Chimeric antigen receptors with card domains

Abstract

The present disclosure relates generally to compositions and methods for improving T cell therapy. In particular, the disclosure provides chimeric and multi-chain receptors including CARD11 polypeptides having mutations capable of altering T cell signaling, cytokine production, and / or in vivo persistence in tumors of therapeutic T cells comprising the mutation. The T cell signaling can be by NFAT, NF-κB and / or AP-1 pathways. The disclosure also provides vectors and cells including the recombinant nucleic acid constructs of the disclosure as well as methods of using the receptors.

Description

CHIMERIC ANTIGEN RECEPTORS WITH CARD DOMAINSCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S. Provisional Patent Application Serial No. 63 / 581,384, filed on September 8, 2023. The contents of the above-referenced application is herein expressly incorporated by reference in its entirety, including any drawings.STATEMENT REGARDING FEDERALLY SPONSORED R&D

[0002] This invention was made with government support under OD025751 awarded by the National Institutes of Health. The government has certain rights in the invention.FIELD

[0003] The present disclosure relates generally to compositions and methods for improving adoptive T cell therapy.BACKGROUND

[0004] Chimeric antigen receptor (CAR)-T cell therapy is a promising immunotherapy approach that involves genetically engineering T cells to express CARs, which enable them to recognize and eliminate tumor cells. CAR-T cell therapy has shown remarkable success in treating hematologic malignancies, but its efficacy in solid tumors has been limited. Solid tumors present unique challenges for CAR-T cell therapy due to the hostile tumor microenvironment, which can inhibit CAR-T cells by promoting T cell exhaustion and apoptosis. In addition, solid tumors often express low or heterogenous levels of target antigens, which can limit CAR-T cell efficacy. Addressing these challenges is critical to developing effective CAR-T cell therapies for solid tumors, and incorporating new signaling domains is one promising approach to improving the functionality of CAR-T cells.

[0005] CARD 11 is a scaffolding protein that plays a critical role in CBM signaling in T cells. The protein is composed of several domains that are important for its function. At the N-terminus, CARD 11 contains a caspase activation and recruitment domain (CARD), which mediates protein-protein interactions with other CARD-containing proteins such as BCL10. Next, CARD 11 contains a coiled-coil domain that mediates CARD11 oligomerization. CARD 11 also contains an inhibitory domain, located after the coiled-coil domain, whichserves to keep CARD11 in an inactive state under basal conditions. Next, CARD11 contains a membrane-associated guanylate kinase (MAGUK) domain that is important for proteinprotein interactions with downstream signaling molecules. At the C-terminal end, CARD11 contains a PDZ-binding motif, which mediates interactions with PDZ domain-containing proteins. Together, these domains and phosphorylation sites allow CARD11 to function as a scaffold for the assembly of the CBM complex and the activation of downstream signaling pathways in T cells.

[0006] Our previous work has identified a novel fusion protein (CARD1 1 -PIK3R3) that enhances CARD11-BCL10-MALT1 signaling to improve CAR-T cell function. CARD11- BCL10-MALT1 (CBM) signaling is a critical pathway in T cells that mediates the activation ofNF-KB and AP-1 transcriptional activity. Upon TCR activation. CARD11 forms a complex with the scaffolding protein BCL10 and the protease MALT1, which activates downstream signaling cascades resulting in NF-KB and AP-1 activation. This signaling pathway is essential for T cell activation, proliferation, and differentiation. Alterations in the CBM pathway have been associated with lymphoma, immunodeficiency and autoimmune disorders.

[0007] CARD11-PIK3R3 expression significantly increased NF-kB and AP-1 signaling and IL -2 production downstream of CAR signaling. Furthermore, we found that expression of CARD11-PIK3R3 in human and mouse T cells significantly improved their ability to eliminate tumor cells in vitro and in vivo when paired with either a CAR or a T cell receptor (TCR). How ever, one limitation of this approach is that we observed accelerated weight loss consistent with xenogeneic graft-versus-host disease in mice treated with human CAR-T cells expressing the CARD11-PIK3R3 protein when the endogenous TCR was present. For this reason, engineering approaches that can incorporate the therapeutic efficacy enhancement of fusion expression while limiting this toxicity are needed. One potential approach to accomplish this is to enhance CBM signaling in CAR-T cells through modification of the CAR construct itself.

[0008] These observ ations point to the need for further development of novel CAR-T cell constructs that incorporate signaling domains to enhance CBM signaling in order to enhance their therapeutic efficacy and prevent toxicities.

[0009] Thus, there remains a need in the art for alternative solutions to address the significant unmet need for effective adoptive T cell therapies and for enhancing engineered T cell fitness.SUMMARY

[0010] This section provides a general summary of the disclosure and is not comprehensive of its full scope or all of its features.

[0011] The disclosure provides chimeric receptor constructs and methods for enhancing adoptive T cell therapies. The chimeric receptor constructs of the disclosure include CARD1 1 polypeptide domains with mutations wherein the mutations enhance the therapeutic efficacy of T cells by altering T cell signaling, decreasing T cell exhaustion and / or by enhancing in vivo persistence and fitness of engineered T cells.

[0012] In one aspect, provided herein are chimeric antigen receptors including, from N- terminus to C-terminus: (a) an extracellular binding domain (ECD) having a binding affinity for a selected ligand; (b) a linking sequence; (c) a transmembrane domain (TMD) having at least about 80% sequence identity to the transmembrane domain of a Type 1 transmembrane receptor and comprising one or more ligand-inducible proteolytic cleavage sites; and (d) an intracellular domain (ICD) comprising, in any order: (i) an intracellular signaling domain (SD) comprising at least one costimulatory domain derived from a signaling molecule and an activation domain, and (ii) a caspase recruitment domain family member 11 (CARD11) or caspase recruitment domain family member 9 (CARD9) polypeptide comprising one or more mutations.

[0013] In one aspect, provided herein are chimeric antigen receptors including, from N- terminus to C-terminus: (a) an extracellular binding domain (ECD) having a binding affinity for a selected ligand; (b) a linking sequence; (c) a transmembrane domain (TMD) having at least about 80% sequence identity to the transmembrane domain of a Type 1 transmembrane receptor and comprising one or more ligand-inducible proteolytic cleavage sites; and (d) an intracellular domain (ICD) comprising, in any order: (i) an intracellular signaling domain (SD) comprising at least one costimulatory domain derived from a signaling molecule and an activation domain, and (ii) a domain comprising caspase recruitment domain (CARD) from caspase recruitment domain family member 11 (CARD 11) or caspase recruitment domain family member 9 (CARD9) polypeptide comprising one or more mutations.

[0014] In some embodiments, binding of the selected ligand induces proteolytic cleavage of the one or more proteolytic cleavage sites in the TMD.

[0015] In some embodiments, the one or mutations in the CARD 11 polypeptide is a point mutation, a gene fusion, a substitution, a gain-of-function mutation, a stop-gain mutation, an insertion mutation, a deletion mutation, a duplication mutation or a translocation.

[0016] In some embodiments, the CARD 11 polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 10 and comprising a substitution at an amino acid position selected from the group consisting of 361, 615, 634, 655, and 357 of SEQ ID NO: 10. In some embodiments, the substitution comprises Y361C, S615F, E634K, D357N, S655C, or combinations thereof.

[0017] In some embodiments, the CARD 9 polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 66 and comprising a substitution at an amino acid position selected from the group consisting of 361, 615, 634, 655, and 357 of SEQ ID NO: 66. In some embodiments, the substitution comprises Y361C, S615F, E634K. D357N, S655C, or combinations thereof.

[0018] In some embodiments, the mutation is a fusion between a CARD 11 polypeptide and a PIK3R3 polypeptide. In some embodiments, the fusion comprises a CARD domain and a coiled coil domain from the CARD11 or CARD9 polypeptide. In some embodiments, the coiled coil domain is a truncated coiled coil domain. In some embodiments, the fusion protein further comprises an inhibitory domain from the CARD11 or CARD9 polypeptide.In some embodiments, the fusion protein further comprises an SH2 domain from PIK3R3. In some embodiments, the chimeric antigen receptor does not comprise an SH2 domain from PIK3R3, or does not comprise an SH2 domain.

[0019] In some embodiments, the chimeric receptor comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 68, 70, 72, 74, 76, or 78.

[0020] In some embodiments, the chimeric antigen receptor further comprises a VI 19E mutation of SEQ ID NO: 45.

[0021] In some embodiments, the extracellular domain comprises an antigen-binding moiety capable of binding to a ligand on the surface of a cell. In some embodiments, the cell is a pathogenic cell. In some embodiments, the cell is a human cell. In some embodiments, the human cell is a tumor cell. In some embodiments, the human cell is a terminally differentiated cell.

[0022] In some embodiments, the ligand comprises a protein or a carbohydrate.

[0023] In some embodiments, the ligand is selected from the group consisting of CD1, CDla, CDlb, CDlc, CDld. CDle, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b. CD19, CD20, CD21, CD22. CD23, CD25, CD27, CD28, CD33, CD34, CD40.CD45, CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80(B7.1), CD86 (B7.2), CD94, CD95, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, 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), Claudin 18.2, DLL3, GCC, EGFR, FGFR2, CEA, AFP, CA125, MUC-1, MAGE, alkaline phosphatase, placental-like 2 (ALPPL2), B-cell maturation antigen (BCMA), LY6G6D, CLDN6, KLK2, KLK3, EGFR806. HER2p95, ADGRE2. MUC16. green fluorescent protein (GFP). blue fluorescent protein (BFP) enhanced green fluorescent protein (EGFP), and signal regulatory protein a (SIRPa).

[0024] In some embodiments, the ligand is selected from the group consisting of CD1, CDla, CDlb, CDlc, CDld, CDle, CD2, CD3d, CD3e, CD3g, CD3s, CD4, CD5, CD7, CD8a. CD8b, CD19, CD20, CD21. CD22, CD23, CD24, CD25, CD27, CD28, CD30. CD33, CD34, CD38, CD40, CD44v6, CD45, CD46, CD47 CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD 179, CD 179a, CD181 (CXCR1), CD 182 (CXCR2). CD 183 (CXCR3), CD210, CD213A2, CD246, CD252. CD253, CD261. CD262, CD272, CD273 (PD-L2), CD274 (PD- Ll), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), CEA, CLECL1, CLL-1, CLDN6, CLDN18.2, CS1, DLL3, LY6G6D, GCC, p53R175H, PRAME, EGFR, EGFRvIII, FGFR2, AFP, CA125, MUC-1, MAGE, ALP1, alkaline phosphatase placental- like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), KLK2, KLK3, Mesothelin, lLf3Ra2, signal regulator}7protein a (SIRPa), TCRalpha, TCRbeta, TSHR, GD2, GD3, Tn Ag, cMET, Axl, R0R1, ROR2, GPC1, GPC2, GPC3, FLT3, TAG72, CEA, EPC AM, KIT (CD117), IL- 13Ra2, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, PDGFR , SSEA-4, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, STEAP1, STEAP2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, ROPN1. GPRC5D, CXORF61, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1. UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE- Al, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, p53R175H, KRAS, mutant KRAS, KRAS G12D, prostein, surviving, telomerase, PCTA-l / Galectin 8. MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NAI 7. PAX3, androgen receptor.cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2,LAIRL FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, FCRL5, IGLL1, PSMA, TROP2, citrullinated vimentin, the extracellular portion of the APRIL protein, and any combinations thereof.

[0025] In some embodiments, the ligand comprises or consists of KIR2D, NRP1, CD221. VEGFR, HGF, integrin a5bl. CD51, CD27, B7H3, NYESO1, EGFR, cMET, BRD4, EpCAM, SSX, Fibronectin EDB, or Integrin al 1 [31 .

[0026] In some embodiments, the ligand is selected from cell surface receptors, adhesion proteins, integrins, mucins, lectins, tumor-associated antigens, and tumor-specific antigens.

[0027] In some embodiments, the ligand is a tumor-associated antigen or a tumor-specific antigen.

[0028] In some embodiments, the extracellular ligand-binding domain comprises the ligand-binding portion of a receptor.

[0029] In some embodiments, the antigen-binding moiety is selected from the group consisting of an antibody, a nanobody, a diabody, a tnabody, a mimbody, an F(ab')2 fragment, an F(ab)v fragment, a single chain variable fragment (scFv), a single domain antibody (sdAb), and a functional fragment thereof. In some embodiments, the antigenbinding moiety comprises an scFv.

[0030] In some embodiments, the antigen-binding moiety specifically binds to a tumor- associated antigen selected from the group consisting of CD19, B7H3 (CD276), BCMA (CD269), ALPPL2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97. CEA, CLDN6, CLECL1, CS- 1, Claudin 18.2. DLL3. GCC, LY6G6D, CLDN6. KLK2, KLK3, EGFR806, HER2p95, ADGRE2, MUC16, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephnn B2, FAP, FLT3, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2 / neu), IGLL1, IL-l lRa, KIT (CD117), MUC1, NCAM, PAP, PDGFR-p, PRSS21, PSCA, PSMA, ROR1, SIRPa, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2. ALP1, citrullinated vimentin. cMet, and Axl.

[0031] In some embodiments, the antigen-binding moiety specifically binds to a tumor- associated antigen selected from the group consisting of CD1, CD la, CD lb, CDlc, CD Id, CDle, CD2. CD3d, CD3e, CD3g, CD3s, CD4, CD5, CD7. CD8a, CD8b, CD19, CD20, CD21. CD22, CD23, CD24, CD25. CD27, CD28, CD30, CD33. CD34, CD38, CD40. CD44v6, CD45, CD46, CD47 CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73,CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94. CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152. CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD213A2, CD246, CD252, CD253, CD261, CD262, CD272, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), CEA, CLECL1, CLL-1, CLDN6, CLDN18.2, CS1, DLL3, LY6G6D, GCC, p53R175H, PRAME, EGFR, EGFRvIII, FGFR2, AFP, CA125, MUC-1, MAGE. ALPI, alkaline phosphatase placental-like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), KLK2, KLK3, Mesothelin, IL13Ra2, signal regulatory protein a (SIRPa), TCRalpha, TCRbeta, TSHR, GD2, GD3, Tn Ag, cMET, Axl, R0R1, R0R2, GPC1. GPC2, GPC3, FLT3. TAG72, CEA, EPCAM, KIT (CD117), IL-13Ra2, IL-l lRa, PSCA, PRSS21. VEGFR2, LewisY, PDGFR0, SSEA-4, folate receptor alpha, ERBB2 (Her2 / neu), MUC 1, MUC16, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, STEAP1, STEAP2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2. folate receptor beta, TEM1 / CD248, TEM7R, R0PN1, GPRC5D. CX0RF61, ALK, Poly sialic acid, PLACE GloboH. NY-BR-1. UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE- Al, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53. p53 mutant, p53R175H, KRAS, mutant KRAS, KRAS G12D. prostein, surviving, telomerase, PCTA-l / Gal ectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp7O- 2,LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, FCRL5, IGLLL PSMA, TROP2, citrullinated vimentin, the extracellular portion of the APRIL protein, and any combinations thereof.

[0032] In some embodiments, the tumor-associated antigen is CD 19, BCMA, CEA, HER2, MUC1, CD20, ALPPL2, SIRPa, or EGFR. In some embodiments, the tumor- associated antigen is CD 19, or ALPPL2.

[0033] In some embodiments, the tumor-associated antigen is KIR2D, NRP1, CD221, VEGFR, HGF, integnn a501, CD51, CD27, B7H3, NYESO1, EGFR, cMET, BRD4, EpCAM, SSX, Fibronectin EDB, integrin al 101, DLL3. CD2. SSTR2, TROP2. KLK2, KLK3, STEAP2, STEAP1, PSMA, PSCA, LY6G6D, IL-1 a, CEA, C242 antigen, CanAg(MUC1 glycoform), CTAA16.88, UCY2C. IGF2BP3, p53R175H, NKG2D ligands, PRAME, MAGEA4, vimentin, HER2 / neu, CCR4, CCR5, ROPN1, R0R1, KK-LC-1, CA- 125, folate receptor 1, folate receptor alpha, MUC1, MUC16, MSLN, CLDN6, WT1, ALPPL2, KRASG12D, KRASG12V, CD70, MSLN, CLDN18.2, HPV E6 / E7, GPC3, IL- 13Ra, EGFRv3, EGFR806, GD2, or CA9.

[0034] In some embodiments, the tumor-associated antigen is KIR2D. NRP1, CD221, VEGFR, HGF, integrin a5bl. CD51, CD27, B7H3, NYESO1, EGFR, cMET, BRD4, EpCAM, SSX, Fibronectin EDB, or Integrin al 1 [31 .

[0035] In some embodiments, the linking polypeptide sequence comprises a hinge domain. In some embodiments, the hinge domain is capable of promoting oligomer formation of the chimeric polypeptide via intermolecular disulfide bonding. In some embodiments, the hinge domain is derived from a CD8a hinge domain, a CD28 hinge domain, a CD152 hinge domain, a PD-1 hinge domain, a CTLA4 hinge domain, an 0X40 hinge domain, an IgGl hinge domain, an IgG2 hinge domain, an IgG3 hinge domain, and an IgG4 hinge domain, or a functional variant of any thereof.

[0036] In some embodiments, the linking sequence is derived from the group selected from a CD8a hinge domain or a functional variant thereof, a CD28 hinge domain or a functional variant thereof, 0X40 hinge domain or a functional variant thereof, and an IgG4 hinge domain or a functional variant thereof.

[0037] In some embodiments, the linking sequence is derived from a CD8a hinge domain or a functional variant thereof.

[0038] In some embodiments, the linking sequence is derived from an CD28 hinge domain or a functional variant thereof.

[0039] In some embodiments, the linking sequence comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 5.

[0040] In some embodiments, the one or more ligand-inducible proteolytic cleavage sites comprises ay secretase cleavage site.

[0041] In some embodiments, the TMD comprises a polypeptide sequence having at least 80% sequence identity7to a transmembrane domain from a Type 1 transmembrane receptor and comprises a y-secretase cleavage site.

[0042] In some embodiments, the chimeric receptor further comprises a stop-transfer- sequence (STS) positioned between the TMD and the ICD.

[0043] In some embodiments, the stop-transfer-sequence comprises a sequence having at least 70% sequence identity to a STS sequence from APLP1, APLP2, APP, TGBR3, CSF1R, CXCL16, CX3CL1, DAG1, DCC, DNER, DSG2, CDH1, GHR, HLA-A, IFNAR2, IGF1R, IL1R1, ERN2, KCNE1, KCNE2, CHL1, LRP1, LRP2, PTPRF, SCN1B, SCN3B, NPR3, NGFR, PLXDC2, PAM, AGER, ROBO1, SORCS3, SORCS1, SORL1, SDC1, SDC2, SPN, TYR, TYRP1, DOT, VASN, FLT1. CDH5. PKHD1, NECTIN1, KL, IL6R, EFNB1, CD44, CLSTN1, LRP8, PCDHGC3. NRG1, LRP1B, JAG2. EFNB2, DLL1, CLSTN2, EPC AM, ErbB4, KCNE3, CDH2, NRG2, PTPRK, BTC, EPHA4, IL1R2, KCNE4, SCN2B, Nradd, PTPRM, Notchl, Notch2, Notch3, andNotch4.

[0044] In some embodiments, the signaling molecule comprises a class 1 or a class 3 human membrane protein.

[0045] In some embodiments, the signaling molecule is selected from the group consisting of CD28, ICOS, CTLA4, PD1, PD1H, BTLA, B71, B7H1, CD226, CRTAM, TIGIT, CD96, TIM1, TIM2, TIM3, TIM4, CD2, SLAM, 2B4, Lyl08, CD84, Ly9, CRACC, BTN1, BTN2, BTN3, LAIR1, LAG3, CD160, 4-1BB, 0X40, CD27, GITR, CD30, TNFR1, TNFR2. HVEM. LT_R, DR3, DCR3, FAS, CD40, RANK, OPG. TRA1LR1, TAC1, BAFFR. BCMA, TWEAKR, EDAR, XEDAR, RELT, DR6, TROY, NGFR, CD22, SIGLEC-3, SIGLEC-5, SIGLEC-7, KLRG1, NKR-P1A, ILT2, KIR2DL1, KIR3DL1, CD94-NKG2A, CD300b, CD300e, TREM1, TREM2. ILT7, ILT3, ILT4, TLT-1, CD200R, CD300a, CD300f, DC- SIGN. B7-2, Allergin-1, LAT, BLNK, LAYN, SLP76. EMB-LMP1, HIV-NEF. HVS-TIP, HVS-ORF5, and HVS-stpC.

[0046] In some embodiments, the signaling molecule is selected from the list consisting of 0X40, ICOS, 4-1BB, CTLA4, CD28, CD30, CD2, CD27, and CD226.

[0047] In some embodiments, the activation domain comprises one or more immunoreceptor tyrosine-based activation motifs (IT AMs).

[0048] In some embodiments, the one or more IT AMs are derived from CD3E CD35, and CD3e.

[0049] In some embodiments, the one or more IT AMs have at least about 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to a CD3^ IT AM.

[0050] In some embodiments, the chimeric receptor comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOS: 68, 70, 72, 74, 76, or 78.

[0051] In another aspect, provided herein are multi-chain chimeric polypeptides comprisinga first polypeptide comprising (i) an extracellular ligand-binding domain having a binding affinity for a selected ligand, (ii) a first transmembrane domain (TMD) comprising a first modified interface and (iii) a first intracellular domain comprising a caspase recruitment domain family member 11 (CARD11) polypeptide comprising one or more mutations or a caspase recruitment domain family member 9 (CARD9) polypeptide comprising one or more mutations: and (b) a second polypeptide comprising (i) a second TMD comprising a second interface and (ii) a second intracellular domain comprising a signaling domain; wherein the first and the second modified interfaces each comprises an amino acid residue having a charged side chain, wherein the amino acid residues in the first and the second modified interfaces have opposite charges, and wherein the first polypeptide is coupled to the second polypeptide via the first modified interface and the second interface and wherein binding of the selected ligand to the extracellular ligand-binding domain induces activity of the signaling domain and release of the CARD 11 or CARD9 polypeptide.

[0052] In another aspect, provided herein are multi-chain chimeric polypeptides comprising a first polypeptide comprising (i) an extracellular ligand-binding domain having a binding affinity for a selected ligand, (ii) a first transmembrane domain (TMD) comprising a first modified interface and (iii) a first intracellular domain comprising a caspase recruitment domain family member 11 (CARD11) polypeptide comprising one or more mutations or a caspase recruitment domain family member 9 (CARD9) polypeptide comprising one or more mutations; and (b) a second polypeptide comprising (i) a second TMD comprising a second interface and (ii) a second intracellular domain comprising a signaling domain; wherein the first and the second modified interfaces each comprises an amino acid residue having a charged side chain, wherein the amino acid residues in the first and the second modified interfaces have opposite charges, wherein the first polypeptide is coupled to the second polypeptide via the first modified interface and the second modified interface and wherein binding of the selected ligand to the extracellular ligand-binding domain induces activity' of the signaling domain and release of the CARD11 or CARD9 polypeptide.

[0053] In some embodiments, the first and the second polypeptides are not covalently connected.

[0054] In some embodiments, the one or mutations in the CARD 11 polypeptide is a point mutation, a gene fusion, a substitution, a gain-of-function mutation, a stop-gain mutation, an insertion mutation, a deletion mutation, a duplication mutation or a translocation.

[0055] In some embodiments, the CARD 11 polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 10 and comprising a substitution at an amino acid position selected from the group consisting of 361, 615, 634, 655, and 357 of SEQ ID NO: 10. In some embodiments, the substitution comprises Y361C, S615F, E634K, D357N, S655C, or combinations thereof.

[0056] 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 and a coiled coil domain. In some embodiments, the coiled coil domain is a truncated coiled coil domain. In some embodiments, the fusion protein further comprises an inhibitory domain.

[0057] In some embodiments, the fusion protein further comprises an SH2 domain from PIK3R3.

[0058] In some embodiments, the first polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 84. 86. 88. 90, 92, or 94.

[0059] In some embodiments, the multi-chain chimeric polypeptide further comprises a V119E mutation of SEQ ID NO: 45.

[0060] In some embodiments, binding of the selected ligand to the extracellular ligandbinding domain simultaneously induces the activity’ of the signaling domain and the release of the CARD11 polypeptide.

[0061] In some embodiments, the first polypeptide comprises (i) the extracellular ligandbinding domain, (ii) the first TMD, and (iii) the first intracellular domain, in order fromN- terminus to C-terminus of the first polypeptide.

[0062] In some embodiments, the first TMD comprises: (i) 10 to 25 contiguous valine residues, or (ii) a Notch 1 transmembrane domain.

[0063] In some embodiments, the second polypeptide comprises (i) the second TMD, and (ii) the second intracellular domain, in order from N-terminus to C-terminus of the second polypeptide.

[0064] In some embodiments, the first modified interface comprises a positively charged residue, and wherein the second interface comprises a negatively charged residue, and wherein the first polypeptide is coupled to the second polypeptide via the electrostatic force between the first and the second modified interfaces.

[0065] In some embodiments, the positively charged residue is lysine or arginine.

[0066] In some embodiments, (i) the first TMD comprises SEQ ID NO:6 or functional variants thereof and the lysine or arginine residue is at position selected from positions 10 to 14 of SEQ ID NO: 6 or (ii) the first TMD comprises SEQ ID NO: 6 and the lysine or arginine is at position selected from positions 8 to 11 of SEQ ID NO: 6.

[0067] In some embodiments, the lysine or arginine residue is (i) at position 12 of SEQ ID NO:6, or (ii) is at position 9 of SEQ ID NO:6.

[0068] In some embodiments, the extracellular domain comprises an antigen-binding moiety capable of binding to a ligand on the surface of a cell.

[0069] In some embodiments, the antigen-binding moiety is selected from the group consisting of an antibody, a nanobody, a diabody, a triabody, a minibody, anF(ab')2 fragment, an F(ab) fragment, a single chain variable fragment (scFv), a single domain antibody (sdAb), and a functional fragment thereof.

[0070] In some embodiments, the ligand comprises a protein or a carbohydrate.

[0071] In some embodiments, the ligand is a tumor-associated antigen or a tumor-specific antigen.

[0072] In some embodiments, the ligand comprises cell-surface receptors, adhesion proteins, integrins, mucins, lectins, tumor associated antigens, or tumor specific antigens.

[0073] In some embodiments, the ligand comprises CD1, CDla, CDlb, CDlc, CDld, CDle, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a. CD8b, CD19, CD20, CD21, CD22. CD23, CD25, CD27, CD28. CD33, CD34, CD40, CD45. CD48, CD52, CD59.CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, 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), EGFR, FGFR2, CEA, AFP, CA125, MUC-1, MAGE, alkaline phosphatase, placental-like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent Protein (eGFP), or signal regulator}7protein a (SIRPa).

[0074] In some embodiments, the cell is a human cell.

[0075] In some embodiments, the cell is a tumor cell.

