transposase (CAST), a Type II or Type V Cas protein, or a functional portion thereof. In some embodiments, the Cas nuclease is Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8a, Cas8b, Cas8c, Cas9, CaslO, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl 2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Cas 12g, Casl2h, Casl2i, Cas 12k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, Cmr6, Csdl, Csd2, Cas5d, Csel, Cse2, Cse3, Cse4, Cas5e, Csfl, Csml, Csm2, Csm3, Csm4, Csm5, Csnl, Csn2, Cstl, Cst2, Cas5t, Cshl, Csh2, Cas5h, Csal, Csa2, Csa3, Csa4, Csa5, Cas5a, CsxlO, Csxl 1, Csyl, Csy2, Csy3, Csy4, Mad7, SpCas9, eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9, SaCas9, NmeCas9, CjCas9, StCas9, TdCas9, LbCasl2a, AsCasl2a, AacCasl2b, BhCasl2b v4, TnpB, dCas (D10A), dCas (H840A), dCasl3a, dCasl3b, or a functional portion thereof. In some embodiments, the RNA-guided nuclease is a Life Edit nuclease (LEG), optionally a LEG 14 nuclease.

[0196] In some embodiments, the method comprises introducing into the cell a ribonucleoprotein (RNP) complex comprising the sequence-specific nuclease or nucleic acid programmable DNA binding.

[0197] In some embodiments, introducing the nucleic acid construct comprises contacting the cell with the particle of the disclosure or the vector of the disclosure.

[0198] In some embodiments, the nucleic acid construct is introduced via electroporation. In some embodiments, the nucleic acid construct is introduced via AAV. In some embodiments, the nucleic acid construct is introduced via lentivirus. In some embodiments, the nucleic acid construct is introduced via LNP.

[0199] In some embodiments, the method further comprises expressing in the cell an immune receptor that binds to an antigen target in a target cell. In some embodiments, expressing the immune receptor comprises administering a nucleic acid construct encoding the immune receptor to the cell; optionally, wherein the nucleic construct is an RNA construct.

[0200] In some embodiments, the immune receptor and the recombinant polypeptide are encoded by the same nucleic acid construct. In some embodiments, the immune receptor and the recombinant polypeptide are encoded by different nucleic acid constructs.

[0201] In some embodiments, expressing the immune receptor comprises introducing to the cell a nucleic acid construct encoding the immune receptor. In some embodiments, the nucleic acid construct encoding the immune receptor is introduced via a viral vector.

[0202] In some embodiments, activating the immune receptor results in the secretion of IFNg by the cell at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, least 30-fold, or at least 100-fold higher than the IFNg secretion level of a control cell lacking the recombinant polypeptide.

[0203] In some embodiments, activating the immune receptor results in the secretion of IL-2 by the cell at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2- fold, at least 5-fold, at least 10-fold, at least 20-fold, least 30-fold, or at least 100-fold higher than the IL-2 secretion level of a control cell lacking the recombinant polypeptide.

[0204] In some embodiments, the nucleic acid construct is inserted into an endogenous locus of the cell.

[0205] In some embodiments, the endogenous locus is or comprises a TRAC gene locus, a TRBC1 gene locus, a TRBC2 gene locus, a TRAJ locus, a CARD9 locus, a CARDIO locus, a CARD11 locus, a CARD14 locus, or a PIK3R3 locus. In some embodiments, the endogenous locus is or comprises a TRAC gene locus. In some embodiments, the endogenous locus is or comprises a TRAJ locus. In some embodiments, the TRAJ locus is or comprises a TRAJ intron splice acceptor locus.

[0206] In some embodiments, the endogenous locus is or comprises a safe harbor locus. In some embodiments, the safe harbor locus is an AAVS1, ABO, CCR5, CLYBL, CXCR4, F3, FUT1, HMGB1, KDM5D, LRP1, MICA, MICB, RHD, ROSA26, or SHS231 locus.

[0207] In some embodiments, the first insertion site is an exon. In some embodiments, the first insertion site is an intron. In some embodiments, the first insertion site is between an intron and an exon. In some embodiments, the first insertion site is in a regulatory region. In some embodiments, the first insertion site is 25 nucleotides or less from a protospacer adjacent motif (PAM) sequence, wherein the PAM sequence is ngg, nag, ngrrt, ngrm, nnnngatt, nnnnryac, nnagaaw, naaaac, tttv, ttn, attn, tttn, gttn, or yttn and wherein: (i) r = a or g, (ii) y = c or t, (iii) w = a or t, (iv) v = a or c or g, and (v) n = a, c, t, or g.

[0208] In some embodiments, the cell comprises a modified TRAC gene locus. In some embodiments, the cell does not express an endogenous TCR.

[0209] In some embodiments, the method further comprises introducing a gene editing machinery into the cell. In some embodiments, the step of introducing comprises homology- directed repair (HDR)-mediated insertion using the gene editing machinery. In some embodiments, the gene editing machinery is CRISPR / Cas9. In some embodiments, the gene editing machinery is introduced via electroporation. In some embodiments, the nucleic acidconstruct is introduced via AAV. In some embodiments, the nucleic acid construct is introduced via LNP.

[0210] In some embodiments, the method thereby produces an engineered cell comprising: (i) the recombinant polypeptide; (ii) the immune receptor (e.g., CAR); and (iii) the modified TRAC gene locus.

[0211] In some embodiments, the cell is for use in a cell therapy.

[0212] In one aspect, the disclosure provides methods of treating a subject, comprising administering to the subject the cell of the disclosure, or the cell prepared by the method of the disclosure. In some embodiments, the subject has a cancer or an autoimmune disease.

[0213] In one aspect, the disclosure provides uses of the cell of the disclsoure, or the cell prepared by the method of the disclsoure, for the manufacture of a medicament for the treatment of a disease. In some embodiments, the disease is a cancer or an autoimmune disease.

[0214] In some embodiments, the autoimmune disease is lupus, systemic lupus erythematosus, rheumatoid arthritis, psoriasis, psoriatic arthritis, multiple sclerosis, Crohn’s disease, ulcerative colitis, Addison’s disease, Graves’ disease, Sjogren’s syndrome, Hashimoto’s thyroiditis, or celiac disease.

[0215] In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is myeloid neoplasm, myelodysplastic syndromes (MDS), myeloproliferative / myelodysplastic syndromes, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), blast crisis chronic myelogenous leukemia (bcCML), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), T-cell lymphoma, or B-cell lymphoma.

[0216] In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is small cell lung cancer, colorectal cancer, testicular cancer, ovarian cancer, melanoma, lymphoma, leukemia, multiple myeloma, prostate cancer, breast cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, liver cancer, kidney cancer, head & neck cancer, glioblastoma, neuroblastoma, soft tissue sarcoma, uterine cancer, brain cancer, skin cancer, renal cancer, bladder cancer, pancreatic cancer, thyroid cancer, eye cancer, gastrointestinal cancer, carcinoma, or sarcoma.

[0217] In some embodiments, the cancer expresses CD1, CDla, CDlb, CDlc, CDld, CDle, CD2, CD3d, CD3e, CD3g, CD3s, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44v6,CD45, CD46, CD47 CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD213A2, CD246, CD252, CD253, CD261, CD262, CD272, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), CDH17, CEA, CLECL1, CLL-1, CLDN6, CLDN18.2, CS1, DLL3, LY6G6D, GCC, p53R175H, PRAME, EGFR, EGFRvIII, FGFR2, AFP, CA125, MUC-1, MAGE, ALPI, alkaline phosphatase placental-like 2 (ALPPL2), B-cell maturation antigen (BCM A), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), KLK2, KLK3, Mesothelin, IL13Ra2, ggcc, signal regulatory protein a (SIRPa), TCRalpha, TCRbeta, TSHR, GD2, GD3, Tn Ag, cMET, Axl, R0R1, ROR2, GPC1, GPC2, GPC3, FLT3, TAG72, CEA, EPC AM, KIT (CD117), IL-13Ra2, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, PDGFRp, SSEA-4, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, STEAP1, STEAP2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, ROPN1, GPRC5D, GPA33, CXORF61, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE- Al, legumain, HPV E6,E7, MAGE-A4, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, p53R175H, KRAS, mutant KRAS, KRAS G12D, prostein, surviving, telomerase, PCTA-l / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70- 2,LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, FCRL5, IGLL1, PSMA, TROP2, citrullinated vimentin, the extracellular portion of the APRIL protein, or any combination thereof. In some embodiments, the cancer expresses B7H3, BCMA, CD19, CD20, CD22, CD70, CD79a, CD79b, CDH17, CLDN6, CLDN18.2, DLL3, GCC, GD2, GD3, GPC3, GPRC5D, GPA33, KLK3, LY6G6D, p53R175H, PRAME, ROPN1, STEAP1, or STEAP2.

[0218] In some embodiments, the subject is not administered a lymphodepletive agent within 7 days prior to administration of the cell. In some embodiments, the subject is not administered cyclophosphamide, fludarabine, or bendamustine within 7 days prior toadministration of the cell. In some embodiments, the subject is not administered at least 600,000 lU / kg of IL-2 every 8 hours.

[0219] In some embodiments, the method does not comprise a checkpoint therapy which blocks PD-1 or CTLA-4 signaling.

[0220] In some embodiments, the cell has reduced exhaustion, increased proliferative capacity, enhanced replicative lifespan, decreased replicative senescence, enhanced antitumor effect, reduced dysfunction, enhanced persistence, and / or increase intratumoral presence in vivo. In some embodiments, the cell has increased or decreased signaling through the CARD11-BCL10-MALT1 complex, NF-KB, AP-1, NF AT, JAK / STAT, and / or MEKZERK pathways.

[0221] In some embodiments, the subject is administered one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are selected from the group consisting of a small molecule inhibitor, a chemotherapeutic agent, a cancer immunotherapy, an antibody, a cellular therapy, a nucleic acid, a surgery, a radiotherapy, an anti-angiogenic therapy, an anti-DNA repair therapy, an anti-inflammatory therapy, an anti- neoplastic agent, a growth inhibitory agent, a cytotoxic agent, a vaccine, a small molecule agonist, a virus-based therapy, an antibody-drug conjugate, a recombinant protein, a fusion protein, a natural compound, a peptide, a PROteolysis-TArgeting Chimera (PROTAC), and any combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0222] FIGS. 1A-1F provide an overview of CARD11-PIK3R3 signaling as well as schematics of the patient derived CARD11-PIK3R3 fusion protein and a selection of the engineered CARD11-PIK3R3 fusion proteins described herein. FIG. 1A shows a schematic of the CARD11-PIK3R3 fusion protein and identifies the portions of the fusion derived from CARD1 1 (i.e., the CARD domain, coiled-coil domain, and inhibitory domain with representative phosphorylation sites) and the portion of the fusion derived from PIK3R3 (i.e., the SH2 domain). FIGS. 1B-1D depict CAR signaling and CARD11-PIK3R3 signaling in different cell states. FIG. IB depicts that, in the absence of tumor antigen, there is minimal CAR signaling and CARD11-PIK3R3 signaling. FIG. 1C depicts that, upon initial binding of tumor antigen, CAR signaling is initiated and results in phosphorylated T cell signaling adapters that become available as substrates for CARD11-PIK3R3 interaction via the PIK3R3 SH2 domain. FIG. ID depicts that, upon further CAR signaling, phosphorylation of the inhibitory domain causes conformational changes in the CARD11-PIK3R3 fusion protein that lead to robust CARD11, Bel 10, Maltl (CBM) complex signaling (culminating in, for example and without limitation, NF-kB activation, JnK activation, and paracaspase signaling). FIG. IE depicts that there is minimal CAR signaling and CARD11- PIK3R3 signaling in the absence of tumor antigen (upon elimination of the tumor cell) due to, without wishing to be bound by theory, the resumption of autoinhibition of the CARD11- PIK3R3 fusion protein. FIG. IF is a schematic depicting the patient derived CARD11- PIK3R3 fusion protein and two optimized CARD11-PIK3R3 fusion proteins. The schematic depicts the locations of the corresponding CARD domains, coiled-coil domains, inhibitory domains (ID), SH2 domains, and fusion breakpoints.

[0223] FIGS. 2A-2B show the T cell phenotype of primary human T cells isolated from human donors transduced with TCR alone, a TCR and a patient-derived CARD11-PIK3R3 fusion (TCR+C11P3), a TCR and an a first optimized CARD11-PIK3R3 fusion (TCR+C1 lP3trl), a TCR and an a second optimized CARD11-PIK3R3 fusion (TCR+C1 lP3tr2), the patient-derived CARD11-PIK3R3 fusion alone (Cl 1P3), the first optimized CARD11-PIK3R3 fusion alone (Cl lP3trl), or the second optimized CARD11- PIK3R3 fusion alone (Cl lP3tr2). FIG. 2A shows the percentage of cells with a particular phenotype, and FIG. 2B shows flow cytometry data showing the percentage of cells expressing CCR7 and CD45RA. FIGS. 2A and 2B demonstrate that T cells transduced with the patient derived and optimized fusions (alone or in combination with a TCR) induce a greater central memory phenotype as compared T cells transduced with a TCR alone

[0224] FIGS. 3A-3B show the level of IFNgamma (FIG. 3A) and IL-2 secretion (FIG. 3B) for T cells transduced with TCR alone, a TCR and a patient-derived CARD11-PIK3R3 fusion (TCR+C11P3), a TCR and an a first optimized CARD11-PIK3R3 fusion (TCR+C1 lP3trl), a TCR and an a second optimized CARD11-PIK3R3 fusion (TCR+C1 lP3tr2), the patient-derived CARD11-PIK3R3 fusion alone (Cl 1P3), the first optimized CARD11-PIK3R3 fusion alone (Cl lP3trl), or the second optimized CARD11- PIK3R3 fusion alone (Cl lP3tr2) after co-culture with two different antigen positive cell lines and one antigen negative cell line. FIG. 3A shows that T cells expressing a TCR and the patient derived or optimized fusions secreted higher levels of IFNgamma compared to T cells express a TCR alone. FIG. 3B shows that T cells expressing a TCR and the patient derived or optimized fusions secreted higher levels of IL-2 compared to T cells express a TCR alone.

[0225] FIGS. 4A-4D show the percentage of primary T cells that are positive for a CAR and negative for CD3 (as a marker for TCR expression) after transduction with lentiviral vectorsencoding the CAR or CAR+fusion constructs and editing the TRAJ intron splice acceptor locus to knockout TCR expression. FIG. 4A shows the percentage of cells that are CAR+, FIG. 4B shows the MFI, FIG. 4C shows the percentage of cells that are CD3 negative, and FIG. 4D shows flow cytometry data showing the percentage of cells expressing CAR and CD3.

[0226] FIGS. 5A-5B show the T cell phenotype of primary human T cells isolated from human donors that were untransduced with no TCR knockout (UTD no KO), untransduced with TCR knockout (UTD KO), transduced with CAR alone (CAR), a CAR and a patient- derived fusion (CAR P2A Cl 1P3), or a CAR and an optimized fusion (CAR P2A Cl lP3trl). FIG. 5A shows the percentage of cells with a particular phenotype, and FIG. 5B shows flow cytometry data showing the percentage of cells expressing CCR7 and CD45RA. FIGS. 5A and 5B show that cells transduced with CAR+C11P3 or CAR+C1 lP3trl had greater central memory phenotype than cells transduced with CAR alone.

[0227] FIGS. 6A-6B show the level of IL-2 secretion (FIG. 6A) and IFNgamma (FIG. 6B) secretion for untransduced T cells or T cells transduced with CAR alone (CAR), a CAR and a patient-derived fusion (CAR+C11P3), or a CAR and an optimized fusion(CAR+C1 lP3trl) after co-culture with an antigen positive cell line and an antigen negative cell line. FIG. 6A shows that T cells expressing a CAR and the patient derived or optimized fusions secreted higher levels of IL-2 compared to T cells express a CAR alone. FIG. 6B shows that T cells expressing a CAR and the patient derived or optimized fusions secreted higher levels of IFNgamma compared to T cells express a CAR alone.

[0228] FIGS. 7A-7B show primary T cells edited with a CAR or a CAR plus an optimized fusion (CAR+C1 lP3trl) knocked-into the TRAJ intron splice acceptor locus with expression under control of the endogenous TRAC promoter. Editing was done via AAV and CRISPR / Cas9 mediated homology directed repair at two AAV MOI doses. FIG. 7A shows the percentage of cells that are CAR+, FIG. 7B shows the MFI, FIG. 7A shows that the engineered CARD11-PIK3R3 fusion proteins, which are shorter than the patient-derived fusions can be delivered with a CAR via a single AAV6 vector. FIG. 7B shows that there is no significant difference in CAR MFI between the different constructs.

[0229] FIG. 8 shows the T cell phenotype of primary human T cells isolated from human donors that were untransduced but had the TCR knockout (UTD KO), transduced with CAR alone (CAR) at two different MOI doses (le5 or 5e5), or a CAR and an optimized fusion (CAR Cl lP3trl) at two different MOI doses (le5 or 5e5). FIG. 8 shows there are notsignificant differences in T cell phenotype when comparing AAV6 delivered CAR T cells and AAV6 delivered CAR+C1 lP3trl cells.

[0230] FIGS. 9A-9B show the level of IFNgamma (FIG. 9A) and IL-2 secretion (FIG. 9B) for untransduced but had the TCR knockout (UTD KO), transduced with CAR alone (CAR) at two different MOI doses (le5 or 5e5), or a CAR and an optimized fusion (CAR Cl lP3trl) at two different MOI doses (le5 or 5e5) after co-culture with an antigen high cell line, and antigen low cell line, and an antigen negative cell line. FIG. 9A shows that T cells expressing a CAR and an optimized fusion secreted IFNgamma in an antigen dependent manner. FIG. 9B shows that T cells expressing a CAR and an optimized fusion secreted IL- 2 in an antigen dependent manner.

[0231] FIGS. 10A-10D show the results of an in vivo experiment where mice treated with CAR T cells harboring the patient derived CARD11-PIK3R3 fusion protein or CAR T cells harboring an engineered CARD11-PIK3R3 fusion protein were able to clear tumors upon multiple tumor challenges. FIG. 10A shows measurements of tumor volumes over time for mice bearing SHP77 tumors treated with untransduced T cells. FIG. 10B shows measurements of tumor volumes over time for mice bearing SHP77 tumors treated with CAR only T cells (dark grey line) compared to untransduced T cells (light grey line). Finally, FIGS. 10C and 10D show measurements of tumor volumes over time for mice bearing SHP77 tumors treated with CAR plus the patient derived CARD11-PIK3R3 fusion protein (FIG. 10C; dark grey line) or treated with CAR plus an engineered CARD11- PIK3R3 fusion protein (FIG. 10D; dark grey line).

[0232] FIGS. 11A-11G show the results of an in vivo experiment where mice treated with OT-I T cells harboring the patient derived CARD11-PIK3R3 fusion protein or OT-I T cells harboring an engineered CARD11-PIK3R3 fusion protein were able to clear tumors at low doses. FIG. 11A shows measurements of tumor volumes over time for mice bearing Bib- Ova cells treated with untransduced OT-I T cells. FIGS. 11B-11D show measurements of tumor volumes over time for mice bearing Bl 6-Ova tumors treated with 50,000 (FIG. 11B), 10,000 (FIG. 11C), or 2,000 (FIG. 11D) OT-I T cells harboring the patient derived CARD11-PIK3R3 fusion protein. FIGS. 11E-11G show measurements of tumor volumes over time for mice bearing Bl 6-Ova tumors treated with 50,000 (FIG. HE), 10,000 (FIG. HF), or 2,000 (FIG. 11G) OT-I T cells harboring an engineered CARD11-PIK3R3 fusion protein.

[0233] FIGS. 12A-12H show the results of in vitro and in vivo assays comparing the performance of CAR T cells co-expressing a TGFbR2-DNR protein and CAR T cells co-expressing an engingeered CARD11-PIK3R3 fusion protein. FIG. 12A shows interferon gamma (ZFNy) expression for untransduced T cells, CAR T cells co-expressing a TGFbR2- DNR protein, or CAR T cells co-expressing an engineered CARD11-PIK3R3 fusion protein co-cultured with two different SCLC tumor cell lines (SHP77 and DMS273). FIG. 12B shows interleukin-2 (IL-2) expression for untransduced T cells, CAR T cells co-expressing a TGFbR2-DNR protein, or CAR T cells co-expressing an engineered CARD11-PIK3R3 fusion protein co-cultured with two different SCLC tumor cell lines (SHP77 and DMS273). FIGS. 12C and 12D show in vitro growth of SHP77 (FIG. 12C) tumor cells or DMS273 tumor cells (FIG. 12D) co-cultured with untransduced T cells, CAR T cells co-expressing a TGFbR2-DNR protein, or CAR T cells co-expressing an engineered CARD11-PIK3R3 fusion protein. FIGS. 12E and 12G show measurements of tumor volumes over time for mice bearing SHP77 tumors treated with either 2xl05(FIG. 12E) or IxlO6(FIG. 12G) untransduced T cells (light grey line) or CAR T cells co-expressing a TGFbR2-DNR protein (dark grey line). FIGS. 12F and 12H show measurements of tumor volumes over time for mice bearing SHP77 tumors treated with either 2xl05(FIG. 12F) or IxlO6(FIG. 12H) untransduced T cells (light grey line) or CAR T cells co-expressing an engineered CARD11- PIK3R3 fusion protein (dark grey line).

