Synthetic t cell receptors for enhancing memory (STEM) t cells using new transmembranes and signaling domains

Abstract

The present invention relates to chimeric antigen receptors (CAR) containing TCR hinge and transmembrane domains. The present invention also relates to using transmembrane pairing to enhance the stability and specificity of dual antigen CARs. The present invention also relates to new CARs containing transmembrane and signaling domains from cytokine receptors.

Description

PATENT Docket No. H3131-00401 SYNTHETIC T CELL RECEPTORS FOR ENHANCING MEMORY (STEM) T CELLS USING NEW TRANSMEMBRANES AND SIGNALING DOMAINS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No.63 / 522,111, filed June 20, 2023, the entire contents of which is incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing that has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 19, 2023, is named H3131-00401_SL.xml and is 139,817 bytes in size. FIELD OF THE INVENTION

[0003] This invention relates to the generation and functional validation of chimeric antigen receptors (CARs) comprising T cell receptor (TCR) hinge and TCR transmembrane domains. This invention also relates to using transmembrane pairing to enhance the stability and specificity of dual antigen CARs. The present invention also relates to CARs engineered to contain transmembrane and signaling domains from cytokine receptors. BACKROUND OF THE INVENTION

[0004] Chimeric antigen receptor (CAR)-engineered T cell immunotherapy has produced clinical responses in blood cancers, and demonstrated the power of synthetic T cell signaling receptors as a promising approach to reprogram immune cells such as T cells to mediate antitumor immunity. Despite some success in using a CD19-CAR for treatment of blood cancer, approximately 30-50% of patients have disease relapse after one year of CAR-T therapy (Park, J.H. et al. Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia. N Engl J Med 378, 449-459 (2018). Majzner, R.G. & Mackall, C.L. Clinical lessons learned from the first leg of the CAR T cell journey. Nat Med 25, 1341-1355 (2019). Current CAR constructs contain four major components: an antigen-binding domain (such as anti- CD19 single-chain fragment variable region, ScFv), a hinge and transmembrane domain, an intracellular co-stimulatory domain, and a T cell signaling domain (Rafiq, S., Hackett, C.S. & 1 DM2\19774561.1PATENT Docket No. H3131-00401 Brentjens, R.J. Engineering strategies to overcome the current roadblocks in CAR T cell therapy. Nat Rev Clin Oncol 17, 147-167 (2020). Sadelain, M., Brentjens, R. & Riviere, I. The basic principles of chimeric antigen receptor design. Cancer Discov 3, 388-398 (2013). Kalos, M. & June, C.H. Adoptive T cell transfer for cancer immunotherapy in the era of synthetic biology. Immunity 39, 49-60 (2013).). The antitumor immunity of CARs could be regulated by the signaling domains, including co-stimulatory domains and a core T cell receptor signaling domain. Among many co-stimulatory signaling domains derived from various proteins such as CD28, 4-1BB (CD137), OX40, CD40, ICOS, and CD27 proteins (Rafiq, S., Hackett, C.S. & Brentjens, R.J. Engineering strategies to overcome the current roadblocks in CAR T cell therapy. Nat Rev Clin Oncol 17, 147-167 (2020).), CD28 and 4-1BB are most used in the CAR constructs. Although CD28 and 4-1BB signaling domain-containing CAR-T cell products are therapeutically effective, there are substantial differences in their proliferation, killing ability, metabolic state and T cell persistence (Kawalekar, O.U. et al. Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells. Immunity 44, 380-390 (2016). van der Stegen, S.J., Hamieh, M. & Sadelain, M. The pharmacology of second-generation chimeric antigen receptors. Nat Rev Drug Discov 14, 499- 509 (2015).). Recent several studies show that the screening of CAR signaling domain libraries derived from other signaling molecules could lead to identification of new costimulatory combinations to replace CD28 and / or 4-1BB signaling in the CARs or generate the next generation of CARs with comparable antitumor activity, or improved cytotoxicity and T cell persistence (Gordon, K.S. et al. Screening for CD19-specific chimaeric antigen receptors with enhanced signalling via a barcoded library of intracellular domains. Nat Biomed Eng 6, 855- 866 (2022). Goodman, D.B. et al. Pooled screening of CAR T cells identifies diverse immune signaling domains for next-generation immunotherapies. Sci Transl Med 14, eabm1463 (2022). Daniels, K.G. et al. Decoding CAR T cell phenotype using combinatorial signaling motif libraries and machine learning. Science 378, 1194-1200 (2022). Castellanos-Rueda, R. et al. speedingCARs: accelerating the engineering of CAR T cells by signaling domain shuffling and single-cell sequencing. Nat Commun 13, 6555 (2022).). By contrast, CD3ζ is the only core T cell signaling transduction domain used in almost all CAR constructs. CD3ζ chain is the 2 DM2\19774561.1PATENT Docket No. H3131-00401 important component of the TCR-CD3 complex, which plays a critical role in signaling transduction. The function of CD3ζ is to recruit Zap70 after phosphorylated by activated LCK in the immunoreceptor tyrosine-based activation motifs (ITAMs), when it is activated after TCR engagement. The ZAP70 binds to CD3ζ after its phosphorylation at Tyrosine (Tyr) 315, Tyr 319, and Tyr493 residuals by LCK, and then phosphorylates two scaffold proteins, LAT and SLP76, leading to the recruitment of downstream signaling molecules and T cell activation (Yan, Q. et al. Structural basis for activation of ZAP-70 by phosphorylation of the SH2-kinase linker. Mol Cell Biol 33, 2188-2201 (2013).).

[0005] The therapeutic efficacy and long-term cancer cure of CAR-T cell therapy is closely associated with T cell persistence. Furthermore, CAR-T cell therapy often cause severe toxicities, including cytokine-release syndrome (CRS) and neurological toxicities mainly triggered by high amounts of serum levels of inflammatory cytokines (Schmidts, A., Wehrli, M. & Maus, M.V. Toward Better Understanding and Management of CAR-T Cell-Associated Toxicity. Annu Rev Med 72, 365-382 (2021). Shah, N.N. et al. CD4 / CD8 T-Cell Selection Affects Chimeric Antigen Receptor (CAR) T-Cell Potency and Toxicity: Updated Results From a Phase I Anti-CD22 CAR T-Cell Trial. J Clin Oncol 38, 1938-1950 (2020). Davila, M.L. et al. Efficacy and toxicity management of 19-28z CAR T cell therapy in B cell acute lymphoblastic leukemia. Sci Transl Med 6, 224ra225 (2014).). SUMMARY

[0006] In one aspect, this disclosure features chimeric antigen receptors and T cells expressing a CAR. In some aspects, the CAR construct comprises an antigen recognition moiety (e.g., a single chain variable fragment (ScFv)), a hinge and transmembrane domain, and an intracellular T-cell activation moiety. Thus, in one aspect, this disclosure features a composition comprising a chimeric antigen receptor (CAR), or cells expressing a CAR, where the CAR comprises an antigen recognition moiety, a hinge and transmembrane (hinge / TM) domain, and an intracellular T-cell activation moiety.

[0007] In one aspect, this disclosure features CARs comprising hinge and transmembrane domains from human or non-human hinge and transmembrane domains, CD4, CD8, CD28, ZAP70, TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ, LCK, LAT, IL-1R, IL-2Rβ, 3 DM2\19774561.1PATENT Docket No. H3131-00401 IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα, or the common cytokine receptor γ chain, murine mTCRα, mTCRβ, mTCRγ, mTCRδ, mCD3γ, mCD3δ, mCD3ε, mCD3ζ, or any combination of any of the foregoing. In some aspects, the hinge / TM domain of the CARs featured herein is derived from a transmembrane domain of CD4, CD8, CD28, ZAP70, TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ, LCK, LAT, IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα, or the common cytokine receptor γ chain, or any combination thereof.

[0008] In some aspects, this disclosure features CARs with different lengths of hinge / TM domains of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ, LCK, LAT, IL-1R, IL- 2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα, the common cytokine receptor γ chain, and 4-1BB-ZAP327, including but not limited to the lengths of 23, 37, 65, 95, and 105 amino acids of the hinge / TM domains.

[0009] In one aspect, this disclosure relates to the development of CARs comprising transmembrane domains derived from cytokine receptors, including but not limited to IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα (such as SEQ ID NO: 49-90, 114-119 and 125-130), and the common cytokine receptor γ chain. In some aspects, this disclosure features CARs comprising IL-7R intracellular domain with or without co-stimulatory and T cell activation signaling domains. In some aspects, this disclosure features CARs comprising IL-15R with or without co-stimulatory and T cell activation signaling domains.

[0010] As featured herein, engineering the hinge and transmembrane domains in CAR constructs improves the therapeutic potential and safety of the CARs.

[0011] In some aspects, the antigen-binding domain of any CARs embodied herein is followed by the hinge and transmembrane domains, and can include or exclude a linker, followed by a costimulatory signaling domain.

[0012] In some aspects of this disclosure, the intracellular T-cell activation moiety of the CARs featured herein comprises a costimulatory signaling domain. In some aspects of this disclosure, the intracellular T-cell activation moiety of the CARs featured herein comprises a costimulatory signaling domain fused with a signaling domain, wherein the costimulatory 4 DM2\19774561.1PATENT Docket No. H3131-00401 signaling domain selected from a CD28, 4-1BB, CD3ζ, IL-2Rβ, IL-4Rα, IL-7R, IL9-R, IL- 12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21R, or a combination thereof. In some aspects, the CARs embodied herein include or exclude a costimulatory signaling domain derived from a CD28, 4-1BB, CD3ζ, signaling domain, or a ZAP70 kinase domain.