[0076] In some embodiments, the signaling domain comprises CD3 zeta, TCR zeta, FcR y, FcRp, CD3y, CD3 , CD3s, CD5, CD22, CD79a, CD79b, CD278 (ICOS), Fee RI, DAP10, DAP 12, or CD66d, signaling domain

[0077] In some embodiments, the first polypeptide further comprises one or more of the following: a hinge domain, a ligand-inducible proteolytic cleavage site, an autoproteolyticpeptide sequence, a nuclear localization signal, a stop-transfer-sequence (STS). In some embodiments, the STS domain is a polybasic domain. In some embodiments, the polybasic domain comprises Notch-1 or Notch-2 STS domains.

[0078] In some embodiments, the autoproteolytic peptide sequence is from a porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), or a combination thereof.

[0079] In some embodiments, the first polypeptide further comprises a hinge domain from CD8, CD28, 0X40, or IgG4. In some embodiments, the hinge domain comprises a truncated CD8a hinge domain.

[0080] In some embodiments, the ligand-inducible proteolytic cleavage site is cleavable by gamma secretase.

[0081] In some embodiments, the multi-chain chimeric polypeptide is an immunoreceptor.

[0082] In some embodiments, the immunoreceptor is a chimeric antigen receptor.

[0083] In another aspect, the present disclosure provides a recombinant nucleic acid comprising a nucleotide sequence encoding the chimeric receptor or multi-chain chimeric polypeptide of the disclosure.

[0084] In some embodiments, the nucleotide sequence is incorporated into an expression cassette or an expression vector. In some embodiments, the expression vector is a viral vector. In some embodiments, the viral vector is a lentiviral vector, an adeno virus vector, an adeno-associated virus vector, or a retroviral vector.

[0085] In another aspect, the present disclosure provides a recombinant cell comprising the chimeric receptor or multi-chain chimeric polypeptide and / or the recombinant nucleic acid of the disclosure. In some embodiments, the recombinant cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some embodiments, the immune cell is a B cell, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, a cytotoxic T cell, or other T cell.

[0086] In some embodiments, the recombinant cell is selected from the group of consisting of a T cell, a CD4+ T cell, a CD8+ T cell, a regulatory T cell (Treg), a gamma delta T cell (y6T). an invariant natural killer T (iNKT) cell, a mucosal associated invariant T (MAIT) cell, a macrophage, a monocyte, a natural killer (NK) cell, a tumor infiltratinglymphocyte (TIL), a cytotoxic T cell, a T helper cell, a memory' T cell, a central memory' T (TCM) cell, a stem memory T (TSCM) cell, a stem-cell-like memory T cell (or stem-like memory T cells), an effector memory T (TEM) cell, a TEMRA (CD45RA+) cell, an effector T cell, a Thl cell, a Th2 cell, a Th9 cell, a Thl7 cell, a Th22 cell, a Tfh (follicular helper) cell, a natural killer T (NKT) cell, a transitional memory' T (TTM) cell, a terminal effector T (TTE) cell, a naive T (TN) cell, a hematopoietic stem cell, and a progenitor cell of the lymphoid lineage.

[0087] In some embodiments, the recombinant is selected from the group consisting of a T cell, a macrophage, a monocyte, and a natural killer (NK) cell.

[0088] In some embodiments, the recombinant cell is a T cell. In some embodiments, the T cell is selected from the group consisting of a regulatory T cell (Treg). a gamma delta T cell, a CD8+ T cell, an invariant iNKT cell, a MAIT cell, a CAR T cell, a tumor-infiltrating lymphocyte, and an engineered T cell comprising a transcriptional receptor.

[0089] In some embodiments, the recombinant cell is an autologous cell.

[0090] In some embodiments, the recombinant cell is an allogeneic cell.

[0091] In some embodiments, the recombinant cell is a primary cell.

[0092] In some embodiments, the recombinant cell is derived from a stem cell.

[0093] Also provided herein is a method for making the recombinant cell according to the present disclosure, comprising a) providing a cell capable of protein expression; b) contacting the provided cell with a recombinant nucleic acid of the present disclosure into the provided cell. In some embodiments, the cell is obtained by leukapheresis performed on a sample obtained from a subject, and the cell is contacted ex vivo.

[0094] In some embodiments, the recombinant nucleic acid is encapsulated in a viral capsid or a lipid nanoparticle.

[0095] Also provided herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier, and one or more of the following: the recombinant nucleic acid of the present disclosure; and the recombinant cell of the present disclosure.

[0096] In some embodiments, the composition comprises a recombinant nucleic acid according to any one of claims 83-86, and a pharmaceutically acceptable carrier.

[0097] In some embodiments, the recombinant nucleic acid is encapsulated in a viral capsid or a lipid nanoparticle.

[0098] In another aspect, provided herein is a system for modulating an activity of a cell, inhibiting a target cancer cell, or treating a health condition in an individual in need thereof, the system comprising one or more of the following: a chimeric receptor of the presentdisclosure; a multi-chain receptor of the present disclosure; a recombinant nucleic acid of the present disclosure; a recombinant cell of the present disclosure; and a pharmaceutical composition of the present disclosure.

[0099] In another aspect, provided herein is a method for modulating an activity of a cell, the method comprising: a) providing a recombinant cell of the present disclosure; and b) contacting the recombinant cell with a selected ligand, wherein binding of the selected ligand to the extracellular ligand-binding domain induces cleavage of a ligand-inducible proteolytic cleavage site and releases the CARD1 1 or CARD9 polypeptide, wherein the released CARD 11 polypeptide modulates an activity of the recombinant cell. In some embodiments, the contacting is carried out in vivo, ex vivo, or in vitro.

[0100] In some embodiments, the activity of the cell to be modulated is selected from the group consisting of expression of a selected gene, proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, secretion of a molecule, cellular adhesion, and cytolytic activity.

[0101] Also provided herein is a method for inhibiting an activity of a target cell in an individual, the method comprising administering to the individual an effective number of the recombinant cells of the present disclosure, wherein the recombinant cells inhibit an activity of the target cell in the individual.

[0102] In some embodiments, inhibiting an activity of a target cell comprises killing the target cell.

[0103] In some embodiments, the target cell is a pathogenic cell. In some embodiments, the pathogenic cell is a cancer cell.

[0104] In another aspect, provided herein is a method for the treatment of a health condition in an individual in need thereof, the method comprising administering to the individual a first therapy comprising an effective number of the recombinant cell of the present disclosure, wherein the recombinant cell treats the health condition in the individual.

[0105] In some embodiments, the health condition is cancer.

[0106] In some embodiments, the method further comprises administering to the individual a second therapy. In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, and toxin therapy.

[0107] Also provided herein is a method for enhancing the in vivo persistence of a T cell in a subject in need thereof, the method comprising administering to the individual aneffective number of the recombinant cells of the present disclosure, wherein the recombinant cells enhance the in vivo persistence of a T cell in the individual.

[0108] In some embodiments, the T cell is selected from the group consisting of a regulatory (Treg), a natural killer (NK), a tumor infiltrating lymphocyte, and a CAR T cell.

[0109] In some embodiments, the subject has cancer or an autoimmune disease.

[0110] In some embodiments, the cancer is solid tumor.

[0111] In some embodiments, the cancer is a hematological cancer.

[0112] In another aspect, provded herein is a method of simultaneously inducing T cell signaling and gene regulation in a T cell, the method comprising: (a) providing a T cell comprising the multi-chain chimeric polypeptide of the present disclosure; and (b) exposing the T cell to a selected ligand, wherein binding of the selected ligand to the extracellular ligand-binding domain simultaneously induces T cell signaling and release of the CARD11 or CARD9 polypeptide.

[0113] In a further aspect, provided herein is a method of simultaneously inducing T cell signaling and gene regulation in a T cell, the method comprising: (a) providing a vector comprising the multi-chain chimeric polypeptide of the present disclosure and (b) transducing a T cell with the vector, wherein binding of the selected ligand to the extracellular ligand-binding domain simultaneously induces intracellular signaling and release of the CARD 11 polypeptide.

[0114] In a further aspect, provided herein is a method of simultaneously inducing T cell signaling and gene regulation in a T cell, the method comprising: (a) providing a vector encoding the multi-chain chimeric polypeptide of the present disclosure and (b) transducing a T cell with the vector, and (c) providing a selected ligand to the T cell, wherein binding of the selected ligand to the extracellular ligand-binding domain of the multi-chain chimeric polypeptide expressed by the T cell simultaneously induces T cell signaling and release of the CARD1 1 or CARD9 polypeptide.

[0115] The foregoing summary' is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0116] FIG. l is a schematic of CAR-CARD11 constructs containing CARD11 domains within the CAR construct. Second-generation CAR constructs containing an scFvrecognizing an antigen of interest, a CD8 hinge / transmembrane domain, 41BB costimulatory domain, and CD3zeta (CD3z) domains were fused with CARD11 sequences of varying lengths, some CARD 11 sequences contained known hypermorphic point mutations (V119E), and containing various functional protein domains of CARD11, or with varying co-stimulatory domains other than 4 IBB.

[0117] FIG. 2 shows CAR-CARD 11 receptors promote antigen-dependent signaling in Jurkat cells. Jurkat cells were transduced with the indicated anti-CD19 CAR-CARD 11 receptor and AP-1 reporter activity was determined. Jurkat cells were co-cultured overnight with an approximately 1: 1 ratio of the indicated cell types, either K562 cells (lacking antigen) or K562-19 (cells overexpressing CD19). Data is represented as mean + / - SEM.

[0118] FIG. 3 shows CAR-CARD 11 receptors express in primary human T cells. Human T cells obtained from a healthy donor were lentivirally transduced with CAR-CARD 11 receptors or with a BBz-CAR as a control. Expression of the CAR was evaluated by staining a FLAG tag on the N-terminal extracellular region of the CAR.

[0119] FIG. 4 shows primary T cells expressing a CAR-CARD 11 receptor can kill tumor cells in vitro. Human T cells obtained from a healthy donor were lentivirally transduced with a CAR-CARD 1 1 receptor or with a BBz-CAR and BBz-CAR with overexpression of the CARD11-PIK3R3 fusion protein as a control. CD 19 overexpressing A549 cells were seeded and co-cultured with CAR-T cells, and A549 target cell count was determined by live cell imaging. Fold change of target cell count from time zero is displayed.

[0120] FIGs. 5A-5B show primary mouse CD8+ T cells expressing a CAR-CARD 11 receptor display improved expansion to CAR, but not T cell receptor, triggering. Primary mouse T cells were either untransduced, transduced with a second-generation ALPPL2 CAR, a CAR-CARD 11 targeting ALPPL2 (FIG. 5A), or an ALPPL2-CAR and CARD11- PIK3R3 fusion protein. The number of CD8+ T cells 48 hours after the indicated conditions (media alone, ALPPL2 expressing K562 cells, or anti-CD3 / CD28 Dynabeads) were then determined by flow7cytometry (FIG. 5B).

[0121] FIG. 6 shows primary mouse CD8+ T cells expressing a CAR-CARD 11 receptor display enhanced target cell killing. Primary mouse T cells were either untransduced, transduced with a second-generation ALPPL2 CAR, or a CAR-CARD 11 and cultured with A549 cells expressing ALPPL2, and target cell count was determined by live cell imaging.

[0122] FIG. 7 shows a schematic of multi-chain receptor format for CAR-CARD 11 receptors. DAP12-based 41BB / CD3zeta chain (BBz) and anti-CD19 scFv CARD11 containing CAR construct in multi -chain architecture.

[0123] FIG. 8 shows multi-chain CAR-CARD11 receptors promote antigen-dependent signaling in Jurkat cells. Jurkat cells were transduced with the indicated anti-CD19 scFv based CAR chain in combination with DAP12-BBz chain, and co-cultured overnight with Jurkat cells expressing CD19. NF-kB reporter activity was determined by flow cytometry. Each data points represents a biological replicate.

[0124] FIGs. 9A-9C shows multi-chain CAR-CARD11 receptors can be expressed in primary human T cells and display enhanced IL-2 secretion. FIG. 9A is a schematic of the tested multi -chain architectures, where GAL4 is employed as a neutral control not expected to enhance function. FIG. 9B show s expression in primary human T cells of receptor (based on Myc or FLAG tag staining) and of the DAP 12 chain (based on mCherry fluorescent protein expression). FIG. 9C shows IL-2 production of multi-chain architecture cells as determined by ELISA. DAP12-BBz and anti-CD19-GAL4 receptor double positive cells were sorted and co-cultured with 1: 1 ratio of CD 19 expressing K562 cells. Supernatants were taken for analysis 48 hours post co-culture. Data points represent technical replicates.

[0125] In the following detailed description, reference is made to the Figures, which form a part hereof. In the Figures, similar symbols generally identify similar components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, draw ings, and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, and designed in a wide variety of different configurations, all of which are explicitly contemplated and make part of this application.DETAILED DESCRIPTION OF THE DISCLOSURE

[0126] In general, the present disclosure relates, inter alia, to compositions and methods for improving adoptive T cell therapy. The inventors have discovered ways to improve the therapeutic efficacy of T cells by altering T cell signaling, decreasing T cell exhaustion and / or by enhancing in vivo persistence and fitness of engineered T cells.

[0127] In particular, some embodiments of the disclosure provide exemplary chimeric receptors that incorporate (i) costimulatory domains and stimulatory domains of a CAR, for example, the cytoplasmic tail of the CD3zeta chain, and a CARD 11 polypeptide having one or more mutations. The architecture of the cytoplasmic tail of these new receptors(costimulatory domain, CD3zeta, CARD 11) can be configured in multiple ways. As described in greater detail below, the present disclosure also identifies multi-chain receptor architectures containing a CARD11 polypeptide that allow more control on where the signaling motifs are located. Additionally, as described in greater detail below, the present disclosure further identifies additional receptor architectures (e.g., “hinge notch"’, “Fn Notch”) that can be engineered to contain the CARD11 polypeptide. The new CARs provided herein can simultaneously stimulate (i) fast time-scale (e.g., from seconds to minutes) proximal signaling and (ii) ligand-dependent activation of a CARD1 1 polypeptide so as to reduce any toxicity effects associated with TCR stimulation of the CARD11 motif while also enhancing cytokine production and elimination of tumors.

[0128] Although various features of the disclosures may be described in the context of a single embodiment, the features may also be provided separately or in any suitable combination. Conversely, although the disclosures may be described herein in the context of separate embodiments for clarity7, the disclosures may also be implemented in a single embodiment. Any published patent applications and any other published references, documents, manuscripts, and scientific literature cited herein are incorporated herein by reference for any purpose. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0129] In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols generally identify similar components, unless context dictates otherwise. The illustrative alternatives described in the detailed description, drawings, and claims are not meant to be limiting. Other alternatives may be used and other changes may be made without departing from the spirit or scope of the subject matter presented here. It will be readily understood that the aspects, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, and designed in a wide variety7of different configurations, all of which are explicitly contemplated and make part of this application.DEFINITIONS

[0130] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent asubstantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0131] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, including mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B.”

[0132] The terms “administration” and “administering”, as used herein, refer to the delivery7of a bioactive composition or formulation by an administration route including, but not limited to, oral, intravenous, intra-arterial, intramuscular, intraperitoneal, subcutaneous, intramuscular, and topical administration, or combinations thereof. The term includes, but is not limited to, administering by a medical professional and self-administering.

[0133] The term “heterologous” refers to a polypeptide sequence or domain which is not native to a flanking sequence, e.g., wherein the heterologous sequence is not found in nature coupled to the polypeptide sequences occurring at one or both ends.

[0134] The term “derived from” as used herein in reference to a protein or polypeptide refers to an origin or source, and may include naturally occurring, recombinant, unpurified or purified polypeptide that is obtained from, is obtained based on a source or original protein or polypeptide. As such, a protein or polypeptide derived from an original protein or polypeptide may include the original protein or polypeptide, in part or in whole, and may be a fragment or variant of the original protein or polypeptide. In some instance, the polypeptide sequence or domain that is derived from a source or origin can be genetically or chemically modified.

[0135] The terms “host cell” and “recombinant cell” are used interchangeably herein. It is understood that such terms, as well as “cell”, “cell culture”, “cell line”, refer not only to the particular subject cell or cell line but also to the progeny or potential progeny of such a cell or cell line, w ithout regard to the number of transfers. It should be understood that not all progeny are exactly identical to the parental cell. This is because certain modifications may occur in succeeding generations due to either mutation (e.g., deliberate or inadvertent mutations) or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein, so long as the progeny retain the same functionality as that of the originally cell or cell line.

[0136] The term "operably linked”,” as used herein, denotes a physical or functional linkage between two or more elements, e.g., polypeptide sequences or polynucleotide sequences, which permits them to operate in their intended fashion.

[0137] The term “percent identity,” as used herein in the context of two or more nucleic acids or proteins, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acids that are the same (e.g., about 60% sequence identity. 65%. 70%. 75%. 80%. 85%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%, 98%, 99%, or higher identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. See. e.g., the NCBI web site at ncbi.nlm.nih.gov / BLAST. Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the complement of a test sequence. This definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. Sequence identity can be calculated over a region that is at least about 20 amino acids or nucleotides in length, or over a region that is 10-100 amino acids or nucleotides in length, or over the entire length of a given sequence. Sequence identity can be calculated using published techniques and widely available computer programs, such as the GCS program package (Devereux et al, Nucleic Acids Res. 12:387, 1984), BLASTP, BLASTN. FASTA (Atschul et al., J Mol Biol 215:403. 1990). Sequence identity can be measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group at the University’ of Wisconsin Biotechnology Center (1710 University' Avenue, Madison, Wis. 53705), with the default parameters thereof.

[0138] As used herein, and unless otherwise specified, a “therapeutically effective amount” of an agent is an amount sufficient to provide a therapeutic benefit in the treatment or management of a health condition, such as a disease (e.g., a cancer), or to delay or minimize one or more symptoms associated with the cancer. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapeutic agents, which provides a therapeutic benefit in the treatment or management of the cancer. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids sy mptoms or causes of the cancer, or enhances therapeutic efficacy of another therapeutic agent. An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of asymptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see. e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 2010); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (2016); Pickar, Dosage Calculations (2012); and Remington: The Science and Practice of Pharmacy, 22nd Edition, 2012, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0139] As used herein, a “subject” or an “individual” includes animals, such as human (e.g., human individuals) and non-human animals. In some embodiments, a “subject” or “individual” is an individual under the care of a physician. Thus, the subject can be a human individual or an individual who has, is at risk of having, or is suspected of having a disease of interest (e.g., cancer) and / or one or more symptoms of the disease. The subject can also be an individual who is diagnosed with a risk of the condition of interest at the time of diagnosis or later. The term “non-human animals” includes all vertebrates, e.g., mammals, e g., rodents, e.g., mice, and non- mammals, such as non-human primates, e.g., sheep, dogs, cows, chickens, amphibians, reptiles, and the like.

[0140] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0141] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0142] As will be understood by one having ordinary skill in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as ‘'up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0143] It is understood that aspects and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments. As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or step.

[0144] Headings, e.g., (a), (b), (i) etc., are presented merely for ease of reading the specification and claims. The use of headings in the specification or claims does not require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented.

[0145] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosureare specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub- combination was individually and explicitly disclosed herein.Notch Receptors

[0146] Notch receptors are large transmembrane proteins that normally communicate signals upon binding to surface-bound ligands expressed on adjacent cells. Notch signals rely on cell- to-cell communication, e.g., communication between two contacting cells, in which one contacting cell is a “receiver” cell and the other contacting cell is a “sender” cell. Notch receptors expressed in a receiver cell recognize their ligands (the delta / serrate / lag, or “DSL” family of proteins) expressed on a sending cell. The engagement of notch and delta on these contacting cells leads to a two-step proteolysis of the notch receptor, which ultimately causes the release of the intracellular portion of the receptor (“ICD”) from the membrane into the cytoplasm. Notch has a matrix metalloprotease cleavage site (denoted “S2”). which, when the receptor is not activated is protected from cleavage by the Notch negative regulatory region (“NRR”). The NRR consists of three LIN-12-Notch repeat (“LNR”) modules and a heterodimerization domain (“HD”). It is believed that this proteolysis is regulated by the force exerted by the sending cell: the DSL ligand pulls on the Notch receptor, which changes the conformation of the NRR and exposes the metalloprotease site. This is cleaved by a constitutively active protease (such as ADAM 10), which releases the extracellular binding portion and negative regulatory region of the receptor. Release of the ligand binding portion of the receptor in turn exposes another cleavage site (denoted “S3”), which is cleaved by y-secretase within the cell membrane: this cleavage releases the nuclear homing ICD from the cell membrane. W.R. Gordon et al., Dev Cell (2015) 33:729-36. This released domain alters receiver cell behavior by regulating transcription. Evolutionary divergence of vertebrates and invertebrates was accompanied by at least two rounds of gene duplication in the Notch lineage: flies possess a single Notch gene, worms two (GLP-1 and LIN-12), and mammals four (NOTCH1-4). Transduction of Notch signals relies on three key events: (i) ligand recognition; (ii) conformational exposure of the ligand-dependent cleavage site; and (iii) assembly of nuclear transcriptional activation complexes.

[0147] Canonical Notch signals are transduced by a process called regulated intramembrane proteolysis. Notch receptors are normally maintained in a resting,proteolytically resistant conformation on the cell surface, but ligand binding initiates a proteolytic cascade that releases the intracellular domain of the receptor (ICD) from the membrane. The critical, regulated cleavage step is affected by ADAM metalloproteases and occurs at a site called S2 immediately external to the plasma membrane. This truncated receptor, dubbed NEXT (for Notch extracellular truncation), remains membrane-tethered until it is processed at site S3 by y-secretase, a multiprotein enzyme complex.

[0148] After y-secretase cleavage, the ICD ultimately enters the nucleus, where it nucleates assembly of a transcriptional activation complex that contains a DNA-binding transcription factor, and a transcriptional coactivator of the Mastermind family. This complex then engages one or more additional coactivator proteins such as p300 to recruit the basal transcription machinery and activate the expression of downstream target genes.

[0149] Notch receptors have a modular domain organization. The ectodomains of Notch receptors consist of a series of N-terminal epidermal growth factor (EGF)-like repeats that are responsible for ligand binding. O-linked glycosylation of these EGF repeats, including modification by O-fucose, Fringe, and Rumi glycosyltransferases, also modulates the activity of Notch receptors in response to different ligand subtypes in flies and mammals.

[0150] The EGF repeats are followed by three LIN-12 / Notch repeat (LNR) modules, which are unique to Notch receptors, and are widely reported to participate in preventing premature receptor activation. The heterodimerization (HD) domain of Notchl is divided by furin cleavage, so that its N-terminal part terminates the extracellular subunit, and its C- terminal half constitutes the beginning of the transmembrane subunit. Following the extracellular region, the receptor has a transmembrane segment and an intracellular domain (ICD), which includes a transcriptional regulator.CARD11-BCL10-MALT Signalsome

[0151] Formation of a CARD 11-BCL10-MALT (CBM) signaling complex is a key event in T- and B cell receptor-induced gene expression. After exposure to distinct immune triggers, these molecules form self-organizing fdaments with MALT1 protease activity to regulate canonical nuclear factor-KB (NF-KB) and mitogen-activated protein kinase (MAPK) signalling pathways and the degradation of mRNA-binding proteins, which provides two layers of control of inflammatory gene expression. Deregulation of CARD11, BCL10 or MALT1 expression or CBM signaling have been associated with cancer, immunodeficiency, and autoimmunity (J. Ruland, L. Hartjes, CARD-BCL-10-MALT1 signalling in protective and pathological immunity. Nat Rev Immunol 19, 118-134 (2019).

[0152] In normal T cells, T cell receptor (TCR) signaling activates PKC0, which in turn promotes the assembly of the CARD11-BCL10-MALT1 (CBM) signalosome. The CBM complex subsequently has three major outputs: NF-KB transcriptional activity, AP-1 transcriptional activity and MALT1 proteolytic activity. The assembly of the CARD1 1- BCL10-MALT1 signalosome complex is an essential step in regulating NF-KB in lymphoid immune cells.

[0153] An inhibitory domain present in CARD 11 may allow for intramolecular autoinhibition which can prevent CARD11 binding to BCL10 in the absence of upstream signals. Upon phosphorylation of the inhibitory domain in normal T cells, CARD11 autoinhibition can be relieved and the CARD11 protein can oligomerize and promote prionlike assembly of the CBM complex with recruitment of BCL10-MALT1 filaments, which then can allow for CBM complex signaling.

[0154] Genes involved in CBM signaling include but are not limited to caspase recruitment domain family member 11 (CARD11), capping protein regulator and myosin 1 linker 2 (CARMIL2), mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), B-cell lymphoma 10 (BCL10). CARD1 1 can be involved in both the innate and adaptive immune systems. CARD1 1 is implicated in the activation of NF-kB by the TCR complex.