[0234] FIGS. 13A-13D show schematics for both non-targeted and targeted methods of expressing a CARD11-PIK3R3 fusion protein using, for example, LVV delivery (FIG. 13A), targeted insertion of a CARD11-PIK3R3 fusion protein using, for example, an RNA targeted nuclease and AAV (FIG. 13B), and re-creation of a CARD11-PIK3R3 fusion protein using, for example, dual / twin prime editing or PASSIGE to insert an SH2 gene fragment into exon 13 or exon 14 (or intron 12 or intron 13) of the CARD11 gene locus (FIGS. 13C and 13D)

[0235] In the following detailed description, reference is made to the Figures, which form a part hereof. In the Figures, similar symbols generally identify similar components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this application.

[0236] In the following detailed description, reference is made to the Figures, which form a part hereof. In the Figures, similar symbols generally identify similar components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects and embodiments, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations (for example, combined with other aspects and / or embodiments described herein), all of which are explicitly contemplated and make part of this application.DETAILED DESCRIPTION OF THE DISCLOSURE

[0237] In general, the present disclosure relates, inter alia, to compositions and methods for improving adoptive T cell therapy. The disclosers have discovered ways to improve the therapeutic efficacy of T cells by altering T cell signaling, decreasing T cell exhaustion and / or by enhancing in vivo persistence and fitness of engineered T cells. T cell cancers can undergo positive selection of beneficial genetic alterations (e.g., mutations). In some embodiments, the present disclosure relates to exploiting such beneficial mutations to improve the effectiveness of T cell therapeutics.I. DEFINITIONS

[0238] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. All publications, patent applications, patents, GenBank or other accession numbers and other references mentioned herein are incorporated by reference in their entirety for all purposes.

[0239] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells,including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B.”

[0240] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0241] Certain ranges are presented herein with numerical values being preceded by the term “approximately” or “about.” The terms “approximately” and “about” are used interchangeably and mean a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1%, of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length, inclusive of the endpoints. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth, unless otherwise apparent from context that it is impossible to extend the boundary beyond certain points (e.g., below 0% or above 100% in some cases). Further, the terms “approximately” and “about’ are used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value.Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.

[0242] As used herein, the term “comparable level” refers to a level that is in between 50% and 200%, inclusive of end points, of the reference level in the control set of conditions or circumstances, unless otherwise apparent from context that it is impossible to extend theboundary beyond certain points. In some embodiments, a comparable level may be preferably between 60% and 170%, more preferably between 70% and 140%, even more preferably between 80% and 125%, or more preferably still between 90% and 110%, of the reference level. Those of ordinary skill in the art will appreciate the appropriate control set in obtaining the reference level. In general, the appropriate control set shares identifical features with the test set except the feature(s) of interest, so that the observed results warrant a reasonable conclusion that any changes in the level observed are caused by or indicative of the variation in the feature(s) that are varied.

[0243] As will be understood by one having ordinary skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0244] It is understood that aspects and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments. As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or step.

[0245] The term “percent identity,” as used herein in the context of two or more nucleic acids or proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. Such sequences are then the to be “substantially identical.” This definition also refers to, or may be applied to, the complement of a test sequence. This definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. Generally, sequence identity can exist over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence.

[0246] If necessary, sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), with the default parameters thereof.

[0247] As used herein, the term “mutation” refers to a point mutation, a gene fusion, a substitution, a gain-of-function mutation, a stop-gain mutation, an insertion mutation, a deletion mutation, a duplication mutation and / or a translocation. The mutation may be in one or more genes. The mutation may be naturally occurring. Alternatively, the mutation may be induced or engineered. As used herein, the term “vector” refers to a nucleic acid construct designed for transfer between host cells, and that may be used for the purpose of transformation, e.g., the introduction of heterologous DNA into a host cell. As such, in some embodiments, the vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. In some embodiments, the expression vector can be an integrating vector.

[0248] As used herein, the term “viral vector” refers either to a nucleic acid molecule (e.g., a transfer plasmid) that includes virus-derived nucleic acid elements that generally facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a viralparticle that mediates nucleic acid transfer. Viral particles will generally include various viral components and sometimes also host cell components in addition to nucleic acid(s). The term viral vector may refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. Viral vectors that can be used in the disclosure include, for example, retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors, lentivirus vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.). For example, a recombinant polypeptide as disclosed herein can be produced in a eukaryotic host, such as a mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, VA). In selecting an expression system, care should be taken to ensure that the components are compatible with one another. Artisans or ordinary skill are able to make such a determination. Furthermore, if guidance is required in selecting an expression system, skilled artisans may consult P. Jones, “Vectors: Cloning Applications”, John Wiley and Sons, New York, N.Y., 2009).

[0249] As used herein, the term “retroviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. The retroviral vector can be a lentiviral vector. As used herein, the term “lentiviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus, which is a genus of retrovirus. Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and nondividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production.

[0250] As used herein, the term “pharmaceutically acceptable carrier” as used herein means any suitable carriers, diluents or excipients. These include all aqueous and non-aqueous isotonic sterile injection solutions, which may contain anti-oxidants, buffers and solutes, which render the composition isotonic with the blood of the intended recipient; aqueous andnon-aqueous sterile suspensions, which may include suspending agents and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic and absorption agents and the like. It will be understood that compositions of the present disclosure may also include other supplementary physiologically active agents. The carrier must be pharmaceutically “acceptable” in the sense of being compatible with the other ingredients of the composition and not injurious to the subject.

[0251] As used herein, the term “PEGylation” refers to modifying a protein by covalently attaching polyethylene glycol (PEG) to the protein, with “PEGylated” referring to a protein having a PEG attached. A range of PEG, or PEG derivative sizes with optional ranges of from about 10,000 Daltons to about 40,000 Daltons may be attached to the recombinant polypeptides of the disclosure using a variety of chemistries. In some embodiments, the average molecular weight of the PEG, or PEG derivative, is about 1 kD to about 200 kD such as, e.g., about 10 kD to about 150 kD, about 50 kD to about 100 kD, about 5 kD to about 100 kD, about 20 kD to about 80 kD, about 30 kD to about 70 kD, about 40 kD to about 60 kD, about 50 kD to about 100 kD, about 100 kD to about 200 kD, or about 150 kD to about 200 kD. In some embodiments, the average molecular weight of the PEG, or PEG derivative, is about 5 kD, about 10 kD, about 20 kD, about 30 kD, about 40 kD, about 50 kD, about 60 kD, about 70 kD, or about 80 kD. In some embodiments, the average molecular weight of the PEG, or PEG derivative, is about 40 kD.

[0252] As used herein, the terms “administration” and “administering” refer to the delivery of a bioactive composition or formulation by an administration route including, but not limited to, oral, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, and topical administration, or combinations thereof. The term includes, but is not limited to, administering by a medical professional and self-administering.

[0253] As used herein, the term “injection” includes intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrastemal injection and infusion.

[0254] The term “cancer” generally refers to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Cancer cells can be in the form of a tumor, but such cells can exist alone within an animal subject, or can be a non-tumorigenic cancer cell, such as a leukemia cell. These terms include a solid tumor, a soft tissue tumor, or a metastatic lesion. As used herein, the term“cancer” includes premalignant, as well as malignant cancers. In some embodiments, the cancer is a solid tumor, a soft tissue tumor, or a metastatic lesion.

[0255] As used herein, and unless otherwise specified, a “therapeutically effective” or “pharmaceutically effective” amount or number of a subject construct, nucleic acid, cell, or composition of the disclosure generally refer to an amount or number sufficient for a construct, nucleic acid, cell, or composition to accomplish a stated purpose relative to the absence of the composition, e.g., to provide a therapeutic benefit in the treatment or management of the cancer, or to delay or minimize one or more symptoms associated with the cancer. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the cancer. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the cancer, or enhances the therapeutic efficacy of another therapeutic agent. An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0256] As used herein, a “subject” or an “individual” includes animals, such as human (e.g., human subjects) and non-human animals. In some embodiments, a “subject” or “individual” is a patient under the care of a physician. Thus, the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. The subject can also be an individual who is diagnosed with a risk of the condition of interest at the time of diagnosis or later. The term “non-human animals” includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, e.g. non-human primates, and non- mammals, e.g., sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0257] The term “engineered cell” as used herein refers to a cell that has been altered in at least some way by human intervention, including, for example, by genetic alterations or modifications such that the engineered cell differs from a wild-type cell.

[0258] The terms “decrease,” “reduced,” “reduction,” and “decreased” are all used herein generally to mean a lowering by a statistically significant amount. However, for avoidance of doubt, “decrease,” “reduced,” “reduction,” “decreased” means a lowering by at least 10% as compared to a reference level, for example a lowering by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% lowering (i.e. absent level as compared to a reference sample), or any lowering between 10- 100% as compared to a reference level. In some embodiments, the cells are engineered to have reduced expression of one or more targets relative to an unaltered or unmodified wildtype cell.

[0259] The terms “increase” and “increased” are all used herein generally to mean an increase by a statistically significant amount. However, for avoidance of doubt, “increase” and “increased” mean an increase by at least 10% as compared to a reference level, for example an increase by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold, or any increase between 10% and 1000-fold as compared to a reference level. In some embodiments, the cells are engineered to have increased expression of one or more targets relative to an unaltered or unmodified wild-type cell.

[0260] As used herein, the terms “conservative amino acid substitution” and “conservative substitution” refer to amino acid substitutions in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0261] Headings, e.g., (a), (b), (i) etc., are presented merely for ease of reading the specification and claims. The use of headings in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented. It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub- combination was individually and explicitly disclosed herein.II. T CELL SIGNALING a. TCR Signaling

[0262] Upon engagement of a peptide-MHC complex, the immunoreceptor tyrosine-based activation motifs (IT AMs) contained within the intracellular tails of the TCR complex become phosphorylated and recruit Zap70. Zap70 then recruits and activates the downstream LAT signalosome containing LAT, PLCy, Slp76. This signalosome causes the activation of the Ras / MAPK / ERK signaling leading to AP-1 transcriptional activity, the activation of PKC0 leading to NF-KB transcriptional activity and finally the influx of calcium which is responsible for NF AT transcriptional activity. Additionally, signaling from costimulatory receptors, such as 4 IBB, and cytokine support from IL-2 enhances these signaling pathways, and is required for T cells to undergo full activation and avoid anergic cell death. When fully activated, T cells rapidly proliferate and differentiate, secrete inflammatory cytokines, and cytotoxic cells begin performing target killing through release of cytotoxic granules. After resolution of this acute response, activated T cells undergo contraction to a form a small population of long-lived memory T cells that surveil the body, ready to respond to future encounters with the antigen. b. T Cell Signaling

[0263] T cells become fully activated through T cell receptor (TCR) engagement, costimulatory signaling and cytokine support. TCR activation induces NF AT, NF-KB and AP-1 transcription factors signaling, IL-2 and other inflammatory cytokine secretion.c. T Cell Exhaustion

[0264] T cell exhaustion is a state of hypo-responsiveness induced in effector T cell populations after chronic exposure to antigen. Exhaustion is marked by the upregulation of surface inhibitory receptors such as PD-1, TIM-3, LAG-3 and CTLA-4 among others, and the inability to proliferate long term, secrete inflammatory cytokines and kill target cells effectively. Exhausted T cells exist in a distinct transcriptional and epigenetic landscape. While exhaustion has been well characterized in chronic viral infections, it is also implicated in cancer, in which tumor infiltrating T cells, and engineered T cell therapies exhibit signs of exhaustion and fail to control tumor growth. To better treat chronic infections or cancer, T cell exhaustion must be prevented or overcome. In fact, checkpoint therapies which block signaling from the PD-1 or CTLA-4 axis of inhibition have proven somewhat effective in reinvigorating T cell responses in certain cancers leading to limited but effective tumor control. d. NF AT Signaling

[0265] The transcription factor nuclear factor of activated T cells (NF AT) is involved in T cell activation as well as programs of T cell exhaustion. At resting state, NF AT transcription factors are found in the cytoplasm in a phosphorylated state, unable to translocate to the nucleus to induce signaling. The extracellular influx of Ca2+that occurs during T cell activation activates the calcium dependent phosphatase calcineurin, resulting in dephosphorylation of NF AT, and its subsequent translocation and signaling in the nucleus. NF AT binds DNA in multiple ways, as a monomer, dimer or as a complex with other transcription factors. In particular NF AT is known to cooperatively bind with the transcription factor AP-1, inducing transcription of activation associated genes critical for effector T cell function.’ In contrast, when NF AT is not bound by AP-1, but is instead partnerless, it has been found to induce the expression of exhaustion associated genes, such as the inhibitory receptor PD-1. Additionally, studies have determined that exhaustion induced by partnerless NF AT can be remedied in CAR T cells by over-expressing the AP-1 family member, eJun. NF AT induces the expression of transcription factors TOX and NR4A1 / 2 / 3, which have been associated with T cell exhaustion and deletion or knockdown of these transcription factors has improved T cell phenotype and in vivo tumor control.

[0266] As NF AT signaling is key to successful T cell activation, but also plays a role in exhaustion, particularly in the absence of AP-1, it can be used to determine how geneticmodifications in T cells influences the dynamics of NF AT signaling. Targeting the NF AT transcriptional pathway can be beneficial for T cell therapeutics. e. NF-kB Signaling

[0267] NF-KB (nuclear factor kappa light chain enhancer of B cells) is a family of transcription factors that induces transcriptional programs critical for T cell activation and effector function. NF-KB signaling occurs in many cell types. In T cells, NF-KB signaling induces a wide range of transcriptional programs responsible for proliferation and memory formation, resistance to apoptosis, cytokine secretion, and the production of a robust effector T cell response. NF-KB signaling is induced through two pathways, the canonical and noncanonical pathways.

[0268] In the canonical pathway NFKB1 is bound in the cytoplasm by IkBa and IkB-like molecule pl05, forming a complex that prevents the nuclear translocation of NKFB1. When activated, TCR signaling induces activation of PKC0, which in turn phosphorylates and activates CARD11. Activated CARD 11 forms a complex with BCL10 and MALT1, and ultimately causes the phosphorylation and degradation of IkBa and IkB-like molecule pl 05, releasing NF-KB to translocate to the nucleus.

[0269] The non-canonical pathway is triggered by signaling through tumor necrosis factor receptor (TNFR) family members, which includes the costimulatory domain 4 IBB. At rest, the NFKB2 is bound by plOO, during activation NF-KB -inducing kinase (NIK) activates IKKa, which in turn phosphorylates pl 00 and leads to the release and translocation of NKFB2 to the nucleus.

[0270] In syngeneic models of solid tumors, endogenous T cells that can respond to the tumors required NF-KB signaling to mediate tumor clearance. While NF-KB is induced through TCR signaling, it is also stimulated through the costimulatory domain 4 IBB (a TNFRS superfamily member), which is used clinically in FDA approved chimeric antigen receptor (CARs) therapies. CAR therapies using 4 IBB costimulatory domains have been found to persist longer in patients as compared to CAR therapies using other costimulatory domains, such as CD28. The persistence of 41BB CARs is directly linked to NF-KB signaling, which improves CAR T resistance to apoptosis through suppression of apoptotic proteins such as Bim. f. AP-1 Signaling

[0271] AP-1 transcription factors are a family of homo or hetero-dimeric proteins formed from complexes of JUN, FOS, ATF or MAF proteins. AP-1 signaling is induced by thephosphorylation cascade called the Mitogen Activated Protein Kinase (MAPK) pathway, triggered by TCR, cytokine / chemokine, or growth factor signaling. AP-1 often signals in complex with NF AT, and as indicated in the NF AT section, the loss of AP-1 causes partnerless NF AT to induce exhaustion programs in T cells, however this exhaustion can be remedied by overexpression of the AP-1 family member c-Jun. Related to this, loss of AP-1 is also known to induce an anergic cell state, where the T cell has partially activated through the TCR, but does not have enough costimulatory and cytokine signaling to induce full activation.

[0272] AP-1 dimers are activated by a plethora of physiological and pathological stimuli. Studies have reported that AP-1 proteins, mostly those that belong to the Jun group, control cell life and death through their ability to regulate the expression and function of cell cycle regulators such as Cyclin DI, p53, p21(cipl / wafl), pl9(ARF) and pl6. Amongst the Jun proteins, c-Jun is unique in its ability to positively regulate cell proliferation through the repression of tumor suppressor gene expression and function, and induction of cyclin DI transcription. These actions are antagonized by JunB, which upregulates tumor suppressor genes and represses cyclin DI. An important target for AP-1 effects on cell life and death is the tumor suppressor p53, whose expression as well as transcriptional activity, are modulated by AP-1 proteins.

[0273] The concomitant induction of NF AT and AP-1 takes concerted activation of two different signaling pathways: calcium / calcineurin, which promotes NF AT dephosphorylation, nuclear translocation and activation; and protein kinase C (PKC) / Ras, which promotes the synthesis, phosphorylation, and activation of members of the Fos and Jun families of transcription factors. (Shaulian E, Karin M. AP-1 as a regulator of cell life and death. Nat Cell Biol 2002 45. 2002;4(5):E131-E136.). g. IL-2 Signaling

[0274] IL-2 is a pleiotropic cytokine required for activation, proliferation differentiation, and maintenance of T cells. Naive T cells express a low affinity IL-2 receptor requiring high volumes of IL-2 to initiate activation, while memory and regulatory T cells express a high affinity IL-2 receptor requiring much lower amounts of IL-2 for effective signaling. The IL- 2 receptor makes use of the JAK / STAT signaling cascade, resulting in wide ranging transcriptional changes. Though CD8+ T cells are responsible for the cytotoxic effector response to foreign antigens, CD8+ T cells themselves cannot effectively produce IL-2 and rely instead on helper CD4+ T cells to produce IL-2 and other cytokine support.

[0275] IL-2 is so critical for T cell persistence and proliferation that high dose IL-2 therapy has been FDA approved for certain cancers, with the hopes of expanding endogenous cytotoxic T cell populations to induce tumor rejection. In metastatic melanoma and renal cell carcinoma trials, high dose IL-2 therapy induces limited (7%) long term response rates.

[0276] However, this therapy is not well tolerated by patients, causing multiple deaths, and likely contributes to the expansion of CD4+CD25+FoxP3+ regulatory T cells. Regulatory T cells are known to be pro-tumorigenic and high numbers of regulatory T cells (Tregs) are correlated with poor prognosis in solid tumor settings. Toxicity and the expansion of protumor Treg populations present so much of a challenge to IL-2 therapy that synthetic orthogonal IL-2 cytokine therapies have recently been developed to target the effects of IL-2 cytokine therapy to cytotoxic T cells and avoid expansion of Tregs.

[0277] IL-2 secretion is highly relevant for the persistence and proliferation of T cell therapeutic products, therefore the inventors / disclosers assessed IL-2 secretion capacity of each mutation when expressed in CAR Jurkat cell lines and after co-culture with target cells. As a follow up for relevant hits, the inventors / disclosers assessed the capacity for mutations to improve CAR T cell in vitro target killing when cultured without supplemental IL-2. This setting, where IL-2 is withheld, better reflects the challenges of the tumor microenvironment, where IL-2 is scarce and autocrine secretion of IL-2 would be beneficial for maintenance of proliferation and long-term killing capabilities. h. CARD11-BCL10-MALT Signalosome

[0278] Formation of a CARD11-BCL10-MALT (CBM) signaling complex is a key event in T- and B cell receptor-induced gene expression. After exposure to distinct immune triggers, these molecules form self-organizing filaments with MALT1 protease activity to regulate canonical nuclear factor-xB (NF-KB) and mitogen-activated protein kinase (MAPK) signalling pathways and the degradation of mRNA-binding proteins, which provides two layers of control of inflammatory gene expression. Deregulation of CARD11, BCL10 or MALT1 expression or CBM signaling have been associated with cancer, immunodeficiency, and autoimmunity (J. Ruland, L. Hartjes, CARD-BCL-10-MALT1 signalling in protective and pathological immunity. Nat Rev Immunol 19, 118-134 (2019).

[0279] In normal T cells, T cell receptor (TCR) signaling activates PKC9, which in turn promotes the assembly of the CARD11-BCL10-MALT1 (CBM) signalosome. The CBM complex subsequently has three major outputs: NF-KB transcriptional activity, AP-1 transcriptional activity and MALT1 proteolytic activity (FIG. 5). The assembly of theCARD1 1-BCL10-MALT1 signalosome complex is an essential step in regulating NF-KB in lymphoid immune cells.

[0280] An inhibitory domain present in CARD 11 may allow for intramolecular autoinhibition which can prevent CARD11 binding to BCL10 in the absence of upstream signals. Upon phosphorylation of the inhibitory domain in normal T cells, CARD 11 autoinhibition can be relieved and the CARD 11 protein can oligomerize and promote prionlike assembly of the CBM complex with recruitment of BCL10-MALT1 filaments, which then can allow for CBM complex signaling.

[0281] Genes involved in CBM signaling include but are not limited to caspase recruitment domain family member 11 (CARD 11), capping protein regulator and myosin 1 linker 2 (CARMIL2), mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), B-cell lymphoma 10 (BCL10). CARD11 can be involved in both the innate and adaptive immune systems. CARD11 is implicated in the activation of NF-kB by the TCR complex.

