Compositions and methods for enhancing adoptive T cell therapy

Abstract

The present disclosure relates generally to compositions and methods for improving T cell therapy. In particular, the disclosure provides polypeptides and recombinant nucleic acid constructs and / or recombinant nucleic acids encoding polypeptides having mutations capable of effecting altering T cell signaling, cytokine production, and / or in vivo persistence of therapeutic T cells comprising the mutations in tumors. The T cell signaling may be carried out by the NFAT, NF-[kappa] B, and / or AP-1 pathway. The disclosure also provides vectors and cells comprising the polypeptides and / or recombinant nucleic acid constructs and / or recombinant nucleic acids of the disclosure, as well as methods of making T cells for use in cell therapy, and methods of identifying mutations useful for improving T cell therapy.

Description

Cross - Reference to Related Applications

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 412,300, filed on September 30, 2022, the entire content of which is incorporated herein by reference for all purposes. Statement Regarding Sequence Listing

[0002] The Sequence Listing XML associated with this application is provided in XML file format and is hereby incorporated by reference into this specification. The name of the XML file containing the Sequence Listing XML is 048536 - 737001WO_ST26.xml. The XML file is 720,329 bytes and was created on October 2, 2023, and was electronically submitted via the USPTO Patent Center. Statement Regarding Federally Sponsored Research and Development

[0003] This invention was made with government support under Grant Nos. OD025751 and 1DP2AI136599 - 01 awarded by the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0004] The present disclosure generally relates to compositions and methods for enhancing T - cell therapies. 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 comprising such recombinant nucleic acids; methods for preparing T - cells for use in cell therapy; and methods for identifying mutations that can be used to improve T - cell therapies. Background Art

[0005] Adoptive T - cell therapies, including chimeric antigen receptor (CAR) T - cells, have revolutionized cancer therapy. However, the impressive responses are limited to a subset of patients with blood cancers and have not been unlocked in patients with solid tumors, which account for 90% of adult cancers. In treatment - resistant blood and solid cancers, adoptive T - cell therapies are limited by a combination of complex factors, including the fitness of engineered T - cells in tumors, T - cell exhaustion, poor in - vivo persistence, and immunosuppressive environmental factors. Despite recent significant progress, rational design has failed to overcome the problems associated with such factors.

[0006] In addition to rational design, another approach to identifying modifications that improve T - cell function in vitro and in vivo is unbiased screening. For example, the vast majority of screening efforts have focused on genome - scale or whole - genome alterations that modify the expression of endogenous wild - type genes via 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) attached 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 CD3ζ, which are designed to mimic T cell receptor (TCR) stimulation and the immunological synapse upon engagement of the antigen specified by the scFv. CAR constructs do not require antigen presentation via MHC molecules and have thus been used to effectively redirect a patient's own T cells against tumor-specific cell surface antigens. To date, five CD19-targeted CAR-T cell therapies have been approved by the FDA for use against blood B cell cancers. Although these therapies have proven highly effective in refractory B cell malignancies, CAR-T cell therapies have not provided robust long-term efficacy against solid tumors. In the solid tumor setting, CAR-T cells can become exhausted and have difficulty proliferating and performing effector functions, ultimately resulting in uncontrolled tumor growth or prevention of recurrence. Therefore, to generate effective targeted cell therapies against solid tumors, it is necessary to improve the proliferative capacity, persistence, and effector functions of CAR-T cells.

[0008] One approach being investigated is to genetically modify 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 non-functional. Interestingly, the second TET2 allele in this patient was also mutated, resulting in a lack of TET2 function in the CD19 CAR-T cells administered to the patient. This single TET2 knockout CAR-T cell clone exhibited altered T cell differentiation and improved overall effector function. Ultimately, this clone expanded to become the majority of the CAR-T cell population and mediated a complete response against the patient's relapsed CLL. In a second example, when the CD22 CAR cassette integrated into the T cell lymphoma tumor suppressor CBL, a similar complete response was mediated in the patient. These case studies suggest that gene knockout of T cell lymphoma tumor suppressors such as TET2 and CBL can have a significant beneficial effect on CAR-T cell therapy. In preclinical studies, genome-wide knockout assays have revealed genes such as REGNASE that improve T cell fitness and in vivo anti-tumor efficacy after knockout. Additionally, other studies have found that knockout of genes associated with T cell exhaustion and memory formation such as the NR4A family of genes can result in improved and prolonged CAR-T cell responses against tumors.

[0009] While these examples demonstrate that CAR-T cell function can be improved by genetic manipulation, particularly through manipulation of tumor suppressor genes, the scope of these studies has generally been very broad (examining the entire genome) and has focused only on the effects of constitutive gene knockouts. Somatic single nucleotide variant (SSNV) mutations, translocations, and gene deletions that occur naturally in cancer provide biologically plausible candidates for genetic manipulation and CAR expression.

[0010] There remains a need in the art for alternative solutions to address the significant unmet needs for effective adoptive T cell therapies and for enhancing the fitness of engineered T cells. SUMMARY OF THE INVENTION

[0011] This section provides a general overview of the present disclosure and does not encompass its full scope or all of its features.

[0012] The present disclosure provides recombinant nucleic acid constructs and / or recombinant nucleic acids and methods for enhancing adoptive T cell therapies. The recombinant nucleic acid constructs and / or recombinant 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 by enhancing the in vivo persistence and fitness of engineered T cells.

[0013] In one aspect, the present disclosure provides a polypeptide comprising: (a) a protein containing a caspase-associated recruitment domain (CARD) or a functional fragment thereof; and

[0014] (b) A domain capable of binding to the following: (i) a substrate located on the inner side of the cytoplasmic membrane, and / or (ii) a target polypeptide containing phosphorylated tyrosine. In some embodiments, the domain in (b) is capable of binding to a substrate that is indirectly located on the inner side of the plasma membrane by binding to another polypeptide or lipid directly located on the inner side of the cell. 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, 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.

[0015] In some embodiments, the function of the CARD-containing protein or its functional fragment is to bind to the 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, macrophage, monocyte or natural killer (NK) cell. In some embodiments, activation of the cell produces the substrate located on the inner side of the plasma membrane. In some embodiments, the substrate located on the inner side of the cytoplasmic membrane is a phosphoinositide. In some embodiments, the phosphoinositide is selected from phosphatidylinositol (3,4,5)-trisphosphate (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, 10 μM, 5 μM, 1 μM, 0.5 μM, 0.1 μM, 0.05 μM or 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 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 at the position corresponding to pY1221 of SEQ ID NO: 302. In some embodiments, the phosphorylated tyrosine is at the position corresponding to pY1346 of SEQ ID NO: 301.

[0018] In some embodiments, the polypeptide binds to the target polypeptide with a Kd of less than 10 μM, 5 μM, 1 μM, 0.5 μM, 0.1 μM, 0.05 μM or 0.01 μM, and wherein the Kd is analyzed by fluorescence polarization. In some embodiments, the polypeptide has a higher affinity for the target polypeptide comprising the phosphorylated tyrosine than for a control polypeptide that is not phosphorylated at the corresponding tyrosine position. In some embodiments, the polypeptide has an affinity for the target polypeptide comprising the phosphorylated tyrosine that is at least 2-fold, at least 5-fold, at least 10-fold, at least 50-fold or at least 100-fold higher (lower Kd) than for a control polypeptide that is not phosphorylated at the corresponding tyrosine position.

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

[0020] In one aspect, the present disclosure provides a polypeptide comprising: (i) a protein containing a caspase-associated recruitment domain (CARD) 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 of SEQ ID NOs: 305 and 307 - 437. In some embodiments, the SH2 domain comprises a motif of a conserved arginine residue in the 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 having 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 the CARD11 protein; and (ii) a second polypeptide portion derived from the 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 NOs: 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 NO: 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, 20, 30, 40, 50, 60, 80, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, or 300 amino acids of the N-terminal portion of the coiled-coil domain.

[0025] In some embodiments, the domain in (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. In some embodiments, the CARD domain is derived from the CARD11 protein, followed by the coiled-coil domain derived from the CARD11 protein.

[0026] In some embodiments, the domain in (b), or the SH2 domain, or the second polypeptide portion is located near the C-terminus of the polypeptide, wherein the polypeptide has no more than 50, 40, 30, 20, 15, 10, or 5 amino acids at the C-terminus of the domain in (b), or the SH2 domain, or the 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, 20, 30, 40, 50, 60, 80, 100, 120, 140, 150, 160, 180, or 200 amino acids of the N-terminal portion of the inhibitory domain (ID).

[0029] In some embodiments, the polypeptide does not comprise a sequence that is 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 in vivo accumulation of the T cells 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 regulatory (Treg), γδ T cells, invariant iNKT cells, MAIT cells, CAR T cells, tumor infiltrating lymphocytes, and engineered T cells comprising a transcriptional receptor.

[0031] In one aspect, the present disclosure provides a polypeptide that comprises mutations capable of: (i) altering T cell signaling via the NFAT, NF-κB, and / or AP-1 pathways, (ii) altering cytokine production, (iii) altering JAK / STAT signaling in T cells, (iv) altering co-stimulatory molecule signaling in T cells, (v) altering RAS / MEK / ERK signaling in T cells, (vi) altering phospholipase γ signaling, (vii) altering transcription factor activity in T cells, and / or (viii) altering or enhancing the in vivo persistence of T cells comprising the mutations in tumors.

[0032] In one aspect, the present disclosure provides a recombinant nucleic acid encoding a polypeptide as 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, IFNγ promoter, TIM3 promoter, IL4 promoter, GAI II A3 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 protein 1 (PD-1) promoter, T cell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, lymphocyte activation gene 3 (LAG-3) promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter, B and T lymphocyte attenuator (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, TGF-β promoter, T-bet promoter, Eomes promoter, GATA3 promoter, CD45RA promoter, 2B4 promoter, type I interferon (IFN)α, type I IFNβ promoter, IFNγ promoter, IRF3 promoter, IRF7 promoter, NFkB promoter, AP-1 promoter, TNF-α promoter, CD130 promoter, NR4A1 promoter, NR4A2 or NR4A3 promoter.

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

[0034] In one aspect, the present disclosure provides a cell comprising a polypeptide as described herein and / or a recombinant nucleic acid as described herein. In some embodiments, the cell is a non-natural cell or has been genetically engineered. In some embodiments, the cell is not a CD4+ T cell. In some embodiments, the cell is not a cancer 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 but no SH2 domain. In some embodiments, the recombinant nucleic acid of the cell is located at the endogenous CARD11-encoding gene locus of the cell or comprises at least a portion of the endogenous CARD11-encoding gene of the cell.

[0036] In some embodiments, the cell comprises a polypeptide, the polypeptide comprising a sequence selected from: 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: 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 IDNO: 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 and functional variants thereof containing at least one mutation listed in Table 1.

[0037] In some embodiments, the cell comprises a nucleic acid sequence or a functional variant thereof containing at least one mutation listed in Table 1, and the nucleic acid sequence has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the following sequences: 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: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ IDNO: 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, SEQ ID NO: 255.

[0038] In some embodiments, the cell further comprises: (i) a chimeric antigen receptor (CAR) specific for a target antigen; and / or (ii) a T cell receptor (TCR) specific for a target antigen. In some embodiments, the cell is selected from 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 as described herein in the T cells. In some embodiments, the method comprises genetically modifying the T cells to express the polypeptide. In some embodiments, the method comprises introducing a recombinant nucleic acid encoding the polypeptide or a vector comprising the recombinant nucleic acid into the T cells. In some embodiments, the method comprises expressing an engineered immune receptor in the T cells that binds to a target in tumor cells. In some embodiments, the method comprises administering to the subject a cell as described herein or a T cell prepared by the method 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 hematologic cancer. In some embodiments, the cancer expresses 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, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2 / neu), IGLL1, IL-11Ra, KIT (CD117), KLK2, LY6G6D, MUC1, NCAM, p53R175H, PAP, PDGFR-B, PRAME, PRSS21, PSCA, PSMA, ROR1, SIRPa, 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 treatment method does not include administering a lymphodepleting agent within 7 days before administering T cell therapy. In some embodiments, the treatment method does not include administering cyclophosphamide, fludarabine, and / or bendamustine within 7 days before administering T cell therapy. In some embodiments, the treatment method does not include administering at least 600,000 IU / kg of IL-2 every 8 hours. In some embodiments, the treatment method does not include checkpoint therapy that blocks PD-1 or CTLA-4 signaling.

[0043] In some embodiments, the cells have reduced exhaustion, enhanced proliferative capacity, increased replicative lifespan, reduced replicative senescence, enhanced anti-tumor effects, reduced dysfunction, enhanced persistence, and / or increased in vivo presence within a tumor. In some embodiments, the cells have increased or reduced signaling via 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 recombinant nucleic acid constructs encoding polypeptides, wherein the polypeptides comprise mutations capable of altering: (i) T cell signaling via the NFAT, NF-κB, and / or AP-1 pathways, (ii) cytokine production, and / or (iii) the in vivo persistence of therapeutic T cells comprising the mutations in a tumor.

[0045] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the mutations are point mutations, gene fusions, substitutions, gain-of-function mutations, stop-gain mutations, insertion mutations, deletion mutations, duplication mutations, or translocations. In some embodiments, the mutations are T cell lymphoma mutations or mutations in a clonally expanded population of T cells. In some embodiments, the mutations are capable of altering / promoting / enhancing CARD11-BCL10-MALT1 complex signaling in T cells.

[0046] In some embodiments, the mutations are located in genes selected from: 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 that has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 25 and comprises substitutions at amino acid positions selected from 361, 615, 634, 655, and 357 of SEQ ID NO: 25. In some embodiments, the substitutions include Y361C, S615F, E634K, D357N, S655C, or combinations thereof.

[0048] In some embodiments, the mutation is a fusion between a CARD11 polypeptide and a PIK3R3 polypeptide. In 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 that has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following sequences: 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 that has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the following sequences: 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 that has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 4, and comprises a substitution at amino acid 647 of SEQ ID NO: 4, SEQ ID NO: 28, and has a substitution at amino acid 575 of SEQ ID NO: 28 or SEQ ID NO: 114, and has 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, wherein the substitution at amino acid 575 of SEQ ID NO: 28 is Q575E, and wherein 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 mutations that can effect: (i) altering JAK / STAT signaling in T cells, (ii) altering cytokine production, and / or (iii) enhancing the in vivo persistence of therapeutic T cells comprising the mutations in tumors. In some embodiments, the polypeptide having the mutations comprises a JAK1, JAK3, STAT3 or STAT5 polypeptide. In some embodiments, the polypeptide comprises an amino acid sequence that has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the following sequences: SEQ ID NO: 90 and has a substitution at amino acid position 1097 of SEQ ID NO: 90, or SEQ ID NO: 94 and has a substitution at amino acid position 573 of SEQ ID NO: 94, or has SEQ ID NO: 176 and has a substitution at amino acid positions 618, 647 or 661 of SEQ ID NO: 176, or SEQ ID NO: 141 and has 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: 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 co-stimulatory molecule signaling in T cells and the persistence of T cells comprising the mutation in a tumor. In some embodiments, the polypeptide having the mutation comprises a TNFRSF1B, CD28, ICOS or CTLA4 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: i) SEQ ID NO: 192 and comprising a substitution at amino acid position 256 or position 377 of SEQ ID NO: 192, or ii) SEQ ID NO: 42 and comprising a substitution at amino acid position 51 or 77 of SEQ ID NO: 42, or iii) SEQ ID NO: 220, or iv) 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 of therapeutic T cells comprising the mutation in a tumor.

[0056] In some embodiments, the mutation comprises a BRAF gene or a RASGRP1 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 the following sequences: i) SEQ ID NO: 16 and comprising an amino acid substitution at amino acid position 469 or position 594 of SEQ ID NO: 16, or SEQ ID NO: 158 and comprising a 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 the following sequences: 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 is capable of altering phospholipase gamma signaling and / or (ii) cytokine production, and / or (iii) the in vivo persistence of the therapeutic T cells comprising the mutation in a tumor. In some embodiments, the mutation is located 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 the following sequences: 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 is capable of altering the transcription factor activity in T cells comprising 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 the following sequences: SEQ ID NO: 118 and comprising a substitution at amino acid 67 of SEQ ID NO: 118, SEQ ID NO: 122 and comprising a substitution at amino acid 565 of SEQ ID NO: 122, or SEQ ID NO: 98 and comprising 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 the following sequences: SEQ ID NO: 115, SEQ ID NO: 119, and SEQ ID NO: 95.

[0059] In some embodiments of the construct polypeptides and nucleic acids of the present disclosure, the mutation is the 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 comprises a sequence selected from the following: 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: 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 IDNO: 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 a functional variant thereof having at least one mutation listed in Table 1.

[0061] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the recombinant nucleic acid construct comprises a nucleic acid sequence or a functional variant thereof containing at least one mutation listed in Table 1, and the nucleic acid sequence has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the following sequences: 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: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ IDNO: 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, SEQ ID NO: 255.

[0062] In one aspect 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 wherein expression of the fusion polypeptide promotes in vivo persistence of therapeutic T cells comprising the fusion polypeptide in a tumor.

[0063] In some embodiments, the T cells are selected from regulatory (Treg), γδ T cells, invariant iNKT cells, macrophages, monocytes, natural killer (NK), CAR T cells, and engineered T cells comprising a transcriptional receptor.

[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 wherein the second polypeptide comprises an SH2 domain from PIK3R3.

[0065] In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the construct 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.

[0066] In some embodiments, the promoter is the MND promoter, EF1a promoter, sEF1a promoter, γ-retroviral 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, IFNγ 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 protein 1 (PD-1) promoter, T cell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, lymphocyte activation gene 3 (LAG-3) promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter, B and T lymphocyte attenuator (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, TGF-β promoter, T-bet promoter, Eomes promoter, GATA3 promoter, CD45RA promoter, 2B4 promoter, type I interferon (IFN) α, type I IFNβ promoter, IFN γ promoter, IRF3 promoter, IRF7 promoter, NFkB promoter, AP-1 promoter, TNF-a promoter, CD130 promoter, NR4A1 promoter, NR4A2 or NR4A3 promoter.

[0067] In one aspect of the present disclosure, there are provided recombinant nucleic acid constructs and / or recombinant nucleic acids, wherein the cytokine is IL-2, IL-4, IL-5, TNFα, IFN-γ, IL-13, and / or a combination of any of them.

[0068] In one aspect of the present disclosure, there are provided vectors comprising any recombinant nucleic acid construct and / or recombinant nucleic acid of the present disclosure. 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 one aspect of the present disclosure, there are provided cells comprising the nucleic acid construct or vector of the present disclosure. In some embodiments, the cell comprises (i) a chimeric antigen receptor (CAR) specific for a target antigen; (ii) a T cell receptor (TCR) specific for a target antigen; and / or (iii) a transcriptional receptor.

[0070] In some embodiments, the cell is selected from immune cells, T cells, regulatory T cells, CD8+ cells, natural killer cells, and tumor-infiltrating lymphocytes.

[0071] In one aspect, the present disclosure provides target antigens. In some embodiments, the target antigen is selected from 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), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), CEA, CLL-1, CS1, EGFR, FGFR2, AFP, CA125, MUC-1, MAGE, placental alkaline phosphatase-like protein 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 alpha (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-β, SSEA-4,, folate receptor α, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostate enzyme, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-ab1, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, 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 A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate cancer-associated protein 6 (prostein), survivin, telomerase, PCTA-1 / galectin 8, Melan-A / 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, intestinal carboxylesterase, muthsp70-2, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, AFP, TRAC, TCRB, 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-β, SSEA-4,, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostate specific antigen, 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, podoplanin, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, KRAS, mutant KRAS, KRAS G12D, prostate cancer associated protein 6, survivin, telomerase, PCTA-1 / galectin 8, Melan-A / 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, intestinal carboxylesterase, mut hsp70-2, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, extracellular portion of APRIL protein or any combination thereof.

[0072] In some embodiments, the target antigen is selected from cell surface receptors, adhesion proteins, integrins, mucins, lectins, tumor-associated antigens, and tumor-specific antigens. In some embodiments, the target antigen is a tumor-associated antigen selected from the following: 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, Ephrin B2, 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 another aspect, the present disclosure provides a method of preparing T cells for use in cell therapy. In some embodiments, the method comprises transcribing the T cells with a recombinant nucleic acid construct comprising a mutation that is capable of altering (i) T cell signaling via the NFAT, NF-κB, and / or AP-1 pathways, (ii) cytokine production, and / or (iii) the in vivo persistence of the T cells in a tumor. In some embodiments, the recombinant nucleic acid construct comprises the recombinant nucleic acid construct of the present disclosure.

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

[0075] In another aspect, the present disclosure provides a method of enhancing the in vivo persistence of T cells in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the T cells of the present disclosure. In some embodiments, the T cells are selected from 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] The present disclosure also provides methods for identifying mutations that can be used to improve T cell therapy, the methods comprising: a) identifying mutations based on a clonal T cell genomic sequencing database; b) identifying the frequency of occurrence of the mutations; and c) applying a statistical test to identify significant differences in hotspot genomic regions where the frequency of occurrence of the mutations is higher than random expectation, 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 other multivariate analysis. In some embodiments, the mutations are T cell lymphoma mutations. In some embodiments, the hotspot regions are located in the coding sequences of genes.

[0078] In some embodiments, the mutations improve T cell therapy by: a) increasing the proliferation of therapeutic T cells comprising the mutations in tumors, and / or b) altering effector function, and / or c) resisting T cell dysfunction, and / or d) enhancing growth. In some embodiments, the mutations are the mutations listed in Table 1. In some embodiments, the mutations promote positive T cell selection and / or T cell clone growth.