[0155] Previous work has identified a novel fusion protein (CARD11-PIK3R3) that enhances CARD11-BCL10-MALT1 signaling to improve CAR-T cell function. CARD11- BCL10-MALT1 (CBM) signaling is a critical pathway in T cells that mediates the activation of NF-KB and AP-1 transcriptional activity. Upon TCR activation, CARD11 forms a complex with the scaffolding protein BCL10 and the protease MALT1, which activates downstream signaling cascades resulting in NF-KB and AP-1 activation. This signaling pathway is essential for T cell activation, proliferation, and differentiation. Alterations in the CBM pathway have been associated with lymphoma, immunodeficiency, and autoimmune disorders.COMPOSITIONS OF THE DISCLOSUREChineric Receptors

[0156] The present disclosure provides, among other things, a new class of chimeric receptors designed to combine fast time-scale intracellular signal transduction and enhanced CARD1 1-BCL10-MALT1 signaling in a controlled manner. In particular, someembodiments of the disclosure provide new receptor architectures that incorporate signaling domains (e.g. co-stimulation, CD3zeta, etc.) also containing a CARD11 polypeptide that can initiate activation of T cells concomitant with enhanced CARD11-BCL10-MALT1 signaling by a CARD11 polypeptide. In some embodiments, activation of the domain comprising CARD 11 polypeptide is not stimulated by TCR activation. In some embodiments, the new receptors provided herein have linear amino acid signaling motif to mediate signaling in T cells added into the cytoplasmic tail of the receptors. As demonstrated in the Examples and figures, these new receptors have enhanced functionality, and improved specificity of functionality in response to CAR and not to T cell receptor signaling as compared to constitutive CARD11-PIK3R3 expression. In the receptors described herein, it is believed that binding of antigen to this chimeric receptor results in a force exerted by the sending cell, which changes the conformation of the receptor and exposes a metalloprotease site. This is cleaved by a constitutively active protease (such as ADAMI 0), which releases the extracellular binding portion and negative regulatory region of the receptor. Release of the ligand binding portion of the receptor in turn exposes another cleavage site within the transmembrane domain of the receptor, which is cleaved by y-secretase. This cleavage releases the intracellular signaling domain comprising a domain from CARDI E

[0157] In one aspect, the present disclosure provides chimeric receptor comprising from N-terminus to C-terminus an extracellular binding domain (ECD) having a binding affinity for a selected ligand; a linking sequence; a transmembrane domain (TMD) having at least about 80% sequence identity to the transmembrane domain of a Type 1 transmembrane receptor and comprising one or more ligand-inducible proteolytic cleavage sites; and an intracellular domain (ICD) comprising, in any order: (i) an intracellular signaling domain (SD) comprising at least one costimulatory domain derived from a signaling molecule and an activation domain, and (ii) a caspase recruitment domain family member 11 (CARD11) polypeptide comprising one or more mutations. In some embodiments, the binding of the selected ligand to the extracellular ligand-binding domain induces cleavage at a ligandinducible proteolytic cleavage site disposed between the intracellular domain and the linking polypeptide. Further, the binding of the selected ligand to the extracellular ligand-binding domain can also induce proximal signaling cascades through the intracellular SD. In some embodiments, the proximal signaling cascades refer to fast time-scale signaling. For instance, the signaling cascades can be induced in seconds to minutes. Alternatively, the signaling cascades can last for seconds to minutes. In some embodiments, such proximal signaling cascades are induced through T-cell receptor costimulatory signals. In someembodiments, the chimeric receptor provided herein does not comprise a LIN-12-Notch repeat (LNR) and / or a heterodimerization domain (HD) of a Notch receptor. In some embodiments, the linking polypeptide is capable of promoting oligomer formation of the chimeric receptor via intermolecular disulfide bonding. In some embodiments, the chimeric receptor of the disclosure completely lacks the NRR of a Notch receptor. In some embodiments, the chimeric receptor of the disclosure comprises neither a LNR nor a HD of a Notch receptor.Extracellular domains (ECD) and ligands

[0158] In some embodiments, the ECD of the chimeric receptors disclosed herein has a binding affinity for one or more target ligands. The target ligand can be expressed on the surface of a cell, or is otherwise anchored, immobilized, or restrained so that it can exert a mechanical force on the chimeric receptor. The cell can be a pathogenic cell or a human cell. In some embodiments, the human cell can be a tumor cell. In some embodiments, the human cell can be a terminally differentiated cell. As such, without being bound to any particular theory, binding of the ECD of a chimeric receptor provided herein to a cell-surface ligand does not necessarily remove the target ligand from the target cell surface, but instead enacts a mechanical pulling force on the chimeric receptor. For example, an otherwise soluble ligand may be targeted if it is bound to a surface, or to a molecule in the extracellular matrix. In some embodiments, the target ligand is a cell-surface ligand. Non-limiting examples of suitable ligand types include cell surface receptors; adhesion proteins; carbohydrates, lipids, glycolipids, lipoproteins, and lipopolysaccharides that are surface-bound; integrins; mucins; and lectins. In some embodiments, the ligand is a protein. In some embodiments, the ligand is a carbohydrate.

[0159] In some embodiments, the ligand is a cluster of differentiation (CD) marker. In some embodiments, the CD marker is selected from the group consisting of CD1, CD la, CDlb, CDlc, CDld, CDle, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD33, CD34, CD40, CD45, CD48. CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD134, CD140 (PDGFR4). CD152, CD154, CD158. CD178, 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), EGFR, FGFR2, CEA, AFP, CA125, MUC-1, and MAGE.

[0160] In some embodiments, the extracellular domain includes the ligand-binding portion of a receptor. In some embodiments, the extracellular domain includes an antigen-bindingmoiety that binds to one or more target antigens. In some embodiments, the antigen-binding moiety includes one or more antigen-binding determinants of an antibody or a functional antigen-binding fragment thereof. One skilled in the art upon reading the present disclosure will readily understand that the term “functional fragment thereof’ or “functional variant thereof’ refers to a molecule having quantitative and / or qualitative biological activity in common with the wild-type molecule from which the fragment or variant was derived. For example, a functional fragment or a functional variant of an antibody is one which retains essentially the same ability to bind to the same epitope as the antibody from which the functional fragment or functional variant was derived. For instance, an antibody capable of binding to an epitope of a cell surface receptor may be truncated at the N-terminus and / or C- terminus, and the retention of its epitope binding activity assessed using assays known to those of skill in the art. In some embodiments, the antigen-binding moiety is selected from the group consisting of an antibody, a nanobody, a diabody, a triabody, or a minibody, an F(ab’)2 fragment, an F(ab) fragment, a single chain variable fragment (scFv), and a single domain antibody (sdAb), or a functional fragment thereof. In some embodiments, the antigen-binding moiety includes an scFv.

[0161] The antigen-binding moiety can include naturally-occurring amino acid sequences or can be engineered, designed, or modified to provide desired and / or improved properties such as, e.g., binding affinity. Generally, the binding affinity of an antigen-binding moiety, e.g., an antibody, for a target antigen (e.g., CD 19 antigen) can be calculated by the Scatchard method described by Frankel et al., Mol. Immunol, 16: 101-06, 1979. In some embodiments, binding affinity is measured by an antigen / antibody dissociation rate. In some embodiments, binding affinity is measured by a competition radioimmunoassay. In some embodiments, binding affinity is measured by ELISA. In some embodiments, antibody affinity is measured by flow cytometry. An antibody that “selectively binds” an antigen (such as CD19) is an antigen-binding moiety that does not significantly bind other antigens but binds the antigen with high affinity, e.g., with an equilibrium dissociation constant (KD) of 100 nM or less, such as 60 nM or less, for example, 30 nM or less, such as. 15 nM or less, or 10 nM or less, or 5 nM or less, or 1 nM or less, or 500 pM or less, or 400 pM or less, or 300 pM or less, or 200 pM or less, or 100 pM or less. In some embodiments, the the ECD selectively binds to its selected ligand.

[0162] A skilled artisan can select an ECD based on the desired localization or function of a cell that is genetically modified to express a chimeric receptor or hybrid SynNotch CAR of the present disclosure. For example, a chimeric receptor or hybrid SynNotch CAR with anECD including an antibody specific for a HER2 antigen can target cells to HER2-expressing breast cancer cells. In some embodiments, the ECD of the disclosed CAR is capable of binding a tumor-associated antigen (TAA) or a tumor-specific antigen (TS A). A skill artisan will understand that TAAs include a molecule, such as e.g., protein, present on tumor cells and on normal cells, or on many normal cells, but at much lower concentration than on tumor cells. In contrast, TSAs generally include a molecule, such as e.g., protein which is present on tumor cells but absent from normal cells.

[0163] In some cases, the antigen-binding moiety is specific for an epitope present in an antigen that is expressed by a tumor cell, i.e., a tumor-associated antigen. The tumor- associated antigen can be an antigen associated with, e.g., a breast cancer cell, a B cell lymphoma, a pancreatic cancer, a Hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell, a non-Hodgkin B-cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a myelogenous leukemia cell, a neuroblastoma cell, a glioma, a glioblastoma, a colorectal cancer cell. etc. It will also be understood that a tumor-associated antigen may also be expressed by a non- cancerous cell. In some embodiments, the antigen-binding domain is specific for an epitope present in a tissue-specific antigen. In some embodiments, the antigen-binding domain is specific for an epitope present in a disease-associated antigen.

[0164] Non-limiting examples of suitable target antigens include CD19. B7H3 (CD276), BCMA (CD269), alkaline phosphatase, placental-like 2 (ALPPL2), green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), signal regulatory protein a (SIRPa), CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38. CD44v6, CD46. CD71, CD97, CEA, CLDN6, CLECL1, CS-1. DLL3, Claudin 18.2, GCC, LY6g6D, CLDN6, KLK2, KLK3, EGFR806, HER2p95, ADGRE2, MUC16, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephrin B2, FAP, FLT3, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2 / neu), IGLL1, IL-l lRa, KIT (CD 117), MUC1, NCAM, PAP, PDGFR-£, PRSS21, PSCA, PSMA, ROR1, SSEA-4, TAG72. TEM1 / CD248. TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, and Axl.

[0165] In some embodiments, the target antigen is selected from CD19, B7H3 (CD276), BCMA (CD269), ALPPL2, 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, Ephnn B2, FAP, FLT3, GD2,GD3, GM3, GPRC5D, HER2 (ERBB2 / neu), IGLL1, IL-l lRa, KIT (CD117), MUC1, NCAM. PAP. PDGFR-p. PRSS21, PSCA, PSMA. R0R1, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, Axl, GPC2, human epidermal growth factor receptor 2 (Her2 / neu), CD276 (B7H3), IL-13Ral, IL-13Ra2, a- fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD123, CD93, CD99, CD 117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), ALK, DLK1. FAP. NY-ESO, WT1, HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, A0C3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 ( a subunit of the heterodimeric IL-2 receptor), CD3, CD4, CD5, IFN-a, IFN-y, IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL- 17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin cc4, integrin a4[37, LFA-1 (CDl la), myostatin, OX-40, scleroscin, SOST, TGFpi, TNF-a, VEGF-A, pyruvate kinase isoenzyme type M2 (tumor M2-PK), CD20, CD5, CD7, CD3, TRBC1, TRBC2. BCMA, CD38, CD123, CD93, CD34, CDla, SLAMF7 / CS1, FLT3, CD33, CD123, T ALLA-1, CSPG4, DLL3, Kappa light chain, Lamba light chain, CD16 / FcyRIII, CD64, FITC, CD22, CD27. CD30, CD70, GD2 (ganglioside G2), GD3, EGFRvIII (epidermal grow th factor variant III), EGFR and isovariants thereof, TEM-8. sperm protein 17 (Spl7), mesothelin.

[0166] Further non-limiting examples of suitable antigens include PAP (prostatic acid phosphatase), prostate stem cell antigen (PSCA), prostein, NKG2D, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of the prostate 1). an abnormal ras protein, an abnormal p53 protein, integrin P3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), Ral-B, GPC2, CD276 (B7H3), or IL-13Ra. In some embodiments, the antigen is Her2. In some embodiments, the antigen is ALPPL2. In some embodiments, the antigen is BCMA. In some embodiments, the antigen-binding moiety of the ECD is specific for a reporter protein, such as BFP, GFP, and eGFP. Non-limiting examples of such antigen binding moiety include a LaG17 anti -GFP nanobody. In some embodiments, the antigen-binding moiety of the ECDincludes an anti-BCMA fully -humanized VH domain (FHVH). In some embodiments, the antigen is signal regulatory protein a (SIRPa).

[0167] Additional antigens suitable for targeting by the chimeric receptors disclosed herein include, but are not limited to GPC2, human epidermal growth factor receptor 2 (Her2 / neu), CD276 (B7H3), IL-13Ral, IL-13Ra2, a-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA). Other suitable target antigens include, but are not limited to, tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD123, CD93, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15). ALK, DLK1, FAP, NY-ESO. WT1. HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin (MSA), neurofilament, neuronspecific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor- 1.

[0168] In some embodiments, the antigen-binding moiety specifically binds to a tumor- associated antigen selected from the group consisting of CD1, CDla, CDlb, CDlc, CDld, CDle, CD2, CD3d, CD3e, CD3g, CD3s, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21. CD22, CD23, CD24, CD25, CD27, CD28, CD30, CD33, CD34, CD38, CD40. CD44v6, CD45, CD46, CD47 CD48. CD52, CD59, CD66, CD70, CD71, CD72, CD73. CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD213A2, CD246, CD252, CD253, CD261, CD262, CD272, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1). CD340 (HER2), CEA. CLECL1, CLL-1, CLDN6, CLDN18.2, CS1, DLL3, LY6G6D, GCC, p53R175H, PRAME, EGFR, EGFRvIII, FGFR2, AFP, CA125, MUC-1 , MAGE, ALPI, alkaline phosphatase placental-like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), KLK2, KLK3, Mesothelin, IL13Ra2. signal regulatory protein a (SIRPa), TCRalpha, TCRbeta, TSHR, GD2, GD3, Tn Ag, cMET, Axl, R0R1, ROR2, GPC 1, GPC2, GPC3, FLT3, TAG72, CEA, EPCAM, KIT (CD117), IL-13Ra2, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, PDGFR , SSEA-4, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, NCAM, prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2. STEAP1. STEAP2, fucosyl GM1. sLe, GM3, TGS5,HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, R0PN1, GPRC5D. CXORF61, ALK, Polysialic acid, PLAC1, GloboH. NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE- Al, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, p53R175H, KRAS, mutant KRAS, KRAS G12D, prostein, surviving, telomerase, PCTA-l / Gal ectin 8, MelanA / MARTl. Ras mutant, hTERT. sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp7O- 2,LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, FCRL5, IGLL1, PSMA, TROP2, citrullinated vimentin, the extracellular portion of the APRIL protein, and any combinations thereof.In some embodiments, the tumor-associated antigen is KIR2D, NRP1, CD221, VEGFR, HGF, integrin a501, CD51, CD27, B7H3, NYESO1, EGFR, cMET, BRD4, EpCAM, SSX, Fibronectin EDB. integrin al 101. DLL3. CD2. SSTR2, TROP2. KLK2. KLK3. STEAP2. STEAP1, PSMA, PSCA, LY6G6D, IL-1 a, CEA, C242 antigen, CanAg (MUC1 glycoform), CTAA16.88, UCY2C, IGF2BP3, p53R175H, NKG2D ligands, PRAME, MAGEA4, vimentin, HER2 / neu, CCR4, CCR5, R0PN1, RORL KK-LC-1, CA-125, folate receptor 1, folate receptor alpha, MUC1, MUC16, MSLN. CLDN6, WTL ALPPL2, KRASG12D, KRASG12V, CD70, MSLN, CLDN18.2, HPV E6 / E7, GPC3, IL-13Ra, EGFRv3, EGFR806, GD2, or CA9.In some embodiments, the tumor-associated antigen is KIR2D, NRP1, CD221, VEGFR, HGF, integrin a5bl, CD51, CD27, B7H3, NYESO1, EGFR, cMET, BRIM, EpCAM, SSX, Fibronectin EDB, or Integrin al 101.

[0169] Additional antigens suitable for targeting by the chimeric receptors disclosed herein include, but are not limited to, those associated with an inflammatory' disease such as, AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor). CD25 (a subunit of the heteromeric of IL-2 receptor), CD3, CD4, CD5, IFN-a, IFN-y, IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL- 17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6. IL-6 receptor, integrin a4, integrin a407, LFA-1 (CDl la). myostatin, OX-40, scleroscin, SOST, TGF0L TNF-a. and VEGF-A.

[0170] Further antigens suitable for targeting by the chimeric receptors disclosed herein include, but are not limited to the pyruvate kinase isoenzyme type M2 (tumor M2-PK), CD20, CD5, CD7, CD3, TRBC1 , TRBC2, BCMA, CD38, CD123, CD93, CD34, CDla, SLAMF7 / CS1, FLT3, CD33, CD123, TALLA-1, CSPG4, DLL3, Kappa light chain, Lamba light chain, CD16 / FcyRIII, CD64, FITC. CD22, CD27, CD30, CD70, GD2 (ganglioside G2). GD3, EGFRvIII (epidermal growth factor variant III), EGFR and isovariants thereof, TEM-8, sperm protein 17 (Spl7), mesothelin. Further non-limiting examples of suitable antigens include PAP (prostatic acid phosphatase), prostate stem cell antigen (PSCA), prostein, NKG2D, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), an abnormal ras protein, an abnormal p53 protein, integrin P3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), and Ral-B. In some embodiments, the antigen is GPC2, CD19, Her2 / neu, CD276 (B7H3), IL-13Ral, or IL-13Ra2. In some embodiments, the antigen is Her2. In some embodiments, the antigen is ALPPL2. In some embodiments, the antigen is BCMA. In some embodiments, the antigen-binding moiety of the ECD is specific for a reporter protein, such as GFP and eGFP. Non-limiting examples of such antigen binding moiety include a LaG17 anti-GFP nanobody. In some embodiments, the antigen-binding moiety of the ECD includes an anti-BCMA fully -humanized VH domain (FHVH).

[0171] In some embodiments, antigens suitable for targeting by the chimeric receptors disclosed herein include ligands derived from a pathogen. For example, the antigen can be HER2 produced by HER2 -positive breast cancer cells. In some embodiments, the antigen can be CD 19 that is expressed on B-cell leukemia. In some embodiments, the antigen can be EGFR that is expressed on glioblastoma multiform (GBM) but much less expressed so on healthy CNS tissue. In some embodiments, the antigen can be CEA that is associated with cancer in adults, for example colon cancer.

[0172] In some embodiments, the antigen-binding moiety of the ECD is specific for a cell surface target, where non-limiting examples of cell surface targets include CD 19, CD30, Her2, CD22, ENPP3, EGFR, CD20, CD52, CD1 la, and a-integrin. In some embodiments, the chimeric receptors disclosed herein include an extracellular domain having an antigenbinding moiety that binds CD 19, CEA, HER2, MUC1, CD20, ALPPL2, BCMA, or EGFR. In some embodiments, the chimeric receptors provided herein include an extracellular domain including an antigen-binding moiety that binds CD19. In some embodiments, the chimeric receptors provided herein include an extracellular domain including an antigen-binding moiety that binds ALPPL2. In some embodiments, the chimeric receptors provided herein include an extracellular domain including an antigen-binding moiety that binds BCMA. In some embodiments, the chimeric receptors provided herein include an extracellular domain including an antigen-binding moiety that binds Her2. In some embodiments, the chimeric receptors and hybrid SynNotch CARs disclosed herein include an extracellular domain including an antigen-binding moiety that binds CD 19. ALPPL2, BCMA. or Her2.

[0173] In some embodiments, the extracellular ligand-binding domain has a binding affinity for a ligand selected from the group consisting of CD1, CDla, CDlb, CDlc, CDld, CDle, CD2. CD3d, CD3e, CD3g, CD3a, CD4, CD5, CD7. CD8a, CD8b, CD19, CD20, CD21. CD22, CD23, CD24, CD25. CD27, CD28, CD30, CD33. CD34, CD38, CD40. CD44v6, CD45, CD46, CD47 CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2). CD183 (CXCR3), CD210, CD213A2, CD246, CD252, CD253, CD261, CD262. CD272. CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3). CD279, CD295, CD339 (JAG1), CD340 (HER2), CEA, CLECL1, CLL-1, CLDN6, CLDN18 2, CS1, DLL3, LY6G6D, GCC, p53R175H, PRAME, EGFR, EGFRvIII, FGFR2, AFP, CA125, MUC-1, MAGE, ALPI, alkaline phosphatase placental-like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), KLK2, KLK3, Mesothelin, IL13Ra2, signal regulatory protein a (SIRPa), TCRalpha, TCRbeta, TSHR, GD2, GD3, Tn Ag, cMET, Axl, ROR1, ROR2, GPC1, GPC2, GPC3, FLT3, TAG72, CEA, EPCAM, KIT (CD117), IL-13Ra2, IL-l lRa, PSCA, PRSS21, VEGFR2, LewisY, PDGFRp. SSEA-4. folate receptor alpha, ERBB2 (Her2 / neu), MUC1. MUC16, NCAM, prostase, PAP, ELF2M, Ephnn B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, STEAP1, STEAP2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, ROPN1, GPRC5D, CXORF61, ALK, Polysialic acid, PLAC1, GloboH. NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3. GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE- Al, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, p53R175H, KRAS, mutant KRAS, KRAS G12D, prostein, surviving, telomerase, PCTA-l / Gal ectin 8, MelanA / MARTl. Ras mutant, hTERT. sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC,TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp7O- 2,LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, FCRL5, IGLLL PSMA, TROP2, citrullinated vimentin, the extracellular portion of the APRIL protein, and any combinations thereof.

[0174] In some embodiments, the extracellular ligand-binding domain has a binding affinity for a ligand that comprises or consists of KIR2D, NRP1. CD221, VEGFR, HGF, integrin a501, CD51, CD27, B7H3, NYESO1, EGFR, cMET, BRD4, EpCAM, SSX, Fibronectin EDB, integrin al 101, DLL3, CD2, SSTR2, TROP2, KLK2, KLK3, STEAP2, STEAP1, PSMA, PSCA, LY6G6D, IL-1 a, CEA, C242 antigen, CanAg (MUC1 glycoform), CTAA16.88, UCY2C, IGF2BP3. p53R175H, NKG2D ligands, PRAME, MAGEA4, vimentin, HER2 / neu, CCR4, CCR5, ROPN1, ROR1, KK-LC-1, CA-125, folate receptor 1, folate receptor alpha, MUC 1, MUC16, MSLN, CLDN6, WT1, ALPPL2, KRASG12D, KRASG12V, CD70, MSLN, CLDN18.2, HPV E6 / E7, GPC3, IL-13Ra, EGFRv3, EGFR806, GD2, or CA9.In some embodiments, the ligand comprises or consists of KJR2D. NRPL CD221, VEGFR. HGF, integrin a5bl, CD51, CD27, B7H3, NYESO1, EGFR, cMET, BRIM, EpCAM, SSX, Fibronectin EDB, or Integrin al 101.

[0175] In some embodiments, the extracellular domain includes an amino acid sequence having at least 70%, at least 75%. at least 80%. at least 85%. at least 90%. at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to a sequence set forth in SEQ ID NOS: 1-4. In some embodiments, the extracellular domain includes an amino acid sequence having at least 90% sequence identity to a sequence set forth in SEQ ID NOS: 1-4. In some embodiments, the extracellular domain includes an amino acid sequence having at least 95% sequence identity to a sequence set forth in SEQ ID NOS: 1-4. In some embodiments, the extracellular domain includes an amino acid sequence having 100% sequence identity to a sequence set forth in SEQ ID NOS: 1-4. In some embodiments, the extracellular domain includes an amino acid sequence set forth in SEQ ID NOS: 1-4, wherein one, two. three, four, or five of the amino acid residues in any one of the SEQ ID NOS: 1-4 is / are substituted by a different amino acid residue.Linking polypeptide

[0176] As described above, the chimeric receptors of the disclosure include a linking polypeptide sequence disposed between the extracellular binding domain (ECD) and the transmembrane domain (TMD).

[0177] In some embodiments, the chimeric receptors of the disclosure can be "S vnNotch" receptors which comprise a heterologous extracellular ligand binding domain, a linking polypeptide having substantial sequence identity with a Notch receptor JMD (e.g., including the NRR), a TMD, and an ICD. In some embodiments, the chimeric receptors of the disclosure can be “Fn Notch” receptors which comprise a heterologous extracellular ligand binding domain, a linking polypeptide having substantial sequence identity with a Robo receptor (such as a mammalian Robol. Robo2. Robo3. or Robo4). followed by 1, 2. or 3 fibronectin repeats ('‘Fn”), a TMD, and an ICD. In some embodiments, the chimeric receptors of the disclosure can be “Mini Notch” receptors which comprise a heterologous extracellular ligand binding domain, a linking polypeptide having substantial sequence identity with a Notch receptor JMD but lacking the NRR (which consists of the LIN- 12- Notch repeat (LNR) modules and the heterodimerization domain (HD)), a TMD, and an ICD. In some embodiments, the chimeric receptors of the disclosure can be “Minimal Linker Notch” receptors which comprise a heterologous extracellular ligand binding domain, a linking polypeptide lacking substantial sequence identity with a Notch receptor (for example, without limitation, having a synthetic (GGS)npolypeptide sequence), a TMD. and an ICD. In some embodiments, the chimeric receptors of the disclosure can be “Hinge Notch” receptors which comprise a heterologous extracellular ligand binding domain, a hinge sequence comprising an oligomerization domain (i.e., a domain that promotes dimerization, trimerization, or higher order multimerization with a synthetic receptor and / or an existing host receptor), a TMD, and an ICD.

[0178] In the Mini Notch receptors, the linking polypeptide is derived from a Notch JMD sequence after deletion of the NRR and HD domain. The Notch JMD sequence may be the sequence from Notchl, Notch2, Notch3, or Notch4, and can be derived from a non-human homolog, such as those from Drosophila, Gallus, Danio, and the like. Four to 50 amino acid residues of the remaining Notch sequence (e.g., 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, amino acid residues) can be used as a polypeptide linker. In some embodiments, the length and amino acid composition of the linker polypeptide sequence are varied to alter the orientation and / or proximity of the ECD and the TMD relative to one another to achieve a desired activity of the chimeric polypeptide, such as the signal transduction level when ligand induced or in the absence of ligand.

[0179] In the Minimal Linker Notch receptors, the linking polypeptide does not have substantial sequence identity to a Notch JMD sequence, including the Notch JMD sequence from Notchl, Notch2, Notch3, or Notch4, or a non-human homolog thereof. Four to 50amino acid residues (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, amino acid residues) can be used as a polypeptide linker. In some embodiments, the length and amino acid composition of the linker polypeptide sequence are varied to alter the orientation and / or proximity of the ECD and the TMD relative to one another to achieve a desired activity of the chimeric polypeptide of the disclosure. The Minimal Linker sequence can be designed to include or omit a protease cleavage site and can include or omit a glycosylation site or sites for other types of post-translational modification. In some embodiments, the Minimal Linker linker does not comprise a protease cleavage site or a glysosylation site.

[0180] In some embodiments, the linking polypeptide has substantial sequence identity with aNotch receptor JMD but lacks the NRR (which consists of the LIN-12-Notch repeat (LNR) modules and the heterodimerization domain), a TMD, and an ICD. Stated differently, in some embodiments, in the receptors of the present disclosure, the linking polypeptide replaces the negative regulatory region (NRR) and heterodimerization (HD) domain of the native Notch. Three to 50 amino acid residues (e.g., 3, 4. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14. 15. 16, 17, 18, 19. 20. etc. amino acid residues) can be used as a polypeptide linker. In some embodiments, the length and amino acid composition of the linker polypeptide sequence can be optimized to vary the orientation and / or proximity’ of the ECD and the TMD relative to one another to achieve a desired activity of the chimeric receptor of the disclosure. All of these sequences can be used as a linking polypeptide for the chimeric receptors of the present disclosure.

[0181] In some embodiments, the linking polypeptide sequence includes a sequence having at least 80% sequence identity, such as, at least 80%, at least 85%, at least 90%, at least 95%. at least 96%. at least 97%, at least 98%, or 99% sequence identity to a sequence set forth in SEQ ID NO: 3. In some embodiments, the linking polypeptide sequence includes an amino acid sequence having at least 90% sequence identity to a sequence set forth in SEQ ID NO: 3. In some embodiments, the linking polypeptide sequence includes an amino acid sequence having at least 95% sequence identity to a sequence set forth in SEQ ID NO: 3. In some embodiments, the linking polypeptide sequence includes an amino acid sequence having at least 99% sequence identity’ to a sequence set forth in SEQ ID NO: 3. In some embodiments, the linking polypeptide sequence includes an amino acid sequence identical to a sequence set forth in SEQ ID NO: 3. In some embodiments, the linking polypeptide sequence includes an amino acid sequence set forth in SEQ ID NO: 3, wherein one, two,three, four, or five of the amino acid residues in any one of the SEQ ID NO: 3 is / are substituted by a different amino acid residue.