[0282] T -cell signaling pathways, including the CARD11-BCL10-MALT signalasome and NF-KB signaling, are key to the activation of chimeric antigen receptor (CAR) T-cells. CAR T-cell therapy has demonstrated significant efficacy for treatment of hematologic cancers; however, some of these treatments are associated with severe side effects such as cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) (see, for example, Chohan et al. Curr Hematol. Malig. Rep. 2023; 18(2): 9- 18 and Hay et al. Blood. 2017; 130(21):2295-2306, which are incorporated by reference herein in their entirety). Without wishing to be bound by theory, modulating the intensity of certain signaling pathways in CAR-T cells is a potential strategy to limit excessive CAR T-cell activation, and could reduce harmful side effects while maintaining CAR T-cell efficacy. Signaling molecules (e.g., CARD11-PIK3R3 fusions and / or CARD domain-containing polypeptides) with reduced signaling intensity could provide this effect. For example, signaling molecules with substitutions at certain amino acid positions and / or truncations of certain domains could provide reduced intensity of particular signaling pathways and thereby reduce side effects of CAR T-cell therapy. In some embodiments, a CARD11- PIK3R3 fusion or a CARD domain-containing polypeptide could provide reduced signaling intensity through the CARD11-BCL10-MALT signalasome and / or through NF-KB. i. JAK / STAT signaling

[0283] The biochemistry of JAK / STAT signaling is well known to a person of skill in the art. Briefly, signaling begins with extracellular association of cytokines or growth factorswith their corresponding transmembrane receptors. This facilitates trans-activation of receptor-bound Janus kinases (JAKS) by putting them in spatial proximity and by prompting conformational changes that distance their kinase domains from inhibitory pseudokinase domains. Activated JAKS then phosphorylate latent STAT monomers, leading to dimerization, nuclear translocation, and DNA binding. In mammals, 4 JAKS (JAK1, JAK2, JAK3, TYK2) and 7 STATs (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6) are employed by more than 50 cytokines and growth factors. (Villarino A V., Kanno Y, O’Shea JJ. Mechanisms and consequences of JAK-STAT signaling in the immune system. Nat Immunol 2017 184. 2017;18(4):374-384). j. Co-Stimulatory Molecule Signaling

[0284] Co-stimulatory and co-inhibitory molecules are cell surface receptors and ligands that are classified into various families on the basis of their structure and functions.

[0285] Co-stimulatory and co-inhibitory receptors determine the functional outcome of T cell receptor (TCR) signaling. The specific recognition of cognate antigenic peptides presented by MHC molecules triggers T cell receptor signaling, but it is co-stimulatory and co-inhibitory receptors on T cells that direct T cell function and determine T cell fate. T cell co-signaling receptors have been broadly defined as cell-surface molecules that can transduce signals into T cells to positively (co-stimulatory receptors) or negatively (co- inhibitory receptors) modulate TCR signaling. Examples of co-stimulatory or co-inhibitory receptors includes CD28 and CTLA-4, both of which bind to ligands B7-1 and 7-2. Other genes involved in co-stimulatory molecule signaling include TNFR2, TNFRS1B, and ICOS (Chen L, Flies DB. Molecular mechanisms of T cell co-stimulation and co-inhibition. Nat Rev Immunol 2013 134. 2013;13(4):227-242.) k. RAS / MEK / ERK Signaling

[0286] As is known by the skilled in the art, the sarcoma / mitogen-activated protein kinase kinase / extracellular receptor kinase (RAS / MEK / ERK) is a conserved signaling pathway that plays pivotal roles in cell proliferation, survival and differentiation. The aberrant activation of the RAS / MEK / ERK signaling pathway induces tumors. Efforts have been dedicated to targeting this signaling pathway for cancer treatment. The aberrant activation of the signaling pathway contributes to tumorigenesis and tumor development.

[0287] It is also known that triggering of a T-cell receptor by its cognate antigen results in nearly immediate activation of downstream signaling cascades, including the RAS / MEK / ERK pathway. Studies have also shown that RAS / MEK / ERK signaling ismemory stage-dependent in human T cells, conferring susceptibility to alloreactive T-cell selective inhibition.

[0288] As an important upstream molecular marker of the RAS-RAF-MEK-ERK pathway, RAS serves as a molecular switch by binding GTP / GDP, and it includes four isoforms:HRAS, KRAS4A, KRAS4B, and NRAS. KRAS is the most frequent isoform in all human cancers. KRAS4A and KRAS4B are the different splicing isoforms of the same gene. When the transmembrane receptors (receptor tyrosine kinase, RTKs) are activated, the complexes of growth-factor-receptor bound protein 2 (GRB2) and son of sevenless (SOS) in the cytoplasm are recruited to the inner surface of cell membrane. (McCubrey JA, Steelman LS, Basecke J, Martelli AM. Raf / mek / erk signaling. Target Ther Acute Myeloid Leuk. January 2015:275-305.)

[0289] Several mutations identified by the inventors / disclosers are in genes having various roles in this signaling pathway.I. Phospholipase C Gamma Signaling

[0290] Phospholipase C (PLC) is an essential mediator of cellular signaling. PLC regulates multiple cellular processes by generating bioactive molecules such as inositol-1,4,5- triphosphate (IP3) and diacylglycerol (DAG). These products propagate and regulate cellular signaling via calcium (Ca2+) mobilization and activation of protein kinase C (PKC), other kinases, and ion channels. PLCyl, one of the primary subtypes of PLC, is directly activated by membrane receptors, including receptor tyrosine kinases (RTKs), and adhesion receptors such as integrin. PLCyl mediates signaling through direct interactions with other signaling molecules via SH domains, as well as its lipase activity. PLCyl is frequently enriched and mutated in various cancers, and is involved in the processes of tumorigenesis, including proliferation, migration, and invasion. (Jang HJ, Suh PG, Lee YJ, Shin KJ, Cocco L, Chae YC. PLCyl : Potential arbitrator of cancer progression. Adv Biol Regul. 2018;67: 179-189, and Patterson RL, Van Rossum DB, Nikolaidis N, Gill DL, Snyder SH. Phospholipase C-y: diverse roles in receptor-mediated calcium signaling. Trends Biochem Sci. 2005;30(12):688- 697.

[0291] The phospholipase C gamma signaling pathway has been implicated in T cell lymphomas and primarily in cutaneous T cell lymphomas (CTCL). Nine PLCG1 mutations (p.R48W, p.S312L, p.D342N, p.S345F, p.S520F, p.R1158H, p.E1163K, p.D1165H, and the in-frame indel p.VYEEDMl 161 V) have been identified in Sezary Syndrome, the leukemic variant of CTCL. (V. M. Patel et al., Frequent and Persistent PLCG1 Mutations in SezaryCells Directly Enhance PLCyl Activity and Stimulate NFKB, AP-1, and NF AT Signaling. J Invest Dermatol 140, 380-389. e384 (2020).III. CARD 11-PIK3R3 FUSION PROTEINSCARD11

[0292] Caspase-associated recruitment domain (CARD) is a conserved homology domain comprising a 6-helix bundle or 5-helix bundle. CARDs can mediate protein-protein interactions between key apoptotic signaling molecules. Non-limiting examples of CARDs include CARD6, CARD8, CARD9, CARDIO, CARD11, CARD 14, CARD 16, CARD 18, and CARD 19. Non-limiting examples of CARD-containing proteins include those described in Boyle and Monie bioRxiv 087908; doi: https: / / doi.org / 10.1101 / 087908; and / or Park. In J Mol Med. 2019 Mar; 43(3): 1119-1127, such as human caspase-1, -2, -4, and -5, mouse caspase-1, -2, -11, and -12, ASC, NODI, N0D2, Apaf-1, BCL-10, and RIG-I.

[0293] As used herein, the term “CARD domain” refers to a caspase-associaed recruitment domain. In some embodiments, the CARD containing protein, or functional fragment thereof, is derived from a CARD protein selected from CARD9 (UniProt # Q9H257), CARDIO (UniProt # Q9BWT7) CARD 11 (UniProt # Q9BXL7), or CARD 14 (UniProt # Q9H257) (Wang et al. J Biol Chem. 2001 Jun 15;276(24):21405-9; Bertin et al. J Biol Chem. 2001 Apr 13;276(15): 11877-82.). In some embodiments, the CARD containing protein, or functional fragment thereof, is derived from a CARD 11 protein (UniProt #Q9BXL7), or a functional fragment thereof. In some embodiments, the CARD containing protein comprises or consists of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 293-323. In some embodiments, the functional fragment of the CARD containing protein is derived from a protein selected from, or wherein the CARD is derived from a protein selected from, CARD9, CARDIO, CARD11, and CARD14. In some embodiments, the functional fragment of the CARD containing protein is derived from CARD 11 and comprises or consists of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 295.

[0294] In some embodiments, the function of the CARD containing protein or the functional fragment thereof is to bind to a CARD domain on BCL10 (Bertin et al. J. Biol Chem. 201 Apr; 276(15): 11877-11882). In some embodiments, the functional fragment thereof comprises at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 300, at least 400, or at least 500 amino acids. Non-limitingexamples of a cell include a T cell, a macrophage, a monocyte, and a natural killer (NK) cell. In some embodiments, activation of the cell produces the substrate localized to the intracellular side of the plasma membrane. In some embodiments, the substrate localized to the intracellular side of the plasma membrane of a cell is a phosphoinositide. Non-limiting examples of phosphoinosities include those described in Posor et al. Nat Rev Mol Cell Biol. 2022 Dec;23(12):797-816. In some embodiments, the phosphoinositide is selected from phosphatidylinositol (3,4,5)-trisphosphate (PIP3), phosphatidylinositol 4, 5 -bisphosphate (PI(4,5)P2) (Hawse and Cattley J. Bio. Chem. 2019 Mar; 294(13):4793-4805; Sun et al. PLoS ONE. 2011 Nov; 6(11): e27227). In some embodiments, the polypeptide binds to the phosphoinositide with a Kd of less than 100 pM, 50 pM, 10 pM, 5 pM, IpM, 0.5 pM, 0.1 pM, 0.05 pM, or 0.01 pM, and wherein the Kd is analyzed using SPR as described in Yu et al. Molecular Cell. 2004 March; 13(5):p677-688.

[0295] CARD11, also known as Caspase Recruitment Domain Family Member 11, CARMA1, Carma 1, BIMP3, Caspase Recruitment Domain-Containing Protein 11, BCL10- Interacting Maguk Protein 3, CARD-Containing MAGUK Protein 1, Card-Maguk Protein 1, IMD11 A, BENT A, IMD11, or PPBL, is encoded by the human CARD11 gene. CARD11 is a membrane-associated guanylate kinase (MAGUK) family protein, a class of proteins that functions as molecular scaffolds for the assembly of multiprotein complexes at specialized regions of the plasma membrane. CARD 11 is also a member of the CARD protein family, defined by the presence of a caspase-associated recruitment domain (CARD). CARD 11 plays a role in various immune responses including, amoung others, lymphocyte activation, proliferation, and differentiation, and plays a role as a positive regulator of NF-kB activation.

[0296] CARD11 is -130 kDa and exhibits a modular structure comprising a CARD domain, a LATCH domain, a coiled-coil domain, an inhibitory domain, and a MAGUK domain (which itself is made up of a PDZ domain, SH3 domain, and GUK domain) CARD 11 is expressed in immune cells, with variations in expression levels observed across different cell types and states (Lu et al. The CBM-opathies-A Rapidly Expanding Spectrum of Human Inborn Errors of Immunity Caused by Mutations in the CARD11-BCL10-MALT1 Complex. Front Immunol. 2018 Sep 19;9:2078).

[0297] CARD11 is a member of the CARD11-BCL10-MALT1 (CBM) signalosome, which is essential for T cell activation and function upon antigen stimulation (Ruland et al. 2019. Nat. Rev. Immunol. 19,118-134). The CBM complex is known to have three important outputs: NF-kB transcriptional activity, AP-1 transcriptional activity, and MALT1proteolytic activity (Kutzner et al. 2022. Sci Signaling. 15: eabk3083). In the absence of signaling from the TCR, the CARD 11 inhibitory domain blocks CARD 11 from forming a complex with BCL10 and MALT1. However, after TCR signaling, the inhibitory domain is phosphorylated and the CARD 11 protein is able to oligimerize and recruite BCL10 and MALT1 to form the CBM complex.

[0298] Human CARD11 (isoform 203) has a CARD domain (SEQ ID NO: 295), a LATCH domain (SEQ ID NO: 342), a coiled-coil domain (SEQ ID NO: 343), an inhibitory domain (SEQ ID NO: 344), and a MAGUK domain (SEQ ID NO: 345). The MAGUK domain is made up of a PDZ domain (SEQ ID NO: 346), SH3 domain (SEQ ID NO: 347), and GUK domain (SEQ ID NO: 348). As used herein, a human CARD 11 CARD domain refers to the CARD domain near the N-terminus of the human CARD 11 protein. In some embodiments, the human CARD 11 CARD domain has an amino acid sequence that corresponds to amino acids 11-103 of SEQ ID NO: 1, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 11-103 of SEQ ID NO: 1. In some embodiments, the human CARD11 CARD domain has an amino acid sequence corresponding to amino acids 11-103 of SEQ ID NO: 1, or an amino acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 11-103 of SEQ ID NO: 1.

[0299] As used herein, a human CARD 11 LATCH domain refers to the LATCH domain between the N-terminal CARD domain and the C-terminal coiled-coil domain of the human CARD 11 protein. In some embodiments, the human CARD 11 LATCH domain has an amino acid sequence that corresponds to amino acids 104-122 of SEQ ID NO: 1, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 104-122 of SEQ ID NO: 1. In some embodiments, the human CARD 11 LATCH domain has an amino acid sequence corresponding to amino acids 104-122 of SEQ ID NO: 1, or an amino acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 104-122 of SEQ ID NO: 1.

[0300] As used herein, a human CARD 11 coiled-coil domain refers to the coiled-coil domain between the N-terminal LATCH domain and the C-terminal inhibitory domain of the human CARD 11 protein. In some embodiments, the human CARD 11 coiled-coil domain has an amino acid sequence that corresponds to amino acids 123-442 of SEQ ID NO: 1, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 123-442 of SEQ ID NO: 1. In someembodiments, the human CARD 11 coiled-coil domain has an amino acid sequence corresponding to amino acids 123-442 of SEQ ID NO: 1, or an amino acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 123-442 of SEQ ID NO: 1.

[0301] As used herein, a human CARD 11 inhibitory domain refers to the inhibitory domain (ID) between the N-terminal coiled-coil domain and the C-terminal MAGUK domain of the human CARD 11 protein. . In some embodiments, the human CARD 11 inhibitory domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 344. In some embodiments, the human CARD11 inhibitory domain has an amino acid sequence that corresponds to amino acids 443-577 of SEQ ID NO: 1, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 443-577 of SEQ ID NO: 1. In some embodiments, the human CARD11 inhibitory domain has an amino acid sequence corresponding to amino acids 443-577 of SEQ ID NO: 1, or an amino acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 443-577 of SEQ ID NO: 1. In some embodiments, the human CARD1 1 inhibitory domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 441.

[0302] As used herein, a human CARD 11 MAGUK domain refers to the MAGUK domain near the C-terminus of the human CARD 11 protein. In some embodiments, the human CARD 11 MAGUK domain has an amino acid sequence that corresponds to amino acids 667-1140 of SEQ ID NO: 518, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 667-1140 of SEQ ID NO: 518. In some embodiments, the human CARD 11 MAGUK domain has an amino acid sequence corresponding to amino acids 667-1140 of SEQ ID NO: 518, or an amino acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 667-1140 of SEQ ID NO: 518.

[0303] The human CARD 11 gene has eight transcripts, three of which encode a protein isoform of CARD11 (Ensemble, Gene: CARD11). The eight transcripts are named CARD 11 -201, CARD 11 -202, CARD 11 -203, CARD 11 -204, CARD 11 -205, CARD 11 -206, CARD1 1-201, and CARD11-208 (Ensembl, Gene: CARD11, ENSG00000198286). The coding DNA sequence (CDS) of the six transcripts are as set forth in SEQ ID NO: 349-354, respectively. The amino acid sequences of the three protein isoforms are as set forth in SEQ ID NO: 516-518, respectively (Table 1).

[0304] Transcript CARD11-203 (SEQ ID NO: 351; Ensembl Transcript ID: ENST00000396946.9; NCBI Reference Sequence: NM_001324281.3) encodes isoform CARD1 1-203 (SEQ ID NO: 518; Uniprot Accession No. Q9BXL7; NCBI Reference Sequence: NP_001311210.1), which has 1154 amino acids. CARD11-203 is the longest transcript of the human CARD 11 gene. It is designated as the representative transcript in the Ensembl database. In identifying the representative transcript, Ensembl aims to identity the transcript that, on balance, has the highest coverage of conserved exons, highest expression, longest coding sequence and is represented in other key resources, such as NCBI and UniProt. All splice junctions of the CARD 11-203 transcript are supported by at least one non-suspect mRNA.

[0305] Transcript CARD11-201 (SEQ ID NO: 349; Ensembl Transcript ID: ENST00000355508.3; NCBI Reference Sequence: NM_001324281.3) encodes isoform CARD 11-201 (SEQ ID NO: 516; Uniprot Accession No. H7BY05), which has 271 amino acids.

[0306] Transcript CARD 11-202 (Ensembl Transcript ID: ENST00000356408.3:) encodes isoform CARD11-202 (SEQ ID NO: 517; Uniprot Accession No. E2QRC0), which has 73 amino acids. The amino acid sequences of the three isoforms are listed in Table 1. The amino acids corresponding to the various domains in the human CARD 11 protein are also identified in Table 1. “Beginning Sequence” refers to the region N-terminal to the CARD domain. “End Sequeunce” refers to the region C-terminal to the MAGUK domain.Table 1. Amino Acid SEQ ID NOs for CARD11 Isoforms

[0307] As used herein, a human CARD 11 PDZ domain refers to the PDZ domain near the N-terminus of the human CARD 11 MAGUK domain. In some embodiments, the human CARD1 1 PDZ domain has an amino acid sequence that corresponds to amino acids 667-755 of SEQ ID NO: 518, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 667-755 of SEQ ID NO: 518. In some embodiments, the human CARD 11 PDZ domain has an amino acid sequence corresponding to amino acids 667-755 of SEQ ID NO: 518, or an amino acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 667-755 of SEQ ID NO: 518.

[0308] As used herein, a human CARD 11 SH3 domain refers to the SH3 domain between the N-terminal PDZ domain and the C-terminal GUK domain of the human CARD 11 MAGUK domain. In some embodiments, the human CARD 11 SH3 domain has an amino acid sequence that corresponds to amino acids 773-806 of SEQ ID NO: 518, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 773-806 of SEQ ID NO: 518. In some embodiments, the human CARD 11 SH3 domain has an amino acid sequence corresponding to amino acids 773-806 of SEQ ID NO: 518, or an amino acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 773- 806 of SEQ ID NO: 518.

[0309] As used herein, a human CARD 11 GUK domain refers to the GUK domain near the C-terminus of the human CARD 11 MAGUK domain. In some embodiments, the human CARD 11 GUK domain has an amino acid sequence that corresponds to amino acids 973- 1140 of SEQ ID NO: 518, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 973-1140 of SEQ ID NO: 518. In some embodiments, the human CARD 11 GUK domain has an amino acid sequence corresponding to amino acids 973-1140 of SEQ ID NO: 518, or an amino acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 973-1140 of SEQ ID NO: 518.

[0310] The amino acid sequences of the CARD11-203 PDZ, SH3, and GUK domains in Table 2. “PDZ to SH3 Connection” refers to the region connecting the PDZ domain and the SH3 domain. “SH3 to GUK Connection” refers to the region connecting the SH3 domain and the GUK domain.Table 2. Amino Acid SEQ ID NOs for CARD11 DomainsPIK3R3

[0311] PIK3R3, also known as phosphatidylinositol 3-kinase regulatory subunit gamma (UniProt Q92569), is encoded by the human PIK3R3 gene (NCBI gene ID 8503).

[0312] PIK3R3, also known as phosphatidylinositol 3-kinase regulatory subunit gamma, p55, p55-GAMMA, p55PIK, phosphoinositide-3 -kinase regulatory subunit 3, is encoded by the human PIK3R3 gene. PIK3R3 is a regulatory subunit of PI3K and phosphorylates phosphatidylinositol (and similar compounds).

[0313] PIK3R3 is ~62kDa and comprises both a catalytic and regulatory subunit including. PIK3R3 comprises two SH2 domains through which it binds activated (phosphorylated) protein-tyrosine kinases to regulate their activity.The human PIK3R3 gene has seven transcripts, five of which encode a protein isoform of PIK3R3 (Ensemble, Gene: PIK3R3, ENSG00000117461). The seven transcripts are named PIK3R3-201, PIK3R3-202, PIK3R3-203, PIK3R3-204, PIK3R3-205, PIK3R3-206, and PIK3R3-207 (Ensemble, Gene: PIK3R3, ENSG00000117461).CARD11-PIK3R3 Fusion Protein

[0314] A CARD11-PIK3R3 fusion protein has previously been derived from a patient with CD4+ T cell lymphoma (Wang et al., Genomic profiling of Sezary syndrome identifies alterations of key T cell signaling and differentiation genes. Nature Genetics 47, 1426-1434(2015). Thereapeutic T cells expressing this CARD11-PIK3R3 fusion protein demonstrate augmented CARD11-BCL10-MALT1 complex signalling and anti-tumour efficacy in several immunotherapy-refractory models in an antigen-dependent manner (Garcia et al., Naturally occurring T cell mutations enhance engineered T cell therapies. Nature. 626, 626- 634 (2024)). Therapeutic T cells expressing the CARD11-PIK3R3 fusion protein showed no evidence of malignant transformation up to 418 days after T-cell transfer, underscoring the safety of such an approach. These references are herein incorporated by reference in their entirety.

