Compositions and methods for enhancing adoptive T cell therapy

Abstract

The present disclosure generally relates to compositions and methods for improving T cell therapy. Specifically, the present disclosure provides polypeptides and recombinant nucleic acid constructs and / or recombinant nucleic acids encoding the polypeptides having mutations that can alter T cell signaling, cytokine production, and / or in vivo persistence in tumors of therapeutic T cells containing the mutations. T cell signaling can be via the NFAT, NF-κB, and / or AP-1 pathways. The present disclosure also provides vectors and cells containing the polypeptides and / or recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, as well as methods for preparing T cells for use in cell therapy, and methods for identifying mutations useful for improving T cell therapy.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 412,300, filed September 30, 2022, the entire contents of which are incorporated herein by reference for all purposes.

[0002] Sequence Listing Description The Sequence Listing XML accompanying this application is provided in XML file format and is hereby incorporated by reference. The XML file containing the Sequence Listing XML is named 048536-737001WO_ST26.xml. The XML file is 720,329 bytes, was created on October 2, 2023, and has been submitted electronically through the USPTO Patent Center.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under grant numbers OD025751 and 1DP2AI136599-01 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0004] Field The present disclosure relates generally to compositions and methods for enhancing T cell therapeutics. The present disclosure provides recombinant nucleic acid constructs and / or recombinant nucleic acids encoding polypeptides that promote T cell signaling, efficacy, and / or in vivo persistence, cells containing such recombinant nucleic acids, methods for preparing T cells for use in cell therapy, and methods for identifying mutations useful for improving T cell therapy. [Background technology]

[0005] background Adoptive T cell therapy, including chimeric antigen receptor (CAR) T cells, has revolutionized cancer treatment. However, impressive responses have been limited to a subset of patients with hematological cancers and have yet to be achieved in patients with solid tumors, which represent 90% of adult cancers. In both treatment-resistant hematological and solid tumors, adoptive T cell therapy is limited by a complex combination of factors, including the compatibility of engineered T cells with the tumor, T cell exhaustion, poor in vivo persistence, and immunosuppressive environmental factors. Despite significant progress in recent years, rational design has yet to overcome the challenges associated with such factors.

[0006] Beyond rational design, another approach to identifying modifications that improve T cell function in vitro and in vivo is unbiased screening. For example, the majority of screening efforts have focused on genome-wide or genome-wide modifications that alter the expression of endogenous wild-type genes by CRISPR-Cas9 or short hairpin RNA (shRNA) or cDNA overexpression.

[0007] Chimeric antigen receptors (CARs) are synthetic receptors that contain an antigen-specific extracellular single-chain variable fragment (scFv) linked to a flexible linker (hinge) region, a transmembrane domain, and an intracellular signaling domain. The intracellular portion of the receptor consists of T cell signaling domains, such as 41BB, CD28, and CD3 zeta, and is designed to mimic T cell receptor (TCR) stimulation and the immunological synapse upon binding to the antigen specified by the scFv. CAR constructs do not require antigen presentation by MHC molecules and have therefore been used to effectively redirect a patient's own T cells to tumor-specific cell surface antigens. To date, five CD19-targeted CAR T cell therapies have been approved by the FDA for use against hematological B-cell cancers. While these treatments have proven highly effective in refractory B-cell malignancies, CAR T cell therapies have yet to provide robust long-term efficacy against solid tumors. In the solid tumor environment, CAR-T cells can become exhausted, have difficulty proliferating and performing effector functions, and ultimately fail to control tumor growth or prevent recurrence. Therefore, to create effective targeted cell therapies for solid tumors, it is necessary to improve the proliferation capacity, persistence, and effector function of CAR-T cells.

[0008] An approach under investigation is genetically modifying CAR-T cells to improve their functionality in solid tumors. A recent case study described a patient with chronic lymphocytic leukemia (CLL) who experienced a delayed but complete response after treatment with CD19 CAR-T cell therapy. It was later discovered that within a single T-cell clone, the CD19 CAR cassette had integrated into one allele of TET2, a known T-cell lymphoma tumor suppressor, rendering it nonfunctional. Interestingly, the patient's second TET2 allele was also mutated, resulting in the loss of TET2 function in the CD19 CAR-T cells administered to this patient. This single TET2 knockout CAR-T cell clone exhibited altered T-cell differentiation and improved overall effector function. Ultimately, this clone expanded to comprise a majority of the CAR-T cell population and mediated the patient's complete response to relapsed CLL. In a second example, a CD22 CAR cassette integrated into the T cell lymphoma tumor suppressor CBL mediated a similar complete response in patients. These case studies demonstrate that gene knockout of T cell lymphoma tumor suppressors, such as TET2 and CBL, can have significant beneficial effects on CAR-T cell therapy. In preclinical studies, genome-wide knockout assays have identified genes, such as REGNASE, that, when knocked out, improve T cell compatibility and in vivo antitumor efficacy. In addition, other studies have found that knockout of genes associated with T cell exhaustion and memory formation, such as genes in the NR4A family, can improve and prolong CAR-T cell responses to tumors.

[0009] While these examples demonstrate that CAR-T cell functionality can be improved through genetic engineering, particularly through manipulation of tumor suppressor genes, these studies are often extremely broad in scope (examining the entire genome) and focus solely on the effects of constitutive gene knockouts. Naturally occurring somatic single nucleotide variant (SSNV) mutations, translocations, and gene deletions in cancer provide biologically rational candidates for genetic engineering along with CAR expression.

[0010] There remains a need in the art for alternative solutions to address the significant unmet need for effective adoptive T cell therapy and enhancing the compatibility of engineered T cells. Summary of the Invention [Means for solving the problem]

[0011] Abstract This section provides a general overview of the disclosure and is not intended to encompass its entire scope or all of its characteristics.

[0012] The present disclosure provides recombinant nucleic acid constructs and / or nucleic acids and methods for enhancing adoptive T cell therapy. The recombinant nucleic acid constructs and / or nucleic acids of the present disclosure encode polypeptides having mutations that enhance the therapeutic efficacy of T cells by altering T cell signaling, reducing T cell exhaustion, and / or enhancing the in vivo persistence and fitness of the engineered T cells.

[0013] In one aspect, the disclosure provides a method for treating a caspase-associated recruitment domain (CARD)-containing protein or a functional fragment thereof, comprising:

[0014] (b) a domain capable of binding to (i) a substrate localized on the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine. In some embodiments, the domain of b) is capable of binding to a substrate indirectly localized on the intracellular side of the plasma membrane through binding to another polypeptide or lipid directly localized on the intracellular side. 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: 261-289. In some embodiments, the CARD-containing protein is selected from CARD9, CARD10, CARD11, and CARD14. 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: 261-264. In some embodiments, a functional fragment of a CARD-containing protein is derived from CARD11 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: 263.

[0015] In some embodiments, the function of the CARD-containing protein or functional fragment thereof is to bind to a CARD domain on BCL10. In some embodiments, the functional fragment 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.

[0016] In some embodiments, the cell is a T cell, a macrophage, a monocyte, or a natural killer (NK) cell. In some embodiments, activation of the cell results in the generation of a substrate localized on the intracellular side of the plasma membrane. In some embodiments, the substrate localized on the intracellular side of the plasma membrane of the cell is a phosphoinositide. In some embodiments, the phosphoinositide is selected from phosphatidylinositol (3,4,5)-triphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). In some embodiments, the polypeptide binds to the phosphoinositide with a Kd of less than 50 μM, less than 10 μM, less than 5 μM, less than 1 μM, less than 0.5 μM, less than 0.1 μM, less than 0.05 μM, or less than 0.01 μM, and the Kd is analyzed using SPR.

[0017] 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 target polypeptide is derived from IGF-1R, CTLA-4, or CD28. In some embodiments, the phosphorylated tyrosine is located at a position corresponding to pY1221 of SEQ ID NO: 302. In some embodiments, the phosphorylated tyrosine is located at a position corresponding to pY1346 of SEQ ID NO: 301.

[0018] In some embodiments, the polypeptide binds to a target polypeptide with a Kd of less than 10 μM, less than 5 μM, less than 1 μM, less than 0.5 μM, less than 0.1 μM, less than 0.05 μM, or less than 0.01 μM, where Kd is analyzed by fluorescence polarization assay. In some embodiments, the polypeptide has a higher affinity for a target polypeptide containing a phosphorylated tyrosine than a control polypeptide that does not have 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 a target polypeptide containing a phosphorylated tyrosine than a control polypeptide that does not have phosphorylation at the corresponding tyrosine position.

[0019] In some embodiments, the (b) domain is or comprises an SH3 domain. In some embodiments, the (b) domain is or comprises a phosphotyrosine binding (PTB) domain. In some embodiments, the (b) domain is or comprises a pleckstrin homology (PH) domain. In some embodiments, the (b) domain is or comprises an SH2 domain.

[0020] In one aspect, the present disclosure provides a polypeptide comprising (i) a caspase-associated recruitment domain (CARD)-containing protein or a functional fragment thereof, and (ii) an SH2 domain. In some embodiments, the CARD-containing protein is CARD11. In some embodiments, the SH2 domain is capable of binding to a polypeptide comprising a phosphorylated tyrosine. 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 comprises a motif of a conserved arginine residue in a FLVR motif.

[0021] 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 SEQ ID NO: 305. In some embodiments, the SH2 domain is an engineered SH2 domain with enhanced affinity for phosphotyrosine. 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: 313-15.

[0022] In one aspect, the present disclosure provides a polypeptide comprising: (i) a CARD domain derived from a CARD11 protein; and (ii) a second polypeptide portion derived from a PIK3R3 protein. In some embodiments, the second polypeptide portion 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:205, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, and SEQ ID NO:255.

[0023] In some embodiments, the polypeptide does not comprise a coiled-coil domain or a portion thereof. In some embodiments, the polypeptide comprises a coiled-coil domain or a portion thereof. In some embodiments, the coiled-coil 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: 290-293. In some embodiments, the coiled-coil 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: 290.

[0024] In some embodiments, the polypeptide comprises or consists of about 10, about 20, about 30, about 40, about 50, about 60, about 80, about 100, about 120, about 140, about 150, about 160, about 180, about 200, about 220, about 240, about 250, about 260, about 280, or about 300 amino acids of the N-terminal portion of the coiled-coil domain. In some embodiments, the polypeptide comprises no more than 10, no more than 20, no more than 30, no more than 40, no more than 50, no more than 60, no more than 80, no more than 100, no more than 120, no more than 140, no more than 150, no more than 160, no more than 180, no more than 200, no more than 220, no more than 240, no more than 250, no more than 260, no more than 280, or no more than 300 amino acids of the N-terminal portion of the coiled-coil domain.

[0025] In some embodiments, the domain of (b), or the SH2 domain, or the second polypeptide portion, is located N-terminal to the CARD domain, between the CARD domain and the coiled-coil domain, or C-terminal to the CARD domain and / or the coiled-coil domain. In some embodiments, the CARD domain is derived from a CARD11 protein and is followed by a coiled-coil domain derived from a CARD11 protein.

[0026] In some embodiments, the (b) domain, or SH2 domain, or second polypeptide portion is located proximal to the C-terminus of the polypeptide, and the polypeptide has no more than 50, no more than 40, no more than 30, no more than 20, no more than 15, no more than 10, or no more than 5 amino acids at the C-terminus of the (b) domain, or SH2 domain, or second polypeptide portion.

[0027] In some embodiments, the polypeptide does not comprise an inhibitory domain (ID) or a portion thereof. In some embodiments, the polypeptide comprises an inhibitory domain (ID) or a portion thereof. In some embodiments, the inhibitory domain (ID) 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: 294. In some embodiments, the inhibitory domain (ID) 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.

[0028] In some embodiments, the polypeptide comprises or consists of about 10, about 20, about 30, about 40, about 50, about 60, about 80, about 100, about 120, about 140, about 150, about 160, about 180, or about 200 amino acids of the N-terminal portion of the inhibitory domain (ID). In some embodiments, the second polypeptide portion comprises no more than 10, no more than 20, no more than 30, no more than 40, no more than 50, no more than 60, no more than 80, no more than 100, no more than 120, no more than 140, no more than 150, no more than 160, no more than 180, or no more than 200 amino acids of the N-terminal portion of the inhibitory domain (ID).

[0029] In some embodiments, the polypeptide does not comprise a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 297. In some embodiments, the polypeptide comprises one or more mutations corresponding to S615F, D357N, Y361C, E634K, and / or S655C of SEQ ID NO: 26.

[0030] In some embodiments, expression of the polypeptide in T cells promotes its in vivo accumulation in tumors. In some embodiments, the T cells express an engineered immune receptor that binds to a target on tumor cells. In some embodiments, the T cells are selected from the group consisting of regulatory (Treg), gamma delta T cells, invariant iNKT cells, MAIT cells, CAR T cells, tumor-infiltrating lymphocytes, and engineered T cells comprising transcriptional receptors.

[0031] In one aspect, the present disclosure provides polypeptides comprising a mutation that can (i) alter T cell signaling through the NFAT, NF-κB, and / or AP-1 pathways; (ii) alter cytokine production; (iii) alter JAK / STAT signaling in T cells; (iv) alter costimulatory molecule signaling in T cells; (v) alter RAS / MEK / ERK signaling in T cells; (vi) alter phospholipase gamma signaling; (vii) alter transcription factor activity in T cells; and / or (viii) alter or enhance in vivo persistence in tumors of T cells comprising the mutation.

[0032] In one aspect, the present disclosure provides a recombinant nucleic acid encoding a polypeptide described herein. In some embodiments, the nucleic acid comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is a CD4 promoter, a CD8a promoter, a CD8b promoter, a TCRa promoter, a TCRb promoter, a CD3d promoter, a CD3g promoter, a CD3e promoter, or a CD3z promoter. In some embodiments, the promoter is a minimal TATA promoter, pGK, actin promoter, CD25 promoter, IL2 promoter, IL7 promoter, IL15 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD11a promoter, CD58 promoter, CD99 promoter, CD62L promoter, CD103 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, IFN gamma promoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL1 promoter, IL5 promoter, IL6 promoter, IL13 promoter, IL10 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, CD127 promoter, PD-1 promoter, CD122 promoter, CD132 promoter, c-Kit promoter, nuclear factor of activated T cells (NFAT) 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 attenuating factor (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, CD130 promoter, NR4A1 promoter, NR4A2 promoter, or NR4A3 promoter.

[0033] In one aspect, the present disclosure provides a vector comprising the recombinant nucleic acid construct described herein.In some embodiments, the vector is a viral vector selected from a retroviral vector, an adenoviral vector, and an adeno-associated viral vector.In some embodiments, the retrovirus is a lentivirus.

[0034] In one aspect, the present disclosure provides a cell comprising a polypeptide described herein and / or a recombinant nucleic acid described herein. In some embodiments, the cell is a non-native cell or is genetically engineered. In some embodiments, the cell is not a CD4+ T cell. In some embodiments, the cell is not a cancerous cell. In some embodiments, the recombinant nucleic acid is exogenous.

[0035] In some embodiments, the cell comprises at least one copy, or at least two copies, of an endogenous nucleic acid sequence encoding a CARD11 protein or a protein comprising a CARD11 CARD domain without any SH2 domain. In some embodiments, the recombinant nucleic acid of the cell is located at an endogenous CARD11-encoding locus or comprises at least a portion of the cell's endogenous CARD11-encoding gene.

[0036] In some embodiments, the cells comprise SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO 8, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO: SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194 Column number 196, SEQ ID NO: 198, SEQ ID NO: 200, SEQ ID NO: 202, SEQ ID NO: 204, SEQ ID NO: 206, SEQ ID NO: 208, SEQ ID NO: 210, SEQ ID NO: 212, SEQ ID NO: 214, SEQ ID NO: 216, SEQ ID NO: 218, SEQ ID NO: 220, SEQ ID NO: 222, SEQ ID NO: 224, SEQ ID NO: 226, SEQ ID NO: 228, SEQ ID NO: 230, SEQ ID NO: 232, SEQ ID NO: 234, SEQ ID NO: 236, SEQ ID NO: 238, SEQ ID NO: 240, SEQ ID NO: 242, SEQ ID NO: 244, SEQ ID NO: 246, SEQ ID NO: 248, SEQ ID NO: 250, SEQ ID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256,and functional variants thereof containing at least one mutation listed in Table 1.

[0037] In some embodiments, the cells comprise SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:66 7, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO: 133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO: No. 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255,or a nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to these functional variants containing at least one mutation listed in Table 1.

[0038] In some embodiments, the cells further comprise (i) a chimeric antigen receptor (CAR) having specificity for a target antigen, and / or (ii) a T cell receptor (TCR) having specificity for a target antigen. In some embodiments, the cells are selected from the group consisting of immune cells, T cells, regulatory T cells, CD8+ cells, natural killer cells, tumor-infiltrating lymphocytes, and MAIT cells. In some embodiments, the target antigen is DLL3, LY6G6D, claudin 6, GCC, p53R175H, or PRAME.

[0039] In one aspect, the present disclosure provides a method of preparing T cells for use in cell therapy, the method comprising expressing a polypeptide described herein in the T cells. In some embodiments, the method comprises genetically modifying the T cells for expression of the polypeptide. In some embodiments, the method comprises introducing into the T cells a recombinant nucleic acid encoding the polypeptide or a vector comprising the recombinant nucleic acid. In some embodiments, the method comprises expressing in the T cells an engineered immune receptor that binds to a target on a tumor cell. In some embodiments, the method comprises administering to a subject the cells described herein or T cells prepared by the methods described herein.

[0040] In some embodiments, the subject has cancer or an autoimmune disease. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is characterized by the expression of CD19, B7H3 (CD276), BCMA (CD269), ALPPL2, claudin 18.2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, claudin 6 (CLDN6), CLECL1, CS-1, DLL-3, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, ephrin B2, F The cancer expresses AP, FLT3, GCC, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2 / neu), IGLL1, IL-11Rα, KIT (CD117), KLK2, LY6G6D, MUC1, NCAM, p53R175H, PAP, PDGFR-β, PRAME, PRSS21, PSCA, PSMA, ROR1, SIRPα, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, and / or Axl. In some embodiments, the cancer expresses DLL3, LY6G6D, claudin 6, GCC, p53R175H, and / or PRAME.

[0041] 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 and 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.

[0042] In some embodiments, the method of treatment does not include administration of a lymphodepleting agent within 7 days prior to administration of T cell therapy. In some embodiments, the method of treatment does not include administration of cyclophosphamide, fludarabine, and / or bendamustine within 7 days prior to administration of T cell therapy. In some embodiments, the method of treatment does not include administration of at least 600,000 IU / kg of IL-2 every 8 hours. In some embodiments, the method of treatment does not include checkpoint therapy that blocks PD-1 or CTLA-4 signaling.

[0043] In some embodiments, the cells have reduced exhaustion, increased proliferative capacity, enhanced replicative lifespan, decreased replicative senescence, enhanced anti-tumor activity, reduced dysfunction, enhanced persistence, and / or increased intratumoral presence in vivo. In some embodiments, the cells have increased or decreased signaling through the CARD11-BCL10-MALT1 complex, NF-κB, AP-1, NFAT, JAK / STAT, and / or MEK / ERK pathways.

[0044] In some embodiments, the present disclosure provides a recombinant nucleic acid construct encoding a polypeptide, wherein the polypeptide comprises a mutation that can alter (i) T cell signaling through the NFAT, NF-κB, and / or AP-1 pathways, (ii) cytokine production, and / or (iii) in vivo persistence in a tumor of a therapeutic T cell that comprises the mutation.

[0045] In some embodiments of the recombinant nucleic acid construct and / or recombinant nucleic acid of the present disclosure, the mutation is a point mutation, gene fusion, substitution, gain-of-function mutation, gain-of-stop mutation, insertion mutation, deletion mutation, duplication mutation, or translocation. In some embodiments, the mutation is a T cell lymphoma mutation or a mutation in a clonally expanded T cell population. In some embodiments, the mutation can alter / promote / enhance CARD11-BCL10-MALT1 complex signaling in T cells.

[0046] In some embodiments, the mutation is in a gene selected from the group consisting of caspase recruitment domain family member 11 (CARD11), capping protein regulator and myosin 1 linker 2 (CARMIL2), mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), B-cell lymphoma 6 (BCL6), B-cell lymphoma 10 (BCL10), and MYCN.

[0047] In some embodiments, the polypeptide comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 25 and comprising a substitution at an amino acid position selected from the group consisting of 361, 615, 634, 655, and 357 of SEQ ID NO: 25. In some embodiments, the substitution comprises Y361C, S615F, E634K, D357N, S655C, or a combination thereof.

[0048] In some embodiments, the mutation is a fusion of a CARD11 polypeptide with a PIK3R3 polypeptide, hi some embodiments, the fusion comprises a CARD domain, a coiled-coil domain, and an SH2 domain from PIK3R3.

[0049] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide is encoded by a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:205, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, or SEQ ID NO:255.

[0050] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:206, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, or SEQ ID NO:256.

[0051] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:4 and including a substitution at amino acid 647 of SEQ ID NO:4; having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:28 and including a substitution at amino acid 575 of SEQ ID NO:28; or having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:114 and including a substitution at amino acid 44 of SEQ ID NO:114. In some embodiments, the substitution at amino acid 647 of SEQ ID NO: 4 is S647R, the substitution at amino acid 575 of SEQ ID NO: 28 is Q575E, and the substitution at amino acid 44 of SEQ ID NO: 114 is P44L.

[0052] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide comprises a mutation that can (i) alter JAK / STAT signaling in T cells, (ii) alter cytokine production, and / or (iii) enhance in vivo persistence in tumors of therapeutic T cells comprising the mutation. In some embodiments, the polypeptide having the mutation comprises a JAK1, JAK3, STAT3, or STAT5 polypeptide. In some embodiments, the polypeptide has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:90 and has a substitution at amino acid position 1097 of SEQ ID NO:90, or has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:94 and has a substitution at amino acid position 573 of SEQ ID NO:94, or has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:94 and has a substitution at amino acid position 573 of SEQ ID NO:176. or an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141 and having a substitution at amino acid position 618, 647, or 661 of SEQ ID NO: 176, or having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141 and having a substitution at amino acid position 628 or amino acid position 665 of SEQ ID NO: 182.

[0053] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from the group consisting of SEQ ID NO:87, SEQ ID NO:91, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:177, or SEQ ID NO:179.

[0054] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide comprises a mutation that can alter costimulatory molecule signaling in T cells and the persistence of T cells in tumors that comprise the mutation. In some embodiments, the polypeptide with the mutation comprises a TNFRSF1B, CD28, ICOS, or CTLA4 polypeptide. In some embodiments, the polypeptide i) has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 192 and includes a substitution at amino acid position 256 or 377 of SEQ ID NO: 192, or ii) has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 42. or iii) comprises a substitution at amino acid position 51 or 77 of SEQ ID NO:42, or iii) has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:220, or iv) has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:218. In some embodiments, the nucleic acid construct comprises a polypeptide having an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:189, SEQ ID NO:189, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:43, SEQ ID NO:217, or SEQ ID NO:219.

[0055] In some embodiments, the polypeptide comprises a mutation that can alter RAS / MEK / ERK signaling in T cells and the in vivo persistence in tumors of therapeutic T cells that comprise the mutation.

[0056] In some embodiments, the mutation comprises a BRAF gene or a RASGRP1 polypeptide. In some embodiments, the polypeptide comprises: i) an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16, and including an amino acid substitution at amino acid position 469 or 594 of SEQ ID NO: 16, or at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 158, and including an amino acid substitution at amino acid position 261 of SEQ ID NO: 261. In some embodiments, the nucleic acid construct comprises a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, or SEQ ID NO:157.

[0057] In some embodiments, the polypeptide comprises a mutation that can alter phospholipase gamma signaling, and / or (ii) alter cytokine production, and / or (iii) alter the in vivo persistence in tumors of therapeutic T cells comprising the mutation. In some embodiments, the mutation is in the phospholipase C gamma 1 (PLCG1) gene. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 142, and having a substitution at amino acid position 47, 48, 520, 1163, or 1165 of SEQ ID NO: 142. In some embodiments, the recombinant nucleic acid construct comprises a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, or SEQ ID NO:143.

[0058] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide comprises a mutation that can alter transcription factor activity in a T cell that comprises the mutation. In some embodiments, the polypeptide having the mutation comprises an NFKB1, NFKB2, or JUNB polypeptide. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 118 and including a substitution at amino acid 67 of SEQ ID NO: 118; having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 122 and including a substitution at amino acid 565 of SEQ ID NO: 122; or having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 98 and including a substitution at amino acid 282. In some embodiments, the nucleic acid construct comprises a nucleic acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:115, SEQ ID NO:119, and SEQ ID NO:95.

[0059] In some embodiments of the constructs, polypeptides, and nucleic acids of the present disclosure, the mutation is a mutation listed in Table 1.

[0060] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide is selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:1 28, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:19 0, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252,The present invention relates to a method for producing a medicament for the treatment of a pulmonary arthritis, the treatment of a pulmonary arthritis, and the method for the treatment of a pulmonary arthritis, the method comprising administering to a patient a medicament ...

[0061] In some embodiments of the recombinant nucleic acid construct and / or recombinant nucleic acid of the present disclosure, the recombinant nucleic acid construct comprises SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59 , SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:1 27, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:188 9, SEQ ID NO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251,The nucleic acid sequences include those having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 253, SEQ ID NO: 255, or functional variants thereof containing at least one mutation listed in Table 1.

[0062] In certain aspects of the present disclosure, provided herein is a recombinant nucleic acid construct encoding a fusion polypeptide, wherein the fusion polypeptide comprises a first polypeptide encoding a partial CARD11 polypeptide and a second polypeptide encoding a partial PIK3R3 polypeptide, and expression of the fusion polypeptide promotes in vivo persistence in a tumor of therapeutic T cells comprising the fusion polypeptide.

[0063] In some embodiments, the T cells are selected from the group consisting of regulatory (Treg), gamma delta T cells, invariant iNKT cells, macrophages, monocytes, natural killer (NK), CAR T cells, and engineered T cells comprising transcriptional receptors.

[0064] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the first polypeptide comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 258. In some embodiments, the second polypeptide comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 260. In some embodiments, the first polypeptide comprises a CARD domain, a coiled-coil domain, and the second polypeptide comprises an SH2 domain derived from PIK3R3.

[0065] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the constructs include a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the constitutive promoter is a CD4 promoter, a CD8a promoter, a CD8b promoter, a TCRa promoter, a TCRb promoter, a CD3d promoter, a CD3g promoter, a CD3e promoter, or a CD3z promoter.

[0066] In some embodiments, the promoter is selected from the group consisting of MND promoter, EF1a promoter, sEF1a promoter, gammaretroviral LTR promoter, minimal TATA promoter, pGK, actin promoter, CD25 promoter, IL2 promoter, IL7 promoter, IL15 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD11a promoter, CD58 promoter, CD99 promoter, CD62L promoter, CD103 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, IFN gamma promoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL1 promoter, IL5 promoter, IL6 promoter, IL13 promoter, IL10 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, CD127 promoter, PD-1 promoter, CD122 promoter, CD132 promoter, c-Kit promoter, nuclear factor of activated T cells (NFAT) 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 attenuating factor (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, CD130 promoter, NR4A1 promoter, NR4A2 promoter, or NR4A3 promoter.

[0067] In certain aspects of the present disclosure, provided herein are recombinant nucleic acid constructs and / or recombinant nucleic acids, wherein the cytokine is IL-2, IL-4, IL-5, TNF-alpha, IFN-gamma, IL-13, and / or any combination thereof.

[0068] In some aspects of the present disclosure, the present disclosure provides a vector comprising any of the recombinant nucleic acid constructs and / or recombinant nucleic acids.In some embodiments, the vector is a retrovirus, an adenovirus, or an adeno-associated virus.In some embodiments, the retrovirus is a lentivirus.

[0069] In some aspects of the present disclosure, a cell is provided herein that comprises a nucleic acid construct or vector of the present disclosure. In some embodiments, the cell comprises (i) a chimeric antigen receptor (CAR) having specificity for a target antigen, (ii) a T cell receptor (TCR) having specificity for a target antigen, and / or (iii) a transcription receptor.