[0182] In the Hinge Notch receptors, hinge linkers of the disclosure can include an oligomerization domain (e.g., a hinge domain) containing one or more polypeptide motifs that promote oligomer formation of the chimeric polypeptides via intermolecular disulfide bonding. In these instances, within the chimeric Notch receptors disclosed herein, the hinge domain generally includes a flexible polypeptide connector region disposed between the ECD and the TMD. Thus, the hinge domain provides flexibility between the ECD and TMD and also provides sites for intermolecular disulfide bonding between two or more chimeric polypeptide monomers to form an oligomeric complex. In some embodiments, the hinge domain includes motifs that promote dimer formation of the chimeric polypeptides disclosed herein. In some embodiments, the hinge domain includes motifs that promote trimer formation of the chimeric polypeptides disclosed herein (e.g., a hinge domain derived from 0X40). Hinge polypeptide sequences suitable for the compositions and methods of the disclosure can be naturally-occurring hinge polypeptide sequences (e.g., those from naturally-occurring immunoglobulins) or can be engineered, designed, or modified so as to provide desired and / or improved properties, e g., modulating transcription. Suitable hinge polypeptide sequences include, but are not limited to, those derived from IgA, IgD, and IgG subclasses, such as IgGl hinge domain, IgG2 hinge domain, IgG3 hinge domain, and IgG4 hinge domain, or a functional variant thereof. In some embodiments, the hinge polypeptide sequence contains one or more CXXC motifs. In some embodiments, the hinge polypeptide sequence contains one or more CPPC motifs. Additional information in this regard can be found in, for example, a recent review by G. Vidarsson et al., Frontiers Immunol (2014) 5:520 (doi: 10.3389 / fimmu.2014.00520), which is hereby incorporated by reference in its entirety.

[0183] Hinge polypeptide sequences can also be derived from a CD8a hinge domain, a CD28 hinge domain, a CD152 hinge domain, a PD-1 hinge domain, a CTLA4 hinge domain, an 0X40 hinge domain, and functional variants thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from a CD8a hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from a CD28 hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an 0X40 hinge domain or a functional variant thereof. In some embodiments, the hingedomain includes a hinge polypeptide sequence derived from an IgG4 hinge domain or a functional variant thereof.

[0184] The Hinge linker can include about 5 to about 60 amino acids from or overlapping with the selected hinge domain, for example at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 12, at least about 15, at least about 17, at least about 20, at least about 22, at least about 24, at least about 26, at least about 28, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, or , at least about 60 amino acids. In embodiments of the invention, the Hinge linker has no more than about 60 amino acids, less than about 55, less than about 50, less than about 45, less than about 40, less than about 35, less than about 32, less than about 30, less than about 29, less than about 28, less than about 27. less than about 26, less than about 25, less than about 24, less than about 23, less than about 22, less than about 21, less than about 20, less than about 18, less than about 16, less than about 14, less than about 12, or less than about 10 amino acids.

[0185] In the Fn Notch receptors, the Fn Notch linking polypeptide is derived from the Robot JMD, which contains a fibronectin repeat (Fn) domain, with a short polypeptide sequence between the Fn repeats and the TMD. The Fn Notch linking polypeptide does not contain a Notch negative regulatory' region (NRR), or the Notch HD domain. The Fn linking polypeptide can contain 1, 2, 3, 4, or 5 Fn repeats. In some embodiments, the chimeric receptor comprises a Fn linking polypeptide having about 1 to about 5 Fn repeats, about 1 to about 3 Fn repeats, or about 2 to about 3 Fn repeats. The short polypeptide sequence between the Fn repeats and the TMD can be from about 2 to about 30 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17. 18, 19, 20, or more, amino acid residues). In some embodiments, the short polypeptide sequence can be between about 5 and about 20 amino acids, of any sequence. In some embodiments, the short polypeptide sequence can be between about 5 and about 20 naturally-occurring amino acids, of any sequence. In some embodiments, the short polypeptide sequence can be between about 5 and about 20 amino acids, of any sequence but having no more than one proline. In some embodiments, the short polypeptide sequence can be between about 5 and about 20 amino acids, and about 50% or more of the amino acids are glycine. In some embodiments, the short polypeptide sequence can be between about 5 and about 20 amino acids, where the amino acids are selected from glycine, serine, threonine, and alanine. In some embodiments, the length and amino acid composition of the Fn linking polypeptide sequence can be variedto alter the orientation and / or proximity of the ECD and the TMD relative to one another to achieve a desired activity of the chimeric polypeptide of the disclosure.

[0186] In some embodiments, the linker polypeptide sequence includes a sequence having at least 80% sequence identity, such as, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 47-65 in the Sequence Listing. In some embodiments, the linker polypeptide sequence includes an amino acid sequence having at least 90% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 47-65. In some embodiments, the linker polypeptide sequence includes an amino acid sequence having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NOS: 47-65. In some embodiments, the linker polypeptide sequence includes an amino acid sequence having at least 100% sequence identity to a sequence selected from the group consisting of SEQ ID NOs: 47-65. In some embodiments, the linker polypeptide sequence includes an amino acid sequence having a sequence selected from the group consisting of SEQ ID NOs: 47-65, wherein one, two, three, four, or five of the amino acid residues in any one of the SEQ ID NOs: 47-65 is / are substituted by a different ammo acid residue.Transmembrane domains (TMD)

[0187] As described above, the chimeric receptors of the disclosure include a TMD comprising one or more ligand-inducible proteolytic cleavage sites.

[0188] Examples of proteolytic cleavage sites in a Notch receptor (e.g., S2 or S3) are as described above. Additional proteolytic cleavage sites suitable for the compositions and methods disclosed herein include, but are not limited to, a metalloproteinase cleavage site for a MMP selected from collagenase- 1, -2, and -3 (MMP-L - 8, and -13), gelatinase A and B (MMP-2 and -9), stromelysin 1, 2, and 3 (MMP-3, -10, and -11), matrilysin (MMP-7), and membrane metalloproteinases (MT 1 -MMP and MT2-MMP). For example, the cleavage sequence of MMP-9 is Pro-X-X-Hy (wherein, X represents an arbitrary residue; Hy, a hydrophobic residue such as Leu. He. Vai, Phe. Trp, Tyr. Vai, Met, and Pro), e.g.. Pro-X-X- Hy-(Ser / Thr), e.g., Pro-Leu / Gln-Gly-Met-Thr-Ser or Pro-Leu / Gln-Gly-Met-Thr. Another example of a suitable protease cleavage site is a plasminogen activator cleavage site, e.g., a urokinase plasminogen activator (uPA) or a tissue plasminogen activator (tPA) cleavage site. Another example of a suitable protease cleavage site is a prolactin cleavage site. Specific examples of cleavage sequences of uPA and tPA include sequences comprising Val-Gly-Arg. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is a tobacco etch virus (TEV) protease cleavage site, e.g., Glu-Asn-Leu-Tyr- Thr-Gln-Ser, where the protease cleaves between the glutamine and the serine. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is an enterokinase cleavage site, e.g., Asp-Asp-Asp-Asp-Lys, where cleavage occurs after the lysine residue. Another example of a protease cleavage site that can be included in a proteolytically cleavable linker is a thrombin cleavage site, e.g., Leu-Val-Pro-Arg. Additional suitable linkers comprising protease cleavage sites include sequences cleavable by the following proteases: a PreScission™ protease (a fusion protein comprising human rhinovirus 3C protease and glutathione-S-transferase), a thrombin, cathepsin B, Epstein-Barr virus protease, MMP -3 (stromelysin), MMP-7 (matrilysin), MMP-9; thermolysin-like MMP, matrix metalloproteinase 2 (MMP -2), cathepsin L; cathepsin D, matrix metalloproteinase 1 (MMP-1), urokinase-type plasminogen activator, membrane type 1 matrix metalloproteinase (MT-MMP), stromelysin 3 (or MMP-11), thermolysin, fibroblast collagenase and stromelysin- 1. matrix metalloproteinase 13 (collagenase-3), tissue-type plasminogen activator(tPA), human prostate-specific antigen, kallikrein (hK.3), neutrophil elastase, and calpain (calcium activated neutral protease). Proteases that are not native to the host cell in which the receptor is expressed (for example, TEV) can be used as a further regulatory mechanism, in which activation of the receptor is reduced until the protease is expressed or otherwise provided. Additionally, a protease may be tumor-associated or disease-associated (expressed to a significantly higher degree than in normal tissue), and serve as an independent regulatory mechanism. For example, some matrix metalloproteases are highly expressed in certain cancer types.

[0189] Generally, the TMD suitable for the chimeric receptors disclosed herein can be any transmembrane domain of a Type 1 transmembrane receptor including at least one y- secretase cleavage site. Detailed description of the structure and function of the y-secretase complex as well as its substrate proteins, including amyloid precursor protein (APP) and Notch, can, for example, be found in a recent review by Zhang et al., Frontiers Cell Neurosci (2014). Non-limiting suitable TMDs from Type 1 transmembrane receptors include those from CLSTN1, CLSTN2, APLP1, APLP2, LRP8, APP, BTC, TGBR3, SPN, CD44, CSF1R, CXCL16, CX3CL1, DCC, DLL1, DSG2, DAG1, CDH1, EPCAM, EPHA4, EPHB2, EFNB1, EFNB2, ErbB4, GHR, HL A- A, and IFNAR2, wherein the TMD includes at least one y-secretase cleavage site. Additional TMDs suitable for the compositions and methods described herein include, but are not limited to, transmembrane domains from Type 1transmembrane receptors IL1R1, IL1R2, IL6R. INSR, ERN1, ERN2, JAG2, KCNE1, KCNE2, KCNE3. KCNE4, KL, CHL1. PTPRF, SCN1B, SCN3B, NPR3, NGFR, PLXDC2, PAM, AGER, R0B01, SORCS3, SORCS1, SORL1, SDC 1, SDC2, SPN, TYR, TYRP1, DCT, VASN, FLT1, CDH5, PKHD1, NECTIN1, PCDHGC3, NRG1, LRP1B, CDH2, NRG2, PTPRK, SCN2B, Nradd, and PTPRM. In some embodiments, the TMD of the chimeric receptors of the disclosure is a TMD derived from the TMD of a member of the calsyntenin family, such as. alcadein alpha and alcadein gamma. In some embodiments, the TMD of the chimeric receptors of the disclosure is a TMD known for Notch receptors. In some embodiments, the TMD of the chimeric receptors of the disclosure is a TMD derived from a different Notch receptor. For example, in a Mini Notch based on human Notchl, the Notchl TMD can be substituted with a Notch2 TMD, Notch3 TMD. Notch4 TMD, or a Notch TMD from a non-human animal such as Danio rerio. Drosophila melanogaster, Xenopus laevis, or Gallus gallus.

[0190] In some embodiments, the amino acid substitution(s) within the TMD includes one or more substitutions within a ‘"GV” motif of the TMD. In some embodiments, at least one of such substitution(s) comprises a substitution to alanine. For example, one, two. three, four, five, or more of the amino acid residues of the sequence FMYVAAAAFVLLFFVGCGVLL (SEQ ID NO: 6) may be substituted by a different amino acid residue. In some embodiments, the amino acid residue at position 18 and / or 19 of the "GV" motif within SEQ ID NO: 4 is substituted by a different amino acid residue. In some embodiments, the glycine residue at position 18 of SEQ ID NO: 6 is substituted by a different amino acid residue. In some embodiments, the valine residue at position 19 of SEQ ID NO: 6 is substituted by a different amino acid residue. In some embodiments, the transmembrane domain comprises an amino acid sequence having a sequence corresponding to SEQ ID NO: 6 with a mutation at the position corresponding to position 18 of SEQ ID NO: 6, such as G18A mutations. In some embodiments, the transmembrane domain comprises an amino acid sequence having a sequence corresponding to SEQ ID NO: 6 with a mutation at the position corresponding to position 19 of SEQ ID NO: 6, such as V19A mutations. The TMD can be derived from but longer or shorter than SEQ ID NO: 6. For instance, the TMD can be one, two, three, four, or more amino acids longer or shorter than SEQ ID NO: 6. In some embodiments, the TMD includes a sequence having at least 80% sequence identity, such as, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity to SEQ ID NO: 6.Stop-transfer sequence (STS)

[0191] The chimeric receptors of the disclosure include an STS which comprises a charged, hydrophilic domain located between the TMD and the ICD. Without being bound to any particular theory, this domain disposed between the TMD and the ICD prevents the ICD from entering the plasma membrane. In some embodiments, a single-chain peptide comprising about 1 to about 40 amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16. 17. 18, 19, 20, or more amino acid residues) in which most of the residues have charged side chains under physiological conditions can be used as a STS. In short STS embodiments (e.g., less than about 6 amino acids), about 5 or 6 of the amino acids will have charged side chains. In some embodiments, the STS includes about 1 to 15, about 5 to 20, about 8 to 25, about 10 to 30, about 12 to 35, about 14 to 40, about 5 to 40, about 10 to 35, about 15 to 30, about 20 to 25, about 20 to 40, about 10 to 30, about 4 to 20. or about 5 to 25 amino acid residues. In some embodiments, the STS includes about 4 to 10, about 5 to 12, about 6 to 14, about 7 to 18, about 8 to 20, about 9 to 22, about 10 to 24, or about 11 to 26 amino acid residues. In some embodiments, the STS includes about 4 to 10 residues, such as. 4, 5, 6, 7, 8, 9, or 10 amino acid residues.

[0192] In some embodiments, the STS includes a sequence having at least about 80% sequence identity, such as, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity to the STS domain of a Type 1 receptor. In some embodiments, the STS includes an amino acid sequence having at least 90% sequence identity to the STS domain of a Type 1 receptor. In some embodiments, the STS includes a sequence having at least 70% sequence identity', such as, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity to a STS sequence from APLP1, APLP2, APP, TGBR3, CSF1R, CXCL16, CX3CL1, DAG1, DCC, DNER, DSG2, CDH1, GHR. HLA-A, IFNAR2, IGF1R, IL1R1, ERN2, KCNE1, KCNE2, CHL1, LRP1, LRP2, PTPRF, SCN1B, SCN3B, NPR3, NGFR, PLXDC2, PAM, AGER, ROBO1, SORCS3, SORCS1, SORL1, SDC1, SDC2, SPN, TYR, TYRP1, DOT, VASN, FLT1, CDH5, PKHD1, NECTIN1, KL, IL6R, EFNB1, CD44, CLSTN1, LRP8, PCDHGC3, NRG1. LRP1B, JAG2, EFNB2, DLL1, CLSTN2, EPCAM. ErbB4, KCNE3, CDH2, NRG2, PTPRK, BTC, EPHA4, IL1R2, KCNE4, SCN2B, Nradd, PTPRM, Notchl, Notch2, Notch3, and Notch4. In some embodiments, the STS includes a sequence comprising only Lys (K) or Arg (R) in the first 4 residues. In some embodiments, the STS includes one, two, three, four, five, or more basic residues. In some embodiments, the STS includes five, four, three, two, one. or zero aromatic residues or residues with hydrophobic and / or bulky side chains.

[0193] In some embodiments, the STS includes a sequence having at least 80% sequence identity, such as, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%. at least 97%, at least 98%, or 99% sequence identity to KRKRKH (SEQ ID NO: 7). In some embodiments, the STS includes an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7. The STS can be derived from but longer or shorter than SEQ ID NO: 7. For instance, the STS can be one, two, three, four, or more amino acids longer or shorter than SEQ ID NO: 7. In some embodiments, the STS includes a sequence having at least 80% sequence identity, such as, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity to SEQ ID NO: 7. In some embodiments, the STS includes an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 7. In some embodiments, the STS includes an amino acid sequence having at least 100% sequence identity to SEQ ID NO: 7. In some embodiments, the STS includes the amino acid sequence of SEQ ID NO: 7, wherein one, two, three, four, or five of the amino acid residues in SEQ ID NO: 7 is / are substituted by a different amino acid residue.Intracellular Domain (ICD)

[0194] The chimeric receptors of the disclosure includes an intracellular domain (ICD) comprising, in any order: (i) an intracellular signaling domain (SD) comprising at least one costimulatory domain derived from a signaling molecule and an activation domain, and (ii) a caspase recruitment domain family member 11 (CARD1 1) polypeptide comprising one or more mutations or a caspase recruitment domain family member 9 (CARD9) polypeptide comprising one or more mutations. In other words, the ICD of the chimeric receptors of the disclosure can have at least three distinct domains, as depicted in FIG. 1. The three distinct domains can be arranged in specific orders and can be operably linked to one another via one or more linkers. In some embodiments, the three distinct domains are linked via (GS)n linkers. The n can be any number selected from 1 to 100. For example, the n can be 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other embodiments, the n can be 11, 12, 13, 14, 15, 16, 17, 18. 19. or 20. An exemplary GS linker can have 4 GS repeats and the sequence of GSGSGSGS (SEQ ID NO: 8). One skilled in the art would know how to modify the linker to suit specific uses.

[0195] In some embodiments, the intracellular domain of the chimeric receptors of the disclosure further comprises an intracellular signaling domain. The intracellular signaling domain can have at least two distinct domains: at least one costimulatory domain and an activation domain.

[0196] In some embodiments, the costimulatory domain comprises a sequence that is derived from a signaling molecule. The signaling molecule can be a protein selected from a class 1 or a class 3 human membrane protein. In some embodiments, the signaling molecule is selected from CD28, ICOS, CTLA4, PD1, PD1H, BTLA, B71, B7H1, CD226, CRTAM, TIGIT, CD96, TIM1, TIM2, TIM3, TIM4, CD2, SLAM, 2B4, Lyl08, CD84, Ly9, CRACC, BTN1, BTN2. BTN3, LAIR1, LAG3, CD160, 4-1BB, 0X40, CD27, GITR, CD30, TNFR1, TNFR2. HVEM, LT R. DR3, DCR3, FAS, CD40. RANK, OPG, TRAILR1, TACI.BAFFR, BCMA, TWEAKR, ED AR, XEDAR, RELT, DR6, TROY, NGFR, CD22, SIGLEC-3, SIGLEC-5, SIGLEC-7, KLRGI, NKR-P1A, ILT2, KIR2DL1, KIR3DL1, CD94-NKG2A, CD300b, CD300e, TREM1, TREM2, ILT7, ILT3, ILT4, TLT-1, CD200R, CD300a, CD300L DC-SIGN. B7-2, Allergin-1, LAT, BLNK, LAYN, SLP76. EMB-LMP1, HIV-NEF, HVS-TIP, HVS-0RF5, and HVS-stpC, and derivatives, mutants, variants, fragments and combinations thereof. In other embodiments, the signaling molecule is selected from the list consisting of 0X40, ICOS, 4-1BB, CTLA4, CD28, CD30, CD2, CD27. and CD226, and derivatives, mutants, variants, fragments and combinations thereof. In some embodiments, the signaling molecule is selected from the group consisting of 4- 1BB, BAFF-R, BCMA, BTLA, CD2, CD200R, CD244, CD28, CD300a, CD300f, CD40, CD7, CD72, CD96, CRACC, CRTAM, CTLA4, CXADR, DC-SIGN, GITR, HAVCR2, ICOS, ILT2, ILT3, ILT4, KIR2DL1, KIR3DL1, KLRGI, LAG3, LAIR1, NKG2D, NKR- P1A, NTB-A. PD1, Siglec-3, TACI, TIGIT, TLT-1, and TNR8 (CD30), and derivatives, mutants, variants, fragments and combinations thereof. In other embodiments, the signaling molecule is CD28 or 4-1BB. In one exemplary embodiment, the costimulatory domain comprises a sequence that is derived from CD28. In another exemplary' embodiment, the costimulatory domain comprises a sequence that is derived from 4-1BB. In another embodiment, the costimulatory domain comprises one of the CD28 signaling motifs appended to the C terminus of a trunc41BB costimulatory domain.

[0197] In some embodiments, the activation domain includes one or more conserv ed amino acid motifs that serve as substrates for phosphorylation such as. for example, immunoreceptor tyrosine-based activation motifs (IT AMs). In some embodiments, the activation domain includes at least 1, at least 2, at least 3, at least 4, or at least 5 specific tyrosine-based motifs selected from IT AM motifs, an ITIM motifs, or related intracellular motifs that serve as a substrate for phosphorylation. In some embodiments of the disclosure, the activation domain of the intracellular signaling domain includes at least 1, at least 2. at least 3, at least 4, or at least 5 IT AMs. Generally, any activation domain including an IT AMcan be suitably used for the construction of the chimeric receptor s as described herein. An IT AM generally includes a conserved protein motif that is often present in the tail portion of signaling molecules expressed in many immune cells. The motif may include two repeats of the amino acid sequence YxxL / I separated by 6-8 amino acids, wherein each x is independently any amino acid, producing the conserved motif YxxL / Ix(6-8)YxxL / I. IT AMs within signaling molecules are important for signal transduction within the cell, which is mediated at least in part by phosphorylation of tyrosine residues in the ITAM following activation of the signaling molecule. IT AMs may also function as docking sites for other proteins involved in signaling pathways

[0198] In some embodiments, the activation domain is derived from CD3^, CD35, and CD3E. For instance, in some embodiments, the IT AMs are derived from CD3^, CD3o, CD3 / , and CD3E. In one exemplary embodiment, the ITAM is derived from CD3^. In certain embodiments, the ITAM comprises a sequence that is at least about 80, 85, 90, 95, 96, 97, 98, 99, or 100% identical to a CD3^ ITAM. In some embodiments, the activation domain comprises at least 1, at least 2, at least 3, at least 4, or at least 5 IT AMs independently selected from the IT AMs derived from CD3C,, FcRy, and combinations thereof. In some embodiments, the activation domain comprises a CD3^ ITAM.

[0199] In some embodiments, the intracellular domain of the chimeric receptors of the disclosure further comprises a caspase recruitment domain family member 11 (CARD11) polypeptide comprising one or more mutations.

[0200] In some embodiments, the intracellular domain of the chimeric receptors of the disclosure further comprises a caspase recruitment domain family member 9 (CARD9) polypeptide comprising one or more mutations.

[0201] In some embodiments, the CARD11 polypeptide comprises 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. The 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.

[0202] In some embodiments, the nucleic acid constructs of the disclosure encode a polypeptide a sequence having at least 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: 10 and comprising a substitution at an amino acid position 361. 615, 634. 655, 357 or combinations thereof. In some embodiments, the substitution is Y361C, S615F, E634K, S655C and / or D357N. In some embodiments the nucleic acidconstructs of the disclosure encode a polypeptide a sequence having at least 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: 19 and compnsing at least one, two or more substitutions at an amino acid position 361, 615, 634, 655, 357. In some embodiments, the nucleic acid constructs encode a polypeptide having E634K and S655C mutations. In some embodiments, the recombinant nucleic acid construct encodes a substitution selected from the group consisting of: Y361C. S615F. E634K, D357N, S655C. and any combination thereof.

[0203] In some embodiments, the nucleic acid constructs of the disclosure encode a polypeptide a sequence having at least 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: 66 and comprising a substitution at an amino acid position 361, 615, 634, 655, 357 or combinations thereof. In some embodiments, the substitution is Y361C, S615F, E634K, S655C and / or D357N. In some embodiments the nucleic acid constructs of the disclosure encode a polypeptide a sequence having at least 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: 66 and compnsing at least one, two or more substitutions at an amino acid position 361, 615, 634, 655, 357. In some embodiments, the nucleic acid constructs encode a polypeptide having E634K and S655C mutations. In some embodiments, the recombinant nucleic acid construct encodes a substitution selected from the group consisting of: Y361C, S615F, E634K, D357N, S655C, and any combination thereof.

[0204] In some embodiments, the mutation is a fusion between a CARD11 poly peptide and a PIK3R3 polypeptide or any truncations thereof. In some embodiments, the nucleic acid constructs include a CARD1 1-PIK3R3 fusion polypeptide or truncations thereof including an amino acid sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOS: 11-28. In some embodiments, the truncations are encoded by a nucleic acid sequence having at least 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: 29-44.

[0205] In some embodiments, the CARD11-PIK3R3 fusion polypeptide includes a CARD domain, a coiled-coil domain, and an SH2 domain from PIK.3R3.

[0206] In some embodiments of the recombinant nucleic acid constructs of the disclosure, includes a CARD11-PIK3R3 fusion polypeptide encoded by a nucleic acid sequence having at least 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: 29-44.

[0207] In some embodiments of the recombinant nucleic acid constructs of the disclosure, the polypeptide includes a first polypeptide encoding a partial CARD 11 polypeptide and a second polypeptide encoding a partial PIK3R3 polypeptide. In some embodiments, the first polypeptide includes an amino acid sequence having at least 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: 45. In s some embodiments, the second polypeptide includes an amino acid sequence having at least 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: 46.Additional Domains

[0208] In some embodiments, the chimeric receptors provided herein can further include an additional region or domain. For example, in some embodiments, the extracellular domains located N-terminally to the TMD can include a membrane localization signal such as a CD8A signal. In other embodiments, the chimeric receptors can include a detectable label, such as a myc tag or His tag, and the like. In additional embodiments, the chimeric receptors provided herein can also include a tumor-specific cleavage site, or a diseasespecific cleavage site. In further embodiments, the chimeric receptors provided herein can include a combination of these additional regions.

[0209] In some embodiments, the chimeric receptor of the disclosure includes an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to a chimeric receptor disclosed herein. In some embodiments, provided herein are chimeric receptors including an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 68. 70, 'll, 74, 76, or 78.Multi-Chain Chimeric Receptors

[0210] The disclosure also provides, inter alia, multi-chain chimeric polypeptides having two distinct polypeptides (chains) such that a CARD 11 polypeptide comprising one or moremutations or a CARD9 polypeptide comprising one or more mutations is located on the first polypeptide close to the membrane and a signaling domain is located on the second polypeptide. The first polypeptide and the second polypeptide can associate to allow, in a ligand dependent manner, the simultaneous modulation of signaling (e.g., T-cell signaling) from one chain and the release of the CARD11 (or CARD9) polypeptide, upon binding of a select ligand to one of the polypeptides.

[0211] Particularly, the disclosure relates to multi-chain chimeric polypeptides with distinct polypeptide chains that can associate post-translationally via residues located within the TMD of the first and second polypeptide. Ligand binding to the extracellular ligand binding domain (ECD) of the first polypeptide, can activate cell signaling on the second DAP12 polypeptide and induce cleavage release of a CARD11 / CARD9 polypeptide from the first polypeptide. The cleaved CARD11 / CARD9 polypeptide can function, therefore, free of any other fused signaling domain and vice versa.