[0315] The present disclosure provides recombinant nucleic acids encoding a recombinant polypeptide as described herein, wherein the recombinant nucleic acid additionally encodes a CARD-containing protein (e.g., a CARD11-PIK3R3 fusion protein) or a functional fragment thereof, for example those described in PCT / US2023 / 075738, which is herein incorporated by reference in its entirety.Engineered CARD11-PIK3R3 Fusion Proteins

[0316] The present disclosure provides recombinant polypeptides comprising a caspase- associated recruitment domain (CARD) or a functional fragment thereof. A functional fragment of a recombinant polypeptide comprising a CARD can be, for example, a fragment that provides NF-KB transcriptional activity, AP-1 transcriptional activity, and / or MALT1 proteolytic activity activity at a level at least 70%, 75%, 80%, 85%, 90%, or 95% of that of a full-length protein, as determined by the in vitro CAR Jurkat assay.

[0317] The disclosure further relates to recombinant polypeptides comprising a CARD- containing protein or a functional fragment thereof, or to recombinant nucleic acids encoding such polypeptides, comprising a domain capable of binding to (i) a substrate localized to the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine (pTyr). In some embodiments, the substrate localized to the intracellular side of the plasma membrane of a cell is a phosphoinositide. Non-limiting examples of phosphoinosities include those described in Posor et al. Nat Rev Mol Cell Biol. 2022 Dec;23(12):797-816. In some embodiments, the phosphoinositide is selected from phosphatidylinositol (3,4,5)-trisphosphate (PIP3), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) (Hawse and Cattley J. Bio. Chem. 2019 Mar; 294(13):4793-4805; Sun et al. PLoS ONE. 2011 Nov; 6(11): e27227). In some embodiments, the polypeptide binds to the phosphoinositide with a Kd of less than 100 pM,50 pM, 10 pM, 5 pM, IpM, 0.5 pM, 0.1 pM, 0.05 pM, or 0.01 pM, and wherein the Kd is analyzed using SPR as described in Yu et al. Molecular Cell. 2004 March; 13(5):p 677-688.

[0318] In some embodiments, the domain is capable of binding to a substrate indirectly localized to the intracellular side of the plasma membrane. Indirect localization can refer to localization of the substrate to the intracellular side of the plasma membrane through, for example, binding of the substrate to another polypeptide or lipid that is directly localized to the intracellular side of the plasma membrane. Indirect localization can refer to localization of the substrate to the intracellular side of the plasma membrane through, for example, interaction of the substrate with another polypeptide or lipid that is directly localized to the intracellular side of the plasma membrane. In some embodiments, the domain is capable of binding to a substrate directly localized to the intracellular side of the plasma membrane. Direct localization can refer to localization of the substrate to the intracellular side of the plasma membrane through, for example, binding of the substrate to the intracellular side of the plasma membrane itself. Direct localization can refer to localization of the substrate to the intracellular side of the plasma membrane through, for example, interaction of the substrate with the intracellular side of the plasma membrane itself.

[0319] The disclosure further relates to recombinant polypeptides comprising a recruitment domain or a functional fragment thereof. The disclosure further relates to recombinant polypeptides comprising (a) a caspase-associated recruitment domain (CARD), and (b) a recruitment domain or a functional fragment thereof. The disclosure further relates to recombinant polypeptides comprising: (a) a first polypeptide capable of binding to a caspase-associated recruitment domain (CARD) on BCL10, and (b) a recruitment domain or a functional fragment thereof. In some embodiments, the recruitment domain is capable of binding to (i) a substrate localized to the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine (pTyr). In some embodiments, the recombinant polypeptide does not comprise a full length sequene of an endogenous protein comprising the recruitment domain. In some embodiments, the recombinant polypeptide comprises no more than 724 amino acids. In some embodiments, the recombinant polypeptide does not comprise a full length sequence of an endogenous protein comprising the CARD and / or a full length sequence of an endogenous protein comprising the recruitment domain. In some embodiments, the recombinant polypeptide does not comprise a full length sequence of an endogenous protein capable of binding the CARD recruitment domain of BCL10.

[0320] Non-limiting examples of recruitment domains capable of binding to (i) and / or (ii) include Src Homology region 2 (SH2) domains, Src Homology region 3 (SH3) domains, pleckstrin homology (PH) domains, and phosphytyrosine-binding (PTB) domains. SH2 and PTB domains mediate protein-protein interactions involved in many signal transduction pathways. SH2, SH3, and PTB domains that can be used in the disclosure include, for example, those disclosed in Schlessinger et al. Sci STKE. 2003 Jul 15;2003(191):RE12. SH2 containing proteins can bind to plasma membrane lipids through a different binding pocket than the pTyr binding pocket; most SH2 domains bind plasma membrane lipids and many have high phosphoinositide specificity (Park et al., Cell. 2016 Apr 7;62(l):7-20). In some embodiments, the recruitment domain is, or comprises, an SH3 domain. In some embodiments, the recruitment domain is, or comprises, a PTB domain. In some embodiments, the recruitment domain is, or comprises, a PH domain. In some embodiments, the recruitment domain is, or comprises, an SH2 domain.

[0321] In some embodiments, the start of the recruitment domain corresponds to amino acid position 622 of SEQ ID NO: 1 and the end of the recruitment domain corresponds to amino acid position 716 of SEQ ID NO: 1. In some embodiments, the recruitment domain comprises at least 90 amino acids.

[0322] In some embodiments, the recombinant polypeptide comprises a functional fragment of the recruitment domain. In some embodiments, the recombinant polypeptide comprises a truncated portion of the recruitment domain having no more than 85, no more than 80, no more than 75, no more than 70, no more than 60, no more than 50, or no more than 40, amino acids.

[0323] The recruitment domain may comprise a truncation at the N-terminus, at the C- terminus, or both. In some embodiments, the recruitment domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 amino acids at the N-terminus. In some embodiments, the recruitment domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 amino acids at the C-terminus. In some embodiments, the recruitment domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 amino acids in between the N-terminus and the C-terminus. In some embodiments, the recruitment domain or the functional fragment thereof comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a segment of at least 30, 40, 50, 60, 70, 80, 90, or 97 amino acids within the region of amino acids 622-716 ofSEQ ID NO: 1. In some embodiments, the recombinant polypeptide does not comprise any recruitment domain having at least 30 amino acids in length.

[0324] In some embodiments, the SH2 domain comprises or consists of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 305 and 307-437. In some embodiments, the SH2 domain is from a PIK3R3 protein (UniProt # Q92569). In some embodiments, the SH2 domain comprises the motif of a conserved arginine residue in the FLVR motif (Arg PB5 or Argl75 in the v-Src SH2 domain). Most conserved resides are clustered on the PB strand, and the conserved arginine residue in the FLVR motif plays the central role in forming a double hydrogen bond with the phosphate group of pTyr. Additional residues that are key for phosphopeptide binding are His PD4, Lys PD6, and Arg aA2, which coordinate and anchor the aromatic ring of the phospho-tyrosine (Diop et al. Int J Mol Sci. 2022 Dec 15;23(24): 15944). In some embodiments, the SH2 domain is an engineered SH2 domain with an enhanced affinity for phosphotyrosine (e.g., Veggiani et al. Protein Sci. 2019 Feb;28(2):403-413).

[0325] Non-limiting examples of target polypeptides comprising a pTyr include Pl 10a / PIK3CA, Pl 10p / PIK3CB, Pl 105 / PIK3CD, IGF-1R, ErbB2, CTLA-4, and CD28. In some embodiments, the target polypeptide is derived from IGF-1R, CTLA-4, or CD28. In some embodiments, the target polypeptide comprises or consists of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 298-304. In some embodiments, the pTyr is located at the position corresponding to pY1346 of SEQ ID NO: 325. In some embodiments, the pTyr is located at the position corresponding to pY1221 of SEQ ID NO: 324. In some embodiments, the polypeptide binds to the target polypeptide with a Kd of less than 100 pM, 50 pM, 10 pM, 5 pM, IpM, 0.5 pM, 0.1 pM, 0.05 pM, or 0.01 pM, wherein the Kd is analyzed by flourescence polarization assay as described in Hause et al. PLoS One. 2012;7(9):e44471. In some embodiments, the polypeptide has a higher affinity for the target polypeptide comprising the pTyr than a control polypeptide without phosphorylation at the corresponding tyrosine position. In some embodiments, the polypeptide has at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold, higher affinity (lower Kd) for the target polypeptide comprising the phosphorylated tyrosine than a control polypeptide without phosphorylation at the corresponding tyrosine position.

[0326] In some embodiments, the domain is capable of binding to a substrate indirectly localized to the intracellular side of the plasma membrane. Indirect localization can refer to localization of the substrate to the intracellular side of the plasma membrane through, forexample, binding of the substrate to another polypeptide or lipid that is directly localized to the intracellular side of the plasma membrane. Indirect localization can refer to localization of the substrate to the intracellular side of the plasma membrane through, for example, interaction of the substrate with another polypeptide or lipid that is directly localized to the intracellular side of the plasma membrane. In some embodiments, the domain is capable of binding to a substrate directly localized to the intracellular side of the plasma membrane. Direct localization can refer to localization of the substrate to the intracellular side of the plasma membrane through, for example, binding of the substrate to the intracellular side of the plasma membrane itself. Direct localization can refer to localization of the substrate to the intracellular side of the plasma membrane through, for example, interaction of the substrate with the intracellular side of the plasma membrane itself.

[0327] Furthermore, the disclosure relates to recombinant polypeptides comprising a CARD containing protein or a functional fragment thereof, and a domain capable of binding to (i) a substrate localized to the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine (also called a CARD fusion polypeptide), as disclosed herein. In some embodiments, the polypeptide comprises a CARD containing protein, or a functional fragment thereof, and an SH2 domain (also called a CARD-SH2 fusion polypeptide). In some embodiments, the polypeptide is a CARD11- PIK3R3 fusion polypeptide. In some embodiments, the recombinant nucleic acid constructs encode a CARD11-PIK3R3 fusion polypeptide or any truncations thereof.

[0328] In some embodiments, the disclosure relates to a polypeptide comprising a functional fragment of a CARD containing protein derived from a CARD 11 protein and an SH2 domain from a PIK3R3 protein (also called a CARD11-PIK3R3 fusion polypeptide).

[0329] The present disclosure further relates to polypeptides wherein the domain capable of binding to the target polypeptide comprising the phosphorylated tyrosine, or the SH2 domain, or the second polypeptide portion, is located at the N-terminus of the CARD containing protein or functional fragment thereof, between the CARD containing protein and the Coiled-coil domain, or at the C-terminus of the CARD containing protein and / or the Coiled-coil domain. In some embodiments, the polypeptide comprises the CARD domain derived from a CARD 11 protein followed by the Coiled-coil domain derived from the CARD 11 protein. The present disclosure further relates to polypeptides wherein the domain capable of binding to the target polypeptide comprising the phosphorylated tyrosine, or the SH2 domain, or the second polypeptide portion, is located close to the C-terminus of the polypeptide, wherein the polypeptide has no more than 50, 40, 30, 20, 15, 10, or 5 aminoacids at the C-terminus of the domain in (b), or the SH2 domain, or the second polypeptide portion.

[0330] In some embodiments, the polypeptide does not comprise an inhibitory domain (ID) or a portion thereof. In other embodiments, the polypeptide comprises an ID or a portion thereof.

[0331] In some embodiments, the CARD11-PIK3R3 fusion polypeptide includes a CARD domain (i.e., containing protein or functional fragment thereof), a CARD inhibitory domain (ID), a coiled-coil domain, and an SH2 domain from PIK3R3. In some embodiments, the CARD1 1-PIK3R3 fusion polypeptide includes a functional fragment of a CARD containing protein derived from CARD 11 domain, a coiled-coil domain, and an SH2 domain from PIK3R3.

[0332] In some embodiments, the polypeptide of the disclosure has a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3-292 and functional variants thereof, optionally comprising at least one mutation listed in Table 3.

[0333] In some embodiments, the polypeptide of the disclosure has a sequence selected from the group consisting of SEQ ID NO: 3-292 and functional variants thereof, optionally comprising at least one mutation listed in Table 3. In some embodiments, the polynucleotide of the disclosure has a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3-292 and functional variants thereof, optionally comprising at least one mutation listed in Table 3. In some embodiments, the polynucleotide of the disclosure has a sequence selected from the group consisting of SEQ ID NO: 3-292 and functional variants thereof, optionally comprising at least one mutation listed in Table 3.

[0334] The present disclosure provides engineered CARD11-PIK3R3 proteins that have fewer amino acids than the patient derived full-length human CARD11-PIK3R3 fusion. In some embodiments, the engineered CARD11-PIK3R3 protein is a recombinant polypeptide comprising a caspase-associated recruitment domain (CARD) containing protein or a functional fragment thereof, wherein the recombinant polypeptide comprises no more than 724 amino acids. In some embodiments, the recombinant polypeptide does not comprise a full length CARD containing protein.

[0335] In some embodiments, the recombinant polypeptide comprises no more than 700, no more than 650, no more than 600, no more than 550, no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than200, or no more than 150 amino acids. In some embodiments, the CARD containing protein or the functional fragment thereof, the CARD, comprises no more than 40, no more than 50, no more than 60, no more than 70, no more than 80, no more than 90, no more than 100, no more than 150, no more than 200, no more than 300, no more than 400, or no more than 500 amino acids.

[0336] In some embodiments, the recombinant polypeptide comprises a full CARD domain. In some embodiments, the full CARD domain comprises at least 90 amino acids. In some embodiments, the recombinant polypeptide comprises a truncated portion of a full CARD domain. In some embodiments, the recombinant polypeptide comprises a truncated portion of the CARD domain having no more than 85, no more than 80, no more than 75, no more than 70, no more than 60, no more than 50, or no more than 40, amino acids.

[0337] The CARD domain may comprise a truncation at the N-terminus, the C-terminus, or both. In some embodiments, the CARD domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 amino acids at the N- terminus. In some embodiments, the CARD domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 amino acids at the C- terminus. In some embodiments, the CARD domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 amino acids in between the N-terminus and the C-terminus. In some embodiments, the recombinant polypeptide does not comprise any full or truncated portion of CARD domain having at least 30 amino acids in length.

[0338] In some embodiments, the recombinant polypeptide comprises or consists of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 225-245. In some embodiments, the recombinant polypeptide does not comprise the polypeptide sequence “MDDY” within 10 amino acids before the N-term of the start of the CARD domain.

[0339] In some embodiments, the recombinant polypeptide comprises a full coiled-coil domain (e.g., a full CARD 11 coiled-coil domain). In some embodiments, the start of the full coiled-coil domain corresponds to amino acid position 123 of SEQ ID NO: 1, and the end of the full coiled-coil domain corresponds to amino acid position 442 of SEQ ID NO: 1. In some embodiments, the full coiled-coil domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 123-442 of SEQ ID NO: 1. In some embodiments, the full coiled-coil domain comprises at least 310 amino acids.

[0340] In some embodiments, the recombinant polypeptide comprises a truncated portion of a coiled-coil domain (e.g., a truncated portion of a CARD 11 coiled-coil domain). In some embodiments, the recombinant polypeptide comprises a truncated portion of the coiled-coil domain having no more than 300, no more than 290, no more than 280, no more than 270, no more than 260, no more than 250, no more than 240, no more than 230, no more than 220, no more than 210, no more than 200, no more than 190, no more than 180, no more than 170, no more than 160, no more than 150, no more than 140, no more than 130, no mor than 120, no more than 110, no more than 100, no more than 95, no more than 90, no more than 85, no more than 80, no more than 75, no more than 70, no more than 60, no more than 50, or no more than 40, amino acids; preferably, wherein the truncated portion of the coiled- coil domain has no more than 260 amino acids.

[0341] The coiled-coil domain may comprise a truncation at the N-terminus, the C-terminus, or both. In some embodiments, the coiled-coil domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, or at least 270 amino acids at the N- terminus. In some embodiments, the coiled-coil domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, or at least 270 amino acids at the C- terminus. In some embodiments, the coiled-coil domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, at least 130, at least 140, at least 150, at least 160, at least 170, at least 180, at least 190, at least 200, at least 210, at least 220, at least 230, at least 240, at least 250, at least 260, or at least 2700270 amino acids in between the N-terminus and the C-terminus; optionally, the coiled-coil domain comprises a truncation of about 60 to about 80 amino acids in between the N-terminus and the C- terminus.

[0342] A region within the coil-coil domain may be removed or replaced. In some embodiments, a region within the coiled-coil domain corresponding to amino acids 253-324 of SEQ ID NO: 1 is removed or replaced by a polypeptide comprising no more than 20, 15, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids. In some embodiments, the region withinthe coiled-coil domain is replaced by a polypeptide comprising a sequence of SGGGGSGGGGS (SEQ ID NO: 431) or QAGKRSLPD (SEQ ID NO: 432), or a sequence having at most 1, 2, or 3 mutations thereto. In some embodiments, the recombinant polypeptide does not comprise any full or truncated portion of coiled-coil domain having at least 30 amino acids in length. In some embodiments, the recombinant polypeptide comprises or consists of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 193-224 and 246-276.

[0343] In some embodiments, the recombinant polypeptide comprises a full inhibitory domain (e.g., a full CARD 11 inhibitory domain). In some embodiments, the inhibitory domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 344. In some embodiments, the start of the inhibitory domain corresponds to amino acid position 443 of SEQ ID NO: 1, and the end of the inhibitory domain corresponds to amino acid position 577 of SEQ ID NO: 1. In some embodiments, the inhibitory domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 443-577 of SEQ ID NO: 1. In some embodiments, the human CARD 11 inhibitory domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 441. In some embodiments, the inhibitory domain comprises at least 130, or at least 125, amino acids.

[0344] In some embodiments, the recombinant polypeptide comprises a truncated portion of an inhibitory domain (e.g., a truncated portion of a CARD 11 inhibitory domain). In some embodiments, the recombinant polypeptide comprises a truncated portion of the inhibitory domain having no more than 120, no more than 110, no more than 100, no more than 95, no more than 90, no more than 85, no more than 80, no more than 75, no more than 70, no more than 60, no more than 50, or no more than 40, amino acids.

[0345] The inhibitory domain may comprise a truncation at the N-terminus, the C-terminus, or both. In some embodiments, the inhibitory domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 amino acids at the C-terminus. In some embodiments, the inhibitory domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 amino acids in between the N-terminus and the C-terminus. In some embodiments, the recombinant polypeptide does not comprise any full or truncated portion of inhibitory domain having at least 30 amino acids in length.

[0346] The inhibitory domain may comprise a certain number of serines capable of being phosphorylated. In some embodiments, the inhibitory domain or the truncated portion thereof comprises at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 serines that are capable of being phosphorylated. In some embodiments, the serines are capable of being phosphorylated by a protein kinase C (PKC). Non-limiting examples of PKC include PKCP and PKC9. In some embodiments, the native serine phosphorylation site(s) in the full-length wildtype inhibitory domain, which would otherwise be removed due to truncation, are inserted into the truncated inhibitory domain sequence. Thus, in some embodiments, the truncated inhibitory domain may comprise more serine phosphorylation sites than the corresponding region of the wildtype inhibitory domain. Such design may provide additional regulation of the activity of the polypeptides of the disclosure. Additional descriptions of serine phosphorylation in the inhibitory domain can be found, for example, in Sommer et al., Immunity. 2005 Dec;23(6):561-74, the content of which is incorporated by reference in its entirety.

[0347] In some embodiments, the inhibitory domain or the truncated portion thereof comprises a sequence of DASPRT (SEQ ID NO: 425), RAKSPI (SEQ ID NO: 426), DASPSS (SEQ ID NO: 427), SRSSIMS (SEQ ID NO: 428), RKFSLER (SEQ ID NO: 429), and / or FRPSVTS (SEQ ID NO: 430), or a sequence having at most 1, 2, or 3 mutations thereto. In some embodiments, the inhibitory domain or the truncated portion thereof comprises DASPRT (SEQ ID NO: 425), RAKSPI (SEQ ID NO: 426), DASPSS (SEQ ID NO: 427), and / or SRSSIMS (SEQ ID NO: 428), or a sequence having at most 1, 2, or 3 mutations thereto. In some embodiments, the inhibitory domain or the truncated portion thereof comprises RAKSPI (SEQ ID NO: 426), DASPSS (SEQ ID NO: 427), and / or SRSSIMS (SEQ ID NO: 428), or a sequence having at most 1, 2, or 3 mutations thereto. In some embodiments, the inhibitory domain or the truncated portion thereof comprises SRSSIMS (SEQ ID NO: 428), RKFSLER (SEQ ID NO: 429), and / or FRPSVTS (SEQ ID NO: 430), or a sequence having at most 1, 2, or 3 mutations thereto. In some embodiments, the inhibitory domain or the truncated portion thereof comprises DASPRT (SEQ ID NO:425), RAKSPI (SEQ ID NO: 426), and DASPSS (SEQ ID NO: 427). In some embodiments, the inhibitory domain or the truncated portion thereof comprises RAKSPI (SEQ ID NO:426), DASPSS (SEQ ID NO: 427) and SRSSIMS (SEQ ID NO: 428). In some embodiments, the inhibitory domain or the truncated portion thereof comprises DASPSS (SEQ ID NO: 427), SRSSIMS (SEQ ID NO: 428) and RKFSLER (SEQ ID NO: 429). In some embodiments, the inhibitory domain or the truncated portion thereof comprisesSRSSIMS (SEQ ID NO: 428), RKFSLER (SEQ ID NO: 429), and FRPSVTS (SEQ ID NO: 430). In some embodiments, the inhibitory domain or the truncated portion thereof comprises DASPRT (SEQ ID NO: 425) and RAKSPI (SEQ ID NO: 426). In some embodiments, the inhibitory domain or the truncated portion thereof comprises RAKSPI (SEQ ID NO: 426), and DASPSS (SEQ ID NO: 427). In some embodiments, the inhibitory domain or the truncated portion thereof comprises DASPSS (SEQ ID NO: 427) and SRSSIMS (SEQ ID NO: 428). In some embodiments, the inhibitory domain or the truncated portion thereof comprises SRSSIMS (SEQ ID NO: 428) and RKFSLER (SEQ ID NO: 429). In some embodiments, the inhibitory domain or the truncated portion thereof comprises RKFSLER (SEQ ID NO: 429), and FRPSVTS (SEQ ID NO: 430).