[0070] In some embodiments, the cell is selected from the group consisting of an immune cell, a T cell, a regulatory T cell, a CD8+ cell, a natural killer cell, and a tumor-infiltrating lymphocyte.

[0071] In some aspects, the present disclosure provides target antigens, DLL3, LY6G6D, claudin 6, GCC, p53R175H, PRAME, CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD3ε, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD33 , CD34, CD38, CD40, CD44v6, CD45, CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80(B7.1), C D86(B7.2), CD94, CD95, CD97, CD123, CD134, CD140(PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179 a, CD181(CXCR1), CD182(CXCR2), CD183(CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273(PD-L2), CD 274(PD-L1), CD276(B7H3), CD279, CD295, CD339(JAG1), CD340(HER2), CEA, CLL-1, CS1, EGFR, FGFR2, AFP, CA12 5, MUC-1, MAGE, placenta-like alkaline phosphatase 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), claudin 18.2, PSMA, ROR1, mesothelin, IL13Ra2, FAP, signal-regulatory protein α (SIRPα), TRAC, TCRβ, BCMA, TSHR, EGFRvIII, GD2, GD3, TnAg, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, PDGFR-beta, SSEA-4, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, 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 Al, ETV6-AML, seminal fluid protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, surviving, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, gastrointestinal carboxylesterase, mut hsp70-2, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, AFP, TRAC, TCRβ, BCMA, TSHR, EGFRvIII, GD2, GD3, TnAg, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, PDGFR-beta, SSEA-4, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr -abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, 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 Al, ETV6-AML, seminal fluid protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, KRAS, mutant KRAS, KRAS G12D, prostein, surviving, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, gastrointestinal carboxylesterase, mut The protein is selected from the group consisting of hsp70-2, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, the extracellular portion of APRIL protein, or any combination thereof.

[0072] In some embodiments, the target antigen is selected from a cell surface receptor, an adhesion protein, an integrin, a mucin, a lectin, a tumor-associated antigen, and a tumor-specific antigen. In some embodiments, the target antigen is selected from CD19, B7H3 (CD276), BCMA (CD269), ALPPL2, claudin 18.2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, CLECL1, CS-1, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, and ephrin B. 2, FAP, FLT3, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2 / neu), IGLL1, IL-11Rα, KIT (CD117), MUC1, NCAM, PAP, PDGFR-β, PRSS21, PSCA, PSMA, ROR1, SIRPα, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, and Axl.

[0073] In a further aspect, the present disclosure provides methods of preparing T cells for use in cell therapy. In some embodiments, the methods comprise transducing T cells with a recombinant nucleic acid construct comprising a mutation capable of altering (i) T cell signaling through the NFAT, NF-κB, and / or AP-1 pathways, (ii) cytokine production, and / or (iii) in vivo persistence of T cells in tumors. In some embodiments, the recombinant nucleic acid construct comprises a recombinant nucleic acid construct of the present disclosure.

[0074] In some embodiments, the T cell further comprises a CAR, a TCR, or a transcriptional receptor.

[0075] In a further aspect, the present disclosure provides a method for enhancing the in vivo persistence of T cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of T cells of the present disclosure. In some embodiments, the T cells are selected from the group consisting of regulatory (Treg), natural killer (NK), tumor-infiltrating lymphocytes, and CAR T cells.

[0076] In some embodiments, the subject has cancer or an autoimmune disease. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a hematological cancer.

[0077] Also provided herein is a method for identifying mutations useful for improving T cell therapy, the method comprising: a) identifying mutations from a clonal T cell genome sequencing database; b) determining the frequency of occurrence of the mutations; and c) applying a statistical test to identify significant differences in hotspot genomic regions where the mutations occur at a frequency greater than expected by chance, thereby identifying mutations in the hotspot regions that can improve T cell therapy. In some embodiments, the statistical test comprises using a binomial distribution, chi-square analysis, or any multivariate analysis. In some embodiments, the mutation is a T cell lymphoma mutation. In some embodiments, the hotspot region is in the coding sequence of a gene.

[0078] In some embodiments, the mutation improves T cell therapy by a) increasing proliferation, and / or b) altering effector function, and / or c) resisting T cell dysfunction, and / or d) enhancing the growth of therapeutic T cells comprising the mutation in the tumor. In some embodiments, the mutation is a mutation listed in Table 1. In some embodiments, the mutation promotes positive T cell selection and / or T cell clonal expansion.

[0079] The foregoing summary is illustrative only and is not to be construed as limiting in any way. In addition to the exemplary embodiments and features described herein, further aspects, embodiments, objects, and features of the present disclosure will become more fully apparent from the drawings and detailed description, and from the claims. [Brief explanation of the drawings]

[0080] [Figure 1] FIG. 1 is a schematic diagram showing the disclosed approach for generating screening libraries of mutations identified in T-cell lymphomas and for in vitro and in vivo screening analysis of the libraries.

[0081] [Figure 2A]Figures 2A-2G show detailed signaling results of the in vitro T cell lymphoma mutation screen. Figures 2A-2D show reporter activity Z-scores for NFAT, NF-κb, and AP-1 for each of the indicated T cell lymphoma mutation constructs. In some cases, PD-1 expression and IL-2 Z-scores are also shown. PD-1 expression was assessed by flow cytometry. IL-2 secretion was assessed by ELISA. Z-scores represent the average Z-score of two independent biological replicates. Figure 2A shows reporter activity for each construct when expressed in CD19-CD28z CAR Jurkat cells and cocultured with the K562 (CD19-negative) cell line. Figure 2B shows reporter activity and IL-2 secretion for each construct when expressed in CD19-CD28z CAR Jurkat cells and cocultured with the CD19-K562-positive cell line. Figure 2C shows reporter activity and PD-1 expression for each construct when expressed in CD19-BBz CAR Jurkat cells and co-cultured with K562 (CD19-negative) cell lines. Figure 2D shows reporter activity, IL-2 secretion, and PD-1 expression for each construct when expressed in CD19-BBz CAR Jurkat cells and co-cultured with CD19-K562-positive cell lines. Figure 2E shows a bar graph demonstrating that point mutation and fusion constructs in both CD19-CD28z CAR and CD19-BBz CAR environments exhibited significantly different effects than controls in the CD19-K562 condition compared to the parent K562 (CD19-negative) line. Figure 2F shows a bar graph indicating the number of T cell lymphoma point mutation or fusion constructs whose expression in CD19-BBz CAR Jurkat resulted in different combinations of up- or down-regulation of signaling upon antigen stimulation. Figure 2G shows a bar graph depicting percent NFAT, NF-κB, and AP-1 signaling in CD19-BBz CAR cells upon stimulation with CD19-K562 of selected mutations, highlighting the high tunability of signaling output. Each point represents a biological replicate. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 2E-F] Same as above. [Figure 2G] Same as above.

[0082] [Figure 3] Figure 3 shows the Z-scores of NFAT, AP-1, and NF-κB signaling for mutations that showed statistically significant differences from wild-type controls. Mutations are ranked from positive (top of y-axis) to negative (bottom of y-axis) based on Z-score. This ranking indicates that some mutations induce gain-of-function (increased signaling output compared to wild-type), while others cause loss-of-function (reduced signaling output compared to wild-type).

[0083] [Figure 4] Figure 4 shows tumor growth curves of CD19-K562 tumor-bearing animals treated with PBS or CD19-BBz CAR T cells.

[0084] [Figure 5] Figures 5A and 5B show the correlation between the results of in vivo screening and PD-1 in vitro studies for identified mutations of the present disclosure. Figure 5A shows a waterfall plot of the in vivo screening log2 fold change of each construct compared to baseline (pre-injection) as determined by MAGeCK. A positive log2 fold change indicates increased persistence in the tumor compared to the input. Figure 5B shows a bar graph plotting the in vivo fold change of each mutation, classifying the constructs into two categories: 1) all constructs that did not significantly increase PD-1 expression in the in vitro jurkat screen, or 2) constructs that significantly increased PD-1 expression in the in vitro jurkat screen. These results indicate that in vitro PD-1 expression can be useful in predicting the in vivo persistence of T-cell lymphoma mutations.

[0085] [Figure 6] Figure 6 is a schematic diagram showing signaling by the CARD11-BCL10-MALT1 (CBM) complex in T cells. In normal T cells, T cell receptor (TCR) signaling activates PKCθ, which promotes the assembly of the CBM signalosome. The CBM complex then has three major outputs: NF-κB transcriptional activity, AP-1 transcriptional activity, and MALT1 proteolytic activity.

[0086] [Figure 7] Figure 7 shows a schematic diagram of the CARD11-PIK3R3 fusion of the present disclosure. The top diagram shows the structure and domains of wild-type CARD11, which contains 1,154 amino acids, and the location of the breakpoint where the C-terminal component is missing and the PIK3R3 polypeptide binds in the fusion protein. The middle diagram shows the structure of wild-type PIK3R3, which contains 461 amino acids, and the location of the breakpoint where the C-terminal component of PIK3R3 binds in the fusion protein. The bottom diagram shows a CARD11-PIK3R3 fusion protein with 725 amino acids, which contains the CARD, coiled-coil, and truncated inhibitory domain from the N-terminus of CARD11, and the SH2 domain from the C-terminus of PIK3R3. The CARD11 truncation is located at the position labeled "breakpoint" in the wild-type gene shown in the top structure, and the PIK3R3 truncation is located at the position labeled "breakpoint" in the wild-type gene shown in the middle structure.

[0087] [Figure 8] Figures 8A-B show various CARD11-PIK3R3 fusion variants and their functions. Figure 8A shows the generated CARD11-PIK3R3 fusion variants with various domain deletions. Figure 8B shows the reporter activity of CD19-BBz CAR CARD11-PIK3R3 fusion variants in Jurkat cells co-cultured with CD19-K562.

[0088] [Figure 9] Figures 9A-C illustrate CARD11-PIK3R3 fusion function in the CBM complex. Figure 9A is a diagram of a BCL10-binding-deficient CARD11-PIK3R3 mutant containing an alanine-to-arginine substitution at amino acid position 28. Figure 9B shows CD19-BBz CAR Jurkat reporter activity of a BCL10-binding-deficient CARD11-PIK3R3 mutant compared to control and CARD11-PIK3R3-expressing cells. **** indicates a P value <0.0001 as determined by one-way ANOVA followed by Tukey's multiple comparison test. Figure 9C shows Western blotting of MALT1 substrates in CD19-BBz CAR Jurkat cells expressing CARD11-PIK3R3 or control cells. P / I indicates phorbol myristate acetate / ionomycin treatment. Ct indicates C-terminal truncation product. Data represent two independent experiments.

[0089] [Figure 10] Figures 10A-B show the phosphorylation signaling kinetics of the CARD11-PIK3R3 fusion. Figure 10A shows a schematic illustrating the experimental approach for studying CAR-dependent signaling in primary human CD3+ T cells according to the present disclosure. Figure 10B shows a heat map of marker expression determined by time-of-flight mass cytometry (CyTOF) for each of the indicated time points in CD19-BBz CAR samples. Values ​​represent the average of three independent T cell donors.

[0090] [Figure 11]Figures 11A-C show the CARD11-PIK3R3 fusion transcriptional landscape. Figure 11A shows principal component (PC) analysis of human CD8+ T cells from three independent donors transduced with the indicated constructs and left unstimulated (unstimulated) or cocultured with CD19-expressing A549 cells for 8 hours (stimulated). Figure 11B shows a volcano plot of differentially expressed genes in CD19-BBz CAR T cells either unstimulated or stimulated with CD19 antigen. Select genes of interest are labeled. Positive log2 fold changes indicate higher expression in CARD11-PIK3R3-expressing CAR-T cells. Figure 11C shows enrichment of NF-κB, AP-1, and MALT1 gene signatures in CARD11-PIK3R3-expressing CD19-BBz CAR cells after stimulation with CD19-expressing A549 cells.

[0091] [Figure 12] Figures 12A-B show the results of an experiment on CD3+ T cell growth after anti-CD3 / anti-CD28 bead stimulation. Cell counts of CD19-BBz CAR (Figure 12A) or CD19-CD28z CAR (Figure 12B) CD3+ T cells over a 9-day period after removal of anti-CD3 / anti-CD28 bead stimulation. Ns indicates no significant difference, and * indicates a P value < 0.05.

[0092] [Figure 13] Figures 13A-B show CD3+ cytokine secretion after coculture with CD19-K562. Cytokine secretion profiles of CD19-BBz CAR (Figure 13A) or CD19-CD28z CAR (Figure 13B) CD3+ T cells cocultured with CD19-K562 at a 1:1 ratio for 48 hours. P values ​​were determined by a paired t-test using ratios. Ns indicates no significant difference, * indicates a P value < 0.05, and ** indicates a P value < 0.01.

[0093] [Figure 14]Figure 14 shows activation and cytokine secretion of CD8+ T cells after co-culture with CD19-K562. Figure 14A shows activation markers expressed by CD19-BBz CAR T cells with or without CARD11-PIK3R3 after 24 hours of 1:1 co-culture with CD19-K562. Figure 14B-14A shows activation markers expressed by untransduced or CARD11-PIK3R3-transduced CD8+ T cells after 24 hours of 1:1 co-culture with CD19-K562. Figure 14C shows cytokine secretion of CD8+ T cells expressing CD19-BBz CAR with or without CARD11-PIK3R3 after 48 hours of 1:1 co-culture with CD19-K562. P values ​​were determined by a paired t-test with proportions. NS indicates no significant difference, and * indicates a P value < 0.05.

[0094] [Figure 15] Figures 15A-B show the results of experiments on long-term coculture persistence, killing, and multi-stimulus growth. Figure 15A is a flow plot showing CD19-BBz CAR-transduced CD8+ T cell and CD19-K562 populations after 14 days of coculture with and without IL-2 supplementation; one donor is shown. Bar graphs summarize population percentages obtained from three donors. P values ​​were determined by unpaired t-test. Figure 15B shows cell counts of CD19-BBz CAR-transduced CD8+ T cells cocultured with CD19-K562 on day 0 and restimulated with targets on day 6. P values ​​were determined by unpaired t-test. Ns indicates no significant difference. * indicates P value <0.05, and **** indicates P value <0.0001.

[0095] [Figure 16]Figures 16A and B show cytotoxicity at various effector-to-target ratios. Growth of CD19-A549 mKate2+ targets co-cultured with CD19-BBz CAR (A) or CD19-CD28z CAR (B) CD8+ T cells over a 108-hour period. Bars represent target cell counts at 108 hours, normalized to the control. P values ​​were calculated by one-way ANOVA followed by Tukey's multiple comparison test. ** indicates a P value < 0.01, and **** indicates a P value < 0.0001.

[0096] [Figure 17] Figure 17 shows a schematic diagram of the xenograft CAR leukemia model used to evaluate the therapeutic function of CARD11-PIK3R3 in T cells in vivo. In this model, NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ (NSG) mice are administered 5x10 Nalm6-GFP-luciferase cells via tail vein injection. Primary human CD3+ T cells are transduced to express CD19-BBz CAR (with or without co-expression of CARD11-PIK3R3) and electroporated with TRAC RNP to knock out human TCR expression. TRAC RNP is a CRISPR / Cas9 ribonucleoprotein complex targeting the human T cell receptor alpha constant region gene. Nalm6-bearing mice are administered 1x10 CD19-BBz CAR T cells, and tumor burden is assessed twice weekly by bioluminescence imaging using an IVIS imaging system. Mice are euthanized at the first sign of hind limb paralysis.

[0097] [Figure 18]Figures 18A-E show that CD19-BBz CAR expressing a CARD11-PIK3R3 fusion controls Nalm6 leukemia in vivo. Figure 18A shows the average brightness of luciferase-expressing Nalm6 tumor cells measured using in vivo imaging, where brightness is used as an approximation of tumor burden. Data represent the mean ± standard deviation. P values ​​were determined using an unpaired t-test of the average brightness values ​​at day 21. Control (n = 5 mice), CAR (n = 6 mice), CAR + CARD11-PIK3R3 (n = 5 mice). ** indicates a P value < 0.01. Figure 18B shows survival analysis of Nalm6-bearing mice treated with untransduced (control) (n = 5 mice), BBz CAR (n = 6 mice), or BBz CAR with CARD11-PIK3R3 (n = 5 mice) T cells. P values ​​were determined using the log-rank (Mantel-Cox) test. *** indicates a P value <0.001.

[0098] [Figure 19] Figure 19 is a schematic diagram of the syngeneic CAR melanoma model used to evaluate CARD11-PIK3R3 therapeutic function in CD19-BBz CAR T cells in vivo. hCD19-B16 = B16 tumor cells expressing human CD19. OT-I cells = T cells harvested from C57BL / 6-Tg(TcraTcrb)1100Mjb / J mice.

[0099] [Figure 20]Figures 20A-C show the results of experiments using CD19-BBz CAR with a CARD11-PIK3R3 fusion in controlling hCD19 B16 tumors. Figure 20A shows the accumulation of CD19-BBz CAR T cells in the spleen and tumor 5 days after adoptive cell transfer. P values ​​were determined using an unpaired Mann-Whitney t-test. Figures 20B and C show tumor volume (B) and survival analysis (C) of CD19-B16 tumor-bearing animals treated with non-transduced (control) (n=5 animals), BBz CAR (n=5 animals), or BBz CAR with CARD11-PIK3R3 (n=5 animals) OT-I T cells. P values ​​were determined using the log-rank (Mantel-Cox) test. * indicates a P value <0.05, and ** indicates a P value <0.01.

[0100] [Figure 21] Figure 21 shows a schematic diagram depicting the syngeneic CAR mesothelioma model used to evaluate CARD11-PIK3R3 therapeutic function in CAR T cells in vivo. hALPPL2-40L = tumor cells expressing hALPPL2.

[0101] [Figure 22] Figure 22 shows tumor growth curves of 40L (mesothelioma) tumors expressing hALPPL2 that were injected subcutaneously and treated with non-transduced, ALPPL2-BBz CAR, or ALPPL2-BBz CAR T cells with CARD11-PIK3R3. The data show that ALPPL2-BBz CAR T cells with CARD11-PIK3R3 expression induce some tumor control in the 40L model.

[0102] [Figure 23] Figure 23 is a schematic diagram showing the syngeneic TCR transgenic melanoma model used to evaluate CARD11-PIK3R3 therapeutic function in T cells in vivo. OT-I CD8+ T cells = CD45.1 T cells harvested from C57BL / 6-Tg(TcraTcrb)1100Mjb / J mice.

[0103] [Figure 24] Figure 24 shows that CARD11-PIK3R3 OT-I cells preferentially expand and accumulate in tumors. This shows the accumulation of TILs in a dual-transfer competition assay 7 days after T cell transfer. Figure 24A shows the expansion by flow cytometry plot. Figure 24B quantifies the fold expansion of CARD11-PIK3R3 OT-I in tumors in vivo relative to the control. P values ​​were determined by a paired T-test with ratios. **** indicates a P value of <0.0001.

[0104] [Figure 25] Figure 25 shows the accumulation of CARD11-PIK3R3 OT-I in tumors, spleens, and draining lymph nodes 7 days after transfer of 1 x 10 CARD11-PIK3R3 or control OT-I. P values ​​were determined by unpaired T-test. * indicates a P value of < 0.05, and **** indicates a P value of < 0.0001.

[0105] [Figure 26] Figure 26A shows TCF-1 expression in OT-I + CARD11-PIK3R3 TILs. TCF-1 expression in TIL cells 7 days after transfer of 1 x 10 CARD11-PIK3R3 or control OT-I. Figure 26B shows the frequency of TCF-1+ OT-I cells in the spleen and tumor-draining lymph nodes (LN) of mice bearing B16-OVA subcutaneous tumors. P values ​​were determined by unpaired T-test. ns indicates no significant difference, and *** indicates a P value < 0.001.

[0106] [Figure 27]Figure 27 shows that CARD11-PIK3R3 OT-I has improved functionality in vivo. This shows the ex vivo cytokine production of TILs isolated 7 days after transfer of 1x10 CARD11-PIK3R3 or control OT-I and restimulated ex vivo with PMA / ionomycin. P values ​​were determined by unpaired T-test. * indicates P value < 0.05, and *** indicates P value < 0.001.

[0107] [Figure 28] Figures 28A and 28B show that CARD11-PIK3R3 OT-I exhibits improved antitumor efficacy in vivo. Figure 28A shows tumor size and Figure 28B shows survival analysis in B16-OVA melanoma-bearing mice treated with PBS or OT-I cells (2x106) 12 days after tumor inoculation. A complete response was defined as the absence of detectable tumors. **** indicates a P value of <0.0001.

[0108] [Figure 29] Figures 29A and 29B show that CARD11-PIK3R3 OT-I exhibits improved functionality in vivo at low doses and upon tumor rechallenge. Tumor size in B16-OVA melanoma-bearing mice treated with OT-I CARD11-PIK3R3 T cells at a T cell dose of 1 x 10 (n = 4) or 2 x 10 (n = 5) compared with 2 x 10 control OT-I T cells (n = 5) (A), and in mice from a dose-response experiment that had tumor clearance and were rechallenged with a B16-OVA melanoma tumor subcutaneously in the opposite flank (B). *** indicates a P value of <0.001, and **** indicates a P value of <0.0001.

[0109] [Figure 30]

[0033] Figures 30A-D show the results of experiments to evaluate the CARD11-PIK3R3 truncated form in CD8+ CD19-BBz CAR T cells in vitro. Figure 30A is a schematic showing the CARD11-PIK3R3 coiled-coil truncation design, where ΔID indicates removal of the inhibitory domain and -XX AA indicates the number of amino acids removed from the coiled-coil region. Figure 30B shows cell counts of CD19-BBz CAR CD8+ T cells harboring the CARD11-PIK3R3 truncated form over a 7-day period after removal from anti-CD3 / anti-CD28 bead stimulation. Figure 30C shows CD19-BBz CAR CD8+ T cells harboring the CARD11-PIK3R3 truncated form co-cultured with CD19 target cells at a 1:1 ratio for 24 hours, after which supernatants were assessed for IL-2 secretion by ELISA. FIG. 30D shows flow cytometry plots showing CD19-BBz or fusion variant CD8+ T cells with CARD11-PIK3R3, as well as CD19-K562 populations after 14 days of co-culture without IL-2 supplementation.

[0110] [Figure 31] Figure 31A shows a lollipop diagram of STAT3 gene point mutations. Figure 31B shows the in vivo screening log2 fold change determined by MAGeCK for each STAT mutation and wild type compared to baseline (before injection). A positive log2 fold change indicates increased persistence in the tumor compared to input.

[0111] [Figure 32] Figure 32A shows a lollipop diagram showing BRAF gene point mutations. Figure 32B shows the CD19-BBz CAR Jurkat reporter activity Z-scores for NFAT, NF-κB, AP-1, and IL-2 for each BRAF mutation or wild-type after co-culture with CD19-K562. The Z-scores represent the average Z-scores of two independent biological replicates.

[0112] [Figure 33]Figure 33 shows CARD11 mutations. Figure 33A shows the CD19-BBz CAR Jurkat reporter activity Z-scores for NFAT, NF-κB, AP-1, and IL-2 for each CARD11 mutation or wild-type after co-culture with CD19-K562. The Z-scores represent the average Z-scores of two independent biological replicates. Figure 33B shows the in vivo screening log2 fold change, as determined by MAGeCK, for each CARD11 mutation or wild-type, compared to baseline (before injection). A positive log2 fold change indicates increased persistence in the tumor compared to the input.

[0113] [Figure 34] Figure 34A shows a lollipop diagram showing RASGRP1 gene point mutations. Figure 39B shows CD19-BBz CAR Jurkat reporter activity Z-scores for NFAT, NF-κB, AP-1, and IL-2 for each RASGRP1 mutation or wild-type after co-culture with CD19-K562. Z-scores represent the average Z-score of two independent biological replicates. Figure 39C shows the in vivo screening log2 fold change, as determined by MAGeCK, for each RASGRP1 mutation or wild-type, compared to baseline (before injection). A positive log2 fold change indicates increased persistence in tumors compared to input.

[0114] [Figure 35] Figure 35A shows a lollipop diagram illustrating various PLCG1 gene point mutations. Figure 35B shows a graph showing the CD19-BBz CAR Jurkat reporter activity Z-scores of NFAT, NF-κB, AP-1, and IL-2 for each PLCG1 mutation after co-culture with CD19-K562. The Z-scores represent the average Z-scores of two independent biological replicates.

[0115] [Figure 36]Figure 36A is a lollipop diagram showing TNFRSF1B gene point mutations. Figure 36B shows a bar graph illustrating the CD19-BBz CAR Jurkat reporter activity Z-scores for NFAT, NF-κB, AP-1, and IL-2 for each TNFRSF1B mutation or wild-type after co-culture with CD19-K562. The Z-scores represent the average Z-scores of two independent biological replicates. Figure 36C shows the in vivo screening log2 fold change, as determined by MAGeCK, for each TNFRSF1B mutation or wild-type, compared to baseline (before injection). A positive log2 fold change indicates increased persistence in the tumor compared to input.

[0116] [Figure 37] Figure 37A is a lollipop diagram showing JAK / JAK3 gene mutations. Figure 37B shows the in vivo screening log2 fold change as determined by MAGeCK for each JAK1 / JAK3 mutation compared to baseline (pre-injection). A positive log2 fold change indicates increased persistence in the tumor compared to input.

[0117] [Figure 38] Figure 38 shows NF-κB reporter activity of control, CARD11, or BCL10 CRISPR knockout BBz-CAR Jurkat cells.

[0118] [Figure 39A-B]Figure 39A shows principal component analysis of the transcriptome of human BBz-CAR T cells. Figure 39B is a heatmap of genes in CARD11-PIK3R3 that are significantly upregulated compared to control BBz-CAR T cells after stimulation with CD19 antigen, which is common between both CD4+ and CD8+ T cells. Figure 39C shows the top five reactome pathways enriched in CD4+ and CD8+ T cells (top), as well as NF-kB, AP-1, and MALT1 gene signature enrichment in CD8+ BBz-CAR T cells expressing CARD11-PIK3R3 after stimulation (bottom). [Figure 39C] Same as above.

[0119] [Figure 40] Figure 40 shows activation markers expressed by transduced CD4 and CD8 T cells 24 hours after 1:1 co-culture with CD19-K562. MFI ratios for CARD11-PIK3R3 compared to control are shown. P values ​​were determined by unpaired T-test.

[0120] [Figure 41] Figure 41 shows cytokine secretion of CD4+ CD19-BBz-CAR T cells and CD19-BBz-CAR+ CARD11-PIK3R3 T cells after 48 hours of stimulation.

[0121] [Figure 42A]Figure 42A shows control (n=6 mice), CD19-BBz-CAR (n=7 mice), CD19-BBz-CAR + CARD11-PIK3R3 (n=7 mice) dosed with 7e6 CAR+ T cells. The dose of CARD11-PIK3R3 T cells (n=7 mice) was equivalent to the total number of CARD11-PIK3R3-expressing T cells dosed in the CD19-BBz-CAR + CARD11-PIK3R3 group. Figure 42B shows control (n=7 mice), CD19-CD28z-CAR (n=7 mice), CD19-CD28z-CAR + CARD11-PIK3R3 (n=7 mice) T cells dosed with 4e5 CAR+ T cells. Figure 42C shows survival analysis of surviving CD19-CD28z-CAR+CARD11-PIK3R3 (n=6) animals or naive mice (n=4) from part (b) re-challenged with 5e5 Nalm6-Luc-GFP tumors. Figure 42D shows tumor volumes of M28 tumor-bearing animals treated with control (n=5), MCAM-CD28z-CAR (n=5), or MCAM-CD28z-CAR+CARD11-PIK3R3 (n=5). T cells were dosed at 5e5 CAR+ cells, with control cells dosed equivalent to the highest total T cell dose in the other treatment groups. [Fig. 42B-C] Same as above. [Figure 42D] Same as above.