[0079] The foregoing summary is illustrative only and is not intended to be limiting in any way. In addition to the illustrative embodiments and features described herein, other aspects, embodiments, objects, and features of the present disclosure will become fully apparent from the drawings and the detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 is a schematic diagram depicting the method of the present disclosure for creating a screening library of mutations identified in T cell lymphoma, and in vitro and in vivo screening analyses of the library.

[0081] Figures 2A - 2G Shows detailed signaling results of in vitro T cell lymphoma mutation screening. Figures 2A - 2D Shows the reporter activity Z scores of NFAT, NF-κB, and AP-1 in each of the indicated T cell lymphoma mutation constructs. In some cases, PD-1 expression and IL-2 Z scores are also depicted. PD-1 expression is evaluated by flow cytometry. IL-2 secretion is evaluated by ELISA. The Z scores indicate the average Z scores of two independent biological replicates. Figure 2A Shows the reporter activity of each construct when expressed in CD19-CD28z CAR Jurkat cells and co-cultured with the K562 (CD19 negative) cell line. Figure 2B Shows the reporter activity and IL-2 secretion of each construct when expressed in CD19-CD28z CAR Jurkat cells and co-cultured with the CD19-K562 positive cell line. Figure 2CShows the reporter activity and PD-1 expression of each construct when expressed in CD19-BBz CAR Jurkat cells and co-cultured with K562 (CD19-negative) cell line. Figure 2D Shows the reporter activity, IL-2 secretion and PD-1 expression of each construct when expressed in CD19-BBz CAR Jurkat cells and co-cultured with CD19-K562 positive cell line. Figure 2E Shows bar graphs that demonstrate that in both the CD19-CD28z CAR and CD19-BBz CAR contexts, point mutations and fusion constructs in the CD19-K562 condition show more significantly different effects compared to the control, as compared to the parental K562 (CD19-negative) line. Figure 2F Shows bar graphs that indicate the number of T cell lymphoma point mutations or fusion constructs for which expression in CD19-BBz CAR Jurkat results in each different combination of upregulation or downregulation of signaling after antigen stimulation. Figure 2G Shows bar graphs that indicate the percentages of NFAT, NF-κB, and AP-1 signaling in CD19-BBz CAR cells for selected mutations after CD19-K562 stimulation, highlighting the highly tunable nature of the signaling output. Each point represents a biological replicate.

[0082] Figure 3 Shows the Z-scores of mutant NFAT, AP-1, and NF-κB signaling, which show a statistically significant difference from their wild-type controls. Mutations are ranked from positive (top of y-axis) to negative (bottom of y-axis) based on the Z-scores. This ranking demonstrates that some mutations induce gain-of-function (increased signaling output compared to wild-type), while other mutations result in loss-of-function (decreased signaling output compared to wild-type).

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

[0084] Figure 5A and Figure 5B Shows the correlation between the results of in vivo screening and in vitro studies of PD-1 for the mutations identified in the present disclosure. Figure 5A Shows a waterfall plot of the in vivo screening log2 fold change for each construct as determined by MAGeCK compared to baseline (before injection). Positive log2 fold changes indicate increased persistence in the tumor compared to the input. Figure 5BDepicts a bar graph that plots the fold change in vivo for each mutation and classifies 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 did significantly increase PD-1 expression in the in vitro Jurkat screen. These results suggest that in vitro PD-1 expression can contribute to predicting the in vivo persistence of T cell lymphoma mutations.

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

[0086] Figure 7 Shows a schematic diagram of the CARD11-PIK3R3 fusion according to the present disclosure text. The topmost figure shows the structure and domains of wild-type CARD11 containing 1154 amino acids, and the position of the breakpoint where the C-terminal component is lost and the PIK3R3 polypeptide is attached in the fusion protein. The middle figure depicts the structure of wild-type PIK3R3 containing 461 amino acids, and the position of the breakpoint where the C-terminal component of PIK3R3 is attached in the fusion protein. The bottom figure depicts the CARD11-PIK3R3 fusion protein with 725 amino acids, which has a CARD, coiled-coil, and truncated inhibitory domain derived from the N-terminus of CARD11 and an SH2 domain derived from the C-terminus of PIK3R3. The truncation of CARD11 is at the position shown in the topmost structure of the wild-type gene and labeled "breakpoint", and the truncation of PIK3R3 is at the position shown in the middle structure of the wild-type gene and labeled "breakpoint".

[0087] Figures 8A - 8B Depicts various CARD11-PIK3R3 fusion variants and their functions. Figure 8A Is a figure of variants containing deletions of various domains of the generated CARD11-PIK3R3 fusion. Figure 8B Depicts the reporter activity of the CD19-BBz CAR CARD11-PIK3R3 fusion variant in Jurkat cells co-cultured with CD19-K562.

[0088] Figures 9A - 9C Illustrates the function of the CARD11-PIK3R3 fusion in the CBM complex. Figure 9ASchematic of the BCL10-binding defective CARD11-PIK3R3 mutant, which contains an alanine-to-arginine substitution at amino acid position 28. Figure 9B Shows CD19-BBz CAR Jurkat reporter activity of the BCL10-binding defective CARD11-PIK3R3 mutant compared to control and cells expressing CARD11-PIK3R3. **** Indicates P value < 0.0001, determined by one-way ANOVA and subsequent Tukey's multiple comparison test. Figure 9C Shows a western blot 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 cleavage product. Data represent 2 independent experiments.

[0089] Figures 10A - 10B Shows the phosphorylation signaling kinetics of the CARD11-PIK3R3 fusion. Figure 10A Shows a schematic indicating the experimental method for studying CAR-dependent signaling in primary human CD3+ T cells according to the present disclosure. Figure 10B Shows a heatmap of marker expression determined by cytometry by time-of-flight mass cytometry (CyTOF) at each indicated time point in CD19-BBz CAR samples. Values indicate the mean of three independent T cell donors.

[0090] Figures 11A - 11C Shows the transcriptional landscape of the CARD11-PIK3R3 fusion. Figure 11A Shows principal component (PC) analysis of human CD8+ T cells from three independent donors, which were transduced with the indicated constructs and kept unstimulated (Unstim) or co-cultured with A549 cells expressing CD19 for 8 hours (Stim). Figure 11B Shows a volcano plot of differentially expressed genes in unstimulated or CD19 antigen-stimulated CD19-BBz CAR T cells. Selected target genes are marked. Positive log2 fold change indicates higher expression in CAR-T cells expressing CARD11-PIK3R3. Figure 11C Shows NF-κB, AP-1, and MALT1 gene signature enrichment in CD19-BBz CAR cells expressing CARD11-PIK3R3 after stimulation with A549 cells expressing CD19.

[0091] Figures 12A - 12BShows the experimental results of CD3+ T cell growth after anti-CD3 / anti-CD28 bead stimulation. During the 9-day period after removal from anti-CD3 / anti-CD28 bead stimulation, the cell counts of CD19-BBz CAR( Figure 12A ) or CD19-CD28z CAR( Figure 12B ) CD3+ T cells. ns indicates not significant, * indicates P value < 0.05.

[0092] Figures 13A - 13B Shows the cytokine secretion of CD3+ cells after co-culture with CD19-K562. The cytokine secretion profiles of CD19-BBz CAR( Figure 13A ) or CD19-CD28z CAR( Figure 13B ) CD3+ T cells co-cultured with CD19-K562 at a 1:1 ratio for 48 hours. P values were determined by paired ratio T-tests. ns indicates not significant, * indicates P value < 0.05, ** indicates P value < 0.01.

[0093] Figure 14 shows the activation and cytokine secretion of CD8+ T cells after co-culture with CD19-K562. Figure 14A Shows the activation markers expressed on CD19-BBz CAR T cells in the presence or absence of CARD11-PIK3R3, 24 hours after co-culture with CD19-K562 at a 1:1 ratio. Figure 14B 14A shows the activation markers expressed on untransduced or CARD11-PIK3R3-transduced CD8+ T cells, 24 hours after co-culture with CD19-K562 at a 1:1 ratio. Figure 14C Shows the cytokine secretion of CD8+ T cells expressing CD19-BBz CAR, 48 hours after co-culture with CD19-K562 at a 1:1 ratio, in the presence or absence of CARD11-PIK3R3. P values were determined by paired ratio T-tests. ns indicates not significant, * indicates P value < 0.05.

[0094] Figures 15A - 15B Shows the experimental results regarding long-term co-culture persistence, killing, and multi-stimulus growth. Figure 15A Shows flow plots depicting CD19-BBz CAR-transduced CD8+ T cells and CD19-K562 populations, depicting one donor, after co-culture together for 14 days in the presence and absence of supplemental IL-2. Bar graphs summarize the population percentages from three donors. P values were determined by unpaired T-tests. Figure 15BShows the cell counts of CD19-BBz CAR-transduced CD8+ T cells co-cultured with CD19-K562 on day 0 and restimulated with the target on day 6. P values were determined by unpaired T test. ns indicates not significant. * indicates P value < 0.05, **** indicates P value < 0.0001.

[0095] Figure 16A and Figure 16B Shows cytotoxicity at different effector-to-target ratios. Growth of CD19-A549mKate2+ targets co-cultured with CD19-BBz CAR (A) or CD19-CD28z CAR (B) CD8+ T cells over 108 hours. Bar graphs indicate target cell counts at hour 108, normalized to control. P values were calculated by one-way ANOVA and subsequent Tukey's multiple comparison test. ** indicates P value < 0.01, **** indicates P value < 0.0001.

[0096] Figure 17 Shows a schematic diagram indicating a xenograft CAR leukemia model for evaluating the therapeutic function of CARD11-PIK3R3 in T cells in vivo. In this model, NOD.Cg-Prkdcscid I12rgtm1Wj1 / SzJ (NSG) mice were injected via the tail vein with 5x10 5 Nalm6-GFP-luciferase cells. Primary human CD3+ T cells were transduced to express CD19-BBz CAR (with or without co-expression of CARD11-PIK3R3), and electroporated with TRAC RNP to knock out the expression of the human TCR. TRAC RNP is a CRISPR / Cas9 ribonucleoprotein complex targeting the human T cell receptor α constant region gene. Mice carrying Nalm6 were administered 1x10 6 CD19-BBz CAR T cells, and tumor burden was evaluated twice a week via bioluminescence imaging using an IVIS imaging system. Mice were euthanized when signs of hind limb paralysis first appeared.

[0097] Figures 18A - 1 8E demonstrates that CD19-BBz CAR expressing the CARD11-PIK3R3 fusion controls Nalm6 leukemia in vivo. Figure 18A Shows the mean radiance of luciferase-expressing Nalm6 tumor cells measured using in vivo imaging. Radiance was used as an indicator of tumor burden. Data indicate mean ± standard deviation. P values were determined by unpaired T test of mean radiance values on day 21. Control (n = 5), CAR (n = 6), CAR + CARD11-PIK3R3 (n = 5). ** indicates P value < 0.01. Figure 18BSurvival analysis of Nalm6-bearing mice treated with untransduced (control) (n = 5), BBz CAR (n = 6), or BBzCAR with CARD11-PIK3R3 (n = 5) T cells. P values were determined using the log-rank (Mantel-Cox) test. *** indicates P value < 0.001.

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

[0099] Figures 20A - 20C Shows the experimental results of using CD19-BBzCAR with CARD11-PIK3R3 fusion to control hCD19B16 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. Figure 20B and Figure 20C Shows tumor volume (B) and survival analysis (C) of animals bearing CD19-B16 tumors treated with untransduced (control) (n = 5), BBz CAR (n = 5), or BBz CAR with CARD11-PIK3R3 (n = 5) OT-1 T cells. P values were determined using the log-rank (Mantel-Cox) test. * indicates P value < 0.05, ** indicates P value < 0.01.

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

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

[0102] Figure 23It is a schematic diagram that indicates an isogeneic TCR transgenic melanoma model for evaluating the therapeutic function of CARD11-PIK3R3 in vivo in T cells. OT-I CD8+ T cells = T cells collected from CD45.1 C57BL / 6-Tg(TcraTcrb)1100Mjb / J mice.

[0103] Figure 24 shows that CARD11-PIK3R3 OT-I cells preferentially expand and accumulate in tumors. It shows the accumulation of TILs in a dual-transfer competition assay 7 days after T cell transfer. Figure 24A Amplification is demonstrated via flow cytometry plots. Figure 24B Quantify the fold expansion of CARD11-PIK3R3 OT-I in tumors in vivo compared to the control. P values were determined by a ratio paired T test. **** indicates P value < 0.0001.

[0104] Figure 25 Shows the accumulation of CARD11-PIK3R3 OT-I in tumors, spleen, and draining lymph nodes 7 days after transfer of 1x10 6 CARD11-PIK3R3 or control OT-I. P values were determined by an unpaired T test. * indicates P value < 0.05, **** indicates P value < 0.0001.

[0105] Figure 26A Shows the TCF-1 expression in OT-I + CARD11-PIK3R3 TILs. TCF-1 expression in TIL cells 7 days after transfer of 1x10 6 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 an unpaired T test. ns indicates not significant, *** indicates P value < 0.001.

[0106] Figure 27 Shows that CARD11-PIK3R3 OT-I has improved in vivo functionality. It depicts the ex vivo cytokine production of TILs isolated 7 days after transfer of 1x10 6 CARD11-PIK3R3 or control OT-I and restimulated ex vivo with PMA / ionomycin. P values were determined by an unpaired T test. * indicates P value < 0.05, *** indicates P value < 0.001.

[0107] Figure 28A and Figure 28B Shows that CARD11-PIK3R3 OT-I demonstrates improved in vivo anti-tumor efficacy. Figure 28Adepicts tumor size, and Figure 28B depicts the survival analysis of mice bearing B16-OVA melanoma treated with PBS or OT-I cells (2x10 6 ) at 12 days post tumor inoculation. Complete response was defined as the absence of detectable tumors. **** indicates P value < 0.0001.

[0108] Figure 29A and Figure 29B shows that CARD11-PIK3R3 OT-I demonstrates improved in vivo functionality at low doses and during tumor rechallenge. Compared with 2x10 6 control OT-I T cells (n = 5) (A) and mice (B) in which tumors from a dose-response experiment had been cleared and were subcutaneously rechallenged with B16-OVA melanoma tumors on the contralateral flank, tumor size of mice bearing B16-OVA melanoma treated with OT-I CARD11-PIK3R3 T cells at a dose of 1x10 5 (n = 4) or 2x10 5 (n = 5) T cells. *** indicates P value < 0.001, **** indicates P value < 0.0001.

[0109] Figures 30A - 30D shows the experimental results for evaluating CARD11-PIK3R3 truncations in vitro in CD8+CD19-BBz CAR T cells. Figure 30A is a schematic diagram depicting the coiled-coil truncation design of CARD11-PIK3R3, where AID indicates removal of the inhibitory domain and -XXAA indicates the number of amino acids removed from the coiled-coil region. Figure 30B shows the cell counts of CD19-BBz CAR CD8+ T cells with CARD11-PIK3R3 truncations at 7 days after removal from anti-CD3 / anti-CD28 bead stimulation. Figure 30C shows CD19-BBz CAR CD8+ T cells with CARD11-PIK3R3 truncations co-cultured with CD19 target cells at a 1:1 ratio for 24 hours, and the IL-2 secretion in the supernatant was evaluated by ELISA after the co-culture. Figure 30D shows flow cytometry plots depicting CD19-BBz or fusion variant CD8+ T cells with CARD11-PIK3R3 and CD19-K562 populations co-cultured together for 14 days in the absence of supplemental IL-2.

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

[0111] Figure 32A Shows a lollipop plot depicting point mutations in the BRAF gene. 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-score indicates the average Z-score of two independent biological replicates.

[0112] 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-score indicates the average Z-score of two independent biological replicates. Figure 33B Shows the in vivo screening log2 fold change for each CARD11 mutation or wild type as determined by MAGeCK compared to the baseline (before injection). A positive log2 fold change indicates an increase in persistence in the tumor compared to the input.

[0113] Figure 34A Shows a lollipop plot depicting point mutations in the RASGRP1 gene. Figure 39B The 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. The Z-score indicates the average Z-score of two independent biological replicates. Figure 39C Shows the in vivo screening log2 fold change for each RASGRP1 mutation or wild type as determined by MAGeCK compared to the baseline (before injection). A positive log2 fold change indicates an increase in persistence in the tumor compared to the input.

[0114] Figure 35A Depicts a lollipop plot showing multiple point mutations in the PLCG1 gene. Figure 35B Is a figure showing: the CD19-BBz CAR Jurkat reporter activity Z-scores for NFAT, NF-κB, AP-1, and IL-2 for each PLCG1 mutation after co-culture with CD19-K562. The Z-score indicates the average Z-score of two independent biological replicates.

[0115] Figure 36AIt is a lollipop plot depicting point mutations in the TNFRSF1B gene. Figure 36B Bar graphs are shown that illustrate the CD19-BBz CAR Jurkat reporter activities 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-score indicates the average Z-score of two independent biological replicates. Figure 36C Shown is the in vivo screening log2 fold change for each TNFRSF1B mutation or wild type as determined by MAGeCK compared to baseline (before injection). A positive log2 fold change indicates an increase in persistence in the tumor compared to the input.

[0116] Figure 37A It is a lollipop plot depicting JAK / JAK3 gene mutations. Figure 37B Shown is the in vivo screening log2 fold change for each JAK1 / JAK3 mutation as determined by MAGeCK compared to baseline (before injection). A positive log2 fold change indicates an increase in persistence in the tumor compared to the input.

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

[0118] Figure 39A Shown is the principal component analysis of the transcriptome of human BBz-CAR T cells. Figure 39B It is a heatmap of genes significantly upregulated in CARD11-PIK3R3 compared to control BBz-CAR T cells after stimulation with the CD19 antigen common to both CD4+ and CD8+ T cells. Figure 39C Shown are the top 5 Reactome pathways enriched in CD4+ and CD8+ T cells (top), and NF-kB, AP-1, and MALT1 gene signature enrichment in CD8+ BBz-CAR T cells expressing CARD11-PIK3R3 after stimulation (bottom).

[0119] Figure 40 Shown are the activation markers expressed on transduced CD4 and CD8 T cells 24 hours after co-culture with CD19-K562 at a 1:1 ratio. The ratio of MFI in CARD11-PIK3R3 relative to control is shown. P values were determined by unpaired T test.

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

[0121] Figure 42A Shows control (n = 6), CD19-BBz-CAR (n = 7), CD19-BBz-CAR+CARD11-PIK3R3 (n = 7) administered with 7e6 CAR+ T cells. The dose of CARD11-PIK3R3 T cells (n = 7) is equivalent to the total number of T cells expressing CARD11-PIK3R3 administered in the CD19-BBz-CAR+CARD11-PIK3R3 group. Figure 42B Shows control (n = 7), CD19-CD28z-CAR (n = 7), CD19-CD28z-CAR+CARD11-PIK3R3 (n = 7) T cells administered 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) re-challenged with 5e5 Nalm6-Luc-GFP tumors in part (b). Figure 42D Shows tumor volumes of animals bearing M28 tumors treated with control (n = 5), MCAM-CD28z-CAR (n = 5), or MCAM-CD28z-CAR+CARD11-PIK3R3 (n = 5). T cells were administered at 5e5 CAR+ cells, and control cells in other treatment groups were administered at an equivalent to the highest total T cell dose.

[0122] Figure 43A Shows flow cytometry plots indicating CAR (FLAG) and CARD11-PIK3R3 (mCherry) expression in human CD3 T cells. Figure 43B and Figure 43C Shows percentage change in body weight compared to baseline of tumor-bearing ( Figure 43B ) or non-tumor-bearing ( Figure 43C ) animals treated with control, CD19-BBz-CAR, CD19-BBz-CAR+CARD11-PIK3R3, or CARD11-PIK3R3. Figure 43D Shows Figure 42B Survival analysis of surviving CD19-BBz-CAR (n = 3), CD19-BBz-CAR+CARD11-PIK3R3 (n = 4) animals or naive mice (n = 4) re-challenged with 5e5 Nalm6-Luc-GFP tumors. P values were determined by log-rank Mantel-Cox. *Indicates P value < 0.05.

[0123] Figure 44A and Figure 44B shows flow cytometry plots indicating FLAG (CAR) and mCherry (CARD11-PIK3R3) expression in human CD3 T cells. Figure 44C shows the percentage change in body weight relative to baseline in animals bearing M28 tumors treated with control, MCAM-CD28z-CAR, or MCAM-CD28z-CAR + CARD11-PIK3R3.

[0124] Figure 45 shows tumor sizes in mice bearing SNU-1 HLA-C*08:02 gastric cancer xenografts treated with control (n = 5), KRAS p.G12D-specific TCR T cells (n = 5), or KRAS p.G12D-specific TCR CARD11-PIK3R3 T cells (n = 6). Complete response was defined as the absence of detectable tumors. * indicates P value < 0.05, *** indicates P value < 0.001, **** indicates P value < 0.0001.

[0125] Figure 46A 、 Figure 46B and Figure 46C shows in vitro expansion of CD19-BBz-CAR T cells with and without CARD11-PIK3R3. Figure 46A and Figure 46B shows sorting for purity of CAR or CAR + CARD11-PIK3R3, followed by 12 days of expansion with IL-2. On day 12,

[0126] the cultures were split and the groups were re-plated in the absence of IL-2 ( Figure 46A ) and in the presence of IL-2 ( Figure 46B ). 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 in the presence of IL-2 were re-plated in the absence of IL-2, and cells were counted and split for an additional 10 days.