[0348] In some embodiments, the serine capable of being phosphorylated in DASPRT (SEQ ID NO: 425), or a sequence having at most 1, 2, or 3 mutations thereto, is located at a position corresponding to S466 according to SEQ ID NO: 518 in the inhibitory domain. In some embodiments, the serine capable of being phosphorylated in RAKSPI (SEQ ID NO: 426), or a sequence having at most 1, 2, or 3 mutations thereto, is located at a position corresponding to S512 according to SEQ ID NO: 518 in the inhibitory domain. In some embodiments, the serine capable of being phosphorylated in DASPSS (SEQ ID NO: 427), or a sequence having at most 1, 2, or 3 mutations thereto, is located at a position corresponding to S535 according to SEQ ID NO: 518 in the inhibitory domain. In some embodiments, the serine capable of being phosphorylated in SRSSIMS (SEQ ID NO: 428), or a sequence having at most 1, 2, or 3 mutations thereto, is located at a position corresponding to S559 according to SEQ ID NO: 518 in the inhibitory domain. In some embodiments, the serine capable of being phosphorylated in RKFSLER (SEQ ID NO: 429), or a sequence having at most 1, 2, or 3 mutations thereto, is located at a position corresponding to S644 according to SEQ ID NO: 518 in the inhibitory domain. In some embodiments, the serine capable of being phosphorylated in FRPSVTS (SEQ ID NO: 430), or a sequence having at most 1, 2, or 3 mutations thereto, is located at a position corresponding to S652 according to SEQ ID NO: 518 in the inhibitory domain.

[0349] In some embodiments, the recombinant polypeptide comprises, from N-term to C- term: the coiled-coil domain or the truncated portion thereof, and the inhibitory domain or the truncated portion thereof, without any additional amino acid in between. In some embodiments, the recombinant polypeptide comprises or consists of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 179-192, 277-288, and 289-292.

[0350] The recombinant polypeptide may comprise the domains described herein in a particular order, from N-terminus to C-terminus, optionally with no more than a particular number of amino acids between any two domains. In some embodiments, the recombinant polypeptide comprises from N-terminus to C-terminus: the inhibitory domain or the truncated portion thereof, and the recruitment domain or the functional fragment thereof, with no more than 5, no more than 10, no more than 15, no more than 20, or no more than 25, amino acids in between. In some embodiments, the recombinant polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 433-438 in between the inhibitory domain or the truncated portion thereof and the recruitment domain or the functional fragment thereof. In some embodiments, the recombinant polypeptide comprises, from N- terminus to C-terminus, the CARD domain, optionally the coiled-coil domain, optionally the inhibitory domain, and the recruitment domain.

[0351] In some embodiments, the recombinant polypeptide comprises no more than 40, no more than 35, no more than 30, no more than 25, no more than 20, no more than 15, no more than 10, or no more than 5, amino acids, in between the CARD containing protein or functional fragment thereof and the recruitment domain. In some embodiments, the recombinant polypeptide comprises or consists of a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 3-150. In some embodiments, the recombinant polypeptide comprises or consists of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 151-178. In some embodiments, the recombinant polypeptide comprises an aspartatic acid acid (D) at the position corresponding to amino acid 602 of SEQ ID NO: 1.

[0352] In some embodiments, the CARD containing protein or functional fragment thereof possesses certain binding capabilities and / or functional abilities.

[0353] In some embodiments, the CARD containing protein or functional fragment thereof the CARD, or the recombinant polypeptide, is capable of binding to a CARD domain on BCL10. In some embodiments, the CARD containing protein or functional fragment thereof the CARD, or the recombinant polypeptide, is capable of forming a complex with BCL10 and MALTE

[0354] In some embodiments, when the recombinant polypeptide is expressed in an immune cell and when the immune cell binds a target antigen, the immune cell exhibits decreased signaling and / or elicits decreased IFNg and / or IL-2 production compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1. In some embodiments, whenthe recombinant polypeptide is expressed in an immune cell and when the immune cell binds a target antigen, the immune cell exhibits equivalent or enhanced signaling and / or elicits equivalent or higher IFNg and / or IL-2 production compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1. In some embodiments, when the is expressed in an immune cell and when the immune cell binds a target antigen, the immune cell exhibits decreased (a) BCL10 binding; (b) BCL10 and MALT1 complex formation, (c) NF AT, NF-KB and / or AP-1 signaling, (d) cytokine production, (e) JAK / STAT signaling, (f) co-stimulatory molecule signaling, (g) phospholipase gamma signaling, and / or (h) transcription factor activity, compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1. In some embodiments, when the is expressed in an immune cell and when the immune cell binds a target antigen, the immune cell exhibits equivalent or enhanced (a) BCL10 binding; (b) BCL10 and MALT1 complex formation, (c) NF-KB and / or AP-1 signaling, (d) MALT1 paracaspase activity; (e) cytokine production, (f) peak tumor expansion, (g) peak peripheral blood expansion; (h) contraction following peak tumor expansion, and / or (i) contraction following peak peripheral blood expansion,, compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1. In some embodiments, the recombinant polypeptide affords certain beneficial technical effects, including delivery via integrating vectors.

[0355] In some embodiments, the cell comprises at least one copy, or at least two copies, of an endogenous nucleic acid sequence encoding a CARD 11 protein, or a protein comprising a CARD domain from CARD 11 without any SH2 domain. In some embodiments, the recombinant nucleic acid of the cell is located at the endogenous CARD 11 -encoding gene locus, or comprises at least a portion of the endogenous CARD 11 -encoding gene, of the cell.

[0356] In some embodiments, the cell comprises at least one copy, or at least two copies, of an endogenous nucleic acid sequence encoding a PIK3R3 protein, or a protein comprising an SH2 domain from PIK3R3 without any CARD domain. In some embodiments, the recombinant nucleic acid of the cell is located at the endogenous PIK3R3 -encoding gene locus, or comprises at least a portion of the endogenous PIK3R3 -encoding gene, of the cell.TABLE 3. CARD11-PIK3R3 Truncationse. Biological Activity of Engineered CARD11-PIK3R3 Fusion Proteins

[0357] In some embodiments the engineered CARD11-PIK3R3 fusion protein is functionally equivalent to a patient derived full length CARD11-PIK3R3 fusion protein. As used herein, “functionally equivalent” refers to having at least one type of biological activity of a patient-derived human CARD11-PIK3R3 fusion protein.

[0358] In some embodiments an engineered cell comprising the engineered CARD11- PIK3R3 fusion protein is functionally equivalent to an engineered cell comprising a patient derived full length CARD11-PIK3R3 fusion protein. As used herein, “functionally equivalent” refers to having at least one type of biological activity of an engineered cell comprising a patient-derived human CARD11-PIK3R3 fusion protein.

[0359] The polypeptides of the disclosure, or the recombinant nucleic acid constructs of the disclosure, can alter T cell signaling in various ways. In some embodiments, the altering of the T cell signaling can be by enhancing, promoting, improving, reducing, regulating, or modulating the signaling pathway within a T cell. In some embodiments, the altering of the T cell signaling can be by activating, increasing, suppressing, inhibiting, or other means of changing the signaling.

[0554] The polypeptides of the disclosure, or the recombinant nucleic acid constructs of the disclosure, can alter T cell signaling through one or more T cell signaling pathways. In some embodiments, the polypeptides or recombinant nucleic acid constructs of the disclosure can alter T cell signaling by increasing one or more pathways and / or reducing one or more pathways, or by a combination of enhancing and / or reducing various pathways, for example as shown in FIG 2F. In some embodiments, the mutation can alter cytokine production. The cytokine can be, without limitation, IL-2, IL-4, IL-5, TNF alpha, IFN- gamma, IL- 13 and / or other cytokines. In some embodiments, the cytokine is IL-2. In some embodiments, the cytokine is IL-2, and the production of IL-2 is increased.

[0555] In some embodiments, the polypeptides or recombinant nucleic acid constructs of the disclosure can alter T cell signaling through one or more T cell signaling pathways. Signaling pathways can be, without limitation, the NF AT pathway, NF-KB pathway, AP-1 pathway, JAK / STAT pathway, RAS / MEK / ERK, and / or phospholipase gamma signaling, or described elsewhere herein.

[0556] In some embodiments, the polypeptides or recombinant nucleic acid constructs of the disclosure can alter CARD11-BCL10-MALT1 complex signaling, co-stimulatory molecule signaling, cytokine production and / or transcription factor activity in T cells.

[0557] The disclosure also relates to polypeptides having one or more mutations that can reduce T cell exhaustion, increase proliferation, alter effector function, resist T cell dysfunction, increase T cell fitness, enhance in vivo persistence, and / or increase intratumoral presence of therapeutic T cells. The disclosure further relates to recombinant nucleic acids encoding polypeptides having one or more mutations, that can reduce T cell exhaustion, increase proliferation, alter effector function, resist T cell dysfunction, increase T cellfitness, enhance in vivo persistence, and / or increase intratumoral presence of therapeutic T cells.

[0558] T cell fitness can refer to the ability of a T cell to generate an immune response. The ability of a T cell to perform T cell functions such as signaling, cytokine production, survival, and persistence in a tumor, may contribute to the fitness of a T cell. T cell exhaustion may contribute to reducing its fitness.

[0559] In some embodiments, polypeptides of the disclosure, or the recombinant nucleic acid constructs of the disclosure can alter in vivo persistence in tumors of therapeutic T cells including the mutation. The in vivo persistence of therapeutic T cells can refer to the length of time that the therapeutic T cells exist within the tumor of the host after infusion. In some embodiments, in vivo persistence of therapeutic T cells is enhanced. Enhanced in vivo persistence of therapeutic T cells can include at least a positive log2 fold change in therapeutic T cells as compared to the input total.

[0560] In some embodiments, the polypeptides of the disclosure, or the recombinant nucleic acid constructs of the disclosure can alter the therapeutic efficacy of engineered T cells. Alteration of therapeutic efficacy can include, without limitation, a decrease in T cell exhaustion, increased proliferative capacity, enhanced anti-tumor effect, enhanced replicative lifespan, decreased replicative senescence, enhanced ability to kill, enhanced fitness of engineered T cells and / or other functions or activities of T cells. In some embodiments, the nucleic acid constructs of the disclosure encodes a polypeptide comprising an amino acid sequence having at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 3-150 or a variant thereof.

[0360] In some embodiments, the nucleic acid constructs of the disclosure include a nucleic acid sequence encoding a polypeptide with at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NOs: 3-150 or a variant thereof.IV. POLYNUCLEOTIDES

[0361] In some embodiments, the engineered polypeptides disclosed herein are encoded by a polynucleotide. In some embodiments, the polynucleotide of the disclosure is a recombinant nucleic acid / nucleic acid construct — for example, a plasmid construct or other recombinant constructs. In some embodiments, the polynucleotide is or comprises DNA. In some embodiments, the polynucleotide is or comprises cDNA. In someembodiments, the polynucleotide is or comprises RNA. In some embodiments, the polynucleotide is or comprises mRNA.

[0362] A polynucleotide encoding the engineered polypeptides disclosed herein can be obtained by methods known in the art. For example, the polynucleotide can be obtained from cloned DNA (e.g., from a DNA library), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA or fragments thereof, purified from the desired cell. When the polynucleotides are produced by recombinant means, any method known to those skilled in the art for identification of nucleic acids that encode desired genes can be used. Polynucleotides produced by recombinant means may also be referred to as “recombinant nucleic acids” herein. Any method available in the art can be used to obtain a full length (i.e. encompassing the entire coding region) cDNA or genomic DNA encoding a desired human CARD 11 protein and PIK3R3 protein, such as from a cell or tissue source. Modified or variant polynucleotides, including truncated forms of CARD11-PIK3R3 fusion proteins such as provided herein, can be engineered from a patient-derived polynucleotide using standard recombinant DNA methods. Polynucleotides can be cloned or isolated using any available methods known in the art for cloning and isolating nucleic acid molecules. Such methods include PCR amplification of nucleic acids and screening of libraries, including nucleic acid hybridization screening, antibody-based screening, and activity-based screening.

[0363] Methods for amplification of polynucleotides can be used to isolate polynucleotides encoding a desired protein, including for example, polymerase chain reaction (PCR) methods. PCR can be carried out using any known methods or procedures in the art. Exemplary methods include use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp). A nucleic acid containing gene of interest can be used as a source material from which a desired polypeptide-encoding nucleic acid molecule can be amplified. For example, DNA and mRNA preparations, cell extracts, tissue extracts from an appropriate source (e.g. testis, prostate, breast), fluid samples (e.g. blood, serum, saliva), samples from healthy and / or diseased subjects can be used in amplification methods. The source can be from any eukaryotic species including, but not limited to, vertebrate, mammalian, human, porcine, bovine, feline, avian, equine, canine, and other primate sources. Nucleic acid libraries also can be used as a source material. Primers can be designed to amplify a desired polynucleotide. For example, primers can be designed based on expressed sequences from which a desired polynucleotide is generated. Primers can be designed based on back- translation of a polypeptide amino acid sequence. If desired, degenerate primers can be used for amplification. Oligonucleotide primers that hybridize to sequences at the 3’ and 5’termini of the desired sequence can be uses as primers to amplify by PCR from a nucleic acid sample. Primers can be used to amplify the entire full-length polynucleotide, or a truncated sequence thereof. Nucleic acid molecules generated by amplification can be sequenced and confirmed to encode a desired polypeptide.

[0364] In some embodiments, the polynucleotide encoding the engineered polypeptides disclosed herein additionally encodes at least one additional polypeptide (e.g., a second polypeptide or a second recombinant polypeptide). In some embodiments, the nucleic acid construct further comprises a polynucleotide sequence encoding one or more additional polypeptides and / or a non-coding RNA. In some embodiments, the non-coding RNA comprises a shRNA or a microRNA. In some embodiments, the one or more additional polypeptides comprise a chimeric antigen receptor (CAR), a recombinant T cell receptor (TCR), an additional potency enhancement polypeptide, a cytokine, a chemokine, a growth factor, a safety switch, or any combination thereof. In some embodiments, the one or more additional polypeptides comprise a CAR or a recombinant TCR. In some embodiments, the CAR or TCR is any CAR or TCR described herein.

[0365] In some embodiments, the polynucleotide sequence encoding the recombinant polypeptide and the polynucleotide sequence encoding the at least one additional polypeptide are separated by a polynucleotide sequence encoding one or more cleavable linkers. In some embodiments, all these polynucleotide sequences form a continuous open reading frame. In some embodiments, the one or more cleavable linkers comprise a P2A, E2A, F2A, or T2A self-cleaving peptide.

[0366] In some embodiments, the polynucleotide sequence encoding the engineered polypeptide and the polynucleotide sequence encoding the at least one additional polypeptide are separated by an internal ribozyme entry site (IRES).

[0367] In some embodiments, the polynucleotide is a DNA. In some embodiments, the polynucleotide is an RNA. In some embodiments, the polynucleotide is a mRNA.

[0368] In some embodiments, the polynucleotide comprises one or more modified nucleotides. In some embodiments, the one or more modified nucleotides are or comprise pseudouridine, N1 -methylpseudouridine, 4’ -thiouridine, 5 -methylcytosine, 2-thio-l-methyl- 1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl- pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5- methoxyuridine, 2’-O-methyl uridine, or any combination thereof. In some embodiments,the one or more modified nucleotides are selected from the group consisting of pseudouridine, N1 -methylpseudouridine, 5-methylcytosine, 5- methoxyuridine, and a combination thereof. In some embodiments, the one or more modified nucleotides are Nl- methylpseudouridines.

[0369] The nucleic acid construct may further comprise homology arms before the 5’ end and / or after the 3’ end of the nucleic acid sequence encoding the recombinant polypeptide. In some embodiments, the nucleic acid construct comprises a first homology arm before the 5’ end of the nucleic acid sequence encoding the recombinant polypeptide and a second homology arm after the 3’ end of the nucleic acid sequence encoding the recombinant polypeptide. In some embodiments, the lengths of the two homology arms are between about 150 bp and about 1500 bp. In some embodiments, the length of at least one of the homology arms is about 200bp, 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, or about 500 bp. In some embodiments, the lengths of both of the homology arms are about 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, about 500 bp, about 525 bp, about 550 bp, about 575 bp, or about 600 bp. In some embodiments, the total length of the homology arms is between about 400 bp and about 500 bp, about 500 bp and about 600 bp, about 600 bp and about 700 bp, about 700 bp and about 800 bp, about 800 bp and about 900 bp, about 900 bp and about 1000 bp, about 1000 bp and about 1100 bp, or about 1100 bp and about 1200 bp.

[0370] In some embodiments, the recombinant nucleic acid is less than 10 kb, less than 9 kb, less than 8 kb, less than 7 kb, less than 6 kb, less than 5 kb, less than 4 kb, or less than 3 kb in length. In some embodiments, the recombinant nucleic acid is less than about 5.0kb in length. In some embodiments, the recombinant nucleic acid is less than about 4.7kb in length.

[0371] In some embodiments, the length of the polynucleotide sequence encoding the one or more additional polypeptides is less than about 2100 bp, less than about 2150 bp, less than about 2200 bp, less than about 2250 bp, less than about 2300 bp, less than about 2350 bp, or less than about 2400 bp. In some embodiments, the length of the one or more additional polypeptides is less than about 700 amino acids, less than about 725 amino acids, less than about 750 amino acids, less than about 775 amino acids, less than about 800 amino acids, less than about 825 amino acids, less than about 850 amino acids, less than about 875 amino acids, or less than about 900 amino acids.

[0372] In some embodiments, the polynucleotide encoding the engineered peptides disclosed herein is comprised with a plasmid. In some embodiments, the plasmid is between about 2 kilobases (kb) and about 10 kb in size, or between about 3 kb and about 8 kb in size. In some embodiments, the plasmid is less than about 8 kb, less than about 7 kb, less than about 6 kb, less than about 5 kb, or less than about 4.5 kb in size. In some embodiments, the plasmid is less than about 8 kb in size. In some embodiments, the plasmid is less than about7.5 kb in size. In some embodiments, the plasmid is less than about 7 kb in size. In some embodiments, the plasmid is less than about 6.5 kb in size. In some embodiments, the plasmid is less than about 6 kb in size. In some embodiments, the plasmid is less than about5.5 kb in size. In some embodiments, the plasmid is less than about 5 kb in size. In some embodiments, the plasmid is less than about 4.5 kb in size. In some embodiments, the plasmid is about 4.5 kb in size. In some embodiments, the plasmid is about 5 kb in size. In some embodiments, the plasmid is about 5.5 kb in size. In some embodiments, the plasmid is about 6.5 kb in size. In some embodiments, the plasmid is about 7 kb in size. In some embodiments, the plasmid is about 6 kb in size. In some embodiments, the plasmid is about7.5 kb in size. In some embodiments, the plasmid is about 8 kb in size.

[0373] In some embodiments, the polynucleotide encoding the polynucleotide encoding the engineered CARD11-PIK3R3 fusion protein contains at least one promoter that is operatively linked to control expression of the polynucleotide encoding the engineered CARD1 1-PIK3R3 fusion protein. In some examples, the polynucleotide contains two, three, or more promoters operatively linked to control expression of the polynucleotide encoding the engineered CARD11-PIK3R3 fusion protein. In some embodiments, polynucleotide can contain regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the polynucleotide is to be introduced, as appropriate and taking into consideration whether the polynucleotide is DNA- or RNA-based. In some embodiments, the polynucleotide can contain regulatory / control elements, such as a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, internal ribosome entry sites (IRES), a 2A sequence, and splice acceptor or donor. In some embodiments, the polynucleotide can contain a non-native promoter operably linked to the nucleotide sequence encoding the polynucleotide encoding the engineered CARD11-PIK3R3 fusion protein and / or one or more additional polypeptide(s). In some embodiments, the promoter is selected from among an RNA pol I, pol II or pol III promoter. In some embodiments, the promoter is recognized by RNA polymerase II (e.g., a CMV, SV40 early region oradenovirus major late promoter). In another embodiment, the promoter is recognized by RNA polymerase III (e.g., a U6 or Hl promoter). In some embodiments, the promoter can be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus. Other known promoters also are contemplated.

[0374] In some embodiments, the promoter is or comprises a constitutive promoter. Exemplary constitutive promoters include, MND promoter, EFla promoter, sEFl promoter, gamma retroviral LTR promoter, CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, CARD9 promoter, CARDIO promoter, CARD 11 promoter, CARD 14 promoter, PIK3R3 promoter, MND promoter or a short EFl promoter.