[0122] [Figure 43A]Figure 43A shows flow cytometry plots showing CAR (FLAG) and CARD11-PIK3R3 (mCherry) expression in human CD3 T cells. Figures 43B and 43C show the percent body weight change from baseline in tumor-bearing animals (Figure 43B) or non-tumor-bearing animals (Figure 43C) treated with control, CD19-BBz-CAR, CD19-BBz-CAR+CARD11-PIK3R3, or CARD11-PIK3R3. Figure 43D shows survival analysis of surviving CD19-BBz-CAR (n=3), CD19-BBz-CAR+CARD11-PIK3R3 (n=4) animals, or naive mice (n=4) from Figure 42B that were re-challenged with 5e5 Nalm6-Luc-GFP tumors. P values ​​were determined by the log-rank (Mantel-Cox) test. * indicates a P value <0.05. [Figure 43B] Same as above. [Figure 43C] Same as above. [Figure 43D] Same as above.

[0123] [Figure 44A] Figures 44A and 44B show flow cytometry plots showing FLAG (CAR) and mCherry (CARD11-PIK3R3) expression in human CD3 T cells. Figure 44C shows the percent body weight change from baseline in M28 tumor-bearing animals treated with control, MCAM-CD28z-CAR, or MCAM-CD28z-CAR+CARD11-PIK3R3. [Figure 44B] Same as above. [Figure 44C] Same as above.

[0124] [Figure 45]Figure 45 shows tumor size in SNU-1 HLA-C*08:02 gastric cancer xenograft-bearing mice treated with control (n=5 mice), KRAS p.G12D-specific TCR T cells (n=5 mice), or KRAS p.G12D-specific TCRCARD11-PIK3R3 T cells (n=6 mice). A complete response was defined as the absence of detectable tumor. * indicates a P value <0.05, *** indicates a P value <0.001, and **** indicates a P value <0.0001.

[0125] [Figure 46] Figures 46A, 46B, and 46C show in vitro expansion of CD19-BBz-CAR T cells with and without CARD11-PIK3R3. Figures 46A and 46B show CAR or CAR + CARD11-PIK3R3 sorted for purity and expanded for 12 days with IL-2. On day 12,

[0126] Cultures were split and each group was replated without (Figure 46A) and with (Figure 46B) IL-2. Cells were counted and split from day 12 to day 30. Figure 46C shows that on day 30, CD19-BBz-CAR+CARD11-PIK3R3 T cells cultured with IL-2 were replated without IL-2, counted, and split for an additional 10 days.

[0127] [Figure 47] Figure 47 shows an in vivo analysis of CAR T cells expressing CARD11-PIK3R3. Histograms are shown showing human CD19 ligand expression in hCD19-B16 tumors that reached the euthanasia endpoint and were treated with CD19-BBz-CAR or CD19-BBz-CAR+CARD11-PIK3R3, compared to known CD19-positive B16 tumor samples.

[0128] [Figure 48A]Figures 48A, 48B, and 48C show in vivo analysis of OT-I T cells expressing CARD11-PIK3R3. Figure 48A shows a schematic diagram of a competition experiment with CARD11-PIK3R3 and CARD11-PIK3R3 R28A pmel-1 CD8 T cells in a B16-F10 tumor model. Figure 48B shows tumor growth curves for the mice described in (a). Figure 48C shows the fold enrichment in tumors of pmel-1 CD8 T cells expressing wild-type CARD11-PIK3R3 or CARD11-PIK3R3(p.R28A) compared to vector controls 7 days after adoptive transfer. All P values ​​were determined by a paired T-test with proportions. [Fig. 48B-C] Same as above.

[0129] [Figure 49A-C]Figures 49A-49J show the long-term evaluation of B6 mice treated with OT-I T cells expressing CARD11-PIK3R3. Figure 49A shows that mice that cleared B16-F10-OVA were monitored for up to 240 days after adoptive T cell transfer. Necropsies were performed as outlined in this schematic. Figure 49B shows that the weights of all CARD11-PIK3R3-injected mice from Figures 29A-29B were measured weekly and compared to the predicted weight curve published by The Jackson Laboratory Research Institute. Figure 49C shows the spleen weights of three animals that underwent necropsy on day 240. This was calculated as a percentage of body weight and compared to the predicted spleen weight published by The Jackson Laboratory Research Institute. Figure 49D shows the necropsy of one representative animal. None of the three animals had gross pathology. Figure 49E shows the percentage of CD8 T cells expressing CARD11-PIK3R3 in the spleen and blood 240 days after adoptive transfer. Figure 49F shows representative hematoxylin and eosin-stained tissue sections from selected organs where nodal or extranodal lymphomas may arise. Animals were subjected to general dissection. Tissues showed no evidence of nuclear atypia, altered cellular architecture, or the presence of neoplastic disease. Representative images at low (left) and high (right) magnifications are shown with size bars. The white box reflects the area of ​​the high-magnification image. Figures 49G and 49I show schematic diagrams of blood collection for the mice shown in Figures 29A-29B at 330 days after adoptive transfer (Figure 49G) or mice treated with 2e6 OT-I CARD11-PIK3R3 at 418 days after adoptive transfer (Figure 49I). Figures 49H and 49J show the percentage of CD8 T cells in the blood that express CARD11-PIK3R3. [Figure 49D-E] Same as above. [Figure 49F] Same as above. [Figure 49G-H] Same as above. [Figure 49I-J] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0130] In the following detailed description, reference is made to the drawings, which form a part of this specification. In the drawings, like symbols generally identify like elements unless context dictates otherwise. The exemplary 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 herein. It will be readily understood that the aspects generally described herein and illustrated in the drawings may be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are expressly contemplated and made a part of this application. Detailed Description of the Disclosure overview

[0131] In general, the present disclosure relates to, among other things, compositions and methods for improving adoptive T cell therapy. The present inventors have discovered a means to improve the therapeutic efficacy of T cells by altering T cell signaling, reducing T cell exhaustion, and / or enhancing the in vivo persistence and fitness of engineered T cells. T cell cancers can undergo positive selection for beneficial genetic alterations (e.g., mutations). In some embodiments, the present disclosure relates to exploring such beneficial mutations to improve the efficacy of T cell therapeutics.

[0132] The present disclosure also relates to polypeptides having one or more mutations that can alter, promote, or enhance signal transduction in T cells. The present disclosure also relates to recombinant nucleic acid constructs and / or recombinant nucleic acids encoding polypeptides having one or more mutations that can alter, promote, or enhance signal transduction in T cells. Non-limiting examples of such mutations are shown in Table 1. Non-limiting examples of signal transduction in T cells include 1) CARD11-BCL10-MALT1 complex signaling, 2) JAK / STAT signaling, 3) costimulatory molecule signaling, 4) RAS / MEK / ERK signaling, 5) phospholipase gamma signaling, 6) transcription factor activity and / or other signaling pathways.

[0133] In some embodiments, the mutation alters TCR transcriptional signaling output, hi some embodiments, the mutation inhibits or reduces TCR transcriptional signaling output, e.g., NFAT, NF-κB, or AP-1 signaling, or cytokine output (e.g., IL-2).

[0134] The present 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 compatibility, enhance in vivo persistence, and / or increase intratumoral presence of therapeutic T cells. The present disclosure further relates to recombinant nucleic acid constructs and / or 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 cell compatibility, enhance in vivo persistence, and / or increase intratumoral presence of therapeutic T cells.

[0135] Reprogramming T cells to proliferate, produce cytokines, and differentiate into effector cells after T cell receptor complex engagement can depend on activating costimulatory signals and can be countered by co-inhibitory molecules. Transcription factors NF-κB, NFAT, and AP-1 play key roles in inducing the transcriptional programs required for T cell activation and differentiation. Measuring the expression of such transcription factors is within the scope of the disclosed methods and can be an indicator of major changes in T cell function related to the disclosed mutations. definition

[0136] Unless otherwise defined, all technical terms, notations, and other scientific terms or terminology used herein are intended to have the meaning commonly understood by those skilled in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein is not necessarily interpreted as representing a substantial difference from what is commonly understood in the art. Many of the techniques and procedures described or referenced herein are well understood by those skilled in the art and are commonly utilized using conventional methods. 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.

[0137] The singular forms "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."

[0138] Where a range of values ​​is provided, unless the context clearly indicates otherwise, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of that range, or 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 within the smaller ranges and are also encompassed within the disclosure, provided that any upper or lower limit in the stated range is specifically excluded. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the disclosure.

[0139] Certain ranges are presented herein with the term "about" before the numerical value.The term "about" is used herein to provide literal support for the exact number immediately following it, as well as for numbers that are close or approximate to the number immediately following it.When determining whether a number is close or approximate to a specifically listed number, the close or approximate unlisted number may be a number that, in the context in which it is presented, provides a number that is substantially equivalent to the specifically listed number.For example, "about" may mean, according to the practice in the art, within 1 or more than 1 standard deviation.Alternatively, "about" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value.Alternatively, particularly with respect to biological systems or processes, this term may mean within one order of magnitude, preferably within 5 times, and more preferably within 2 times of a value.

[0140] As will be understood by those skilled in the art, for any and all purposes, e.g., with respect to providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges. It can be readily recognized that all recited ranges fully describe and allow for the same range to be broken down into at least one-half, one-third, one-quarter, one-fifth, one-tenth, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, and upper third, etc. Also, as will be understood by those skilled in the art, all terms such as "up to," "at least," "greater than," and "less than" are inclusive of the recited numbers and refer to ranges that can be subsequently broken down into subranges as described above. Finally, as will be understood by those skilled in the art, ranges include each individual member. Thus, for example, a group having 1 to 3 elements refers to groups having 1, 2, or 3 elements. Similarly, a group having 1 to 5 elements refers to groups having 1, 2, 3, 4, or 5 elements, etc.

[0141] Aspects and embodiments of the present disclosure described herein are understood to include "comprising," "consisting of," and "consisting essentially of" aspects and embodiments. As used herein, the term "comprising" is used interchangeably with "including," "containing," or "characterized by," is inclusive or expansive, and does not exclude additional, unstated elements or method steps. As used herein, the phrase "consisting of" excludes any element, step, or ingredient not recited 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. In particular, any recitation herein of the term "comprising" in a description of a component of a composition or a description of a step of a method is understood to encompass compositions and methods that consist essentially of and consist of the recited components or steps.

[0142] 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 the same nucleotides or amino acids (e.g., about 60% sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity over a specified region when compared and aligned for maximum correspondence over the comparison window or specified region) as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection. See, e.g., the NCBI website, ncbi.nlm.nih.gov / BLAST. Such sequences are therefore "substantially identical." This definition also refers to or can be applied to the complement of a test sequence. This definition 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.

[0143] 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, University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wis. 53705), using its default parameters.

[0144] As used herein, the term "mutation" refers to point mutations, gene fusions, substitutions, gain-of-function mutations, gain-of-stop mutations, insertion mutations, deletion mutations, duplication mutations, and / or translocations. Mutations can be in one or more genes. Mutations can be naturally occurring. Alternatively, mutations can be induced or engineered. As used herein, the term "vector" refers to a recombinant polynucleotide construct designed for transfer between host cells and can be used for transformation, e.g., introducing heterologous DNA into a host cell. Thus, in some embodiments, a vector can be a replicon, e.g., a plasmid, phage, or cosmid, into which another DNA segment can be inserted to result in replication of the inserted segment. In some embodiments, an expression vector can be an integrating vector.

[0145] As used herein, the term "viral vector" generally refers to either a nucleic acid molecule (e.g., a transfer plasmid) containing viral-derived nucleic acid elements that facilitate the transfer or integration of a nucleic acid molecule into a cellular genome, or a viral particle that mediates nucleic acid transfer. Viral particles generally contain various viral components and may contain host cell components in addition to the nucleic acid. The term viral vector can refer to either a virus or viral particle that can transfer a nucleic acid to a cell, or the nucleic acid itself that is to be transferred. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from viruses. Viral vectors that can be used in the present disclosure include, for example, retroviral vectors, adenoviral vectors and adeno-associated viral vectors, lentiviral vectors, herpesviruses, simian virus 40 (SV40), and bovine papillomavirus vectors (see, e.g., Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY). For example, the recombinant polypeptides disclosed herein can be produced in eukaryotic hosts, such as mammalian cells (e.g., COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from a number of suppliers, including the American Type Culture Collection (Manassas, VA). When selecting an expression system, care must be taken to ensure that the components are compatible with each other. Those skilled in the art are able to make such decisions. Furthermore, for guidance regarding the selection of an expression system, those skilled in the art can refer to P. Jones, "Vectors: Cloning Applications", John Wiley and Sons, New York, NY, 2009).

[0146] As used herein, the term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof derived primarily from retroviruses. The retroviral vector may be a lentiviral vector. As used herein, the term "lentiviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof, including LTRs derived primarily from the retrovirus genus lentivirus. Lentiviral vectors offer several attractive properties as gene delivery vehicles, including: (i) sustained gene delivery through stable vector integration into the host genome; (ii) the ability to infect both dividing and non-dividing cells; (iii) broad tissue tropism, including cell types that are important targets for gene and cell therapy; (iv) no viral protein expression after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) potentially safer integration site profiles; and (vii) a relatively simple system for vector manipulation and production.

[0147] As used herein, the term "pharmaceutically acceptable carrier" refers to any suitable carrier, diluent, or excipient. These include all aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, and solutes that render the composition isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic and absorbing agents, and the like. It will be understood that the compositions of the present disclosure may also contain other auxiliary physiologically active agents. A carrier must be pharmaceutically "acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to the subject.

[0148] As used herein, the term "PEGylation" refers to modifying a protein by covalently attaching polyethylene glycol (PEG) to the protein, and "PEGylated" refers to a protein to which PEG has been attached. PEG or PEG derivatives of a size range, optionally ranging from about 10,000 daltons to about 40,000 daltons, can be attached to recombinant polypeptides of the present 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, 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 1,150 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, hi some embodiments, the average molecular weight of the PEG or PEG derivative is about 40 kD.

[0149] As used herein, the terms "administration" and "administering" refer to the delivery of a bioactive composition or formulation by a route of administration, including, but not limited to, oral, intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, and topical administration, or a combination thereof. This term includes, but is not limited to, administration by a healthcare professional and self-administration.

[0150] 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 intrasternal injection and infusion.

[0151] The term "cancer" generally refers to the presence of cells that have characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic phenotypic properties. Cancer cells can be in the form of a tumor, but such cells may exist alone in an animal subject or may be non-tumorigenic cancer cells, such as leukemia cells. These terms include solid tumors, soft tissue tumors, or metastatic lesions. As used herein, the term "cancer" includes pre-malignant as well as malignant cancers. In some embodiments, the cancer is a solid tumor, soft tissue tumor, or metastatic lesion.

[0152] 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 present disclosure generally refers to an amount or number of the construct, nucleic acid, cell, or composition sufficient to achieve the stated purpose, e.g., providing a therapeutic benefit in the treatment or management of cancer or delaying or minimizing one or more symptoms associated with cancer, compared to the absence of the composition. A therapeutically effective amount of a compound refers to an amount of a therapeutic agent, alone or in combination with other therapeutic agents, that provides a therapeutic benefit in the treatment or management of cancer. The term "therapeutically effective amount" can encompass an amount that improves overall treatment, reduces or avoids the symptoms or causes of cancer, or enhances the therapeutic effectiveness of another therapeutic agent. An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or reduction of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount." A "reduction" of a symptom refers to a decrease in the severity or frequency of the symptom, or elimination of the symptom. The exact amount of a composition that comprises a "therapeutically effective amount" will depend on the purpose of the treatment, and can be ascertained 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).

[0153] As used herein, "subject" or "individual" includes animals, e.g., humans (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, a subject may be a human patient or individual who has, is at risk of, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. A subject may also be an individual who is diagnosed as being at risk for a condition of interest at the time of diagnosis or thereafter. The term "non-human animal" includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, e.g., non-human primates, as well as non-mammals, e.g., sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0154] Headings, such as (a), (b), (i), etc., are provided solely for ease of reading the specification and claims. The use of headings in the specification or claims does not require that the steps or elements be performed in the alphabetical or numerical order in which they are presented. It is understood that certain features of the present disclosure, which are described for clarity in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the present disclosure, which are described for brevity in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of the embodiments pertaining to the present disclosure are specifically embraced by the present disclosure and are disclosed herein as if each and every combination were individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are specifically embraced by the present disclosure and are disclosed herein as if each and every such subcombination were individually and explicitly disclosed herein. TCR Signaling

[0155] Upon peptide-MHC complex binding, immunoreceptor tyrosine-based activation motifs (ITAMs) contained within the intracellular tail of the TCR complex become phosphorylated and recruit Zap70. Zap70 then recruits and activates the downstream LAT signalosome, which includes LAT, PLCy, and Slp76. This signalosome activates Ras / MAPK / ERK signaling, which leads to AP-1 transcriptional activation, PKCθ activation, which leads to NF-κB transcriptional activation, and ultimately calcium influx, which is responsible for NFAT transcriptional activation. In addition, signaling from costimulatory receptors, such as 41BB, and cytokine support from IL-2 enhance these signaling pathways, which are necessary for T cells to undergo full activation and avoid anergic cell death. Once fully activated, T cells rapidly proliferate and differentiate, secrete proinflammatory cytokines, and cytotoxic cells begin to execute target killing through the release of cytotoxic granules. After resolution of this acute response, activated T cells undergo contraction to form a small population of long-lived memory T cells that monitor the body and prepare to respond to future antigen encounters. T Cell Signaling

[0156] T cells become fully activated through T cell receptor (TCR) engagement, costimulatory signaling, and cytokine support. TCR activation induces NFAT, NF-κB, and AP-1 transcription factor signaling and secretion of IL-2 and other proinflammatory cytokines. T cell exhaustion

[0157] T cell exhaustion is a state of hyporesponsiveness induced in effector T cell populations after chronic antigen exposure. Exhaustion is characterized by upregulation of surface inhibitory receptors, such as PD-1, TIM-3, LAG-3, and CTLA-4, among others, as well as prolonged proliferation, secretion of inflammatory cytokines, and an inability to effectively kill target cells. Exhausted T cells exist in distinct transcriptional and epigenetic states. Exhaustion has been well characterized in chronic viral infections but is also implicated in cancer, where tumor-infiltrating T cells and engineered T cell therapies exhibit signs of exhaustion and are unable to control tumor growth. To better treat chronic infections or cancer, it is necessary to prevent or overcome T cell exhaustion. Indeed, checkpoint therapies that block signaling from the PD-1 or CTLA-4 inhibitory axis have proven somewhat effective in restoring T cell responses in certain cancers, resulting in limited but effective tumor control. NFAT Signaling:

[0158] The transcription factor nuclear factor of activated T cells (NFAT) is involved in the program of T cell activation as well as T cell exhaustion. In the resting state, the NFAT transcription factor is found in the cytoplasm in a phosphorylated state and is unable to translocate to the nucleus to induce signal transduction. Ca generated during T cell activation 2+The extracellular influx of NFAT activates the calcium-dependent phosphatase calcineurin, leading to the dephosphorylation of NFAT and subsequent translocation to the nucleus and signal transduction there. NFAT binds to DNA in multiple ways, as a monomer, a dimer, or a complex with other transcription factors. Specifically, NFAT is known to bind cooperatively to the transcription factor AP-1 and induce the transcription of activation-related genes crucial for effector T cell function. In contrast, when NFAT is not bound to AP-1 and instead acts without a partner, it has been found to induce the expression of exhaustion-related genes, such as the inhibitory receptor PD-1. In addition, studies have shown that the exhaustion induced by unattached NFAT can be reversed in CAR T cells by overexpressing the AP-1 family member cJun. NFAT induces the expression of the transcription factors TOX and NR4A1 / 2 / 3, which are associated with T cell exhaustion, and deletion or knockdown of these transcription factors improved T cell phenotype and in vivo tumor control.

[0159] Because NFAT signaling is not only important for successful T cell activation but also plays a role in exhaustion, particularly in the absence of AP-1, this can be used to determine how genetic modifications in T cells affect NFAT signaling dynamics. Targeting the NFAT transcriptional pathway may be beneficial for T cell therapeutics. NF-κB signaling:

[0160] NF-κB (nuclear factor kappa-light-chain-enhancer of B cells) is a family of transcription factors that induce transcriptional programs critical for T cell activation and effector function. NF-κB signaling occurs in many cell types. In T cells, NF-κB signaling induces a broad transcriptional program responsible for proliferation and memory formation, resistance to apoptosis, cytokine secretion, and the generation of potent effector T cell responses. NF-κB signaling is induced through two pathways: canonical and non-canonical.

[0161] In the canonical pathway, NFKB1 binds to the cytoplasm via IkBa and the IkB-like molecule p105, forming a complex that prevents NKFB1 from translocating to the nucleus. Upon activation, TCR signaling induces the activation of PKCθ, which phosphorylates and activates CARD11. Activated CARD11 forms a complex with BCL10 and MALT1, ultimately leading to the phosphorylation and degradation of IkBa and the IkB-like molecule p105, releasing NF-κB and translocating it to the nucleus.

[0162] The noncanonical pathway is triggered by signaling through tumor necrosis factor receptor (TNFR) family members, which contain the costimulatory domain 41BB. In the resting state, NFKB2 is bound by p100, and during activation, NF-κB-inducing kinase (NIK) activates IKKa, which phosphorylates p100 and causes the release and translocation of NKFB2 to the nucleus.

[0163] In syngeneic models of solid tumors, endogenous T cells capable of responding to tumors require NF-κB signaling to mediate tumor clearance. NF-κB is induced through TCR signaling, but it is also stimulated through the costimulatory domain 41BB (a member of the TNFRS superfamily), which is used clinically in FDA-approved chimeric antigen receptor (CAR) therapy. CAR therapy using the 41BB costimulatory domain has been found to persist longer in patients compared with CAR therapy using other costimulatory domains, such as CD28. The persistence of 41BB CARs is directly related to NF-κB signaling, which improves CAR T resistance to apoptosis through the suppression of apoptotic proteins, such as Bim. AP-1 signaling:

[0164] AP-1 transcription factors are a family of homodimeric or heterodimeric proteins formed from complexes of JUN, FOS, ATF, or MAF proteins. AP-1 signaling is induced by a phosphorylation cascade called the mitogen-activated protein kinase (MAPK) pathway, which is triggered by TCR, cytokine / chemokine, or growth factor signaling. AP-1 often signals in complex with NFAT. As shown in the NFAT section, loss of AP-1 causes uncoupled NFAT to induce an exhaustion program in T cells. However, this exhaustion can be reversed by overexpression of the AP-1 family member c-Jun. In this context, loss of AP-1 is also known to induce an anergic cell state, in which T cells are partially activated through the TCR but lack sufficient costimulatory and cytokine signaling to induce full activation.

[0165] AP-1 dimers are activated by numerous physiological and pathological stimuli. Studies have reported that AP-1 proteins, most of which belong to the Jun family, control cell life and death through their ability to regulate the expression and function of cell cycle regulators, such as cyclin D1, p53, p21(cip1 / waf1), p19(ARF), and p16. Among 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 the induction of cyclin D1 transcription. These effects are antagonized by JunB, which upregulates tumor suppressor genes and represses cyclin D1. A key target of AP-1 action on cell life and death is the tumor suppressor p53, the expression and transcriptional activity of which is modulated by AP-1 proteins.

[0166] The simultaneous induction of NFAT and AP-1 results in the coordinate activation of two distinct signaling pathways: calcium / calcineurin, which promotes NFAT 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.) IL-2 Signaling:

[0167] IL-2 is a pleiotropic cytokine required for T cell activation, proliferation, differentiation, and maintenance. Naive T cells express low-affinity IL-2 receptors and require large amounts of IL-2 to initiate activation, whereas memory and regulatory T cells express high-affinity IL-2 receptors and require much lower amounts of IL-2 for effective signaling. The IL-2 receptor utilizes the JAK / STAT signaling cascade, resulting in widespread transcriptional changes. CD8+ T cells are responsible for the cytotoxic effector response to foreign antigens, but they themselves cannot effectively produce IL-2 and instead depend on IL-2-producing helper CD4+ T cells and other cytokine support.

[0168] Because IL-2 is crucial for T cell persistence and proliferation, high-dose IL-2 therapy has been approved by the FDA for certain cancers to expand endogenous cytotoxic T cell populations to induce tumor rejection. In studies of metastatic melanoma and renal cell carcinoma, high-dose IL-2 therapy induced limited (7%) long-term response rates.

[0169] However, this therapy was poorly tolerated by patients and caused multiple deaths, likely contributing to the expansion of CD4+CD25+FoxP3+ regulatory T cells. Regulatory T cells are known to be pro-tumorigenic, and large numbers of regulatory T cells (Tregs) correlate with poor prognosis in solid tumor settings. Because the toxicity and expansion of pro-tumorigenic Treg populations pose significant challenges to IL-2 therapy, 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 Treg expansion.

[0170] IL-2 secretion is highly relevant to the persistence and proliferation of T cell therapeutic products, therefore, the inventors / disclosers evaluated the IL-2 secretion ability of each mutation when expressed in a CAR Jurkat cell line and after co-culture with target cells. As a follow-up to relevant hits, the inventors / disclosers evaluated the ability of mutations to improve in vitro target killing of CAR T cells when cultured without IL-2 supplementation. This IL-2-deprived environment better reflects the challenges of the tumor microenvironment, where IL-2 is scarce and autocrine secretion of IL-2 would be beneficial for sustained proliferation and long-term killing capacity. In vivo screening for mutations:

[0171] The main obstacle to the success of cell therapy is the accumulation and persistence of T cells in tumors. The present inventors / disclosers attempted to systematically screen mutations in primary human CAR T cells in xenograft models. To find mutations that improve persistence under highly harmful conditions, the present inventors / disclosers utilized the difficult-to-control K562 subcutaneous tumor model, which has limited T cell efficacy.

[0172] The present inventors / disclosers found that clinically relevant CD19-targeted BBz CAR T cells were ineffective at controlling CD19-K562 tumors in a subcutaneous xenograft model, likely failing due to low CAR T cell infiltration and lack of persistence and expansion within the tumor microenvironment. These challenges mirror some of the clinical failures of CAR T cells observed when treating solid tumors. The present inventors / disclosers chose to screen mutations in this challenging "failure" model, aiming to find mutations that significantly improve CAR T cell persistence within solid tumors.

[0173] Although adoptive cell therapy has proven highly effective in refractory B-cell malignancies, CAR-T cell therapy has yet to provide robust long-term efficacy against solid tumors. In the solid tumor environment, CAR-T cells become exhausted, making it difficult for them to proliferate and perform effector functions, ultimately leading to their inability to control tumor growth or prevent recurrence. Therefore, the present inventors / disclosers chose to create an effective targeted cell therapy for solid tumors by improving the proliferation capacity, persistence, and effector function of CAR-T cells. CARD11-BCL10-MALT signalosome

[0174] Formation of the CARD11-BCL10-MALT (CBM) signaling complex is a key event in T- and B-cell receptor-induced gene expression. Following exposure to different immune triggers, these molecules form self-assembling filaments with MALT1 protease activity to regulate the canonical nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways and the degradation of mRNA-binding proteins, providing two layers of control over inflammatory gene expression. Dysregulation of CARD11, BCL10, or MALT1 expression or CBM signaling has been associated with cancer, immune deficiency, and autoimmunity (J. Ruland, L. Hartjes, CARD-BCL-10-MALT1 signaling in protective and pathological immunity. Nat Rev Immunol 19, 118-134 (2019)).

[0175] In normal T cells, T cell receptor (TCR) signaling activates PKCθ, which promotes the assembly of the CARD11-BCL10-MALT1 (CBM) signalosome. The CBM complex subsequently has three major outputs: NF-κB transcriptional activity, AP-1 transcriptional activity, and MALT1 proteolytic activity (Figure 5). Assembly of the CARD11-BCL10-MALT1 signalosome complex is an essential step for NF-κB regulation in lymphoid immune cells.

[0176] The inhibitory domain present in CARD11 may enable intramolecular autoinhibition, preventing CARD11 binding to BCL10 in the absence of upstream signals. When the inhibitory domain is phosphorylated in normal T cells, CARD11 autoinhibition may be relieved, allowing the CARD11 protein to oligomerize and promote prion-like assembly of the CBM complex, involving the recruitment of BCL10-MALT1 filaments, which may then enable CBM complex signaling.