[0127] Figure 47 shows in vivo analysis of CAR T cells expressing CARD11-PIK3R3. A histogram is shown indicating human CD19 ligand expression on hCD19-B16 tumors treated with CD19-BBz-CAR or CD19-BBz-CAR + CARD11-PIK3R3 that had reached the euthanasia endpoint, compared to a known CD19-positive B16 tumor sample.

[0128] Figure 48A , Figure 48B and Figure 48C show in vivo analysis of OT-I T cells expressing CARD11-PIK3R3. Figure 48A Shows a schematic diagram of a competition experiment using CARD11-PIK3R3 and CARD11-PIK3R3 R28A pmel-1 CD8 T cells in a B16-F10 tumor model. Figure 48B Shows the tumor growth curve of 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.R 28A) compared to vector control 7 days after adoptive transfer. All P values were determined by ratio paired T-test.

[0129] Figures 49A - 49J Shows long-term evaluation of B6 mice treated with OT-I T cells expressing CARD11-PIK3R3. Figure 49A Shows monitoring of mice cleared of B16-F10-OVA for up to 240 days after adoptive T cell transfer. Autopsies were performed as outlined in this schematic diagram. Figure 49B Shows measuring the body weight of all mice infused with CARD11-PIK3R3 Figures 29A - 29B weekly and comparing it with the expected body weight curve published by The Jackson Laboratory Research Institute. Figure 49C Shows the spleen weights of three animals that underwent autopsy on day 240. It was calculated as a percentage of body weight and compared with the expected spleen weight published by The Jackson Laboratory Research Institute. Figure 49D Shows the autopsy of a 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 lymphoma or extranodal lymphoma may occur. The animals were subjected to a full body autopsy. The tissues did not show nuclear atypia, changes in cell architecture, or evidence of neoplastic disease. Representative images and scale bars at low magnification (left) and high magnification (right) are shown. The white box reflects the location of the high magnification image. Figure 49G and Figure 49I Show at 330 days after adoptive transfer Figures 29A - 29B of the mice presented in Figure 49G) or blood sampling of 2e6 OT-I CARD11-PIK3R3-treated mice 418 days after adoptive transfer( Figure 49I ) Schematic diagram. Figure 49H and Figure 49J show the percentage of CD8 T cells expressing CARD11-PIK3R3 in the blood.

[0130] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, like symbols generally identify like components unless the context dictates otherwise. The illustrative alternatives described in the detailed description, the drawings, and the claims are not intended 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 can be arranged, substituted, combined, and designed in a variety of different configurations, all of which are expressly covered and form a part of this application. Detailed Description Overview

[0131] Generally, the present disclosure particularly relates to compositions and methods for improving adoptive T cell therapy. The present inventors have discovered ways 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 exploiting such beneficial mutations to improve the effectiveness of T cell therapy.

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

[0133] In some embodiments, the mutation alters TCR transcriptional signaling output. In 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 of therapeutic T cells, increase proliferation, alter effector function, resist T cell dysfunction, increase T cell fitness, enhance in vivo persistence, and / or increase intratumoral presence. The present disclosure also relates to recombinant nucleic acid constructs and / or recombinant nucleic acids encoding polypeptides having one or more mutations that can reduce T cell exhaustion of therapeutic T cells, increase proliferation, alter effector function, resist T cell dysfunction, increase T cell fitness, enhance in vivo persistence, and / or increase intratumoral presence.

[0135] After T cell receptor complex engagement, reprogramming T cells for proliferation, cytokine production, and differentiation into effector cells can rely on activation of co-stimulatory signals and can be counteracted by co-inhibitory molecules. The transcription factors NF-κB, NFAT, and AP-1 have a major role in inducing the transcriptional programs required for T cell activation and differentiation. Measuring the expression of such transcription factors is within the methods of the present disclosure and can indicate significant changes in the function of the mutations of the present disclosure in T cells. Definitions

[0136] Unless otherwise defined, all technical terms, symbols, and other scientific terms or nomenclature used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this application belongs. In some instances, terms having a commonly understood meaning are defined herein for clarity and / or for ease of reference, and these definitions included herein need not be construed as representing a substantial difference from the meaning commonly understood in the art. Many of the techniques and procedures described or referenced herein are well understood by those of ordinary skill in the art and are commonly employed 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] Unless the context clearly dictates otherwise, the singular forms “a,” “an,” and “the” include plural referents. For example, the term “a cell” includes one or more cells, including mixtures thereof. The use of “A and / or B” herein is intended to include all of the following alternative forms: “A,” “B,” “A or B,” and “A and B.”

[0138] Where a range of values is provided, it is to be understood that every intermediate value between the upper and lower limits of that range (to the tenth of the unit of the lower limit unless the context clearly dictates otherwise) and any other stated value or intermediate value within the stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limitation within the stated range. Where the stated range includes one or both of the recited limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0139] Certain ranges are presented herein by numerical values preceded by the term "about". The term "about" is used herein to provide literal support for the exact number that follows and for numbers that are close to or approximate the number that follows. When determining whether a number is close to or approximates a specifically recited number, an unrecited number that is close to or approximates the specifically recited number may be a number that provides a substantially equivalent form of the specifically recited number in the context in which it is presented. For example, in accordance with the practice in the art, "about" may mean within 1 or more standard deviations. Alternatively, "about" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and still more preferably up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude of the value, preferably within 5-fold, and more preferably within 2-fold.

[0140] As will be understood by one of ordinary skill in the art, for any and all purposes, such as in providing a written description, all ranges disclosed herein also encompass any and all possible subranges thereof and combinations of such subranges. Any listed range can be readily identified as fully describing the same range and enabling the same range to be broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. By way of 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 of skill in the art, all such terms as "up to", "at least", "greater than", "less than", etc. include the recited number and refer to ranges that can then be broken down into the subranges as discussed above. Finally, as will be understood by those of skill in the art, a range includes each individual member. Thus, for example, a group having 1-3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1-5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0141] It should be understood that aspects and embodiments of the present disclosure described herein include "comprising aspects and embodiments", "consisting of aspects and embodiments", and "consisting essentially of aspects and embodiments". As used herein, "comprising" is synonymous with "including", "containing", or "characterized by", and is inclusive or open-ended and does not exclude additional unrecited elements or method steps. As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claimed composition or method. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation of the term "comprising" in this application, particularly in the description of the components of a composition or the steps of a method, should be understood to encompass compositions and methods that consist essentially of and consist of the recited components or steps.

[0142] As used herein in the context of two or more nucleic acids or proteins, the term "percent identity" 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 higher identity in a specified region when compared and aligned to obtain maximum correspondence in a comparison window or specified region), as measured using the BLAST or BLAST 2.0 sequence comparison algorithms with the default parameters described below or by manual alignment and visual inspection. See, e.g., the NCBI website at ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be "substantially identical". This definition also relates to or can be applied to the complement of a test sequence. This definition also includes sequences having deletions and / or additions as well as those having substitutions. In general, sequence identity can exist over a region of at least about 20 amino acids or nucleotides in length, or over a region of 10 - 100 amino acids or nucleotides in length, or over the entire length of a given sequence.

[0143] If desired, published techniques and publicly available computer programs can be used to calculate sequence identity, 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 with its default parameters, such as the sequence analysis software package of the Genetics Computer Group of the University of Wisconsin Biotechnology Center (1710 University Avenue, Madison, Wisconsin 53705).

[0144] As used herein, the term "mutation" refers to point mutations, gene fusions, substitutions, gain-of-function mutations, stop-gain mutations, insertion mutations, deletion mutations, duplication mutations, and / or translocations. Mutations can be located 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 such a recombinant polynucleotide construct that is designed to transfer between host cells and can be used for transformation purposes, such as introducing heterologous DNA into a host cell. Thus, in some embodiments, a vector can be a replicon (such as a plasmid, phage, or cosmid) into which another DNA segment can be inserted to cause replication of the inserted segment. In some embodiments, an expression vector can be an integrating vector.

[0145] As used herein, the term "viral vector" refers to a nucleic acid molecule (e.g., a transfer plasmid) that contains nucleic acid elements of viral origin, which typically facilitate the transfer or integration of the nucleic acid molecule into the genome of a cell; or refers to a viral particle that mediates nucleic acid transfer. In addition to one or more nucleic acids, the viral particle will typically contain a variety of viral components and sometimes also host cell components. The term viral vector can refer to a virus or viral particle capable of transferring nucleic acid into a cell, or to the nucleic acid itself that is transferred. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are mainly derived from viruses. Viral vectors that can be used in the present disclosure include, for example, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, herpesviruses, simian virus 40 (SV40), and bovine papillomavirus vectors (see, for example, Gluzman (ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.). 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 can be obtained from many sources, including the American Type Culture Collection (Manassas, Va.). When selecting an expression system, care should be taken to ensure that the components are compatible with each other. A person skilled in the art or an ordinary technician can make such a decision. Additionally, if guidance is needed in selecting an expression system, a skilled technician can consult P. Jones, "Vectors: Cloning Applications", John Wiley and Sons, New York, N.Y., 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 that are mainly derived from a retrovirus. A retroviral vector can 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) that are mainly derived from a lentivirus, which is a genus of retroviruses. Lentiviral vectors offer several attractive features as gene delivery vehicles, including: (i) sustained gene delivery by stable integration of the vector into the host genome; (ii) the ability to infect both dividing and non-dividing cells; (iii) broad tissue tropism, including important gene therapy target cell types and cell therapy target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic sequences or intron-containing sequences; (vi) a potentially safer integration site profile; and (vii) relatively easy systems for vector manipulation and production.

[0147] As used herein, the term "pharmaceutically acceptable carrier" 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 contain suspending and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic agents, and absorbents, among others. It will be understood that the compositions of the present disclosure may also contain other supplementary physiologically active agents. The carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the composition and not 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 is attached. A series of PEGs or PEG derivatives having an optional range of sizes from about 10,000 daltons to about 40,000 daltons can be attached to the 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 from about 1 kD to about 200 kD, such as, for example, about 10 kD to about 150 kD, about 50 kD to about 100 kD, about 5 kD to about 100 kD, about 20 kD to about 80 kD, about 30 kD to about 70 kD, about 40 kD to about 60 kD, about 50 kD to about 100 kD, about 100 kD to about 200 kD, or about 150 kD to about 200 kD. In some embodiments, the average molecular weight of the PEG or PEG derivative is about 5 kD, about 10 kD, about 20 kD, about 30 kD, about 40 kD, about 50 kD, about 60 kD, about 70 kD, or about 80 kD. In some embodiments, the average molecular weight of the PEG or PEG derivative is about 40 kD.

[0149] As used herein, the terms "administration" ("administration" and "administering") refer to delivering a bioactive composition or formulation by an administration route, which includes but is not limited to oral, intravenous, intraarterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, and topical administration or combinations thereof. The terms include but are not limited to administration by medical professionals and self-administration.

[0150] As used herein, the term "injection" includes intravenous, intramuscular, intraarterial, intrathecal, intracavitary, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, intrathecal, and intrasternal injection and infusion.

[0151] The term "cancer" generally refers to the presence of cells having the typical characteristics of cancer cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rates, and certain characteristic morphological features. Cancer cells can be in the form of tumors, but such cells can exist alone in an animal subject or can 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-cancerous as well as malignant cancers. In some embodiments, the cancer is a solid tumor, a soft tissue tumor, or a metastatic lesion.

[0152] As used herein and unless otherwise indicated, a "therapeutically effective" or "pharmacologically effective" amount or quantity of a subject construct, nucleic acid, cell, or composition of the present disclosure generally refers to an amount or quantity sufficient to achieve the stated purpose (e.g., provide a therapeutic benefit in the treatment or management of cancer, or delay or minimize one or more symptoms associated with cancer) relative to the absence of the composition. A therapeutically effective amount of a compound means an amount of the therapeutic agent that, alone or in combination with other therapeutic agents, provides a therapeutic benefit in the treatment or management of cancer. The term "therapeutically effective amount" can encompass an amount that improves the overall therapy of cancer, alleviates or avoids the symptoms or causes of cancer, or enhances the therapeutic efficacy of another therapeutic agent. Examples of "effective amounts" are amounts sufficient to effect the treatment, prevention, or alleviation of one or more symptoms of a disease, which may also be referred to as a "therapeutically effective amount." "Alleviation" of a symptom means a reduction in the severity or frequency of one or more symptoms or the elimination of one or more symptoms. The exact amount of the composition, including a "therapeutically effective amount," will depend on the purpose of the treatment and will be determinable by those 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 ed., 2003, Gennaro ed., Lippincott, Williams & Wilkins).

[0153] As used herein, "subject" or "individual" includes animals, such as 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 can be a human patient or individual suffering from, at risk of developing, or suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. A subject can also be an individual diagnosed as being at risk of a condition of interest at or after diagnosis. The term "non-human animal" includes all vertebrates, such as mammals, e.g., rodents (e.g., mice), e.g., non-human primates; and non-mammals, e.g., sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0154] The provision of headings (e.g., (a), (b), (i), etc.) is for ease of reading the specification and claims only. The use of headings in the specification or claims does not require steps or elements to be carried out in alphabetical or numerical order or the order in which they are presented. It should be understood that certain features of the present disclosure described in the context of separate embodiments can also be provided in combination in a single embodiment. Conversely, various features of the present disclosure described in the context of a single embodiment for brevity can also be provided separately or in any suitable sub-combination. All combinations that are within the scope of the embodiments of the present disclosure are exactly covered by the present disclosure and are disclosed herein as if each combination were separately and explicitly disclosed. Additionally, all sub-combinations of the various embodiments and their elements are exactly covered by the present disclosure and are disclosed herein as if each such sub-combination were separately and explicitly disclosed herein. TCR Signaling

[0155] Upon engagement of the peptide-MHC complex, immunoreceptor tyrosine-based activation motifs (ITAMs) contained within the intracellular tails of the TCR complex are phosphorylated and recruit Zap70. Zap70 then recruits and activates the downstream LAT signalingosome containing LAT, PLCγ, and Slp76. This signalingosome causes activation of Ras / MAPK / ERK signaling, leading to AP-1 transcriptional activity; causes activation of PKCθ, leading to NF-κB transcriptional activity; and finally causes calcium influx, leading to NFAT transcriptional activity. Additionally, signaling from co-stimulatory receptors such as 41BB and cytokine support from IL-2 enhance these signaling pathways and are required for T cells to undergo full activation and avoid anergic cell death. Upon full activation, T cells rapidly proliferate and differentiate, secrete inflammatory cytokines, and cytotoxic cells begin target killing by releasing cytotoxic granules. After this acute response subsides, activated T cells undergo contraction, forming a small population of long-lived memory T cells that surveil the body and are primed to respond upon future encounter with antigen. T Cell Signaling

[0156] T cells are fully activated through T cell receptor (TCR) engagement, co-stimulatory signaling, and cytokine support. TCR activation induces NFAT, NF-κB, and AP-1 transcription factor signaling, and IL-2 and other inflammatory cytokine secretion. T Cell Exhaustion

[0157] T cell exhaustion is a state of hyporesponsiveness induced in effector T cell populations upon chronic exposure to antigen. Hallmarks of exhaustion are upregulation of surface inhibitory receptors such as PD-1, TIM-3, LAG-3, and CTLA-4, among others, and the inability to proliferate long term, secrete inflammatory cytokines, and effectively kill target cells. Exhausted T cells exist with a unique transcriptional and epigenetic landscape. While exhaustion has been well characterized in chronic viral infections, it is also associated with 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, T cell exhaustion must be prevented or overcome. Indeed, checkpoint therapies that block signaling from the PD-1 or CTLA-4 inhibitory axes have proven marginally effective in rejuvenating T cell responses in some cancers, leading to limited but effective tumor control. NFAT Signaling:

[0158] The transcription factor nuclear factor of activated T cells (NFAT) is involved in T cell activation and the T cell exhaustion program. In the resting state, the NFAT transcription factor is found in the cytoplasm in a phosphorylated state and cannot translocate to the nucleus to induce signal transduction. During T cell activation, the extracellular influx of Ca 2+ activates the calcium-dependent phosphatase calcineurin, resulting in the dephosphorylation of NFAT and its subsequent translocation and signal transduction in the nucleus. NFAT binds to DNA in multiple ways (as a monomer, dimer, or as a complex with other transcription factors). Specifically, NFAT is known to bind cooperatively with the transcription factor AP-1 to induce the transcription of activation-related genes crucial for effector T cell function. In contrast, when NFAT does not bind to AP-1 but is unliganded, it has been found to induce the expression of exhaustion-related genes such as the inhibitory receptor PD-1. Additionally, studies have determined that the exhaustion induced by unliganded NFAT can be rescued 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 associated with T cell exhaustion, and the deletion or knockdown of these transcription factors improves the T cell phenotype and tumor control in vivo.

[0159] Since NFAT signaling is crucial for the successful activation of T cells but also plays a role in exhaustion, particularly in the absence of AP-1, it can be used to determine how genetic modifications in T cells affect the kinetics of NFAT signaling. Targeting the NFAT transcriptional pathway can be beneficial for T cell therapies. NF-κB signaling:

[0160] NF-κB (nuclear factor κ-light-chain enhancer of B cells) is a family of transcription factors that induce transcriptional programs crucial 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 a robust effector T cell response. NF-κB signaling is induced through two pathways (canonical and non-canonical).

[0161] In the canonical pathway, NFKB1 is bound in the cytoplasm by IkBa and the IkB-like molecule p105, forming a complex that prevents the nuclear translocation of NKFB1. Upon activation, TCR signaling induces the activation of PKCθ, which in turn phosphorylates and activates CARD11. The activated CARD11 forms a complex with BCL10 and MALT1, and ultimately leads to the phosphorylation and degradation of IkBa and the IkB-like molecule p105, releasing NF-κB for translocation to the nucleus.

[0162] The non-canonical pathway is triggered by signaling via members of the tumor necrosis factor receptor (TNFR) family, which includes the co-stimulatory domain 41BB. At rest, NFKB2 binds to p100, and during activation, NF-κB-inducing kinase (NIK) activates IKKα, which in turn phosphorylates p100 and leads to the release and translocation of NKFB2 to the nucleus.

[0163] In syngeneic models of solid tumors, endogenous T cells that can respond to tumors require NF-κB signaling to mediate tumor clearance. Although NF-κB is induced by TCR signaling, it is also stimulated by the co-stimulatory domain 41BB (a member of the TNFRS superfamily), which is used clinically in FDA-approved chimeric antigen receptor (CAR) therapies. It has been found that CAR therapies using the 41BB co-stimulatory domain last longer in patients compared to CAR therapies using other co-stimulatory domains such as CD28. The persistence of 41BB CAR is directly related to NF-κB signaling, which increases the resistance of CAR T cells to apoptosis by inhibiting apoptosis proteins such as Bim. AP - 1 Signaling:

[0164] AP-1 transcription factors are a family of homo- or heterodimeric proteins formed by 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 typically signals in a complex with NFAT, and as indicated in the NFAT section, deletion of AP-1 leads to the induction of an exhaustion program in T cells by unpaired NFAT, which can however be rescued by overexpression of the AP-1 family member c-Jun. In this context, it is also known that deletion of AP-1 induces an anergic cell state in which T cells have been partially activated by the TCR but do not have sufficient co-stimulation and cytokine signaling to induce full activation.

[0165] AP-1 dimers are activated by an array of physiological and pathological stimuli. Studies have reported that AP-1 proteins (primarily those belonging to the Jun group) control cell life and death through their ability to regulate the expression and function of cell cycle regulators such as cyclin D1, p53, p21(cip1 / waf1), p19(ARF), and p16. Among the Jun proteins, c-Jun is unique in its ability to positively regulate cell proliferation through repression of tumor suppressor gene expression and function and induction of cyclin D1 transcription. These effects are antagonized by JunB, which upregulates tumor suppressor genes and represses cyclin D1. An important target of the actions of AP-1 on cell life and death is the tumor suppressor p53, whose expression and transcriptional activity are regulated by AP-1 proteins.

[0166] The concomitant induction of NFAT and AP-1 employs the co-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 transcription factor families. (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 activation of these receptors requires large amounts of IL-2, while memory T cells and regulatory T cells express high-affinity IL-2 receptors, and effective signaling through these receptors requires much lower amounts of IL-2. The IL-2 receptor utilizes the JAK / STAT signaling cascade, resulting in widespread transcriptional changes. Although CD8+ T cells are responsible for the cytotoxic effector response to foreign antigens, CD8+ T cells themselves cannot efficiently produce IL-2 and instead rely on helper CD4+ T cells to produce IL-2 and other cytokines for support.

[0168] IL-2 is so important for T cell persistence and proliferation that the FDA has approved high-dose IL-2 therapy for certain cancers in the hope of expanding the endogenous cytotoxic T cell population to induce tumor rejection. In metastatic melanoma and renal cell carcinoma trials, high-dose IL-2 therapy induced limited (7%) long-term response rates.

[0169] However, patients had poor tolerance to this therapy, resulting in multiple deaths and potentially promoting the expansion of CD4+CD25+FoxP3+ regulatory T cells. Regulatory T cells are known to be tumor-promoting, and a large number of regulatory T cells (Tregs) are associated with poor prognosis in the solid tumor microenvironment. The toxicity and expansion of the tumor-promoting Treg population pose such a great challenge to IL-2 therapy that synthetic orthogonal IL-2 cytokine therapies have recently been developed to target the effects of IL-2 cytokine therapy to cytotoxic T cells and avoid the expansion of Tregs.