[0013] In some aspects of this disclosure, the costimulatory signaling domain comprises the replacement of CD3ζ with a ZAP70 kinase domain or a mutant or a variant thereof. In certain aspects, the intracellular T-cell activation moiety comprises or consists of CD28 or 4- 1BB costimulatory signaling domain fused with a signaling domain, such as, a ZAP70 kinase domain or a mutant or a variant thereof. In one aspect of this disclosure, any of the CARs embodied herein comprise a costimulatory signaling domain wherein CD3 is replaced by a ZAP70 kinase domain or a mutant or a variant thereof, which comprises or consists of, e.g., ZAP300 (SEQ ID NO:1), ZAP327 (SEQ ID NO: 2), ZAP338 (SEQ ID NO:3), ZAP255 (SEQ ID NO: 4), or ZAP280 (SEQ ID NO: 5) signaling domain or other signaling domain derived from ZAP70. In another aspect, any of the CARs embodied herein comprise a costimulatory signaling domain wherein the costimulatory signaling domain is derived from a 4-1BB signaling domain and a ZAP70 kinase domain or a mutant or a variant thereof, e.g. ZAP300 (SEQ ID NO:1), ZAP327 (SEQ ID NO: 2), ZAP338 (SEQ ID NO:3), ZAP255 (SEQ ID NO: 4), or ZAP280 (SEQ ID NO: 5), or a mutant or variant of . wherein the N-terminal amino acid is any of 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279. 280, 281, 282, 283, 284, 285, 286. 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338), and the C-terminal amino acid is 610, 611, 612, 613, 614, 615, 616, 617, 618 or 619 a.a.) of ZAP70 kinase.

[0014] In one aspect, this disclosure provides a CAR comprising: at least one extracellular binding domain comprising at least one antigen recognition moiety such as SEQ ID NO: 132 and SEQ id NO: 133; at least one hinge domain and at least one transmembrane domain or a fragment or variant thereof such as SEQ ID: 7-29, 91-98; and 5 DM2\19774561.1PATENT Docket No. H3131-00401 at least one intracellular signaling domain (such as ZAP300 (SEQ ID NO: 1), ZAP327 (SEQ ID NO: 2), ZAP338 (SEQ ID NO: 3), ZAP255 (SEQ ID NO: 4) or ZAP280 (SEQ ID NO: 5).

[0015] In certain aspects, the CAR may comprise a T-cell activation moiety.

[0016] In certain aspects of this disclosure, the extracellular binding domain of a CAR described herein is an antibody or an antigen-binding fragment thereof or receptor or ligand binding fragment thereof. In particular embodiments, the extracellular binding domain is a single chain variable fragment (scFv) such as SEQ ID NO: 133-144. In certain aspects, the extracellular binding domain may specifically bind, for example, an autoantigen or a B cell antigen, or a receptor ligand.

[0017] In one aspect, this disclosure relates to methods for generating and functionally validating CARs with hinge and transmembrane domains derived from the T cell receptors and related signaling molecules, e.g., cytokines, for increased T cell signaling, activation, function and persistence, but with low amounts of cytokines produced by CAR-T cells. The strategies to improve or prolong T cell persistence as featured herein are fundamentally important to T- cell immunotherapy, because therapeutic efficacy and long-term cure of cancer, autoimmune diseases, graft vs. host, aging, and other conditions by CAR-T cell therapy is closely associated with T cell persistence. T cell persistence relies on several key factors or mechanisms, including cell population size of stem-like memory and effector memory T cells, T cell proliferation, and resistance to T cell apoptosis and exhaustion. In one aspect, the CARs of this disclosure feature hinge and transmembrane domains derived from TCRs, signaling molecules and cytokine receptors are directly involved in T cell signaling, activation and cytokine production. For example, in some aspects, this disclosure relates to generating and functionally validating hinge and transmembrane (hinge / TM) domains derived from T cell receptors and their subunits (TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ), as well as membrane-anchored TCR signaling molecules (such as LCK, LAT), and the second panel of the hinge and transmembrane domains derived from cytokine receptors, including but not limited to IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα, and the common cytokine receptor γ chain. 6 DM2\19774561.1PATENT Docket No. H3131-00401

[0018] In one aspect, this disclosure includes CARs comprising a variable region fused to a modified human constant region, or to a non-human constant region which can be modified or unmodified. In some aspects the CAR comprises an antigen, e.g., cancer, autoimmune, or donor antigen specific variable region fused to a non-human, e.g., murine TCR constant region. In some aspects the TCR variable region comprises an alpha and a beta chain variable region fused to an alpha and a beta TCR constant region, respectively and can include any alpha and / or beta chain of any other aspect of this disclsoure. In some aspects, fusion of the TCR variable region to a modified or non-human constant region reduces mispairing between the chimeric TCR and an endogenous TCR.

[0019] In one aspect, the antigen-binding domain of any CAR embodied herein is followed by the hinge and transmembrane domains, with or without a linker. In some aspects, the antigen-binding domain of any CARs embodied herein is followed by a linker that can be 15- 25 amino acids in length. In one aspect the linker is, for example, (X1X2X3X4S)3-5 wherein any of X1-X4 is G or A. In one aspect the linker is, for example, (GGGGS)3-5 (SEQ ID NO: 148), such as SEQ ID NO: 131). In one aspect, for example, a CD19 ScFv and hinge and transmembrane domain with the linker (GGGGS)3 (SEQ ID NO: 131), followed by a 4-1BB costimulatory signaling domain and ZAP327 T cell activation domain (CD19TMαBBZAP327 CAR).

[0020] In one aspect, this disclosure relates to the functional analysis of CARs comprising TCR hinge and transmembrane domains. In yet another aspect, this disclosure relates to CARs expressing a single TCR transmembrane domain directing the CAR to form a complex with endogenous TCRs.

[0021] In one aspect, this disclosure relates to dual antigen CARs using TCR TMα-TMβ and TMγ-TMδ pairing. In some aspects, this disclosure features pairs of CARs, wherein a first CAR comprising a first ScFv that binds to a first target, TCR hinge / TMα, and T cell signaling domain; and a second CAR comprising a second ScFv that binds to a second target different from the first target, TCR hinge / TMβ, and no signaling domain, such that when paired together, the CARs pair then function to target each ScFv target individually or both antigen-expressing tumor cells. Similarly, in another aspect, this disclosure features pairs of CARs, wherein a first 7 DM2\19774561.1PATENT Docket No. H3131-00401 CAR comprising a first ScFv that binds to a first target, TCR hinge / TMγ, and T cell signaling domain; and a second CAR is comprising a second ScFv that binds to a second target different from the first target, TCR hinge / TMδ, and no signaling domain, such that when paired together, the CARs pair then function to target each ScFv target individually or both antigen-expressing tumor cells. In some aspects, the dual antigen CARs use non-human, e.g. murine, TCR α / β and TCR γ / δ pairing.

[0022] In one aspect, this disclosure relates to the development of dual antigen CARs using leucine-zipper dimerization. In some aspects, the leucine zipper is designed downstream of the TCR hinge / TM domain: the pairs of CARs include a first CAR comprising a first ScFv that binds to a first target, TCR hinge / TMα, a leucine zipper, and T cell signaling domain and a second CAR comprising a second ScFv that binds to a second target different from the first target, TCR hinge / TMβ, a leucine zipper, and no signaling domain, such that when paired together, the CARs pair then function to target each ScFv target individually or both antigen- expressing tumor cells; or the pairs of CARs include a first CAR comprising a first ScFv that binds to a first target, TCR hinge / TMγ, a leucine zipper, and T cell signaling domain and a second CAR comprising a second ScFv that binds to a second target different from the first target, TCR hinge / TMδ, a leucine zipper, and no signaling domain, such that when paired together, the CARs pair then function to target each ScFv target individually or both antigen- expressing tumor cells. In some aspects, the leucine zipper is designed upstream of the TCR hinge / TM domain.

[0023] In some aspects, the CARs or TCRs include or exclude, for example, antigen recognition moieties, e.g., ScFvs and / or antibody or antibody fragments and / or receptor or receptor fragmentsthat specifically bind to one or more proteins or antigens set forth in the detailed description herein, or any combination thereof.