[0177] Genes involved in CBM signaling include, but are not limited to, caspase recruitment domain family member 11 (CARD11), capping protein regulator and myosin 1 linker 2 (CARMIL2), mucosal-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), and B-cell lymphoma 10 (BCL10). CARD11 may be involved in both the innate and adaptive immune systems. CARD11 is involved in the activation of NF-kB by the TCR complex. JAK / STAT signaling

[0178] The biochemistry of JAK / STAT signal transduction is well known to those skilled in the art. Briefly, signal transduction begins with the extracellular association of cytokines or growth factors with their corresponding transmembrane receptors. This promotes the transactivation of receptor-bound Janus kinases (JAKS) by bringing them into spatial proximity and promoting a conformational change that distances their kinase domains from the inhibitory pseudokinase domain. Activated JAKS then phosphorylates latent STAT monomers, leading to dimerization, nuclear translocation, and DNA binding. In mammals, four JAKS (JAK1, JAK2, JAK3, TYK2) and seven STATs (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6) are utilized 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). Costimulatory Molecule Signaling

[0179] Costimulatory and co-inhibitory molecules are cell surface receptors and ligands that are classified into different families based on their structure and function.

[0180] Costimulatory and co-inhibitory receptors determine the functional outcome of T cell receptor (TCR) signaling. While specific recognition of cognate antigenic peptides presented by MHC molecules triggers T cell receptor signaling, it is the co-stimulatory and co-inhibitory receptors on T cells that guide T cell function and determine T cell fate. T cell co-signaling receptors are broadly defined as cell surface molecules that can transmit signals to T cells to positively (co-stimulatory receptors) or negatively (co-inhibitory receptors) modulate TCR signaling. Examples of costimulatory or co-inhibitory receptors include CD28 and CTLA-4, both of which bind to the ligands B7-1 and 7-2. Other genes involved in costimulatory 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). RAS / MEK / ERK

[0181] As known to those skilled in the art, sarcomeric / mitogen-activated protein kinase kinase / extracellular receptor kinase (RAS / MEK / ERK) is a conserved signal transduction pathway that plays a central role in cell proliferation, survival, and differentiation. Abnormal activation of the RAS / MEK / ERK signal transduction pathway induces tumors. Efforts are being made to target this signal transduction pathway for cancer treatment. Abnormal activation of the signal transduction pathway contributes to tumorigenesis and tumor progression.

[0182] It is also known that triggering of the T cell receptor by its cognate antigen results in the almost immediate activation of downstream signaling cascades, including the RAS / MEK / ERK pathway. Studies have also shown that RAS / MEK / ERK signaling is memory stage-dependent in human T cells and confers sensitivity to alloreactive T cell-selective inhibition.

[0183] As a key upstream molecular marker of the RAS-RAF-MEK-ERK pathway, RAS functions as a molecular switch by binding GTP / GDP and includes four isoforms: HRAS, KRAS4A, KRAS4B, and NRAS. KRAS is the most prevalent isoform in all human cancers. KRAS4A and KRAS4B are alternatively spliced ​​isoforms of the same gene. Activation of transmembrane receptors (receptor tyrosine kinases, RTKs) recruits the cytoplasmic complex of growth factor receptor-bound protein 2 (GRB2) and son of sevenless (SOS) to the inner surface of the plasma membrane. (McCubrey JA, Steelman LS, Basecke J, Martelli AM. Raf / mek / erk signaling. Target Ther Acute Myeloid Leuk. January 2015:275-305.)

[0184] Several of the mutations identified by the present inventors are in genes with different roles in this signaling pathway. Phospholipase C gamma signaling

[0185] Phospholipase C (PLC) is an essential mediator of intracellular signal transduction. 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 cell signaling through calcium (Ca2+) mobilization and activation of protein kinase C (PKC), other kinases, and ion channels. PLCγ1, one of the major subtypes of PLC, is directly activated by membrane receptors, including receptor tyrosine kinases (RTKs) and adhesion receptors, such as integrins. PLCγ1 mediates signal transduction through direct interaction with other signaling molecules via its SH domain as well as its lipase activity. PLCγ1 is frequently enriched and mutated in various cancers and is involved in tumorigenic processes, including proliferation, migration, and invasion. (Jang HJ, Suh PG, Lee YJ, Shin KJ, Cocco L, Chae YC. PLCγ1: 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-γ: diverse roles in receptor-mediated calcium signaling. Trends Biochem Sci. 2005;30(12):688-697

[0186] The phospholipase C gamma signaling pathway is involved in T-cell lymphoma, primarily subcutaneous T-cell lymphoma (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.VYEEDM1161V) have been identified in Sézary syndrome, a leukemic variant of CTCL. (VM Patel et al., Frequent and Persistent PLCG1 Mutations in Sezary Cells Directly Enhance PLCγ1 Activity and Stimulate NFκB, AP-1, and NFAT Signaling. J Invest Dermatol 140, 380-389.e384 (2020)). Recombinant Nucleic Acid Constructs and / or Recombinant Nucleic Acids and Polypeptides

[0187] Certain aspects of the present disclosure are directed to polypeptides, recombinant nucleic acid constructs and recombinant nucleic acids encoding the polypeptides, wherein the polypeptides comprise mutations capable of altering T cell signaling.

[0188] Exemplary mutations in polypeptides encoded by recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure are listed in Table 1. In some embodiments of recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide may contain more than one mutation. For example, one, two, three, four, five, or more mutations. In some embodiments, the mutations are a combination of different types of mutations. The different types of mutations may be point mutations, gene fusions, substitutions, gain-of-function mutations, gain-of-stop mutations, insertion mutations, deletion mutations, duplication mutations, or translocations. The mutations may be T-cell lymphoma mutations or mutations in clonally expanded T-cell populations. In some embodiments, the mutations are point mutations or substitutions.

[0189] In some embodiments, the mutation is a gene fusion. In some embodiments, the gene fusion comprises a polypeptide comprising a caspase-associated recruitment domain (CARD). In some embodiments, the gene fusion comprises a polypeptide comprising a CARD-containing protein or a functional fragment thereof. A functional fragment of a CARD-containing protein can be a fragment that provides NF-κB transcriptional activity, AP-1 transcriptional activity, and / or MALT1 proteolytic activity at a level at least 70%, 75%, 80%, 85%, 90%, or 95% of that of the full-length protein, as determined, for example, by the in vitro CAR Jurkat assay shown in FIG. 1 and described herein. In some embodiments, the gene fusion comprises a domain capable of binding to (i) a substrate localized on 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 gene fusion comprises a CARD-containing protein and a domain capable of binding to (i) a substrate localized on the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a pTyr.

[0190] In some embodiments, the domain can bind to a substrate that is indirectly localized on the intracellular side of the plasma membrane. Indirect localization refers to the localization of the substrate on the intracellular side of the plasma membrane, for example, through binding of the substrate to another polypeptide or lipid that is directly localized on the intracellular side of the plasma membrane. Indirect localization can refer to the localization of the substrate on the intracellular side of the plasma membrane, for example, through interaction of the substrate with another polypeptide or lipid that is directly localized on the intracellular side of the plasma membrane. In some embodiments, the domain can bind to a substrate that is directly localized on the intracellular side of the plasma membrane. Direct localization can refer to the localization of the substrate on the intracellular side of the plasma membrane, for example, through binding of the substrate to the intracellular side of the plasma membrane itself. Direct localization refers to the localization of the substrate on the intracellular side of the plasma membrane, for example, through interaction of the substrate with the intracellular side of the plasma membrane itself.

[0191] In some embodiments, the mutation is a T-cell lymphoma mutation. The T-cell lymphoma mutation can be a mutation that occurs or is identified in T-cell lymphoma. T-cell lymphoma is a heterogeneous group of lymphoid malignancies that occur in intranodal or extranodal sites. There are two major types of T-cell lymphoma: T-lymphoblastic lymphoma and peripheral T-cell lymphoma, which are classified based on clinical symptoms and cytogenetic mutations. Peripheral T-cell lymphoma can be further divided into subcutaneous T-cell lymphoma, adult T-cell lymphoma, angioimmunoblastic T-cell lymphoma, natural killer T-cell lymphoma, enteropathy-associated T-cell lymphoma, and anaplastic large cell lymphoma. In some embodiments, the T-cell lymphoma mutation occurs in T-lymphoblastic lymphoma. In some embodiments, the T-cell lymphoma mutation occurs in peripheral T-cell lymphoma.

[0192] In some embodiments, the mutation is a mutation in clonally expanded T cell population.As used herein, clonally expanded T cell population can be a population of T cells originating from a single progenitor cell.The T cells in clonally expanded population can express the same T cell receptor.

[0193] The polypeptides of the present disclosure, or the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, can alter T cell signaling in various ways. In some embodiments, alteration of T cell signaling can be achieved by enhancing, promoting, improving, reducing, regulating, or modulating signaling pathways in T cells. In some embodiments, alteration of T cell signaling can be by activating, increasing, suppressing, inhibiting, or other altering means of signaling.

[0194] A polypeptide of the present disclosure, or a recombinant nucleic acid construct and / or recombinant nucleic acid of the present disclosure, can alter T cell signaling through one or more T cell signaling pathways. In some embodiments, a polypeptide of the present disclosure, or a recombinant nucleic acid construct and / or recombinant nucleic acid can alter T cell signaling by increasing one or more pathways and / or decreasing one or more pathways, or by a combination of enhancing and / or decreasing 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 IL-2 production is increased.

[0195] In some embodiments, the polypeptides or recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure can alter T cell signaling through one or more T cell signaling pathways, which may be, without limitation, the NFAT pathway, the NF-κB pathway, the AP-1 pathway, the JAK / STAT pathway, the RAS / MEK / ERK, and / or phospholipase gamma signaling, or may be described elsewhere herein.

[0196] In some embodiments, the polypeptides or recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure can alter CARD11-BCL10-MALT1 complex signaling, costimulatory molecule signaling, cytokine production, and / or transcription factor activity in T cells.

[0197] The present 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 compatibility, enhance in vivo persistence, and / or increase intratumoral presence of therapeutic T cells. The present 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 cell compatibility, enhance in vivo persistence, and / or increase intratumoral presence of therapeutic T cells.

[0198] T cell fitness can refer to the ability of T cells to generate immune responses.The ability of T cells to perform T cell functions such as signal transduction, cytokine production, survival and persistence in tumors can contribute to T cell fitness.T cell exhaustion can contribute to the reduction of its fitness.

[0199] In some embodiments, the polypeptides of the present disclosure or the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure can alter the in vivo persistence of therapeutic T cells containing mutations in tumors. The in vivo persistence of therapeutic T cells can refer to the length of time that therapeutic T cells reside in a host tumor after injection. In some embodiments, the 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 compared to the total input number.

[0200] In some embodiments, the polypeptides or recombinant nucleic acid constructs and / or recombinant nucleic acids of the disclosure may alter the in vivo persistence in tumors or other function or activity of T cells described herein through one or more of the following genes: BCL6, BCOR, BRAF, CARD11, CARMIL2, CCND3, CD28, CD3E, CSNK1A1, CSNK2B, ECSIT, EIFS1, FYN, GATA, GNAQ, IRF4, ITGB2, JAK1, JAK3, JUNB, KCNQ1, LATS1, MSC, MYCN, NFKB1, NFKB2, NRAS, PDCD1, PLCG1, PRKCB1, RARA, RASGRP1, RHOA, SMARCB1, STAT3, STAT5, TBL1XR1, TNFRSF1B, TP53, and VAV1.

[0201] In some embodiments, the gene comprises caspase recruitment domain family member 11 (CARD11). In some embodiments, the gene includes capping protein regulator and myosin 1 linker 2 (CARMIL2), mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), B-cell lymphoma 6 (BCL6), B-cell lymphoma 10 (BCL10), and MYCN. In some embodiments, the gene may be a STAT3, STAT5B, JAK1, JAK2, or JAK3 gene. In some embodiments, the gene may be a BRAF or RASGRP1 gene. In some embodiments, the gene may be a phospholipase C gamma 1 (PLCG1) gene. In some embodiments, the gene may be a NFKB1, NFKB2, or JUNB gene. In some embodiments, the gene may be TNFRSF1B.

[0202] In some embodiments, a polypeptide of the present disclosure or a recombinant nucleic acid construct and / or recombinant nucleic acid of the present disclosure may alter the therapeutic efficacy of an engineered T cell, which may include, without limitation, decreased T cell exhaustion, increased proliferative capacity, enhanced anti-tumor activity, increased replicative lifespan, decreased replicative senescence, enhanced killing capacity, enhanced fitness of the engineered T cell, and / or other function or activity of the T cell. In some embodiments, the nucleic acid constructs and / or recombinant nucleic acids of the disclosure are selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:10 6, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156 , SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206,SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256 or 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 a variant of the same gene whose amino acid sequence contains one or more of the mutations listed in Table 1.

[0203] In some embodiments, the nucleic acid constructs and / or recombinant nucleic acids of the present disclosure are selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ No. 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129 , SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191 , SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253,or a nucleic 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% identity to SEQ ID NO: 255 or a variant thereof containing one or more of the mutations listed in Table 1 for the same gene.

[0204] In some embodiments, a polypeptide of the present disclosure has a polypeptide sequence that is 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% identical to SEQ ID NO: 25 and includes a substitution at amino acid positions 361, 615, 634, 655, 357, or a combination thereof, or a nucleic acid construct and / or recombinant nucleic acid of the present disclosure encodes such a polypeptide sequence. In some embodiments, the substitution is Y361C, S615F, E634K, S655C, and / or D357N. In some embodiments, the nucleic acid constructs and / or recombinant nucleic acids of the present disclosure encode a polypeptide 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% identity to SEQ ID NO: 25 and containing at least one, two, or more substitutions at amino acid positions 361, 615, 634, 655, 357. In some embodiments, the nucleic acid constructs and / or recombinant nucleic acids encode a polypeptide having an E634K and S655C mutation. In some embodiments, the recombinant nucleic acid construct encodes a substitution selected from the group consisting of Y361C, S615F, E634K, D357N, S655C, and any combination thereof.

[0205] In some embodiments, the present disclosure relates to a polypeptide comprising a caspase-associated recruitment domain (CARD)-containing protein or a functional fragment thereof, or a recombinant nucleic acid encoding the polypeptide. CARD is a conserved homology domain containing a six-helix bundle or a five-helix bundle. CARD can mediate protein-protein interactions between important apoptosis signaling molecules. Non-limiting examples of CARDs include CARD6, CARD8, CARD9, CARD10, CARD11, CARD14, CARD16, CARD18, and CARD19. Non-limiting examples of proteins that comprise a CARD 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, NOD1, NOD2, Apaf-1, BCL-10, and RIG-I. In some embodiments, 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 NOs: 261-289. In some embodiments, the CARD-containing protein or functional fragment thereof is derived from a CARD protein selected from CARD9 (UniProt No. Q9H257), CARD10 (UniProt No. Q9BWT7), CARD11 (UniProt No. Q9BXL7), or CARD14 (UniProt No. 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 CARD11 protein (UniProt No. 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: 261-264. In some embodiments, a functional fragment of a CARD-containing protein is derived from CARD11 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: 263.

[0206] In some embodiments, the function of the CARD-containing protein or 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 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-limiting examples of cells include T cells, macrophages, monocytes, and natural killer (NK) cells. In some embodiments, activation of the cells results in a substrate localized on the intracellular side of the plasma membrane. In some embodiments, the substrate localized on the intracellular side of the plasma membrane of the cells is a phosphoinositide. Non-limiting examples of phosphoinositides 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)-triphosphate (PIP3) and 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 phosphoinositides with a Kd of less than 100 μM, less than 50 μM, less than 10 μM, less than 5 μM, less than 1 μM, less than 0.5 μM, less than 0.1 μM, less than 0.05 μM, or less than 0.01 μM, where Kd is analyzed using SPR as described in Yu et al. Molecular Cell. 2004 March; 13(5):p677-688.

[0207] The present disclosure further relates to polypeptides comprising a domain capable of binding to (i) a substrate localized on 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 domain can bind to a substrate indirectly localized on the intracellular side of the plasma membrane. Indirect localization refers to localization of a substrate on the intracellular side of the plasma membrane, for example, through binding of the substrate to another polypeptide or lipid directly localized on the intracellular side of the plasma membrane. Indirect localization may refer to localization of a substrate on the intracellular side of the plasma membrane, for example, through interaction of the substrate with another polypeptide or lipid directly localized on the intracellular side of the plasma membrane. In some embodiments, the domain can bind to a substrate directly localized on the intracellular side of the plasma membrane. Direct localization may refer to localization of a substrate on the intracellular side of the plasma membrane, for example, through binding of the substrate to the intracellular side of the plasma membrane itself. Direct localization refers to the localization of a substrate to the intracellular side of the plasma membrane, for example, through interaction of the substrate with the intracellular side of the plasma membrane itself.

[0208] Non-limiting examples of domains that can bind to (i) and / or (ii) include Src homology region 2 (SH2) domain, Src homology region 3 (SH3) domain, pleckstrin homology (PH) domain, and phosphytyrosine binding (PTB) domain. SH2 and PTB domains mediate protein-protein interactions involved in numerous signal transduction pathways. SH2, SH3, and PTB domains that can be used in the present 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 binding pockets that are different from the pTyr binding pocket, and most SH2 domains bind to plasma membrane lipids, with many having high phosphoinositide specificity (Park et al., Cell. 2016 Apr 7;62(1):7-20). In some embodiments, the domain is or comprises an SH3 domain. In some embodiments, the domain is or comprises a PTB domain. In some embodiments, the domain is or comprises a PH domain. In some embodiments, the domain is or comprises an SH2 domain. 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 derived from the PIK3R3 protein (UniProt number Q92569). In some embodiments, the SH2 domain comprises a conserved arginine residue motif in the FLVR motif (ArgβB5 or Arg175 in the v-Src SH2 domain). Most of the conserved residues are clustered on the βB strand, and the conserved arginine residue in the FLVR motif plays a central role in forming a double hydrogen bond with the phosphate group of pTyr.Additional residues important for phosphopeptide binding are His βD4, Lys βD6, and Arg αA2, which coordinate and anchor the aromatic ring of phospho-tyrosine (Diop et al. Int J Mol Sci. 2022 Dec 15;23(24):15944). 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 SEQ ID NO: 305. In some embodiments, the SH2 domain is an engineered SH2 domain with enhanced affinity for phosphotyrosine. 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: 313-315 (e.g., Veggiani et al. Protein Sci. 2019 Feb;28(2):403-413).

[0209] Non-limiting examples of target polypeptides comprising pTyr include P110α / PIK3CA, P110β / PIK3CB, P110δ / 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 a position corresponding to pY1346 of SEQ ID NO: 301. In some embodiments, the pTyr is located at a position corresponding to pY1221 of SEQ ID NO: 302. In some embodiments, the polypeptide binds to a target polypeptide with a Kd of less than 100 μM, less than 50 μM, less than 10 μM, less than 5 μM, less than 1 μM, less than 0.5 μM, less than 0.1 μM, less than 0.05 μM, or less than 0.01 μM, where Kd is analyzed by a fluorescence polarization assay as described in Hause et al. PLoS One. 2012;7(9):e44471. In some embodiments, the polypeptide has a higher affinity for a target polypeptide containing a pTyr than a control polypeptide that does not have 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 a target polypeptide containing a phosphorylated tyrosine than a control polypeptide that does not have phosphorylation at the corresponding tyrosine position.

[0210] Furthermore, the present disclosure relates to a polypeptide (also referred to as a CARD fusion polypeptide) comprising a CARD-containing protein or a functional fragment thereof as disclosed herein and a domain capable of binding to (i) a substrate localized on the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide containing a phosphorylated tyrosine. In some embodiments, the polypeptide comprises a CARD-containing protein or a functional fragment thereof and an SH2 domain (also referred to as a CARD-SH2 fusion polypeptide). In some embodiments, the present disclosure relates to a polypeptide (also referred to as a CARD11-PIK3R3 fusion polypeptide) comprising a functional fragment of a CARD-containing protein derived from a CARD11 protein and an SH2 domain derived from a PIK3R3 protein.

[0211] Furthermore, the present disclosure relates to a recombinant nucleic acid encoding a polypeptide comprising a CARD-containing protein or a functional fragment thereof and a domain capable of binding to (i) a substrate localized on the intracellular side of the plasma membrane of a cell and / or (ii) a target polypeptide comprising a phosphorylated tyrosine, as disclosed herein. In some embodiments, the recombinant nucleic acid encodes a polypeptide comprising a CARD-containing protein or a functional fragment thereof and an SH2 domain. In some embodiments, the recombinant nucleic acid encodes a functional fragment of a CARD-containing protein derived from a CARD11 protein and an SH2 domain derived from a PIK3R3 protein. In some embodiments, the recombinant nucleic acid encodes a functional fragment of a CARD-containing protein derived from a CARD9 protein and an SH2 domain derived from a PIK3R3 protein.

[0212] In some embodiments, the polypeptide is a CARD11-PIK3R3 fusion polypeptide. In some embodiments, the recombinant nucleic acid construct and / or the recombinant nucleic acid encodes a CARD11-PIK3R3 fusion polypeptide or any truncated form thereof. In some embodiments, the polypeptide comprises, or the nucleic acid construct and / or recombinant nucleic acid encodes, a CARD11-PIK3R3 fusion polypeptide or a truncated form thereof 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 NO:206, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, or SEQ ID NO:256. In some embodiments, the truncated forms are encoded by a nucleic 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 SEQ ID NO:205, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, or SEQ ID NO:255.

[0213] The present disclosure further relates to a second polypeptide portion derived from PIK3R3. Non-limiting examples of polypeptides derived from PIK3R3 include two SH2 domains, a catalytic subunit, and a regulatory subunit. In some embodiments, the second polypeptide portion 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:205, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, and SEQ ID NO:255. In some embodiments, the polypeptide does not comprise a coiled-coil domain or a portion thereof. In some embodiments, the polypeptide comprises a coiled-coil domain or a portion thereof. In some embodiments, the coiled-coil 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: 290-294. In some embodiments, the coiled-coil domain 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: 290. In some embodiments, the polypeptide comprises or consists of about 10, about 20, about 30, about 40, about 50, about 60, about 80, about 100, about 120, about 140, about 150, about 160, about 180, about 200, about 220, about 240, about 250, about 260, about 280, or about 300 amino acids of the N-terminal portion of the coiled-coil domain.In some embodiments, the polypeptide comprises no more than 10, no more than 20, no more than 30, no more than 40, no more than 50, no more than 60, no more than 80, no more than 100, no more than 120, no more than 140, no more than 150, no more than 160, no more than 180, no more than 200, no more than 220, no more than 240, no more than 250, no more than 260, no more than 280, or no more than 300 amino acids of the N-terminal portion of the coiled-coil domain.

[0214] The present disclosure further relates to a polypeptide, wherein the domain capable of binding to a target polypeptide comprising a phosphorylated tyrosine, or the SH2 domain, or the second polypeptide moiety, is located at the N-terminus of the CARD-containing protein or a 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 a CARD domain derived from a CARD11 protein followed by a coiled-coil domain derived from a CARD11 protein. The present disclosure further relates to a polypeptide, wherein the domain capable of binding to a target polypeptide comprising a phosphorylated tyrosine, or the SH2 domain, or the second polypeptide moiety, is located proximal to the C-terminus of the polypeptide, and wherein the polypeptide has no more than 50, no more than 40, no more than 30, no more than 20, no more than 15, no more than 10, or no more than 5 amino acids at the C-terminus of the domain (b), or the SH2 domain, or the second polypeptide moiety.

[0215] 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. In some embodiments, the ID 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: 294. In some embodiments, the inhibitory domain (ID) 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. In some embodiments, the polypeptide comprises or consists of about 10, about 20, about 30, about 40, about 50, about 60, about 80, about 100, about 120, about 140, about 150, about 160, about 180, or about 200 amino acids of the N-terminal portion of the ID. In some embodiments, the second polypeptide portion comprises no more than 10, no more than 20, no more than 30, no more than 40, no more than 50, no more than 60, no more than 80, no more than 100, no more than 120, no more than 140, no more than 150, no more than 160, no more than 180, or no more than 200 amino acids of the N-terminal portion of the ID. In some embodiments, the polypeptide does not comprise a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 297. In some embodiments, the polypeptide comprises one or more mutations corresponding to S615F, D357N, Y361C, E634K, and / or S655C of SEQ ID NO: 26.

[0216] In some embodiments, a CARD11-PIK3R3 fusion polypeptide comprises a CARD domain (i.e., a CARD-containing protein or a functional fragment thereof), a CARD inhibitory domain (ID), a coiled-coil domain, and an SH2 domain derived from PIK3R3. In some embodiments, a CARD11-PIK3R3 fusion polypeptide comprises a functional fragment of a CARD-containing protein derived from a CARD11 domain, a coiled-coil domain, and an SH2 domain derived from PIK3R3.

[0217] In some embodiments, a recombinant nucleic acid construct and / or recombinant nucleic acid of the present disclosure comprises a CARD11-PIK3R3 fusion polypeptide encoded by a nucleic 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 SEQ ID NO:205, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, or SEQ ID NO:296.

[0218] In some embodiments, a polypeptide of the disclosure comprises, or a recombinant nucleic acid construct and / or recombinant nucleic acid of the disclosure encodes, a polypeptide comprising a BCL3 polypeptide having a substitution at amino acid 647. In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the disclosure, the polypeptide comprises 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 SEQ ID NO: 4 and comprising a substitution at amino acid 647. In some embodiments, the substitution at amino acid 647 of SEQ ID NO: 4 is S647R.

[0219] In some embodiments, a polypeptide of the disclosure comprises, or a recombinant nucleic acid construct and / or recombinant nucleic acid of the disclosure encodes, a polypeptide comprising a CARMIL2 polypeptide having a substitution at amino acid 575. In some embodiments, a polypeptide comprises 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 SEQ ID NO: 28, and comprising a substitution at amino acid 575. In some embodiments, the substitution at amino acid 575 of SEQ ID NO: 28 is Q575E.

[0220] In some embodiments, a polypeptide of the disclosure comprises, or a recombinant nucleic acid construct and / or recombinant nucleic acid of the disclosure encodes, a polypeptide comprising a MYCN polypeptide having a substitution at amino acid 44. In some embodiments, the polypeptide comprises 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 SEQ ID NO: 114, and including a substitution at amino acid 44. In some embodiments, the substitution at amino acid 44 of SEQ ID NO: 114 is P44L.

[0221] In some embodiments, the polypeptide is encoded by a nucleic acid sequence comprising 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 SEQ ID NO:1, SEQ ID NO:111, or SEQ ID NO:129. In some embodiments, the polypeptide comprises a mutation that can (i) alter JAK / STAT signaling in T cells, (ii) alter cytokine production, and / or (iii) enhance in vivo persistence in tumors of therapeutic T cells comprising the mutation. In some embodiments, the mutation is in the JAK1, JAK3, STAT3, or STAT5 gene.In some embodiments, the polypeptide (i) has 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 SEQ ID NO: 90 and includes a substitution at amino acid position 1097 of SEQ ID NO: 90; i) 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 SEQ ID NO: 94 and including a substitution at amino acid position 573 of SEQ ID NO: 94; (iii) at least about (iv) has 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 SEQ ID NO: 176 and includes a substitution at amino acid position 618, 647, or 661, or a combination thereof, of SEQ ID NO: 182; 182, and includes 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 SEQ ID NO: 182, and including a substitution at amino acid position 628 or amino acid position 665, or a combination thereof, of SEQ ID NO: 182.

[0222] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 87, and the nucleic acid sequence encodes a JAK1 polypeptide having a G1097A substitution.

[0223] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 91, and the nucleic acid encodes a JAK3 polypeptide having an A573V substitution.

[0224] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 169 and encoding a STAT3 polypeptide having an N647I substitution.

[0225] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 171 and encoding a STAT3 polypeptide having a G618R substitution.

[0226] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 173 and encoding a STAT3 polypeptide having a D661I substitution.

[0227] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 177 and encoding a STAT5 polypeptide having a T628S substitution.

[0228] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 179 and encoding a STAT5 polypeptide having a Y665F substitution.

[0229] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide comprises a mutation that can alter costimulatory molecule signaling in T cells and the persistence of T cells in tumors, including the mutation. In some embodiments, the mutation is in a TNFR2, TNFRS1B, CD28, ICOS, CTLA4 gene, or a combination thereof.

[0230] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid encodes a polypeptide, including a TNFRSF1B polypeptide having a substitution at amino acid 256 and / or 377. In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide comprises 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 SEQ ID NO: 192, and including a substitution at amino acid position 256 and / or 377 of SEQ ID NO: 192, or a combination thereof.