[0375] In some embodiments, the promoter is a minimal TATA promoter, a pGK, actin promoter, CD25 promoter, IL2 promoter, IL7 promoter, IL 15 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD1 la promoter, CD58 promoter, CD99 promoter, CD62L promoter, CD 103 promoter, CCR4 promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter, CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme A promoter, Granzyme B promoter, Perforin promoter, CD57 promoter, CD161 promoter, IL-18Ra promoter, CD69 promoter, GzmB promoter, T-bet promoter, IFNgamma promoter, TIM3 promoter, IL4 promoter, GAT A3 promoter, IL1 promoter, IL5 promoter, IL6 promoter, IL 13 promoter, IL 10 promoter, IL17A promoter, IL6 promoter, IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25 promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter, CD95 promoter, CD28 promoter, CD27 promoter, CD 127 promoter, PD-1 promoter, CD122 promoter, CD132 promoter, c-Kit promoter, nuclear factor of activated T cells (NF AT) promoter, programmed death 1 (PD-1) promoter, T cell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, lymphocyte-activation protein 3 (LAG-3) promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, TGF-beta promoter, T-bet promoter, Eomes promoter, GATA3 promoter, CD45RA promoter, 2B4 promoter, Type I interferon (IFN) alpha, Type I IFN beta promoter, IFN gamma promoter, IRF3 promoter, IRF7 promoter, NF-KB promoter, AP-1 promoter, TNF- alpha promoter, and CD 130 promoter, NR4A1 promoter, NR4A2, NR4A3 promoter, MND promoter, EF-1 alpha promoter, short EF-1 alpha promoter, CAG promoter, ubiquitin / S27apromoter, SV40 promoter, SV40 early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, Moloney murine leukemia virus (MMLV) long terminal repeat (LTR) region, CMV promoter, immunoglobulin promoter, heat shock promoter, polyoma virus promoter, fowlpox virus promoter, bovine papilloma virus promoter, avian sarcoma virus promoter, retrovirus promoter, hepatitis-B virus promoter, PGK promoter, vaccinia virus 7.5K promoter, TK promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of moloney virus, Epstein Barr virus (EBV) promoter, Rous sarcoma virus (RSV) promoter, U6 promoter, or UBC promoter. Exemplary constitutive promoters include, e.g., simian virus 40 early promoter (SV40), cytomegalovirus immediate-early promoter (CMV), human Ubiquitin C promoter (UBC), human elongation factor la promoter (EFla), mouse phosphoglycerate kinase 1 promoter (PGK), and chicken P-Actin promoter coupled with CMV early enhancer (CAG). In some embodiments, the constitutive promoter is a synthetic or modified promoter. In some embodiments, the promoter is or comprises an MND promoter, a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer (see Challita et al. (1995) J. Virol. 69(2): 748-755). In some embodiments, the promoter is a tissue-specific promoter. In another embodiment, the promoter is a viral promoter. In another embodiment, the promoter is a non-viral promoter. In some embodiments, exemplary promoters can include, but are not limited to, human elongation factor 1 alpha (EFla) promoter or a modified form thereof or the MND promoter.

[0376] In another embodiment, the promoter is a regulated promoter (e.g., inducible promoter). In some embodiments, the promoter is an inducible promoter or a repressible promoter. In some embodiments, the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence or a doxycycline operator sequence, or is an analog thereof or is capable of being bound by or recognized by a Lac repressor or a tetracycline repressor, or an analog thereof. In some embodiments, the recombinant nucleic acid does not include a regulatory element, e.g. promoter. In some embodiments, the polynucleotide sequence encoding the recombinant polypeptide is not operably linked to a promoter.

[0377] In some embodiments, the polynucleotide sequence further comprises a polynucleotide sequence encoding one or more cleavable linkers. Non-limiting examples of cleavable linkers include P2A, E2A, F2A, and T2A self-cleaving peptides. In some embodiments, the self-cleaving peptide comprises a sequence at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to any one of SEQ ID NO: 376-379.

[0378] In some embodiments, the recombinant nucleic acid further comprises one or more additional nucleic acid coding or regulatory sequences. Non-limiting examples of control sequences are ribosomal binding sites, enhancer elements, activator elements, translational start sequences, translational termination sequences, transcription start sequences, transcription termination sequences, polyadenylation signal sequences, a 70 bp poly(A) tract, a 100 bp poly(A) tract, a 172 bp poly(A) tract, a 200 bp poly(A) tract, a 300 bp poly(A) tract, a 325 bp poly(A) tract, replication elements, RNA processing and export elements, transposon sequences, transposase sequences, insulator sequences, internal ribosome entry sites (IRES), 5’UTRs, 3’UTRs, mRNA 3’ end processing sequences, boundary elements, locus control regions (LCR), matrix attachment regions (MAR), recombination or cassette exchange sequences, linker sequences, cleavable linker sequences, secretion signals, resistance markers, anchoring peptides, localization signals, fusion tags, affinity tags, chaperonins, proteases, or any combination thereof.

[0379] In some cases, the nucleic acid sequence encoding the polypeptide disclosed herein contains a signal sequence that encodes a signal peptide. In some aspects, the signal sequence may encode a signal peptide derived from a native polypeptide. In other aspects, the signal sequence may encode a heterologous or non-native signal peptide, such as the exemplary signal peptide of a GMCSFR alpha chain. In some cases, the signal peptide is comprised within the polypeptide.

[0380] In some embodiments, the polynucleotide sequence further comprises a polynucleotide sequence encoding one or more additional polypeptides and / or a non-coding RNA, optionally wherein the non-coding RNA comprises a shRNA or a microRNA. In some embodiments, the one or more additional polypeptides comprise an additional potency enhancement polypeptide, a tolerogenic factor, a cytokine, a chemokine, a growth factor, or any combination thereof. Non-limiting examples of additional potency enhancement polypeptides include a dominant negative form of an inhibitor of a cell-mediated immune response of the immune cell (e.g., TGFPR2 DNR), c-Jun, CCL19, CCL21, IL2R, IL7, IL7Ralpha, IL15, IL15RA, IL18, decoy-resistant IL18 (DR-18), MyD88 / CD40, PD1-CD28 switch receptor, PD1-41BB switch receptor, CD40L-CD28 switch receptor, CTBR12 switch receptor, or CD8alpha / beta.

[0381] In some embodiments, the tolerogenic factor is or comprises A20 / TNFAIP3, B2M- HLA-E, CD16, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD47, CD52, CD55,CD59, CD64, CD200, CCL21, CCL22, CTLA4-Ig, Cl inhibitor, CR1, DUX4, FASL, HLA- C, HLA-E, HLA-E heavy chain, HLA-F, HLA-G, H2-M3, ID01, IL-10, IL15-RF, IL-35, IL-39, MANF, Mfge8, PD-L1, Serpinb9, or any combination thereof.

[0382] In some embodiments, the polynucleotide contains a nucleic acid sequence encoding one or more additional polypeptides, e.g., one or more marker(s) and / or one or more effector molecules. In some embodiments, the disclosure provides a combination of polynucleotides, in which the engineered polypeptides are encoded by separate polynucleotides. In some embodiments, the one or more marker(s) includes a transduction marker, a surrogate marker and / or a selection marker. Among additional nucleic acid sequences introduced, e.g., encoding for one or more additional polypeptide(s), include nucleic acid sequences that encode a chimeric antigen receptor or heterologous TCR, nucleic acid sequences that can improve the efficacy of therapy, such as by promoting viability and / or function of transferred cells; nucleic acid sequences to provide a genetic marker for selection and / or evaluation of the cells, such as to assess in vivo survival or localization; nucleic acid sequences to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11 :6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also WO 1992008796 and WO 1994028143 describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker, and US Patent No. 6,040,177.

[0383] Any of the polynucleotide encoding the engineered CARD11-PIK3R3 fusion proteins and / or the additional polypeptide(s) described herein can be encoded by one or more polynucleotides containing one or more nucleic acid sequences encoding polynucleotide encoding the engineered CARD11-PIK3R3 fusion proteins, in any combinations, orientation or arrangements. For example, one, two, three or more polynucleotides can encode one, two, three or more different polypeptides, e.g., polynucleotide encoding the engineered CARD11-PIK3R3 fusion proteins or portions or components thereof, and / or one or more additional polypeptide(s), e.g., a marker and / or an effector molecule. In some embodiments, one polynucleotide contains a nucleic acid sequence encoding a polynucleotide encoding the engineered CARD11-PIK3R3 fusion protein, or portion or components thereof, and a nucleic acid sequence encoding one or more additional polypeptide(s). In some embodiments, one vector or construct contains a nucleic acid sequence encoding a polynucleotide encoding the engineered CARD11-PIK3R3 fusion protein, or portion or components thereof, and a separate vector or construct contains anucleic acid sequence encoding one or more additional polypeptide(s). In some embodiments, the nucleic acid sequence encoding the polynucleotide encoding the engineered CARD11-PIK3R3 fusion protein and the nucleic acid sequence encoding the one or more additional polypeptide(s) are operably linked to two different promoters. In some embodiments, the nucleic acid encoding the polynucleotide encoding the engineered CARD1 1-PIK3R3 fusion protein is present upstream of the nucleic acid encoding the one or more additional polypeptide(s). In some embodiments, the nucleic acid encoding the polynucleotide encoding the engineered CARD11-PIK3R3 fusion protein is present downstream of the nucleic acid encoding one or more additional polypeptide(s).

[0384] In some embodiments, polynucleotides can be engineered as a bicistronic unit containing an IRES, which allows coexpression of gene products (e.g. encoding the recombinant receptor and the additional polypeptide) by a message from a single promoter. Alternatively, in some cases, a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three genes (e.g. encoding the marker and encoding the recombinant receptor) separated from one another by sequences encoding a self-cleavage peptide (e.g., 2A sequences) or a protease recognition site (e.g., furin). The ORF thus encodes a single polypeptide, which, either during (in the case of 2A) or after translation, is processed into the individual proteins. In some cases, the peptide, such as a T2A, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream (see, e.g., de Felipe, Genetic Vaccines and Ther. 2: 13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)). Various 2A elements are known. Examples of 2A sequences that can be used in the methods and system disclosed herein, without limitation, 2A sequences from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A), and porcine teschovirus-1 (P2A) as described in U.S. Patent Pub. No. 20070116690.

[0385] In some embodiments, the polynucleotide comprises one or more modified nucleotides. Non-limiting examples of modified nucleotides include pseudouridine, Nl- methylpseudouridine, 4’ -thiouridine, 5-methylcytosine, 2-thio-l-methyl-l -deazapseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl- pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5- methoxyuridine, 2’-O-methyl uridine. In some embodiments, the one or more modifiednucleotides are selected from the group consisting of pseudouridine, Nl- methylpseudouridine, 5-methylcytosine, 5- methoxyuridine, and a combination thereof. In some embodiments, the one or more modified nucleotides are Nl- methylpseudouridines.

[0386] In some aspects, nucleic acid sequences of interest, including coding and / or noncoding sequences and / or partial coding sequences, that are inserted or integrated at the target location in the genome can also be referred to as “transgene,” “transgene sequences,” “exogenous nucleic acids sequences,” “heterologous sequences” or “donor sequences.” In some aspects, the transgene is a nucleic acid sequence that is exogenous or heterologous to an endogenous genomic sequences, such as the endogenous genomic sequences at a specific target locus or target location in the genome, of a T cell, e.g., a human T cell. In some aspects, the transgene is a sequence that is modified or different compared to an endogenous genomic sequence at a target locus or target location of a T cell, e.g., a human T cell. In some aspects, the transgene is a nucleic acid sequence that originates from or is modified compared to nucleic acid sequences from different genes, species and / or origins. In some aspects, the transgene is a sequence that is derived from a sequence from a different locus, e.g., a different genomic region or a different gene, of the same species.

[0387] Polynucleotides for insertion can also be referred to as “transgene” or “exogenous sequences” or “donor” polynucleotides or molecules. The template polynucleotide can be DNA, single-stranded and / or double-stranded and can be introduced into a cell in linear or circular form. The template polynucleotide can be RNA single-stranded and / or doublestranded and can be introduced as a RNA molecule (e.g., part of an RNA virus). See also, U.S. Patent Publication Nos. 20100047805 and 20110207221. The template polynucleotide can also be introduced in DNA form, which may be introduced into the cell in circular or linear form. If introduced in linear form, the ends of the template polynucleotide can be protected (e.g., from exonucleolytic degradation) by known methods. For example, one or more dideoxynucleotide residues are added to the 3’ terminus of a linear molecule and / or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. If introduced in doublestranded form, the template polynucleotide may include one or more nuclease target site(s),for example, nuclease target sites flanking the transgene to be integrated into the cell’s genome. See, e.g., U.S. Patent Publication No. 20130326645.V. VECTORS

[0388] In another aspect, the present disclosure provides a vector comprising the polynucleotide that encodes the engineered polypeptides disclosed herein. In some embodiments, the vector can be used to transform or transfect a host cell, e.g., a cell for engineering. Exemplary vectors include vectors designed for introduction, propagation and expansion or for expression or both, such as DNA vectors (e.g., a plasmid or closed-end vector) and viral vectors. In some aspects, the vector is an expression vector, e.g., a recombinant expression vector. In some embodiments, the recombinant expression vectors can be prepared using standard recombinant DNA techniques.

[0389] The nucleic acid molecules can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transformed / transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available, or readily prepared by a skilled artisan. See for example, Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D.W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference).

[0390] Any methods known to those skilled in the art for the insertion of nucleic acid fragments into a vector can be used to construct expression vectors comprising a polynucleotide disclosed herein. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo (genetic) recombination. The polynucleotide disclosed herein can be operably linked to control sequences in the expression vector(s) to ensure the expression of the polypeptide of the disclosure. Such control sequences may include, but are not limited to, leader or signal sequences, promoters (e.g., naturally associated or heterologous promoters), ribosomal binding sites, enhancer or activator elements, translational start and termination sequences, and transcription start and termination sequences, and are chosen to be compatible with the host cell chosen to express the polypeptide of the disclosure. Constitutive or inducible promoters as known in the art are also contemplated. The promoters may be either naturally occurring promoters, hybrid promoters that combine elements of more than one promoter, or synthetic promoters. An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome such as in a gene locus. In some embodiment, the expression vector includes a selectable marker gene to allow the selection of transformed host cells. Some embodiments, include an expression vector comprising a nucleotide sequence encoding a variant polypeptide operably linked to at least one regulatory control sequence. Regulatory control sequence for use herein include promoters, enhancers, and other expression control elements. In some embodiments, an expression vector is designed for the choice of the host cell to be transformed, the particular variant polypeptide desired to be expressed, the vector's copy number, the ability to control that copy number, and / or the expression of any other protein encoded by the vector, such as antibiotic markers.

[0391] Examples of suitable mammalian promoters include, for example, MND promoter, EFla promoter, sEFl promoter, gamma retroviral LTR promoter, CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, CD3z promoter, MND promoter or a short EFl promoter. In some embodiments, the promoter is a minimal TATA promoter, a pGK, actin promoter, CD25 promoter, IL2 promoter, IL7 promoter, IL 15 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CDl la promoter, CD58 promoter, CD99 promoter, CD62L promoter, CD 103 promoter, CCR4 promoter, CCR5 promoter, CCR6 promoter, CCR9 promoter, CCR10 promoter, CXCR3 promoter, CXCR4 promoter, CLA promoter, Granzyme A promoter, Granzyme B promoter, Perforin promoter,CD57 promoter, CD161 promoter, IL-18Ra promoter, CD69 promoter, GzmB promoter, T- bet promoter, IFNgamma promoter, TIM3 promoter, IL4 promoter, GAT A3 promoter, IL1 promoter, IL5 promoter, IL6 promoter, IL 13 promoter, IL 10 promoter, IL17A promoter, IL6 promoter, IL21 promoter, IL23R promoter, FoxP3 promoter, CTLA4 promoter, CD25 promoter, PD1 promoter, CD45RO promoter, CCR7 promoter, CD28 promoter, CD95 promoter, CD28 promoter, CD27 promoter, CD 127 promoter, PD-1 promoter, CD 122 promoter, CD 132 promoter, c-Kit promoter, nuclear factor of activated T cells (NF AT) promoter, programmed death 1 (PD-1) promoter, T cell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, lymphocyte-activation protein 3 (LAG-3) promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter, B- and T-lymphocyte attenuator (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, TGF-beta promoter, T-bet promoter, Eomes promoter, GATA3 promoter, CD45RA promoter, 2B4 promoter, Type I interferon (IFN) alpha, Type I IFN beta promoter, IFN gamma promoter, IRF3 promoter, IRF7 promoter, NF-KB promoter, AP-1 promoter, TNF-alpha promoter, and CD130 promoter, NR4A1 promoter, NR4A2, or NR4A3 promoter. Exemplary constitutive promoters include, e.g., simian virus 40 early promoter (SV40), cytomegalovirus immediate- early promoter (CMV), human Ubiquitin C promoter (UBC), human elongation factor la promoter (EFla), mouse phosphoglycerate kinase 1 promoter (PGK), and chicken P-Actin promoter coupled with CMV early enhancer (CAG). In some embodiments, the constitutive promoter is a synthetic or modified promoter. In some embodiments, the promoter is or comprises an MND promoter, a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer (see Challita et al. (1995) J. Virol. 69(2):748-755). In some embodiments, the promoter is a tissue-specific promoter. In another embodiment, the promoter is a viral promoter. In another embodiment, the promoter is a non-viral promoter. In some embodiments, exemplary promoters can include, but are not limited to, human elongation factor 1 alpha (EFla) promoter or a modified form thereof or the MND promoter.

[0392] Examples of suitable mammalian promoters include, for example: TRAC promoter, TRBC1 promoter, TRBC2 promoter, CD3alpha promoter, CD3beta promoter, CD3epsilon promoter, CD3delta promoter, CD3zeta promoter, CARD11 promoter, PIK3R3 promoter, B2M promoter, CIITA promoter, PD-1 promoter, CXCR4 promoter, an albumin promoter, an F3 promoter (also known as CD142), a MICA promoter, a MICB promoter, a LRP1 promoter (also known as CD91), a HMGB1 promoter, an ABO promoter, a RHD promoter,a FUT1 promoter, a KDM5D promoter (also known as HY), a CCR5 promoter, a PDGFRa promoter, a OLIG2 promoter, and / or a GFAP promoter, elongation factor 1 alpha (EFla) promoter, CAG promoter, ubiquitin / S27a promoter of the hamster (WO 97 / 15664), Simian vacuolating virus 40 (SV40) early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, the long terminal repeat region of Rous Sarcoma Virus (RSV), mouse mammary tumor virus promoter (MMTV), Moloney murine leukemia virus Long Terminal repeat region, and the early promoter of human Cytomegalovirus (CMV). Examples of other heterologous mammalian promoters are the actin, immunoglobulin or heat shock promoter(s). In additional embodiments, promoters for use in mammalian host cells can be obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40). In further embodiments, heterologous mammalian promoters are used. Examples include the actin promoter, an immunoglobulin promoter, and heat-shock promoters. The early and late promoters of SV40 are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature 273: 113-120 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll restriction enzyme fragment (Greenaway et al., Gene 18: 355-360 (1982)). The foregoing references are incorporated by reference in their entirety.

[0393] In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector that carries the polypeptide of the disclosure. In some embodiments, the vector is a selfinactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope, and which carries the polynucleotide encoding the polypeptide of the disclosure.

[0394] In some of any embodiments, the viral vector is an AAV vector that carries the polypeptide of the disclosure. In some of any embodiments, the AAV vector is an AAV6 vector that carries the encoding the polypeptide of the disclosure.

[0395] The vector can include, but is not limited to, viral vectors, plasmid DNA, and closed- end DNA. Viral vectors can include, but are not limited to, adenoviral vectors, lentiviral vectors, retroviral vectors, adeno-associated viral vectors, vaccinia viruses, poxviruses, and herpes simplex viruses, or any of the viruses described elsewhere herein. Commonly, expression vectors contain selection markers such as ampicillin-resistance, hygromycin-resi stance, tetracycline resistance, kanamycin resistance, or neomycin resistance to permit detection of those cells transformed with the desired DNA sequences. Suitable vectors, promoter, and enhancer elements are known in the art; many are commercially available forgenerating subject recombinant constructs. In some embodiments, the vector is a polycistronic vector. In some embodiments, the vector is a bicistronic vector or a tricistronic vector. Bicistronic or multi ci str onic expression vectors may include (1) multiple promoters fused to each of the open reading frames; (2) insertion of splicing signals between genes; (3) fusion of genes whose expressions are driven by a single promoter; and (4) insertion of proteolytic cleavage sites between genes (self-cleavage peptide) or insertion of internal ribosomal entry sites (IRESs) between genes.

[0396] A polycistronic vector is used to co-express multiple genes in the same cell. Two strategies are most commonly used to construct a multi cistronic vector. First, an Internal Ribosome Entry Site (IRES) element is typically used for bi-cistronic vectors. The IRES element, acting as another ribosome recruitment site, allows initiation of translation from an internal region of the mRNA. Thus, two proteins are translated from one mRNA. IRES elements are quite large (usually 500-600 bp) (Pelletier et al., 1988; Jang et al., 1988). In some embodiments, the polypeptide of the disclosure disclosed herein have a smaller size compared to the endogenous or patient-derived counterpart polypeptides, and thus could be expressed using IRES element in a multi cistronic vectors having limited packaging capacity.