[0024] In some aspects, the CARs or TCRs include or exclude, for example, an intracellular T-cell activation moiety comprising a costimulatory signaling domain that includes or excludes or is selected from CD28, 4-1BB (CD137), ICOS (CD278), CD27, OX40 (CD134), MyD88, EphB6, TSLP-R, HLA-DR, CO2, CD4, CD5, CD7, CDS, CD8alpha, CD8beta, CD11a, CD11b, CD11e, CD11d, CD18, CD19, CD19a, CD29, CD30, CD30L, CD40, CD40L 8 DM2\19774561.1PATENT Docket No. H3131-00401 (CD154), CD48, CD49a, CD49D, CD49f, CD58, CD53, ICAM-1 (CD54), CD69, CD70, CD80 (B7-l), CD82, CD83, CD84, CD86 (B7-2), CD90, CD96, CD100, CD103, CD122, CD132, CD150 (SLAMFl), CD160 (BY55), CD162 (DNAMl), CD223 (LAG3), CD226, CD229, CD244, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-Ll), CD278, LAT, lymphocyte function-associated antigen-I (LFA-1), LIGHT, NKG2C, NKG2D, NK.p30, NKp44, NKp46, NKp80 (KLRFl), DAPl0, DAP12, LAG-3, 2B4, CARDI, CTLA-4 (CD152), TRIM, ZAP70, FcERIγ, 4-lBBL, BAFF, GADS, GITR, GITR-L, BAFF-R, HVEM, CD27L, OX40L, TACl, BLAME, CRACC, CD2F-10, NTB-A, integrin α4, integrin α4β1, integrin α4β7, IA4, ICAM- 1, IL-2Ralpha, IL-2Rbeta, IL-2Rgamma, IL-4Ralpha, IL-7Ralpha, IL-9Ralpha, IL-12R, IL- 21Ralpha, B7-H2, B7-H3, CD83 ligand, PD-1, SLP-76, Toll-like receptors (TLRs, such as TLR2), ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGBl, ITGB2, ITGB7, KIRDS2, LTBR, PAG / Cbp, PSGLI, SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLAl, VLA-6, BTLA, ikaros, LAG-3, LMIR, CEACAMl, CRTAM, TCLIA, DAP12, TIM-1, Dectin-1, PDCD6, PD-1, TIM-4, TSLP, or any combination thereof.

[0025] In one aspect this disclosure also relates to methods of using any CAR-T cells of any preceding aspect or any aspect or embodiment disclosed herein in the treatment of a disease or disorder selected from the group consisting of cancer, an inflammatory disease, an autoimmune disease, an allergic disease, an organ transplantation condition, an infectious disease and symptoms of aging. In some aspects, the present disclosure features a population of immune cells expressing one or more CARs, dual antigen CARs, TCRs, bispecific antibodies and / or bispecific, trispecific, or multivalent multispecific binding proteins as featured herein. In some aspects, the immune cells are selected from the group consisting of T cells, natural killer (NK) cells, ab T cells, γδ T cells, double negative (DN) cells, regulatory immune cells, regulatory T (Treg) cells, effector immune cells, effector T cells, B cells, and myeloid-derived cells, and any combination thereof, wherein the immune cells are optionally human cells. In particular embodiments, the population comprises Treg cells, wherein the Treg cells are optionally human cells.

[0026] Generally, in one aspect, the T cells (e.g., Treg cells) of the invention are expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex 9 DM2\19774561.1PATENT Docket No. H3131-00401 associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the cells. In particular, the T cells (e.g., Treg cells) may be stimulated as described herein, such as by contact with an anti-CD3 antibody or antigen-binding fragment thereof. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD19 antibody, under conditions appropriate for stimulating proliferation of the T cells. Other expansion methods known in the art can be used (Berg et al, Transplant Proc. 30(8):3975-3977 (1998); Haanen et al, J. Exp. Med. 190(9): 1319-1328 (1999); Garland et al, J. Immunol Meth. 227(l-2):53-63 (1999)).

[0027] In some aspects, this disclosure features T cells that are genetically modified by expression of one or more CARs as described herein, which may also have been genetically modified. In some aspects, the genetically modified immune cell of this disclosure is an allogeneic immune cell. In such aspects, the cell may be engineered to reduce host rejection to the cell (graft rejection) and / or the cell’s potential attack on the host (graft- versus-hostdisease). By way of example, in some aspects the cell is engineered to have a null genotype forone or more of the following: (i) T cell receptor (TCR alpha chain or beta chain); (ii) a polymorphic major histocompatibility complex (MHC) class I or II molecule (e.g., HLA-A, HLA-B, or HLA-C; HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR; or 2-microglobulin (B2M)); (iii) a transporter associated with antigen processing (e.g., TAP-l or TAP-2); (iv) Class II MHC transactivator (CUT A); (v) a minor histocompatibility antigen (MiHA; e.g., HA-1 / A2, HA-2, HA-3, HA-8, HB-1H, or HB-1Y); and (vi) any combination thereof.

[0028] In one aspect an immune cell as featured herein is engineered such that cell surface expression of TCR molecules is downregulated. For example, in some aspects, the allogeneic immune cell is engineered such that it does not express any functional TCR on its surface, engineered such that it does not express one or more subunits that comprise a functional TCR, or engineered such that it produces very little functional TCR on its surface. In some aspects, the T cell can express a substantially impaired TCR, e.g., by expression of mutated or truncated 10 DM2\19774561.1PATENT Docket No. H3131-00401 forms of one or more of the subunits of the TCR. The term "substantially impaired TCR" as used herein, means that this TCR will not elicit an adverse immune reaction in a host.

[0029] In some aspects, the allogeneic immune cell is engineered such that it does not express a functional HLA on its surface. For example, an immune cell as featured herein is engineered such that cell surface expression of HLA, e.g., HLA class 1 and / or HLA class II and / or non-classical HLA molecules is downregulated.

[0030] In some aspects, the T cell can lack a functional TCR and / or a functional HLA such as HLA class I and / or HLA class II.

[0031] Modified immune cells, for example, immune cells that lack expression of a functional TCR and / or HLA, can be obtained by any suitable means, including a knock out or knock down of one or more subunit of TCR and / or HLA. For example, the Treg cell can include a knock down of TCR and / or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR) transcription-activator like effector nuclease (TALEN), zinc finger endonuclease (ZFN), meganuclease (mn, also known as homing endonuclease), or megaTAL (combining a TAL effector with a mn cleavage domain).

[0032] In some aspects of this disclosure, the nucleic acid encoding a CAR as described herein is inserted at a specific locus in the genome of an immune cell, such as, for example, at the locus of a gene to be deleted. In some aspects, the nucleic acid encoding a CAR as described herein is inserted within a TCR and / or HLA locus, thereby resulting in the inhibition of TCR and / or HLA expression.

[0033] In some aspects of this disclosure, TCR and / or HLA expression is inhibited using siRNAs and / or shRNAs that target one or more of a nucleic acid encoding a TCR and / or HLA in a T cell. Expression of siRNAs and shRNAs in T cells can be achieved using any suitable expression system, e.g., such as a lentiviral expression system. Exemplary siRNAs and shRNAs that downregulate expression of HLA class I and / or HLA class II genes are described, e.g., in U.S. Patent Publication 2007 / 0036773. Exemplary shRNAs that downregulate expression of components of the TCR are described, e.g., in U.S. Patent Publication 2012 / 0321667. 11 DM2\19774561.1PATENT Docket No. H3131-00401

[0034] “CRISPR” as used herein refers to a set of clustered regularly interspaced short palindromic repeats, or a system comprising such a set of repeats. “Cas,” as used herein, refers to a CRISPR-associated protein. A“CRISPR / Cas” system refers to a system derived from CRISPR and Cas that can be used, for example, to introduce an edit or to engineer a TCR and / or HLA. Such edits can, for example, silence or mutate a TCR and / or HLA gene.

[0035] It is understood that the above procedures may also be carried out by CRISPR systems that utilize endonucleases other than Cas, such as Cpfl and C2cl / 2 / 3.

[0036] In some aspects, a T cell population expressing a CAR of the invention is one that lacks an endogenous T cell receptor (TCR) or has been altered to reduce or eliminate the expression or activity of the endogenous TCR. It is understood that any of the methods described herein for inhibiting or eliminating HLA expression may also be used to target one or more components of the endogenous TCR.

[0037] Although T cell recognition of a target antigen is HLA-restricted, chimeric antigen receptor (CAR)-T cell recognition of a target is not HLA-dependent. As disclosed herein, the modulation of CAR-T signaling and function in vivo is critically important to prolong T cell persistence (and reduce T cell exhaustion) by direct regulation of CAR signaling domains or knockdown / knockout of negative signaling molecules which can include or exclude PD-1, VHL, PPP2R2D and epigenetic factors which can include or exclude JMJD3 and LSD1.

[0038] Engineered T cell receptors (TCRs) from tumor antigen specific T cells; modulation of TCR-T cells to increase and prolong T cell persistence and reduce T cell exhaustion by direct manipulation of TCR signaling domains are reported, for example in U.S. Application 18 / 002,969, which is a 371 National Stage Entry of PCT / US2021 / 039262, which is incorporated by reference as if set forth fully herein.

[0039] In one aspect, this disclosure also features methods and strategies to prolong CAR- T cell persistence or TCR-T cell persistence by direct manipulation of CAR signaling domains or by knockdown / knockout of negative signaling molecules. In some aspects, this disclosure also features methods to enhance CAR-T cell persistence by expression of chemokine receptor and shRNA knockout in CAR constructs. In some aspects, the present disclosure provides an engineered immune (TCR-T or CAR-T) cell comprising an knockdown, knockout, inactivating 12 DM2\19774561.1PATENT Docket No. H3131-00401 mutation in one or more endogenous genes selected from a group of negative regulators that include or exclude, or is selected from, e.g., ANKRDJ 1, ARID1A, BACH2, BCL2Ll 1, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3Hl (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2DJA, SMAD2,SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, ZC3H12A, indoleamine (2,3)-dioxygenase (IDO) (including isoforms IDO1 and IDO2), OX40, CTLA-4 (programmed cytotoxic T-lymphocyte antigen 4), PD-1 (programmed death 1), PD-L1 (programmed death ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), B7 homolog 3 (B7-H3), VHL, PPP2R2D and epigenetic factors which can include or exclude JMJD3 and LSD1. In some aspects the negative signaling molecules / regulators selected for knockdown, knockout, inactivating mutation are, for example, indoleamine (2,3)-dioxygenase (IDO) (including isoforms IDO1 and IDO2), OX40, CTLA-4 (programmed cytotoxic T-lymphocyte antigen 4), PD-1 (programmed death 1), PD- L1 (programmed death ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), B7 homolog 3 (B7-H3), VHL, PPP2R2D and epigenetic factors which can include or exclude JMJD3 and LSD1. In certain aspects, the negative signaling molecules or regulators selected for knockout, knockdown or inactivating mutation are, for example, PD-1, VHL, PPP2R2D and epigenetic factors which can include or exclude JMJD3 and LSD1.