[0231] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid encodes a polypeptide, including a TNFRSF1B polypeptide having a substitution at amino acid 337. In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 187 and encoding a TNFRSF1B polypeptide having a T337I substitution.

[0232] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid encodes a polypeptide, including a TNFRSF1B polypeptide having a substitution at amino acid 256. In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 189 and encoding a TNFRSF1B polypeptide having a G256C substitution.

[0233] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid encodes a polypeptide, including a CD28 polypeptide having a substitution at amino acid 51 and / or 77. In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide comprises 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 SEQ ID NO:42 and encoding a CD28 polypeptide comprising a substitution at amino acid position 51 and / or 77 of SEQ ID NO:42, or a combination thereof.

[0234] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 35 and encoding a CD28 polypeptide having an F51V substitution.

[0235] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 37 and encoding a CD28 polypeptide having an F51I substitution.

[0236] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 39 and encoding a CD28 polypeptide having a Q77P substitution.

[0237] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 43 and encoding a CD28 polypeptide having a T195P substitution.

[0238] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid encodes a polypeptide comprising an ICOS-CD28 polypeptide. In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide comprises 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 SEQ ID NO:220.

[0239] In some embodiments of the recombinant nucleic acid construct of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO:219.

[0240] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid encodes a polypeptide comprising a CD28-CTLA4 polypeptide. In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide comprises 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 SEQ ID NO:218.

[0241] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO:217.

[0242] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide comprises a mutation that can alter RAS / MEK / ERK signaling in T cells and the in vivo persistence in tumors of therapeutic T cells comprising the mutation. In some embodiments, the mutation is in the BRAF gene. In some embodiments, the polypeptide comprises an amino acid sequence having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 16 and comprising an amino acid substitution at amino acid position 469 or 594, or a combination thereof. In some embodiments, the amino acid substitution is G469R. In some embodiments, the amino acid substitution is G469A. In some embodiments, the substitution is D594N.

[0243] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 9 and encoding a BRAF polypeptide having a G469R substitution. In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 11 and encodes a BRAF polypeptide having a G469A substitution.

[0244] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 13 and encodes a BRAF polypeptide having a D594N substitution.

[0245] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide comprises a mutation that can alter RAS / MEK / ERK signaling in T cells and the in vivo persistence in tumors of therapeutic T cells comprising the mutation. In some embodiments, the mutation is in the RASGRP1 gene. In some embodiments, the polypeptide comprises 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 SEQ ID NO: 158, and comprising a substitution at amino acid position 261.

[0246] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid construct comprises a nucleic 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 SEQ ID NO: 157 and encoding a RASGRP1 polypeptide having an M261I substitution.

[0247] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide comprises a mutation that can alter phospholipase gamma signaling, and / or (ii) alter cytokine production, and / or (iii) alter the in vivo persistence in tumors of therapeutic T cells comprising the mutation. In some embodiments, the mutation is in the phospholipase C gamma 1 (PLCG1) gene. In some embodiments, the polypeptide comprises 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 SEQ ID NO: 142, and comprising a substitution at amino acid position 47, 48, 520, 1163, 1165, or any combination thereof. In some embodiments, the substitution is E47K. In some embodiments, the substitution is R48W. In some embodiments, the substitution is S520F. In some embodiments, the substitution is E1163K. In some embodiments, the substitution is D1165H.

[0248] In some embodiments, the recombinant nucleic acid construct comprises a nucleic 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 SEQ ID NO: 133 and encodes a PLCG1 polypeptide having an E47K substitution.

[0249] In some embodiments, the recombinant nucleic acid construct comprises a nucleic 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 SEQ ID NO: 135, and encodes a PLCG1 polypeptide having an S520F substitution.

[0250] In some embodiments, the recombinant nucleic acid construct comprises a nucleic 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 SEQ ID NO: 137 and encodes a PLCG1 polypeptide having the substitution E1163K.

[0251] In some embodiments, the recombinant nucleic acid construct comprises a nucleic 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 SEQ ID NO: 139 and encodes a PLCG1 polypeptide having a D1165H substitution.

[0252] In some embodiments, the recombinant nucleic acid construct comprises a nucleic 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 SEQ ID NO: 143, and encodes a PLCG1 polypeptide having an R48W substitution.

[0253] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide comprises a mutation that can alter transcription factor activity in a T cell that contains the mutation. In some embodiments, the mutation is in the NFKB1, NFKB2, or JUNB gene.

[0254] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid encodes a polypeptide, including an NFKB1 polypeptide having a substitution at amino acid 67. In some embodiments, the polypeptide comprises 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 SEQ ID NO: 118, and comprising a substitution at amino acid 67 of SEQ ID NO: 118. In some embodiments, the substitution is H67Y.

[0255] In some embodiments, the recombinant nucleic acid construct comprises a nucleic 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 SEQ ID NO: 115 and encodes an NFKB1 polypeptide having an H67Y substitution.

[0256] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid encodes a polypeptide, including an NFKB2 polypeptide having a substitution at amino acid 656. In some embodiments, the polypeptide comprises 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 SEQ ID NO: 122, and comprising a substitution at amino acid 656 of SEQ ID NO: 122. In some embodiments, the substitution is K656X.

[0257] In some embodiments, the recombinant nucleic acid construct comprises a nucleic 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 SEQ ID NO: 119 and encodes an NFKB2 polypeptide having a K656X mutation.

[0258] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the nucleic acid encodes a polypeptide, including a JUNB polypeptide having a substitution at amino acid 282. In some embodiments, the polypeptide comprises 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 SEQ ID NO: 98, and comprising a substitution at amino acid 282 of SEQ ID NO: 98. In some embodiments, the substitution is A282V.

[0259] In some embodiments, the recombinant nucleic acid construct comprises a nucleic 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 SEQ ID NO: 95 and encodes a JUNB polypeptide having an A282V substitution.

[0260] In some embodiments, the polypeptides comprise a first polypeptide encoding a partial CARD 11 polypeptide and a second polypeptide encoding a partial PIK3R3 polypeptide. In some embodiments, the first polypeptide comprises 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 SEQ ID NO:258. In some embodiments, the first polypeptide comprises 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 SEQ ID NO:260.

[0261] In some embodiments, the recombinant nucleic acid construct and / or recombinant nucleic acid of the present disclosure comprises a promoter. The promoter can be any promoter. The promoter can be a T cell promoter or an inducible promoter. The promoter can be active in a subset of T cells.

[0262] In some embodiments, the promoter can be a constitutive promoter.Examples of constitutive promoters include but are not limited to MND promoter, EF1a promoter, sEF1 promoter, gamma retrovirus LTR promoter, CD4 promoter, CD8a promoter, CD8b promoter, TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, or CD3z promoter. In some embodiments, the promoter is a minimal TATA promoter, pGK, actin promoter, CD25 promoter, IL2 promoter, IL7 promoter, IL15 promoter, KLRG-1 promoter, HLA-DR promoter, CD38 promoter, CD69 promoter, Ki-67 promoter, CD11a promoter, CD58 promoter, CD99 promoter, CD62L promoter, CD103 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, IFN gamma promoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL1 promoter, IL5 promoter, IL6 promoter, IL13 promoter, IL10 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, CD127 promoter, PD-1 promoter, CD122 promoter, CD132 promoter, c-Kit promoter, nuclear factor of activated T cells (NFAT) 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 attenuating factor (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-κB promoter, AP-1 promoter, TNF-alpha promoter, and CD130 promoter, NR4A1 promoter, NR4A2 promoter, or NR4A3 promoter.

[0263] The present disclosure also provides vectors comprising the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure.

[0264] The nucleic acid molecule can be contained within a vector, for example, that is capable of directing its expression in a cell that the vector has been transformed / transduced into. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available or readily prepared by one skilled in the art. See, for example, Sambrook, J., & Russell, DW (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russell, DW (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to herein as "Sambrook"); Ausubel, FM (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (with supplements up to 2014); Bollag, DM 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, MG 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).See Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley. Mullis, KB, Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher. Greenfield, EA (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press. Beaucage, SL et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements up to 2014), and Makrides, SC (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences BV, the disclosures of which are incorporated herein by reference.

[0265] DNA vector can be introduced into eukaryotic cells by conventional transformation or transfection techniques.Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (2012, above) and other standard molecular biology laboratory manuals, and include, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrape-loading, gene gun transfection, nucleoporation, hydrodynamic shock and infection.

[0266] In some embodiments, the expression vector may be a viral vector. host cell

[0267] The nucleic acids of the present disclosure can be introduced into a host cell, such as, for example, a human T lymphocyte, to produce a recombinant or engineered cell comprising the nucleic acid molecule. Accordingly, some embodiments of the present disclosure relate to a method for making a recombinant or engineered cell, comprising: (a) providing a cell capable of protein expression; and (b) contacting the provided cell with a recombinant nucleic acid of the present disclosure.

[0268] Introduction of the nucleic acid molecules of the present disclosure into cells can be achieved by methods known to those of skill 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 microinjection, nanoparticle-mediated nucleic acid delivery, and the like.

[0269] Therefore, in some embodiments, nucleic acid molecules can be delivered by viral or non-viral delivery vehicles known in the art.For example, nucleic acid molecules can be stably integrated into host genome, or can be episomal replicated in recombinant host cells, or exist as minicircle expression vectors for transient expression.Therefore, in some embodiments, nucleic acid molecules are maintained and replicated in recombinant host cells as episomal units.In some embodiments, nucleic acid molecules are stably integrated into the genome of recombinant cells.Stable integration can be achieved by using classical random genome recombination technology, or by more precise techniques, such as CRISPR / Cas9 genome editing directed by guide RNA, or DNA-guided endonuclease genome editing using NgAgo (Natronobacterium gregoryi Argonaute), or TALEN genome editing (transcription activator-like effector nuclease).In some embodiments, nucleic acid molecules exist as minicircle expression vectors for transient expression in recombinant host cells.

[0270] Nucleic acid molecules can be encapsulated in viral capsids or lipid nanoparticles, or can be delivered by viral or non-viral delivery means known in the art, such as electroporation.For example, the introduction of nucleic acid into cells can be achieved by viral transduction.In a non-limiting example, adeno-associated virus (AAV) is engineered to deliver nucleic acid to target cells by viral transduction.Several AAV serotypes have been described, and all known serotypes can infect cells from multiple diverse tissue types.AAV can transduce a wide range of species and tissues without causing toxicity, and generates relatively mild innate and adaptive immune responses.

[0271] Lentivirus-derived vector systems are also useful for nucleic acid delivery and gene therapy by viral transduction.Lentivirus vectors offer several attractive properties as gene delivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the ability to infect both dividing and non-dividing cells; (iii) broad tissue tropism, including the cell types that are important gene and cell therapy targets; (iv) no viral protein expression after vector transduction; (v) the ability to deliver complex gene elements, such as polycistronic or intron-containing sequences; (vi) potentially safer integration site profile; and (vii) a relatively simple system for vector manipulation and production.

[0272] In some embodiments, a host cell can be genetically engineered (e.g., transduced or transformed or transfected) with a vector construct of the present application, which can be, for example, a viral vector or a vector for homologous recombination comprising a nucleic acid sequence that is homologous to a portion of the host cell's genome, or an expression vector for expression of a polypeptide of interest. The host cell can be an untransformed cell or a cell that has already been transfected with at least one nucleic acid molecule.

[0273] Certain aspects of the present disclosure relate to cells comprising the constructs and / or recombinant nucleic acids or vectors of the present disclosure. In some embodiments, the recombinant cell is a prokaryotic or eukaryotic cell. In some embodiments, the cell is in vivo. In some embodiments, the cell is ex vivo. In some embodiments, the cell is in vitro. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the recombinant cell is an animal cell. In some embodiments, the animal cell is a mammalian cell. In some embodiments, the animal cell is a human cell. In some embodiments, the cell is a non-human primate cell. In some embodiments, the cell is a non-native cell or genetically engineered. In some embodiments, the cell is not a cancerous cell. In some embodiments, the recombinant nucleic acid is exogenous. In some embodiments, the mammalian cell is an immune cell, neuron, epithelial cell, and endothelial cell, or stem cell. In some embodiments, the cell is not a CD4+ T cell. In some embodiments, the cell may be an immune cell, a T cell, a CD4+ cell, a CD8+ cell, a regulatory T cell, a gamma delta T cell, an invariant iNKT cell, a MAIT cell, a macrophage, a monocyte, a natural killer cell (NK), or a tumor-infiltrating lymphocyte (TIL). In some embodiments, the cell comprises at least one copy, or at least two copies, of an endogenous nucleic acid sequence encoding a CARD11 protein or a protein comprising a CARD11 CARD domain without any SH2 domain. In some embodiments, the recombinant nucleic acid of the cell is located at an endogenous CARD11-encoding locus or comprises at least a portion of the cell's endogenous CARD11-encoding gene.

[0274] The T cells or their precursors of the present disclosure may be lymphoid immune cells. In some embodiments, the T cells express an engineered immune receptor that binds to a target on a tumor cell. In some embodiments, the T cells may express a T cell receptor (TCR). As known to those skilled in the art, a TCR may be composed of two distinct alpha and beta chains, each consisting of a constant region that anchors the chain to the inside of the T cell surface membrane and a variable region that can recognize and bind to an antigen presented by MHC. The TCR complex may associate with six polypeptides that form two heterodimers, CD3γε and CD3δε, and one homodimer, CD3ζ, which together form the CD3 complex. TCRs can be engineered to specifically target antigens expressed by specific tumor cells by utilizing modified forms of T cells that harbor these complexes. As used herein, a TCR may be a naturally occurring TCR or an engineered TCR.

[0275] In some embodiments, T cells may be CD4+ or CD8+ and may include, but are not limited to, regulatory T cells, cytotoxic T lymphocytes, helper T cells, and central memory T cells (TCM), stem memory T cells (TSCM), stem cell-like memory T cells (or stem-like memory T cells), and effector memory T cells, such as memory T cells including, by way of example, TEM cells and TEMRA (CD45RA+) cells, effector T cells, Th1 cells, Th2 cells, Th9 cells, Th17 cells, Th22 cells, Tfh (follicular helper) cells, regulatory T cells, natural killer T cells, mucosal-associated invariant T cells (MAIT), and γδ T cells. Major T cell subtypes include TSCM (stem cell memory), TCM (central memory), TTM (transitional memory), TEM (effector memory), TTE (terminal effector), and TN (naive).

[0276] In some embodiments, the T cells or precursors thereof of the present disclosure can be cells that mediate immune responses, i.e., immunostimulatory cells, including, but not limited to, helper T cells (CD4+), cytotoxic T cells, and central memory T cells (TCM), stem memory T cells (TSCM), stem cell-like memory T cells (or stem-like memory T cells), effector memory T cells, including, for example, TEM cells and TEMRA (CD45RA+) cells, effector T cells, Th1 cells, Th2 cells, Th9 cells, Th17 cells, Th22 cells, Tfh (follicular helper) cells, natural killer T cells, mucosal-associated invariant T cells (MAIT), and γδ T cells.

[0277] In some embodiments, the T cells of the present disclosure can be immunoinhibitory cells, i.e., cells that inhibit an immune response. Immunoinhibitory T cells include regulatory T cells (regulatory T cells, Treg) and follicular regulatory T cells (Tfh) cells.

[0278] In some embodiments, the T cells of the present disclosure can be hematopoietic stem and / or progenitor cells of the lymphoid lineage that can differentiate into T cells. Hematopoietic stem and / or progenitor cells can be derived from bone marrow, umbilical cord blood, and adult peripheral blood.

[0279] In some embodiments, the cells further comprise (i) a chimeric antigen receptor (CAR) with specificity for a target antigen, and / or (ii) a T cell receptor (TCR) with specificity for a target antigen.

[0280] In some embodiments, the T cells of the present disclosure may be engineered to express a transgene, such as a CAR or a transcriptional regulator, such as a synthetic receptor, such as the synthetic Notch receptor described in U.S. Patent No. 11,202,801, or other synthetic receptors, such as those described in, for example, Provisional Applications Nos. 62 / 905,251, 63 / 15,428, 62 / 905,268, 62 / 905,263, 62 / 935,024, and 62 / 905,248, which are incorporated herein by reference in their entireties.

[0281] T cells may be genetically engineered to recombinantly express transgenes. Such T cells are already target antigen-specific, and their immune response (e.g., cytotoxicity) is specifically stimulated by such target antigens, so they may, but do not necessarily, express CAR or transcriptional receptors that bind to the target antigen. Such T cells that recognize and are sensitized to target antigens can be obtained by known methods, for example, in vitro sensitization methods using naive T cells or hematopoietic progenitor cells (e.g., as described by Wolfl et al., Nat. Protocols 9:950-966 (2014) or van Lent et al., J. Immunol. 179:4959-4968 (2007)), or can be obtained from subjects that have been exposed to target antigens and have an immune response to them (i.e., in vivo sensitized T cells).

[0282] In some embodiments, cells, such as T cells, NK cells, and / or TILs, comprise a CAR. In some embodiments, cells that can be prepared to express a CAR (e.g., CAR T cells) are, for example, CD8+ T cells or CD4+ T cells. In some embodiments, cells that express a CAR disclosed herein are CAR T cells, such as mono-CAR T cells, genome-edited CAR T cells, dual-CAR T cells, or tandem CAR T cells.

[0283] In some embodiments, the target antigen may be a cell surface receptor, an adhesion protein, an integrin, a mucin, a lectin, a tumor-associated antigen, or a tumor-specific antigen.

[0284] In some embodiments, the target antigen may be a tumor-associated antigen. Non-limiting exemplary tumor-associated antigens suitable for the compositions and methods of the present disclosure include CD19, B7H3 (CD276), BCMA (CD269), ALPPL2, claudin 18.2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, CLDN6, CLECL1, CS-1, DLL-3, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, These include ephrinB2, FAP, FLT3, GCC, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2 / neu), IGLL1, IL-11Rα, KIT (CD117), KLK2, LY6G6D, MUC1, NCAM, p53R175H, PAP, PDGFR-β, PRAME, PRSS21, PSCA, PSMA, ROR1, SIRPα, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, and Axl.

[0285] In some embodiments, the target antigen is CD1, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD3ε, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40, CD44v6, CD45, CD D48, 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), CD18 2(CXCR2), CD183(CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273(PD-L2), CD274(PD-L1), CD2 76(B7H3), CD279, CD295, CD339(JAG1), CD340(HER2), CEA, CLL-1, CS1, EGFR, FGFR2, AFP, CA125, MUC-1, M AGE, placenta-like alkaline phosphatase 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), claudin 18.2, PSMA, ROR1, mesothelin, IL13Ra2, FAP, signal-regulatory protein α (SIRPα), TRAC, TCRβ, BCMA, TSHR, EGFRvIII, GD2, GD3, TnAg, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, PDGFR-beta, SSEA-4, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-a bl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, 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 Al, ETV6-AML, seminal fluid protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, KRAS, mutant KRAS, KRAS G12D, prostein, surviving, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, gastrointestinal carboxylesterase, mut hsp70-2, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, AFP, TRAC, TCRβ, BCMA, TSHR, EGFRvIII, GD2, GD3, TnAg, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, PDGFR-beta, SSEA-4, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr -abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, 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 Al, ETV6-AML, seminal fluid protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, surviving, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, gastrointestinal carboxylesterase, mut It may be hsp70-2, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, the extracellular portion of the APRIL protein, or any combination thereof.

[0286] In some embodiments, the TCR is selected from the group consisting of AFP, CD19, TRAC, TCRβ, BCMA, CLL-1, CS1, CD38, CD19, TSHR, CD123, CD22, CD30, CD171, CD33, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, 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 Al, ETV6-AML, seminal fluid protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, KRAS, mutant KRAS, KRAS G12D, prostein, surviving, telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, gastrointestinal carboxylesterase, mutThe target is hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, the extracellular portion of the APRIL protein, or any combination thereof.

[0287] CAR can contain, for example, a costimulatory signaling domain to increase signaling strength.See U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (above), Song et al., Blood 119:696-706 (2012), Kalos et al., Sci Transl. Med. 3:95 (2011), Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016).The signal generated through TCR alone may be insufficient to fully activate T cells, and secondary or costimulatory signals can increase activation. Thus, in some embodiments, the signaling domain further comprises one or more additional signaling domains (e.g., a costimulatory signaling domain) that activate one or more immune cell effector functions (e.g., a natural immune cell effector function described herein). In some embodiments, a portion of such a costimulatory signaling domain can be used, so long as that portion transmits an effector function signal. In some embodiments, the cytoplasmic domain described herein comprises one or more cytoplasmic sequences of a T cell co-receptor (or a fragment thereof). Non-limiting examples of such T cell co-receptors include ligands that bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), MYD88, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. An exemplary costimulatory protein has the amino acid sequence of a costimulatory protein naturally found on T cells, the complete native amino acid sequence of which is set forth in NCBI Reference Sequence: NP_006130.1.

[0288] In various embodiments, a mechanism for modulating (e.g., reducing) the binding activity of a target antigen is desired, for example, to minimize or reduce adverse events caused by binding activity. It may also be desirable to include an inducible "on" or "acceleration" switch in immune cells. Suitable techniques include the use of inducible caspase-9 (U.S. Application No. 2011 / 0286980) or thymidine kinase before, after, or simultaneously with transducing cells with the CAR construct of the present disclosure. Additional methods for introducing suicide genes and / or "on" switches include TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technology, and other techniques.

[0289] According to the present disclosure, on-off or other types of control switch techniques can be incorporated herein.These techniques can include the use of dimerization domains and optionally the dimerization activators of such domains, such as the use of the FKBP / rapalog dimerization system in certain cells disclosed by Wu et al., Science 2015 Oct; 350(6258):aab4077, the contents of which are incorporated herein by reference in their entirety.Additional dimerization techniques are described, for example, in Fegan et al. Chem. Rev. 2010, 110, 3315-3336, and U.S. Patent Nos. 5,830,462, 5,834,266, 5,869,337, and 6,165,787, the contents of which are also incorporated herein by reference with respect to dimerization techniques. Additional dimerization pairs may include cyclosporin-A / cyclophilin receptor, estrogen / estrogen receptor (optionally using tamoxifen, 4-hydroxytamoxifen, or endoxifen), glucocorticoid / glucocorticoid receptor, tetracycline / tetracycline receptor, and / or vitamin D / vitamin D receptor. Further examples of dimerization techniques can be found, for example, in International Publication Nos. WO 2014 / 127261, WO 2015 / 090229, US 2014 / 0286987, US 2015 / 0266973, US 2016 / 0046700, U.S. Patent No. 8,486,693, US 2014 / 0171649, and US 2012 / 0130076, the contents of which are further incorporated herein by reference in their entireties.

[0290] In some embodiments, T cells can contain bicistronic CARs. Bicistronic CARs can contain two CARs that bind to different targets and are encoded by a single vector. Bicistronic CARs can contain a first CAR sequence and a second CAR sequence that are expressed as a single polypeptide with a cleavable linker between the first CAR and the second CAR. Non-limiting examples of the first and / or second CAR sequences include CD19, CD20, BCMA, CD22, CD70, DLL3, LY6G6D, claudin 6, GCC, p53R175H, and PRAME. An exemplary cleavable linker is furin-GSG-T2A (see, e.g., Chng et al. MAbs. 2015 Mar-Apr; 7(2): 403-412, which is incorporated by reference herein regarding cleavable linkers; see also Guedan et al. Mol Ther Methods Clin Dev. 2019 Mar 15; 12: 145-156, which is incorporated by reference herein regarding bicistronic CAR design).

[0291] In some embodiments, the T cell may comprise a bispecific CAR. In some embodiments, the first binding motif and the second binding motif (e.g., different anti-CD20 and anti-CD19 binding motifs) are both contained in a single bispecific CAR. In such bispecific CARs, the CAR molecule itself may be engineered to recognize more than one antigen. In tandem bispecific CARs, the first and second binding motifs are extracellular and may be characterized as membrane-proximal and membrane-distal binding motifs.

[0292] Sources of T cells include, but are not limited to, peripheral blood, bone marrow, or other hematopoietic cell sources. T cells can be isolated by methods well known in the art, including commercially available isolation methods (e.g., Rowland-Jones et al., Lymphocytes: A Practical Approach, Oxford University Press, New York (1999); Su et al., Methods Mol. Biol. 806:287-299 (2012); Bluestone et al., Sci. Transl. Med. 7(315) (doi: 10.1126 / scitranslmed.aad4134)(2015); Miyara et al., Nat. Rev. Rheumatol. 10:543-551 (2014); Liu et al., J. Exp. Med. 203:1701-1711 (2006); Seddiki et al., J. Exp. Med. 203:1693-1700 (2006), Ukena et al., Exp. Hematol. 39:1152-1160 (2011), Chen et al., J. Immunol. 183:4094-4102 (2009), Putnam et al., Diabetes 58:652-662 (2009), Putnam et al., Am. Tranplant. 13:3010-3020 (2013), Lee et al., Cancer Res. 71:2871-2881 (2011), MacDonald et al., J Clin. Invest. 126:1413-1424 (2016)).

[0293] Various known techniques can be used to isolate or enrich desired immune cells, such as T cells. When trying to isolate a specific type of T cell, various cell surface markers or combinations of markers can be used to separate cells, as is well known in the art, including but not limited to CD3, CD4, CD8, CD34 (for hematopoietic stem and progenitor cells) (see Kearse, T Cell Protocols: Development and Activation, Humana Press, Totowa NJ (2000); De Libero, T Cell Protocols, Vol. 514 of Methods in Molecular Biology, Humana Press, Totowa NJ (2009)). Negative selection methods can be used to remove cells that are not desired immune cells. In addition, positive selection methods can be used to isolate or enrich desired T cells. In some cases, a combination of both positive and negative selection methods can be used. Pharmaceutical Composition

[0294] In some embodiments, the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, and the recombinant cells of the present disclosure, can be incorporated into compositions, including pharmaceutical compositions. Such compositions generally comprise the recombinant nucleic acids, recombinant polypeptides, and / or recombinant cells of the present disclosure and a pharmaceutically acceptable excipient, e.g., a carrier.

[0295] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition should be sterile and fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants, such as sodium dodecyl sulfate. Protection from the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be common to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0296] Sterile injection solution can be prepared by incorporating active compound in the required amount with one or combination of above-mentioned components in suitable solvent, and if necessary, subsequently sterilize by filtration.Generally, dispersion solution is prepared by incorporating active compound into sterile vehicle, which comprises basic dispersion medium and other necessary components other than above-mentioned.For the sterile powder that is used to prepare sterile injection solution, the preferred preparation method is vacuum drying and freeze-drying, which obtains powder from the solution that active compound plus any other desired components are previously sterilized and filtered.

[0297] The systemic administration of the recombinant nucleic acid constructs and / or recombinant nucleic acids and recombinant cells of the subject of the present disclosure can also be via transmucosal or transdermal means.For transmucosal or transdermal administration, a permeant appropriate to the barrier to be permeated is used in the formulation.Such permeant agents are generally known in the art, and for example, for transmucosal administration, include surfactants, bile salts, and fusidic acid derivatives.Transmucosal administration can be achieved through the use of nasal sprays or suppositories.For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams, as is generally known in the art.

[0298] In some embodiments, recombinant nucleic acid constructs and / or recombinant nucleic acids and recombinant cells of the present disclosure can also be administered by transfection or infection using methods known in the art, including, but not limited to, those described in McCaffrey et al. (Nature 418:6893, 2002), Xia et al. (Nature Biotechnol. 20: 1006-1010, 2002), or Putnam (Am. J. Health Syst. Pharm. 53: 151-160, 1996, erratum at Am. J. Health Syst. Pharm. 53:325, 1996).