[0170] IL-2 secretion is highly correlated with the persistence and proliferation of T cell therapy products. Therefore, the inventors / Disclosers evaluated the IL-2 secretion capacity of each mutation when expressed in the CAR Jurkat cell line and after co-culture with target cells. As a follow-up of relevant hits, the inventors / Disclosers evaluated the ability of the mutations to improve CAR T cell in vitro target killing when cultured in the absence of supplemental IL-2. This IL-2-free environment better reflects the challenges of the tumor microenvironment, where IL-2 is scarce and autocrine IL-2 is beneficial for maintaining proliferation and long-term killing capacity. In Vivo Screening of Mutations:

[0171] The major obstacle to the success of cell therapy is the accumulation and persistence of T cells within tumors. The inventors / Disclosers sought to systematically screen for mutations in primary human CAR T cells in a xenograft model. To find mutations that improve persistence under highly adverse conditions, the inventors / Disclosers utilized the difficult-to-control K562 subcutaneous tumor model, where T cell efficacy is limited.

[0172] The inventors / Disclosers found that clinically relevant CD19-targeting BBz CAR T cells were ineffective in 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 reflect some of the CAR T cell clinical failures observed in the treatment of solid tumors. The inventors / Disclosers chose to screen for mutations in this high-bar "failure" model, hoping to find mutations that significantly improve the persistence of CAR T cells within solid tumors.

[0173] Although adoptive cell therapy has proven highly effective in refractory B-cell malignancies, CAR-T cell therapy has not provided robust long-term efficacy against solid tumors. In the solid tumor context, CAR-T cells become exhausted and have difficulty proliferating and performing effector functions, ultimately resulting in uncontrolled tumor growth or prevention of recurrence. Accordingly, the inventors / Disclosers chose to generate an effective targeted cell therapy against solid tumors by improving the proliferative capacity, persistence, and effector functions of CAR-T cells. CARD11 - BCL10 - MALT Signalosome

[0174] The formation of the CARD11-BCL10-MALT (CBM) signaling complex is a key event in T cell- and B cell-receptor-induced gene expression. Upon exposure to diverse immune triggers, these molecules form self-organizing filaments that have MALT1 protease activity to regulate the canonical nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways as well as the degradation of mRNA-binding proteins, thereby providing a two-tiered control of inflammatory gene expression. Dysregulation of CARD11, BCL10, or MALT1 expression or CBM signaling is associated with cancer, immunodeficiency, and autoimmunity (J. Ruland, L. Hartjes, CARD-BCL-10-MALT1 signalling in protective and pathological immunity. Nat Rev Immunol 19, 118-134 (2019)).

[0175] In normal T cells, T cell receptor (TCR) signaling activates PKCθ, which in turn promotes the assembly of the CARD11-BCL10-MALT1 (CBM) signalosome. The CBM complex then 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 a necessary step for regulating NF-κB in lymphoid immune cells.

[0176] An inhibitory domain present in CARD11 allows for intramolecular autoinhibition, which can prevent CARD11 from binding to BCL10 in the absence of upstream signals. After phosphorylation of the inhibitory domain in normal T cells, CARD11 autoinhibition can be relieved, and the CARD11 protein can oligomerize and promote prion-like assembly of the CBM complex with recruitment of BCL10-MALT1 filaments, which can then allow 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 I linker 2 (CARMIL2), mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), and B-cell lymphoma 10 (BCL10). CARD11 can participate 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 signaling is well-known to those skilled in the art. Briefly, signaling begins with the extracellular association of a cytokine or growth factor with its corresponding transmembrane receptor. This facilitates the transactivation of receptor-associated Janus kinases (JAKs), achieved by bringing the kinases spatially closer and promoting conformational changes that move their kinase domains away from inhibitory pseudokinase domains. The activated JAKs then phosphorylate latent STAT monomers, leading to dimerization, nuclear translocation, and DNA binding. In mammals, 4 JAKs (JAK1, JAK2, JAK3, TYK2) and 7 STATs (STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6) are 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). Co - stimulatory Molecule Signaling

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

[0180] Costimulatory and coinhibitory receptors determine the functional outcome of T cell receptor (TCR) signaling. Specific recognition of cognate antigenic peptides presented by MHC molecules triggers TCR signaling, but costimulatory and coinhibitory receptors on T cells guide T cell function and determine T cell fate. T cell co-signaling receptors have been broadly defined as cell surface molecules that can transduce signals into T cells to positively (costimulatory receptors) or negatively (coinhibitory receptors) regulate TCR signaling. Examples of costimulatory or coinhibitory receptors include CD28 and CTLA-4, both of which bind to ligands B7-1 and B7-2. 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 is known to those skilled in the art, the sarcoma / mitogen-activated protein kinase kinase / extracellular receptor kinase (RAS / MEK / ERK) is a conserved signaling pathway that plays a key role in cell proliferation, survival, and differentiation. Aberrant activation of the RAS / MEK / ERK signaling pathway induces tumors. Efforts have been made to target this signaling pathway for cancer treatment. Aberrant activation of the said signaling pathway promotes tumorigenesis and tumor development.

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

[0183] As an important upstream molecular marker of the RAS-RAF-MEK-ERK pathway, RAS acts as a molecular switch by binding GTP / GDP, and it includes four isoforms: HRAS, KRAS4A, KRAS4B, and NRAS. KRAS is the most common isoform in all human cancers. KRAS4A and KRAS4B are different splice isoforms of the same gene. When transmembrane receptors (receptor tyrosine kinases, RTKs) are activated, a complex of growth factor receptor-bound protein 2 (GRB2) and son of sevenless (SOS) in the cytoplasm is recruited to the inner surface of the cell membrane. (McCubrey JA, Steelman LS, J, Martelli AM. Raf / mek / erk signaling. Target Ther Acute Myeloid Leuk. January 2015: 275-305.)

[0184] Several mutations identified by the inventors / Disclosers are located in genes that have multiple roles in this signaling pathway. Phospholipase Cγ Signaling

[0185] Phospholipase C (PLC) is an essential mediator of cell signaling. PLC regulates multiple cellular processes by generating bioactive molecules such as inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). These products propagate and regulate cell signaling via calcium (Ca2+) mobilization and activation of protein kinase C (PKC), other kinases, and ion channels. PLCγ1, one of the major isoforms of PLC, is directly activated by membrane receptors including receptor tyrosine kinases (RTKs) and adhesion receptors such as integrins. PLCγ1 mediates signaling through direct interaction with other signaling molecules via its SH domains and its lipase activity. PLCγ1 is frequently enriched and mutated in multiple 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 Cγ signaling pathway is involved in T cell lymphoma and mainly in cutaneous T cell lymphoma (CTCL). Nine PLCG1 mutations (p.R48W, p.S312L, p.D342N, p.S345F, p.S520F, p.R1158H, p.E1163K, p.D1165H, and in-frame indel p.VYEEDM1161V) have been identified in Sézary syndrome (the leukemic variant of CTCL). (V.M. Patel et al., Frequent and Persistent PLCG1 Mutations in Sézary 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 relate to polypeptides, recombinant nucleic acid constructs encoding the polypeptides, and recombinant nucleic acids, wherein the polypeptides comprise mutations capable of altering T cell signaling.

[0188] Exemplary mutations in the polypeptides encoded by the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure are described in Table 1. In some embodiments of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure, the polypeptide may comprise more than one mutation. For example, one or two or three or four or five or more mutations. In some embodiments, the mutations are a combination of different types of mutations. Different types of mutations can be point mutations, gene fusions, substitutions, gain-of-function mutations, stop-gain mutations, insertion mutations, deletion mutations, duplication mutations, or translocations. The mutations can 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 containing a caspase-associated recruitment domain (CARD). In some embodiments, the gene fusion comprises a polypeptide that comprises a protein containing CARD or a functional fragment thereof. A functional fragment of a protein containing CARD can be, for example, a fragment that provides NF-κB transcriptional activity, AP-1 transcriptional activity, and / or MALT1 proteolytic activity at a level of at least 70%, 75%, 80%, 85%, 90%, or 95% of the activity of the full-length protein, as determined by Figure 1Determined by the in vitro CAR Jurkat assays shown herein and described herein. In some embodiments, the gene fusion comprises a domain capable of binding to: (i) a substrate located on the inner side of the plasma membrane of a cell, and / or (ii) a target polypeptide comprising phosphorylated tyrosine (pTyr). In some embodiments, the gene fusion comprises a CARD-containing protein and a domain capable of binding to: (i) a substrate located on the inner side of the plasma membrane of a cell, and / or (ii) a target polypeptide comprising pTyr).

[0190] In some embodiments, the domain is capable of binding to a substrate indirectly located on the inner side of the plasma membrane. Indirect localization can refer to localizing the substrate to the inner side of the plasma membrane by, for example, the binding of the substrate to another polypeptide or lipid directly located on the inner side of the plasma membrane. Indirect localization can refer to localizing the substrate to the inner side of the plasma membrane by, for example, the interaction of the substrate with another polypeptide or lipid directly located on the inner side of the plasma membrane. In some embodiments, the domain is capable of binding to a substrate directly located on the inner side of the plasma membrane. Direct localization can refer to localizing the substrate to the inner side of the plasma membrane, for example, by the binding of the substrate to the inner side of the plasma membrane itself. Direct localization can refer to localizing the substrate to the inner side of the plasma membrane, for example, by the interaction of the substrate with the inner side of the plasma membrane itself.

[0191] In some embodiments, the mutation is a T-cell lymphoma mutation. A 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 lymph nodes and extranodal sites. There are two main types of T-cell lymphoma, namely T-lymphoblastic lymphoma and peripheral T-cell lymphoma, which are classified based on clinical manifestations and cytogenetic mutations. Peripheral T-cell lymphoma can be further divided into cutaneous 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 a clonally expanded population of T cells. As used herein, a clonally expanded population of T cells can be a population of T cells descended from a single progenitor cell. T cells within the 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 a variety of ways. In some embodiments, the alteration of T cell signaling can be carried out by enhancing, promoting, improving, reducing, regulating, or modulating the signaling pathways within T cells. In some embodiments, the alteration of T cell signaling can be carried out by activating, increasing, suppressing, inhibiting, or otherwise altering the signaling.

[0194] 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 that occurs via one or more T cell signaling pathways. In some embodiments, the polypeptides or recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure can alter T cell signaling by increasing one or more pathways and / or reducing one or more pathways, or by a combination of enhancing and / or reducing multiple pathways (such as Figure 2F shown). In some embodiments, the mutation can alter cytokine production. The cytokine can be, but is not limited to, IL-2, IL-4, IL-5, TNFα, IFN-γ, IL-13, and / or other cytokines. In some embodiments, the cytokine is IL-2. In some embodiments, the cytokine is IL-2, and the production of IL-2 is increased.

[0195] 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 T cell signaling that occurs via one or more T cell signaling pathways. The signaling pathways can be, but are not limited to, the NFAT pathway, the NF-κB pathway, the AP-1 pathway, the JAK / STAT pathway, the RAS / MEK / ERK, and / or phospholipase γ signaling, or as described elsewhere herein.

[0196] 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 CARD11-BCL10-MALT1 complex signaling, co-stimulatory 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 of therapeutic T cells, increase proliferation, alter effector function, resist T cell dysfunction, increase T cell fitness, enhance in vivo persistence, and / or increase intratumoral presence. The present disclosure also relates to recombinant nucleic acids encoding polypeptides having one or more mutations that can reduce T cell exhaustion of therapeutic T cells, increase proliferation, alter effector function, resist T cell dysfunction, increase T cell fitness, enhance in vivo persistence, and / or increase intratumoral presence.

[0198] T cell fitness can refer to the ability of T cells to mount an immune response. The ability of T cells to perform T cell functions (such as signal transduction, cytokine production, survival, and persistence) in tumors may contribute to T cell fitness. T cell exhaustion can contribute to a reduction in its fitness.

[0199] In some embodiments, the polypeptides or 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 are present within the host tumor following infusion. In some embodiments, the in vivo persistence of therapeutic T cells is enhanced. The 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.

[0200] In some embodiments, the polypeptides or recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure can alter the in vivo persistence or other functions or activities of the T cells described herein in tumors 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 includes 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 can be the STAT3, STAT5B, JAK1, JAK2, or JAK3 gene. In some embodiments, the gene can be BRAF or RASGRP1. In some embodiments, the gene can be the phospholipase Cγ1 (PLCG1) gene. In some embodiments, the gene can be the NFKB1, NFKB2, or JUNB gene. In some embodiments, the gene can be TNFRSF1B.

[0202] 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 therapeutic efficacy of engineered T cells. The alteration of therapeutic efficacy can include, but is not limited to, a reduction in T cell exhaustion, increased proliferative capacity, enhanced anti-tumor effect, increased replicative lifespan, reduced replicative senescence, enhanced killing ability, enhanced fitness of engineered T cells, and / or other functions or activities of T cells. In some embodiments, the nucleic acid constructs and / or recombinant nucleic acids of the present disclosure encode polypeptides that comprise 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 the following sequences or variants thereof: 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, SEQID 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, SEQID NO: 252, SEQ ID NO: 254, SEQ ID NO: 256, wherein the amino acid sequence comprises one or more of the mutations listed in Table 1 for the same gene.

[0203] In some embodiments, the nucleic acid constructs and / or recombinant nucleic acids of the present disclosure comprise a nucleic acid sequence or a variant thereof that 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% identity to the following sequences: 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: 127, SEQ IDNO: 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 SEQ ID NO: 255, wherein said variant comprises one or more of the mutations listed for the same gene in Table 1.

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

[0205] In some embodiments, the present disclosure relates to a polypeptide or a recombinant nucleic acid encoding a polypeptide, the polypeptide comprising a protein containing a caspase-associated recruitment domain (CARD) or a functional fragment thereof. A CARD is a conserved homology domain containing a 6-helix bundle or a 5-helix bundle. A CARD can mediate protein-protein interactions between key apoptosis signaling molecules. Non-limiting examples of CARDs include CARD6, CARD8, CARD9, CARD10, CARD11, CARD14, CARD16, CARD18, and CARD19. Non-limiting examples of proteins containing a CARD include those described in Boyle and Monie bioRxiv 087908; doi: https: / / doi.org / 10.110I / 087908; and / or Park. In J Mol Med. March 2019; 43(3):1119-1127, such as human caspase-1, caspase-2, caspase-4, and caspase-5, mouse caspase-1, caspase-2, caspase-11, and caspase-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 protein containing a CARD or a functional fragment thereof is derived from a CARD protein selected from CARD9 (UniProt#Q9H257), CARD10 (UniProt#Q9BWT7), CARD11 (UniProt#Q9BXL7), or CARD14 (UniProt#Q9H257) (Wang et al. J Biol Chem. June 15, 2001; 276(24):21405-9; Bertin et al. J Biol Chem. April 13, 2001; 276(15):11877-82.). In some embodiments, the protein containing a CARD or a functional fragment thereof is derived from the CARD11 protein (UniProt#Q9BXL7) or a functional fragment thereof. In some embodiments, the protein containing a 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: 261-264.In some embodiments, the functional fragment of the CARD-containing protein is derived from CARD11 and comprises or consists of the following: 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 its functional fragment is to bind to the CARD domain on BCL10 (Bertin et al. J. Biol Chem. April 201; 276(15): 11877-11882). In some embodiments, its 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 cell produces a substrate that localizes to the inner side of the plasma membrane of the cell. In some embodiments, the substrate that localizes to the inner side of the plasma membrane of the cell is a phosphoinositide. Non-limiting examples of phosphoinositides include those described in Posor et al. Nat Rev Mol Cell Biol. December 2022; 23(12): 797-816. In some embodiments, the phosphoinositide is selected from phosphatidylinositol (3,4,5)-trisphosphate (PIP3), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) (Hawse and Cattley J. Bio. Chem. March 2019; 294(13): 4793-4805; Sun et al. PLoS ONE. November 2011; 6(11): e27227). In some embodiments, the polypeptide binds to the phosphoinositide with a Kd of less than 100 μM, 50 μM, 10 μM, 5 μM, 1 μM, 0.5 μM, 0.1 μM, 0.05 μM or 0.01 μM, and wherein the Kd is analyzed using SPR as described in Yu et al. Molecular Cell. March 2004; 13(5): pages 677-688.

[0207] The present disclosure further relates to polypeptides comprising domains capable of binding to: (i) a substrate located on the inner side of the plasma membrane and / or (ii) a target polypeptide comprising phosphorylated tyrosine (pTyr). In some embodiments, the domain is capable of binding to a substrate indirectly located on the inner side of the plasma membrane. Indirect localization may refer to the localization of a substrate to the inner side of the plasma membrane by, for example, the binding of the substrate to another polypeptide or lipid directly located on the inner side of the plasma membrane. Indirect localization may refer to the localization of a substrate to the inner side of the plasma membrane by, for example, the interaction of the substrate with another polypeptide or lipid directly located on the inner side of the plasma membrane. In some embodiments, the domain is capable of binding to a substrate directly located on the inner side of the plasma membrane. Direct localization may refer to the localization of a substrate to the inner side of the plasma membrane by, for example, the binding of the substrate to the inner side of the plasma membrane itself. Direct localization may refer to the localization of a substrate to the inner side of the plasma membrane by, for example, the interaction of the substrate with the inner side of the plasma membrane itself.

[0208] Non-limiting examples of domains capable of binding to (i) and / or (ii) include Src homology 2 (SH2) domains, Src homology 3 (SH3) domains, pleckstrin homology (PH) domains, and phosphotyrosine-binding (PTB) domains. SH2 and PTB domains mediate protein-protein interactions involved in many signal transduction pathways. SH2, SH3, and PTB domains that can be used in the present disclosure include, for example, those disclosed in Schlessinger et al., Sci STKE. July 15, 2003; 2003(191):RE12. SH2-containing proteins can bind to plasma membrane lipids through a binding pocket different from the pTyr-binding pocket; most SH2 domains bind plasma membrane lipids and many are highly phosphoinositide-specific (Park et al., Cell. April 7, 2016; 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 from the PIK3R3 protein (UniProt#Q92569). In some embodiments, the SH2 domain comprises a motif of conserved arginine residues in the FLVR motif (ArgβB5 or Arg175 in the v-Src SH2 domain). Most 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 critical for phosphopeptide binding are HisβD4, LysβD6, and ArgαA2, which coordinate and anchor the aromatic ring of phosphotyrosine (Diop et al., Int J Mol Sci. December 15, 2022; 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 of SEQ ID NOs: 313 - 315 (e.g., Veggiani et al. Protein Sci. February 2019; 28(2):403 - 413).

[0209] Non - limiting examples of target polypeptides containing 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 of SEQ ID NOs: 298 - 304. In some embodiments, pTyr is at the position corresponding to pY1346 of SEQ ID NO: 301. In some embodiments, pTyr is at the position corresponding to pY1221 of SEQ ID NO: 302. In some embodiments, the polypeptide binds to the target polypeptide with a Kd of less than 100 μM, 50 μM, 10 μM, 5 μM, 1 μM, 0.5 μM, 0.1 μM, 0.05 μM or 0.01 μM, wherein the Kd is analyzed by fluorescence polarization as described by Hause et al. PLoS One. 2012; 7(9):e44471. In some embodiments, the polypeptide has a higher affinity for the target polypeptide containing pTyr than for a control polypeptide that is not phosphorylated at the corresponding tyrosine position. In some embodiments, the polypeptide has an affinity for the target polypeptide containing phosphorylated tyrosine that is at least 2 - fold, at least 5 - fold, at least 10 - fold, at least 50 - fold or at least 100 - fold higher (lower Kd) than for a control polypeptide that is not phosphorylated at the corresponding tyrosine position.

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

[0211] In addition, 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 located on the inner side of the cytoplasmic membrane, and / or (ii) a target polypeptide containing 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 the CARD11 protein and an SH2 domain from the PIK3R3 protein. In some embodiments, the recombinant nucleic acid encodes a functional fragment of a CARD-containing protein derived from the CARD9 protein and an SH2 domain from the 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 truncation thereof. In some embodiments, the polypeptide comprises or the nucleic acid construct and / or the recombinant nucleic acid encodes a CARD11-PIK3R3 fusion polypeptide or a truncation thereof, the fusion polypeptide or the truncation 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 the following sequences: 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 truncation is 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 the following sequences: 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 either of the two SH2 domains, the catalytic subunit, and the 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, 20, 30, 40, 50, 60, 80, 100, 120, 140, 150, 160, 180, 200, 220, 240, 250, 260, 280, or 300 amino acids of the N-terminal portion of the coiled-coil domain.

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

[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 moiety comprises no more than 10, 20, 30, 40, 50, 60, 80, 100, 120, 140, 150, 160, 180, or 200 amino acids of the N-terminal portion of the ID. In some embodiments, the polypeptide does not comprise a sequence that is 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, the CARD11-PIK3R3 fusion polypeptide comprises a CARD domain (i.e., containing a protein or a functional fragment thereof), a CARD inhibitory domain (ID), a coiled-coil domain, and an SH2 domain from PIK3R3. In some embodiments, the CARD11-PIK3R3 fusion polypeptide comprises a functional fragment of a CARD-containing protein derived from the CARD11 domain, a coiled-coil domain, and an SH2 domain from PIK3R3.