[0040] In some aspects, treatment with any of the engineered TCR T cells of this disclosure, where the T cells comprise and / or express a signaling component and / or having negative signaling molecule knockdown surprisingly reduces relapse / cancer recurrence following initial treatment and initial reduction of the cancer / tumor burden. In some aspects, treatment with any of the engineered TCR T cells of this disclosure, where the T cells comprise and / or express a signaling component and / or having negative signaling molecule knockdown surprisingly reduces relapse of a treated inflammatory disease, autoimmune disease, allergic disease, organ transplantation condition, an infectious disease and / or symptoms of aging 13 DM2\19774561.1PATENT Docket No. H3131-00401

[0041] In some aspects, the present disclosure provides an engineered immune (TCR-T or CAR-T) cell comprising knockdown, knockout, inactivating mutations in two or more target genes selected from a group of negative regulators that can include or exclude, or is selected from, e.g., ANKRDJ 1, ARID1A, BACH2, BCL2Ll 1, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3Hl (ROQUIN- 1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2DJA, SMAD2,SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, ZC3H12A, indoleamine (2,3)-dioxygenase (IDO) (including isoforms IDO1 and IDO2), OX40, CTLA-4 (programmed cytotoxic T-lymphocyte antigen 4), PD-1 (programmed death 1), PD-L1 (programmed death ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), and B7 homolog 3 (B7-H3), In some aspects the negative signaling molecules are selected from, for example, indoleamine (2,3)-dioxygenase (IDO) (including isoforms IDO1 and IDO2), OX40, CTLA-4 (programmed cytotoxic T-lymphocyte antigen 4), PD-1 (programmed death 1), PD-L1 (programmed death ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), and B7 homolog 3 (B7-H3), VHL, PPP2R2D and epigenetic factors which can include or exclude JMJD3 and LSD1.

[0042] In some aspects, knockdown of target gene expression is achieved through an siRNA or an shRNA nucleic acid molecule by targeting one or more genes, where the one or more genes code for negative regulators that can include or exclude, or is selected from, e.g., ANKRDJ 1, ARID1A, BACH2, BCL2Ll 1, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3Hl (ROQUIN- 1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2DJA, SMAD2,SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, 14 DM2\19774561.1PATENT Docket No. H3131-00401 ZC3H12A, indoleamine (2,3)-dioxygenase (IDO) (including isoforms IDO1 and IDO2), OX40, CTLA-4 (programmed cytotoxic T-lymphocyte antigen 4), PD-1 (programmed death 1), PD-L1 (programmed death ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), and B7 homolog 3 (B7-H3), B7 homolog 3 (B7-H3), VHL, PPP2R2D and epigenetic factors which can include or exclude JMJD3 and LSD1. and wherein the siRNA or shRNA molecule comprises about 20-30 nucleotides that bind to an RNA sequence encoded by a DNA sequence of the selected genes.

[0043] In some aspects, knockout or inactivation of target gene expression is achieved through a CRISPR / Cas9 system comprising a single-strand guide RNA (sgRNA) molecule and Cas endonuclease and is capable of reducing or knocking out gene expression of one or more target genes that can include or exclude, or is selected from, e.g., ANKRDJ 1, ARID1A, BACH2, BCL2Ll 1, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3Hl (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2DJA, SMAD2,SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6 and ZC3H12A, and wherein the sgRNA molecule comprises a targeting RNA sequence that binds to a target DNA sequence selected from a group of negative regulators ANKRDJ 1, ARID1A, BACH2, BCL2Ll 1, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3Hl (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2DJA, SMAD2,SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, ZC3H12A, indoleamine (2,3)-dioxygenase (IDO) (including isoforms IDO1 and IDO2), OX40, CTLA-4 (programmed cytotoxic T-lymphocyte antigen 4), PD-1 (programmed death 1), PD-L1 (programmed death ligand 1), PD-L2, 15 DM2\19774561.1PATENT Docket No. H3131-00401 lymphocyte activation gene 3 (LAG3), and B7 homolog 3 (B7-H3). VHL, PPP2R2D and epigenetic factors which can include or exclude JMJD3 and LSD1.

[0044] In one aspect, this disclosure also features methods to enhance T cell trafficking into tumor cells in vivo by forced expression of chemokine receptors. In some aspects, expression of chemokine receptors is forced by fusing any of the CAR constructs of any of the preceding aspects or embodiments described herein withi a chemokine receptor. In some aspects the chemokine receptor is CCR5, CXCR3, and / or CCR2. In some aspects the chemokine receptor is CCR5.

[0045] In some aspects knockout, knockdown, or inactivating mutatinos in any negative signaling molecule, and / or expression of the chemokine receptor and / or shRNA knockout can be used in any of the CAR-T or TCR-T cells, or CAR constructs in any of the preceding aspects or embodiments described herein. For example, the CAR-T-cells subject to knockout, knockdown, or inactivating mutations in any negative signaling molecule, and / or expression of the chemokine receptor and / or shRNA knockout may express one or more CARs featured herein, e.g., comprising: at least one extracellular binding domain comprising at least one antigen recognition moiety as disclosed and recited herein; at least one hinge domain and at least one transmembrane domain or a fragment or variant thereof as disclosed and recited herein; and at least one intracellular signaling domain as disclosed and recited herein. For example, in one aspect, the CAR-T-cells subject to knockout, knockdown, or inactivating mutations in any negative signaling molecule, and / or expression of the chemokine receptor and / or shRNA knockout may express one or more CARs featured herein, comprising at least one extracellular binding domain comprising at least one antigen recognition moiety such as SEQ. ID NO: 132 and SEQ. ID NO: 133; at least one hinge domain and at least one transmembrane domain or a fragment or variant thereof such as SEQ ID: 7-29, 91-98; and at least one intracellular signaling domain (such as ZAP300 (SEQ. ID NO: 1), ZAP327 (SEQ. ID NO: 2), ZAP338 (SEQ. ID NO: 3), ZAP255 (SEQ. ID NO: 4) or ZAP280 (SEQ. ID NO: 5).

[0046] Also featured are nucleic acids encoding any of the above-mentioned CARs, CAR domains, ScFvs, antibodies or antigen binding fragments thereof, receptors or receptor binding fragments thereof, bispecific antibodies, trispecific antibodies, or multivalent multispecific 16 DM2\19774561.1PATENT Docket No. H3131-00401 binding proteins, and / or any other polypeptide discosed in any aspect or embodiment herein; recombinant nucleic acids comprising the above-mentioned nucleic acids; vectors or constructs comprising the above-mentioned recombinant nucleic acids; and cells into which one or a plurality of the above-mentioned nucleic acids or vectors is transduced. In one aspect, also disclosed herein are nucleic acids encoding a polypeptide comprising the epitope of any preceding aspect.

[0047] Nature killer (NK) cells and nature killer T (NKT) cells are considered as innate immune cells. NKT cells are a subset of T cells expressing T cell receptor a and b chains, but share properties of NK cells, including several NK cell markers such as NK1.1. NKT cells recognized various foreign or self-lipid antigens presented by CD1d presenting molecules.

[0048] In some aspects CARs, bispecific antibodies and / or bispecific, trispecific or multivalent, multi-specific binding proteins are engineered to be expressed in immune cells, including any of, e.g., T-cells, NK cells, NKT cells, and macrophages. The engineered cell products are CAR-T, CAR-NK, CAR-NKT and CAR-M cells. Similarly, in some aspects TCRs are engineered to be expressed in immune cells, including any of, e.g., T cells and NKT cells. However, NK cells cannot directly be engineered to express TCRs because NK cells do not express their own TCR chains. Thus, NK cells are first modified to express TCR complex subunits before introduction of TCRs featured herein. The TCR complex that is responsible for antigen recognition contains six CD3 subunits and a TCR heterodimer (α and β chains or γ and δ chains).

[0049] In some aspects, this disclosure features a pharmaceutical composition comprising a pharmaceutically acceptable excipient and an immune cell, a host cell, or an immune cell population engineered to express any CAR featured herein. Also provided in some aspects is a method for treating a disease or disorder in a human subject in need thereof, comprising administering to the subject any pharmaceutical composition featured herein.

[0050] In one aspect, this disclosure also provides a chimeric antigen receptor or immune cell population described herein, for use in the treatment of a disease or disorder in a human subject in need thereof. 17 DM2\19774561.1PATENT Docket No. H3131-00401

[0051] In one aspect, this disclosure features the use of any chimeric antigen receptor or immune cell population featured herein for the manufacture of a medicament for the treatment of a disease or disorder in a human subject in need thereof. In one aspect, the disease or disorder that is treated is selected from the group consisting of an inflammatory disease, an autoimmune disease, an allergic disease, an organ transplantation condition, a cancer, and an infectious disease.