[0299] In some embodiments, the recombinant nucleic acid constructs and / or recombinant nucleic acids and recombinant cells of the present disclosure are prepared with carriers that will protect the recombinant polypeptide from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes infected and targeted to cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. As described in more detail below, the polypeptides of the present disclosure can also be modified to achieve a prolonged duration of action by pegylation, acylation, Fc fusion, linkage to molecules such as albumin, and the like. In some embodiments, recombinant polypeptides may be further modified to extend their half-life in vivo and / or ex vivo. Non-limiting examples of known strategies and techniques suitable for modifying recombinant polypeptides of the present disclosure include (1) chemical modification of the recombinant polypeptides using highly soluble macromolecules, such as polyethylene glycol ("PEG"), which prevent the recombinant polypeptides described herein from contacting proteases, and (2) covalently linking or conjugating the recombinant polypeptides described herein to stable proteins, such as albumin. Thus, in some embodiments, the polypeptides of the present disclosure may be fused to stable proteins, such as albumin. For example, human albumin is known as one of the most effective proteins for enhancing the stability of polypeptides fused thereto, and many such fusion proteins have been reported.

[0300] In some embodiments, the pharmaceutical composition of the present disclosure comprises one or more PEGylation reagents. In some embodiments, the PEGylation reagent is selected from methoxypolyethylene glycol-succinimidyl propionate (mPEG-SPA), mPEG-succinimidyl butyrate (mPEG-SBA), mPEG-succinimidyl succinate (mPEG-SS), mPEG-succinimidyl carbonate (mPEG-SC), mPEG-succinimidyl glutarate (mPEG-SG), mPEG-N-hydroxyl-succinimide (mPEG-NHS), mPEG-tresylate, and mPEG-aldehyde. In some embodiments, the PEGylation reagent is polyethylene glycol. In some embodiments, the PEGylation reagent is polyethylene glycol having an average molecular weight of 20 kD covalently attached to the N-terminal methionine residue of a recombinant polypeptide of the present disclosure, or of approximately 80 kD covalently attached to the N-terminal methionine residue of a polypeptide of the present disclosure. In some embodiments, the PEGylation reagent is polyethylene glycol having an average molecular weight of about 40 kD covalently attached to the N-terminal methionine residue of a polypeptide of the disclosure.

[0301] Thus, in some embodiments, the recombinant nucleic acid constructs and / or recombinant nucleic acids and recombinant cells of the present disclosure are chemically modified with one or more polyethylene glycol moieties, e.g., PEGylated, or similar modifications, e.g., PASylated. In some embodiments, a PEG or PAS molecule is conjugated to one or more amino acid side chains of the disclosed recombinant polypeptide. In some embodiments, a PEGylated or PASylated polypeptide contains a PEG or PAS moiety on only one amino acid. In other embodiments, a PEGylated or PASylated polypeptide contains PEG or PAS moieties on two or more amino acids, for example, attached to two or more, five or more, ten or more, fifteen or more, or twenty or more different amino acid residues. In some embodiments, the PEG or PAS chain is 2000 Da, greater than 2000 Da, 5000 Da, greater than 5,000 Da, 10,000 Da, greater than 10,000 Da, greater than 10,000 Da, 20,000 Da, greater than 20,000 Da, and 30,000 Da. The PASylated polypeptide can be linked directly (e.g., without a linking group) to PEG or PAS through an amino group, sulfhydryl group, hydroxyl group, or carboxyl group. In some embodiments, the recombinant polypeptide of the present disclosure is covalently linked to polyethylene glycol having an average molecular weight of 20,000 daltons. In some embodiments, the recombinant polypeptides of the present disclosure are covalently linked to polyethylene glycol having an average molecular weight ranging from about 1 kD to about 200 kD, such as, for example, from about 10 kD to about 150 kD, from about 50 kD to about 100 kD, from about 5 kD to about 100 kD, from about 20 kD to about 80 kD, from about 30 kD to about 70 kD, from about 40 kD to about 60 kD, from about 50 kD to about 100 kD, from about 100 kD to about 200 kD, or from about 1,150 kD to about 200 kD.In some embodiments, the recombinant polypeptides of the present disclosure are covalently linked to polyethylene glycol having an average molecular weight of 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, hi some embodiments, the recombinant polypeptides of the present disclosure are covalently linked to polyethylene glycol having an average molecular weight of about 40 kD. Methods of the present disclosure

[0302] The present disclosure also relates to methods comprising a polypeptide, a recombinant nucleic acid construct and / or a recombinant nucleic acid of the present disclosure, or a cell of the present disclosure. Methods for identifying mutations useful for improving T cell therapy

[0303] In one aspect, the present disclosure relates to a method for identifying beneficial mutations useful for improving T cell therapy (e.g., as described in Example 1). Mutations can be identified from genome sequencing data from T cell lymphomas or clonal or other T cells. The method can apply a statistical test to determine mutations that occur at a frequency greater than that expected by chance in hotspot regions of coding sequences. As used herein, a hotspot region can be a segment of DNA that is susceptible to genetic alteration. In some embodiments, the hotspot region is in the coding sequence of a gene. In some embodiments, the statistical test utilizes a binomial distribution. In some embodiments, the statistical test utilizes chi-square analysis or other multivariate analysis.

[0304] A binomial distribution may be utilized across the entire length of the gene in the entire genome of the cell. Statistical tests may utilize various false discovery rates. In some embodiments, the discovery rate may be 0.05. In some embodiments, this may be controlled by the Benjamini-Hochberg algorithm. In some embodiments, heterogeneity due to transcription couple repair may be considered.

[0305] In some embodiments, mutations useful or beneficial for improving T cell therapy can be identified by selecting mutations occurring in patients. In some embodiments, mutations can be identified by statistical analysis of genome sequencing data from clonal T cells, including T cell lymphomas, public and / or private databases. In some embodiments, mutations are identified using a binomial distribution across the length of genes in the entire genome and a false discovery rate of 0.05 controlled by the Benjamini-Hochberg algorithm. In some embodiments, mutations are identified in hotspot regions where mutations occur at a frequency above that expected by chance alone. In some embodiments, mutations are identified by permuting the background mutation rate across the genome to account for heterogeneity due to transcription-coupled repair, thereby identifying genes with mutations occurring at a frequency above that expected by chance using the permuted background mutation rate.

[0306] The identified mutations may be mutations that increase proliferation of therapeutic T cells, alter effector function, resist T cell dysfunction and / or enhance T cell growth, reduce T cell exhaustion, or promote in vivo persistence of T cells. Increasing proliferation of therapeutic T cells may include clonal expansion, increase T cell replication rate, and / or increase T cell numbers. The mutations may be any of the mutations listed in Table 1.

[0307] T cell effector function can involve the interaction of armed effector T cells with target cells that present a specific antigen. Effector proteins released by these T cells are concentrated at appropriate target cells by a mechanism activated by recognition of the antigen on the target cell.

[0308] In some embodiments, a method for identifying beneficial mutations useful for improving T cell therapy includes various steps, such as: a) identifying mutations from a clonal T cell genome sequencing database; b) determining the frequency of occurrence of the mutations; and c) applying statistical tests to identify significant differences in hotspot genomic regions where the mutations occur at a frequency greater than expected by chance, thereby identifying mutations in the hotspot regions that can improve T cell therapy. The mutations improve T cell therapy by increasing proliferation, altering effector function, resisting T cell dysfunction, and / or enhancing growth of therapeutic T cells containing the mutations in tumors. The mutations can promote positive T cell selection and / or T cell clonal expansion. The mutations can be any of the mutations listed in Table 1. Methods for preparing T cells for use in cell therapy

[0309] The present disclosure also provides a method for preparing T cells for use in cell therapy. The T cells may further comprise a CAR, a TCR, and a transcriptional receptor. The T cells may be NK cells or tumor-infiltrating lymphocytes derived from a patient with cancer. Preparing the cells may include introducing (e.g., by transduction) into the cells any one or more of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure. Preparing the cells may include introducing (e.g., by transduction) into the cells any one or more vectors containing any one or more of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure. Preparing the cells may include expressing in the cells any one or more of the polypeptides of the present disclosure. The cells may be genetically modified for expression of the polypeptide. The cells may include expression of an engineered immune receptor that binds to a target in the tumor cell.

[0310] Cells can be transduced with recombinant nucleic acid constructs that can alter T cell signaling through the NFAT pathway, the NF-κB pathway, the AP-1 pathway, the JAK / STAT pathway, RAS / MEK / ERK, phospholipase gamma signaling, or other T cell signaling pathways.

[0311] Methods of preparing T cells according to the present disclosure may include transducing T cells with a recombinant nucleic acid construct and / or recombinant nucleic acid that can enhance or promote or improve or reduce or regulate or modulate a pathway, or activate or increase or suppress or inhibit or otherwise alter T cell signaling. Methods of preparing T cells according to the present disclosure may include transducing cells with a recombinant nucleic acid construct and / or recombinant nucleic acid that can alter CARD11-BCL10-MALT1 complex signaling, costimulatory molecule signaling, and cytokine production, and / or transcription factor activity in T cells.

[0312] In certain embodiments, a method of preparing T cells for use in cell therapy comprises transducing T cells with a recombinant nucleic acid construct and / or recombinant nucleic acid having a mutation that can alter (i) T cell signaling through the NFAT, NF-κB, and / or AP-1 pathways, (ii) cytokine production, and / or (iii) in vivo persistence of T cells in a tumor.

[0313] In some embodiments, methods of preparing T cells for use in cell therapy include a polypeptide and / or a recombinant nucleic acid construct and / or a recombinant nucleic acid that can alter the in vivo persistence of therapeutic T cells containing a mutation in a tumor. In some embodiments, the recombinant nucleic acid construct and / or recombinant nucleic acid of the present disclosure can alter therapeutic efficacy, reduce T cell exhaustion, increase proliferative capacity, enhance anti-tumor activity, enhance replicative lifespan, reduce replicative senescence, and enhance killing capacity, enhance the fitness of the engineered T cells, and / or other functions or activities of the T cells.

[0314] In some embodiments, the polypeptide or recombinant nucleic acid construct and / or recombinant nucleic acid of the present disclosure can alter the in vivo persistence in tumors, in vivo accumulation in tumors, or other function or activity of T cells described herein through mutations in one or more of the 41 genes listed in Table 1 or FIG. 2A. In some embodiments, the gene includes caspase recruitment domain family member 11 (CARD11). In some embodiments, the genes include capping protein regulator and myosin 1 linker 2 (CARMIL2), mucosal-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), B-cell lymphoma 6 (BCL6), B-cell lymphoma 10 (BCL10), and MYCN. The gene can be a signal transducer and activator of transcription (STAT) gene, e.g., STAT3, STAT5B. The gene can be a Janus kinase gene, e.g., a JAK1, JAK2, or JAK3 gene. The gene can be v-raf murine sarcoma viral oncogene homolog B1 (BRAF). The gene may be RAS guanyl-releasing protein 1 (RASGRP1). The gene may be tumor necrosis factor receptor superfamily member 1B (TNFRSF1B). The gene may be phospholipase C gamma 1 (PLCG1) gene. The gene may be nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB1) or (NF-κB2), or JunB proto-oncogene, AP-1 transcription factor subunit (JUNB) gene. Methods for Treating a Subject

[0315] The present disclosure also provides a method of treating a subject in need of cell therapy, comprising administering to the subject the cells described herein or T cells prepared by any of the methods described herein. The present disclosure also provides a method for enhancing the in vivo persistence of T cells (e.g., therapeutic T cells) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the T cells of the present disclosure. The T cells may comprise any of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure.

[0316] This administering step can be accomplished using any method of implantation delivery known in the art, for example, the recombinant cells of the present disclosure can be injected directly into the individual's bloodstream or otherwise administered to the individual.

[0317] In some embodiments, the methods disclosed herein include administering recombinant cells to an individual by a method or route that results in at least partial localization of the introduced cells at a desired site to produce a desired effect; this term is used interchangeably with the terms "introducing," "implanting," and "transplanting." The recombinant cells and their differentiated progeny can be administered by any suitable route that results in delivery to a desired location in an individual, where at least a portion of the administered cells or components of the cells remain viable. The survival period of the cells after administration to an individual can be on the order of a few hours, e.g., 24 hours, to several days, years, or even the lifespan of the individual, i.e., long-term engraftment.

[0318] When provided prophylactically, the recombinant cells described herein can be administered to an individual prior to any symptoms of the disease or condition to be treated. Thus, in some embodiments, prophylactic administration of the recombinant cell population prevents the onset of symptoms of the disease or condition.

[0319] When provided therapeutically, in some embodiments, the recombinant cells are provided at (or after) the onset of a symptom or indicator of a disease or condition, e.g., at the onset of the disease or condition.

[0320] For use in the various embodiments described herein, an effective amount of the recombinant cells disclosed herein is at least 10 2 cells, at least 5 x 10 2 cells, at least 10 3 cells, at least 5 x 10 3 cells, at least 10 4 cells, at least 5 x 10 4 cells, at least 10 5 cells, at least 2 x 10 5 cells, at least 3 x 10 5 cells, at least 4 x 10 5 cells, at least 5 x 10 5 cells, at least 6 x 10 5 cells, at least 7 x 10 5 cells, at least 8 x 10 5 cells, at least 9 x 10 5 cells, at least 1 x 10 6 cells, at least 2 x 10 6 cells, at least 3 x 10 6 cells, at least 4 x 10 6 cells, at least 5 x 10 6 cells, at least 6 x 10 6 cells, at least 7 x 10 6 cells, at least 8 x 10 6 cells, at least 9 x 10 6 The recombinant cells may be derived from one or more donors or may be obtained from an autologous source. In some embodiments, the recombinant cells are expanded in culture prior to administration to an individual in need thereof.

[0321] In some embodiments, delivery of a recombinant cell composition (e.g., a composition comprising a plurality of recombinant cells, any of the cells described herein) to an individual by a method or route results in at least partial localization of the cell composition at a desired site. The composition comprising recombinant cells can be administered by any suitable route that results in effective treatment in the individual, for example, administration results in delivery to a desired location in the individual, with at least a portion of the delivered composition, e.g., at least 1×10 4 The cells are delivered to the desired site for a period of time. Modes of administration include injection, infusion, and infusion. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intravesical, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous. For cell delivery, delivery by injection or infusion is a preferred mode of administration.

[0322] In some embodiments, the recombinant cells are administered systemically, e.g., by infusion or injection. For example, the recombinant cell population is administered such that, other than by direct administration to a target site, tissue, or organ, it enters the circulatory system of an individual and is therefore subject to metabolic and other similar biological processes.

[0323] The effectiveness of a treatment, including any of the compositions provided herein, for treating a disease or condition can be determined by a skilled clinician. However, those skilled in the art will understand that a treatment is considered effective if any one or all of the signs or symptoms or markers of the disease are improved or alleviated. Efficacy can also be measured by the individual's lack of deterioration, as assessed by a reduction in the need for hospitalization or medical intervention (e.g., the progression of the disease is stopped or at least slowed). Methods for measuring these indicators are known to those skilled in the art and / or described herein. Treatment includes any treatment of disease in an individual or animal (some non-limiting examples include humans or mammals), including (1) inhibiting the disease, e.g., stopping or slowing the progression of symptoms, or (2) alleviating the disease, e.g., causing the regression of symptoms, and (3) preventing or reducing the likelihood of the onset of symptoms.

[0324] As noted above, a therapeutically effective amount includes an amount of a therapeutic composition that is sufficient to promote a particular beneficial effect when administered to an individual, e.g., one having, suspected of having, or at risk for a disease. In some embodiments, an effective amount also includes an amount sufficient to prevent or delay the onset of disease symptoms, alter the course of disease symptoms (e.g., but not limited to, delaying the progression of disease symptoms), or reverse disease symptoms. It will be understood that for any given case, the appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

[0325] In some embodiments of the disclosed methods, the individual is a mammal. In some embodiments, the mammal is a human. In some embodiments, the individual has or is suspected of having a disease associated with the inhibition of cell signaling mediated by a cell surface ligand or antigen. Diseases suitable for treatment by the compositions and methods of the present disclosure include, but are not limited to, cancer, autoimmune diseases, inflammatory diseases, and infectious diseases. In some embodiments, the disease is cancer or a chronic infectious disease.

[0326] The methods for CAR design, delivery and expression in T cells, and the production of clinical grade CAR-T cell populations are known in the art.See, for example, Lee et al., Clin Cancer Res (2012) 18(10):2780-90, which is incorporated herein by reference in its entirety.For example, engineered CAR can be introduced into T cells using retrovirus, and the nucleic acid sequence encoding chimeric antigen receptor is efficiently and stably integrated into target cell genome.

[0327] Other methods known in the art include lentiviral transduction, transposon-based systems, direct RNA transfection, and CRISPR / Cas systems (e.g., Type I, II, or III systems) containing suitable Cas proteins, such as Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, Cas10d, Cas12a (Cpf1), Cas13a (C2c2), Cas13b, Cas13d, CasF, CasG, Cas1b ... Examples of suitable caspases include, but are not limited to, H, Csy1, Csy2, Csy3, Cse1 (or CasA), Cse2 (or CasB), Cse3 (or CasE), CasX, CasY, Cse4 (or CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csz1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966.

[0328] In some embodiments, recombinant adeno-associated virus (AAV) vectors can be used for delivery.The technique of producing rAAV particles is standard in the art, in which the AAV genome to be packaged, containing the polynucleotide to be delivered, the rep and cap genes, and helper virus functions are provided in cells.The production of rAAV requires the following components to be present in a single cell (referred to herein as packaging cell): the rAAV genome, the AAV rep and cap genes that are separate from (e.g., not present in) the rAAV genome, and the helper virus functions. The AAV rep and cap genes can be derived from any AAV serotype from which a recombinant virus can be derived, and can be derived from an AAV serotype different from 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. The production of pseudotyped rAAV is disclosed, for example, in International Patent Application Publication No. WO 01 / 83692.

[0329] CAR-T cells can be expanded ex vivo, for example, in response to an autoimmune disease antigen, and then re-infused into the subject in therapeutically effective amounts.

[0330] The exact amount of CAR T cells to be administered can be determined by a physician, taking into account individual differences in the subject's age, weight, extent of disease and condition.

[0331] Administration of T cell therapy may be defined by total number of cells per infusion or number of cells per kilogram of body weight, particularly for pediatric subjects (e.g., patients). Because T cells replicate and expand after transfer, the administered cell dose may not approximate the final steady-state cell number. In some embodiments, pharmaceutical compositions comprising CAR T cells of the present disclosure are administered in doses of 10 4 ~10 10In another embodiment, the pharmaceutical composition comprising the CAR T cells of the present disclosure may be administered in a dosage of 10 3 ~10 8 The cells may be administered at a dosage of 100 cells / kg of body weight (including all integer values ​​within these ranges).

[0332] The compositions comprising the CAR T cells of the present disclosure can also be administered multiple times at these dosages. The cells can be administered by using injection techniques known in the art (see, for example, Rosenberg et al., New Engl J Med, (1988) 319:1676). The optimal dosage and treatment regimen for a particular subject can be determined by those skilled in the art by monitoring the subject for signs of disease and adjusting the treatment accordingly.

[0333] In some embodiments, administration of any of the compositions embodied herein, e.g., for the treatment of autoimmune or inflammatory diseases, can be combined with other cell-based therapies, e.g., stem cells, antigen-presenting cells, pancreatic islets, etc.

[0334] The compositions of the present disclosure may be prepared in a manner known in the art and suitable for oral administration to mammals, particularly humans, comprising a therapeutically effective amount of the composition alone together with one or more pharmaceutically acceptable carriers or diluents.

[0335] The term "pharmaceutically acceptable carrier" as used herein means any suitable carrier, diluent, or excipient. These include all aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, and solutes that render the composition isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic and absorbing agents, and the like. It will be understood that the compositions of the present disclosure may also include other supplementary physiologically active agents.

[0336] The carrier must be pharmaceutically "acceptable" in the sense that it is compatible with other components of the composition and is not harmful to the subject.The composition includes those suitable for parenteral administration, including subcutaneous, intramuscular, intravenous, and intradermal administration.The composition can conveniently be presented in unit dosage form and can be prepared by any method well known in the field of pharmacy.Such a method includes preparing a carrier for association with CAR-T cells.Generally, the composition is prepared by bringing any active ingredient into uniform and intimate association with a liquid carrier.

[0337] In some embodiments, the compositions are suitable for parenteral administration, hi other embodiments, the compositions are suitable for intravenous administration.

[0338] Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bactericides, and solutes which render the composition isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0339] Enhancing the in vivo persistence of T cells in a subject can enhance the fitness, function, and / or efficacy of therapeutic T cells, which can be measured by determining the accumulation of T cell numbers in the tumor (e.g., as described in Example 13 below).

[0340] In some embodiments, in vivo persistence may be achieved by promoting intratumoral increases in effector cytokines. In some embodiments, in vivo persistence may be achieved by increasing the expression of the stemness-associated transcription factor TCF1 in TILs. In some embodiments, in vivo persistence may be achieved by increasing the expression of TNF-α, IFN-γ, and / or IL-2.

[0341] In some embodiments, enhanced in vivo persistence can be measured by measuring differences in gene expression, including but not limited to activation markers (IL2RA, CD69), cytotoxic and effector molecules (IFNG, TNF, IL4, IL5, IL13, GZMA, GZMB), chemokines (CCL4, CCL20, CCL8), and costimulatory molecules (ICOS, OX40, 4-1BB, GITR). In some embodiments, the subject has cancer or an autoimmune disease. In some embodiments, the cancer may be a solid tumor. In some embodiments, the cancer may be a hematological cancer. In some embodiments, the cancer expresses a tumor-associated antigen as described herein. In some embodiments, the cancer expresses DLL3, LY6G6D, claudin 6, GCC, p53R175H, and / or PRAME.

[0342] Exemplary solid tumors include, without limitation, small cell lung cancer, colorectal cancer, testicular cancer, ovarian cancer, or melanoma, lymphoma, leukemia, multiple myeloma, prostate cancer, breast cancer, non-small cell lung cancer, gastric cancer, esophageal cancer, liver cancer, kidney cancer, head and 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, and sarcoma.

[0343] In some embodiments, the method of treatment does not include administration of a lymphodepleting agent within 7 days prior to administration of T cell therapy. Non-limiting examples of lymphodepleting agents include cyclophosphamide, fludarabine, and / or bendamustine within 7 days prior to administration of T cell therapy. In some embodiments, the method of treatment does not include administration of at least 600,000 IU / kg of IL-2 every 8 hours. In some embodiments, the method of treatment does not include checkpoint therapy that blocks PD-1 or CTLA-4 signaling.

[0344] In some embodiments, methods of treatment include cells with reduced exhaustion, increased proliferative capacity, enhanced replicative lifespan, decreased replicative senescence, enhanced anti-tumor activity, reduced dysfunction, enhanced persistence, and / or increased intratumoral presence in vivo. In some embodiments, methods of treatment include cells with increased or decreased signaling through the CARD11-BCL10-MALT1 complex, NF-κB, AP-1, NFAT, JAK / STAT, and / or MEK / ERK pathways.

[0345] The cells of the disclosed methods can be regulatory (Treg), natural killer (NK) cells, gamma delta T cells, invariant iNKT cells, macrophages, monocytes, TILs, or engineered T cells. In some embodiments, the engineered T cells can express recombinant TCRs or CARs or transcriptional receptors.

[0346] The engineered T cells can be autologous or allogeneic / non-autologous to the subject to whom they are administered in the methods of the present disclosure. For example, autologous cells can be isolated from the subject to whom the T cells are to be administered. Autologous cells can be isolated from the subject to whom the engineered cells recombinantly expressing a CAR or transcriptional receptor are to be administered. Optionally, the cells can be obtained by leukapheresis, in which white blood cells are selectively removed from collected blood, engineered, and recombined before being reintroduced into the donor. Alternatively, allogeneic cells derived from a non-autologous donor that is not allogeneic / non-autologous can be used. In the case of an allogeneic / non-autologous donor, the cells are sorted and human leukocyte antigen (HLA) matched to determine the appropriate level of compatibility, as is well known in the art. Methods for genetic engineering and / or administration to a subject for both autologous and allogeneic cells are well known in the art. In some situations, the cells can be stored (e.g., cryopreserved) as needed until ready for use.

[0347] Procedures for cell separation include, but are not limited to, flow cytometry, affinity chromatography, density gradient centrifugation, magnetic separation using antibody-coated magnetic beads, conjugation to particles that modify cell density, cytotoxic agents conjugated to or used in conjunction with monoclonal antibodies (mAbs), or any other suitable technique.

[0348] In some embodiments, the isolated T cells are genetically engineered ex vivo for recombinant expression of a transgene. In some embodiments, the isolated T cells are genetically engineered ex vivo for recombinant expression of a CAR or transcriptional receptor, as described in more detail above. In some embodiments, the cells can be genetically engineered for recombinant expression. Suitable methods for genetically engineering cells for recombinant expression are well known in the art. Systems and Kits

[0349] The disclosed systems or kits include one or more of any of the recombinant nucleic acids, recombinant cells, or pharmaceutical compositions disclosed herein, as well as syringes (including pre-filled syringes) and / or catheters (including pre-filled syringes) used to administer any of the recombinant nucleic acids, recombinant cells, or pharmaceutical compositions to a subject. The kits also include any of the recombinant nucleic acids, recombinant cells, or pharmaceutical compositions disclosed herein, as well as instructions for using the syringes and / or catheters for use in administering them.

[0350] Any of the above-described systems and kits may further comprise one or more additional reagents, where such additional reagents may be selected from a dilution buffer, a renaturing solution, a wash buffer, a control reagent, a control expression vector, a negative control polypeptide, a positive control polypeptide, and reagents for in vitro production of a chimeric receptor polypeptide.

[0351] In some embodiments, the components of the system or kit may be in separate containers. In some other embodiments, the components of the system or kit may be combined in a single container.

[0352] In some embodiments, the system or kit may further include instructions for practicing the method using the components of the kit. The instructions for practicing the method are generally recorded on a suitable recording medium. For example, the instructions may be printed on a substrate, such as paper or plastic. The instructions may be present in the kit as a package insert, on a label on the container (i.e., associated with the packaging or subpackaging) of the kit or its components, or the like. The instructions may be present as an electronic storage data file present on a suitable computer-readable storage medium, such as a CD-ROM, diskette, flash drive, etc. In some cases, the actual instructions are not present in the kit, but a means for obtaining the instructions from a remote source (e.g., via the Internet) may be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions may be recorded on a suitable substrate.

[0353] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0354] No admission is made that any reference cited herein constitutes prior art. The discussion of references states what their authors assert, and the inventors / disclosers reserve the right to challenge the accuracy and pertinence of the cited documents. Although numerous sources of information, including scientific journal articles, patent documents, and textbooks, are referenced herein, it is expressly understood that this reference does not constitute an admission that any of these documents form part of the general general knowledge in the art.

[0355] The discussion of general methods provided herein is intended for illustrative purposes only. Other alternative methods and substitutes will be apparent to those skilled in the art upon consideration of this disclosure, and will be within the spirit and scope of this application. [Example]

[0356] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those of skill in the art, and such techniques are described in detail in the above-referenced documents.

[0357] Further embodiments are disclosed in more detail in the following examples, which are provided for illustrative purposes and are not intended to limit the scope of the disclosure or the claims in any way. Example 1 Mutation identification

[0358] This example describes the design and implementation of statistical tests to identify mutations to improve T cell therapy by altering signaling pathways, reducing T cell exhaustion, increasing therapeutic T cell proliferation, altering T cell effector function, resisting T cell dysfunction, enhancing growth in a harsh tumor microenvironment, increasing in vivo persistence of therapeutic T cells, and / or other means.

[0359] To identify mutations useful or beneficial for improving T cell therapy, the inventors / disclosers performed statistical analysis of genome sequencing data from public and private databases, either 1) selecting mutations that occurred in patients or 2) clonal T cells, including T cell lymphomas, to identify hotspot regions and mutations occurring at frequencies above those predicted by chance alone, using a binomial distribution across the length of genes in the entire genome and a false discovery rate of 0.05 controlled by the Benjamini-Hochberg algorithm. Specifically, the inventors / disclosers permuted the entire genome against background mutation rates, taking into account heterogeneity due to transcription-coupled repair. Genes with mutations occurring at frequencies above those predicted by chance alone, assuming background mutation rates, were then identified.

[0360] Using this method, we identified and cloned 62 point mutations (encoding nonsynonymous amino acid substitutions and putative gain-of-function truncation mutations) in 40 different genes. In addition, we identified 10 gene fusions. Example 2 Library construction and in vitro mutation screening

[0361] This example describes the design and construction of a library of mutations shown in Table 1 and the screening of the constructs to identify those that can improve the in vivo persistence of human CAR T cells.