[0217] In some embodiments, the 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 the following sequences: 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, the polypeptide of the present disclosure has or the recombinant nucleic acid construct and / or recombinant nucleic acid of the present disclosure encodes a polypeptide that comprises a BCL3 polypeptide having a substitution at amino acid 647. In some embodiments of the recombinant nucleic acid construct and / or recombinant nucleic acid 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: 4 and comprises 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, the polypeptides of the present disclosure, or recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure encode polypeptides that comprise a CARMIL2 polypeptide having a substitution at amino acid 575. 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: 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, the polypeptides of the present disclosure, or recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure encode polypeptides that comprise 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 comprising 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 that comprises 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 129. In some embodiments, the polypeptide comprises a mutation that is capable of (i) altering JAK / STAT signaling in T cells, (ii) altering cytokine production, and / or (iii) enhancing the in vivo persistence of therapeutic T cells comprising the mutation in tumors. In some embodiments, the mutation is located in the JAK1, JAK3, STAT3, or STAT5 gene. In some embodiments, the polypeptide comprises an amino acid sequence that 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 the following sequences: (i) SEQ ID NO: 90 and comprises a substitution at amino acid position 1097 of SEQ ID NO: 90, (ii) SEQ ID NO: 94 and comprises a substitution at amino acid position 573 of SEQ ID NO: 94, (iii) SEQ ID NO: 176 and comprises a substitution at amino acid position 618, 647, or 661, or a combination thereof, of SEQ ID NO: 176, (iv) SEQ ID NO: 182 and comprises 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 that 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: 87, wherein 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 that 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: 91, wherein 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 that 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: 169 and encodes 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 that 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: 171 and encodes 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 that 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: 173 and encodes 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 that 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: 177 and encodes 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 that 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: 179 and encodes 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 co-stimulatory molecule signaling in T cells and the persistence of T cells comprising the mutation in a tumor. In some embodiments, the mutation is in 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 that comprises 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 that 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: 192 and comprises a substitution at amino acid position 256 and / or position 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 that comprises 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 that 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: 187 and encodes 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 that comprises 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 that 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: 189 and encodes 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 that comprises 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 that 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: 42 and encodes 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 that 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: 35 and encodes 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 that 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: 37 and encodes 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 that 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: 39 and encodes 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 that 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: 43 and encodes 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 constructs 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 of therapeutic T cells comprising the mutation in tumors. In some embodiments, the mutation is located 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 comprises 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 encodes 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 of therapeutic T cells comprising the mutation in a tumor. In some embodiments, the mutation is located in the RASGRP1 gene. In some embodiments, the polypeptide comprises an amino acid sequence that 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: 158 and comprises 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 that 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: 157 and encodes a RASGRP1 polypeptide having an M261T 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 (i) phospholipase γ signaling, and / or (ii) cytokine production, and / or (iii) the in vivo persistence of therapeutic T cells comprising the mutation in a tumor. In some embodiments, the mutation is located in the phospholipase Cγ1 (PLCG1) gene. In some embodiments, the polypeptide comprises an amino acid sequence that 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: 142 and comprises a substitution at amino acid position 47, 48, 520, 1163, 1165 or a 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 that 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: 133 and encodes a PLCG1 polypeptide having an E47K substitution.

[0249] In some embodiments, the recombinant nucleic acid construct comprises a nucleic acid sequence that 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: 135 and encodes a PLCG1 polypeptide having an S520F substitution.

[0250] In some embodiments, the recombinant nucleic acid construct comprises a nucleic acid sequence that 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: 137 and encodes a PLCG1 polypeptide having a substitution of E1163K.

[0251] In some embodiments, the recombinant nucleic acid construct comprises a nucleic acid sequence that 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: 139 and encodes a PLCG1 polypeptide having a D1165H substitution.

[0252] In some embodiments, the recombinant nucleic acid construct comprises a nucleic acid sequence that 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: 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 the transcription factor activity in a T cell comprising 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 that comprises an NFKB1 polypeptide having a substitution at amino acid 67. In some embodiments, the polypeptide comprises an amino acid sequence that 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: 118 and comprises 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 that 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: 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 that comprises an NFKB2 polypeptide having a substitution at amino acid 656. In some embodiments, the polypeptide comprises an amino acid sequence that 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: 122 and comprises 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 that 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: 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 that comprises a JUNB polypeptide having a substitution at amino acid 282. In some embodiments, the polypeptide comprises an amino acid sequence that 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: 98 and comprises 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 that 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: 95 and encodes a JUNB polypeptide having an A282V substitution.

[0260] In some embodiments, the polypeptide comprises a first polypeptide encoding a portion of the CARD 11 polypeptide and a second polypeptide encoding a portion of the 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 subsets of T cells.

[0262] In some embodiments, the promoter can be a constitutive promoter. Examples of constitutive promoters include, but are not limited to, the MND promoter, the EF1a promoter, the sEF1 promoter, the γ-retroviral LTR promoter, the CD4 promoter, the CD8a promoter, the CD8b promoter, the TCRa promoter, the TCRb promoter, the CD3d promoter, the CD3g promoter, the CD3e promoter, or the 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, IFNγ 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 protein 1 (PD-1) promoter, T cell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, lymphocyte activation gene 3 (LAG-3) promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter, B and T lymphocyte attenuator (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, TGF-β promoter, T-bet promoter, Eomes promoter, GATA3 promoter, CD45RA promoter, 2B4 promoter, type I interferon (IFN)α, type I IFNβ promoter, IFNγ promoter, IRF3 promoter, IRF7 promoter, NF-κB promoter, AP-1 promoter, TNF-α promoter, and CD130 promoter, NR4A1 promoter, NR4A2 or NR4A3 promoter.

[0263] The present disclosure also provides a vector comprising the recombinant nucleic acid construct and / or recombinant nucleic acid of the present disclosure.

[0264] Nucleic acid molecules can be contained within a vector that is capable of directing the expression of the nucleic acid molecule in, for example, a cell that has been transformed / transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available, or can be readily prepared by a person skilled in the art. See, for example, Sambrook, J. and Russell, D.W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J. and Russel, D.W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (collectively referred to herein as "Sambrook"); Ausubel, F.M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements up to 2014); Bollag, D.M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M.G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K.B., Ferré, F and Gibbs, R. (1994). PCR: The Polymerase Chain Reaction.Boston: Birkhauser Publisher; Greenfield, E.A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S.L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements up to 2014); and Makrides, S.C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference).

[0265] DNA vectors can be introduced into eukaryotic cells via conventional transformation or transfection techniques. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (2012, supra) and other standard molecular biology laboratory manuals, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, transfection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, biolistic introduction, nuclear perforation, hydrodynamic shock, and infection.

[0266] In some embodiments, the expression vector can 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 containing the nucleic acid molecule. Accordingly, some embodiments of the present disclosure relate to methods for preparing a recombinant or engineered cell, the method comprising (a) providing a cell capable of expressing a protein and (b) contacting the provided cell with the recombinant nucleic acid of the present disclosure.

[0268] Introduction of the nucleic acid molecules of the present disclosure into cells can be carried out by methods known to those skilled in the art, such as viral infection, transfection, conjugation, protoplast fusion, liposome transfection, electroporation, nuclear transfection, 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, etc.

[0269] Thus, 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 the host genome, or can replicate episomally, or exist as minicircle expression vectors for transient expression in recombinant host cells. Thus, in some embodiments, nucleic acid molecules are maintained and replicated as episomal units in recombinant host cells. In some embodiments, nucleic acid molecules are stably integrated into the genome of recombinant cells. Stable integration can be achieved using classical random genomic recombination techniques or with more precise techniques such as guide RNA-guided CRISPR / Cas9 genome editing, or DNA-guided endonuclease genome editing using NgAgo (Natronobacterium gregoryi Argonaute), or TALEN genome editing (transcription activator-like effector nucleases). 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 and methods known in the art such as electroporation. For example, introduction of nucleic acids into cells can be achieved by viral transduction. In a non-limiting example, adeno-associated virus (AAV) is engineered to deliver nucleic acids to target cells via viral transduction. Several AAV serotypes have been described, and all known serotypes can infect cells from a variety of different tissue types. AAV is capable of transducing a wide range of species and tissues in vivo without signs of toxicity, and it generates a relatively mild innate and adaptive immune response.

[0271] Lentivirus-derived vector systems can also be used for nucleic acid delivery and gene therapy via viral transduction. Lentiviral vectors offer several attractive features as gene delivery vehicles, including: (i) sustained gene delivery by stable integration of the vector into the host genome; (ii) the ability to infect both dividing and non-dividing cells; (iii) broad tissue tropism, including important gene therapy target cell types and cell therapy target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements such as polycistronic sequences or intron-containing sequences; (vi) a potentially safer integration site profile; and (vii) relatively easy systems for vector manipulation and production.

[0272] In some embodiments, a host cell can be genetically engineered (e.g., transduced or transformed or transfected) with, for example, a vector construct of the present application, which can be, for example, a viral vector or a vector for homologous recombination (which contains a nucleic acid sequence homologous to a part of the host cell genome), or can be an expression vector for expressing a target polypeptide. The host cell can be an untransformed cell or a cell that has 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 cell or a 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-natural cell or has been genetically engineered. In some embodiments, the cell is not a cancer cell. In some embodiments, the recombinant nucleic acid is exogenous. In some embodiments, the mammalian cell is an immune cell, a neuron, an epithelial cell, an endothelial cell, or a stem cell. In some embodiments, the cell is not a CD4+ T cell. In some embodiments, the cell can be an immune cell, a T cell, a CD4+ cell, a CD8+ cell, a regulatory T cell, a γδ 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 the CARD11 CARD domain but not any SH2 domain. In some embodiments, the recombinant nucleic acid of the cell is located at the endogenous CARD11-encoding gene locus of the cell or comprises at least a part of the endogenous CARD11-encoding gene of the cell.

[0274] The T cells or precursors thereof of the present disclosure may be immune cells of the lymphoid lineage. 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). The TCRs known to those skilled in the art can be composed of two different chains (α chain and β chain), each chain consisting of a constant region that anchors the chain inside the cell surface membrane of the T cell and a variable region that can recognize and bind to an antigen presented by MHC. The TCR complex can associate with six polypeptides to form two heterodimers (CD3γε and CD3δε) and one homodimer (CD3ζ), which together form the CD3 complex. The TCR can be engineered to specifically target an antigen expressed by a particular tumor cell using modifications of the T cell that retain these complexes. As used herein, the TCR can be a naturally occurring or engineered TCR.

[0275] In some embodiments, the T cells can be CD4+ or CD8+ and can include, but are not limited to, regulatory T cells, cytotoxic T lymphocytes, T helper cells, and memory T cells, including central memory T cells (TCM), stem cell memory T cells (TSCM), stem-cell-like memory T cells (or stem-like memory T cells), and effector memory T cells (such as, 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, T regulatory cells, natural killer T cells, mucosa-associated invariant T cells (MAIT), and γδ T cells. The 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 an immune response (i.e., immune-stimulatory cells). Immune-stimulatory T cells include, but are not limited to, T helper cells (CD4+), cytotoxic T cells, and memory T cells, including central memory T cells (TCM), stem cell memory T cells (TSCM), stem-cell-like memory T cells (or stem-like memory T cells), and effector memory T cells (e.g., 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 immunosuppressive cells (i.e., cells that inhibit an immune response). Immunosuppressive T cells include regulatory T cells (T regulatory cells, Treg) and follicular regulatory T cells (Tfh) cells.

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

[0279] In some aspects, the cell further comprises (i) a chimeric antigen receptor (CAR) specific for a target antigen; and / or (ii) a T cell receptor (TCR) specific for a target antigen.

[0280] In some embodiments, the T cells of the present disclosure can be engineered to express a transgene, such as a CAR or a transcriptional regulator. Transcriptional regulators include synthetic receptors, such as the synthetic notch receptor described in US 11,202,801 or other synthetic receptors, such as, for example, those described in provisional application numbers: 62 / 905,251, 63 / 15,428, 62 / 905,268, 62 / 905,263, 62 / 935,024, and 62 / 905,248, which are hereby incorporated by reference in their entirety.

[0281] T cells can be genetically engineered for recombinant expression of transgenes. Such T cells can, but need not, express a CAR or transcriptional receptor that binds to a target antigen, as the cells are already target antigen-specific, such that their immune response (e.g., cytotoxicity) is specifically stimulated by this target antigen. Such T cells that recognize and are sensitized to a target antigen can be obtained by known methods, for example, using in vitro sensitization methods of naive T cells or hematopoietic progenitor cells (e.g., as described by Wolff et al., Nat. Protocols 9:950-966 (2014) or by van Lent et al., J Immunol. 179:4959-4968 (2007)), or obtained from a subject that has been exposed to the target antigen and is mounting an immune response against the target antigen (i.e., in vivo-sensitized T cells).

[0282] In some aspects, the cells (e.g., T cells, NK cells, and / or TILs) comprise a CAR. In some aspects, the 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 aspects, the CAR-expressing cells disclosed herein are CAR T cells, e.g., single CAR T cells, genome-edited CAR T cells, dual CAR T cells, or tandem CAR T cells.

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

[0284] In some embodiments, the target antigen can be a tumor-associated antigen. Non-limiting exemplary tumor-associated antigens applicable to 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, Ephrin B2, 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 can be 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), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD246, CD252, CD253, CD261, CD262, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), CEA, CLL-1, CS1, EGFR, FGFR2, AFP, CA125, MUC-1, MAGE, placental alkaline phosphatase-like protein 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 alpha (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-β, SSEA-4,, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostate enzyme, 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, Podocalyxin, HPV E6, E7, MAGE Al, ETV6-AML, Spermatogenic Protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related Antigen 1, p53, p53 Mutant, KRAS, Mutant KRAS, KRAS G12D, Prostate Cancer Associated Protein 6, Survivin, Telomerase, PCTA-1 / Galectin 8, Melan-A / 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, Intestinal Carboxylesterase, mut hsp70-2, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, AFP, TRAC, TCRβ, BCMA, TSHR, EGFRvIII, GD2, GD3, Tn Ag, PSMA, ROR1, ROR2, GPC1, GPC2, FLT3, FAP, TAG72, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, PDGFR-β, SSEA-4, Folate Receptor α, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, Prostate-Specific Antigen, 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 β, 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-1a, MAGE-A1, Podocalyxin, HPV E6, E7, MAGE Al, ETV6-AML, Spermatogenic Protein 17, XAGE1, Tie 2,MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutants, prostate cancer-associated protein 6, survivin, telomerase, PCTA-1 / galectin 8, Melan-A / MART1, Ras mutants, 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, intestinal carboxylesterase, mut hsp70-2, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, the extracellular portion of APRIL protein or any combination thereof.

[0286] In some aspects, the TCR targets 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-β, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, EGFR, NCAM, prostate enzyme, 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-1a, MAGE-A1, podoplanin, HPVE6, E7, MAGEAl, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, KRAS, mutant KRAS, KRAS G12D, prostate cancer-associated protein 6, survivin, telomerase, PCTA-1 / galectin 8, Melan-A / 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, intestinal carboxylesterase, mut 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] The CAR can include a co-stimulatory signaling domain, e.g., to increase signaling efficacy. See U.S. Patent Nos. 7,741,465 and 6,319,494, and Krause et al. and Finney et al. (supra), 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). Signals generated solely through the TCR may be insufficient to fully activate T cells, and secondary or co-stimulatory signals can increase activation. Thus, in some embodiments, the signaling domain further includes one or more additional signaling domains (e.g., co-stimulatory signaling domains) that activate one or more immune cell effector functions (e.g., the innate immune cell effector functions described herein). In some embodiments, a portion of such co-stimulatory signaling domains can be used, provided that the portion transduces effector function signals. In some embodiments, the cytoplasmic domain described herein includes 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 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 the ligand that binds to CD83. Exemplary co-stimulatory proteins have the amino acid sequence of a co-stimulatory protein that naturally occurs on T cells, the complete native amino acid sequence of which is described in NCBI reference sequence: NP_006130.1.

[0288] In various embodiments, a mechanism for modulating (e.g., reducing) the binding activity of the target antigen is desired, e.g., to minimize or abate adverse events caused by the binding activity. It may also be desirable to include an inducible "on" or "accelerator" switch in the immune cell. Suitable techniques include using inducible caspase-9 (U.S. Application 2011 / 0286980) or thymidine kinase before, after, or simultaneously with transducing cells with the CAR constructs 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 technologies can be incorporated herein. These technologies can include the use of dimerization domains and optional activators for dimerization of such domains, for example, utilization of the FKBP / Rapalog dimerization system in certain cells as disclosed by Wu et al., Science October 2015; 350(6258): aab4077, the content of which is incorporated herein by reference in its entirety. Additional dimerization technologies 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 content of each of which regarding dimerization technologies is also incorporated herein by reference. Additional dimerization pairs can include cyclosporin-A / cyclophilin, receptors, estrogen / estrogen receptor (optionally using tamoxifen, 4-hydroxytamoxifen, or endoxifen), glucocorticoid / glucocorticoid receptor, tetracycline / tetracycline receptor, and / or vitamin D / vitamin D receptor. Additional examples of dimerization technologies can be found, for example, in WO 2014 / 127261, WO 2015 / 090229, US2014 / 0286987, US 2015 / 0266973, US 2016 / 0046700, U.S. Patent No. 8,486,693, US 2014 / 0171649, and US 2012 / 0130076, the content of which is further incorporated herein by reference in its entirety.

[0290] In some embodiments, a T cell can comprise a bicistronic CAR. The bicistronic CAR can comprise two CARs that bind different targets and are encoded by a single vector. The bicistronic CAR can comprise a first CAR sequence and a second CAR sequence expressed as a single polypeptide, the single polypeptide comprising 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, for example, Chng et al. MAbs. March-April 2015; 7(2): 403-412, the content of which regarding cleavable linkers is incorporated herein by reference; also see Guedan et al. Mol Ther Methods Clin Dev. March 15, 2019; 12: 145-156, the content of which regarding bicistronic CAR design is incorporated herein by reference).

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

[0292] Sources of T cells include, but are not limited to, peripheral blood, bone marrow, or other sources of hematopoietic cells. T cells can be isolated by methods well known in the art, including commercially available isolation methods (see, 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] A variety of known techniques can be used to isolate or enrich desired immune cells (such as T cells). If a specific type of T cell is to be isolated, a variety of cell surface markers or combinations of markers (including but not limited to CD3, CD4, CD8, CD34 (for hematopoietic stem cells and progenitors), etc.) can be used to isolate the cells, as is well known in the art (see Kearse, T Cell Protocols: Development and Activation, Humana Press, Totowa N.J. (2000); De Libero, T Cell Protocols, Methods in Molecular Biology Vol. 514, Humana Press, Totowa N.J. (2009)). Negative selection methods can be used to remove cells that are not the desired immune cells. Additionally, positive selection methods can be used to isolate or enrich the desired T cells. In some cases, a combination of 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 typically contain the recombinant nucleic acids, recombinant polypeptides, and / or recombinant cells of the present disclosure, as well as pharmaceutically acceptable excipients (e.g., carriers).

[0295] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions, as well as sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor EL TM. (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy injection is possible. 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 a dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size (in the case of a dispersion), and by using surfactants (e.g., sodium lauryl sulfate). The action of microorganisms can be prevented by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, isosmotic agents such as sugars, polyols (such as mannitol, sorbitol) and sodium chloride are commonly included in the composition. Prolonged absorption of the composition can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the injectable composition.

[0296] Sterile injectable solutions can be prepared by incorporating the active compound in an appropriate solvent, with or without one or a combination of the ingredients enumerated above as required, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound in a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying which yield a powder of the active ingredient and any additional required ingredients from a previously sterile filtered solution.

[0297] Systemic administration of the recombinant nucleic acid constructs and / or recombinant nucleic acids and recombinant cells of the subject matter of the present disclosure can also be effected by transmucosal or transdermal means. For transmucosal or transdermal administration, permeating agents appropriate to the barrier to be permeated are used in the formulation. Such permeating agents are generally known in the art and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated as an ointment, paste, gel, or cream as is commonly known in the art.