[0397] In some embodiments, the polycistronic construct comprises two expression cassettes, i.e., is bicistronic. In some embodiments, the polycistronic construct comprises three expression cassettes, i.e., is tricistronic. In some embodiments, the polycistronic construct comprises four expression cassettes, i.e., is quadci stronic. In some embodiments, the polycistronic construct comprises more than four expression cassettes. In any of these embodiments, each of the expression cassettes comprises a nucleotide sequence encoding a protein of interest (e.g., a polypeptide of the disclosure and / or a CAR). In certain embodiments, the two or more genes being expressed are under the control of a single promoter and are separated from one another by one or more cleavage sites to achieve coexpression of the proteins of interest from one transcript. In other embodiments, the two or more genes may be under the control of separate promoters. As the name suggests, a polycistronic construct allows simultaneous expression of two or more separate proteins from one mRNA transcript in a host cell. Cleavage sites can be used in the design of a polycistronic construct to achieve such co-expression of multiple genes.

[0398] In some embodiments, the one or more cleavage sites comprise one or more selfcleaving sites. The second strategy relies on “self-cleaving” 2A peptides. These peptides, first discovered in picomaviruses, are short (about 20 amino acids) and produce equimolarlevels of multiple genes from the same mRNA. The term "self-cleaving" is not entirely accurate, as these peptides are thought to function by making the ribosome skip the synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream (Kim et al., 2011). The "cleavage" occurs between the glycine and proline residues found on the C-terminus. Thus, the upstream cistron will have a few additional residues added to the end, while the downstream cistron will start with the proline. There are four 2A peptides commonly employed in molecular biology, T2A, P2A, E2A, and F2A, the sequences of which are summarized in Table 4. A glycine-serine-glycine (GSG) linker is optionally added to the N-terminal of a 2A peptide to increase cleavage efficiency. The use of “()” around a sequence in the present disclosure means that the enclosed sequence is optional.Table 4. Sequences of 2A Peptides

[0399] In some embodiments, the one or more cleavage sites additionally comprise one or more protease sites. The one or more protease sites can either precede or follow the selfcleavage sites (e.g., 2 A sites) in the 5’ to 3’ order. The protease site may be cleaved by a protease after translation of the full transcript or after translation of each expression cassette such that the first expression product is released prior to translation of the next expression cassette. In these embodiments, having a protease site in addition to the 2A site, especially preceding the 2 A site in the 5’ to 3’ order, may reduce the number of extra amino acid residues attached to the expressed proteins of interest. In some embodiments, the protease site comprises a furin site, also known as a Paired basic Amino acid Cleaving Enzyme (PACE) site. There are at least three furin cleavage sequences, FC1, FC2, and FC3, the amino acid sequences of which are summarized in Table 5. Similar to the 2A sites, one or more optional glycine-serine-glycine (GSG) sequences can be included for cleavage efficiency.Table 5. Sequences of Turin Sites

[0400] In some embodiments, the one or more cleavage sites comprise one or more selfcleaving sites, one or more protease sites, and / or any combination thereof. For example, the cleavage site can include a 2A site alone. For another example, the cleavage site can include a FC2 or FC3 site, followed by a 2A site. In these embodiments, the one or more selfcleaving sites may be the same or different. Similarly, the one or more protease sites may be the same or different.

[0401] In some embodiments, the polycistronic construct may be in the form of a vector. Any type of vector suitable for introduction of nucleotide sequences into a host cell can be used, including, for example, plasmids, adenoviral vectors, adenoviral-associated vectors, retroviral vectors, lentiviral vectors, phages, and homology-directed repair (HDR)-based donor vectors.

[0402] In some embodiments, the vector herein is a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule, including into the cell or into the genome of a cell. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell or may include sequences sufficient to allow integration into host cell DNA. In some embodiments, the vector herein is a plasmid. In some embodiments, the vector herein is a DNA vector. In some embodiments, the vector herein is a closed linear DNA vector. In some embodiments, the vector is a synthetic RNA vector. In some embodiments, the vector herein is a phagemid vector. In some embodiments, the vector herein is a synthetic DNA vector. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, e.g., replication defective retroviruses and lentiviruses. Non-viral vectors may require a delivery vehicle to facilitate entry of the nucleic acid molecule into a cell.

[0403] A viral vector can comprise a nucleic acid molecule that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule orintegration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles will typically include various viral components and sometimes also host cell components in addition to nucleic acid(s). A viral vector can comprise, e.g., a virus or viral particle capable of transferring a nucleic acid into a cell, or to the transferred nucleic acid (e.g., as naked DNA). Viral vectors and transfer plasmids can comprise structural and / or functional genetic elements that are primarily derived from a virus. A retroviral vector can comprise a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus.

[0404] In some embodiments, the vector is a viral vector, such as a retroviral vector. In some embodiments, the polynucleotide encoding the polypeptide of the disclosure are introduced into the cell via retroviral or lentiviral vectors, or via transposons (see, e.g., Baum et al. (2006) Molecular Therapy: The Journal of the American Society of Gene Therapy. 13: 1050-1063; Frecha et al. (2010) Molecular Therapy 18: 1748-1757; and Hackett et al. (2010) Molecular Therapy 18:674-683).

[0405] In some vectors described herein, at least part of one or more protein coding regions that contribute to or are essential for replication may be absent compared to the corresponding wild-type virus. This makes the viral vector replication-defective. In some embodiments, the vector is capable of transducing a target non-dividing host cell and / or integrating its genome into a host genome.

[0406] In some embodiments, the vector is a retroviral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is a VSV-G pseudotyped lentivirus. In some embodiments, the vector is an AAV vector. In some embodiments, the vector is an AAV6 vector. In some embodiments, the vector is an AAV9 vector. In some embodiments, the vector is a split-intein dual AAV vector. In some embodiments, the vector is a redirected lentiviral vector. In some embodiments, the vector is a fusosome. In some embodiments, the vector is a lentiviral particle engineered with the anti-CD3 Cocal glycoprotein. In some embodiments, the vector is an enveloped delivery vehicle. In some embodiments, the vector is a self-replicating RNA virus. In some embodiments, the vector is an mRNA-packaging virus-like particle. In some embodiments, the vector is RNP-packaging virus-like particle. In some embodiments, the vectors used in the context of this disclosure are the vectors described in WO2023193015A1; WO2022164935A1; Raguram, Aditya, Samagya Banskota, and David R. Liu. "Therapeutic in vivo delivery of gene editing agents." Cell (2022); Hamilton, Jennifer R., et al. "In vivo human T cell engineering withenveloped delivery vehicles." Nature Biotechnology (2024): 1-9; Lundstrom, Kenneth. "Self-replicating RNA viruses for RNA therapeutics." Molecules 23.12 (2018): 3310.

[0407] In some embodiments, the retroviral nucleic acid comprises one or more of (e.g., all of): a 5’ promoter (e.g., to control expression of the entire packaged RNA), a 5’ LTR (e.g., that includes R (polyadenylation tail signal) and / or U5 which includes a primer activation signal), a primer binding site, a psi packaging signal, a RRE element for nuclear export, a promoter directly upstream of the transgene to control transgene expression, a transgene (or other exogenous agent element), a polypurine tract, and a 3’ LTR (e.g., that includes a mutated U3, a R, and U5). In some embodiments, the retroviral nucleic acid further comprises one or more of a cPPT, a WPRE, and / or an insulator element (e.g., as described in Browning et al., “Insulators to Improve the Safety of Retroviral Vectors for HIV Gene Therapy,” Biomedicines, 4(1):4 (2016)).

[0408] A retrovirus typically replicates by reverse transcription of its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. The structure of a wild-type retrovirus genome often comprises a 5' long terminal repeat (LTR) and a 3' LTR, between or within which are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome and gag, pol and env genes encoding the packaging components which promote the assembly of viral particles. More complex retroviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell. In the provirus, the viral genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are involved in proviral integration and transcription. LTRs also serve as enhancerpromoter sequences and can control the expression of the viral genes. Encapsidation of the retroviral RNAs occurs by virtue of a psi sequence located at the 5' end of the viral genome.

[0409] The LTRs themselves are typically similar (e.g., identical) sequences that can be divided into three elements, which are called U3, R and U5. U3 is derived from the sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA and U5 is derived from the sequence unique to the 5' end of the RNA. The sizes of the three elements can vary considerably among different retroviruses.

[0410] For the viral genome, the site of transcription initiation is typically at the boundary between U3 and R in one LTR and the site of poly (A) addition (termination) is at the boundary between R and U5 in the other LTR. U3 contains most of the transcriptionalcontrol elements of the provirus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins. Some retroviruses comprise any one or more of the following genes that code for proteins that are involved in the regulation of gene expression: tot, rev, tax and rex.

[0411] With regard to the structural genes gag, pol and env themselves, gag encodes the internal structural protein of the virus. Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid). The pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (IN), which mediate replication of the genome. The env gene encodes the surface (SU) glycoprotein and the transmembrane (TM) protein of the virion, which form a complex that interacts specifically with cellular receptor proteins. This interaction promotes infection, e.g., by fusion of the viral membrane with the cell membrane.

[0412] In a replication-defective retroviral vector genome gag, pol and env may be absent or not functional. The R regions at both ends of the RNA are typically repeated sequences. U5 and U3 represent unique sequences at the 5' and 3' ends of the RNA genome respectively. Retroviruses may also contain additional genes which code for proteins other than gag, pol and env. Examples of additional genes include (in HIV), one or more of vif, vpr, vpx, vpu, tat, rev and nef. El AV has (amongst others) the additional gene S2.

[0413] Illustrative retroviruses suitable for use in particular embodiments, include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV) and human immunodeficiency virus (HIV).

[0414] In some embodiments the retrovirus is a Gammretrovirus. In some embodiments the retrovirus is an Epsilonretrovirus. In some embodiments the retrovirus is an Alpharetrovirus. In some embodiments the retrovirus is a Betaretrovirus. In some embodiments the retrovirus is a Deltaretrovirus. In some embodiments the retrovirus is a Spumaretrovirus. In some embodiments the retrovirus is an endogenous retrovirus. In some embodiments the retrovirus is a lentivirus.

[0415] In some embodiments, a retroviral or lentivirus vector further comprises one or more insulator elements, e.g., an insulator element described in Browning et al., “Insulators to Improve the Safety of Retroviral Vectors for HIV Gene Therapy,” Biomedicines, 4(1):4 (2016). In various embodiments, the vectors comprise a promoter operably linked to apolynucleotide encoding an exogenous agent. The vectors may have one or more LTRs, wherein either LTR comprises one or more modifications, such as one or more nucleotide substitutions, additions, or deletions. The vectors may further comprise one of more accessory elements to increase transduction efficiency (e.g., a cPPT / FLAP), viral packaging (e.g., a Psi (Y) packaging signal, RRE), and / or other elements that increase exogenous gene expression (e.g., poly (A) sequences), and may optionally comprise a WPRE or HPRE. In some embodiments, a lentiviral nucleic acid comprises one or more of, e.g., all of, e.g., from 5’ to 3’, a promoter (e.g., CMV), an R sequence (e.g., comprising TAR), a U5 sequence (e.g., for integration), a PBS sequence (e.g., for reverse transcription), a DIS sequence (e.g., for genome dimerization), a psi packaging signal, a partial gag sequence, an RRE sequence (e.g., for nuclear export), a cPPT sequence (e.g., for nuclear import), a promoter to drive expression of the exogenous agent, a gene encoding the exogenous agent, a WPRE sequence (e.g., for efficient transgene expression), a PPT sequence (e.g., for reverse transcription), an R sequence (e.g., for polyadenylation and termination), and a U5 signal (e.g., for integration).

[0416] Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In some embodiments, HIV based vector backbones (i.e., HIV cis-acting sequence elements) are used. A lentivirus vector can comprise a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus.

[0417] In embodiments, a lentivirus vector (e.g., lentiviral expression vector) may comprise a lentiviral transfer plasmid (e.g., as naked DNA) or an infectious lentiviral particle. With respect to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is to be understood that the sequences of these elements can be present in RNA form in lentiviral particles and can be present in DNA form in DNA plasmids.

[0418] In embodiments, a lentivirus vector is a vector with sufficient retroviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle capable of infecting a target cell. Infection of the target cell can comprise reverse transcription and integration into the target cell genome. The recombinant lentivirus vector (RLV) typically carries non-viral coding sequences which are to be delivered by the vector to the target cell. In embodiments, an RLV is incapable ofindependent replication to produce infectious retroviral particles within the target cell. Usually, the RLV lacks a functional gag-pol and / or env gene and / or other genes involved in replication. The vector may be configured as a split-intron vector, e.g., as described in PCT patent application WO 99 / 15683, which is herein incorporated by reference in its entirety.

[0419] In some embodiments, the lentivirus vector comprises a minimal viral genome, e.g., the viral vector has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell, e.g., as described in WO 98 / 17815, which is herein incorporated by reference in its entirety.

[0420] A minimal lentiviral genome may comprise, e.g., (5')R-U5-one or more first nucleotide sequences-U3-R(3'). However, the plasmid vector used to produce the lentiviral genome within a source cell can also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a source cell. These regulatory sequences may comprise the natural sequences associated with the transcribed retroviral sequence, e.g., the 5' U3 region, or they may comprise a heterologous promoter such as another viral promoter, for example the CMV promoter. Some lentiviral genomes comprise additional sequences to promote efficient virus production. For example, in the case of HIV, rev and RRE sequences may be included.

[0421] In some embodiments, the vector can be a vector of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), or the pEX series (Clontech, Palo Alto, Calif.). In some cases, bacteriophage vectors, such as ZG I O. XGT11, XZapII (Stratagene), XEMBL4, and ZNM 1 149, also can be used. In some embodiments, plant expression vectors can be used and include pBIOl, pBH01.2, pBH01.3, pBH21 and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech).

[0422] In some embodiments, the vector (e.g., a viral vector) is a double stranded DNA vector. In some embodiments, the DNA vector is a closed-end vector. In some embodiments, the closed-end vector is Doggybone™ DNA (dbDNA). Closed-end DNA vectors, including dbDNA, are known in the art and have been described in e.g., Karda et al., Gene Ther (2019) 26:86-92.

[0423] In some cases, a vector may be used that does not require that the cells, e.g., T cells, are activated. In some such instances, the cells may be selected and / or transduced prior toactivation. Thus, the cells may be engineered prior to, or subsequent to culturing of the cells, and in some cases at the same time as or during at least a portion of the culturing.

[0424] In some embodiments, the cells are activated prior to introduction of the polynucleotide or vector. In some embodiments, the T cells are incubated with e.g., anti- CD3 / anti-CD28 antibodies. In some embodiments, the T cells are incubated with apololipoprotein E (ApoE) prior to, during, and / or subsequent to introduction of the polynucleotide or vector. For example, in some instances, the T cells are incubated with ApoE (e.g. ApoE4) prior to incubation with LNPs containing the polynucleotide or a composition thereof. In some embodiments, the T cells are incubated with about 1 pg / mL ApoE4 prior to incubation with LNPs containing the polynucleotide or a composition thereof.VI. DELIVERY OF NUCLEIC ACIDS

[0425] Introduction of the nucleic acid molecules or amino acid molecules of the disclosure into cells can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle- mediated nucleic acid delivery, naked nucleic acid delivery, as nucleic acid complexed with materials such as a liposome, nanoparticle or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)), and the like. Introduction of the nucleic acid molecules or amino acid molecules of the disclosure into cells can be ex vivo or in vivo.

[0426] In some embodiments, introducing the polynucleotides encoding the engineered polypeptides described herein into cells can be achieved by any suitable technique. Suitable techniques include calcium phosphate, lipid-mediated transfection, electroporation, nucleofection, fusogens, transposons (e.g., a Sleeping Beauty system), and transduction or infection using a viral vector, as discussed herein. In some embodiments, the polynucleotides are introduced into a cell via viral transduction (e.g., AAV transduction, lentiviral transduction) or otherwise delivered on a viral vector (e.g., fusogen-mediated delivery). In some embodiments, the polynucleotides are introduced into a cell via a fusogen-mediated delivery or a transposase system selected from the group consisting of conditional or inducible transposases, conditional or inducible PiggyBac transposons,conditional or inducible Sleeping Beauty (SB11) transposons, conditional or inducible Mosl transposons, and conditional or inducible Tol2 transposons.

[0427] The process of introducing the nucleic acid molecule into the cell can be achieved by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector. In some embodiments, the nucleic acid molecule comprises DNA. In some embodiments, the nucleic acid molecule comprises a modified DNA. In some embodiments, the nucleic acid molecule comprises RNA. In some embodiments, the nucleic acid molecule comprises mRNA. In some embodiments, the nucleic acid molecule comprises a modified mRNA.

[0428] In some embodiments, the nucleic acid molecules can be delivered by conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals, such as, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, nucleoporation, hydrodynamic shock, and infection.

[0429] In some embodiments, one or more polynucleotide(s) are introduced into a cell using electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). For examples, in some embodiments, one or more RNA molecules is introduced into a T cell using electroporation. Methods and systems for electroporation are known in the art, including e.g. Nucleofection® Technology (Lonza). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material, e.g., polynucleotides and / or vectors, into immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle- facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987) and other approaches described in, e.g., International Pat. App. Pub. No. WO 2014055668, and U.S. Patent No. 7,446,190.

[0430] In some embodiments, the nucleic acid molecules can be delivered by viral or non- viral delivery vehicles known in the art. For example, the nucleic acid molecule can bestably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for transient expression. Accordingly, in some embodiments, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can be achieved using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR / Cas9 genome editing, or DNA-guided endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the nucleic acid molecule is present in the recombinant host cell as a minicircle expression vector for transient expression.

[0431] In some embodiments, the nucleic acid molecules can be encapsulated in a viral capsid or a lipid nanoparticle, or can be delivered by viral or non-viral delivery means and methods known in the art, such as electroporation. For example, introduction of nucleic acids into cells may be achieved by viral transduction. In a non-limiting example, adeno- associated virus (AAV) is engineered to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.

[0432] In some embodiments, the polypeptide of the disclosure is delivered using viral transduction, for example, with a vector. In some embodiments, the vector is a pseudotyped, self- inactivating lentiviral vector that carries the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope, and which carries the exogenous polynucleotide.

[0433] In some embodiments, a lentiviral vector can be used for delivery. Lentiviral-derived vector systems are useful for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive properties as gene-delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy -target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production.I l l

[0434] Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101 : 1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497-505. In some embodiments, the polynucleotide encoding the recombinant receptor and / or one or more additional polypeptide(s), is introduced into a population containing cultured cells, such as by retroviral transduction, transfection, or transformation.

[0435] In some embodiments, a recombinant adeno-associated virus (AAV) vector can be used for delivery. Techniques to produce rAAV particles, in which an AAV genome to be packaged that includes the polynucleotide to be delivered, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (e.g., not in) the rAAV genome, and helper virus functions. The AAV rep and cap genes can be from any AAV serotype for which recombinant virus can be derived, and can be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13 and AAV rh.74. Production of pseudotyped rAAV is disclosed in, for example, international patent application publication number WO 01 / 83692.

[0436] In some embodiments, the nucleic acid encoding the engineered protein of the present disclosure is delivered with a retrovirus. In some embodiments, the nucleic acid encoding the engineered protein of the present disclosure is delivered with a lentivirus. In some embodiments, the nucleic acid encoding the engineered protein of the present disclosure is delivered with a VSV-G pseudotyped lentivirus. In some embodiments, the nucleic acid encoding the engineered protein of the present disclosure is delivered with an AAV. In some embodiments, the nucleic acid encoding the engineered protein of the present disclosure is delivered with an AAV6. In some embodiments, the nucleic acid encoding the engineered protein of the present disclosure is delivered with an AAV9. 1 In some embodiments, the nucleic acid encoding the engineered protein of the present disclosure is delivered with a split-intein dual AAV. In some embodiments, the nucleic acid encoding the engineered protein of the present disclosure is delivered with a redirected lentivirus. In some embodiments, the nucleic acid encoding the engineered protein of the present disclosure is delivered with a fusosome. In some embodiments, the nucleic acid encoding the engineered protein of the present disclosure is delivered with a lentiviral particle engineered with theanti-CD3 Cocal glycoprotein. In some embodiments, the nucleic acid encoding the engineered protein of the present disclosure is delivered with a enveloped delivery vehicle. In some embodiments, the nucleic acid encoding the engineered protein of the present disclosure is delivered with a self-replicating RNA virus. In some embodiments, the nucleic acid encoding the engineered protein of the present disclosure is delivered with an mRNA- packaging virus-like particle. In some embodiments, the nucleic acid encoding the engineered protein of the present disclosure is delivered with an RNP -packaging virus-like particle. In some embodiments, the delivery used in the context of this disclosure are described in WO2023193015A1; WO2022164935A1; Raguram, Aditya, Samagya Banskota, and David R. Liu. "Therapeutic in vivo delivery of gene editing agents." Cell (2022); Hamilton, Jennifer R., et al. "In vivo human T cell engineering with enveloped delivery vehicles." Nature Biotechnology (2024): 1-9; Lundstrom, Kenneth. "Self-replicating RNA viruses for RNA therapeutics." Molecules 23.12 (2018): 3310.