[0362] The inventors / disclosers cloned 62 identified point mutations and 10 fusion polypeptides. For each point mutation construct, a wild-type control of the same gene was generated to control for the effect of overexpressing the wild-type form of the gene. Five control constructs were also cloned. These targets were cloned into barcoded lentiviral constructs to allow for pooled screening. In total, the library for the T cell lymphoma mutation screen contained 117 unique constructs (Figures 1 and 2A-D).

[0363] Wild-type genes for the mutant library were ordered as plasmids through DNASU, and point mutations were introduced via PCR site-directed mutagenesis. For some mutant genes, genes were synthesized by Twist Biosciences (South San Francisco, CA). Wild-type or mutant gene fragments were cloned into a modified pHR'SIN:CSW vector containing the PGK promoter followed by a T2A self-cleaving sequence, a unique barcode, and the fluorescent tag mCherry, used to identify transduced cells. Wild-type or mutant gene fragments were cloned into a multiple cloning site 3' to the PGK promoter sequence via an Sbf1 site. All constructs were cloned by infusion cloning (Clontech #ST0345) or Gibson assembly.

[0364] The intracellular domain, including the appropriate costimulatory domain and CD3 zeta domain, was synthesized using Twist. Receptors were constructed by fusing CD19 scFv to the corresponding receptor scaffold and intracellular tail. All receptors contained an N-terminal CD8α signal peptide (MALPVTALLLPLALLLHAARP, SEQ ID NO: 261) for membrane targeting and a flag tag (DYKDDDDK, SEQ ID NO: 262) for easy determination of surface expression with α-flag PE (Biolegend 637310). In some cases, the receptors also contained a T2A self-cleaving sequence followed by a tNGFR sequence used in downstream applications for T cell isolation. Receptors were cloned into a modified pHR'SIN:CSW vector containing the PGK promoter for all primary T cell experiments.

[0365] Triple reporter Jurkat cells (a human T cell line) were generated that stably express NFAT-eGFP, NF-kB-eCFP, and AP-1-iRFP fluorescent protein reporter constructs.

[0366] These triple reporter cells were then transduced to express CD19-CD28z or CD19-BBz CAR, resulting in CD19 antigen-dependent signaling and IL-2 production.

[0367] The effects of most of the mutations have not been fully characterized in the context of T cell signaling and effector function. To elucidate their function, the inventors / disclosers transduced each construct in the library into a triple reporter system. Collectively, the reporter cells allowed for elucidation of the effects of mutations on biochemical signaling pathways, the effects of antigen, and evaluation of effector cytokine production. A schematic diagram of in vitro and in vivo screening of mutations to reveal their effects on T cell signaling pathways using triple reporter Jurkat cells (a human T cell line) stably expressing NFAT-eGFP, NF-kB-eCFP, and AP-1-iRFP fluorescent protein reporter constructs is illustrated in Figure 1.

[0368] To screen the effects of T cell lymphoma mutations on CAR signaling, triple reporter CAR cells were transduced with CD19-CD28z or CD19-BBz chimeric antigen receptor (CAR). They were then cocultured with K652 or K562-CD19 cells, and NFAT, NF-κB, and AP-1 reporter activity was determined by flow cytometry. As a readout of effector function, supernatants were collected from the K562-CD19 condition and analyzed for IL-2 by ELISA. Each screen was performed twice with independent transductions, and this was highly reproducible across biological replicates. In vitro screening revealed numerous mutations with significant effects on CAR signaling and cytokine production (Figure 2, Tables 3-5). For the CD19-BBz CAR screen, PD-1 levels were assessed by flow cytometry.

[0369] The mutations altered signaling in a manner that could not be achieved through expression of the wild-type form of the gene. The 25 point mutation constructs showed significant differences compared to their wild-type counterparts, indicating that the substantial increases or decreases in signaling resulting from the mutations were not attributable to overexpression of the wild-type gene (Figure 3). Thus, this mutation library allows for the alteration of T cell signaling in a manner not possible with overexpression of the wild-type gene. This feature of the mutation library distinguishes it from approaches that enhance or increase expression of the wild-type gene.

[0370] In vitro screening revealed numerous mutations with significant effects on CAR signaling and cytokine production. These screens were highly reproducible across two biological replicates. Similar effects were observed when the mutations were paired with either the CD19-CD28z CAR or the CD19-BBz CAR. Mutations previously reported to upregulate TCR-dependent signaling (e.g., PLCG1) had effects consistent with these previous findings, suggesting that our assay efficiently captures known positives. In addition, expression of the negative control construct DGKZ, encoding diacylglycerol kinase zeta, a known inhibitor of T cell receptor signaling, significantly reduced CAR-dependent signaling, as expected.

[0371] The mutant constructs demonstrated a significant degree of antigen specificity: for both the CD19-CD28z CAR and the CD19-BBz CAR, the mutant constructs exhibited significantly greater activity upon antigen stimulation than in the absence of antigen (Figure 2E).

[0372] The mutations had highly diverse effects on TCR-dependent signaling (Figure 2F). For the CD19-BBz CAR, a total of 10 different combinations of mutation-induced up- or down-regulation of signaling pathways were observed. Some of these modifications may be desirable. For example, an imbalance in signaling between NFAT and AP-1 may contribute to T cell exhaustion. Therefore, mutations that increase AP-1 but not NFAT may be beneficial for T cell therapy. The 25 mutant constructs showed significant differences compared to their wild-type counterparts, indicating that the substantial increases or decreases in signaling caused by the mutations were not attributable to overexpression of the wild-type gene (Figure 3).

[0373] Furthermore, these mutations result in effects of magnitudes not achievable by altering gene expression alone, allowing for tunable changes over a wide dynamic range. As an example of this tunability, AP-1 reporter expression could be down- or up-regulated over a range from 0.4-fold to nearly 3-fold above the level induced by CAR activation in controls (Figure 2G). Example 3 In vivo mutation screening

[0374] The inventors then systematically screened T cell lymphoma mutations to identify constructs that could improve the in vivo persistence of human CAR-T cells as follows: Primary human CD3+ T cells were co-transduced with the CD19-BBz CAR and T cell lymphoma mutation constructs. Cells expressing both the CAR and the mutation constructs were pooled, sorted, and then injected into immunodeficient mice bearing subcutaneous CD19-K562 tumors (Figure 1). Samples of this pooled library were obtained before injection for barcode sequencing. Barcode frequencies in the tumors were compared with the pre-injection T cell pool to determine constructs that were depleted or enriched in vivo. The single most depleted construct in this library was PDCD1, which encodes the co-inhibitory receptor and immunotherapy target PD-1, suggesting that in vivo screening could identify therapeutically relevant targets (Figure 5A). The K562 subcutaneous xenograft model generally exhibits low T cell infiltration and persistence, as well as a lack of antitumor efficacy of CAR-T cells (Figure 4). This model was utilized to identify constructs that enable T cells to overcome the harsh solid tumor microenvironment that limits therapeutic T cell efficacy. 1 x 10 6 CD19-K562 were injected subcutaneously into NSG mice on day 0, followed by 5 × 10 6 CD19-BBz CAR cells or PBS vector control cells were intravenously injected. These data indicate that CD19-BBz CAR cells exhibit poor control of CD19-K562 growth, suggesting that T cell function is limited in this model, and provide a rationale for using this model to screen for mutant constructs that improve T cell function. Constructs that improve invasion and / or persistence in this model may be useful for improving the antitumor efficacy of cell therapy against solid tumors.

[0375] In vivo screening identified 35 mutant constructs with positive log2 fold changes in vivo (Figure 5A). Positive log2 fold changes indicate mutations that are enriched in tumors compared to pre-injection samples. Tumor enrichment suggests that these constructs are capable of accumulating, persisting, or expanding T cells in tumors in vivo, a desirable feature for cell therapy. These include the following mutant constructs: CARD11-PIK3R3, MYCN_P44L, CCND3_P284S, GATA3_Y63X, STAT3_G618R, TNFRSF1B_G256C, CARMIL2_Q575E, RHOA_C16R, JAK1_G1097A, PLCG1_S520F, STAT3_N647I, JAK3_A573V, NPM-TYK, ITK-SYK, LATS1_P165T, NFKB2_K656X, CD3E_S41C, STA T3_D661I, ITK-FER, PRKCB1_D427N, VAV1_R798Q, KCNQ1_R583C, CARD11_Y361C, ECSIT_V140A, EIFS1_R89I, PDCD1_R231X, TP53_R273 P, NFKB1_H67Y, TNFRSF1B_T377I, CARD11_S615F, CARD11_D357N, CARD11_E634K:S655C, CSNK1A1_S27C, CD28_T195P, RASGRP1_M261I.

[0376] The mutant constructs altered in vivo persistence in a manner not achievable by overexpression of the wild-type gene (Table 3). For example, wild-type CARD11 showed a negative log2 fold change, whereas the CARD11-PIK3R3 fusion and four CARD11 point mutations (CARD11 p.Y361C, p.S615F, p.D357N, and p.E634K:S655C) all showed positive log2 fold changes. Similarly, in vivo in tumors, the TNFRSF1B p.G256C and p.T377I mutations showed positive log2 fold changes, whereas the TNFRSF1B wild-type showed a negative log2 fold change. Thus, mutations can enable improved in vivo persistence in tumors beyond that possible by expression of the wild-type gene.

[0377] The in vitro screening results correlated with the in vivo screening results. Consistent with the role of PD-1 levels in regulating the in vivo persistence of CAR cells, staining of PD-1 on CD19-BBz CAR Jurkat cells showed that constructs that significantly upregulated PD-1 in response to antigen had poor persistence (Figure 5B). Example 4 Analysis of different domains of CARD11-PIK3R3 gene fusions

[0378] This experiment was performed to elucidate which domain of the CARD11-PIK3R3 gene fusion was responsible for the gain of function of CARD11-PIK3R3.

[0379] The CARD11-PIK3R3 fusion was first identified in a patient with CD4+ leukemia-related subcutaneous T-cell lymphoma (L. Wang et al., Genomic profiling of Sezary syndrome identifies alterations of key T cell signaling and differentiation genes. Nature Genetics 47, 1426-1434 (2015)).

[0380] The product of the translocation between CARD11 and PIK3R3 (SEQ ID NO: 160) results in a genetic fusion of the N-terminal CARD11 protein CARD domain, the coiled-coil domain, and part of the inhibitory domain with the SH2 domain derived from the C-terminus of PIK3R3 (Figure 7). Due to the important role of CARD11 in mediating antigen-dependent signaling in T cells (Figure 6) and the partial lack of the inhibitory domain in the fusion protein, the CARD11-PIK3R3 fusion was tested to determine whether it altered CBM complex signaling.

[0381] We generated variants lacking specific domains and tested them for their ability to induce NF-κB and AP-1 signaling (Figures 8A-8B). Deletion of the PIK3R3 component of the fusion abolished signaling activity, but the PIK3R3 component by itself was not sufficient to induce these pathways. Thus, CARD11-PIK3R3 fusions do not gain function simply by truncating a portion of the CARD11 protein; rather, the C-terminal PIK3R3 is required to activate downstream signaling cascades. The remainder of the inhibitory domain was not required for fusion gain of function; however, removing the coiled-coil domain or the entire CARD11 portion of the fusion component prevented function. Example 5 Truncating the CARD11-PIK3R3 gene fusion

[0382] This experiment was performed to characterize the structure-function relationship of the CARD11-PIK3R3 gene fusion (SEQ ID NO: 206).

[0383] The present inventors / disclosers have introduced various truncated forms of the fusion polypeptide as shown in Figure 30A (SEQ ID NOs: 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256). SEQ ID NOs: 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256 comprise the sequence of SEQ ID NO: 206 with 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 amino acid deletions, respectively. The various truncated forms were evaluated in CD8+CD19-BBz CAR T cells over a 7-day period after removal of anti-CD3 / anti-CD28 bead stimulation (Figure 30B). IL-2 secretion was analyzed by ELISA after co-culture of CD19-BBz CAR CD8+ T cells carrying CARD11-PIK3R3 truncated forms with CD19 target cells at a 1:1 ratio for 24 hours. CD8+ and CD19K562 populations were evaluated after 14 days of co-culture without IL-2 supplementation, and the results are shown in Figure 30D. A long-term co-culture killing assay (Figure 30D) showed that several truncated versions of the fusion (-ID, -20, -60, -100, -140, -220) induced sustained target cell killing similar to that of the wild-type (full-length) version of the fusion without IL-2 supplementation. In contrast, several truncated versions of the fusion (-180, -200, -260, -300) were unable to induce sustained target cell killing without IL-2 supplementation, appearing to more closely resemble conditions with CAR alone. These results indicate that the coiled-coil domain of CARD11 can be shortened without losing the established phenotype of the full-length fusion, but that certain truncated forms result in loss of function. Additionally, IL-2 secretion data (Figure 30C) show that only the -ID fusion variants produce similar amounts of IL-2 compared to the wild-type (full-length) version of the fusion (dashed line labeled wild-type IL-2 secretion).However, when comparing the IL-2 secretion data with the long-term coculture killing data, the inventors hypothesized that there exists a minimum threshold of IL-2 secretion required to observe long-term coculture killing without an IL-2 phenotype. In Figure 30C, this minimum threshold is indicated by the labeled dashed line. Fusion variants that failed to kill CD19-K562 targets in long-term coculture killing without the IL-2 assay (Figure 30D) are variants that fall below the hypothesized minimum threshold of IL-2 secretion in Figure 30C. Example 6 CARD11-PIK3R3 promotes CBM complex assembly and signaling

[0384] To test whether CARD11-PIK3R3 promotes CBM complex assembly and signaling through binding to BCL10, we generated a mutant version of CARD11-PIK3R3 with an amino acid substitution (R28A) at the BCL10-binding interface of the CARD domain of CARD11, previously reported to be essential for CARD11-BCL10 binding. We observed that BCL10 binding was important for both NF-κB and AP-1 induced by tonic NF-κB and CAR signaling (Figures 9A and 9B). In addition, Western blotting of the canonical MALT1 substrates CYLD and HOIL-1 showed increased tonic- and stimulatory-induced cleavage of these proteins in the presence of the fusion (Figure 9C). Treatment with a MALT1 inhibitor demonstrated that cleavage of these proteins was indeed MALT1-dependent (Figure 9C). Collectively, these data demonstrate that the CARD11-PIK3R3 fusion protein enhances CBM complex signaling.

[0385] To test whether CARD11 or BCL10 was required for CARD11-PIK3R3 function, we performed CRISPR knockout experiments. Using CRISPR / Cas9, we knocked out CARD11 or BCL10 in the BBz-CAR Jurkat signaling cell line. Under conditions of no stimulation, CAR stimulation, and pharmacological TCR stimulation, we observed the dependence of CARD11-PIK3R3 on BCL10, but not wild-type CARD11 (Figure 38). In contrast, control cells were dependent on both CARD11 and BCL10 for TCR-triggered NF-κB activation, as expected. Interestingly, cells lacking CARD11-PIK3R3 were not dependent on CARD11 or BCL10 for CAR-dependent signaling, indicating that CBMs may not be optimally coupled during CAR-T cell signaling (Figure 38). Example 7 Effect of CARD11-PIK3R3 expression on signal transduction in primary human T cells

[0386] This example was to test the expression of CARD11-PIK3R3 fusion in primary human T cells.

[0387] Human CD3+ T cells from three healthy donors were lentivirally transduced with CD19-CD28z or BBz CARs with or without CARD11-PIK3R3 fusion. CAR T cells were analyzed by CyTOF at 0, 7, 15, 30, 60, and 120 minutes of coculture with CD19-expressing cells (Figure 10A). Consistent with the induced activation of NF-κB signaling in Jurkat cells, both CD19-CD28z and CD19-BBz CAR cells expressing the CARD11-PIK3R3 fusion showed significantly reduced levels of IκBα, a negative regulator of NF-κB that is degraded downstream of CBM complex activation (Figure 10B). Furthermore, CD19-BBz CAR cells showed enhanced signaling through two pathways downstream of the CBM complex, PI3K / Akt (p4EBP1) and MAPK, which were not significantly different in CD19-CD28z CAR cells. No differences in proximal signaling events were observed, consistent with signaling differences being mediated through the fusion itself rather than differences in transduction from the CAR.

[0388] This data indicated that CARD11-PIK3R3 expression altered downstream signaling events in primary cells after CAR binding. Example 8 CARD11-PIK3R3 enhances gene expression

[0389] To examine possible differences in gene expression, bulk RNA sequencing of human CD8+ T cells from three healthy donors was performed with and without CAR stimulation through coculture with CD19-expressing targets. Principal component analysis revealed strong transcriptional differences due to stimulation, but minimal overall differences in the transcriptomes of control-derived CARD11-PIK3R3 cells (Figure 11A). Differential expression analysis revealed that genes significantly upregulated in CARD11-PIK3R3 CD19-BBz CAR cells compared with controls under stimulated conditions were genes normally induced in control cells by CAR signaling and were enhanced in the presence of the fusion (Figure 11B). This suggests that, rather than inducing a globally distinct transcriptional state, CARD11-PIK3R3 expression increases the expression of a subset of CAR-induced genes. These include activation markers (IL2RA, CD69), cytotoxic and effector molecules (IFNG, TNF, IL4, IL5, IL13, GZMA, GZMB), chemokines (CCL4, CCL20, CCL8), and costimulatory molecules (ICOS, OX40, 4-1BB, GITR). Consistent with our biochemical analysis, gene set enrichment utilizing previously reported gene sets downregulated in response to MALT1 paracaspase inhibition in human T cells revealed significant enrichment of these genes, as well as genes induced by NF-κB and AP-1, in CARD11-PIK3R3-expressing cells (Figure 11C).

[0390] RNA sequencing of human CD4+ and CD8+ T cells from three healthy donors with and without antigen stimulation (Supplementary Table 2). Principal component analysis showed that the most dramatic transcriptional differences were induced by CAR-dependent stimulation, suggesting that CARD11-PIK3R3 expression was not sufficient to allow primary cells to acquire a fully antigen-activated phenotype (Figure 39A). By comparing the transcripts upregulated in both CD4+ and CD8+ T cells upon CAR stimulation, we identified a core group of 43 genes modulated by CARD11-PIK3R3 (Figure 39B). These included several transcripts with important roles in CAR T cell function and effector cytokine production (Figure 39B), including activation markers (IL2RA), cytotoxic and effector molecules (IFNG, TNF, IL4, IL5, IL13), chemokines (CCL4), and costimulatory molecules (ICOS, TNFRSF4 [OX40]). Some of these genes, such as ICOS48 and OX4049, have previously been suggested to favorably influence antitumor responses. Interestingly, CD4+ T cells expressing CARD11-PIK3R3 were enriched for gene signatures related to the cell cycle compared with CD8+ T cells, with these being most enriched for RNA metabolism, cytokine signaling, and translation signatures (Figure 39C). Consistent with our biochemical analysis, gene set enrichment identified enrichment for NF-κB, AP-1, and MALT1 paracaspase signatures (Figure 39C). Example 9 CARD11-PIK3R3 provides a proliferative advantage to CD3 T cells

[0391] The following experiments demonstrate how increased CBM complex signaling affects the activation state and effector phenotype in primary CAR T cells.

[0392] Primary T cells were co-transduced with CD19-BBz or CD19-CD28z CAR and CARD11-PIK3R3 fusion protein. After removing the anti-CD3 / CD28 beads and sorting for purified populations, the cells were expanded and allowed to settle in culture for approximately one week. In transduced CD3 T cells, a greater expansion of CD19-BBZ CAR T cells bearing CARD11-PIK3R3 was observed compared to CD19-BBz CAR T cells alone (Figure 12A). However, this observation was not replicated in a population of CD19-CD2z T cells alone (Figure 12B), suggesting that the fusion provides a growth advantage, especially when paired with BBz CAR.

[0393] Further experiments were performed to determine whether CARD11-PIK3R3 could induce cytokine-independent growth in primary CAR T cells. To do this, we co-transduced primary T cells with CD19-BBz-CAR and CARD11-PIK3R3. After removing anti-CD3 / CD28 stimulation and sorting for purified populations, the cells were expanded and allowed to settle in culture with IL-2. CARD11-PIK3R3 improved CAR-T cell expansion in the presence of IL-2; however, removing IL-2 either early or late in culture caused a rapid T cell population decline (Figures 46A-46C). Example 10 Antigen-induced activation state

[0394] To assess antigen-induced activation status, CD8+ T cells were co-cultured with K562-CD19 targets for 24 hours, and then expression of activation markers was assessed by flow cytometry.

[0395] Most short-term activation markers (CD25, CD69, PD-1, CD39) were upregulated by both CD19-BBz CAR T cells and CD19-BBz CAR T cells carrying the CARD11-PIK3R3 fusion protein (Figure 14A). Consistent with the bulk RNA-seq data, ICOS was found to be significantly upregulated in CD19-BBz CAR T cells carrying the CARD11-PIK3R3 fusion compared to CAR T cells alone. These observations were similar in the context of the CD19-CD28z CAR. Interestingly, although Jurkat signaling data suggested that the CARD11-PIK3R3 fusion protein caused higher basal expression of NF-κB, the inventors / disclosers did not find that CD8+ T cells expressing only the CARD11-PIK3R3 fusion had significantly higher expression of activation markers compared to control (untransduced) T cells (Figure 14B). This suggested that the fusion induces some basal NF-κB signaling, but not significantly enough to cause antigen-independent activation of cells.

[0396] Further experiments were performed using both CD8+ and CD4+ T cells. Comparable expression of some activation markers (PD-1, CD39) was observed in CD19-BBz-CAR T cells with and without CARD11-PIK3R3 (Figure 40). Consistent with the bulk RNAseq data, CD25 (IL2RA) and ICOS were found to be significantly upregulated in CARD11-PIK3R3 CD19-BBz CAR T cells compared to CAR T cells alone (Figure 40). Example 11 CARD11-PIK3R3 upregulates cytokines

[0397] This example demonstrates how CARD11-PIK3R3 upregulates various cytokines.

[0398] Bulk RNAseq analysis of activated CD19-BBz CAR T cells harboring the CARD11-PIK3R3 fusion identified upregulation of multiple cytokines (TNFα, IFN-γ, IL-5, and IL-13).

[0399] To confirm these findings at the protein level, the present inventors / disclosers co-cultured transduced CD8+ T cells with CD19-K562 targets for 48 hours and evaluated the supernatants for various cytokines. By verifying the results of Jurkat ELISA, the present inventors found that the CARD11-PIK3R3 fusion induced higher secretion of IL-2 in addition to other inflammatory cytokines, such as IFN-γ, TNFα, and GMCSF (although not significantly increased) (Figure 14C). Surprisingly, cytokine secretion was substantially increased in CD8+ CARD11-PIK3R CAR T cells, but to a lesser extent in CD4+ CARD11-PIK3R CAR T cells, despite this having been originally observed in CD4+ T-cell cancers (Figure 41). Interestingly, the Th2 cytokine IL-5 was also significantly increased by fusion expression, indicating that the fusion induces a unique cytokine profile. Similar trends were observed in CD8+ T cells bearing the CD19-CD28z CAR and bulk CD3 T cells bearing either the CD19-BBz or CD19-CD28z CAR; however, in some cases, the increases observed in fusion-transduced cells were not significant (Figures 13A-B). Example 12 CARD11-PIK3R3, cytotoxicity, and growth

[0400] To understand how expression of the CARD11-PIK3R3 fusion shaped the cytotoxicity and growth of CAR T cells, the following experiments were performed.

[0401] Because tumor microenvironments often lack pro-survival signaling, such as IL-2, required by cytotoxic T cells, the inventors / disclosers conducted experiments to determine how CARD11-PIK3R3 fusion affects the killing of engineered T cells under IL-2 starvation conditions. Long-term assays were established, and transduced T cells were mixed with CD19-K562 at a 1:1 ratio and maintained in culture for 2 weeks with or without IL-2 supplementation. Interestingly, after 2 weeks of culture, both CD19-BBz CAR alone and CAR with CARD11-PIK3R3 were able to efficiently clear CD19-K562 targets; however, when cultured without IL-2 supplementation, only CD19-BBz CAR with CARD11-PIK3R3 was able to efficiently clear CD19-K562 targets (Figure 15A). For the CD19-CD28z CAR, a less dramatic difference in killing was observed in the long-term co-culture killing assay.

[0402] When stimulated twice over a two-week period, the inventors / disclosers confirmed that CD19-BBz CARs alone decreased in number, while CD19-BBz CARs carrying CARD11-PIK3R3 efficiently expanded (with IL-2) or maintained higher cell numbers (without IL-2) over time, regardless of IL-2 supplementation (Figure 15B). Finally, the inventors / disclosers evaluated target killing at various ratios over time and found that CD19-BBz CARs carrying CARD11-PIK3R3 better controlled target cell growth compared to CD19-BBz CARs alone (Figure 16). A similar trend was confirmed for CD19-28z CARs with and without CARD11-PIK3R3 fusion. Example 13 CARD11-PIK3R3 improves the fitness, function, and efficacy of therapeutic T cells

[0403] In this example, we demonstrate how CARD11-PIK3R3 fusions improved the fitness, function, and efficacy of therapeutic T cells in a fully immunocompetent syngeneic environment.

[0404] Transgenic TCR-expressing mouse OT-I CD8+ T cells specific for chicken ovalbumin (OVA) were harvested from CD45.1 C57BL / 6-Tg(TcraTcrb)1100Mjb / J(OT-I) mice and used in the B16-OVA mouse model in which B16 melanoma cells express the OVA antigen (Figure 23).

[0405] CD45.1+ OT-I CD8+ T cells were transduced with either control (GFP+) or CARD11-PIK3R3 (mCherry+) retroviruses, allowing for tracking of adoptively transferred cells in CD45.2+ C57BL / 6J hosts bearing B16-OVA melanoma tumors. To analyze in vivo accumulation, we used a dual transfer system, which allows for the assessment of competition between transferred T cells in the same tumor. We generated a mixture of CD45.1+ OT-I T cells, in which approximately 10% of the cells were mCherry-positive (corresponding to CARD11-PIK3R3-expressing cells), and directly compared them with GFP+ control cells in vivo. Notably, 7 days after transfer, we observed a 145-fold increase in the number of CARD11-PIK3R3-expressing cells (normalized to input) among TILs compared with control cells (Figures 24A and B).

[0406] We confirmed enhanced competitive accumulation of CARD11-PIK3R3 in a second transgenic TCR mouse model, pmel-1 T cells (which recognize the endogenous melanoma antigen gp100), against B16-F10 tumors (Figures 48A-48C). We performed this assay with both wild-type CARD11-PIK3R3 and the R28A mutant to assess the dependence of the in vivo phenotype on BCL10 interaction. Mutating the R28A binding site reduced in vivo accumulation by 1257-fold (Figures 48A-48C).

[0407] This corresponded to a significant increase in the fraction of transferred cells among all CD8+ T cells, even without normalization to input numbers. Thus, within the same tumor microenvironment, CARD11-PIK3R3-expressing T cells have a dramatically improved accumulation compared to control cells. Example 14 Phenotypic characterization of cells after adoptive transfer of CARD11-PIK3R3

[0408] The following tests were used to characterize the phenotype of cells after adoptive transfer of OT-I cells transduced with either CARD11-PIK3R3 or control.

[0409] Data obtained from competitive double transfection assays showed that there was enhanced accumulation of CARD11-PIK3R3-expressing cells in tumors when transfected separately. In addition, the inventors / disclosers detected higher production of CARD11-PIK3R3 OT-I cells in the spleen and tumor-draining lymph nodes compared with control OT-I cells, but to a lesser extent than that observed in tumors (Figure 25). Example 15 Effector function of CARD11-PIK3R3

[0410] The following tests were used to characterize the effector functions of the CARD11-PIK3R3 fusion protein.

[0411] Ex vivo restimulation and intracellular cytokine staining of CARD11-PIK3R3 OT-I cells revealed enhanced effector function in fusion-expressing cells, including higher production of TNF-α, IFN-γ, and IL-2 (Figure 27). Furthermore, the proportion of multipotent cells producing all three of these effector cytokines was significantly elevated in CARD11-PIK3R3 OT-I cells. In addition, CARD11-PIK3R3 expression substantially increased the expression of the stemness-associated transcription factor TCF1 in TILs (Figure 26A). Consistent with tumor-specific reprogramming, TCF1 expression was not significantly different in tumor-draining lymph nodes or the spleen (Figure 26B).