[0052] In some embodiments, the inflammatory disease is selected from the group consisting of rheumatoid arthritis, inflammatory bowel disease (Crohn's disease and ulcerative colitis), psoriasis, systemic lupus erythematosus (SLE), vasculitis, osteoarthritis, gout, ankylosing spondylitis, Sjögren's syndrome, Behçet's disease, polymyalgia rheumatica, and juvenile idiopathic arthritis.

[0053] In some embodiments, the autoimmune disease is selected from the group consisting of multiple sclerosis, type 1 diabetes mellitus, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, Addison's disease, pemphigus, scleroderma, Goodpasture syndrome, autoimmune hepatitis, and autoimmune hemolytic anemia.

[0054] In some embodiments, the allergic disease is selected from the group consisting of asthma, allergic rhinitis (hay fever), atopic dermatitis (eczema), food allergies (e.g., peanut allergy, shellfish allergy, milk allergy, egg allergy, wheat allergy), drug allergies (e.g., penicillin allergy, sulfonamide allergy), latex allergy, insect sting allergies, and urticaria.

[0055] In some embodiments, the organ transplantation condition is selected from the group consisting of graft-versus-host disease (GvHD), acute rejection, chronic rejection, transplant vasculopathy, post-transplant lymphoproliferative disorder (PTLD), and delayed graft function.

[0056] In some embodiments, the cancer is selected from the group consisting of lung cancer, breast cancer, colorectal cancer, prostate cancer, melanoma, leukemia (e.g., acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia), lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma), multiple myeloma, brain cancer (e.g., glioblastoma), ovarian cancer, pancreatic cancer, liver cancer, kidney cancer, bladder cancer, and thyroid cancer. 18 DM2\19774561.1PATENT Docket No. H3131-00401

[0057] In some embodiments, the infectious disease is selected from the group consisting of human immunodeficiency virus (HIV) infection, hepatitis B, hepatitis C, tuberculosis, pneumococcal pneumonia, influenza, malaria, dengue fever, Zika virus infection, Ebola virus disease, Chikungunya virus infection, Lyme disease, and sepsis.

[0058] In one aspect, the human subject is in need of immunosuppression and the CAR is expressed in Treg cells in the human subject.

[0059] In one aspect, the disease or disorder is an inflammatory disease, an autoimmune disease, an allergic disease, or an organ transplantation condition (e.g., graft rejection or graft- versus -host disease).

[0060] In one aspect, this disclosure also provides a chimeric antigen receptor or immune cell population described herein for use as a medicament. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The following detailed description, given by way of example, but not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

[0062] FIG. 1. Construction of CARs containing different transmembrane domains derived from T cell receptors (TCR) and their related signaling molecules. (A) CAR constructs containing hinge and transmembrane domains from hTCR-TMα, hTCR-TMβ, hTCR-TMγ, hTCR-TMδ fused to the CD28-CD3ζ signaling domain. (B) CAR constructs containing transmembrane domains from hTCR-TMα, hTCR-TMβ, hTCR-TMγ, hTCR-TMδ fused to the 4-1BB-CD3z signaling domain. (C) CAR constructs containing hinge and transmembrane domains from hTCR-TMα, hTCR-TMβ, hTCR-TMγ, hTCR-TMδ fused to the CD28-ZAP327 signaling domain. (D) CAR constructs containing hinge and transmembrane domains from hTCR-TMα, hTCR-TMβ, hTCR-TMγ, hTCR-TMδ fused to the 4-1BB-ZAP327 signaling domain. For the simplicity, hinge and transmembrane domains are labeled as either H / TM or TM through the entire application. The amino acid sequences of both human and mouse TCR hinge and transmembranes with different lengths are listed in SEQ ID NO 7-48 and SEQ ID NO 110-113. 19 DM2\19774561.1PATENT Docket No. H3131-00401

[0063] FIG. 2. Construction and functional analysis of CD19 CARs (19TMα and 19 TMβ). (A) A schematic presentation of 19TMAs and 19TMBs. Each construct contains a FMC063 anti-CD19 ScFv, (G4S)3 (SEQ ID NO: 131), a Not I site, different lengths of hinge and transmembrane from TCR-TMα or TMβ chain, short tail after transmembrane, 4-1BB and ZAP70 kinase domain from 327 aa (ZAP327). (B) IFN-gamma release from T cells transduced different CARs with or without tumor cells. (C) Specific lysis measured by LDH cytotoxicity assay of different CARs containing different lengthes of hinge and TM domains. The amino acid sequences of the truncated forms of human ZAP-70 kinase are listed as SEQ. ID NO 1 (ZAP300, starting amino acid 300), SEQ. ID NO 2 (ZAP327, starting amino acid 327), SEQ. ID NO 3 (ZAP338, starting amino acid 338), SEQ. ID NO 4 (ZAP255, starting amino acid 255) and SEQ. ID NO 5 (ZAP280, starting amino acid 280). The amino acid sequences of mouse TCR-h / TMα and TCR-h / TMβ are listed in the SEQ. ID s: 91-98. The amino acid sequences of the (G4S)3 (SEQ ID NO: 131) linker and anti-CD19 scFv are listed in the SEQ. ID NOs: 131- 132.

[0064] FIG. 3. Expression of 19TMAs and 19TMBs. (A) Expression of CAR-T cells with different length of hinge and transmembrane from TCR alpha or beta chain was detected by flow cytometry. (B) A diagram of GFP tagged CAR. 19TMA-GFP with fusion expression of CAR and GFP; 19TMA-T2A-GFP with separated expression of CAR and GFP-T2A (a self- cleaved peptide). (C) Expression of 19TMA-GFP and 19TMA-T2A-GFP was analyzed by using a confocal microscope. (D) Expression of 19TMA-GFP and 19TMA-2A-GFP was determined by flow cytometry. (E) TCR-TM domain-containing CARs can be expressed in a complex with endogenous T cell subunits besides previously known CAR molecule alone.

[0065] FIG. 4. Construction and validation of CARs containing human TCR hinge and transmembrane domains (hTCR-TMα, -TMβ, −TMγ and -TMδ) derived from human TCRα, - β, -γ and -δ chains (SEQ. ID NOs: 7-17). (A and B) A diagramic presentation of 19-hTCR- TMA, 19-hTCR-TMB, 19-hTCR-TMG and 19-hTCR-TMD. (C) Different hTCR-TM domain- containing CD19 CARs specifically responded to MDA-MB-231-CD19 (MM231-CD19) cells for cytokine interferon-damma release. (D) Different hTCR-TM domain-containing CD19 CARs specifically recognized and lysed MM231-CD19 cells. (E) The hTCR-TM domain- 20 DM2\19774561.1PATENT Docket No. H3131-00401 containing CD19 CARs can be further engineered through P2A sequence to express MyD88, Toll-like receptors (TLR), TRAF, TAK1, IKK or MALT-1 molecules in the downstream of hTCR-TM domain to activate NF-κB signaling for enhancing CAR-T cell function and persistence.

[0066] FIG. 5. Construction of dual antigens CARs paired through either TMα and TMβ interaction or TMγ and TMδ interaction. (A) Dual antigens CARs paired through either TMα and TMβ interaction or TMγ and TMδ interaction, and then TMα- or TMγ fused to CD28-CDz signaling domain. The amino acid sequences of both human and mouse TCR- TMα, TMβ, TMγ and TMδ are listed in SEQ. ID NOs: 7-29. (B) Dual antigens CARs paired through either TMα and TMβ interaction or TMγ and TMδ interaction, and then TMα- or TMγ fused to 4-1BB-CDz signaling domain. (C) Dual antigens CARs paired through either TMα and TMβ interaction or TMγ and TMd interaction, and then TMα- or TMγ fused to CD28-ZAP327 signaling domain. (D) Dual antigens CARs paired through either TMα and TMβ interaction or TMγ and TMδ interaction, and then TMα- or TMγ fused to 4-1BB-ZAP327 signaling domain.

[0067] FIG. 6. Construction of CARs containing leucine zipper (LZ)-signaling domains. (A) CARs containing TM-leucine zipper (LZ)-CD28-CDz signaling domains. (B) CARs containing TM-leucine zipper (LZ)-4-1BB-CDz signaling domains. (C) CARs containing TM-leucine zipper (LZ)-CD28-ZAP327 signaling domains. (D) CARs containing TM-leucine zipper (LZ)-4-1BB-ZAP327 signaling domains. The amino acid sequences of LZ- TM and TM-LZ are listed as SEQ. ID NOs: 99-104.

[0068] FIG.7. Construction and functional analysis of leucine zipper-linked CAR. (A) A diagram of leucine zipper linked ZAP327 CARs. 19TMA-RR contains leucine zipper RR fused after TCR alpha transmembrane.19TMB-EE contains leucine zipper EE fused after TCR beta transmembrane. (B) IFN-gamma release from leucine zipper CAR-T cells with or without tumor cells. (C) Specific lysis of CAR-T cells measured by LDH cytotoxicity assay. The amino acid sequences of TM-LZ-RR and TM-LZ-EE are listed as SEQ. ID NOs: 103-104.

[0069] FIG. 8. Construction of CARs containing leucine zipper (LZ)-TM-signaling domains. (A) CARs containing LZ-TM-CD28-CDζ signaling domains. (B) CARs containing 21 DM2\19774561.1PATENT Docket No. H3131-00401 LZ-TM-4-1BB-CD3ζ signaling domains. (C) CARs containing LZ-TM-CD28-ZAP327 signaling domains. (D) CARs containing LZ-TM-4-1BB-ZAP327 signaling domains. The linker sequence is listed in the SEQ. ID NOs: 131.