[0212] In some embodiments, the multi-chain chimeric poly peptides presented herein can have the following unique architecture in which the first polypeptide may include (i) an extracellular ligand-binding domain having a binding affinity (specificity) for a selected ligand, (ii) a first TMD with a first modified interface and (iii) a first intracellular domain with a CARD11 polypeptide having one or more mutations or a CARD9 polypeptide having one or more mutations. The second polypeptide may include (i) a second TMD having a second interface and (ii) a second intracellular domain with a signaling domain. The first and the second polypeptide can associate via charged residues within the first modified interface and the second interface. Binding of the select ligand to the extracellular ligandbinding domain can simultaneously induce activity of the signaling domain and release of the CARD11 (or CARD9) polypeptide.

[0213] The design of the multi-chain polypeptides of the disclosure can improve the functionality of reviously described multi-chain chimeric receptors. By incorporating signaling domains in the multi-chain format of the disclosure, a skilled in the art can effectively couple auxiliary functions to a CARD11 (or CARD9) domain response and have both activated at the same time and in response to the same antigen. Auxiliary functions can include, for example, destroying cells (through the use of immunoreceptor tyrosine-based activation motif (IT AM) domains on the signaling chain) or promoting survival signaling through the use of costimulatory domains (such as 41BB, CD28 costimulatory domains, etc).

[0214] In some embodiments of the multi-chain chimeric polypeptide of the disclosure, the first modified interface comprises a positively charged residue, the second interface comprises a negatively charged residue, and the first polypeptide is coupled to the second polypeptide via the electrostatic force between the first and the second modified interfaces.

[0215] In a further aspect, provided herein are multi-chain chimeric polypeptides that can enable simultaneous transcriptional regulation via CARD 11 or CARD9 poly peptides and cellular activation (e.g.. T cell activation), upon binding of a selected ligand to the extracellular-ligand binding domain of the first polypeptide of the multi-chain chimeric polypeptides of the disclosure.

[0216] In some embodiments, the multi-chain chimeric polypeptides of the disclosure can be immunoreceptors. In some embodiments, the immunoreceptors are chimeric antigen receptors (CARS).First Polypeptides

[0217] The multi-chain polypeptides of the disclosure include a first chimeric polypeptide having an extracellular ligand-binding domain (moiety) with a binding affinity' for a selected ligand, a first TMD having a first modified interface, a first intracellular domain and a CARD1 1 polypeptide comprising one or more mutations or a CARD9 polypeptide comprising one or more mutations.

[0218] In some embodiments, the first polypeptides of the disclosure include in order from the N-terminus to the C-terminus (i) the extracellular ligand-binding domain, (ii) the first TMD, and (iii) the first intracellular domain.

[0219] In some embodiments, the first polypeptides of the disclosure include an amino acid sequence that is has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 84, 86, 88, 90, 92, or 94 or functional variants thereof.Extracellular Ligand-Binding Domains (ECD)

[0220] In some embodiments, the extracellular domains (ECDs) of the first polypeptide of the multi-chain chimeric polypeptides disclosed herein have a binding affinity' for one or more target ligands. The target ligand can be expressed on a cell surface, or otherwise be anchored, immobilized, or restrained so that it can exert a mechanical force on the chimeric receptor. As such, without being bound to any particular theory, binding of the ECD of the fist polypeptide of the multi-chain chimeric polypeptides provided herein to a cell-surface ligand does not necessarily remove the target ligand from the target cell surface, but instead enacts a mechanical pulling force on the chimeric receptor. For example, an otherwisesoluble ligand may be targeted if it is bound to a surface, or to a molecule in the extracellular matrix.

[0221] In some embodiments, the target ligand is a cell-surface ligand. Non-limiting examples of suitable ligand types include cell surface receptors; adhesion proteins; carbohydrates, lipids, glycolipids, lipoproteins, and lipopolysaccharides that are surfacebound; integrins, mucins; and lectins. In some embodiments, the ligand is a protein. In some embodiments, the ligands include tumor associated antigens or tumor-specific antigens. In some embodiments, the ligand is a cluster of differentiation ligand (CD) and includes but is not limited to CD1, CDla, CDlb, CDlc, CDld, CDle, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21. CD22, CD23, CD25, CD27, CD28, CD33, CD34. CD40, CD45, CD48, CD52. CD59, CD66, CD70, CD71. CD72, CD73, CD79A. CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD178, 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). EGFR, FGFR2, CEA, AFP, CA125. MUC-1, MAGE, alkaline phosphatase, placental-like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), or signal regulatory protein a (SIRPa).

[0222] In some embodiments, the extracellular domains of the first polypeptide of the multi-chain chimeric polypeptides of the disclosure includes an antigen-binding moiety. In some embodiments, the antigen-binding moiety can bind to a ligand on the surface of a cell. In some embodiments, the antigen-binding moiety can bind one or more targets. In some embodiments, the antigen-binding moiety includes one or more antigen-binding determinants of an antibody or a functional antigen-binding fragment thereof. One skilled in the art upon reading the present disclosure will readily understand that the term "functional fragment thereof' or "functional variant thereof refers to a molecule having quantitative and / or qualitative biological activity in common with the wild-type molecule from which the fragment or variant was derived. For example, a functional fragment or a functional variant of an antibody is one which retains essentially the same ability to bind to the same epitope as the antibody from which the functional fragment or functional variant was derived. For instance, an antibody that can bind to an epitope of a cell surface receptor may be truncated at the N-terminus and / or C-terminus, and the retention of its epitope binding activity assessed using assays known to those of skill in the art. The antigen-binding moiety can be, but is not limited to, an antibody, a nanobody, a diabody, a triabody, a minibody, anF(ab')2 fragment, an F(ab) fragment, a single chain variable fragment (scFv), a single domain antibody (sdAb), and any functional fragment thereof.

[0223] The antigen-binding moiety can include naturally-occurring amino acid sequences or can be engineered, designed, or modified to provide desired and / or improved properties such as, e.g., binding affinity. Generally, the binding affinity of an antigen-binding moiety, e.g., an antibody, for a target antigen (e.g., CD 19 antigen) can be calculated by the Scatchard method described by Frankel et al.. Mol. Immunol. 16: 101-06. 1979. In some embodiments, binding affinity is measured by an anti gen / anti body dissociation rate. In some embodiments, binding affinity is measured by a competition radioimmunoassay. In some embodiments, binding affinity is measured by ELISA. In some embodiments, antibody affinity is measured by flow cytometry.

[0224] An antibody that "selectively binds" an antigen (such as CD19) is an antigenbinding moiety that does not significantly bind other antigens but binds the antigen with high affinity, e.g., with an equilibrium dissociation constant (KD) of 100 nM or less, such as 60 nM or less, for example, 30 nM or less, such as. 15 nM or less, or 10 nM or less, or 5 nM or less, or 1 nM or less, or 500 pM or less, or 400 pM or less, or 300 pM or less, or 200 pM or less, or 100 pM or less.

[0225] A skilled artisan can select an ECD based on the desired localization or function of a cell that is genetically modified to express a multi-chain chimeric polypeptide or a first polypeptide of the present disclosure. For example, a first polypeptide with an ECD including an antibody specific for a HER2 antigen can target cells to HER2-expressing breast cancer cells. In some embodiments, the ECD of the disclosed first polypeptide is capable of binding a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). A skill artisan will understand that TAAs include a molecule, such as e.g., protein, present on tumor cells and on normal cells, or on many normal cells, but at much lower concentration than on tumor cells. In contrast, TSAs generally include a molecule, such as e.g., protein which is present on tumor cells but absent from normal cell.

[0226] In some cases, the antigen-binding moiety is specific for an epitope present in an antigen that is expressed by a tumor cell, i.e.. a tumor-associated antigen. The tumor- associated antigen can be an antigen associated with, e.g., a breast cancer cell, a B cell lymphoma, a pancreatic cancer, a Hodgkin lymphoma cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma, a lung cancer cell, a non-Hodgkin B-cell lymphoma (B-NHL) cell, an ovarian cancer cell, a prostate cancer cell, a mesothelioma cell, a melanoma cell, a chronic lymphocytic leukemia cell, an acute lymphocytic leukemia cell, a neuroblastomacell, a glioma, a glioblastoma, a colorectal cancer cell, etc. It will also be understood that a tumor-associated antigen may also be expressed by a non-cancerous cell. In some embodiments, the antigen-binding domain is specific for an epitope present in a tissuespecific antigen. In some embodiments, the antigen-binding domain is specific for an epitope present in a disease-associated antigen.

[0227] Non-limiting examples of suitable target antigens include CD19, B7H3 (CD276), BCMA (CD269), alkaline phosphatase, placental-like 2 (ALPPL2), green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), signal regulatory protein alpha (SIRPa), CD123, CD171, CD179. alpha., 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- HR. alpha., KIT (CD 117), MUC 1, NCAM, PAP, PDGFR-.beta., PRSS21, PSCA, PSMA, ROR1, SSEA-4, TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, and Axl.

[0228] In some embodiments, the target antigen is selected from CD19, B7H3 (CD276), BCMA (CD269), ALPPL2. CD123, CD171, CD179.alpha„ 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-l lRa, KIT (CD117), MUC1, NCAM. PAP. PDGFR-beta, PRSS21, PSCA. PSMA, ROR1, SSEA-4. TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, Axl, GPC2, human epidermal growth factor receptor 2 (Her2 / neu), CD276 (B7H3), IL- 13R. alpha.1, IL- 13R.alpha.2, .alpha. -fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen- 125 (CA-125), CA19-9. calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD123, CD93, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), ALK, DLK1, FAP, NY- ESO, WT1, HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin (MSA), neurofilament, neuronspecific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (a subunit of the heterodimeric IL-2 receptor), CD3, CD4, CD5, IFN-alpha, IFN-gamma, IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6receptor, integrin alpha.4, integrin ,alpha.4.beta.7, LFA-1 (CD11. alpha.), myostatin, OX-40, scleroscin, SOST. TGF beta 1, TNF-alpha, VEGF-A, pyruvate kinase isoenzyme type M2 (tumor M2-PK), CD20, CD5, CD7, CD3, TRBC1, TRBC2, BCMA, CD38, CD123, CD93, CD34, CDla, SLAMF7 / CS1, FLT3, CD33, CD123, T ALLA-1, CSPG4, DLL3, Kappa light chain, Lamba light chain, CD16 / Fc gamma RIII, CD64, FITC, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), GD3, EGFRvIII (epidermal growth factor variant III), EGFR and isovariants thereof, TEM-8. sperm protein 17 (Spl7), mesothelin.

[0229] Further non-limiting examples of suitable antigens include PAP (prostatic acid phosphatase), prostate stem cell antigen (PSCA), prostein, NKG2D, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of the prostate 1). an abnormal ras protein, an abnormal p53 protein, integrin (33 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), Ral-B, GPC2, CD276 (B7H3), or IL- 13R. alpha.. In some embodiments, the antigen is Her2. In some embodiments, the antigen is ALPPL2. In some embodiments, the antigen is BCMA. In some embodiments, the antigen-binding moiety of the ECD is specific for a reporter protein, such as GFP and eGFP. Non-limiting examples of such antigen-binding moiety include a LaG17 anti-GFP nanobody. In some embodiments, the antigen-binding moiety of the ECD includes an anti-BCMA fully -humanized VH domain (FHVH). In some embodiments, the antigen is signal regulatory' protein alpha (SIRP. alpha).

[0230] Additional antigens suitable for targeting by the first chimeric polypeptide and the multi-chain CARs disclosed herein include, but are not limited to GPC2, human epidermal growth factor receptor 2 (Her2 / neu), CD276 (B7H3), IL-13R.alphal, IL-13Ralpha.2, alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA). Other suitable target antigens include, but are not limited to, tyrosinase, melanoma- associated antigen (MAGE), CD34, CD45, CD123, CD93, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), ALK. DLK1, FAP, NY-ESO, WT1, HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor- 1.

[0231] Additional antigens suitable for targeting by the polypeptides disclosed herein include, but are not limited to, those associated with an inflammatory disease such as, AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD125, CD147 (basigin), CD154 (CD40L),CD2, CD20, CD23 (IgE receptor), CD25 (a subunit of the heteromeric of IL-2 receptor), CD3, CD4, CD5, IFN-alpha, IFN-gamma, IgE. IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL- 17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrm .alpha.4, integrin .alpha.4. beta.7, LFA-1 (CD1 1. alpha.), myostatin, OX-40, scleroscin, SOST, TGF.beta.1, TNF-.alpha., and VEGF-A.

[0232] Further antigens suitable for targeting by the first chimeric polypeptides and the multi-chain CARs disclosed herein include, but are not limited to the pyruvate kinase isoenzyme type M2 (tumor M2-PK), CD20, CD5, CD7, CD3, TRBC1 , TRBC2, BCMA, CD38, CD123, CD93, CD34, CDla, SLAMF7 / CS1, FLT3, CD33, CD123, TALLA-1, CSPG4, DLL3, Kappa light chain, Lamba light chain, CD16 / Fc gamma RIII, CD64, FITC, CD22. CD27, CD30, CD70, GD2 (ganglioside G2), GD3, EGFRvIII (epidermal growth factor variant III), EGFR and isovariants thereof, TEM-8, sperm protein 17 (Spl7), mesothelin. Further non-limiting examples of suitable antigens include PAP (prostatic acid phosphatase), prostate stem cell antigen (PSCA), prostein, NKG2D, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of the prostate 1). an abnormal ras protein, an abnormal p53 protein, integnn .beta.3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), and Ral-B. In some embodiments, the antigen is GPC2, CD19, Her2 / neu, CD276 (B7H3), IL- 13R. alpha. 1, or IL-13R.alpha.2. In some embodiments, the antigen is Her2. In some embodiments, the antigen is ALPPL2. In some embodiments, the antigen is BCMA. In some embodiments, the antigen-binding moiety of the ECD is specific for a reporter protein, such as GFP and eGFP. Non-limiting examples of such antigen-binding moiety7include a LaG17 anti-GFP nanobody. In some embodiments, the antigen-binding moiety7of the ECD includes an anti- BCMA fully -humanized VH domain (FHVH).

[0233] In some embodiments, the antigen can be HER2 produced by HER2 -positive breast cancer cells. In some embodiments, the antigen can be CD 19 that is expressed on B-cell leukemia. In some embodiments, the antigen can be EGFR that is expressed on glioblastoma multiform (GBM) but much less expressed so on healthy CNS tissue. In some embodiments, the antigen can be CEA that is associated with cancer in adults, for example colon cancer.

[0234] In some embodiments, the antigen-binding moiety of the ECD is specific for a cell surface target, where non-limiting examples of cell surface targets include CD 19, CD30, Her2, CD22, ENPP3, EGFR, CD20, CD52, CD11 a, and a -integrin. In some embodiments, the chimeric first polypeptides and the multi-chain CARs disclosed herein include an extracellular domain having an antigen-binding moiety that binds CD 19, CEA, HER2,MUC1, CD20, ALPPL2, BCMA, or EGFR. In some embodiments, the multi-chain CARs provided herein include an extracellular domain including an antigen-binding moiety that binds CD 19. In some embodiments, the chimeric first polypeptides provided herein include an extracellular domain including an antigen-binding moiety that binds ALPPL2. In some embodiments, the chimeric first polypeptides provided herein include an extracellular domain including an antigen-binding moiety that binds BCMA. In some embodiments, the chimeric first polypeptides include an extracellular domain including an antigen-binding moiety that binds Her2.

[0235] In some embodiments, antigens suitable for targeting by the chimeric first polypeptides disclosed herein include ligands derived from a pathogen.Linking Sequences between ECD and First Transmembrane domain (TMD)

[0236] The multi-chain chimeric polypeptides and receptors of the disclosure can include a linking sequence disposed between the ECD and the TMD. The linking sequence can be a natural or a synthetic polypeptide. The linking sequence can be a flexible connector that provides structural flexibility and spacing to flanking polypeptide regions. The linking sequence can be a hinge domain inserted N-terminally to the TMD. In some embodiments, the hinge domain is a CD8 hinge domain. In some embodiments, the CD8 hinge domain is a truncated CD8a hinge domain.. In some embodiments, the truncated CD8a is encoded by an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 3. In some embodiments, the truncated CD8a comprises an amino acid sequence comprising SEQ ID NO: 3. In some embodiments, the truncated CD8a is encoded by SEQ ID NO: 3.

[0237] Hinge polypeptide sequences suitable for the compositions and methods of the disclosure can be naturally-occurring hinge polypeptide sequences (e.g., those from naturally-occurring immunoglobulins). Alternatively, a hinge polypeptide sequence can be a synthetic sequence that corresponds to a naturally-occurring hinge polypeptide sequence, or can be an entirely synthetic hinge sequence, or can be engineered, designed, or modified to provide desired and / or improved properties, e.g.. modulating transcription. Suitable hinge polypeptide sequences include, but are not limited to, those derived from IgA, IgD, and IgG subclasses, such as IgGl hinge domain, IgG2 hinge domain, IgG3 hinge domain, and IgG4 hinge domain, or a functional variant thereof. In some embodiments, the hinge polypeptide sequence contains one or more CXXC motifs. In some embodiments, the hinge polypeptide sequence contains one or more CPPC motifs. Additional information in this regard can befound in, for example, a recent review by Vidarsson G. et al., Frontiers Immunol. Oct. 20, 2014, which is hereby incorporated by reference in its entirety.

[0238] Accordingly, in some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an IgGl hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an IgG2 hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an IgG3 hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an IgG4 hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an IgA hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an IgD hinge domain or a functional variant thereof.

[0239] Additional hinge polypeptide sequences suitable for the compositions and methods disclosed herein include, but are not limited to, hinge polypeptide sequences derived from a CD8a hinge domain, a CD28 hinge domain, a CD 152 hinge domain, a PD-1 hinge domain, a CTLA4 hinge domain, an 0X40 hinge domain, an Fey Rllla hinge domain, and functional variants thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from a CD8. alpha hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from a CD28 hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an 0X40 hinge domain or a functional variant thereof. In some embodiments, the hinge domain includes a hinge polypeptide sequence derived from an IgG4 hinge domain or a functional variant thereof.

[0240] In principle, there are no particular limitations to the length and / or amino acid composition of the hinge domain other than it confers flexibility. However, one skilled in the art will readily appreciate that the length and amino acid composition of the hinge polypeptide sequence can be optimized to vary the orientation and / or proximity of the ECD and the TMD relative to one another, as well as of the first polypeptide and second polypeptide to one another, to achieve a desired activity of the chimeric multi-chain polypeptides of the disclosure. In some embodiments, any arbitrary single-chain peptide including about one to 100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. 13, 14, 15, 16, 17, 18. 19. 20. etc. amino acid residues) can be used as a hinge domain. In some embodiments, the hinge domain includes about 5 to 50, about 10 to 60, about 20 to 70, about30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the hinge domain includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the hinge domain includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the hinge domain includes about 1 to 10. about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues. In some embodiments, the hinge domain includes a sequence having at least 80% sequence identity, such as, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity to SEQ ID NO: 3.First Transmembrane Domains

[0241] The multi-chain chimeric polypeptides of the present disclosure include a transmembrane domain located in the first polypeptide (e.g., first TMD). In some embodiments of the first polypeptide of the disclosure, the first TMD is fully synthetic. In some embodiments the first TMD includes a contiguous stretch of valine residues. In some embodiments, the contiguous stretch of valine residues includes 5 to 25 valine residues. In some embodiments, the first TMD includes a contiguous stretch of 5 to 25 valine residues. In some embodiments, the first TMD includes a contiguous stretch of 5, or 6, or 7, or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 consecutive valine residues.

[0242] In some embodiments, the first TMD includes a positively charged residue. In some embodiments, the positively charged residue is lysine or arginine. In some embodiments, the positively charged residue is within the contiguous stretch of valine residues. In some embodiments, the positively charged residue is located after the first valine residue from the N-terminus. In some embodiments, the positively charged residue is located after the second or third or fourth or fifth or sixth or seventh or eighth or ninth or tenth or eleventh or twelfth or thirteenth or fourteenth or fifteenth or sixteenth or seventeenth or eighteenth or nineteenth or twentieth or twenty first or twenty second or twenty third valine residue from the N-terminus. In some embodiments, the lysine or arginine residue replaces the sixth or seventh or eighth or ninth or tenth or eleventh or twelfth valine residue from the N-terminus. In some embodiments, the TMD of the first polypeptide includes a sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 9. In some embodiments, the TMD is SEQ ID NO: 9. In some embodiments, the lysine or arginine residue is residue number 8 or residue number 9 or residue number 10 orresidue number 11 of SEQ ID NO:9 or at a position corresponding to residue number 8 or residue number 9 or residue number 10 or residue number 11 of SEQ ID NO:9.

[0243] The first TMD of the multi-chain chimeric polypeptides of the disclosure can include a TMD from a Notch receptor. In some embodiments, the TMD includes the Notch 1 receptor. In some embodiments, the Notch 1 receptor is a human Notch 1 receptor.

[0244] The first TMD can include one or more ligand-inducible proteolytic cleavage sites. Examples of ligand-inducible proteolytic cleavage sites in aNotch receptor (e.g., S2 or S3) are described in US Patent No. 11 ,202,801 , incorporated herein by reference in its entirety. In some embodiments, the ligand-inducible proteolytic cleavage site is cleavable by gamma secretase, a multiprotein enzyme complex.

[0245] The first polypeptide of the multi-chain chimeric polypeptides of the disclosure can include a stop-transfer-sequence (STS). The STS can be located C-terminally to the TMD. The STS can include a highly-charged domain. In some embodiments, the STS is aNotch 2 JMD.

[0246] In principle, there are no particular limitations to the length and / or amino acid composition of the STS. Any arbitrary single-chain peptide that includes about 4 to about 40 amino acid residues (e g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, etc. amino acid residues) can be used as a STS. In some embodiments, the STS includes about 4 to 15, about 6 to 20, about 8 to 25, about 10 to 30, about 12 to 35, about 14 to 40, about 5 to 40, about 10 to 35, about 15 to 30, about 20 to 25, about 20 to 40, about 10 to 30, about 4 to 20, or about 5 to 25 amino acid residues. In some embodiments, the STS includes about 4 to 10, about 5 to 12, about 6 to 14, about 7 to 18, about 8 to 20, about 9 to 22, about 10 to 24, or about 11 to 26 amino acid residues. In some embodiments, the STS includes about 4 to 10 residues, such as, 4, 5, 6, 7, 8. 9, or 10 amino acid residues.

[0247] In some embodiments, the STS is a polybasic domain. In some embodiments, the polybasic domain includes Notch -1 or Notch-2. In some embodiments, the polybasic domain comprises an amino acid sequence where the majority' (i.e., at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%. at least 95%. at least 96%. at least 97%. at least 98%. at least 99%. or 100%.) of the residues are lysine and / or arginine and / or histidine and / or any combination thereof.

[0248] The STS can include a sequence having at least 70% sequence identity, such as, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% sequence identity to a JMD sequence from Notchl, Notch2, Notch3, Notch4, CLSTN1, CLSTN2, CSF1R, CXCL16, DAG1, GHR, PTPRF,AGER, KL, NRG1, LRP1B, Jag2, EPCAM, KCNE3, CDH2, CDH5, NRG2, PTPRK, BTC, EPHA3. EPHA4, IL1R2, or PTPRM. In some embodiments, the STS includes a sequence having only Lys (K) or Arg (R) in the first 4 residues. In some embodiments, the STS includes one, two, three, four, five, or more basic residues. In some embodiments, the STS includes five, four, three, two, one, or zero aromatic residues or residues with hydrophobic and / or bulky side chains.First Intracellular Domains and Transcriptional Regulators

[0249] The first polypeptides of the multi-chain chimeric polypeptides of the disclosure can include a CARD 11 polypeptide comprising one or more mutations.

[0250] CARD11 poypeptides comprising one or mutations for use in the multi-chain chimeric receptors described herein are described in more detail above in relation to hybrid receptors.

[0251] Various embodiments of the first polypeptides of the disclosure can include the following non-limiting examples of components. The first polypeptide can include an extracellular domain, a TMD. an intracellular domain and other sequences from known receptors such as. for example, the triggering receptor expressed on myeloid cells 2 (TREM 2). TREM2 can undergo proteolytic cleavage between amino acids H157 and S158 by members of the disintegrin and metalloproteinase (ADAM) family. This cleavage results in the release of a soluble TREM2 ectodomain (sTREM2) into extracellular fluids, and generation of a membrane tethered C-terminal fragment (CTF). The TREM2 CTF represents a substrate for intramembrane proteolysis by y-secretase. In some embodiments of the disclosure, the first polypeptide can include TREM2 TMD without its cytoplasmic domain. In some embodiments, the first polypeptide can includes a TREM2 extracellular domain, and a TMD with a Notch 1 y-secretase cleavage site and / or a Notch-2 JMD.

[0252] The first polypeptide of the multi-chain chimeric polypeptides of the disclosure can include a first modified interface via which the first polypeptide associates with the second polypeptide. The first modified interface can be from a Notch 1 TMD. The first modified interface can have a positively charged residue via which the first polypeptide associates with the second polypeptide of the multiple-chimeric polypeptides of the disclosure. The positively charged residue can be a lysine or arginine.Other components

[0253] In some embodiments of the disclosure, the first polypeptide can further include one or more of the following: an autoproteolytic peptide sequence or a nuclear localization signal.

[0254] In some embodiments, the autoproteolytic peptide sequence is from a porcine teschovirus-1 2A (P2A), a foot-and-mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna virus 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Virus 2A (BmIFV2A), or a combination thereof.

[0255] The first polypeptide can further include a proteolytic cleavage site. In some embodiments, the proteolytic cleavage site is cleavable by gamma secretase. In some embodiments, the proteolytic cleavage site is ligand-inducible proteolytic cleavage site. The proteolytic cleavage site can be disposed between the CARD11 polyeptide and the hinge domain. The proteolytic cleavage site can be ligand-inducible wherein binding of the selected ligand to the extracellular ligand-binding domain induces cleavage at the ligandinducible proteolytic cleavage site.Second Polypeptides

[0256] The multi-chain chimeric polypeptides of the disclosure include a second polypeptide having (i) a second TMD with a second interface, and (ii) a second intracellular domain in order from N -terminus to C-terminus of the second polypeptide.

[0257] In some embodiments, the second polypeptides of the disclosure include an amino acid sequence that is has 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 80 or SEQ ID NO:082 or functional variants thereof.Second Transmembrane Domains

[0258] The second polypeptides of the multi-chain chimeric polypeptides of the disclosure can include a second TMD having a second interface. The second TMD can include any domain that is capable of associating with the first polypeptide of the multi-chain chimeric polypeptides of the disclosure via a charged residue in the TMD and that has a signaling domain that can be activated upon binding of a target ligand to the extracellular ligandbinding domain of the first polypeptide. The second interface is the region in the second polypeptide can include a negatively charged residue via which it associates with a positively charged residue in the TMD of the first polypeptide of the disclosure. In some embodiments, the negatively charged residue can be arginine or lysine.