[0412] These results indicate that CARD11-PIK3R3 promotes the intratumoral accumulation of highly functional stem-like T cells. Example 16 Antitumor efficacy of CARD11-PIK3R3 in a syngeneic TCR transgenic melanoma mouse model

[0413] The following experiment was performed to determine the effect of CARD11-PIK3R3 on the therapeutic efficacy of T cells in a syngeneic immune-competent system.

[0414] On day 12 after subcutaneous inoculation of B16-OVA tumors, mice were treated with PBS control or 2 × 10 transduced with either control or CARD11-PIK3R3 retroviruses without preconditioning or lymphodepletion. 6The mice were treated with OT-I cells. CARD11-PIK3R3 cells mediated significantly enhanced tumor volume control (Figure 28A). In addition, CARD11-PIK3R3 OT-I cells promoted extended overall survival (Figure 28B). Sixty percent (3 / 5) of mice receiving CARD11-PIK3R3 OT-I achieved complete tumor clearance 85 days after tumor challenge, a time point at which none of the PBS- or control OT-I-treated mice survived. Collectively, these data indicate that T cells expressing CARD11-PIK3R3 possess superior therapeutic function in vivo. Cell number reduction is routinely used as a "stress test" to quantify the relative efficacy of engineered T cell therapies. Furthermore, these data suggest that CARD11-PIK3R3 fusions may overcome one important limitation of cell therapy: the ability to produce sufficient cells for therapeutic efficacy in humans. To test this, we compared the therapeutic efficacy of 20,000 and 100,000 CARD11-PIK3R3-transduced OT-I cells with 2 million control OT-I cells in B16-OVA tumor-bearing mice. Remarkably, CARD11-PIK3R3 was able to control tumors at both 20- and 100-fold lower doses than control cells (Figure 29A).

[0415] Current T cell therapies often lack long-term persistence and fail to form memory populations after primary tumor clearance, resulting in a high incidence of relapse. After more than two weeks of initial tumor clearance, CARD11-PIK3R3 OT-I-treated animals treated with a low cell dose or naive controls were re-challenged with B16-OVA tumor cells in the contralateral flank. CARD11-PIK3R3 OT-I-treated animals were protected from tumor development compared with naive untreated mice (Figure 29B). Rejection of secondary tumor challenge suggests that CARD11-PIK3R3 OT-I generates a long-term memory phenotype capable of responding to tumors and suppressing relapse. This phenotype is highly desirable in cancer therapy, allowing a single engineered therapy to respond to primary tumors and prevent relapse, inducing long-term clinical remission. Collectively, these data demonstrate that CARD11-PIK3R3-expressing T cells have superior therapeutic function in vivo. Example 17 Enhancement of cell types other than the original cell type by mutant constructs

[0416] The CARD11-PIK3R3 fusion was first discovered in patients 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)). The OT-I data described in Example 16 is a CD8+-only cell therapy model, and the CARD11-PIK3R3 fusion dramatically enhanced the function of these CD8+ T cells in vivo, despite the sequence being derived from CD4+ T cells. Thus, the mutations described herein may have therapeutic value in cell types different from those in which the mutations originally arose.

[0417] Example 18

[0418] CARD11-PIK3R3 improves the antitumor efficacy of murine CAR T cells in a syngeneic melanoma model

[0419] T cells were harvested from C57BL / 6-Tg(TcraTcrb)1100Mjb / J(OT-I) mice and transduced with hCD19-CAR (CD19-BBz CAR) with or without the CARD11-PIK3R3 fusion (Figure 19). T cells were transferred into mice bearing hCD19-B16 melanoma tumors. In this model, B6.SJL-Ptprca Pepcb / BoyJ mice received 1x10 5 1 x 10 hCD19-expressing B16 tumor cells were injected subcutaneously into the posterior flank. 11 days after tumor inoculation, 1 x 10 6 CAR+ mouse T cells were administered to tumor-bearing animals, and tumor growth was tracked by taking caliper measurements three times a week. Tumor volume was calculated using the following formula: (length x width x width) / 2, where length is the longest measurement. Mice were euthanized when tumors reached 2000 mm3 or when either the length or width reached 2 cm.

[0420] CARD11-PIK3R3 CAR T cells had significantly increased accumulation in tumors and spleens, significantly increased ability to control tumor volume, and significantly increased overall survival (Figure 20). Thus, CARD11-PIK3R3 enhances the efficacy of CAR T cells in vivo in a syngeneic setting without lymphodepletion, radiation, or chemotherapy.

[0421] In a repeat study, tumors treated with CD19-BBz-CAR and CARD11-PIK3R3 CD19-BBz-CAR T cells were evaluated at tumor endpoints by flow cytometry. CD19-BBz-CAR-treated tumors maintained CD19 expression, whereas CARD11-PIK3R3 CD19-BBz-CAR T cell-treated tumors were consistently CD19-negative, suggesting antigen loss as a recurrence mechanism (Figure 47). Example 19 CARD11-PIK3R3 improves the antitumor efficacy of human CAR T cells in a xenograft leukemia model.

[0422] Human CD3+ T cells were transduced with CD19-BBz CAR with or without CARD11-PIK3R3 fusion. The T cells were then electroporated with Cas9 RNP targeting the TCR alpha locus to knock out the endogenous T cell receptor. CAR T cells were injected into NSG mice bearing Nalm-6 luciferase leukemia (Figure 17). Mice treated with CARD11-PIK3R3 CAR T cells had a significant reduction in tumor burden and improved survival (Figure 18).

[0423] The improved efficacy of CD19-BBz-CAR T cells also demonstrated a 7 × 10 6 This was also observed at high doses of CAR+ T cells, where 4 of 7 animals treated with CD19-BBz-CAR began to relapse with NALM-6 disease from initially controlled tumors, whereas 7 of 7 animals treated with CARD11-PIK3R3 CD19-BBz-CAR T cells did not relapse (Figure 42A, Figure 43A). We also showed that CARD11-PIK3R3-expressing T cells alone did not have an anti-tumor effect, indicating that the observed anti-tumor effect was antigen-specific and dependent on CAR activity (Figure 42A). To address safety concerns, we also determined that NALM-6-bearing animals that controlled tumors by treatment with CAR or CARD11-PIK3R3 CAR T cells gained weight over the course of 100 days and only lost weight when NALM-6 relapse occurred (Figure 42A, Figure 43B). Similarly, non-tumor-bearing mice tolerated high doses of control T cells, CAR T cells, and CAR + CARD11-PIK3R3 T cells (Figure 43C). 5Upon NALM-6 tumor rechallenge (in animals that had eliminated leukemia and did not develop symptoms of GVHD), we found that CD19-BBz-CAR-treated animals succumbed to NALM-6 disease at a rate similar to naive age-matched controls, whereas CARD11-PIK3R3 CD19-BBz-CAR T cells prevented leukemia growth (Figure 43D). These data indicate that CARD11-PIK3R3 maintains safety in vivo, even at high T cell doses, while enhancing therapeutic efficacy.

[0424] We next determined whether CARD11-PIK3R3 could improve CD19-CD28z-CAR T cell activity at low doses in the NALM6 leukemia model described above. We therefore administered 4x10 CARD11-PIK3R3 to NALM6-bearing mice. 5 We administered CD19-CD28z-CAR T cells or CARD11-PIK3R3 CD19-CD28z-CAR T cells (Figure 44A) and found that while animals treated with CAR T cells succumbed to disease, CARD11-PIK3R3 CAR T cells survived two consecutive NALM6 challenges (Figures 42B-42C). Thus, the antitumor efficacy of CARD11-PIK3R3 expression was not limited to CARs with a 4-1BB costimulatory domain; those with a CD28 domain also improved, suggesting that CARD11-PIK3R3 expression may be broadly beneficial for CAR T cell therapy.

[0425] Finally, we attempted to determine how CARD11-PIK3R3 might function in a xenograft solid tumor model. Here, we used a subcutaneous model of mesothelioma (M28), which naturally expresses the tumor-associated antigen MCAM55. We produced 5 x 10 MCAM-targeted CD28z-CAR T cells or control T cells with or without CARD11-PIK3R3 and administered them to tumor-bearing animals (Figure 44B). The animals maintained their weight throughout the study (Figure 44C), indicating that the therapy was well tolerated. MCAM-CD28z-CAR treatment delayed tumor growth compared to the control, but both the control and CAR T cell-treated tumors progressively increased in size (Figure 42D). In contrast, animals treated with CARD11-PIK3R3 MCAM CD28z-CAR T cells controlled M28 tumor growth over the course of approximately 70 days (Figure 42D). Thus, CARD11-PIK3R3 safely improves the long-term therapeutic efficacy of human CAR T cells in vivo in both hematological and solid tumor settings. Furthermore, CARD11-PIK3R3 CAR T cells protect against relapse (rechallenge) compared to CAR T cells alone. Example 20 CARD11-PIK3R3 improves the antitumor efficacy of human CAR T cells in a human TCR-based xenograft model

[0426] To further extend these TCR efficacy findings to human engineered TCR T cells, we developed a xenograft model based on a human TCR. KRAS p.G12D, a common mutation present in human solid tumors, can be presented on various human HLA alleles and has been targeted by adoptive T cell therapy in small-scale human studies. We developed a xenograft model based on the human TCR, which harbors a clinically validated TCR against the KRAS p.G12D mutation and HLA-C*08:02, which presents KRAS p.G12D. *Using SNU-1 gastric cancer cells overexpressing 08:02, we observed a significant enhancement of tumor clearance with CARD11-PIK3R3 expression (Figure 45). Thus, CARD11-PIK3R3 expression enhances the function of therapeutic TCR cells in both humans and mice. Collectively, these data demonstrate that CARD11-PIK3R3-expressing T cells have superior in vivo therapeutic function in multiple immunotherapy-refractory tumor models, including those based on CAR and TCR transgenics. Example 21 CARD11-PIK3R3 improves the antitumor efficacy of murine CAR T cells in a syngeneic mesothelioma model

[0427] Mouse T cells were transduced with hAPPL2-CAR with or without CARD11-PIK3R3 fusion (Figure 21). T cells were transferred into mice bearing hAPPL2-40L mesothelioma tumors. In this model, C57BL / 6J mice received 2x10 6 2 × 10 hALPPL2-expressing 40L (mesothelioma) tumor cells were injected subcutaneously into the posterior flank of mice. T cells were harvested from B6.SJL-Ptprca Pepcb / BoyJ (CD45.1) mice and transduced to express the ALPPL2-BBz CAR with or without co-expression of CARD11-PIK3R3. 13 days after tumor inoculation, 2 × 10 6 CAR+ mouse T cells were administered to tumor-bearing animals, and tumor growth was tracked by taking caliper measurements three times a week. Tumor volume was calculated using the following formula: (length x width x width) / 2, where length is the longest measurement. Mice were euthanized when tumors reached 2000 mm3 or when either the length or width reached 2 cm. CARD11-PIK3R3 CAR T cells had a significantly increased ability to control tumor volume (Figure 22).

[0428] The approach used by the present inventors / disclosers enabled the identification of a CARD11-PIK3R3 gene fusion that dramatically enhances therapeutic T cell function and efficacy through binding to the CBM complex. Furthermore, expression of the fusion enabled superior tumor control in fully immunocompetent mouse models without lymphodepletion at a cell dose 100-fold lower than control cells. Notably, the gene fusion identified by the present inventors / disclosers would not be accessible through previous T cell screening efforts, such as loss-of-function, CRISPR activation, or wild-type gene overexpression screening. Therefore, naturally occurring mutations that undergo positive selection in vivo in humans represent a novel and powerful toolset for enhancing cell therapy. The results obtained by the present i...

Claims

1. A polypeptide comprising: (a) a caspase-associated recruitment domain (CARD)-containing protein or a functional fragment thereof; (b) 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 containing a phosphorylated tyrosine; A polypeptide comprising:

2. 2. The polypeptide of claim 1, wherein the domain b) is capable of binding to a substrate indirectly localized on the intracellular side of the plasma membrane through binding to another polypeptide or lipid directly localized on the intracellular side.

3. 3. The polypeptide of claim 1 or 2, wherein 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: 261-289.

4. The polypeptide of any one of claims 1 to 3, wherein the CARD-containing protein is selected from CARD9, CARD10, CARD11, and CARD14.

5. 5. The polypeptide of claim 4, wherein 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:261-264.

6. 6. The polypeptide of any one of claims 1 to 5, wherein the functional fragment of the CARD-containing protein is derived from CARD11 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:

263.

7. The polypeptide of any one of claims 1 to 6, wherein the function of the CARD-containing protein or the functional fragment thereof is to bind to a CARD domain on BCL10.

8. 8. The polypeptide of any one of claims 1 to 7, wherein 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.

9. The polypeptide of any one of claims 1 to 8, wherein the cell is a T cell, a macrophage, a monocyte, or a natural killer (NK) cell.

10. The polypeptide of any one of claims 1 to 9, wherein activation of the cell results in the substrate being localized on the intracellular side of the plasma membrane.

11. The polypeptide of any one of claims 1 to 10, wherein the substrate localized on the intracellular side of the plasma membrane of a cell is a phosphoinositide.

12. The polypeptide of claim 11, wherein the phosphoinositide is selected from phosphatidylinositol (3,4,5)-triphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2).

13. 13. The polypeptide of claim 11 or 12, wherein the polypeptide binds to the phosphoinositide with a Kd of less than 50 μM, less than 10 μM, less than 5 μM, less than 1 μM, less than 0.5 μM, less than 0.1 μM, less than 0.05 μM, or less than 0.01 μM, and the Kd is analyzed using SPR.

14. 10. The polypeptide of any one of claims 1 to 9, wherein 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.

15. The polypeptide of any one of claims 1 to 9 or 14, wherein the target polypeptide is derived from IGF-1R, CTLA-4, or CD28.

16. 15. The polypeptide of any one of claims 1 to 9 or 14, wherein the phosphorylated tyrosine is located at a position corresponding to pY1221 of SEQ ID NO:

302.

17. 16. The polypeptide of claim 15, wherein the phosphorylated tyrosine is located at a position corresponding to pY1346 of SEQ ID NO:

301.

18. 18. The polypeptide of any one of claims 1-9 or 14-17, wherein the polypeptide binds to the target polypeptide with a Kd of less than 10 μM, less than 5 μM, less than 1 μM, less than 0.5 μM, less than 0.1 μM, less than 0.05 μM, or less than 0.01 μM, wherein the Kd is analyzed by a fluorescence polarization assay.

19. 19. The polypeptide of any one of claims 1 to 9 or 14 to 18, having a higher affinity for the target polypeptide containing the phosphorylated tyrosine than a control polypeptide that does not have phosphorylation at the corresponding tyrosine position.

20. 20. The polypeptide of claim 19, which 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 containing the phosphorylated tyrosine than a control polypeptide that does not have phosphorylation at the corresponding tyrosine position.

21. The polypeptide according to any one of claims 1 to 20, wherein the domain (b) is or comprises an SH3 domain.

22. The polypeptide of any one of claims 1 to 20, wherein the domain (b) is or comprises a phosphotyrosine binding (PTB) domain.

23. The polypeptide according to any one of claims 1 to 20, wherein the domain (b) is or comprises a pleckstrin homology (PH) domain.

24. The polypeptide according to any one of claims 1 to 20, wherein the domain (b) is or comprises an SH2 domain.

25. A polypeptide comprising: (i) a caspase-associated recruitment domain (CARD)-containing protein or a functional fragment thereof; (ii) an SH2 domain; A polypeptide comprising:

26. 26. The polypeptide of claim 25, wherein the CARD-containing protein is CARD11.

27. A polypeptide according to any one of claims 24 to 26, wherein the SH2 domain is capable of binding to a polypeptide containing a phosphorylated tyrosine.

28. 28. The polypeptide of any one of claims 24 to 27, wherein 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.

29. A polypeptide according to any one of claims 24 to 28, wherein the SH2 domain comprises a motif of the conserved arginine residue in the FLVR motif.

30. 30. The polypeptide of any one of claims 24 to 29, wherein 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 SEQ ID NO:

305.

31. A polypeptide according to any one of claims 24 to 30, wherein the SH2 domain is an engineered SH2 domain with enhanced affinity for phosphotyrosine.

32. 32. The polypeptide of any one of claims 24 to 31, wherein 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: 313 to 15.

33. A polypeptide comprising: (i) a CARD domain derived from the CARD11 protein; and (ii) a second polypeptide portion derived from a PIK3R3 protein; and A polypeptide comprising:

34. 34. The polypeptide of claim 33, wherein the second polypeptide portion 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:205, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:246, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, and SEQ ID NO:

255.

35. 10. A polypeptide according to any preceding claim, which does not comprise a coiled-coil domain or part thereof.

36. 36. A polypeptide according to any one of claims 1 to 35, comprising a coiled-coil domain or a portion thereof.

37. 37. The polypeptide of claim 36, wherein the coiled-coil 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: 290-293.

38. 38. The polypeptide of claim 37, wherein the coiled-coil domain 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:

290.

39. 39. The polypeptide of any one of claims 36-38, comprising or consisting of about 10, about 20, about 30, about 40, about 50, about 60, about 80, about 100, about 120, about 140, about 150, about 160, about 180, about 200, about 220, about 240, about 250, about 260, about 280, or about 300 amino acids of the N-terminal portion of the coiled-coil domain.

40. 40. The polypeptide of any one of claims 35 to 39, comprising no more than 10, no more than 20, no more than 30, no more than 40, no more than 50, no more than 60, no more than 80, no more than 100, no more than 120, no more than 140, no more than 150, no more than 160, no more than 180, no more than 200, no more than 220, no more than 240, no more than 250, no more than 260, no more than 280, or no more than 300 amino acids in the N-terminal portion of the coiled-coil domain.

41. 2. A polypeptide according to any one of the preceding claims, wherein the domain (b), or the SH2 domain, or the second polypeptide portion is located at the N-terminus of the CARD domain, between the CARD domain and the coiled-coil domain, or at the C-terminus of the CARD domain and / or the coiled-coil domain.

42. 10. A polypeptide according to any one of the preceding claims, comprising the CARD domain derived from a CARD11 protein followed by the coiled-coil domain derived from the CARD11 protein.

43. 10. The polypeptide of any one of the preceding claims, wherein the (b) domain, or the SH2 domain, or the second polypeptide portion is located close to the C-terminus of the polypeptide, and the polypeptide has no more than 50, no more than 40, no more than 30, no more than 20, no more than 15, no more than 10, or no more than 5 amino acids at the C-terminus of the (b) domain, or the SH2 domain, or the second polypeptide portion.

44. 10. The polypeptide of any one of the preceding claims, which does not comprise an inhibitory domain (ID) or a portion thereof.

45. 45. The polypeptide of any one of claims 1 to 44, comprising an inhibitory domain (ID) or a portion thereof.

46. 46. ​​The polypeptide of claim 45, wherein the inhibitory domain (ID) 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:

294.

47. 47. The polypeptide of claim 46, wherein the inhibitory domain (ID) 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.

48. 48. The polypeptide of any one of claims 45-47, comprising or consisting of about 10, about 20, about 30, about 40, about 50, about 60, about 80, about 100, about 120, about 140, about 150, about 160, about 180, or about 200 amino acids of the N-terminal portion of the inhibitory domain (ID).

49. 49. The polypeptide of any one of claims 45-48, wherein the second polypeptide portion comprises no more than 10, no more than 20, no more than 30, no more than 40, no more than 50, no more than 60, no more than 80, no more than 100, no more than 120, no more than 140, no more than 150, no more than 160, no more than 180, or no more than 200 amino acids of the N-terminal portion of the inhibitory domain (ID).

50. 50. The polypeptide of any one of claims 1 to 49, which does not comprise a sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:

297.

51. 51. The polypeptide of any one of claims 1 to 50, comprising one or more mutations corresponding to S615F, D357N, Y361C, E634K, and / or S655C of SEQ ID NO:

26.

52. 52. The polypeptide of any one of claims 1 to 51, wherein expression of said polypeptide in T cells promotes its in vivo accumulation in tumors.

53. 53. The polypeptide of claim 8 or 52, wherein the T cell expresses an engineered immune receptor that binds to a target on a tumor cell.

54. 54. The polypeptide of any one of claims 9 and 52-53, wherein the T cells are selected from the group consisting of regulatory (Treg), gamma delta T cells, invariant iNKT cells, MAIT cells, CAR T cells, tumor infiltrating lymphocytes, and engineered T cells comprising transcriptional receptors.

55. A polypeptide comprising: (i) altering T cell signaling through the NFAT, NF-κB, and / or AP-1 pathways; (ii) altering cytokine production; (iii) altering JAK / STAT signaling in T cells; (iv) altering costimulatory molecule signaling in T cells; (v) altering RAS / MEK / ERK signaling in T cells; (vi) altering phospholipase gamma signaling; (vii) altering transcription factor activity in T cells; and / or (viii) altering or enhancing the in vivo persistence of T cells containing said mutation in tumors. A polypeptide comprising a mutation that allows

56. A recombinant nucleic acid encoding a polypeptide according to any one of claims 1 to 55.

57. 57. The recombinant nucleic acid of claim 56, comprising a promoter.

58. 58. The recombinant nucleic acid of claim 56 or claim 57, wherein the promoter is a constitutive promoter.

59. 59. The recombinant nucleic acid of any one of claims 56 to 58, wherein the constitutive promoter is a CD4 promoter, a CD8a promoter, a CD8b promoter, a TCRa promoter, a TCRb promoter, a CD3d promoter, a CD3g promoter, a CD3e promoter, or a CD3z promoter.

60. The promoter is selected from the group consisting of a minimal TATA promoter, pGK, an actin promoter, a CD25 promoter, an IL2 promoter, an IL7 promoter, an IL15 promoter, a KLRG-1 promoter, an HLA-DR promoter, a CD38 promoter, a CD69 promoter, a Ki-67 promoter, a CD11a promoter, a CD58 promoter, a CD99 promoter, a CD62L promoter, a CD103 promoter, a CCR4 promoter, a CCR5 promoter, a CCR6 promoter, a CCR9 promoter, and a CCR10 promoter. 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, IFN gamma promoter, TIM3 promoter, IL4 promoter, GATA3 promoter, IL1 promoter, IL5 promoter, IL6 promoter, IL13 promoter, IL10 promoter, IL 17A 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, CD127 promoter, PD-1 promoter, CD122 promoter, CD132 promoter, c-Kit promoter, nuclear factor of activated T cells (NFAT) promoter, programmed death 1 (PD- 1) promoters, 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 attenuating factor (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, TGF-beta promoter, T-bet promoter, Eomes promoter,The recombinant nucleic acid according to any one of claims 56 to 59, which is a GATA3 promoter, a CD45RA promoter, a 2B4 promoter, a type I interferon (IFN) alpha, a type I IFN beta promoter, an IFN gamma promoter, an IRF3 promoter, an IRF7 promoter, an NFkB promoter, an AP-1 promoter, a TNF-alpha promoter, a CD130 promoter, an NR4A1 promoter, an NR4A2 promoter, or an NR4A3 promoter.

61. A vector comprising the recombinant nucleic acid construct of any one of claims 56 to 60.

62. 62. The vector of claim 61, which is a viral vector selected from a retroviral vector, an adenoviral vector, and an adeno-associated viral vector.

63. 63. The vector of claim 62, wherein the retrovirus is a lentivirus.

64. A cell comprising a polypeptide according to any one of claims 1 to 55 or a recombinant nucleic acid according to any one of claims 56 to 60.

65. 65. The cell of claim 64, which is a non-naturally occurring cell or which is genetically engineered.

66. 66. The cell of claim 64 or 65, which is not a CD4+ T cell.

67. The cell of any one of claims 64 to 66, which is not a cancerous cell.

68. 68. The cell of any one of claims 64 to 67, wherein the recombinant nucleic acid is exogenous.

69. 69. A cell according to any one of claims 64 to 68, comprising at least one copy, or at least two copies, of an endogenous nucleic acid sequence encoding a CARD11 protein or a protein comprising a CARD11 CARD domain without any SH2 domain.

70. 70. The cell of any one of claims 64 to 69, wherein the recombinant nucleic acid of the cell is located at an endogenous CARD11-encoding locus or comprises at least a portion of the endogenous CARD11-encoding gene of the cell.

71. SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, Sequence number 72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO: No. 136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 162, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO: 182, SEQ ID NO: 184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 192, SEQ ID NO: 194, SEQ ID NO: 1 96, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256,71. The cell of any one of claims 64 to 70, comprising a polypeptide comprising a sequence selected from the group consisting of: and functional variants thereof comprising at least one mutation listed in Table 1.

72. SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO: No. 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: No. 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 1 95, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255,or a functional variant thereof comprising at least one mutation listed in Table 1.

73. (i) a chimeric antigen receptor (CAR) having specificity for a target antigen, and / or (ii) a T cell receptor (TCR) with specificity for a target antigen; 73. The cell of any one of claims 64 to 72, further comprising:

74. 74. The cell of claim 72 or 73, selected from the group consisting of immune cells, T cells, regulatory T cells, CD8+ cells, natural killer cells, tumor-infiltrating lymphocytes, and MAIT cells.

75. The cell of any one of claims 72 to 74, wherein the target antigen is DLL3, LY6G6D, claudin 6, GCC, p53R175H, or PRAME.

76. 56. A method of preparing T cells for use in cell therapy, the method comprising the step of expressing in said T cells a polypeptide according to any one of claims 1 to 55.

77. 77. The method of Claim 76, comprising genetically modifying said T cells for expression of said polypeptide.

78. 78. The method of claim 76 or 77, comprising introducing into said T cell a recombinant nucleic acid encoding said polypeptide or a vector comprising said recombinant nucleic acid.

79. 79. The method of any one of claims 76 to 78, further comprising expressing in the T cells an engineered immune receptor that binds to a target on a tumor cell.

80. 80. A method of treating a subject in need of cell therapy, comprising administering to the subject the cells of any one of claims 64-75 or T cells prepared by the method of any one of claims 76-79.

81. 81. The method of claim 80, wherein the subject has cancer or an autoimmune disease.

82. 82. The method of claim 81, wherein the cancer is a solid tumor.

83. 83. The method of claim 82, wherein the cancer is a hematological cancer.

84. The cancer is characterized by the expression of CD19, B7H3 (CD276), BCMA (CD269), ALPPL2, claudin 18.2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97, CEA, claudin 6 (CLDN6), CLECL1, CS-1, DLL-3, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, ephrin B2, FAP, FLT3, GCC, GD 2, GD3, GM3, GPRC5D, HER2 (ERBB2 / neu), IGLL1, IL-11Rα, KIT (CD117), KLK2, LY6G6D, MUC1, NCAM, p53R175H, PAP, PDGFR-β, PRAME, PRSS21, PSCA, PSMA, ROR1, SIRPα, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, and / or Axl.

85. 85. The method of claim 84, wherein the cancer expresses DLL3, LY6G6D, claudin 6, GCC, p53R175H, and / or PRAME.

86. 86. The method of any of claims 81 to 85, wherein 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 and 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.

87. 87. The method of any of claims 81-86, wherein said method of treatment does not include administration of a lymphodepleting agent within 7 days prior to administration of said T cell therapy.

88. 88. The method of claim 87, wherein said method of treatment does not include administration of cyclophosphamide, fludarabine, and / or bendamustine within 7 days prior to administration of said T cell therapy.

89. 89. The method of any of claims 81-88, wherein the method of treatment does not include administration of at least 600,000 IU / kg of IL-2 every 8 hours.

90. 90. The method of any of claims 81-89, wherein the method of treatment does not include checkpoint therapy that blocks PD-1 or CTLA-4 signaling.

91. 91. The method of any of claims 81-90, wherein the cells have reduced exhaustion, increased proliferative capacity, enhanced replicative lifespan, reduced replicative senescence, enhanced anti-tumor activity, reduced dysfunction, enhanced persistence, and / or increased intratumoral presence in vivo.

92. 92. The method of any of claims 81-91, wherein the cells have increased or decreased signaling through the CARD11-BCL10-MALT1 complex, NF-κB, AP-1, NFAT, JAK / STAT, and / or MEK / ERK pathways.

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