[0437] In some embodiments, the nucleic acid is or comprises mRNA encoding the engineered protein of the disclosure. The mRNA may be delivered to the cell using any appropriate technique, such as electroporation or using a lipid nanoparticle (LNP). In some embodiments, the nucleic acid is delivered with a lipid nanoparticle (LNP). In some embodiments, the nucleic acid is delivered with a selective organ targeting (SORT) LNP). In some embodiments, the nucleic acid is delivered with an antibody targeted LNP. In some embodiments, the LNP comprises: (i) an ionizable lipid (e.g., an amino lipid), (ii) a sterol or other structural lipid, (iii) a non-cationic helper lipid or phospholipid, and (iv) a PEG-lipid (e.g., a PEG-modified lipid). In some embodiments, the nucleic acid is delivered with a polymer nanoparticle. In some embodiments, the nucleic acid is delivered with a protein nanoparticle. In some embodiments, the delivery used in the context of this disclosure are described in W02024006960A1; WO2019152557A1; Raguram, Aditya, Samagya Banskota, and David R. Liu. "Therapeutic in vivo delivery of gene editing agents." Cell (2022); Rurik, Joel G., et al. "CAR T cells produced in vivo to treat cardiac injury." Science 375.6576 (2022): 91-96.

[0438] In some embodiments, the polynucleotides disclosed herein can be delivered or introduced using one or more agent(s), capable of inducing a genetic disruption (e.g., CRISPR / Cas and / or gRNA components), to a cell, using any of a number of known delivery methods or vehicles for introduction or transfer to cells, for example, using viral delivery vectors, or any of the known methods or vehicles for delivering CRISPR / Cas molecules and gRNAs. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9):689-701; Cooper et al. (2003) Blood. 101 : 1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497- 505. In some embodiments, nucleic acid sequences encoding one or more components of one or more agent(s) capable of inducing a genetic disruption is introduced into the cells, e.g., by any methods for introducing nucleic acids into a cell described herein or known. In some embodiments, a vector encoding components of one or more agent(s) capable of inducing a genetic disruption such as a CRISPR guide RNA and / or a Cas enzyme can be delivered into the cell.

[0439] In some embodiments, the one or more agent(s) capable of inducing a genetic disruption (e.g., one or more agent(s) that is a CRISPR / Cas / gRNA), is introduced into the cell as a ribonucleoprotein (RNP) complex. RNP complexes include a sequence of ribonucleotides, such as an RNA or a gRNA molecule, and a protein, such as a Cas9 protein or variant thereof. For example, the Cas9 protein is delivered as RNP complex that comprises a Cas9 protein and a gRNA molecule targeting the target sequence, e.g., using electroporation or other physical delivery method. In some embodiments, the RNP is delivered into the cell via electroporation or other physical means, e.g., particle gun, Calcium Phosphate transfection, cell compression or squeezing. In some embodiments, the RNP can cross the plasma membrane of a cell without the need for additional delivery agents (e.g., small molecule agents, lipids, etc.). In some embodiments, delivery of the one or more agent(s) capable of inducing genetic disruption, e.g., CRISPR / Cas9, as an RNP offers an advantage that the targeted disruption occurs transiently, e.g., in cells to which the RNP is introduced, without propagation of the agent to cell progenies. For example, delivery by RNP minimizes the agent from being inherited to its progenies, thereby reducing the chance of off-target genetic disruption in the progenies. In such cases, the genetic disruption and the integration of transgene can be inherited by the progeny cells, but without the agent itself, which may further introduce off-target genetic disruptions, being passed on to the progeny cells.

[0440] Agent(s) and components capable of inducing a genetic disruption (e.g., a CRISPR / Cas molecule and gRNA molecule), can be introduced into target cells in a variety of forms using a variety of delivery methods and formulations, as set forth in Tables 6 and 7, or methods described in, e.g., WO 2015 / 161276; US 2015 / 0056705, US 2016 / 0272999, US 2017 / 0211075; or US 2017 / 0016027. As described further herein, the delivery methods and formulations can be used to deliver template polynucleotides and / or other agents to the cell (such as those required for engineering the cells) in...

Claims

CLAIMS1. A recombinant polypeptide comprising a caspase-associated recruitment domain (CARD domain) containing protein or a functional fragment thereof, wherein the recombinant polypeptide comprises no more than 724 amino acids.

2. A recombinant polypeptide comprising:(a) a CARD domain, and(b) a recruitment domain or a functional fragment thereof, wherein the recruitment domain is capable of binding to (i) a substrate localized to the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine, wherein the recombinant polypeptide comprises no more than 724 amino acids.

3. A recombinant polypeptide comprising:(a) a first polypeptide capable of binding to a CARD domain on BCL10, and(b) a recruitment domain or a functional fragment thereof, wherein the recruitment domain is capable of binding to (i) a substrate localized to the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine, wherein the recombinant polypeptide comprises no more than 724 amino acids.

4. The recombinant polypeptide of any one of claims 1-3, wherein when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits decreased Interferon gamma (IFNg) and / or Interleukin-2 (IL-2) production compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

5. The recombinant polypeptide of any one of claims 1-4, wherein when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits equivalent or enhanced:(a) BCL10 binding,(b) BCL10 and MALT1 complex formation,(c) NF-KB and / or AP-1 signaling,(d) MALT1 paracaspase activity,(e) cytokine production,(f) peak tumor expansion,(g) peak peripheral blood expansion,(h) contraction following peak tumor expansion, and / or(i) contraction following peak peripheral blood expansion, compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

6. The recombinant polypeptide of any one of claims 1-5, wherein when the recombinant polypeptide is expressed in an immune cell and the immune cell binds a target antigen, the immune cell exhibits equivalent or higher IFNg and / or IL-2 production compared to an immune cell expressing a control polypeptide consisting of SEQ ID NO: 1.

7. The recombinant polypeptide of any one of claims 1-6, wherein the recombinant polypeptide comprises no more than 700, no more than 650, no more than 600, no more than 550, no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, or no more than 150 amino acids.

8. The recombinant polypeptide of any one of claims 1-7, wherein the CARD domaincontaining protein or the functional fragment thereof, the CARD domain, or the recombinant polypeptide, is capable of binding to a CARD domain on BCL10.

9. The recombinant polypeptide of any one of claims 1-8, wherein the CARD domaincontaining protein or the functional fragment thereof, the CARD domain, or the recombinant polypeptide, is capable of forming a complex with BCL10 and MALT1.

10. The recombinant polypeptide of any one of claims 1-9, wherein the CARD domaincontaining protein or the functional fragment thereof, or the CARD domain, comprises or consists of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to any one of SEQ ID NO: 293-323.

11. The recombinant polypeptide of any one of claims 1-10, wherein the CARD domaincontaining protein is selected from, or wherein the CARD domain is derived from a protein selected from, CARD9, CARDIO, CARD11, and CARD14.

12. The recombinant polypeptide of claim 11, wherein the CARD domain-containing protein is, or wherein the CARD domain is derived from, CARD11.

13. The recombinant polypeptide of any one of claims 1-12, wherein the recombinant polypeptide comprises a full coiled-coil domain or a truncated portion thereof, wherein thestart of the full coiled-coil domain corresponds to amino acid position 123 of SEQ ID NO: 1, and wherein the end of the full coiled-coil domain corresponds to amino acid position 442 of SEQ ID NO: 1; optionally, wherein the full coiled-coil domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 123-442 of SEQ ID NO: 1.

14. The recombinant polypeptide of claim 13, wherein the full coiled-coil domain comprises at least 310 amino acids.

15. The recombinant polypeptide of claim 13 or claim 14, wherein the recombinant polypeptide comprises the full coiled-coil domain.

16. The recombinant polypeptide of claim 13, wherein the recombinant polypeptide comprises a truncated portion of the coiled-coil domain having no more than 300, no more than 290, no more than 280, no more than 270, no more than 260, no more than 250, no more than 240, no more than 230, no more than 220, no more than 210, no more than 200, no more than 190, no more than 180, no more than 170, no more than 160, no more than 150, no more than 140, no more than 130, no mor than 120, no more than 110, no more than 100, no more than 95, no more than 90, no more than 85, no more than 80, no more than 75, no more than 70, no more than 60, no more than 50, or no more than 40 amino acids; preferably, wherein the truncated portion of the coiled-coil domain has no more than 260 amino acids.

17. The recombinant polypeptide of claim 16, wherein a region within the coiled-coil domain corresponding to amino acids 253-324 of SEQ ID NO: 1 is removed or replaced by a polypeptide comprising no more than 20, 15, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acids.

18. The recombinant polypeptide of claim 17, wherein the region within the coiled-coil domain is replaced by a polypeptide comprising a sequence of SGGGGSGGGGS (SEQ ID NO:431) or QAGKRSLPD (SEQ ID NO: 432), or a sequence having at most 1, 2, or 3 mutations thereto.

19. The recombinant polypeptide of any one of claims 1-18, wherein the recombinant polypeptide comprises an inhibitory domain or a truncated portion thereof, wherein the start of the inhibitory domain corresponds to amino acid position 443 of SEQ ID NO: 1, andwherein the end of the inhibitory domain corresponds to amino acid position 577 of SEQ ID NO: 1; optionally, wherein the inhibitory domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 443-577 of SEQ ID NO: 1.

20. The recombinant polypeptide of any one of claims 1-19, wherein the recombinant polypeptide comprises an inhibitory domain or a truncated portion thereof, wherein the inhibitory domain comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 441.

21. The recombinant polypeptide of claim 19 or 20, wherein the inhibitory domain comprises at least 130, or at least 125, amino acids.

22. The recombinant polypeptide of any one of claims 19-21, wherein the recombinant polypeptide comprises the full inhibitory domain.

23. The recombinant polypeptide of any one of claims 19-22, wherein the recombinant polypeptide comprises a truncated portion of the inhibitory domain having no more than 120, no more than 110, no more than 100, no more than 95, no more than 90, no more than 85, no more than 80, no more than 75, no more than 70, no more than 60, no more than 50, or no more than 40 amino acids.

24. The recombinant polypeptide of claim 23, wherein the inhibitory domain comprises N-terminal and / or C-terminal truncation(s).

25. The recombinant polypeptide of claim 24, wherein the inhibitory domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 amino acids at the N-terminus.

26. The recombinant polypeptide of claim 24 or 25, wherein the inhibitory domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 amino acids at the C-terminus.

27. The recombinant polypeptide of any one of claims 24-26, wherein the inhibitory domain comprises a truncation of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 amino acids in between the N-terminus and the C-terminus.

28. The recombinant polypeptide of any one of claims 24-26, wherein the inhibitory domain or the truncated portion thereof comprises at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 serines that are capable of being phosphorylated; optionally wherein the serines are capable of being phosphorylated by a protein kinase C (PKC); optionally, wherein the PKC comprises PKCP and / or PKC9.

29. The recombinant polypeptide of claim 28, wherein the inhibitory domain or the truncated portion thereof comprises a sequence of DASPRT (SEQ ID NO: 425), RAKSPI (SEQ ID NO: 426), DASPSS (SEQ ID NO: 427), SRSSIMS (SEQ ID NO: 428), RKFSLER (SEQ ID NO: 429), and / or FRPSVTS (SEQ ID NO: 430), or a sequence having at most 1, 2, or 3 mutations thereto; optionally, where the inhibitory domain or the truncated portion thereof comprises the sequence of RAKSPI (SEQ ID NO: 426), DASPSS (SEQ ID NO: 427), and / or SRSSIMS (SEQ ID NO: 428).

30. The recombinant polypeptide of any one of claims 20-29, comprising, from N-term to C-term: the coiled-coil domain or the truncated portion thereof, and the inhibitory domain or the truncated portion thereof, without any additional amino acid in between.

31. The recombinant polypeptide of any one of claims 1-30, wherein the recombinant polypeptide comprises (b) a recruitment domain or a functional fragment thereof, wherein the recruitment domain is capable of binding to (i) a substrate localized to the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine.

32. The recombinant polypeptide of claim 31, wherein the recruitment domain is capable of binding to a substrate indirectly localized to the intracellular side of the plasma membrane, and wherin the recruitment domain binds a polypeptide or lipid that is directly localized to the intracellular side of the plasma membrane of a cell.

33. The recombinant polypeptide of any one of claim 31 or 32, wherein the recruitment domain is a Src Homology region 2 (SH2) domain, a Src Homology region 3 (SH3) domain, a phosphotyrosine-binding (PTB) domain, or a pleckstrin homology (PH) domain.

34. The recombinant polypeptide of any one of claims 31-33, wherein the recruitment domain is a Src Homology region 2 (SH2) domain.

35. The recombinant polypeptide of any one of claims 31-34, wherein the recruitment domain or the functional fragment thereof comprises a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a segment of at least 30, 40, 50, 60, 70, 80, 90, or 97 amino acids within the region of amino acids 622-716 of SEQ ID NO: 1.

36. The recombinant polypeptide of any one of claims 31-35, comprising, from N-term to C-term: the inhibitory domain or the truncated portion thereof, and the recruitment domain or the functional fragment thereof, with no more than 5, no more than 10, no more than 15, no more than 20, or no more than 25, amino acids in between.

37. The recombinant polypeptide of claim 36, comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 433-438 in between the inhibitory domain or the truncated portion thereof and the recruitment domain or the functional fragment thereof.

38. The recombinant polypeptide of any one of claims 1-37, wherein the recombinant polypeptide comprises, from N-terminus to C-terminus, the CARD domain, optionally the coiled-coil domain, optionally the inhibitory domain, and the recruitment domain.

39. A nucleic acid construct comprising a polynucleotide sequence encoding the recombinant polypeptide of any one of claims 1-38.

40. The nucleic acid construct of claim 39, wherein the nucleic acid construct further comprises a polynucleotide sequence encoding one or more additional polypeptides and / or a non-coding RNA.

41. The nucleic acid construct of claim 40, wherein the one or more additional polypeptides comprise a chimeric antigen receptor (CAR), a recombinant T cell receptor (TCR), an additional potency enhancement polypeptide, a cytokine, a chemokine, a growth factor, a safety switch, or any combination thereof.

42. The nucleic acid construct of claim 41, wherein the one or more additional polypeptides comprise a CAR or a recombinant TCR.

43. The nucleic acid construct of any one of claims 39-42, comprising a first homology arm before the 5’ end of the nucleic acid sequence encoding the recombinant polypeptideand a second homology arm after the 3’ end of the nucleic acid sequence encoding the recombinant polypeptide; optionally, wherein the lengths of the two homology arms are between about 150 bp and about 1500 bp.

44. The nucleic acid construct of claim 43, wherein the length of at least one of the homology arms is about 200bp, 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, or about 500 bp; optionally, wherein the lengths of both of the homology arms are about 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, about 500 bp, about 525 bp, about 550 bp, about 575 bp, or about 600 bp.

45. The nucleic acid construct of claim 43 or 44, wherein the total length of the homology arms is between about 400 bp and about 500 bp, about 500 bp and about 600 bp, about 600 bp and about 700 bp, about 700 bp and about 800 bp, about 800 bp and about 900 bp, about 900 bp and about 1000 bp, about 1000 bp and about 1100 bp, or about 1100 bp and about 1200 bp.

46. The nucleic acid construct of any one of claims 39-45, wherein the nucleic acid construct is less than 10 kb, less than 9 kb, less than 8 kb, less than 7 kb, less than 6 kb, less than 5 kb, less than 4 kb, or less than 3 kb in length.

47. The nucleic acid construct of claim 46, wherein the nucleic acid construct is less than about 5.0 kb in length.

48. The nucleic acid construct of claim 46, wherein the nucleic acid construct is less than about 4.7 kb in length.

49. A vector comprising the nucleic acid construct of any one of claims 39-48.

50. The vector of claim 49, wherein the vector is a viral vector.

51. The vector of claim 50, wherein the viral vector is selected from a retrovirus vector, an adenovirus vector, and an adeno-associated virus (AAV) vector; optionally, wherein the viral vector is an AAV vector.

52. An engineered cell comprising the recombinant polypeptide of any one of claims 1- 38 or the nucleic acid construct of any one of claims 39-48.

53. The engineered cell of claim 52, wherein activating the engineered cell results in the secretion of IFNg by the cell at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, least 30-fold, or at least 100-fold higher than the IFNg secretion level of a control cell lacking the recombinant polypeptide.

54. The engineered cell of claim 52 or 53, wherein activating the engineered cell results in the secretion of IL-2 by the cell at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5 -fold, at least 10-fold, at least 20-fold, least 30-fold, or at least 100-fold higher than the IL-2 secretion level of a control cell lacking the recombinant polypeptide.

55. The engineered cell of claim 52, wherein activation of the engineered cell and subsequent removal of the target antigen results in CARD11-PIK3R3 signaling by the cell at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, least 30-fold, or at least 100-fold lower than the CARD1 1-PIK3R3 signaling of a control cell comprising a CARD11-PIK3R3 fusion protein lacking the inhibitory domain.

56. The engineered cell of claim 52 or 55, wherein activation of the engineered cell and subsequent removal of the target antigen results in CARD11-PIK3R3 signaling by the cell at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, least 30-fold, or at least 100-fold lower than the CARD1 1-PIK3R3 signaling of a control cell comprising a CARD11-PIK3R3 fusion protein lacking one of more of S466, S512, S535, S559, S644, and S652 according to SEQ ID NO: 518 in the inhibitory domain.

57. The engineered cell of any of claims 52-56, wherein CARD11-PIK3R3 signaling in the cell in the absence of a target antigen is at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, least 30-fold, or at least 100-fold lower than the CARD11-PIK3R3 signaling of a control cell comprising a control CARD11-PIK3R3 fusion protein lacking the inhibitory domain.

58. The engineered cell of any of claims 52-57, wherein CARD11-PIK3R3 signaling in the cell in the absence of a target antigen is at a level that is at least 10%, at least 20%, at least 50%, at least 1-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, least 30-fold, or at least 100-fold lower than the CARD11-PIK3R3 signaling of a control cell comprising a control CARD11-PIK3R3 fusion protein lacking one of more of S466, S512, S535, S559, S644, and S652 according to SEQ ID NO: 518 in the inhibitory domain.

59. The engineered cell of claim any one of claims 52-58, wherein the cell is selected from the group consisting of a T cell, a macrophage, a monocyte, and a natural killer (NK) cell.

60. The engineered cell of claim 59, wherein the cell is a T cell.

61. The engineered cell of any one of claims 52-60, wherein the cell further comprises:(i) a chimeric antigen receptor (CAR) having specificity for a target antigen; or(ii) a recombinant T cell receptor (TCR) having specificity for a target antigen.

62. A composition comprising the engineered cell of any one of claims 52-60, or a plurality of the engineered cell of any one of claims 52-60.

63. A method of preparing an engineered cell, the method comprising expressing in the cell the recombinant polypeptide of any one of claims 1-38, thereby making the engineered cell.

64. The method of claim 63, comprising introducing into the cell the nucleic acid construct of any one of claims 39-48.

65. A method of preparing an engineered cell, the method comprising introducing into the cell the nucleic acid construct of any one of claims 39-48, thereby making the engineered cell.

66. The method of any one of claims 63-65, wherein the step of introducing comprises homology-directed repair (HDR)-mediated insertion using a gene editing machinery.

67. The method of claim 66, where the HDR occurs in at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 99%, of a population of the cells; optionally, wherein the HDR occursin between about 20%-30%, about 30%-40%, about 40%-50%, about 50%-60%, about 60%- 70%, about 70%-80%, about 80%-90%, or about 90%-100%, of the cells.

68. The method of claim 66 or claim 67, wherein the step of introducing comprises HDR-mediated insertion in the absence of an HDR enhancer; optionally, wherein the HDR enhancer is M3814 or AZD7648.

69. The method of any one of claims 66-68, wherein the nucleic acid construct comprises a first homology arm before the 5’ end of the nucleic acid sequence encoding the recombinant polypeptide and a second homology arm after the 3’ end of the nucleic acid sequence encoding the recombinant polypeptide; optionally, wherein the lengths of the two homology arms are between about 150 bp and about 1500 bp.

70. The method of claim 69, wherein the length of each of the homology arms is at least 200 bp.

71. The method of claim 69 or 70, wherein the length of at least one of the homology arms is about 200bp, 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, or about 500 bp; optionally, wherein the lengths of both of the homology arms are about 225 bp, about 250 bp, about 275 bp, about 300 bp, about 325 bp, about 350 bp, about 375 bp, about 400 bp, about 425 bp, about 450 bp, about 475 bp, about 500 bp, about 525 bp, about 550 bp, about 575 bp, or about 600 bp.

72. The method of any one of claims 66-71, wherein the total length of the homology arms is between about 400 bp and about 500 bp, about 500 bp and about 600 bp, about 600 bp and about 700 bp, about 700 bp and about 800 bp, about 800 bp and about 900 bp, about 900 bp and about 1000 bp, about 1000 bp and about 1100 bp, or about 1100 bp and about 1200 bp.

73. The method of any one of claims 66-72, wherein the nucleic acid construct comprises, from 5’ to 3’, the first homology arm, a promoter, a CAR encoding sequence, a polynucleotide sequence encoding the recombinant polypeptide, and the second homology arm.

74. The method of any one of claims 64-73, wherein the nucleic acid construct is introduced via AAV.

75. The method of any one of claims 63-74, wherein the method thereby produces an engineered cell comprising: (i) the recombinant polypeptide; (ii) the immune receptor (e.g., CAR); and (iii) the modified TRAC gene locus.

76. The method of any one of claims 63-74, wherein the method thereby produces an engineered cell comprising: (i) the recombinant polypeptide; (ii) the immune receptor (e.g., CAR); and (iii) the modified TRAJ intron splice acceptor locus.

77. A method of treating a subject, comprising administering to the subject the engineered cell of any one of claims 52-61, or the engineered cell prepared by the method of any one of claims 63-76.

78. The method of claim 77, wherein the subject has a cancer or an autoimmune disease.

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