[0298] In some embodiments, the recombinant nucleic acid constructs and / or recombinant nucleic acids and recombinant cells of the present disclosure may also be administered by transfection or infection using methods known in the art, including but not limited to the methods 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 in 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 a vector that will protect the recombinant polypeptide from rapid elimination by the body, such as controlled - release formulations, including implants and microencapsulation delivery systems. Biodegradable and 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. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeted to infected 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 of skill in the art, for example as described in U.S. Patent No. 4,522,811. As described in more detail below, the polypeptides of the present disclosure can also be modified to achieve an extended duration of action, such as by pegylation, acylation, Fc - fusion, linkage to a molecule such as albumin, etc. In some embodiments, the recombinant polypeptides can be further modified to extend their in - vivo and / or ex - vivo half - lives. Non - limiting examples of known strategies and methods suitable for modifying the recombinant polypeptides of the present disclosure include (1) chemically modifying the recombinant polypeptides described herein with highly soluble macromolecules such as polyethylene glycol (“PEG”), which prevent the recombinant polypeptides from contacting proteases; and (2) covalently linking or conjugating the recombinant polypeptides described herein to a stabilizing protein such as, for example, albumin. Thus, in some embodiments, the polypeptides of the present disclosure can be fused to a stable protein such as albumin. For example, human albumin is known to be 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 compositions of the present disclosure comprise one or more polyethylene glycolating reagents. In some embodiments, the polyethylene glycolating reagents are 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-hydroxysuccinimide (mPEG-NHS), mPEG-trifluoromethanesulfonate (mPEG-tresylate), and mPEG-aldehyde. In some embodiments, the polyethylene glycolating reagent is polyethylene glycol. In some embodiments, the polyethylene glycolating reagent is polyethylene glycol with an average molecular weight of 20 kD covalently bound to the N-terminal methionine residue of the recombinant polypeptide of the present disclosure, or polyethylene glycol with an average molecular weight of about 80 kD covalently bound to the N-terminal methionine residue of the polypeptide of the present disclosure. In some embodiments, the polyethylene glycolating reagent is polyethylene glycol with an average molecular weight of about 40 kD covalently bound to the N-terminal methionine residue of the polypeptide of the present 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 (e.g., PEGylated) with one or more polyethylene glycol moieties; or have a similar modification (e.g., PASylated). In some embodiments, PEG molecules or PAS molecules are conjugated to one or more amino acid side chains of the disclosed recombinant polypeptides. In some embodiments, the PEGylated or PASylated polypeptide contains a PEG or PAS moiety on only one amino acid. In other embodiments, the PEGylated or PASylated polypeptide contains PEG or PAS moieties on two or more amino acids, e.g., the PEG or PAS moieties are 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 directly coupled to PEG or PAS (e.g., without a linker group) via an amino, thiol, hydroxyl, or carboxyl group. In some embodiments, the recombinant polypeptide of the present disclosure is covalently bound to polyethylene glycol having an average molecular weight of 20,000 daltons. In some embodiments, the recombinant polypeptide of the present disclosure is covalently bound to polyethylene glycol, and the average molecular weight of the polyethylene glycol ranges from about 1 kD to about 200 kD, such as, for example, about 10 kD to about 150 kD, about 50 kD to about 100 kD, about 5 kD to about 100 kD, about 20 kD to about 80 kD, about 30 kD to about 70 kD, about 40 kD to about 60 kD, about 50 kD to about 100 kD, about 100 kD to about 200 kD, or about 150 kD to about 200 kD. In some embodiments, the recombinant polypeptide of the present disclosure is covalently bound 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. In some embodiments, the recombinant polypeptide of the present disclosure is covalently bound to polyethylene glycol having an average molecular weight of about 40 kD. The methods of the present disclosure

[0302] The present disclosure also relates to methods comprising the polypeptides, recombinant nucleic acid constructs and / or recombinant nucleic acids or cells of the present disclosure. Methods for Identifying Mutations Useful for Improving T Cell Therapy

[0303] In one aspect, the present disclosure relates to methods for identifying mutations that are useful (beneficial) for improving T cell therapy (e.g., as described in Example 1). The mutations can be identified based on genomic sequencing data of T cell lymphoma or based on clonal T cells or other T cells. The method can include applying a statistical test to determine mutations that occur at a frequency higher than random expectation in hot spot regions of the coding sequence. As used herein, a hot spot region can be a DNA segment that is prone to genetic alteration. In some embodiments, the hot spot region is located in the coding sequence of a gene. In some embodiments, the statistical test uses a binomial distribution. In some embodiments, the statistical test uses chi-square analysis or any other multivariate analysis.

[0304] The binomial distribution can be used across the gene length of the entire genome of the cell. The statistical test can use a variety of false discovery rates. In some embodiments, the discovery rate can be 0.05. In some embodiments, it can be controlled by the Benjamini-Hochberg algorithm. In some embodiments, the heterogeneity due to transcription-coupled repair can be considered.

[0305] In some embodiments, mutations that are useful or beneficial for improving T cell therapy can be identified by selecting mutations that occur in a patient. In some embodiments, mutations can be identified by performing a statistical analysis on genomic sequencing data of clonal T cells (including T cell lymphoma) from public and / or private databases. In some embodiments, mutations are identified by using a binomial distribution across the gene length of the entire genome and a false discovery rate of 0.05 controlled by the Benjamini-Hochberg algorithm. In some embodiments, mutations are identified in hot spot regions where the occurrence frequency of the mutation in the hot spot region is higher than only random expectation. In some embodiments, mutations are identified by altering the background mutation rate in the genome (considering the heterogeneity due to transcription-coupled repair), and thus using the altered background mutation rate to identify genes containing mutations whose occurrence frequency is higher than random expectation.

[0306] The identified mutations can be mutations that increase the proliferation of therapeutic T cells, or alter effector function, or resist T cell dysfunction, and / or enhance T cell growth, or reduce T cell exhaustion, or promote the in vivo persistence of T cells. Increasing the proliferation of therapeutic T cells can include clonal expansion, an increase in the T cell replication rate, and / or an increase in the number of T cells. The mutations can 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 presenting a specific antigen. The mechanisms by which effector proteins released by these T cells are activated by recognizing antigens on target cells are focused on appropriate target cells.

[0308] In some embodiments, methods for identifying mutations that are useful (beneficial) for improving T cell therapy include multiple steps such as a) identifying mutations from a clonal T cell genomic sequencing database; b) identifying the frequency of occurrence of the mutations; and c) applying statistical tests to identify significant differences in hotspot genomic regions where the frequency of occurrence of mutations is higher than random expectation, thereby identifying mutations in the hotspot regions that can improve T cell therapy. Mutations can improve T cell therapy by increasing the proliferation of therapeutic T cells containing the mutation in tumors, altering effector function, resisting T cell dysfunction, and / or enhancing growth. Mutations can promote positive T cell selection and / or T cell clone growth. 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 methods for preparing T cells for use in cell therapy. The T cells can further include CAR, TCR, and transcriptional receptors. The T cells can be NK cells or tumor-infiltrating lymphocytes from cancer patients. Preparing the cells can include introducing (e.g., by transduction) any one or more of the recombinant nucleic acid constructs and / or recombinant nucleic acids of the present disclosure into the cells. Preparing the cells can include introducing (e.g., by transduction) 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 into the cells. Preparing the cells can include expressing any one or more of the polypeptides of the present disclosure in the cells. The cells can be genetically modified to express the polypeptides. The cells can further include the expression of an engineered immune receptor that binds to a target in tumor cells.

[0310] The cells can be transduced with a recombinant nucleic acid construct that can alter T cell signaling via the NFAT pathway, NF-κB pathway, AP-1 pathway, JAK / STAT pathway, RAS / MEK / ERK, phospholipase γ signaling, or other T cell signaling pathways.

[0311] The method for preparing T cells according to the present disclosure can include transducing the 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 the pathway, or activate or increase or suppress or inhibit or otherwise alter T cell signaling. The method for preparing T cells according to the present disclosure can include transducing the cells with a recombinant nucleic acid construct and / or recombinant nucleic acid that can alter CARD11-BCLl0-MALT1 complex signaling, co-stimulatory molecule signaling, and cytokine production and / or transcription factor activity in T cells.

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

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

[0314] In some embodiments, the polypeptide or the recombinant nucleic acid construct and / or the recombinant nucleic acid of the present disclosure can alter the in vivo persistence in a tumor, the in vivo accumulation in a tumor, or other functions or activities of the T cells described herein by a mutation in one or more of the forty-one genes listed in Table 1 or Figure 2A In some embodiments, the genes include caspase recruitment domain family member 11 (CARD11). In some embodiments, the genes include 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. The genes can be signal transducer and activator of transcription genes (STAT), including STAT3, STAT5B. The genes can be janus kinase genes, such as the JAK1, JAK2, or JAK3 genes. The genes can be v-raf murine sarcoma viral oncogene homolog B1 (BRAF). The genes can be RAS guanine nucleotide releasing protein 1 (RASGRP1). The genes can be tumor necrosis factor receptor superfamily member 1B (TNFRSF1B). The genes can be phospholipase C gamma 1 (PLCG1) gene. The genes can 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 methods for treating a subject in need of cell therapy, the methods comprising administering to the subject cells as described herein or T cells prepared by any of the methods described herein. The present disclosure also provides methods for enhancing the in vivo persistence of T cells (e.g., therapeutic T cells) in a subject by administering a therapeutically effective amount of the T cells of the present disclosure to a subject in need thereof. The T cells can comprise any recombinant nucleic acid construct and / or recombinant nucleic acid of the present disclosure.

[0316] This administration step can be accomplished using any implantation delivery method in the art. For example, the recombinant cells of the present disclosure can be directly infused into the bloodstream of an individual or otherwise administered to the individual.

[0317] In some embodiments, the methods disclosed herein comprise administering (the term is used interchangeably with the terms “introducing,” “implanting,” and “transplanting”) recombinant cells into an individual, by a method or route that results in at least partial localization of the introduced cells at a desired site, thereby producing one or more desired effects. The recombinant cells or their differentiated progeny can be administered by any suitable route that results in delivery to a desired location in the individual, where at least a portion of the administered cells or cell components remain viable. The period of viability of the cells after administration to the individual can be as short as a few hours, e.g., twenty-four hours, to several days, to up to several years, or even for the lifetime of the individual, i.e., long-term transplantation.

[0318] When provided prophylactically, the recombinant cells described herein can be administered to an individual before the appearance of any symptoms of the disease or disorder to be treated. Thus, in some embodiments, prophylactic administration of a population of recombinant cells prevents the occurrence of symptoms of the disease or disorder.

[0319] When provided therapeutically in some embodiments, the recombinant cells are provided at the onset (or after) of symptoms or indications of the disease or disorder, e.g., at the onset of the disease or disorder.

[0320] For use in the various embodiments described herein, an effective amount of the recombinant cells as disclosed herein can be at least 10 2 cells, at least 5×10 2 cells, at least 10 3 cells, at least 5×10 3 cells, at least 10 4 cells, at least 5×10 4 cells, at least 10 5 cells, at least 2×10 5 cells, at least 3×10 5 cells, at least 4×10 5 cells, at least 5×105 cells, at least 6×10 5 cells, at least 7×10 5 cells, at least 8×10 5 cells, at least 9×10 5 cells, at least 1×10 6 cells, at least 2×10 6 cells, at least 3×10 6 cells, at least 4×10 6 cells, at least 5×10 6 cells, at least 6×10 6 cells, at least 7×10 6 cells, at least 8×10 6 cells, at least 9×10 6 cells or a multiple thereof. The recombinant cells can be derived from one or more donors or can be obtained from autologous sources. In some embodiments, the recombinant cells are expanded in culture prior to administration to an individual in need.

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

[0322] In some embodiments, the recombinant cells are administered systemically, e.g., via infusion or injection. For example, the recombinant cell population is not administered directly to the target site, tissue, or organ such that it enters the individual's circulatory system and thus undergoes metabolism and other similar biological processes.

[0323] The efficacy of a treatment with any composition provided herein for treating a disease or disorder can be determined by a skilled clinician. However, one of ordinary skill 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 improve or are ameliorated. Efficacy can also be measured by the individual not worsening, as evaluated by, for example, hospitalization or a decrease in the need for medical intervention (e.g., disease progression stops or at least slows). Methods for measuring these metrics are known to those of ordinary skill in the art and / or are described herein. Treatment includes any treatment of a disease in an individual or animal (some non-limiting examples include humans or mammals) and includes: (1) inhibiting the disease, e.g., stopping or slowing the progression of symptoms; or (2) alleviating the disease, e.g., causing symptoms to resolve; and (3) preventing the development of symptoms or reducing the likelihood of symptom development.

[0324] As discussed 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 such as an individual having, suspected of having, or at risk of developing a disease. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of disease symptoms, alter the course of disease symptoms (e.g., but not limited to slowing the progression of disease symptoms), or reverse disease symptoms. It should be understood that for any given case, one of ordinary skill in the art can determine the appropriate effective amount 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 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 infection.

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

[0327] Other methods known in the art include, but are not limited to, lentiviral transduction, transposon-based systems, direct RNA transfection, and CRISPR / Cas systems (e.g., type I, type II, or type III systems that use 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, CasH, 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, etc.).

[0328] In some embodiments, recombinant adeno-associated virus (AAV) vectors can be used for delivery. Techniques for generating rAAV particles are standard in the art, where an AAV genome to be packaged is provided to a cell, and the AAV genome includes a polynucleotide to be delivered, the rep and cap genes, and helper virus functions. Generation of rAAV requires the presence of the following components within a single cell (herein referred to as a packaging cell): an rAAV genome, AAV rep and cap genes isolated from the rAAV genome (e.g., not within the rAAV genome), and helper virus functions. The AAV rep and cap genes can be from any AAV serotype from which a recombinant virus can be derived, and can be from an AAV serotype different from the rAAV genome ITR, 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 AAVrh.74. Generation of pseudotyped rAAV is disclosed, for example, in International Patent Application Publication No. WO 01 / 83692.

[0329] Once the CAR-T cells are expanded ex vivo in response to, for example, an autoimmune disease antigen, the CAR-T cells can be reinfused into a subject in a therapeutically effective amount.

[0330] An exact amount of CAR T cells to be administered can be determined by a physician after considering the individual differences of the subject in terms of age, weight, degree of disease and condition.

[0331] The administration of T cell therapy can be defined by the total number of cells per infusion or the number of cells per kilogram of body weight, especially for pediatric subjects (e.g., patients). As the T cells replicate and expand after transfer, the administered cell dose may be different from the final steady-state cell number. In some embodiments, a pharmaceutical composition comprising the CAR T cells of the present disclosure can be administered at a dose of 10 4 to 10 10 total cells. In another embodiment, a pharmaceutical composition comprising the CAR T cells of the present disclosure can be administered at a dose of 10 3 to 10 8 cells / kg body weight (including all integer values within those ranges).

[0332] Compositions comprising the CAR T cells of the present disclosure can also be administered multiple times at these doses. The cells can be administered by using infusion techniques known in the art (see, for example, Rosenberg et al., New Engl J Med, (1988) 319:1676). The optimal dose and treatment regimen for a particular subject can be determined by those skilled in the art by monitoring the subject's disease signs and adjusting the treatment accordingly.

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

[0334] The compositions of the present disclosure can be prepared in a manner known in the art and in a manner suitable for parenteral administration to mammals, especially humans, including a therapeutically effective amount of the composition alone, and one or more pharmaceutically acceptable carriers or diluents.

[0335] As used herein, the term "pharmaceutically acceptable carrier" 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 contain suspending agents and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic agents and absorbents, etc. It will be understood that the compositions of the present disclosure may also contain other supplementary physiologically active agents.

[0336] The carrier must be pharmaceutically "acceptable" in the sense of being compatible with the other components of the composition and harmless to the subject. The compositions include those suitable for parenteral administration (including subcutaneous, intramuscular, intravenous, and intradermal administration). The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the pharmaceutical art. Such methods include preparing a carrier for association with the CAR-T cells. Generally, the compositions are prepared by uniformly and intimately associating any active ingredient with a liquid carrier.

[0337] In some embodiments, the compositions are suitable for parenteral administration. In another embodiment, 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, bacteriostats, and solutes which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents.

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

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

[0341] In some embodiments, enhancing the in vivo persistence can be measured by measuring differences in gene expression. In some embodiments, the genes include, but are 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 can be a solid tumor. In some embodiments, the cancer can 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, but are not limited to, 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 treatment method does not include administering a lymphodepleting agent within 7 days before administering T cell therapy. Non-limiting examples of lymphodepleting agents include cyclophosphamide, fludarabine, and / or bendamustine within 7 days before administering T cell therapy. In some embodiments, the treatment method does not include administering at least 600,000 IU / kg of IL-2 every 8 hours. In some embodiments, the treatment method does not include checkpoint therapy that blocks PD-1 or CTLA-4 signaling.

[0344] In some embodiments, the treatment method includes cells that have reduced exhaustion, enhanced proliferative capacity, increased replicative lifespan, reduced replicative senescence, enhanced anti-tumor effects, reduced dysfunction, enhanced persistence, and / or increased in vivo presence within the tumor. In some embodiments, the treatment method includes cells that have increased or reduced signaling via the CARD11-BCL10-MALT1 complex, NF-κB, AP-1, NFAT, JAK / STAT, and / or MEK / ERK pathways.

[0345] The cells of the methods of the present disclosure can be regulatory (Treg), natural killer (NK) cells, γδ T cells, invariant iNKT cells, macrophages, monocytes, TILs, or engineered T cells. In some embodiments, the engineered T cells can express a recombinant TCR or CAR or transcriptional receptor.

[0346] In the methods of the present disclosure, the engineered T cells can be autologous or allogeneic / non-autologous to the subject to whom they are administered. 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 engineered cells recombinantly expressing a CAR or transcriptional receptor are to be administered. Optionally, the cells can be obtained by leukapheresis (where white blood cells are selectively removed from the drawn blood), engineered and reconstituted, and then re-infused into the donor. Alternatively, allogeneic cells from an allogeneic / non-autologous donor who is not the subject can be used. In the case of an allogeneic / non-autologous donor, as is well known in the art, the cells are typed and human leukocyte antigen (HLA) matched to determine an appropriate level of compatibility. For both autologous and allogeneic cells, the methods for genetic manipulation and / or administration to a subject are well known in the art. In some cases, the cells can optionally be preserved (e.g., cryopreserved) until ready for use.

[0347] Procedures for isolating cells include, but are not limited to, flow cytometry, affinity chromatography, density gradient centrifugation, magnetic separation using antibody-coated magnetic beads, conjugation with particles that alter cell density, conjugation with a cytotoxic agent in combination with a monoclonal antibody (mAb), 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. Methods suitable for genetically engineering cells for recombinant expression are well known in the art. Systems and Kits

[0349] The systems or kits of the present disclosure include one or more of the following: any recombinant nucleic acid, recombinant cell, or pharmaceutical composition disclosed herein, and a syringe (including a drug-loaded syringe) and / or catheter (including a drug-loaded syringe) for administering any recombinant nucleic acid, recombinant cell, or pharmaceutical composition to a subject. The kit further includes written instructions for using any recombinant nucleic acid, recombinant cell, or pharmaceutical composition disclosed herein and the syringe and / or catheter for its administration.

[0350] Any of the above systems and kits can further comprise one or more additional reagents, where such additional reagents can be selected from: dilution buffer, reconstitution solution, wash buffer, control reagent, control expression vector, negative control polypeptide, positive control polypeptide, reagents for in vitro generation of chimeric receptor polypeptides.

[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. Instructions for practicing the method are typically 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, in the label of the container of the kit or its components (i.e., associated with the packaging or sub-packaging), etc. The instructions may exist as an electronic storage data file present on a suitable computer-readable storage medium (e.g., CD-ROM, floppy disk, flash drive, etc.). In some cases, the actual instructions are not present in the kit, but means may be provided for obtaining the instructions from a remote source (e.g., via the Internet). An example of this embodiment is a kit that includes a website address where the instructions can be viewed and / or downloaded. Like the instructions, such means for obtaining the instructions may be recorded on a suitable substrate.

[0353] All publications and patent applications mentioned in this disclosure are incorporated herein 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] It is not admitted that any reference cited herein constitutes prior art. The discussion of references states the claims of their authors, and the inventors / owners reserve the right to challenge the accuracy and relevance of the cited documents. It will be clearly understood that although many sources of information are mentioned herein, including scientific journal articles, patent documents, and textbooks; such mention does not amount to an admission that any of these documents forms part of the common general knowledge in the art.

[0355] The discussion of the general methods given herein is intended for illustrative purposes only. After reviewing this disclosure, other alternative methods and alternatives will be clear to those skilled in the art and will be included within the spirit and scope of this application. Examples

[0356] Unless otherwise indicated, the practice of this disclosure will employ conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology that are well known to those skilled in the art. Such techniques are fully explained in the references cited above.

[0357] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration only and are not intended to limit the scope of the present disclosure or claims in any way. Example 1 Identification of Mutations

[0358] This example describes the design and implementation of a statistical test for identifying mutations that can 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 the in vivo persistence of therapeutic T cells, and / or by other means.

[0359] To identify mutations that can be used or are beneficial for improving T cell therapy, the inventors / Disclosers: 1 - selected mutations that occur in patients, or 2 - performed a statistical analysis of genomic sequencing data of cloned T cells (including T cell lymphomas), and have identified hotspots and mutations that occur at a frequency higher than random expectation in the hotspots from public and private databases using the binomial distribution of gene lengths across the entire genome and a false discovery rate of 0.05 controlled by the Benjamini-Hochberg algorithm. Specifically, the inventors / Disclosers varied the background mutation rate in the genome, taking into account the heterogeneity due to transcription-coupled repair. Then, genes containing mutations that occur at a frequency higher than random expectation were identified under the assumption of the background mutation rate.

[0360] Using these methods, the inventors / Disclosers identified and cloned 62 point mutations (encoding non-synonymous amino acid substitutions and putative gain-of-function truncating mutations) in 40 different genes. Additionally, the inventors / Disclosers identified 10 gene fusions. Example 2 Library Construction and In Vitro Mutation Screening

[0361] This example describes the design and construction of the mutation library shown in Table 1 and the screening of constructs to identify constructs 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 the effect of overexpression of the wild-type form of the gene. Five control constructs were also cloned. These targets were cloned into barcoded lentiviral constructs to enable pooled screening. In total, the library for T cell lymphoma mutation screening included 117 unique constructs ( Figure 1 andFigures 2A - 2D )。

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

[0364] The intracellular domain containing the appropriate co-stimulatory domain and CD3ζ domain was synthesized as synthesized by Twist. The receptor was constructed by fusing CD19 scFv with the corresponding receptor scaffold and intracellular tail. All receptors contain an N-terminal CD8a signal peptide (MALPVTALLLPLALLLHAARP SEQ ID NO: 261) for membrane targeting and a flag tag (DYKDDDDK SEQ ID NO: 262) with α-flag PE (Biolegend 637310) for easily determining surface expression. In some cases, the receptor additionally contains a T2A self-cleaving sequence and a subsequent tNGFR sequence, which is used in downstream applications for T cell isolation. For all primary T cell experiments, the receptor was cloned into the modified pHR'SIN:CSW vector containing a PGK promoter.