[0259] In some embodiments, the second polypeptide includes a DAP 12 TMD. In some embodiments, the second polypeptide includes a DAP12 domain as well as a DAP12 intracellular signaling domain ( / . e. cytoplasmic domain) as explained in more details below.Second Intracellular Domain

[0260] The second polypeptide of the multi-chain chimeric polypeptides of the disclosure include an intracellular domain. The intracellular domain can be responsible for propagation of downstream signaling upon ligand binding by the first polypeptide chain. In some embodiments, the intracellular domain is from DAP 12 (DNAZ activation protein of 12 kDa, TYROBP). The DAP12 intracellular domain, also known as the cytoplasmic domain of DAP 12, includes an immunoreceptor tyrosine-based activation motif (IT AM), which gets phosphorylated upon ligand-binding to TREM2, and thereon regulate several intracellular signaling pathways that control cell proliferation and differentiation, survival, phagocytosis, cytoskeletal remodeling, calcium mobilization and / or cytokine production. In some embodiments, the signaling domain can be a CD3 zeta. TCR zeta, FcR y, FcR . CD3 y, CD3 A, CD3 s, CD5, CD22, CD79a, CD79b, CD278 (ICOS), Fc8 RL DAP10, DAP12, or CD66d, signaling domain.Nucleic Acid Molecules

[0261] In another aspect, provided herein are various nucleic acid molecules including nucleotide sequences encoding the chimeric receptors and multi-chain receptors of the disclosure, including expression cassettes, and expression vectors containing these nucleic acid molecules operably linked to heterologous nucleic acid sequences such as, for example, regulatory sequences which facilitate in vivo expression of the receptor in a host cell.

[0262] Nucleic acid molecules of the present disclosure can be of any length, including for example, betw een about 1.5 Kb and about 50 Kb, between about 5 Kb and about 40 Kb, betw een about 5 Kb and about 30 Kb, between about 5 Kb and about 20 Kb, or between about 10 Kb and about 50 Kb, for example between about 15 Kb to 30 Kb, between about 20 Kb and about 50 Kb, between about 20 Kb and about 40 Kb, about 5 Kb and about 25 Kb, or about 30 Kb and about 50 Kb.

[0263] In some embodiments, provided herein is a nucleic acid molecule including a nucleotide sequence encoding a chimeric receptor including, from N-terminus to C- terminus: (a) an extracellular ligand-binding domain having a binding affinity for a selected ligand; (b) a linking sequence; (c) a transmembrane domain including one or more ligandinducible proteolytic cleavage sites; and (d) an intracellular domain including (i) an intracellular signaling domain (SD) comprising at least one costimulatory domain derived from a signaling molecule and an activation domain, and (ii) a caspase recruitment domain family member 11 (CARD11) polypeptide comprising one or more mutations or a caspaserecruitment domain family member 9 (CARD9) polypeptide comprising one or more mutations.

[0264] In some embodiments, provided herein is a nucleic acid molecule including a nucleotide sequence encoding a multi-chain chimeric polypeptide including (a) a first polypeptide comprising (i) an extracellular ligand-binding domain having a binding affinity' for a selected ligand, (ii) a first transmembrane domain (TMD) comprising a first modified interface and (iii) a first intracellular domain comprising a caspase recruitment domain family member 11 (CARD11 ) polypeptide comprising one or more mutations or a caspase recruitment domain family member 9 (CARD9) polypeptide comprising one or more mutations; and (b) a second polypeptide comprising (i) a second TMD comprising a second interface and (ii) a second intracellular domain comprising a signaling domain; wherein the first and the second modified interfaces each comprises an amino acid residue having a charged side chain, wherein the amino acid residues in the first and the second modified interfaces have opposite charges, and wherein the first polypeptide is coupled to the second polypeptide via the first modified interface and the second interface and wherein binding of the selected ligand to the extracellular ligand-binding domain induces activity of the signaling domain and release of the CARD11 or CARD9 polypeptide..

[0265] In some embodiments, the nucleotide sequence is incorporated into an expression cassette or an expression vector. It will be understood that an expression cassette generally includes a construct of genetic material that contains coding sequences and enough regulatory information to direct proper transcription and / or translation of the coding sequences in a recipient cell, in vivo and / or ex vivo. Generally, the expression cassette may be inserted into a vector for targeting to a desired host cell and / or into an individual. As such, in some embodiments, an expression cassette of the disclosure include a coding sequence for the chimeric receptor as disclosed herein, which is operably linked to expression control elements, such as a promoter, and optionally, any7or a combination of other nucleic acid sequences that affect the transcription or translation of the coding sequence.

[0266] In some embodiments, the nucleotide sequence is incorporated into an expression vector. It will be understood by one skilled in the art that the term “vector” generally refers to a recombinant polynucleotide construct designed for transfer between host cells, and that may be used for the purpose of transformation, e.g., the introduction of heterologous DNA into a host cell. As such, in some embodiments, the vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bringabout the replication of the inserted segment. In some embodiments, the expression vector can be an integrating vector.

[0267] In some embodiments, the expression vector can be a viral vector. As will be appreciated by one of skill in the art, the term “viral vector” is widely used to refer either to a nucleic acid molecule (e.g., a transfer plasmid) that includes virus-derived nucleic acid elements that generally facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles will generally include various viral components and sometimes also host cell components in addition to nucleic acid(s). The term viral vector may refer either to a virus or viral particle capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from a virus. The term “retroviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus. The term “lentiviral vector” refers to a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus, which is a genus of retrovirus.

[0268] In some embodiments, provided herein are nucleic acid molecules encoding a polypeptide with an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to a chimeric receptor disclosed herein. In some embodiments, provided herein are nucleic acid molecules encoding a polypeptide with an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOS: 68, 70, 72, 74, 76, and 78. In some embodiments, the nucleic acid molecules encode a polypeptide with an amino acid sequence having at least about 80%, 90%, 95%, 96%, 97, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOS: 84, 86, 88, 90, 92, and 94.

[0269] The nucleic acid sequences encoding the chimeric receptors and multi-chain receptors can be optimized for expression in the host cell of interest. For example, the G-C content of the sequence can be adjusted to average levels for a given cellular host, as calculated by reference to known genes expressed in the host cell. Methods for codon usage optimization are known in the art. Codon usages within the coding sequence of the chimeric receptor disclosed herein can be optimized to enhance expression in the host cell, such that about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons within the coding sequence have been optimized for expression in a particular host cell.

[0270] Some embodiments disclosed herein relate to vectors or expression cassettes including a recombinant nucleic acid molecule encoding the chimeric receptors disclosed herein. The expression cassette generally contains coding sequences and sufficient regulatory information to direct proper transcription and / or translation of the coding sequences in a recipient cell, in vivo and / or ex vivo. The expression cassette may be inserted into a vector for targeting to a desired host cell and / or into an individual. An expression cassette can be inserted into a plasmid, cosmid. virus, autonomously replicating polynucleotide molecule, phage, as a linear or circular, single-stranded or double-stranded, DNA or RNA polynucleotide molecule, derived from any source, capable of genomic integration or autonomous replication, including a nucleic acid molecule where one or more nucleic acid sequences has been linked in a functionally operative manner, i.e., operably linked.

[0271] Also provided herein are vectors, plasmids, or viruses containing one or more of the nucleic acid molecules encoding any chimeric receptor or multi-chain receptor disclosed herein. The nucleic acid molecules can be contained within a vector that is capable of directing their expression in, for example, a cell that has been transformed / transduced with the vector. Suitable vectors for use in eukaryotic and prokaryotic cells are known in the art and are commercially available, or readily prepared by a skilled artisan. See for example, Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J.. & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology' Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego. CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology'. New York, NY: Wiley; Mullis, K. B., Ferre, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY : Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S.C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference).

[0272] 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, ballistic introduction, nucl eoporation, hydrodynamic shock, and infection.

[0273] Viral vectors that can be used in the disclosure include, for example, retrovirus vectors, adenovirus vectors, and adeno-associated virus vectors, lentivirus vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.). For example, a chimeric receptor as disclosed herein can be produced in a eukary otic host, such as a mammalian cells (e.g., COS cells. NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, VA). In selecting an expression system, care should be taken to ensure that the components are compatible with one another. Artisans or ordinary7skill are able to make such a determination. Furthermore, if guidance is required in selecting an expression system, skilled artisans may consult P. Jones. “Vectors: Cloning Applications”, John Wiley and Sons, New York, N.Y., 2009).

[0274] The nucleic acid molecules provided can contain naturally occurring sequences, or sequences that differ from those that occur naturally, but, due to the degeneracy of the genetic code, encode the same polypeptide, e.g., antibody. These nucleic acid molecules can consist of RNA or DNA (for example, genomic DNA, cDNA, or synthetic DNA, such as that produced by phosphorami dite-based synthesis), or combinations or modifications of the nucleotides within these types of nucleic acids. In addition, the nucleic acid molecules can be double-stranded or single-stranded (e.g.. either a sense or an antisense strand).

[0275] The nucleic acid molecules are not limited to sequences that encode polypeptides (e.g., antibodies); some or all of the non-coding sequences that he upstream or downstream from a coding sequence (e.g., the coding sequence of a chimeric receptor) can also be included. Those of ordinary skill in the art of molecular biology7are familiar with routine procedures for isolating nucleic acid molecules. They can, for example, be generated by treatment of genomic DNA with restriction endonucleases, or by performance of thepolymerase chain reaction (PCR). In the event the nucleic acid molecule is a ribonucleic acid (RNA). molecules can be produced, for example, by in vitro transcription.Host Cells

[0276] The nucleic acid 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 disclosure relate to methods for making a recombinant or engineered cell, including (a) providing a cell capable of protein expression and (b) contacting the provided cell with a recombinant nucleic acid of the disclosure.

[0277] Introduction of the nucleic acid molecules of the disclosure into cells can be achieved by methods known to those skilled in the art such as, for example, viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, nucleofection, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE- dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro-injection, nanoparticle-mediated nucleic acid delivery, and the like.

[0278] Accordingly, in some embodiments, the nucleic acid molecules can be delivered by viral or non-viral delivery vehicles known in the art. For example, the nucleic acid molecule can be stably integrated in the host genome, or can be episomally replicating, or present in the recombinant host cell as a mini-circle expression vector for transient expression. Accordingly, in some embodiments, the nucleic acid molecule is maintained and replicated in the recombinant host cell as an episomal unit. In some embodiments, the nucleic acid molecule is stably integrated into the genome of the recombinant cell. Stable integration can be achieved using classical random genomic recombination techniques or with more precise techniques such as guide RNA-directed CRISPR / Cas9 genome editing, or DNA-guided endonuclease genome editing with NgAgo (Natronobacterium gregoryi Argonaute), or TALENs genome editing (transcription activator-like effector nucleases). In some embodiments, the nucleic acid molecule is present in the recombinant host cell as a minicircle expression vector for transient expression.

[0279] The nucleic acid molecules can be encapsulated in a viral capsid or a lipid nanoparticle, or can be delivered by viral or non-viral delivery means and methods known in the art, such as electroporation. For example, introduction of nucleic acids into cells may be achieved by viral transduction. In anon-limiting example, adeno-associated virus (AAV) is engineered to deliver nucleic acids to target cells via viral transduction. Several AAVserotypes have been described, and all of the known serotypes can infect cells from multiple diverse tissue types. AAV is capable of transducing a wide range of species and tissues in vivo with no evidence of toxicity, and it generates relatively mild innate and adaptive immune responses.

[0280] Lentiviral-derived vector systems are also useful for nucleic acid delivery and gene therapy via viral transduction. Lenti viral vectors offer several attractive properties as genedelivery vehicles, including: (i) sustained gene delivery through stable vector integration into host genome; (ii) the capability of infecting both dividing and non-dividing cells; (iii) broad tissue tropisms, including important gene- and cell-therapy-target cell types; (iv) no expression of viral proteins after vector transduction; (v) the ability to deliver complex genetic elements, such as polycistronic or intron-containing sequences; (vi) a potentially safer integration site profile; and (vii) a relatively easy system for vector manipulation and production.

[0281] In some embodiments, host cells can be genetically engineered (e.g., transduced or transformed or transfected) with, for example, a vector construct of the present application that can be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or can be an expression vector for the expression of the polypeptides of interest. Host cells can be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.

[0282] 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 anon-human primate cell. In some embodiments, the cell is an autologous cell. In some embodiments, the cell is an allogeneic cell. In some embodiments, the cell is a primary cell. In some embodiments, the mammalian cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell. In some embodiments, the recombinant cell is an immune system cell, e g., a lymphocyte (e.g., a T cell or NK cell), or a dendritic cell. In some embodiments, the immune cell is a B cell, a monocyte, a natural killer (NK) cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell (TH), a cytotoxic T cell (TCTL), or other T cell. In some embodiments, the immune system cell is a T lymphocyte.

[0283] In some embodiments, the cell is a stem cell or is derived from a stem cell. In some embodiments, the cell is a hematopoietic stem cell. In some embodiments of the cell, the cell is a lymphocyte. In some embodiments, the cell is a precursor T cell or a T regulatory (Treg) cell. In some embodiments, the cell is a CD34+, CD8+, or a CD4+ cell. In some embodiments, the cell is a CD8+ T cytotoxic lymphocyte cell selected from the group consisting of naive CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cells, and bulk CD8+ T cells. In some embodiments of the cell, the cell is a CD4+ T helper lymphocyte cell selected from the group consisting of naive CD4+ T cells, central memory CD4+ T cells, effector memory7CD4+ T cells, and bulk CD4+ T cells. In some embodiments, the cell is a CD4-CD8- double negative cell. In some embodiments, the cell is selected from the group of consisting of a T cell, a CD4+ T cell, a CD8+ T cell, a regulatory7T cell (Treg), a gamma delta T cell (y8T), an invariant natural killer T (iNKT) cell, a mucosal associated invariant T (MAIT) cell, a macrophage, a monocyte, a natural killer (NK) cell, a tumor infiltrating lymphocyte (TIL), a cytotoxic T cell, a T helper cell, a memory T cell, a central memory T (TCM) cell, a stem memory T (TSCM) cell, a stem-celllike memory7T cell (or stem-like memory T cells), an effector memory T (TEM) cell, a TEMRA (CD45RA+) cell, an effector T cell, a Thl cell, a Th2 cell, a Th9 cell, a Thl7 cell, a Th22 cell, a Tfh (follicular helper) cell, a natural killer T (NKT) cell, a transitional memory T (TTM) cell, a terminal effector T (TTE) cell, a naive T (TN) cell, a hematopoietic stem cell, and a progenitor cell of the lymphoid lineage. In some embodiments, the cell is a T cell selected from the group consisting of a regulatory T cell (Treg), a gamma delta T cell, a CD8+ T cell, an invariant iNKT cell, a MAIT cell, a CAR T cell, a tumor-infiltrating lymphocy te, and an engineered T cell comprising a transcriptional receptor. In some embodiments, the cell can be obtained by leukapheresis performed on a sample obtained from a subject. In some embodiments, the subject is a human patient.

[0284] In another aspect, provided herein are cell cultures including at least one recombinant cell as disclosed herein, and a culture medium. Generally, the culture medium can be any suitable culture medium for culturing the cells described herein. Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application. Methods and systems suitable for generating and maintaining cell cultures are known in the art.Pharmaceutical Compositions

[0285] In some embodiments, the nucleic acids, and recombinant cells of the disclosure can be incorporated into compositions, including pharmaceutical compositions. Such compositions generally include the nucleic acids, and / or recombinant cells, and a pharmaceutically acceptable excipient, e.g., carrier.

[0286] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™. (BASF, Parsippany, N.J.), or phosphate buffered saline (PBS). In all cases, the composition should be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants, e.g., sodium dodecyl sulfate. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal. and the like. In many cases, it will be generally to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0287] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0288] In some embodiments, the chimeric receptors and Notch receptors of the disclosure can 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: 1006-10, 2002), or Putnam (Am. J. Health Syst. Pharm. 53: 151-60, 1996, erratum at Am. J. Health Syst. Pharm. 53:325, 1996).Methods of the Disclosure

[0289] Administration of any one of the therapeutic compositions described herein, e.g., nucleic acids, recombinant cells, and pharmaceutical compositions, can be used to treat patients for relevant health conditions or diseases, such as cancers and chronic infections. In some embodiments, the nucleic acids, recombinant cells, and pharmaceutical compositions described herein can be incorporated into therapeutic agents for use in methods of treating an individual who has, who is suspected of having, or who may be at high risk for developing one or more autoimmune disorders or diseases associated with checkpoint inhibition. Exemplary' autoimmune disorders and diseases can include, without limitation, celiac disease, t pe 1 diabetes, Graves’ disease, inflammatory' bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0290] Accordingly, in one aspect, some embodiments of the disclosure relate to methods for inhibiting an activity of a target cell in an individual, the methods include administering to the individual a first therapy including one or more of nucleic acids, recombinant cells, and pharmaceutical compositions as disclosed herein, wherein the first therapy inhibits the target cell. For example, the target cell may be inhibited if its proliferation is reduced, if its pathologic or pathogenic behavior is reduced, if it is destroyed or killed, etc. Inhibition includes a reduction of the measured pathologic or pathogenic behavior of at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%. In some embodiments, the methods include administering to the individual an effective number of the recombinant cells disclosed herein, wherein the recombinant cells inhibit an activity' of the target cells in the individual. Generally, the target cells of the disclosed methods can be any cell ty pe in an individual and can be, for example an acute myeloma leukemia cell, an anaplastic lymphoma cell, an astrocytoma cell, a B-cell cancer cell, a breast cancer cell, a colon cancer cell, an ependymoma cell, an esophageal cancer cell, a glioblastoma cell, a glioma cell, a leiomyosarcoma cell, a liposarcoma cell, a liver cancer cell, a lung cancer cell, a mantle cell lymphoma cell, a melanoma cell, a neuroblastoma cell, a non-small cell lung cancer cell, an oligodendroglioma cell, an ovarian cancer cell, a pancreatic cancer cell, a peripheral T-cell lymphoma cell, a renal cancer cell, a sarcoma cell, a stomach cancer cell, a carcinoma cell, a mesothelioma cell, or a sarcoma cell. In some embodiments, the target cell is a pathogenic cell.

[0291] In another aspect, some embodiments of the disclosure relate to methods for the treatment of a health condition (e.g., disease) in an individual in need thereof, the methods include administering to the individual a first therapy including one or more of therecombinant cells including a chimeric receptor as disclosed herein, and / or pharmaceutical compositions as disclosed herein, wherein the first therapy treats the health condition in the individual. In some embodiments, the methods include administering to the individual a first therapy including an effective number of the recombinant cells as disclosed herein, wherein the recombinant cells treat the health condition.

[0292] In another aspect, some embodiments of the disclosure relate to methods for assisting in the treatment of a health condition (e.g., disease) in an individual in need thereof, the methods including administering to the individual a first therapy including one or more of chimeric receptor s, Hinge-Notch receptors, nucleic acids, recombinant cells, and pharmaceutical compositions as disclosed herein, and a second therapy, wherein the first and second therapies together treat the disease in the individual. In some embodiments, the methods include administering to the individual a first therapy including an effective number of the recombinant cells as disclosed herein, wherein the recombinant cells treat the health condition.Administration of recombinant cells to an individual

[0293] In some embodiments, the methods of the disclosure involve administering an effective amount of the recombinants cells of the disclosure to an individual in need of such treatment. This administering step can be accomplished using any method of implantation delivery in the art. For example, the recombinant cells of the disclosure can be infused directly in the individual’s bloodstream or otherwise administered to the individual.

[0294] In some embodiments, the methods disclosed herein include administering, which 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 such that a desired effect(s) is / are produced. The recombinant cells or their differentiated progeny can be administered by any appropriate route that results in delivery to a desired location in the individual where at least a portion of the administered cells or components of the cells remain viable. The period of viability of the cells after administration to an individual can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the lifetime of the individual, i.e., long-term engraftment.

[0295] When provided prophylactically, the recombinant cells described herein can be administered to an individual in advance of any symptom of a disease or condition to be treated. Accordingly, in some embodiments the prophylactic administration of arecombinant cell population prevents the occurrence of symptoms of the disease or condition.

[0296] When provided therapeutically in some embodiments, recombinant cells are provided at (or after) the onset of a symptom or indication of a disease or condition, e.g., upon the onset of disease or condition.

[0297] For use in the various embodiments described herein, an effective amount of recombinant cells as disclosed herein, can be at least 102cells, at least 5 * 102cells, at least 103cells, at least 5 x io3cells, at least 104cells, at least 5 x io4cells, at least 105cells, at least 2 x io5cells, at least 3 x io5cells, at least 4 x io5cells, at least 5 x io5cells, at least 6 x 105cells, at least 7 x io5cells, at least 8 x io5cells, at least 9 x io5cells, at least 1 x io6cells, at least 2 x io6cells, at least 3 x io6cells, at least 4 x io6cells, at least 5 x io6cells, at least 6 x io6cells, at least 7 x io6cells, at least 8 x io6cells, at least 9 x io6cells, or multiples thereof. The recombinant cells can be derived from one or more donors or can be obtained from an autologous source. In some embodiments, the recombinant cells are expanded in culture prior to administration to an individual in need thereof.

[0298] In some embodiments, the delivery of a recombinant cell composition (e.g., a composition including a plurality of recombinant cells according to any of the cells described herein) into an individual by a method or route results in at least partial localization of the cell composition at a desired site. A composition including recombinant cells can be administered by any appropriate 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 delivered, e.g., at least 1 x io4cells, is delivered to the desired site for a period of time. Modes of administration include injection, infusion, instillation. "‘Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrastemal injection and infusion. In some embodiments, the route is intravenous. For the delivery' of cells, delivery by injection or infusion is a preferred mode of administration.

[0299] In some embodiments, the recombinant cells are administered systemically, e g., via infusion or injection. For example, a population of recombinant cells are administered other than directly into a target site, tissue, or organ, such that it enters, the individual’s circulatory’ system and, thus, is subject to metabolism and other similar biological processes.

[0300] The efficacy of a treatment including any of the compositions provided herein for the treatment of a disease or condition can be determined by a skilled clinician. However, one skilled in the art will appreciate that a treatment is considered effective if any one or all of the signs or symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of an individual to worsen as assessed by decreased hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g.. causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

[0301] 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 one who has, is suspected of having, or is at risk for a disease. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

[0302] 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. The diseases suitable for being treated by the compositions and methods of the disclosure include, but are not limited to, cancers, autoimmune diseases, inflammatory diseases, and infectious diseases. In some embodiments, the disease is a cancer or a chronic infection.

[0303] Methods for CAR design, delivery and expression in T cells, and the manufacturing of clinical -grade CAR-T cell populations are known in the art. See, for example, Lee et al., Clin Cancer Res (2012) 18(10):2780-90, hereby incorporated by reference in its entirety. For example, the engineered CARs may be introduced into T cells using retroviruses, which efficiently and stably integrate a nucleic acid sequence encoding the chimeric antigen receptor into the target cell genome.

[0304] 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 using a suitable Cas protein such Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, CaslOd, Casl2a (Cpfl), Casl3a (C2c2), Casl3b, Casl3d, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), CasX, CasY, Cse4 (or CasC). Cscl, Csc2, Csa5. Csn2. Csm2, Csm3, Csm4. Csm5, Csm6, Cmrl. Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csxl O, Csxl6, CsaX, Csx3, Cszl , Csxl5, Csfl , Csf2, Csf3, Csf4, and Cui 966, etc.).

[0305] In some embodiments, a recombinant adeno-associated virus (AAV) vector can be used for delivery. Techniques to produce rAAV particles, in which an AAV genome to be packaged that includes the polynucleotide to be delivered, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (e.g.. not in) the rAAV genome, and helper virus functions. The AAV rep and cap genes can be from any AAV serotype for which recombinant virus can be derived, and can be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV -2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV -9, AAV-10, AAV-11, AAV-12, AAV-13 and AAV rh.74. Production of pseudotyped rAAV is disclosed in, for example, international patent application publication number WO 01 / 83692.

[0306] The CAR-T cells, once they have been expanded ex vivo in response to, for example, an autoimmune disease antigen, can be reinfused into the subject in a therapeutically effective amount.

[0307] The precise amount of CAR T cells to be administered can be determined by a physician with consideration of individual differences in age, weight, extent of disease and condition of the subject.

[0308] Administration of T cell therapies may be defined by number of total cells per infusion or number of cells per kilogram of body weight, especially for pediatric subjects (e.g., patients). As T cells replicate and expand after transfer, the administered cell dose may not resemble the final steady-state number of cells. In some embodiments, a pharmaceutical composition including the CAR T cells of the present disclosure may be administered at a dosage of 104 to 1010 total cells. In another embodiment, a pharmaceutical compositionincluding the CAR T cells of the present disclosure may be administered at a dosage of 103 to 108 cells / kg body weight, including all integer values within those ranges.

[0309] Compositions including the CAR T cells of the present disclosure may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are known in the art (see, for example, Rosenberg et al., New Engl J Med, (1988) 319: 1676). The optimal dosage and treatment regimen for a particular subject can be determined by one skilled in the art by monitoring the subject for signs of disease and adjusting the treatment accordingly.

[0310] In some embodiments, administration of any of the compositions embodied herein, for the treatment of, for example, an autoimmune or inflammatory disease, can be combined with other cell-based therapies, for example, stem cells, antigen presenting cells, pancreatic islets etc.

[0311] The composition of the present disclosure may be prepared in a manner known in the art and in a manner suitable for parenteral administration to mammals, particularly humans, including a therapeutically effective amount of the composition alone, with one or more pharmaceutically acceptable carriers or diluents.

[0312] The term “pharmaceutically acceptable carrier” as used herein means any suitable carriers, diluents or excipients. These include all aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers and solutes, which render the composition isotonic with the blood of the intended recipient; aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents, dispersion media, antifungal and antibacterial agents, isotonic and absorption agents and the like. It will be understood that compositions of the present disclosure may also include other supplementary physiologically active agents.

[0313] The carrier must be pharmaceutically “acceptable” in the sense of being compatible with the other ingredients of the composition and not injurious to the subject. 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 method well known in the art of pharmacy. Such methods include preparing the carrier for association with the CAR-T cells. In general, the compositions are prepared by uniformly and intimately bringing into association any active ingredients with liquid carriers.

[0314] In some embodiments, the composition is suitable for parenteral administration. In another embodiment, the composition is suitable for intravenous administration.

[0315] Compositions suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bactericides and solutes, which render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.Additional Therapies

[0316] As discussed above, any one of the compositions as disclosed herein, e.g., the chimeric receptors, recombinant nucleic acids, recombinant cells, cell cultures, and pharmaceutical compositions described herein can be administered to a subject in need thereof as a single therapy (e.g., monotherapy). In addition or alternatively, in some embodiments of the disclosure, the chimeric receptors, recombinant nucleic acids, recombinant cells, cell cultures, and pharmaceutical compositions described herein can be administered to the subject in combination with one or more additional therapies, e.g.. at least one, two, three, four, or five additional therapies. Suitable therapies to be administered in combination with the compositions of the disclosure include, but are not limited to chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, targeted therapy, and surgery. Other suitable therapies include therapeutic agents such as chemotherapeutics, anti-cancer agents, and anti-cancer therapies.