[0070] FIG. 9. Construction and functional analysis of LZ-TM-signaling domain CAR. (A) A diagram of LZ-TM-BBZAP327 CAR. 19RRTMA: Leucine zipper RR was fused in front of TCR alpha hinge. 19EETMB: Leucine zipper EE was fused in front of TCR beta hinge. (1) and (2) represent different length of TCR hinge and transmembrane. (B) IFN-gamma release from LZ-TM-BBZAP327 CAR-T cells with or without tumor cells. (C) Specific lysis of CAR-T cells measured by LDH cytotoxicity assay.

[0071] FIG. 10. Construction of dual antigens CARs containing leucine zipper (LZ)- paired CARs. (A) Dual antigens CARs containing either TMα-TMβ pairing or TMγ-TMδ pairing through LZEE and LZRR interaction, with or without ZAP327. (B) Dual antigens CARs containing TMα-TMβ pairing through LZ-EE and LZ-RR interaction, fused to CD28- CDζ, 4-BB-CDζ, CD28-ZAP327 or 4-1BB-ZAP327 signaling domains. (C) Dual antigens CARs containing TMγ-TMδ pairing through LZ-EE and LZ-RR interaction, fused to CD28- CDζ, 4-BB-CDζ, CD28-ZAP327 or 4-1BB-ZAP327 signaling domains. LZ-EE, LZ-RR and TM are listed as SEQ. ID NOs: 99-104.

[0072] FIG. 11. Construction and functional validation of dual antigen CARs with or without signaling domains. (A) 19-B7TMAB CAR consists of CD19 scFv, TCR-TMα (hinge-TM), P2A, B7H3 scFV, and TCR-TMβ (hinge-TM); 19-B7TMABZAP CAR consists of CD19 scFv, TCR-TMα (hinge-TM), in frame fusion with 4-1BB-ZAP327, P2A, B7H3 scFV, TCR-TMβ (hinge-TM); 19-B7TMABLZ CAR consists of CD19 scFv, TCR-TMα (hinge-TM), LZ-RR, P2A, B7H3 scFV, and TCR-TMβ (hinge-TM)-LZ-EE. (B) 19-B7TMAB, 19-B7TMABZAP and 19-B7TMABLZ CARs form a TMα-TMβ paired complex or a TCR complex (containing other TCR subunits) for CAR-TCR signaling. However, it should be moted that there are no signaling domains in the TMα-TMβ paired complexes of 19-B7TMAB and 19-B7TMABLZ CARs. (C and D) 19-B7TMAB, 19-B7TMABZAP and 19-B7TMABLZ CAR-T cells recognized both Raji (CD19+ B7H3-) and U937 (CD19- B7H3+) tumor cells and specifically killed tumor cells, suggesting that these dual antigens CAR-T cells are functional 22 DM2\19774561.1PATENT Docket No. H3131-00401 for antigen recognition, signaling and killing of target cells. The amino acid sequences of anti- CD19 scFv and anti-B7H3 scFv are listed in the SEQ ID NO 132-133.

[0073] FIG.12. Construction of CARs containing TM and intracellular domains from IL-7R or IL-9R linked to 4-1BB-CD3ζ, ZAP327 or 4-1BB-ZAP327 signaling domains. (A) CARs containing TM and intracellular domains from IL-7R or IL-9R linked to 4-1BB-CD3ζ, ZAP327 or 4-1BB-ZAP327 signaling domains. (B) CARs containing TMα-TMβ, followed by intracellular domains from IL-7R or IL-9R linked to CD28-CD3ζ or 4-1BB-CD3ζ signaling domains. (C) CARs containing TCR- TMα-TMβ, followed by intracellular domains from IL- 7R or IL-9R linked to 4-1BB-ZAP327 signaling domains. The amino acid sequences of human and mouse cytokine receptors are listed in SEQ ID NO 114-119 and 125-130.

[0074] FIG.13. Construction and functional analysis of IL-7R or IL15R intracellular domain-contained CARs. (A) A diagram of CARs with different common gamma chain receptors. 19BBZ: conventional CAR with 4-1BB and CD3zeta. 197: CD19 ScFv, hinge and transmembrane with intracellular domain of interleukin 7 receptor. 197Z: 197 with CD3zeta. 197BBZ: 19BBZ with intracellular domain of interleukin 7 receptor between TM and 4-1BB. 1915: CD19 ScFv, hinge and transmembrane with intracellular domain of interleukin 15 receptor alpha. 1915BBZ: 19BBZ with intracellular domain of interleukin 15 receptor alpha between TM and 4-1BB. (B) IFN-gamma release from different CAR-T cells with or without tumor cells. (C) Specific lysis of different CAR-T cells measured by LDH cytotoxicity assay.

[0075] FIG. 14. Construction and functional analysis of CARs containing truncated CD3 gamma hinges and transmembrane. (A) Diagram of CAR constructs with different lengths of CD3 gamma hinge and transmembrane, fused to IL-7R-BBZAP327 signaling domains. (B) IFN-gamma release from CD3G CAR-T cells with or without tumor cells. (C) Specific lysis of CAR-T cells measured by LDH cytotoxicity assay. The amino acid sequences of TCR subunits (CD3E, CD3D, CD3G) are listed in the SEQ ID NO 30-48, 105-109 and 120- 124.

[0076] FIG. 15. Designs of 19BBZ CAR with different lengths and locations of intracellular domain of IL-9R and IL-21R. The amino acid sequences of human and mouse 23 DM2\19774561.1PATENT Docket No. H3131-00401 cytokine receptors (such as IL-7R, IL-9R and IL-21R) are listed in SEQ ID NO 49-90, 114- 119 and 125-130.

[0077] FIG. 16. Designs of 1928Z CAR with different lengths and locations of intracellular domain of IL-9R and IL-21R. The amino acid sequences of human and mouse cytokine receptors (such as IL-7R, IL-9R and IL-21R) are listed in SEQ ID NO 49-90, 114- 119 and 125-130.

[0078] FIG. 17. Construction and functional validation of 19BBZ CAR with different lengths and locations of intracellular domain of IL-7R, IL9R and IL21R. (A) Construction of 19BBZ CAR with different lengths and locations of intracellular domain of IL-7R, IL9R and IL21R. (B) 197RBBZ-C1, 197RBBZ-C2, 199RBBZ-C1, 199RBBZ-C2, 1921RBBZ-C1 and 1921RBBZ-C2 CAR-T cells specifically recognized CD19-positive Raji cells. (C) 197RBBZ-C1, 197RBBZ-C2, 199RBBZ-C1, 199RBBZ-C2, 1921RBBZ-C1 and 1921RBBZ- C2 CAR-T cells specifically lysed CD19-positive Raji cells. The amino acid sequences of human and mouse cytokine receptors are listed in the SEQ ID NO 49-90, 114-119 and 125- 130.

[0079] FIG. 18. Screening and identification of a phage-display scFv library for their ability to bind to HLA-A2 / NY-ESO-1157-165complex. (A) A diagram of phage-display scFv library screening. Human scFv phage-display antibody library (~1011clones) was constructed and screened. The scFv phage library was incubated first with HLA-A2 / PP65495-503 complexto remove any nonspecific binders, and were then incubated with HLA-A2 / NY-ESO-1157-165complex for panning. (B) Positive A2 / ESO scFV clones were further tested for their binging in ELISA. Strong positive clones were used for DNA isolation from phage clones and sequenced. Among them, 11 positive scFv clones were selected and listed as SEQ NO.134- 144.

[0080] FIG. 19. Construction and functional validation of A2 / ESO-TMBZAP327 CARs with TCR-TMβ transmembrane domain. (A) A diagramic presentation of A2 / ESO- TMBZAP327 CARs containing one of the 11 selected A2 / ESO scFv, TCR-TMβ domain and 4-1BB-ZAP327 signaling domain. (B) A2 / ESO-TMBZAP327 CAR-T cells specifically 24 DM2\19774561.1PATENT Docket No. H3131-00401 recognized EO771 / A2-ESO cells. (C). A2 / ESO-TMBZAP327 CAR-T cells specifically lysed EO771 / A2-ESO cells. A2 / ESO scFv is the one selected from SEQ NO.134-144.

[0081] FIG. 20. BI-specific antibodies (enganers) using A2 / ESO scFv for stimulating immune cells. A2 / ESO scFv antibodies are engineered as shown to fuse with CD3ζ as part of the TCR on T cells, NKG2D or CD16 on NK cells, PD-1, CTLA4 on T cells, PD-L1 on tumor cells, and other molecules on immune cells. INCORPORATION BY REFERENCE

[0082] Throughout this application, various publications are referenced. All documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains. The references disclosed are also individually and specifically incorporated by reference herein for the material contained in them that is discussed in the sentence in which the reference is relied upon. Incorporated by reference as if set forth in full herein is PCT publication WO / 2021 / 263211 and U.S. Patent Application 18 / 002,969, the corresponding US counterpart of WO / 2021 / 263211. DETAILED DESCRIPTION A. Definitions

[0083] Before the present compounds, compositions, articles, devices, and / or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods unless otherwise specified, or to particular reagents unless otherwise specified, as such may, of course, vary. It is also to be 25 DM2\19774561.1PATENT Docket No. H3131-00401 understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0084] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

[0085] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as values between 10 and 15. For example, it is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed. In this application, if a data point range is disclosed, it is understood that each unit from the lowest data point to the highest stated datapoint, including the first (lowest) and last (highest) data point is disclosed. For example, if a data point range 1-20 is disclosed, it is 26 DM2\19774561.1PATENT Docket No. H3131-00401 understood that data points 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20, and each unit between any two particular units in the range are also disclosed. It is also understood that whenever a series of values are disclosed, that any range falling between any two of the recited values is also understood by one of skill in the art.