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

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

[0367] The functions of most of these mutations have not been fully characterized in the context of T cell signaling and effector functions. To elucidate their functions, the inventors / Disclosers transduced a triple reporter system with each construct in the library. In general, the reporter cells enabled the elucidation of the effects of the mutations on biochemical signaling pathways, the effects of antigens, and the assessment of effector cytokine production. A schematic diagram of the in vitro and in vivo screening of the mutations using a triple reporter Jurkat cell (human T cell line) stably expressing NFAT-eGFP, NF-kB-eCFP, and AP-1-iRFP fluorescent protein reporter constructs to reveal the effects of the mutations on T cell signaling pathways is shown in Figure 1 Figure 1.

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

[0369] The mutations alter signaling in a manner that cannot be achieved by expression of the wild-type form of the gene. Twenty-five point mutation constructs showed significant differences relative to their wild-type counterparts, demonstrating a significant increase or decrease in signaling conferred by the mutations rather than attributable to wild-type gene overexpression ( Figure 3 )). Thus, this mutant library allows for the alteration of T cell signaling in a manner that is not possible with overexpression of the wild-type gene. This feature of the mutant library distinguishes it from methods that enhance or increase wild-type gene expression.

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

[0371] The mutant constructs showed a significant degree of antigen specificity. For both the CD19-CD28z CAR and the CD19-BBz CAR, the mutant constructs showed a significantly greater effect after antigen stimulation than in the absence of antigen( Figure 2E ).

[0372] The mutations had a significantly diverse effect on TCR-dependent signaling( Figure 2F ). For the CD19-BBzCAR, a total of 10 different combinations of upregulation or downregulation of the signaling pathway induced by the mutations were observed. Some of these modifications may be desirable. For example, an imbalance between NFAT and AP-1 signaling may contribute to T cell exhaustion. Thus, mutations that increase AP-1 rather than NFAT may be beneficial for T cell therapy. Twenty-five mutant constructs showed significant differences relative to their wild-type counterparts, thus showing a significant increase or decrease in signaling conferred by the mutations rather than attributable to wild-type gene overexpression( Figure 3 ).

[0373] Furthermore, these mutations provide an effect size that cannot be achieved solely by changes in gene expression, thus enabling tunable changes over a wide dynamic range. As an example of this tunability, AP-1 reporter expression can be downregulated or upregulated in the range of 0.4-fold to nearly 3-fold of the level induced by CAR activation in the control( Figure 2G ). Example 3 In Vivo Mutation Screening

[0374] Next, the inventors / specifiers systematically screened T cell lymphoma mutations to identify constructs that could improve the in vivo persistence of human CAR-T cells, as described below. Primary human CD3+ T cells were co-transduced with the CD19-BBz CAR and T cell lymphoma mutant constructs. Cells expressing both the CAR and the mutant constructs were pooled, sorted, and then injected into immunodeficient mice bearing subcutaneous CD19-K562 tumors( Figure 1 ). Samples of the pre-injection pooled library were collected for barcode sequencing. The barcode frequencies in the tumors were compared with the pre-injection T cell pool to identify constructs that were depleted or enriched in vivo. The construct with the highest single depletion level in the library was PDCD1, which encodes the co-inhibitory receptor and immunotherapy target PD-1, thus indicating that in vivo screening can identify therapeutically relevant targets( Figure 5A ). The K562 subcutaneous xenograft model generally showed poor T cell infiltration and persistence, as well as a lack of anti-tumor efficacy of CAR-T cells( Figure 4)。This model was used to identify constructs that enable T cells to overcome the harsh solid tumor microenvironment that limits the efficacy of therapeutic T cells. On day 0, 1x10 6 CD19-K562 cells were subcutaneously injected into NSG mice, and then on day 4, 5x10 6 CD19-BBz CAR cells or PBS vehicle control cells were intravenously injected. These data show that CD19-BBz CAR cells poorly controlled the growth of CD19-K562, indicating that T cell function is limited in this model and providing a rationale for using this model to screen for mutant constructs that improve T cell function. Constructs that improve infiltration and / or persistence in this model may be valuable for improving the anti-tumor efficacy of cell therapy against solid tumors.

[0375] In vivo screening identified 35 mutant constructs with positive log2 fold changes in vivo ( Figure 5A ). A positive log2 fold change indicates a mutant that is enriched in the tumor compared to the pre-injection sample. Enrichment in the tumor indicates that these constructs enable T cells to accumulate, persist, or expand in the tumor in vivo, which is a favorable 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, STAT3_D661I, ITK-FER, PRKCB1_D427N, VAV1_R798Q, KCNQ1_R583C, CARD11_Y361C, ECSIT_V140A, EIFS1_R89I, PDCD1_R231X, TP53_R273P, NFKB1_H67Y, TNFRSF1B_T377I, CARD11_S615F, CARD11_D357N, CARD11_E634K:S655C, CSNK1A1_S27C, CD28_T195P, RASGRP1_M261I.

[0376] The mutant constructs alter in vivo persistence in a manner that cannot be achieved by wild-type gene overexpression (Table 3). For example, wild-type CARD11 shows a negative log2 fold change, while the CARD11-PIK3R3 fusion and four CARD11 point mutants (CARD11 p.Y361C, p.S615F, p.D357N, and p.E634K:S655C) all show positive log2 fold changes. Similarly, in vivo in tumors, the TNFRSF1B p.G256C and p.T377I mutants show positive log2 fold changes, while wild-type TNFRSF1B shows a negative log2 fold change. Thus, mutations can permit an improvement in in vivo persistence in tumors beyond the levels permitted by wild-type gene expression.

[0377] In vitro screening results show a correlation with in vivo screening results. Consistent with the role of PD-1 levels in regulating the in vivo persistence of CAR cells, staining for PD-1 in CD19-BBz CAR Jurkat cells showed that constructs that significantly upregulated PD-1 in response to antigen had worse persistence ( Figure 5B ). Example 4 Analysis of Multiple Domains of the CARD11 - PIK3R3 Gene Fusion

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

[0379] The CARD11-PIK3R3 fusion was initially identified in a patient with CD4+ leukemia cutaneous T cell lymphoma (L. Wang et al., Genomic profiling of Sézary 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) gives rise to the following gene fusion: the N-terminal CARD11 protein CARD domain, coiled-coil domain, and a portion of the inhibitory domain with the C-terminal SH2 domain from PIK3R3 ( Figure 7 ). Due to the important role of CARD11 in mediating antigen-dependent signaling in T cells ( Figure 6) and partial lack of inhibitory domains in the fusion protein to test the CARD11-PIK3R3 fusion to determine whether it alters CBM complex signaling.

[0381] The inventors / Disclosers generated variants lacking specific domains and tested the ability of said variants to induce NF-κB and AP-1 signaling ( Figures 8A - 8B ). Deletion of the PIK3R3 component in the fusion eliminated signaling activity, but the PIK3R3 component alone was not sufficient to induce these pathways. Thus, the CARD11-PIK3R3 fusion does not simply acquire function by truncating a portion of the CARD11 protein, but requires the C-terminal PIK3R3 to activate downstream signaling cascades. The remainder of the inhibitory domain is not required for gain of function of the fusion; however, removal of the coiled-coil domain of the fusion component or the entire CARD11 portion impairs function. Example 5 Truncation of the CARD11 - PIK3R3 Gene Fusion

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

[0383] The inventors / Disclosers introduced multiple truncations of the fusion polypeptide (SEQ ID NO: 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256), as Figure 30A shown. SEQ ID NO: 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256 contain the sequence of SEQ ID NO: 206 and have deletions of 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 amino acids, respectively. Multiple truncations were evaluated in CD8+CD19-BBz CAR T cells during a 7-day period after removal from anti-CD3 / anti-CD28 bead stimulation ( Figure 30B ). After co-culturing CD19-BBz CAR CD8+ T cells with CARD11-PIK3R3 truncations and CD19 target cells at a 1:1 ratio for 24 hours, IL-2 secretion was analyzed by ELISA. CD8+ and CD19K562 populations were evaluated after 14 days of co-culture together in the absence of supplemental IL-2, and the results are shown in Figure 30D Long-term co-culture killing assay ( Figure 30D)Show that some truncated forms of the fusion body (-ID, -20, -60, -100, -140, -220) induce persistent target cell killing in the absence of IL-2 supplementation, similar to the WT (full-length) form of the fusion body. In contrast, some truncated forms of the fusion body (-180, -200, -260, -300) cannot induce persistent target cell killing in the absence of IL-2 supplementation and appear more similar to the condition of only CAR. These results suggest that the coiled-coil domain of CARD11 can be truncated without loss of the established phenotype of the full-length fusion body, but certain truncations result in loss of function. Additionally, IL-2 secretion data ( Figure 30C ) indicate that the -ID fusion variant alone produces a similar amount of IL-2 compared to the WT (full-length) form of the fusion body (dashed line marks WT IL-2 secretion). However, when comparing the IL-2 secretion data with the long-term co-culture killing data, the inventors / Disclosers hypothesized that a minimum threshold of IL-2 secretion is required to observe long-term co-culture killing in the absence of the IL-2 phenotype. In Figure 30C , this minimum threshold is indicated by the marked dashed line. Fusion variants that cannot kill CD19-K562 targets in the long-term co-culture killing assay in the absence of IL-2 ( Figure 30D ) are variants that fall below the hypothesized minimum threshold of IL-2 secretion in Figure 30C . Example 6 CARD11-PIK3R3 Promotes the Assembly and Signaling of the CBM Complex

[0384] To test whether CARD11-PIK3R3 promotes the assembly and signaling of the CBM complex by binding to BCL10, mutant forms of CARD11-PIK3R3 were generated that had an amino acid substitution (R28A) at the BCL10-binding site in the CARD domain of CARD11, which was previously reported to be required for CARD11-BCL10 binding. It was observed that BCL10 binding is important for both basal NF-κB and NF-κB and AP-1 induced by CAR signaling ( Figure 9A and Figure 9B ). Additionally, Western blots of the canonical MALT1 substrates CYLD and HOIL-1 indicate that the basal and stimulus-induced cleavage of these proteins is increased in the presence of the fusion body ( Figure 9C ). Treatment with a MALT1 inhibitor demonstrated that the cleavage of these proteins is indeed MALT1-dependent ( Figure 9C ). In summary, these data suggest that the CARD11-PIK3R3 fusion protein enhances CBM complex signaling.

[0385] To test whether CARD11 or BCL10 is required for CARD11-PIK3R3 function, CRISPR knockout experiments were performed. CARD11 or BCL10 in the BBz-CAR Jurkat signaling cell line was knocked out using CRISPR / Cas9. Under unstimulated, CAR-stimulated, and pharmacological TCR-stimulated conditions, CARD11-PIK3R3 was observed to be dependent on BCL10 but not wild-type CARD11 ( Figure 38 ). In contrast, TCR-triggered NF-κB activation in control cells was dependent on both CARD11 and BCL10, as expected. Interestingly, CAR-dependent signaling in cells lacking CARD11-PIK3R3 was independent of CARD11 or BCL10, indicating that the CBM may not be optimally engaged during CAR-T cell signaling ( Figure 38 ). Example 7 Effect of CARD11-PIK3R3 Expression on Signaling in Primary Human T Cells

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

[0387] Human CD3+ T cells from three healthy donors were lentivirally transduced with CD19-CD28z or BBZ CAR in the presence or absence of the CARD11-PIK3R3 fusion. CAR T cells were analyzed by CyTOF at 0, 7, 15, 30, 60, and 120 minutes time points during co-culture with CD19-expressing cells ( Figure 10A ). Consistent with the activation of NF-κB signaling induced in Jurkat cells, both CD19-CD28z and CD19-BBz CAR cells expressing the CARD11-PIK3R3 fusion exhibited significantly reduced levels of IκBα, a negative regulator of NF-κB that is degraded downstream of CBM complex activation ( Figure 10B ). In addition, CD19-BBz CAR cells showed enhanced signaling via two pathways (PI3K / Akt (p4EBP1) and MAPK) downstream of the CBM complex, but these were not significantly different in CD19-CD28z CAR cells. Consistent with the signal transduction differences being mediated by the fusion itself and no differences in transduction from the CAR, no differences were observed in proximal signaling events.

[0388] This data indicates that after CAR engagement, CARD11-PIK3R3 expression altered downstream signaling events in primary cells. Example 8 CARD11-PIK3R3 Enhances Gene Expression

[0389] To examine potential differences in gene expression, bulk RNA sequencing was performed on human CD8+ T cells from three healthy donors in the presence and absence of CAR stimulation by co-culture with CD19-expressing targets. Principal component analysis revealed strong transcriptional differences due to stimulation, but minimal overall differences from controls in the transcriptome of CARD11-PIK3R3 cells ( Figure 11A ). Differential expression analysis revealed that genes significantly upregulated in CARD11-PIK3R3 CD19-BBz CAR cells compared to controls in the stimulated state were genes normally induced by CAR signaling in control cells, which were enhanced in the presence of the fusogen ( Figure 11B ). This indicates that CARD11-PIK3R3 expression does not induce a completely distinct transcriptional state, but rather enhances the expression of a subset of genes induced by CAR. These include activation markers (IL2RA, CD69), cytotoxic and effector molecules (IFNG, TNF, IL4, IL5, IL13, GZMA, GZMB), chemokines (CCL4, CCL20, CCL8), and co-stimulatory molecules (ICOS, OX40, 4-1BB, GITR). Consistent with the biochemical analysis of the inventors / Disclosers, gene set enrichment using a previously reported gene set downregulated in response to MALT1 paracaspase inhibition in human T cells revealed significant enrichment of these genes as well as NF-κB- and AP-1-induced genes in cells expressing CARD11-PIK3R3 ( Figure 11C ).

[0390] RNA sequencing of human CD4+ and CD8+ T cells from three healthy donors was performed in the presence and absence of antigen stimulation (Supplementary Table 2). Principal component analysis showed that CAR-dependent stimulation induced the most significant transcriptional differences, thus indicating that CARD11-PIK3R3 expression is not sufficient for primary cells to adopt a fully antigen-activated phenotype ( Figure 39A ). By comparing transcripts upregulated in both CD4+ and CD8+ T cells after CAR stimulation, a core set of 43 genes regulated by CARD11-PIK3R3 was identified ( Figure 39B ). These include several transcripts that play 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]). It has been previously shown that several of these genes, such as ICOS48 and OX4049, favorably affect the anti-tumor response. Interestingly, CD4+ T cells expressing CARD11-PIK3R3 are enriched for gene signatures related to the cell cycle, whereas CD8+ T cells are most enriched for RNA metabolism, cytokine signaling, and translation signatures ( Figure 39C ). Consistent with our biochemical analysis, gene set enrichment identified enrichment of NF-κB, AP-1, and MALT1 paracaspase signatures ( Figure 39C ). Example 9 CARD11-PIK3R3 Confers a Proliferative Advantage to CD3 T Cells

[0391] The following experiments show how increased CBM complex signaling affects the activation status 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 removal of anti-CD3 / CD28 beads and sorting for the purified population, the cells were expanded and rested in culture for approximately one week. In transduced CD3 T cells, greater expansion of CD19-BBZ CAR + CARD11-PIK3R3 T cells was observed compared to CD19-BBz CAR T cells alone ( Figure 12A ). However, this observation was not reproduced in the CD19-CD2z T cell population alone ( Figure 12B ), indicating that the fusion provides a proliferative advantage, particularly when paired with the BBz CAR.

[0393] Further experiments were performed to determine whether CARD11-PIK3R3 could induce cytokine-independent growth in primary CAR T cells. For this purpose, primary T cells were co-transduced with CD19-BBz-CAR and CARD11-PIK3R3. After removal of anti-CD3 / CD28 stimulation and sorting for the purified population, the cells were expanded and rested in cultures with IL-2. CARD11-PIK3R3 improved the expansion of CAR-T cells in the presence of IL-2; however, removal of IL-2 early or late in culture led to rapid contraction of the T cell population ( Figures 46A-46C ). Example 10 Antigen-Induced Activation Status

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

[0395] Most short-term activation markers (CD25, CD69, PD-1, CD39) were upregulated in both CD19-BBz CAR T cells and CD19-BBz CAR + CARD11-PIK3R3 fusion protein T cells ( Figure 14A ). Consistent with bulk RNA sequencing data, ICOS was found to be significantly upregulated in CD19-BBz CAR + CARD11-PIK3R3 fusion T cells compared to CAR T cells alone. These observations were similar in the CD19-CD28z CAR context. Interestingly, although Jurkat signaling data indicated that the CARD11-PIK3R3 fusion protein led to higher basal NF-κB expression, 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 indicates that while the fusion does induce some basal NF-κB signaling, it is not significant enough to cause non-antigen-dependent activation of the cells.

[0396] Further experiments were performed using both CD8+ and CD4+ T cells. Equivalent 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 bulk RNA sequencing 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 shows how CARD11-PIK3R3 upregulates multiple cytokines.

[0398] Bulk RNA sequencing analysis of activated CD19-BBz CAR + CARD11-PIK3R3 fusion T cells identified many upregulated cytokines (TNFα, IFN-γ, IL-5, and IL-13).

[0399] To confirm these findings at the protein level, the inventors / Disclosers co-cultured the transduced CD8+ T cells with CD19-K562 targets for 48 hours and evaluated various cytokines in the supernatant. Validating the Jurkat ELISA results, they found that in addition to other inflammatory cytokines such as IFN-γ, TNFα, and GMCSF (although not significantly increased), the CARD11-PIK3R3 fusion also induced higher secretion of IL-2( Figure 14C ). Surprisingly, while cytokine secretion was significantly increased in CD8+ CARD11-PIK3R CAR T cells, it was less increased in CD4+ CARD11-PIK3R CAR T cells, although initially observed in CD4+ T cell cancers( Figure 41 ). Interestingly, the Th2 cytokine IL-5 was also significantly increased by the expression of the fusion, indicating that the fusion induced a unique cytokine profile. Similar trends were observed in CD8+ T cells with CD19-CD28zCAR and bulk CD3 T cells with CD19-BBz or CD19-CD28z CAR, however, in some cases, the increases observed in the fusion-transduced cells were not significant( Figures 13A-13B ). Example 12 CARD11-PIK3R3, Cytotoxicity, and Growth

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

[0401] Since the tumor microenvironment typically lacks the pro-survival signaling (such as IL-2) required by cytotoxic T cells, the inventors / Disclosers conducted experiments to reveal how the CARD11-PIK3R3 fusion would affect engineered T cell killing in the state of IL-2 starvation. A long-term assay was established in which the transduced T cells were mixed with CD19-K562 at a 1:1 ratio and maintained in culture for two weeks in the presence or absence of supplemental IL-2. Interestingly, after two weeks of culture, both the CD19-BBz CAR alone and CAR + CARD11-PIK3R3 were able to effectively clear the CD19-K562 targets, however, when cultured in the absence of supplemental IL-2, only CD19-BBz CAR + CARD11-PIK3R3 was able to effectively clear the CD19-K562 targets( Figure 15A ). In the long-term co-culture killing assay, the killing differences of CD19-CD28z CAR were less significant.

[0402] When stimulated twice over a two-week period, the inventors / Disclosers noted that the numbers of CD19-BBz CAR alone decreased regardless of supplemental IL-2, while CD19-BBz CAR + CARD11-PIK3R3 effectively expanded over time (in the presence of IL-2), or maintained higher cell numbers (in the absence of IL-2)( Figure 15B ). Finally, the inventors / Disclosers evaluated target killing at different ratios over time and found that at each tested ratio, CD19-BBz CAR + CARD11-PIK3R3 better controlled target cell growth compared to CD19-BBz CAR alone (Figure 16). Similar trends were observed for CD19-28z CAR with and without the CARD11-PIK3R3 fusion. Example 13 CARD11-PIK3R3 Improves the Fitness, Function, and Efficacy of Therapeutic T Cells

[0403] This example demonstrates how the CARD11-PIK3R3 fusion improves the fitness, function, and efficacy of therapeutic T cells in a fully immunocompetent syngeneic environment.

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

[0405] CD45.1+ OT-I CD8+ T cells were transduced with control (GFP+) or CARD11-PIK3R3 (mCherry+) retrovirus to enable tracking of adoptively transferred cells in CD45.2+ C57BL / 6J hosts bearing B16-OVA melanoma tumors. To analyze in vivo accumulation, a dual transfer system was used where competition between transferred T cells in the same tumor could be determined. The inventors / Disclosers generated a mixture of CD45.1+ OT-I T cells, where approximately 10% of the cells were mCherry positive (corresponding to cells expressing CARD11-PIK3R3), for direct comparison to GFP+ control cells in vivo. Strikingly, at 7 days post-transfer, in TIL, the cell number (normalized to input) of cells expressing CARD11-PIK3R3 was increased approximately 145-fold compared to control cells( Figure 24A and Figure 24B ).

[0406] We confirmed enhanced competitive accumulation of CARD11-PIK3R3 in a second transgenic TCR mouse model against B16-F10 tumors (pmel-1 T cells, which recognize gp100, an endogenous melanoma antigen). 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 1257-fold Figures 48A-48C .