[0317] Administration ‘’in combination with” one or more additional therapies includes simultaneous (concurrent) and consecutive administration in any order. In some embodiments, the one or more additional therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery. The term chemotherapy as used herein encompasses anti-cancer agents. Various classes of anti-cancer agents can be suitably used for the methods disclosed herein. Non-limiting examples of anti-cancer agents include: alkylating agents, antimetabolites, anthracy clines, plant alkaloids, topoisomerase inhibitors, podophyllotoxin. antibodies (e.g., monoclonal or polyclonal), tyrosine kinase inhibitors (e.g., imatinib mesylate (Gleevec® or Glivec®)), hormone treatments, soluble receptors and other antineoplastics.

[0318] The present disclosure also contemplates the combination of the composition of the disclosure with other drugs and / or in addition to other treatment regimens or modalities such as surgery. When the composition of the present disclosure is used in combination with known therapeutic agents the combination may be administered either in sequence (either continuously or broken up by periods of no treatment) or concurrently or as an admixture. Inthe case of, for example, autoimmune diseases, treatment includes administering to the subject the compositions embodied herein, e.g. autologous T cells transduced or contacted with a CAR embodied herein and one or more anti-inflammatory agents and / or therapeutic agents. The anti-inflammatory agents include one or more antibodies which specifically bind to pro-inflammatory cytokines, e.g., pro-inflammatory cytokines such as IL-1, TNF, IL-6, GM-CSF, and IFN-y. In some embodiments, the antibodies are anti-TNFa, anti-IL-6 or combinations thereof. In some embodiments, one or more agents, other than antibodies can be administered which decrease pro-inflammatory cytokines, e.g. non-steroidal antiinflammatory drugs (NSAIDs). Any combination of antibodies and one or more agents can be administered which decrease pro-inflammatory cytokines.

[0319] Treatment in combination is also contemplated to encompass the treatment with either the composition of the disclosure followed by a known treatment, or treatment with a known agent followed by treatment with the composition of the disclosure, for example, as maintenance therapy. For example, in the treatment of autoimmune diseases, excessive and prolonged activation of immune cells, such as T and B lymphocytes, and overexpression of the master pro-inflammatory cytokine tumor necrosis factor alpha (TNF), together with other mediators such as interleukin-6 (IL-6), interleukin-1 (IL-1), and interferon gamma (IFN-y), play a central role in the pathogenesis of autoimmune inflammatory responses in rheumatoid arthritis (RA), inflammatory bowel disease (IBD), Crohn’s disease (CD), and ankylosing spondylitis (AS).

[0320] Non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, diseasemodifying anti-rheumatic drugs (DMARDs) are traditionally used in the treatment of autoimmune inflammatory diseases. NSAIDs and glucocorticoids are effective in the alleviation of pain and inhibition of inflammation, while DMARDs have the capacity of reducing tissue and organ damage caused by inflammatory responses. More recently, treatment for RA and other autoimmune diseases has been revolutionized with the discovery that TNF is critically important in the development of the diseases. Anti-TNF biologies (such as infliximab, adalimumab, etanercept, golimumab, and certolizumab pepol) have markedly improved the outcome of the management of autoimmune inflammatory diseases.

[0321] Non-steroidal anti-inflammatory drugs have the analgesic, antipyretic, and antiinflammatory effect, frequently used for the treatment of conditions like arthritis and headaches. NSAIDs relieve pain through blocking cyclooxygenase (COX) enzymes. COX promotes the production of prostaglandins, a mediator which causes inflammation and pain. Although NSAIDs have different chemical structures, all of them have the similartherapeutic effect, e g., inhibition of autoimmune inflammatory responses. In general, NSAIDs can be divided into two broad categories: traditional non-selective NSAIDs and selective cyclooxygenase-2 (COX-2) inhibitors (For a review, see, P. Li et al.. Front Pharmacol (2017) 8:460).

[0322] In addition to anti-TNF agents, the biologies targeting other proinflammatory cytokines or immune competent molecules have also been extensively studied and actively developed. For example, abatacept. a fully humanized fusion protein of extracellular domain of CTLA-4 and Fc fraction of IgGl , has been approved for the RA patients with inadequate response to anti-TNF therapy. The major immunological mechanism of abatacept is selective inhibition of co-stimulation pathway (CD80 and CD86) and activation of T cells. Tocilizumab, a humanized anti-IL-6 receptor monoclonal antibody was approved for RA patients intolerant to DMARDs and / or anti-TNF biologies. This therapeutic mAb blocks the transmembrane signaling of IL-6 through binding with soluble and membrane forms of IL-6 receptor. Biological drugs targeting IL-1 (anakinra), Thl immune responses (IL-12 / IL-23, ustekinumab). Thl7 immune responses (IL-17, secukinumab) and CD20 (rituximab) have also been approved for the treatment of autoimmune diseases (For a review see. P. Li et al.. Front Pharmacol (2017) 8:460).

[0323] Accordingly, in some embodiments, the methods of the disclosure include administration of a composition disclosed herein to a subject individually as a single therapy (e.g., monotherapy). In some embodiments, a composition of the disclosure is administered to a subject as a first therapy in combination with a second therapy. In some embodiments, the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery. In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy is administered at the same time as the second therapy. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and / or after the second therapy. In some embodiments, the first therapy and the second therapy are administered in rotation. In some embodiments, the first therapy and the second therapy are administered together in a single formulation.Methods for modulating an activity of a cell

[0324] In another aspect, provided herein are various methods for modulating an activity of a cell. The methods include the steps of (a) providing an effective amount of any of the recombinant cells provided herein, and (b) contacting it with a selected ligand, wherein binding of the selected ligand to the extracellular ligand-binding domain induces cleavage of a ligand-inducible proteolytic cleavage site and releases the intracellular domain comprising the intracellular signaling domain and the CARD11 polypeptide, wherein the CARD 11 polypeptide modulates an activity of the recombinant cell. One skilled in the art upon reading the present disclosure will appreciate that the disclosed methods can be carried out in vivo, ex vivo, or in vitro.

[0325] Non-limiting exemplary cellular activities that can be modulated using the methods provide herein include, but are not limited to. gene expression, proliferation, apoptosis, non- apoptotic death, differentiation, dedifferentiation, migration, secretion of a gene product, cellular adhesion, and cytolytic activity.

[0326] In some embodiments, the CARD11 polypeptide modulates T cell signaling through NF AT, NF-KB and / or AP-1 pathways and / or cytokine production.Methods for Simultaneously Inducing T Cell Signaling and Gene Regulation

[0327] The present disclosure also provides methods for simultaneously inducing T cell signaling and gene regulation in a T cell including (a) providing a T cell including the multichain chimeric polypeptides or the chimeric antigen receptors of the disclosure and (b) exposing the T cell to the selected ligand, wherein binding of a selected ligand to the extracellular ligand-binding domain simultaneously induces intracellular signaling and release of the CARD11 polypeptide.

[0328] Also provided herein are methods for simultaneously inducing T cell signaling and gene regulation in a T cell, the method including (a) providing a vector comprising a multichain chimenc polypeptide or CAR of the disclosure and (b) transducing a T cell with the vector, wherein binding of a selected ligand to the extracellular ligand-binding domain simultaneously induces intracellular signaling and release of the CARD11 polypeptide

[0329] In some embodiments, the induced intracellular signaling of the T cell modulates expression of a selected gene involved in proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, secretion of a molecule, cellular adhesion, and / or a cytolytic activity. In some embodiments, the released CARD11 polypeptide modulates IL-2 expression in the T cell.Systems and Kits

[0330] Also provided herein are systems and kits including the chimeric receptors and multi-chain receptors, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions provided and described herein as well as written instructions for making and using the same. For example, provided herein, in some embodiments, are systems and / or kits that include one or more of: a hybrid receptor and / or multi-chain receptor as described herein, a recombinant nucleic acids as described herein, a recombinant cell as described herein, or a pharmaceutical composition as described herein. In some embodiments, the systems and / or kits of the disclosure further include one or more syringes (including prefilled syringes) and / or catheters (including pre-filled syringes) used to administer one any of the provided hybrid receptor and / or multi-chain receptors, recombinant nucleic acids, recombinant cells, or pharmaceutical compositions to an individual. In some embodiments, a kit can have one or more additional therapeutic agents that can be administered simultaneously or sequentially with the other kit components for a desired purpose, e.g., for modulating an activity' of a cell, inhibiting a target cancer cell, or treating a health condition (e.g., disease) in an individual in need thereof.

[0331] Any of the above-described systems and kits can further include one or more additional reagents, where such additional reagents can be selected from: dilution buffers; reconstitution solutions, wash buffers, control reagents, control expression vectors, negative control polypeptides, positive control polypeptides, reagents for in vitro production of the chimeric receptor polypeptides.

[0332] In some embodiments, the components of a system or kit can be in separate containers. In some other embodiments, the components of a system or kit can be combined in a single container.

[0333] In some embodiments, a system or kit can further include instructions for using the components of the kit to practice the methods. The instructions for practicing the methods are generally recorded on a suitable recording medium. For example, the instructions can be printed on a substrate, such as paper or plastic, etc. The instructions can be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub-packaging), etc. The instructions can be present as an electronic storage data file present on a suitable computer readable storage medium, e g. CD-ROM, diskette, flash drive, etc. In some instances, the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source (e.g., via the internet), can be provided. An example of this embodiment is a kit that includes a web address where the instructions can be viewed and / or from which the instructions can bedownloaded. As with the instructions, this means for obtaining the instructions can be recorded on a suitable substrate.

[0334] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0335] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the inventors reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific j oumal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0336] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure and are to be included within the spirit and purview of this application.

[0337] Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. The disclosure of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purpose.

[0338] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the inventors reserve the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific j oumal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0339] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure and are to be included within the spirit and purview of this application.EXAMPLES

[0340] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology’, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature cited above.

[0341] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.EXAMPLE 1Materials and Methods

[0342] This Example describes the materials and methods used in Examples 2-9.

[0343] Lentiviral production. Pantropic VSV-G pseudotyped lentivirus was produced via transfection of Lenti-X 293T cells (Clontech # 11 131 D) with a transgene expression vector and the viral packaging plasmids pCMVdR8.91 and pMD2.G using Mirus TransIT-Lenti (Mirus #MIR 6606).

[0344] Cell lines. The cell lines used include K562 myelogenous leukemia cells (ATCC #CCL-243), Jurkat cells (Clone E6-1, ATCC# TIB-152), and A549 lung epithelial carcinoma cells (ATCC #CCL-18). K562s and A549s were lentivirally transduced to stably express human CD 19 or ALPPL2. A549s w ere additionally transduced to express the nuclear stain mKate2 to enable live cell imaging. Jurkat cells were transduced with retroviruses encoding fluorescent reporter constructs (Addgene 118095, 118094, and 118031) and stimulated with PMA / ionomycin to sort a line with high induction of each reporter. An iRFP fluorescent reporter was subcloned to replace mCherry in the previously described API -mCherry construct. Jurkat and K562 cells were cultured in RPMI + 10% FBS with penicillin / streptomycin and sodium pyruvate. A549 cells were cultured in DMEM + 10% FBS with penicillin / streptomycin.

[0345] Flow Cytometry. Cells were washed with PBS 2% FBS twice, stained with surface staining markers at room temperature for 20 minutes, washed twice, and resuspended in PBS 2% FBS before analysis on a BD FACSymphony X-50 Flow Cytometer.

[0346] Mouse T cell purification and retroviral transduction. CD8+ or CD3+ T cells were isolated from the spleens of C57BL / 6 or CD45.1+ OT-I mice using a mouse CD8+ T cell isolation kit (Stemcell Technologies) or Mouse Pan CD3+ T cell isolation kit (Biolegend). T cells were then cultured with 100 U / mL recombinant human IL-2(Peprotech), anti-CD3e (1 ug / mL) and anti-CD28 (0.5 ug / mL) overnight. For CAR experiments, T cells were stimulated overnight with anti-CD3 / CD28 beads (ThermoFisher). Retroviral supernatants were added to T cells in Retronectin (Takara) coated plates and spinduction w as performed for 1 hour at 2000 rpm at 30°C. Following transduction, T cells were resuspended and cultured in fresh media containing 100 U / mL IL-2 until adoptive transfer. Transduction efficiency was determined by flow cytometry prior to adoptive transfer.

[0347] Primary Human T Cell Isolation and Culture. Primary CD3+ T cells were isolated from anonymous donor blood after apheresis by negative selection (STEMCELL Technologies #15062 & 15063 or Biolegend #480131). Blood was obtained from Blood Centers of the Pacific (San Francisco, CA) as approved by the University Institutional Review Board. T cells were cryopreserved in RPMI-1640 (UCSF cell culture core) with 20% human AB serum (Valley Biomedical Inc., #HP1022) and 10% DMSO. After thawing, T cells were cultured in human T cell medium consisting ofX-VIVO 15 (Lonza #04-418Q), 5% Human AB serum and 10 mM neutralized N-acetyl L-Cysteine (Sigma- Aldrich #A9165) supplemented with 30 units / mL IL-2.

[0348] Human T cell lentiviral transduction. Primary T cells were thawed, and after 24 hours in culture, w ere stimulated with Human T-Activator CD3 / CD28 Dynabeads (Life Technologies #11131D). T cells were exposed to virus for 24 hours. At day 5 post T cell stimulation, the Dynabeads were removed and the T cells were expanded prior to use in assavs.EXAMPLE 2Generation of CAR-CARD 11 Constructs

[0349] This Example describes the design and construction of CAR-CARD 11 constructs.

[0350] Anti -human CD19-CAR constructs were generated which contain various components of the CARD 11 protein on the intracellular, C-terminus side of the CAR construct, after CD3zeta. Several constructs were generated with varying lengths of the CARD 11 protein included, which in each case included the N-terminal CARD domain and varying amounts of the coiled-coil domain. In addition, a previously reported gain-of- function mutation (VI 19E) was introduced into the CARD 11 sequence. Constructs were generated containing diverse co-stimulatory molecules aside from 41BB, including BAFFRand TACI, which have been previously shown have unique signaling capabilities in CAR-T cells.

[0351] Exemplar}- sequences of CAR-CARD 11 constructs generated are shown in Table 1 below.Table 1IllEXAMPLE 3CAR-CARD 11 Receptors Promote Antigen-Dependent Signaling in Jurkat Cells

[0352] This experiment was done to test the functionality of the CAR-CARD11 receptors.

[0353] In order to test the functionality of these receptors, Jurkat cells were transduced and CAR signaling activity was determined via fluorescent protein-based reporter outputs. These data indicated that across a multitude of lengths of CARD 11 protein incorporated and across distinct co-stimulatory domains, CAR-CARD 11 receptors were capable of transmitting antigen-dependent signals (FIG. 2).EXAMPLE 4CAR-CARD 11 Receptors Express in Primary Human T Cells

[0354] This experiment was done to test the expression of the CAR-CARD11 receptors.

[0355] Accordingly, the ability of our CAR-CARD 11 receptors to express in primary human T cells was tested. Importantly, we observed expression of these receptors on the surface of the cells across numerous lengths of CARD11 tested (FIG. 3).EXAMPLE 5Primary T Cells Expressing a CAR-CARD11 Receptor Can Kill Tumor Cells In Vitro

[0356] This experiment was done to analyze the functionality of the CAR-CARD 11 receptors.

[0357] Having established expression in primary human T cells, we tested functionality using an in vitro cancer cell killing assay. By measuring the number of CD 19+ tumor cells overtime by live cell imaging, we observed similar killing ability of the BBz-CAR, the CARD11-PIK3R3 overexpressing BBz-CAR, and the CAR-CARD11 receptor T cells (FIG. 4) Therefore, CAR-CARD 11 constructs are expressed and functional in primary human T cells.EXAMPLE 6Primary Mouse CD8+ T Cells Expressing a CAR-CARD 11 Receptor Display Improved Expansion to CAR, but not T Cell Receptor, Triggering

[0358] This experiment was done to analyze the functionality of the CAR-CARD 11 receptors.

[0359] Specifically, we tested the functionality of these receptors in mouse primary T cells. Using ALPPL2 as a target antigen and murine 41BB / CD3zeta. we generated a CARCARD 1 1 construct (FIG. 5A) and compared its ability to promote the in vitro expansion of mouse CD8+ T cells to various stimuli. Importantly, we observed that CAR-CARD11 T cells expanded only when stimulated by ALPPL2-expressing K562 cells and not when stimulated through the T cell receptor stimulation using CD3 / CD28 Dynabeads. In contrast, the CARD11-PIK3R3 expressing CAR T cells expanded in the presence of both ALPPL2- expressing K562s and T cell receptor stimulation using CD3 / CD28 Dynabeads (FIG. 5B). These data indicate that CAR-CARD11 constructs have enhanced in vitro functionality, and improved specificity of functionality in response to CAR and not to T cell receptor signaling as compared to CARD11-PIK3R3 expression in CAR T cells.EXAMPLE 7Primary Mouse CD8+ T Cells Expressing a CAR-CARD11 Receptor Display Enhanced Target Cell Killing

[0360] This experiment was done to analyze the functionality of the CAR-CARD 11 receptors.

[0361] CAR-CARD11 expressing T cells displayed enhanced cytotoxicity against ALPPL2 expressing target cells, as determined by live cell imaging (FIG. 6). Altogether, these data support the utility of incorporating novel CARD11 domains into CAR constructs.EXAMPLE 8Multi-chain CAR-CARD 11 Receptors Promote Antigen-Dependent Signaling in Jurkat Cells

[0362] We generated multiple iterations of CARD1 1 -containing anti-CDl 9 CAR constructs in the multi-chain architecture format of varying lengths, with or without PIK3R3 domains from the CARD11-PIK3R3 fusion protein. Jurkat cells transduced with these constructs in combination with a DAP12-BBz chain displayed antigen-specific upregulation of NF-kB activity when co-cultured with CD19 expressing K562 cells, supporting the potential utility of these receptors (FIG. 8).EXAMPLE 9Multi-chain CAR-CARD11 Receptors Express in Primary Human T Cells and Display Enhanced Functionality

[0363] The following experiment demonstrates the expression of the multi-chain receptors.

[0364] Having established multi-chain architecture based CARD11 receptors can signal in Jurkat cells in vitro, we next sought to characterize them in primary human T cells (FIG. 9A). Multi-chain based CARD11 containing receptor was expressed in primary T cells and could be co-transduced with DAP12-BBz chain (FIG. 9B). As compared to control GAL4 containing multi-chain cells, CAR-CARD 11 based multi-chain cells produced higher quantities of IL-2 in response to antigen (FIG. 9C). Therefore, incorporating CARD1 1 domains into multi-chain based signaling architectures improves their functionality.

[0365] While particular alternatives of the present disclosure have been disclosed, it is to be understood that various modifications and combinations are possible and are contemplated within the true spirit and scope of the appended claims. There is no intention, therefore, of limitations to the exact abstract and disclosure herein presented.INFORMAL SEQUENCE LISTING

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A chimeric antigen receptor comprising, from N-terminus to C-terminus: a) an extracellular binding domain (ECD) having a binding affinity for a selected ligand; b) a linking sequence; c) a transmembrane domain (TMD) having at least about 80% sequence identity to the transmembrane domain of a Type 1 transmembrane receptor and comprising one or more ligand-inducible proteolytic cleavage sites; and d) an intracellular domain (ICD) comprising, in any order:(i) an intracellular signaling domain (SD) comprising at least one costimulatory domain derived from a signaling molecule and an activation domain, and(ii) domain comprising a caspase recruitment domain (CARD) from caspase recruitment domain family member 11 (CARD! 1) or caspase recruitment domain family member 9 (CARD9) polypeptide comprising one or more mutations.

2. The chimeric antigen receptor of claim 1, wherein binding of the selected ligand induces proteolytic cleavage of the one or more proteolytic cleavage sites in the TMD.

3. The chimeric antigen receptor of claim 1, wherein the one or mutations in the CARD 11 polypeptide is a point mutation, a gene fusion, a substitution, a gain-of-function mutation, a stop-gain mutation, an insertion mutation, a deletion mutation, a duplication mutation or a translocation.

4. The chimeric antigen receptor of claim 1, wherein the CARD 11 polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 10 and comprising a substitution at an amino acid position selected from the group consisting of 361, 615, 634, 655, and 357 of SEQ ID NO: 10.

5. The chimeric antigen receptor of claim 1, wherein the CARD 9 polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,99% or 100% identity to SEQ ID NO: 66 and comprising a substitution at an amino acid position selected from the group consisting of 361, 615, 634, 655. and 357 of SEQ ID NO: 66.

6. The chimeric antigen receptor of any one of claims 4 or 5. wherein the substitution comprises Y361C, S615F, E634K. D357N, S655C, or combinations thereof.

7. The chimeric antigen receptor of any one of claims 1 to 6, wherein the mutation is a fusion between a CARD 11 polypeptide and a PIK3R3 polypeptide.

8. The chimeric antigen receptor of claim 7, wherein the fusion comprises a CARD domain and a coiled coil domain from the CARD11 or CARD9 polypeptide.

9. The chimeric antigen receptor of claim8, wherein the coiled coil domain is a truncated coiled coil domain.

10. The chimeric antigen receptor of claim 8 or 9, wherein the fusion protein further comprises an inhibitory domain from the CARD11 or CARD9 polypeptide.

11. The chimeric antigen receptor of any one of claims 7 to 10, wherein the fusion protein comprises an SH2 domain from PIK3R3.

12. The chimeric antigen receptor of any one of claims 1 to 11, wherein the chimeric antigen receptor does not comprise an SH2 domain from PIK3R3, or does not comprise an SH2 domain.

13. The chimeric antigen receptor of any one of claims 1 to 12, wherein the chimeric receptor comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 68, 70, 72, 74, 76, or 78.

14. The chimeric antigen receptor of any one of claims 1 to 12 comprises a mutation in the CARD domain corresponding to VI 19E of SEQ ID NO:45.

15. The chimeric antigen receptor of any one of claims 1 to 14, wherein the extracellular domain comprises an antigen-binding moiety capable of binding to a ligand on the surface of a cell.

16. The chimeric antigen receptor of claim 15, wherein the cell is a pathogenic cell.

17. The chimeric antigen receptor of claim 15, wherein the cell is a human cell.

18. The chimeric antigen receptor of claim 17, wherein the human cell is a tumor cell.

19. The chimeric antigen receptor of claim 18, wherein the human cell is a terminally differentiated cell.

20. The chimeric antigen receptor of any one of the preceding claims, wherein the ligand comprises a protein or a carbohydrate.

21. The chimeric antigen receptor of any one of the preceding claims, wherein the ligand is selected from the group consisting of CD1, CDla, CDlb, CDlc, CDld, CDle, CD2, CD3d, CD3e, CD3g, CD3e, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD24. CD25, CD27, CD28, CD30. CD33, CD34, CD38. CD40, CD44v6, CD45, CD46, CD47 CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD213A2, CD246, CD252, CD253, CD261, CD262, CD272, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295, CD339 (JAG1), CD340 (HER2), CEA, CLECL1, CLL-1, CLDN6, CLDN18.2, CS1, DLL3, LY6G6D, GCC, p53R175H, PRAME, EGFR, EGFRvIII, FGFR2, AFP, CA125, MUC-1, MAGE, ALPI, alkaline phosphatase placental-like 2 (ALPPL2). B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), KLK2, KLK3, Mesothelin, IL13Ra2, signal regulatory protein a (SIRPa), TCRalpha, TCRbeta, TSHR, GD2, GD3, Tn Ag, cMET, Axl, ROR1, ROR2, GPC1, GPC2, GPC3, FLT3, TAG72, CEA, EPCAM, KIT(CD 117). IL-13Ra2, IL-HRa, PSCA, PRSS21, VEGFR2. LewisY, PDGFRp, S SEA-4, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, NCAM, prostase, PAP,ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gplOO, bcr-abl, ty rosinase, EphA2, STEAP1. STEAP2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2. folate receptor beta, TEM1 / CD248, TEM7R, R0PN1, GPRC5D, CX0RF61, ALK, Polysialic acid, PLACE GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53. p53 mutant, p53R175H, KRAS, mutant KRAS, KRAS G12D, prostein, surviving, telomerase, PCTA-l / Galectin 8, MelanA / MARTl , Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2.CYP1B1. BORIS, SART3, PAX5. OY-TES1, LCK. AKAP-4, SSX2. RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70- 2,LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, FCRL5, IGLL1, PSMA, TROP2, citrullinated vimentin, the extracellular portion of the APRIL protein, and any combinations thereof.

22. The chimeric antigen receptor of any one of the preceding claims, wherein the ligand comprises or consists of KIR2D, NRP1, CD221, VEGFR, HGF, integrin a501, CD51, CD27, B7H3, NYES01, EGFR, cMET, BRD4, EpCAM, SSX, Fibronectin EDB, integrin al 101. DLL3, CD2. SSTR2, TROP2. KLK2, KLK3, STEAP2. STEAP1, PSMA, PSCA, LY6G6D, IL-1 a, CEA, C242 antigen, CanAg (MUC1 glycoform), CTAA16.88, UCY2C, IGF2BP3, p53R175H, NKG2D ligands, PRAME, MAGEA4, vimentin, HER2 / neu. CCR4, CCR5, R0PN1, R0R1, KK-LC-1, CA-125, folate receptor 1, folate receptor alpha, MUC 1. MUC16, MSLN, CLDN6. WT1, ALPPL2, KRASG12D, KRASG12V, CD70, MSLN, CLDN18.2, HPV E6 / E7, GPC3, IL-13Ra, EGFRv3, EGFR806, GD2, or CA9.

23. The chimeric antigen receptor of any one of the preceding claims, wherein the ligand comprises or consists of KIR2D, NRP1, CD221, VEGFR, HGF, integrin a5bl, CD51, CD27, B7H3, NYES01, EGFR, cMET, BRD4, EpCAM, SSX, Fibronectin EDB, or Integrin al 101.

24. The chimeric antigen receptor of any one of the preceding claims, wherein the ligand is selected from cell surface receptors, adhesion proteins, integrins, mucins, lectins, tumor-associated antigens, and tumor-specific antigens.

25. The chimeric antigen receptor of any one of the preceding claims, wherein the ligand is a tumor-associated antigen or a tumor-specific antigen.

26. The chimeric antigen receptor of any one of the preceding claims, wherein the extracellular ligand-binding domain comprises the ligand-binding portion of a receptor.

27. The chimeric antigen receptor of any one of the preceding claims, wherein the antigenbinding moiety is selected from the group consisting of an antibody, a nanobody, a diabody, a triabody, a minibody, an F(ab')2 fragment, an F(ab)v fragment, a single chain variable fragment (scFv), a single domain antibody (sdAb), and a functional fragment thereof.