[0086] In this specification and in the claims which follow, reference will be made to a number of terms which shall be defined to have the following meanings:

[0087] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0088] The term “immunoglobulin” refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See for instance Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain (abbreviated“HC”) typically is comprised of a heavy chain variable region (abbreviated herein as VH orVH) and a heavy chain constant region (abbreviated herein as CH or CH). The heavy chain constant region typically is comprised of three domains, C H1, C H2, and C H3. The heavy chain may typically further comprise a hinge region. Each light chain (abbreviated “LC”) typically is comprised of a light chain variable region (abbreviated herein as VL or VL) and a light chainconstant region (abbreviated herein as CL or CL). The light chain constant region typically iscomprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and / or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). EachVH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminusto carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Other CDR systems include sequence alignment based schemes such as Kabat and IMGT. 27 DM2\19774561.1PATENT Docket No. H3131-00401

[0089] As used herein, the term “antibody” refers to an immunoglobulin molecule which specifically binds with an antigen. It includes both polyclonal and monoclonal antibodies; primatized (e.g., humanized); murine; mouse-human; mouse-primate; and chimeric; and may be an intact molecule, a fragment thereof (which can include or exclude ScFv, Fv, Fd, Fab, Fab′ and F(ab)′2 fragments), or multimers or aggregates of intact molecules and / or fragments; and may occur in nature or be produced, e.g., by immunization, synthesis or genetic engineering; an “antibody fragment,” as used herein, refers to fragments, derived from or related to an antibody, which bind antigen and which in some embodiments may be derivatized to exhibit structural features that facilitate clearance and uptake, e.g., by the incorporation of galactose residues. “Antibodies” includes, e.g., F(ab), F(ab)′2, ScFv, light chain variable region (VL), heavy chain variable region (VH), monoclonal antibodies, polyclonal antibodies, nanobodies, dual or tri-antibody fusion proteins, and combinations thereof.

[0090] The term “multispecific antibody” in the context of the present invention refers to an antibody having different antigen-binding regions defined by different antibody sequences. Thus a multispecific antibody may have two, three, four, five or more different antigen-binding regions. Examples of multispecific antibodies include antibodies having two different antigen- binding regions; i.e. a bispecific antibody.

[0091] The terms “bivalent binding protein,” “trivalent binding protein: or “multivalent multispecific binding protein” refer to a polypeptide comprising binding moieties that bind to two, three, or two or more binding sites, respectively, (which may be on one or more binding targets) through interactions between an antibody fragment or polypeptide derived therefrom (incorporated into the binding protein) and an antigen binding target, or through intereactions between receptor fragments or polypeptide derived therefrom (incorporated into the binding protein) and a ligand, or through other binding moiety – binding target interactions. In some aspects, binding moieties and binding targets may be polypeptides, polynucleotides, carbohydrates, or lipids.

[0092] The term “bispecific antibody” in the context of the present invention refers to an antibody having two different antigen-binding regions defined by different antibody sequences. 28 DM2\19774561.1PATENT Docket No. H3131-00401

[0093] When used herein, unless contradicted by context, the term “Fab-arm” or “arm” refers to one heavy chain-light chain pair and is used interchangeably with “half molecules” herein.

[0094] The term “binding arm comprising an antigen-binding region” means an antibody molecule or fragment that comprises an antigen-binding region. Thus, a binding arm can comprise, e.g., the six VH and VL CDR sequences, the VH and VL sequences, a Fab or Fab′ fragment, or a Fab-arm.

[0095] When used herein, unless contradicted by context, the term “Fc region” refers to an antibody region comprising at least a hinge region, a CH2 domain, and a CH3 domain.

[0096] As used herein, the term “isotype” refers to the specific type of immunoglobulin encoded by the HC (for instance IgG, IgD, IgA, IgE, and IgM) or LC (kappa, K or lambda, A) genes. Within each isotype, there may be several subclasses, such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, etc.

[0097] The term “monovalent antibody” means in the context of the present invention that an antibody molecule is capable of binding a single molecule of the antigen, and thus is not capable of antigen cross-linking.

[0098] A “CD3 antibody” or “anti-CD3 antibody” is an antibody as described above, which binds specifically to the antigen CD3. Anti-CD3 antibodies include muromonab-CD3, otelixizumab, teplizumab, visilizumab, and foralumab.

[0099] The term “specifically binds”, “specifically binding”, “specific binding” or other similar wording refers to the ability of an antibody to preferentially bind to a particular antigen compared to other antigens, or to a particular part (epitope) of an antigen compared to other parts of the same antigen.

[0100] As used herein, the term “binding” in the context of the binding of an antibody to apredetermined antigen or epitope typically is a binding with an affinity corresponding to a KD ofabout 10−7M or less, such as about 10−8M or less, such as about 10−9M or less, about 10−10M or less, or about 10−11M or even less when determined by for instance surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antibody as the ligand and the antigen as the analyte (or vice versa), and binds to the predetermined antigen with an 29 DM2\19774561.1PATENT Docket No. H3131-00401 affinity corresponding to a KD that is at least ten-fold lower, such as at least 100-fold lower, for instance at least 1,000-fold lower, such as at least 10,000-fold lower, for instance at least 100,000-fold lower than the KD for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is higher is dependent on the KD of the antibody, so that when the KD of the antibody is very low (that is, the antibody is highly specific), then the amount with which the affinity for the antigen is higher than the affinity for a non-specific antigen may be at least 10,000 fold.

[0101] The term “kd” (sec−1), as used herein, refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the koff value.

[0102] The term “KD” (M), as used herein, refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[0103] Two antibodies have the “same specificity” if they bind to the same antigen and to the same epitope. Whether an antibody to be tested recognizes the same epitope as a certain antigen-binding antibody, i.e., the antibodies bind to the same epitope, may be tested by different methods well known to a person skilled in the art.

[0104] Checkpoint inhibiting agents are agents that target checkpoint proteins or a derivative thereof, and may be referred to as “checkpoint inhibitors.” Checkpoint inhibitors can include or exclude proteins, polypeptides, amino acid residues, and monoclonal or polyclonal antibodies. The polyvalent vaccine can include or be administered along with one or more checkpoint inhibitor. The checkpoint inhibitors can bind, for example, to ligands or proteins that are found on any of the family of T cell regulators, such CD28 / CTLA-4. Targets of checkpoint inhibitors include, but are not limited to, receptors or co-receptors (e.g., CTLA-4; CD8) expressed on immune system effector or regulator cells (e.g., T cells); proteins expressed on the surface of antigen-presenting cells (e.g.., expressed on the surface of activated T cells, including PD-1, PD-2, PD-L1 PD-L2, 4-1BB, and OX40); metabolic enzymes or metabolic enzymes that are expressed by both tumor and tumor-infiltrating cells (e.g., indoleamine (IDO), including isoforms, such as IDO1 and IDO2); proteins that belong to the immunoglobulin superfamily (e.g., lymphocyte-activation gene 3, also known as LAG3); proteins that belong 30 DM2\19774561.1PATENT Docket No. H3131-00401 to the B7 superfamily (e.g., B7-H3 or homologs thereof). B7 proteins can be found on both activated antigen presenting cells and T cells.

[0105] “Immune effector cells” are immune cells that can perform immune effector functions. In some embodiments, the immune effector cells perform ADCC effector function. Examples of immune effector cells which mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic Tcells, neutrophils, and eosinophils. In some embodiments, the immune effector cells are Tcells.

[0106] The term "autoimmune disease" as used herein is defined as a disorder that results from an autoimmune response. An autoimmune disease is the result of an inappropriate and excessive response to a self-antigen. Examples of autoimmune diseases include but are not limited to, Addision's disease, alopecia greata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, among others.

[0107] As used herein, the term “separation”, includes any means of substantially purifying one component from another (e.g., by filtration, magnetic attraction, etc.).

[0108] As used herein, the term “isolation” or “isolating”, includes any means of sorting one species of a type of genus from another species of a type of genus.

[0109] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. 31 DM2\19774561.1PATENT Docket No. H3131-00401

[0110] The terms “preventing” or “inhibiting” the development of a cancer or cancer cells” as used herein, refers to the occurrence of the cancer being prevented or the onset of the cancer being delayed.

[0111] The terms “preventing” or “inhibiting” a disease or condition such as, e.g, an inflammatory disease, an autoimmune disease, an allergic disease, an organ transplantation condition, an infectious disease and / or symptoms of aging refers to the occurrence of the inflammatory disease, autoimmune disease, allergic disease, organ transplantation condition, e.g., rejection, infectious disease and / or symptoms of aging being prevented or the onset of the disease or condition being delayed.

[0112] The term “treating” or “reducing the presence of a cancer or cancer cells” as used herein, means that the cancer growth is inhibited, which is reflected by, e.g., tumor volume or numbers of malignant cells. Tumor volume may be determined by various known procedures, e.g., measuring the observed image and comparing the average cross-sectional diameter of the tumor with a calibration line (e.g., as done in ImageJ).