[0407] This corresponded to a significantly increased fraction of transferred cells in total CD8+ T cells, even without normalization relative to the input number. Thus, within the same tumor microenvironment, T cells expressing CARD11-PIK3R3 had significantly improved accumulation compared to control cells. Example 14 Characterization of Cell Phenotype after Adoptive Transfer of CARD11-PIK3R3

[0408] After adoptive transfer of CARD11-PIK3R3- or control-transduced OT-I cells, the following assays were used to characterize the phenotype of the cells.

[0409] Based on data obtained from a competitive dual-transfer assay, enhanced accumulation of cells expressing CARD11-PIK3R3 was also present in tumors when transferred alone. Additionally, in the spleen and tumor-draining lymph nodes, the inventors / Disclosers detected a higher proportion of CARD11-PIK3R3 OT-I cells compared to 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 assays 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 functions of cells expressing the fusion, including higher production of TNF-α, IFN-γ, and IL-2 Figure 27 . Additionally, the proportion of multifunctional cells producing all three of these effector cytokines was significantly elevated in CARD11-PIK3R3 OT-I cells. Further, CARD11-PIK3R3 expression significantly increased the expression of the stemness-related 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 cell-like T cells. Example 16 Antitumor Efficacy of CARD11-PIK3R3 in a Syngeneic TCR-Transgenic Melanoma Mouse Model

[0413] The following experiments were performed to determine the effect of CARD11-PIK3R3 on the therapeutic efficacy of T cells in an allogeneic immune-competent system.

[0414] On day 12 after subcutaneous inoculation of B16-OVA tumors, without preconditioning or lymphodepletion, mice were treated with PBS control or 2x10 6 OT-I cells transduced with control or CARD11-PIK3R3 retrovirus. CARD11-PIK3R3 cells mediated significantly enhanced control of tumor volume ( Figure 28A ). In addition, CARD11-PIK3R3 OT-I cells promoted an extended overall survival ( Figure 28B ). 60% of the mice receiving CARD11-PIK3R3 OT-I (3 / 5) achieved complete tumor clearance 85 days after tumor challenge, at which time no mice treated with PBS or control OT-I survived. Collectively, these data indicate that T cells expressing CARD11-PIK3R3 have excellent therapeutic functions in vivo. Reduced cell numbers were routinely used as a "stress test" to quantify the relative efficacy of improved T cell therapies. In addition, these data indicate that the CARD11-PIK3R3 fusion can overcome a key limitation of cell therapy, namely the ability to manufacture sufficient cells for therapeutic efficacy in humans. To test this, the therapeutic efficacy of 20,000 and 100,000 CARD11-PIK3R3-transduced OT-I cells was compared to 2 million control OT-I cells in mice bearing B16-OVA tumors. Strikingly, CARD11-PIK3R3 enabled tumor control at two doses 20-fold and 100-fold lower than the control cells ( Figure 29A )

[0415] Current T cell therapies often lack long-term persistence and fail to form a memory population after primary tumor clearance, resulting in a high incidence of relapse. Animals treated with CARD11-PIK3R3 OT-1 at a low cell dose or naive controls were rechallenged with B16-OVA tumor cells in the contralateral flank more than two weeks after initial tumor clearance. CARD11-PIK3R3 OT-I-treated animals were protected from tumor development ([ Figure 29B ) compared to naive untreated mice. Rejection of secondary tumor challenge indicates that CARD11-PIK3R3 OT-I forms a long-term memory phenotype capable of responding to and suppressing tumor relapse. This phenotype is highly desirable in cancer therapy, allowing a single engineered therapy to respond to the primary tumor and prevent relapse, thereby inducing durable clinical remission. Collectively, these data demonstrate that T cells expressing CARD11-PIK3R3 have excellent therapeutic functions in vivo. Example 17 Enhancement of Mutant Constructs in Cell Types Other than Progenitor Cells

[0416] The CARD11-PIK3R3 fusion was initially identified in patients with CD4+ T cell lymphoma (Wang et al., Genomic profiling of Sézary 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+ cell-only therapy model, and the CARD11-PIK3R3 fusion significantly enhanced the function of these CD8+ T cells in vivo, even though it is a sequence derived from CD4+ T cells. Thus, the mutations described herein can have therapeutic value in cell types different from the cell type in which the mutations initially occurred.

[0417] Example 18

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

[0419] T cells were collected from C57BL / 6-Tg(TcraTcrb)1100Mjb / J (OT-I) mice and transduced with hCD19-CAR (CD19-BBzCAR) in the presence or absence of 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 a subcutaneous injection of 1x10 5 expressing hCD19 B16 tumor cells on the posterior flank. Eleven days after tumor inoculation, 1x10 6 CAR+ mouse T cells were administered to tumor - bearing animals, and tumor growth was tracked by caliper measurements performed three times a week. Tumor volume was calculated using the following equation: (length x width x width) / 2, where the length is the longest measurement. Mice were euthanized when the tumor reached 2000 mm3 or the length or width reached 2 cm.

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

[0421] In a repeat study, tumors treated with CD19 - BBz - CAR and CARD11 - PIK3R3 CD19 - BBz - CAR T cells were evaluated at the tumor endpoint by flow cytometry. Tumors treated with CD19 - BBz - CAR maintained CD19 expression, while tumors treated with CARD11 - PIK3R3 CD19 - BBz - CAR T cells were uniformly CD19 - negative, indicating that antigen loss is a mechanism of relapse ( 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 the CARD11 - PIK3R3 fusion. The T cells were then electroporated with Cas9 RNP targeting the TCR α 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 significantly lower tumor burdens and significantly improved survival (Figure 18).

[0423] At 7x10 6Improved efficacy of CD19-BBz-CAR T cells was also observed at higher doses of CAR+T cells, where 4 out of 7 animals treated with CD19-BBz-CAR that initially controlled the tumor began to relapse with NALM-6 disease, while none of the 7 animals treated with CARD11-PIK3R3 CD19-BBz-CAR T cells relapsed ( Figure 42A 、 Figure 43A ). We also determined that T cells expressing CARD11-PIK3R3 alone had no anti-tumor effect, demonstrating that the observed anti-tumor effect was antigen-specific and dependent on CAR activity ( Figure 42A ). To address safety concerns, we also determined that animals bearing NALM-6 tumors with controlled tumors treated with CAR or CARD11-PIK3R3 CAR T cells gained weight over the course of 100 days and lost weight only when NALM-6 relapse occurred ( Figure 42A 、 Figure 43B ). Similarly, non-tumor-bearing mice tolerated high doses of control T cells, CAR T cells, CAR + CARD11-PIK3R3 T cells ( Figure 43C ). Finally, after re-challenge with 5x10 5 NALM-6 tumors (for animals that had eliminated leukemia and had not yet developed symptoms of GVHD), we found that animals treated with CD19-BBz-CAR died of NALM-6 disease at a rate similar to that of untreated age-matched controls, while CARD11-PIK3R3 CD19-BBz-CAR T cells prevented leukemia growth ( Figure 43D ). These data indicate that even at high T cell doses, CARD11-PIK3R3 can enhance therapeutic efficacy while maintaining safety in vivo.

[0424] We next determined whether CARD11-PIK3R3 could improve CD19-CD28z-CAR T cell activity at low doses in the above NALM6 leukemia model. Therefore, we administered 4x10 5 CD19-CD28z-CAR T cells or CARD11-PIK3R3 CD19-CD28z-CAR T cells to NALM6-bearing mice ( Figure 44A ), and found that animals treated with CAR T cells died of the disease, while CARD11-PIK3R3 CAR T cells cleared two consecutive NALM6 challenges ( Figures 42B-42C)。Therefore, the anti-tumor efficacy of CARD11-PIK3R3 expression is not limited to CARs with 4-1BB co-stimulatory domains, but also improves CARs with CD28 domains, indicating that CARD11-PIK3R3 expression can be broadly beneficial for CAR T cell therapy.

[0425] Finally, we sought to determine how CARD11-PIK3R3 would perform in a xenograft solid tumor model. Here, we used a subcutaneous model of mesothelioma (M28) that naturally expresses the tumor-associated antigen MCAM55. We generated tumor-bearing animals and administered 5x105 MCAM-targeted CD28z-CAR T cells with or without CARD11-PIK3R3 or control T cells ( Figure 44B ). Animals maintained their body weight throughout the study ( Figure 44C ), indicating that the therapy was well tolerated. Although MCAM-CD28z-CAR treatment delayed tumor growth compared to the control, the size of tumors in both the control and CAR T cell-treated groups increased progressively ( 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. In addition, compared to CAR T cells alone, CARD11-PIK3R3 CAR T cells prevent relapse (re-challenge). 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 human TCR-based xenograft model. KRAS p.G12D (a common mutation present in human solid tumors) can be present on multiple human HLA alleles and has been the target of adoptive T cell therapy in small human studies. Using SNU-1 gastric cancer cells overexpressing HLA-C*08:02 with the KRAS p.G12D mutation and a clinically validated TCR against KRAS p.G12D presented by HLA-C*08:02, we observed a significant enhancement of tumor clearance by CARD11-PIK3R3 expression ( Figure 45)。Therefore, CARD11-PIK3R3 expression enhanced the function of both human and murine therapeutic TCR cells. Collectively, these data indicate that T cells expressing CARD11-PIK3R3 have excellent in vivo therapeutic functions in multiple immunotherapy-refractory tumor models, including CAR- and TCR-transgenic-based models. Example 21 CARD11-PIK3R3 Improves the Antitumor Efficacy of Mouse CAR T Cells in a Syngeneic Mesothelioma Model

[0427] Transduce murine T cells with hAPPL2-CAR with or without the CARD11-PIK3R3 fusion Figure 21 )。Transfer the T cells into mice bearing hAPPL2-40L mesothelioma tumors. In this model, C57BL / 6J mice received a subcutaneous injection of 2x10 6 40L (mesothelioma) tumor cells expressing hALPPL2 in the posterior flank. Collect T cells from B6.SJL-Ptprca Pepcb / BoyJ (CD45.1) mice and transduce them to express ALPPL2-BBz CAR (with or without co-expression of CARD11-PIK3R3). Thirteen days after tumor inoculation, administer 2x10 6 CAR+ murine T cells to the tumor-bearing animals and track tumor growth by caliper measurements performed three times a week. Calculate tumor volume using the following equation: (length x width x width) / 2, where length is the longest measurement. Euthanize the mice when the tumor reaches 2000 mm3 or the length or width reaches 2 cm. CARD11-PIK3R3 CAR T cells have a significantly increased ability to control tumor volume ( Figure 22 ).

[0428] The method used by the inventors / Disclosers allows the identification of CARD11-PIK3R3 gene fusions, which significantly enhance therapeutic T cell function and efficacy by engaging the CBM complex. Additionally, in a fully immunocompetent mouse model without lymphodepletion, expression of the fusion was able to achieve excellent tumor control at a 100-fold lower cell dose compared to control cells. Notably, the gene fusions identified by the inventors / Disclosers would not have been accessible through prior T cell screening efforts such as loss-of-function, CRISPR activation, or wild-type gene overexpression screens. Thus, naturally occurring mutations that have undergone positive selection in the human body represent a novel and effective toolset for enhancing cell therapies. The results obtained by the inventors / Disclosers suggest that further testing of additional mutations identified in T cell lymphomas, or even other somatic mutations occurring in T cells including autoimmune diseases, may hold promise for identifying additional ways to enhance T cell function. Additionally, the method provides a platform for modifying other immune cell types used in cell therapies, such as macrophages, NK cells, γδ T cells, or B cells, with naturally occurring mutations. The potential utility of the method is highlighted by the fact that, although found in CD4+ T cells, the CARD11-PIK3R3 fusion significantly enhances CD8+ T cell function.

[0429] The results presented herein suggest that the CBM signalosome is a key regulator of therapeutic T cell function. Several individual outputs of CBM signaling, such as induction of AP-1 and NF-κB transcriptional activity and downregulation of the MALT1 cleavage substrates REGNASE-1 and ROQUIN, have each independently been considered as ways to improve T cell therapy. This raises the possibility that the unifying feature of these diverse approaches is that they each partially address the relative lack of CBM signaling. CARD11-PIK3R3 expression represents an effective engineered solution to simultaneously enhance each of these individual CBM outputs. Further study of T cell lymphoma mutations in the context of adoptively transferred T cells could improve cell therapy and elucidate new T cell biology. Example 22 Safety in OT-I Mice

[0430] To address concerns about toxicity or transformation, we monitored the mice from Figure 29 for up to 418 days after T cell transfer ( Figure 49A ). During this time, animals treated with CARD11-PIK3R3 OT-I T cells gained weight similarly to the open control ( Figure 49B ). To assess occult disease, we performed necropsies on three mice at day 240 after T cell transfer. The weights and overall appearance of their spleens were normal ( Figures 49C-49D)。In addition, CARD11-PIK3R3 OT-I T cells accounted for less than 1% of the total CD8+ T cells in the spleen and blood ( Figure 49E )。We performed hematoxylin and eosin (H&E) staining on common extranodal sites of lymphoma as well as the spleen and lymph nodes. Pathological examination failed to identify any evidence of nuclear atypia, disruption of normal cellular architecture, or neoplastic disease ( Figure 49F )。

[0431] For the remaining 9 animals, we monitored the blood for evidence of leukemia disease 330 - 418 days after adoptive transfer. There were no atypical cells in the blood. In addition, tail bleeds from CARD11-PIK3R3 OT-I-treated animals revealed that CARD11-PIK3R3 OT-I T cells were present at less than 1% of the total CD8 population ( Figures 49G-49J )。This data indicates that the CARD11-PIK3R3 OT-I T cell population contracts after exposure to naive or re-challenged tumors. Collectively, these data demonstrate a favorable safety profile of anti-tumor T cells expressing CARD11-PIK3R3 and no evidence of in vivo malignant transformation over long time periods. Example 23 Putative Gain-of-Function Mutation in STAT...

Claims

1. A polypeptide, the polypeptide comprising: (a) a protein containing a caspase-associated recruitment domain (CARD) or a functional fragment thereof; and (b) a domain capable of binding to: (i) a substrate located on the inner side of the cytoplasmic membrane, and / or (ii) a target polypeptide containing phosphorylated tyrosine.

2. The polypeptide according to claim 1, wherein the domain in b) is capable of binding to a substrate that is indirectly located on the inner side of the plasma membrane by binding to another polypeptide or lipid directly located on the inner side of the cell.

3. The polypeptide according to claim 1 or claim 2, wherein the CARD-containing protein comprises or consists of the following: 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 according to any one of claims 1-3, wherein the CARD-containing protein is selected from CARD9, CARD10, CARD11 and CARD14.

5. The polypeptide according to claim 4, wherein the CARD-containing protein comprises or consists of the following: 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. The polypeptide according to any one of claims 1-5, wherein the functional fragment of the CARD-containing protein is derived from CARD11 and comprises or consists of the following: 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 according to any one of claims 1-6, wherein the function of the CARD-containing protein or its functional fragment is to bind to the CARD domain on BCL10.

8. The polypeptide according to any one of claims 1-7, 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 according to any one of claims 1-8, wherein the cell is a T cell, macrophage, monocyte or natural killer (NK) cell.

10. The polypeptide according to any one of claims 1-9, wherein activation of the cell produces the substrate located on the inner side of the plasma membrane.

11. The polypeptide according to any one of claims 1-10, wherein the substrate located on the inner side of the cytoplasmic membrane is phosphoinositide.

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

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

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

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

302.

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

301.

18. The polypeptide according to 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, 5 μM, 1 μM, 0.5 μM, 0.1 μM, 0.05 μM or 0.01 μM, and wherein the Kd is analyzed by fluorescence polarization assay.

19. The polypeptide according to any one of claims 1 - 9 or 14 - 18, wherein the polypeptide has a higher affinity for the target polypeptide containing the phosphorylated tyrosine than for a control polypeptide that is not phosphorylated at the corresponding tyrosine position.

20. The polypeptide according to claim 19, wherein the polypeptide has an affinity for the target polypeptide containing the phosphorylated tyrosine that is at least 2 - fold, at least 5 - fold, at least 10 - fold, at least 50 - fold or at least 100 - fold higher (lower Kd) than for a control polypeptide that is not phosphorylated at the corresponding tyrosine position.

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

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

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

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

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

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

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

28. The polypeptide according to any one of claims 24-27, wherein the SH2 domain comprises or consists of the following: 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. The polypeptide according to any one of claims 24 - 28, wherein the SH2 domain comprises a motif of a conserved arginine residue in the FLVR motif.

30. The polypeptide according to any one of claims 24-29, wherein the SH2 domain comprises or consists of the following: A sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:

305.

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

32. The polypeptide according to any one of claims 24-31, wherein the SH2 domain comprises or consists of the following: A sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NO:313 - 15.

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

34. The polypeptide according to claim 33, wherein the second polypeptide moiety comprises or consists of the following: 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. The polypeptide according to any one of the preceding claims, wherein the polypeptide does not comprise a coiled - coil domain or a portion thereof.

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

37. The polypeptide according to claim 36, wherein the coiled-coil domain comprises or consists of the following: A sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NO:290 - 293.

38. The polypeptide according to claim 37, wherein the coiled-coil domain comprises or consists of the following: A sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NO:

290.

39. The polypeptide according to any one of claims 36 - 38, wherein 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.

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

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

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

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

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

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

46. The polypeptide according to claim 45, wherein the inhibitory domain (ID) comprises or consists of the following: A sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:

294.

47. The polypeptide according to claim 46, wherein the inhibitory domain (ID) comprises or consists of the following: A sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:

295.

48. The polypeptide according to any one of claims 45-47, wherein 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 amino acids of the N-terminal portion of the inhibitory domain (ID).

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

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

297.

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

26.

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

53. The polypeptide according to claim 8 or 52, wherein the T cells express an engineered immune receptor that binds to a target on tumor cells.

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

55. A polypeptide, the polypeptide comprising mutations capable of effecting the following: (i) altering T cell signaling via the NFAT, NF-κB, and / or AP-1 pathways, (ii) altering cytokine production, (iii) altering JAK / STAT signaling in T cells, (iv) altering co-stimulatory molecule signaling in T cells, (v) altering RAS / MEK / ERK signaling in T cells, (vi) altering phospholipase γ signaling, (vii) altering the activity of transcription factors in T cells, and / or (viii) altering or enhancing the in vivo persistence of T cells comprising the mutations in tumors.

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

57. The recombinant nucleic acid according to claim 56, wherein the nucleic acid comprises a promoter.

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

59. The recombinant nucleic acid according to any one of claims 56-58, 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 recombinant nucleic acid according to any one of claims 56-59, wherein 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, IFNγ 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 protein 1 (PD-1) promoter, T cell immunoglobulin mucin-3 (TIM-3) promoter, cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, lymphocyte activation gene 3 (LAG-3) promoter, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) promoter, B and T lymphocyte attenuator (BTLA) promoter, CD25 promoter, CD69 promoter, Fas ligand (FasL) promoter, TIGIT promoter, TGF-β promoter, T-bet promoter, Eomes promoter, GATA3 promoter, CD45RA promoter, 2B4 promoter, type I interferon (IFN)α, type I IFNβ promoter, IFNγ promoter, IRF3 promoter, IRF7 promoter, NFkB promoter, AP-1 promoter, TNF-α promoter, CD130 promoter, NR4A1 promoter, NR4A2 or NR4A3 promoter.

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

62. The vector according to claim 61, wherein the vector is a viral vector selected from retroviral vectors, adenoviral vectors, and adeno-associated viral vectors.

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

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

65. The cell according to claim 64, wherein the cell is a non-natural cell or has been genetically engineered.

66. The cell according to claim 64 or 65, wherein the cell is not a CD4+ T cell.

67. The cell according to any one of claims 64-66, wherein the cell is not a cancer cell.

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

69. The cell according to any one of claims 64-68, wherein 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 but not any SH2 domain.

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

71. A cell according to any one of claims 64 - 70, wherein the cell comprises a polypeptide, the polypeptide comprising a sequence selected from the following: 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: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, SEQID 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, and functional variants thereof containing at least one mutation listed in Table 1.

72. A cell according to any one of claims 64 - 71, wherein the cell comprises a nucleic acid sequence or a functional variant thereof containing at least one mutation listed in Table 1, the nucleic acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the following sequences: 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 IDNO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ IDNO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ IDNO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ IDNO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ IDNO: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 IDNO: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, SEQID 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, SEQ ID NO:

255.

73. The cell according to any one of claims 64-72, wherein the cell further comprises: (i) a chimeric antigen receptor (CAR) specific for a target antigen; and / or (ii) a T cell receptor (TCR) specific for a target antigen.

74. The cell according to claim 72 or 73, wherein the cell is selected from immune cells, T cells, regulatory T cells, CD8+ cells, natural killer cells, tumor-infiltrating lymphocytes, and MAIT cells.

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

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

77. The method according to claim 76, the method comprising genetically modifying the T cells to express the polypeptide.

78. The method according to claim 76 or 77, the method comprising introducing a recombinant nucleic acid encoding the polypeptide or a vector comprising the recombinant nucleic acid into the T cells.

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

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

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

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

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

84. The method according to any one of claims 81-83, wherein the cancer expresses 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, 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.

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

86. The method according to any one of claims 81-85, 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. The method according to any one of claims 81-86, wherein the treatment method does not include administering a lymphodepleting agent within 7 days before administering the T cell therapy.

88. The method according to claim 87, wherein the treatment method does not include administering cyclophosphamide, fludarabine, and / or bendamustine within 7 days before administering the T cell therapy.

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

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

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

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

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