28. The chimeric antigen receptor of claim 23, wherein the antigen-binding moiety comprises an scFv.

29. The chimeric antigen receptor of any one of the preceding claims, wherein the antigenbinding moiety specifically binds to a tumor-associated antigen selected from the group consisting of CD19, B7H3 (CD276), BCMA (CD269), ALPPL2, CD123, CD171, CD179a, CD20, CD213A2, CD22, CD24, CD246, CD272, CD30, CD33, CD38, CD44v6, CD46, CD71, CD97. CEA, CLDN6. CLECL1, CS-1, Claudin 18.

2. DLL3, GCC, LY6G6D, CLDN6, KLK2, KLK3, EGFR806, HER2p95, ADGRE2, MUC16, EGFR, EGFRvIII, ELF2M, EpCAM, EphA2, Ephrin B2, FAP, FLT3, GD2, GD3, GM3, GPRC5D, HER2 (ERBB2 / neu), IGLL1, IL-l lRa, KIT (CD 117), MUC1, NCAM, PAP, PDGFR-P, PRSS21, PSCA. PSMA, R0R1, SIRPa, SSEA-4. TAG72, TEM1 / CD248, TEM7R, TSHR, VEGFR2, ALPI, citrullinated vimentin, cMet, and Axl.

30. The chimeric antigen receptor of any one of the preceding claims, wherein the antigenbinding moiety specifically binds to a tumor-associated antigen selected from the group consisting of CD1, CD la, CD lb, CDlc, CD Id, CDle, CD2, CD3d, CD3e, CD3g,CD3e, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD28. CD30, CD33, CD34, CD38, CD40, CD44v6, CD45, CD46. CD47 CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95, CD97, CD123, CD134, CD140 (PDGFR4), CD152, CD154, CD158, CD171, CD178, CD179, CD179a, CD181 (CXCR1), CD182 (CXCR2), CD183 (CXCR3), CD210, CD213A2, CD246, CD252, CD253, CD261, CD262, CD272, CD273 (PD-L2), CD274 (PD-L1), CD276 (B7H3), CD279, CD295. CD339 (JAG1), CD340 (HER2), CEA, CLECL1, CLL-1, CLDN6, CLDN18.2, CS1, DLL3, LY6G6D, GCC, p53R175H, PRAME, EGFR, EGFRvIII, FGFR2, AFP, CA125, MUC-1, MAGE, ALPI, alkaline phosphatase placental-like 2 (ALPPL2), B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), KLK2, KLK3, Mesothelin, IL13Ra2, signal regulatory protein a (SIRPa), TCRalpha, TCRbeta, TSHR, GD2, GD3, Tn Ag, cMET, Axl, R0R1, R0R2, GPC1, GPC2, GPC3, FLT3, TAG72, CEA, EPCAM, KIT (CD117), IL-13Ra2, IL-HRa, PSCA, PRSS21, VEGFR2, LewisY, PDGFR , SSEA-4, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, MUC16, NCAM. prostase. PAP. ELF2M, Ephrin B2, IGF- I receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, STEAP1, STEAP2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, R0PN1, GPRC5D. CX0RF61, ALK, Polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1. ADRB3. PANX3, GPR20, LY6K. OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, p53R175H, KRAS, mutant KRAS, KRAS G12D, prostein, surviving, telomerase, PCTA-l / Gal ectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2,LAIRl, FCAR, LILRA2. CD300LF, CLEC12A. BST2, EMR2, LY75, FCRL5, IGLL1. PSMA, TROP2, citrullinated vimentin, the extracellular portion of the APRIL protein, and any combinations thereof.

31. The chimeric antigen receptor of claim 21, wherein the tumor-associated antigen is CD 19, BCMA, CEA, HER2, MUC1, CD20, ALPPL2, SIRPa, or EGFR.

32. The chimeric antigen receptor of claim 21, wherein the tumor-associated antigen is KIR2D, NRP1, CD221, VEGFR, HGF, integrm a501, CD51, CD27, B7H3, NYESO1, EGFR, cMET, BRD4, EpCAM, SSX, Fibronectin EDB, integrin al 101, DLL3, CD2, SSTR2, TROP2, KLK2, KLK3, STEAP2, STEAP1, PSMA, PSCA, LY6G6D, IL-la, CEA. C242 antigen, CanAg (MUC1 glycoform), CTAA16.88, UCY2C, IGF2BP3, p53R175H, NKG2D ligands, PRAME, MAGEA4, vimentin, HER2 / neu, CCR4, CCR5, ROPN1 , ROR1 , KK-LC-1 , CA-125, folate receptor 1 , folate receptor alpha, MUC1 , MUC16, MSLN, CLDN6, WT1, ALPPL2, KRASG12D, KRASG12V, CD70, MSLN, CLDN18.2, HPV E6 / E7, GPC3, IL-13Ra, EGFRv3, EGFR806, GD2, or CA9.

33. The chimeric antigen receptor of claim 21, wherein the tumor-associated antigen is KIR2D, NRP1, CD221, VEGFR, HGF, integrin a5bl, CD51, CD27, B7H3, NYES01, EGFR, cMET, BRD4, EpCAM, SSX, Fibronectin EDB, or Integrin alipi.

34. The chimeric antigen receptor of claim 31, wherein the tumor-associated antigen is CD 19, or ALPPL2.

35. The chimeric antigen receptor of any one of the preceding claims, wherein the linking polypeptide sequence comprises a hinge domain.

36. The chimeric antigen receptor of claim 35, wherein the hinge domain is capable of promoting oligomer formation of the chimeric polypeptide via intermolecular disulfide bonding.

37. The chimeric antigen receptor of claim 35, wherein the hinge domain is derived from a CD8a hinge domain, a CD28 hinge domain, a CD152 hinge domain, a PD-1 hinge domain, a CTLA4 hinge domain, an 0X40 hinge domain, an IgGl hinge domain, an IgG2 hinge domain, an IgG3 hinge domain, and an IgG4 hinge domain, or a functional variant of any thereof.

38. The chimeric antigen receptor of any one of the preceding claims, wherein the linking sequence is derived from the group selected from: a CD8a hinge domain or a functionalvariant thereof, a CD28 hinge domain or a functional variant thereof, 0X40 hinge domain or a functional variant thereof, and an IgG4 hinge domain or a functional variant thereof.

39. The chimeric antigen receptor of any one of the preceding claims, wherein the linking sequence is derived from a CD8a hinge domain or a functional variant thereof.

40. The chimeric antigen receptor of any one of the preceding claims, wherein the linking sequence is derived from a CD28 hinge domain or a functional variant thereof.

41. The chimeric antigen receptor of any one of the preceding claims, wherein the linking sequence comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 5.

42. The chimeric antigen receptor of any one of the preceding claims, wherein the one or more ligand-inducible proteolytic cleavage sites comprises a y secretase cleavage site.

43. The chimeric antigen receptor of any one of the preceding claims, wherein the TMD comprises a polypeptide sequence having at least 80% sequence identity to a transmembrane domain from a Type 1 transmembrane receptor and comprises a y- secretase cleavage site.

44. The chimeric antigen receptor of any one of the preceding claims, further comprising a stop-transfer-sequence (STS) positioned between the TMD and the ICD.

45. The chimeric antigen receptor of any one of the preceding claims, wherein the stoptransfer-sequence comprises a sequence having at least 70% sequence identity to a STS sequence from APLP1. APLP2, APP, TGBR3, CSF1R, CXCL16. CX3CL1, DAG1, DCC, DNER, DSG2, CDH1, GHR, HLA-A, IFNAR2, IGF1R, IL1R1, ERN2, KCNE1, KCNE2, CHL1, LRP1, LRP2, PTPRF, SCN1B, SCN3B, NPR3, NGFR, PLXDC2, PAM, AGER, ROBO1, SORCS3, SORCS1, SORL1, SDC1. SDC2, SPN, TYR, TYRP1, DOT, VASN, FLT1, CDH5, PKHD1, NECTIN1. KL, IL6R, EFNB1. CD44, CLSTN1, LRP8, PCDHGC3, NRG1, LRP1B, JAG2, EFNB2, DLL1, CLSTN2,EPCAM, ErbB4, KCNE3, CDH2, NRG2, PTPRK, BTC, EPHA4, IL1R2, KCNE4, SCN2B, Nradd, PTPRM, Notchl, Notch2, Notch3, or Notch4.

46. The chimeric antigen receptor of any one of the preceding claims, wherein the signaling molecule comprises a class 1 or a class 3 human membrane protein.

47. The chimeric antigen receptor of any one of the preceding claims, wherein the signaling molecule is selected from the group consisting of CD28, ICOS, CTLA4, PD1 , PD1H, BTLA, B71, B7H1, CD226, CRTAM, TIGIT, CD96, TIM1, TIM2, TIM3, TIM4, CD2, SLAM, 2B4, Lyl08, CD84, Ly9, CRACC, BTN1, BTN2, BTN3, LAIR1, LAG3,CD 160, 4- IBB, 0X40. CD27, GITR, CD30, TNFR1, TNFR2, HVEM, LT_R, DR3, DCR3, FAS, CD40, RANK, OPG, TRAILR1, TACI, BAFFR, BCMA, TWEAKR, EDAR, XEDAR, RELT, DR6, TROY, NGFR, CD22, SIGLEC-3, SIGLEC-5, SIGLEC- 7, KLRG1, NKR-P1A, ILT2, KIR2DL1, KIR3DL1, CD94-NKG2A, CD300b, CD300e, TREM1, TREM2, ILT7, ILT3. ILT4, TLT-1, CD200R, CD300a, CD300f, DC-SIGN, B7-2. Allergin- 1. LAT, BLNK, LAYN, SLP76, EMB-LMP1. HIV-NEF, HVS-TIP, HVS-ORF5, and HVS-stpC.

48. The chimeric antigen receptor of any one of the preceding claims, wherein the signaling molecule is selected from the list consisting of 0X40. ICOS, 4-1BB, CTLA4, CD28. CD30, CD2, CD27, and CD226.

49. The chimeric antigen receptor of any one of the preceding claims, wherein the activation domain comprises one or more immunoreceptor tyrosine-based activation motifs (IT AMs).

50. The chimeric antigen receptor of any one of the preceding claims, wherein the one or more IT AMs are derived from CD3L„ CD38, and / or CD3E.

51. The chimeric antigen receptor of any one of the preceding claims, wherein the one or more IT AMs have at least about 80, 85, 90, 95, 96, 97, 98, 99, or 100% sequence identity to a CD3^ IT AM.

52. The chimeric antigen receptor of any one of the preceding claims, comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOS: 68, 70, 72, 74, 76, or 78.

53. A multi-chain chimeric polypeptide comprising:(a) a first polypeptide comprising (i) an extracellular ligand-binding domain having a binding affinity for a selected ligand, (ii) a first transmembrane domain (TMD) comprising a first modified interface and (iii) a first intracellular domain comprising a caspase recruitment domain family member 11 (CARD11) polypeptide comprising one or more mutations or a caspase recruitment domain family member 9 (CARD9) polypeptide comprising one or more mutations: and(b) a second polypeptide comprising (i) a second TMD comprising a second interface and (ii) a second intracellular domain comprising a signaling domain; wherein the first and the second modified interfaces each comprises an amino acid residue having a charged side chain, wherein the amino acid residues in the first and the second modified interfaces have opposite charges, wherein the first polypeptide is coupled to the second polypeptide via the first modified interface and the second modified interface and wherein binding of the selected ligand to the extracellular ligand-binding domain induces activity of the signaling domain and release of the CARD 11 or CARD9 polypeptide.

54. The multi-chain chimeric polypeptide of claim 53, wherein the first and the second polypeptides are not covalently connected.

55. The multi-chain chimeric polypeptide of claim 53, wherein the one or mutations in the CARD 11 polypeptide is a point mutation, a gene fusion, a substitution, a gain-of- function mutation, a stop-gain mutation, an insertion mutation, a deletion mutation, a duplication mutation or a translocation.

56. The multi-chain chimeric polypeptide of claim 53, wherein the CARD 11 polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 10 and comprising a substitution at an amino acid position selected from the group consisting of 361, 615. 634, 655. and 357 of SEQ ID NO: 10.

57. The multi-chain chimeric polypeptide of claim 56, wherein the substitution comprises Y361C, S615F, E634K, D357N, S655C, or combinations thereof.

58. The multi-chain chimeric polypeptide of any one of claims 53 to 57, wherein the mutation is a fusion between a CARD 11 polypeptide and a PIK3R3 polypeptide.

59. The multi-chain chimeric polypeptide of claim 58, wherein the fusion comprises a CARD domain and a coiled coil domain from the CARD11 or CARD9 polypeptide.

60. The multi-chain chimeric polypeptide of claim 59, wherein the coiled coil domain is a truncated coiled coil domain.

61. The multi-chain chimeric polypeptide of claim 59 or 60, wherein the fusion protein further comprises an inhibitory domain from the CARD11 or CARD9 polypeptide.

62. The multi-chain chimeric polypeptide of any one of claims 58 to 61, wherein the fusion protein further comprises an SH2 domain from PIK3R3.

63. The multi-chain chimeric polypeptide of any one of claims 53 to 62, wherein the first polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 84, 86, 88, 90, 92, or 94.

64. The multi-chain chimeric polypeptide of any one of claims 53 to 62 further comprising a mutation in the CARD domain corresponding to VI 19E of SEQ ID NO: 45.

65. The multi-chain chimeric polypeptide of claim 53, wherein binding of the selected ligand to the extracellular ligand-binding domain simultaneously induces the activity of the signaling domain and the release of the CARD11 or CARD9 polypeptide.

66. The multi-chain chimeric polypeptide of any one of claims 53 to 65, wherein the first polypeptide comprises (i) the extracellular ligand-binding domain, (ii) the first TMD.and (iii) the first intracellular domain, in order fromN-terminus to C-terminus of the first polypeptide.

67. The multi-chain chimeric polypeptide of any one of claims 53 to 66, wherein the first TMD comprises: (i) 10 to 25 contiguous valine residues, or (ii) a Notch 1 transmembrane domain.

68. The multi-chain chimeric polypeptide of any one of claims 53 to 57, wherein the second polypeptide comprises (i) the second TMD, and (ii) the second intracellular domain, in order from N-terminus to C-terminus of the second polypeptide.

69. The multi-chain chimeric polypeptide of any one of claims 53 to 68, wherein the first modified interface comprises a positively charged residue, and wherein the second interface comprises a negatively charged residue, and wherein the first polypeptide is coupled to the second polypeptide via the electrostatic force between the first and the second modified interfaces.

70. The multi-chain chimeric polypeptide of claim 69, wherein the positively charged residue is lysine or arginine.

71. The multi-chain chimeric polypeptide of claim 70, wherein (i) the first TMD comprises SEQ ID NO:9 or functional variants thereof and the lysine or arginine residue is at position selected from positions 10 to 14 of SEQ ID NO: 9 or (ii) the first TMD comprises SEQ ID NO: 9 and the lysine or arginine is at position selected from positions 8 to 11 of SEQ ID NO:9.

72. The multi-chain chimeric polypeptide of claim 71, wherein the lysine or arginine residue is (i) at position 12 of SEQ ID NO:9, or (ii) is at position 9 of SEQ ID NO:9.

73. The multi-chain chimeric polypeptide of any one of claims 53 to 72, wherein the extracellular domain comprises an antigen-binding moiety capable of binding to a ligand on the surface of a cell.

74. The multi-chain chimeric polypeptide of claim 73, wherein the antigen-binding moiety is selected from the group consisting of an antibody, a nanobody, a diabody, a triabody, a minibody, an F(ab')2 fragment, an F(ab) fragment, a single chain variable fragment (scFv), a single domain antibody (sdAb), and a functional fragment thereof.

75. The multi-chain chimeric polypeptide of any one of claims 53 to 74, wherein the ligand comprises a protein or a carbohydrate.

76. The multi-chain chimeric polypeptide of any one of claims 53 to 75, wherein the ligand is a tumor-associated antigen or a tumor-specific antigen.

77. The multi-chain chimeric polypeptide of any one of claims 53 to 76, wherein the ligand comprises cell-surface receptors, adhesion proteins, integrins, mucins, lectins, tumor associated antigens, or tumor specific antigens.

78. The multi-chain chimeric polypeptide of claim 77, wherein the ligand comprises CD1, CDla, CDlb, CDlc, CDld, CDle, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD7, CD8a, CD8b, CD19, CD20, CD21, CD22, CD23, CD25, CD27, CD28, CD33, CD34, CD40, CD45, CD48, CD52, CD59, CD66, CD70, CD71, CD72, CD73, CD79A, CD79B, CD80 (B7.1), CD86 (B7.2), CD94, CD95. CD134, CD140 (PDGFR4), CD152. CD154, CD158, CD178, 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), EGFR, FGFR2, CEA, AFP, CA125, MUC-1, MAGE, alkaline phosphatase, placental-like 2 (ALPPL2). B-cell maturation antigen (BCMA), green fluorescent protein (GFP), enhanced green fluorescent Protein (eGFP), or signal regulatory protein a (SIRPa).

79. The multi-chain chimeric polypeptide of any one of claims 73 to 78, wherein the cell is a human cell.

80. The multi-chain chimeric polypeptide of any one of claims 73 to 79, wherein the cell is a tumor cell.

81. The multi-chain chimeric polypeptide of any one of claims 53 to 80, wherein the signaling domain comprises CD3 zeta. TCR zeta, FcR y, FcR0, CD3y, CD3A, CD3e. CD5, CD22, CD79a, CD79b, CD278 (ICOS), Fc£ RI, DAP10, DAP12, or CD66d, signaling domain82. The multi-chain chimeric polypeptide of any one of claims 53 to 81, wherein the first polypeptide further comprises one or more of the following: a hinge domain, a ligandinducible proteolytic cleavage site, an autoproteolytic peptide sequence, a nuclear localization signal, a stop-transfer-sequence (STS).

83. The multi-chain chimeric polypeptide of claim 82, wherein the STS domain is a polybasic domain.

84. The multi-chain chimeric polypeptide of claim 83, wherein the polybasic domain comprises Notch-1 or Notch-2 STS domains.

85. The multi-chain chimeric polypeptide of any one of claims 82 to 84, wherein the autoproteolytic peptide sequence is from a porcine tescho virus- 1 2A (P2A), a foot-and- mouth disease virus (FMDV) 2A (F2A), an Equine Rhinitis A Virus (ERAV) 2A (E2A), a Thosea asigna vims 2A (T2A), a cytoplasmic polyhedrosis virus 2A (BmCPV2A), a Flacherie Vims 2A (BmIFV2A), or a combination thereof.

86. The multi-chain chimeric polypeptide of any one of claims 82 to 85 wherein the first polypeptide further comprises a hinge domain from CD8, CD28, 0X40, or IgG4.

87. The multi-chain chimeric polypeptide of claim 86, wherein the hinge domain comprises a truncated CD8a hinge domain.

88. The multi-chain chimeric polypeptide of any one of claims 83 to 87, wherein the ligandinducible proteolytic cleavage site is cleavable by gamma secretase.

89. The multi-chain chimeric polypeptide of any one of claims 53 to 88, wherein the multichain chimeric polypeptide is an immunoreceptor.

90. The multi-chain chimeric polypeptide of claim 89, wherein the immunoreceptor is a chimeric antigen receptor.

91. A recombinant nucleic acid comprising a nucleotide sequence encoding the chimeric receptor or multi-chain chimeric polypeptide of any one of the preceding claims.

92. The recombinant nucleic acid of claim 91, wherein the nucleotide sequence is incorporated into an expression cassette or an expression vector.

93. The recombinant nucleic acid of claim 92, wherein the expression vector is a viral vector.

94. The recombinant nucleic acid of claim 93. wherein the viral vector is a lentiviral vector, an adeno virus vector, an adeno-associated virus vector, or a retroviral vector.

95. A recombinant cell comprising the chimeric receptor or multi-chain chimeric polypeptide and / or the recombinant nucleic acid according to any one of the preceding claims.

96. The recombinant cell of claim 95, wherein the recombinant cell is a eukaryotic cell.

97. The recombinant cell of claim 96, wherein the eukaryotic cell is a mammalian cell.

98. The recombinant cell of claim 97, wherein the mammalian cell is an immune cell, a neuron, an epithelial cell, and endothelial cell, or a stem cell.

99. The recombinant cell of any one of claims 95-98, wherein the recombinant cell is selected from the group of consisting of a T cell, a CD4+ T cell, a CD8+ T cell, a regulatory' T cell (Treg), a gamma delta T cell (y5T), an invariant natural killer T (iNKT) cell, a mucosal associated invariant T (MAIT) cell, a macrophage, a monocyte, a natural killer (NK) cell, a tumor infiltrating lymphocyte (TIL), a cytotoxic T cell, a Thelper cell, a memory' T cell, a central memory T (TCM) cell, a stem memory T (TSCM) cell, a stem-cell-like memory T cell (or stem-like memory T cells), an effector memory T (TEM) cell, a TEMRA (CD45RA+) cell, an effector T cell, a Thl cell, a Th2 cell, a Th9 cell, a Thl 7 cell, a Th22 cell, a Tfh (follicular helper) cell, a natural killer T (NKT) cell, a transitional memory T (TTM) cell, a terminal effector T (TTE) cell, a naive T (TN) cell, a hematopoietic stem cell, and a progenitor cell of the lymphoid lineage.

100. The recombinant cell of any one of claims 95-98, wherein the recombinant is selected from the group consisting of a T cell, a macrophage, a monocyte, and a natural killer (NK) cell.

101. The recombinant cell of any one of claims 95-98, wherein the recombinant cell is a T cell.

102. The recombinant cell of claim 101, wherein the T cell is selected from the group consisting of a regulatory' T cell (Treg), a gamma delta T cell, a CD8+ T cell, an invariant iNKT cell, a MAIT cell, a CAR T cell, a tumor-infiltrating lymphocyte, and an engineered T cell comprising a transcriptional receptor.

103. The recombinant cell of any one of claims 95-102, wherein the recombinant cell is an autologous cell.

104. The recombinant cell of any one of claims 95-102, wherein the recombinant cell is an allogeneic cell.

105. The recombinant cell of any one of claims 95-104, wherein the recombinant cell is a primary cell.

106. The recombinant cell of any one of claims 95-104, wherein the recombinant cell is derived from a stem cell.

107. The recombinant cell of claim 98, wherein the immune cell is a B cell, a monocyte, a natural killer cell, a basophil, an eosinophil, a neutrophil, a dendritic cell, a macrophage, a regulatory T cell, a helper T cell, a cytotoxic T cell, or other T cell.

108. A method for making the recombinant cell according to any one of claims 95-107, comprising: a) providing a cell capable of protein expression; b) delivering the recombinant nucleic acid according to any one of claims 91-94 into the provided cell.

109. The method of claim 108. wherein the cell is obtained by leukapheresis performed on a sample obtained from a subject, and the cell is contacted ex vivo.

110. The method of claim 108, wherein the recombinant nucleic acid is encapsulated in a viral capsid or a lipid nanoparticle.

111. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, and one or more of the following: a) the recombinant nucleic acid according to any one of claims 91-94; and b) the recombinant cell according to any one of claims 95-107.

112. The pharmaceutical composition of claim 11, wherein the composition comprises a recombinant nucleic acid according to any one of claims 91-94, and a pharmaceutically acceptable carrier.

113. The pharmaceutical composition of claim 112, wherein the recombinant nucleic acid is encapsulated in a viral capsid or a lipid nanoparticle.

114. A system for modulating an activity of a cell, inhibiting a target cancer cell, or treating a health condition in an individual in need thereof, the system comprising one or more of the following: a) a chimeric receptor according to any one of claims 1-52; b) a multi-chain receptor according to any one of claims 53-90; c) a recombinant nucleic acid according to any one of claims 91-94;d) a recombinant cell according to any one of claims 95-107; and e) a pharmaceutical composition according to any one of claims 111-113.

115. A method for modulating an activity of a cell, the method comprising: a) providing a recombinant cell according to any one of claims 95-107; and b) contacting the recombinant cell with a selected ligand, wherein binding of the selected ligand to the extracellular ligand-binding domain induces cleavage of the ligand-inducible proteolytic cleavage site and releases the CARD1 1 or C ARD9 polypeptide, wherein the released CARD11 or CARD9 polypeptide modulates an activity of the recombinant cell.1 16. The method of claim 115, the contacting is carried out in vivo, ex vivo, or in vitro.

117. The method of any one of the preceding claims, wherein the activity of the cell to be modulated is selected from the group consisting of: expression of a selected gene, proliferation, apoptosis, non-apoptotic death, differentiation, dedifferentiation, migration, secretion of a molecule, cellular adhesion, and cytolytic activity.

118. A method for inhibiting an activity of a target cell in an individual, the method comprising administering to the individual an effective number of the recombinant cells according to any one of claims 95-107, wherein the recombinant cells inhibit an activity of the target cell in the individual.

119. The method of claim 118, wherein inhibiting an activity of a target cell comprises killing the target cell.

120. The method of claim 118, wherein the target cell is a pathogenic cell.

121. The method of claim 119, wherein the pathogenic cell is a cancer cell.

122. A method for the treatment of a health condition in an individual in need thereof, the method comprising administering to the individual a first therapy comprising an effective number of the recombinant cell according to any one of claims 95-107, wherein the recombinant cell treats the health condition in the individual.

123. The method of claim 122, further comprising administering to the individual a second therapy.

124. The method of claim 122, wherein the health condition is cancer.

125. The method of claim 123, wherein the second therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, and toxin therapy.

126. A method for enhancing the in vivo persistence of a T cell in a subject in need thereof, the method comprising administering to the individual an effective number of the recombinant cells according to any one of claims 95-107, wherein the recombinant cells enhance the in vivo persistence of a T cell in the individual.

127. The method of claim 126. wherein the T cell is selected from the group consisting of a regulatory (Treg), a natural killer (NK), a tumor infiltrating lymphocyte, and a CAR T cell.

128. The method of claim 126 or 127, wherein the subject has cancer or an autoimmune disease.

129. The method of claim 128, wherein the cancer is solid tumor.

130. The method of claim 128, wherein the cancer is a hematological cancer.

131. A method of simultaneously inducing T cell signaling and gene regulation in a T cell, the method comprising: (a) providing a T cell comprising the multi-chain chimeric polypeptide of any one of claims 53-90; and (b) exposing the T cell to a selected ligand, wherein binding of the selected ligand to the extracellular ligand-binding domain simultaneously induces the T cell signaling and release of the CARD11 or CARD9 polypeptide.

132. A method of simultaneously inducing T cell signaling and gene regulation in a T cell, the method comprising:(a) providing a vector encoding the multi-chain chimeric polypeptide of any one of claims 53-90,(b) transducing a T cell with the vector, and(c) providing a selected ligand to the T cell. wherein binding of the selected ligand to the extracellular ligand-binding domain of the multi-chain chimeric polypeptide expressed by the T cell simultaneously induces T cell signaling and release of the CARD11 or CARD9 polypeptide.

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