[0113] The term “treating a disease or condition such as, e.g, an inflammatory disease, an autoimmune disease, an allergic disease, an organ transplantation condition, an infectious disease and / or symptoms of aging refers to decreasing or ameliorating the severity of signs or symptoms of the condition.

[0114] “Preventing or inhibiting the development of an infectious disease” as used herein, means the occurrence of the infectious disease is prevented or the onset of the infectious disease is delayed, or the spread of an existing infection is reversed.

[0115] As used herein, the term “activation”, refers to the state of a cell following sufficient cell surface moiety ligation to induce a noticeable biochemical or morphological change. Within the context of T-cells, such activation, refers to the state of a T-cell that has been sufficiently stimulated to induce cellular proliferation. Activation of a T-cell may also induce cytokine production and performance of regulatory or cytolytic effector functions. Within the context of other cells, this term infers either up or down regulation of a particular physico- chemical process. 32 DM2\19774561.1PATENT Docket No. H3131-00401

[0116] The term "cancer" as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body, Examples of various cancers include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like.

[0117] As used herein, the terms “cancer antigen” or “tumor antigen” encompasses tissue- specific differentiation antigens, tumor-specific shared antigens, and mutated tumor-specific and unique antigens, and any portion, peptide, or polypeptide of those antigens capable of eliciting an immune response of CD4+ or CD8+ T cells. Tumor antigens or cancer antigens that are recognized by CD8+ or CD4+ T cells can be classified into several categories ( Wang, R. F. & Wang, H. Y. Immune targets and neoantigens for cancer immunotherapy and precision medicine. Cell research 27, 11-37, doi:10.1038 / cr.2016.155 (2017)): 1) The tissue-specific differentiation antigens, which include MART-1 (Kawakami, Y. et al. Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes. J. experimental medicine 180, 347-352 (1994); Schneider, J., Brichard, V., Boon, T., Meyer zum Buschenfelde, K. H. & Wolfel, T. Overlapping peptides of melanocyte differentiation antigen Melan-A / MART-1 recognized by autologous cytolytic T lymphocytes in association with HLA-B45.1 and HLA- A2.1. International journal of cancer 75, 451-458 (1998), TRP-1 / gp75 (Wang, R. F., Parkhurst, M. R., Kawakami, Y., Robbins, P. F. & Rosenberg, S. A. Utilization of an alternative open reading frame of a normal gene in generating a novel human cancer antigen. J. experimental medicine 183, 1131-1140 (1996)), TRP-2 (Wang, R. F., Appella, E., Kawakami, Y., Kang, X. & Rosenberg, S. A. Identification of TRP-2 as a human tumor antigen recognized by cytotoxic T lymphocytes. J. experimental medicine 184, 2207-2216 (1996); Parkhurst, M. R. et al. Identification of a shared HLA-A*0201-restricted T-cell epitope from the melanoma antigen tyrosinase-related protein 2 (TRP2). Cancer research 58, 4895-4901 (1998); Sun, Y. et al. Identification of a new HLA-A(*)0201-restricted T-cell epitope from the tyrosinase- related protein 2 (TRP2) melanoma antigen. International journal of cancer 87, 399-404 33 DM2\19774561.1PATENT Docket No. H3131-00401 (2000)), and gp100 (Kawakami, Y. et al. Recognition of multiple epitopes in the human melanoma antigen gp100 by tumor-infiltrating T lymphocytes associated with in vivo tumor regression. J. immunology 154, 3961-3968 (1995); Bakker, A. B. et al. Melanocyte lineage- specific antigen gp100 is recognized by melanoma-derived tumor-infiltrating lymphocytes. J. experimental medicine 179, 1005-1009 (1994); Skipper, J. C. et al. Shared epitopes for HLA- A3-restricted melanoma-reactive human CTL include a naturally processed epitope from Pmel- 17 / gp100. J. Immunol. 157, 5027-5033 (1996); Tsai, V. et al. Identification of subdominant CTL epitopes of the GP100 melanoma-associated tumor antigen by primary in vitro immunization with peptide-pulsed dendritic cells. J. Immunol.158, 1796-1802 (1997)) have higher expression in cancer cells compared with normal cells; 2) Tumor-specific shared antigens which can include or exclude MAGE-A1 (Traversari, C. et al. A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E. J. experimental medicine 176, 1453-1457 (1992); Fujie, T. et al. A MAGE-1-encoded HLA-A24-binding synthetic peptide induces specific anti-tumor cytotoxic T lymphocytes. International journal of cancer 80, 169-172 (1999)). and NY-ESO- 1 (Jager, E. et al. Simultaneous humoral and cellular immune response against cancer-testis antigen NY-ESO-1: definition of human histocompatibility leukocyte antigen (HLA)-A2- binding peptide epitopes. J. experimental medicine 187, 265-270 (1998); Rimoldi, D. et al. Efficient simultaneous presentation of NY-ESO-1 / LAGE-1 primary and nonprimary open reading frame-derived CTL epitopes in melanoma. J. Immunol. 165, 7253-7261 (2000); Valmori, D. et al. Naturally occurring human lymphocyte antigen-A2 restricted CD8+ T-cell response to the cancer testis antigen NY-ESO-1 in melanoma patients. Cancer research 60, 4499-4506 (2000); Wang, R.-F., Johnston, S. L., Zeng, G., Schwartzentruber, D. J. & Rosenberg, S. A. A breast and melanoma-shared tumor antigen: T cell responses to antigenic peptides translated from different open reading frames. J. Immunol. 161, 3596-3606 (1998)) are expressed in cancer and testis, but not in other normal tissues. These antigens are also called cancer-testis (CT) antigens; 3) Tumor-specific and unique antigens that are mutated antigens, including CDK4 (Wolfel, T. et al. A p16INK4a-insensitive CDK4 mutant targeted by cytolytic T lymphocytes in a human melanoma. Science 269, 1281-1284 (1995)), catenin (Robbins, P. 34 DM2\19774561.1PATENT Docket No. H3131-00401 F. et al. A mutated beta-catenin gene encodes a melanoma-specific antigen recognized by tumor infiltrating lymphocytes. J. experimental medicine 183, 1185-1192 (1996)), and caspase- 8 (Mandruzzato, S., Brasseur, F., Andry, G., Boon, T. & van der Bruggen, P. A CASP-8 mutation recognized by cytolytic T lymphocytes on a human head and neck carcinoma. J. experimental medicine 186, 785-793 (1997)) antigens; and 4) Overexpressed tumor antigens that are overexpressed in cancer cells compared with in normal cells.

[0118] The term“zeta” or alternatively“zeta chain,”“CD3 zeta” or“TCR-zeta” is defined as the protein provided as GenBank Acc. No. BAG36664.1, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like, and a “zeta stimulatory domain” or alternatively a“CD3 zeta stimulatory domain” or a“TCR-zeta stimulatory domain” is defined as the amino acid residues from the cytoplasmic domain of the zeta chain, or functional derivatives thereof, that are sufficient to functionally transmit an initial signal necessary for T cell activation. In some embodiments, the cytoplasmic domain of zeta comprises residues 52 through 164 of GenBank Acc. No. BAG36664.1 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like, that are functional orthologs thereof.

[0119] The term "co-stimulatory ligand," as used herein, includes a molecule on an antigen presenting cell (e.g., an aAPC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule on a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR / CD3 complex with an MHC molecule loaded with peptide, mediates a T ceii response, including, but not limited to, proliferation, activation, differentiation, and the like. A co-stimulatory ligand can include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costiinulatory ligand (ICOS-L), intercellular adhesion molecule (rCAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, lymphotoxin beta receptor, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7- H3. A co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as, but not limited to, CD27, CD28, 4- IBB, OX40, CD30, CD40, PD-1, 1COS, MALT-1 (Mucosa-associated lymphoid tissue 35 DM2\19774561.1PATENT Docket No. H3131-00401 lymphoma translocation protein 1), Toll-like receptors (TLR1, TLR2, TLR3,TLR4, TLR5, TLR7, TLR8, TLR9), MyD88, TRAF (TRAF1, TRAF2, TRAF3, TRAF4, TRAF5 and TRAF6), TAK1, IKKα, IKKβ, NEMO, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

[0120] A "co-stimulatory molecule" refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory Hgand, thereby mediating a co- stimulatory response by the T cell, such as, but not limited to, proliferation, Co- stimulatory molecules include, but are not Iimited to an MHC class I molecule, BTLA and a Toll ligand receptor.

[0121] A "co-stimulatory signal", as used herein, refers to a signal, which in combination with a primary signal, such as TCR / CD3 ligation, leads to T cell proliferation and / or upregulation or downregulation of key molecules.

[0122] As used herein "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

[0123] “Fragment” or “portion” as the term is used herein means any segment of a protein or gene, having at least 5 or 6 amino acids in the case of a protein fragment and at least 15-18 nucleotides in the case of a gene.

[0124] The “presented” as the term is used herein, encompasses the procedure of transfection of the DNA encoding the full-length or any portion of the tumor antigen into antigen-presenting cells or loading of the peptide of the full-length or any portion of the tumor antigen onto antigen-presenting cells.

[0125] The term “antigen” or “Ag,” as used herein, refers to a molecule that provokes an immune response. This immune response may involve antibody production, the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. In some embodiments, the term ““antigen” or “Ag” refers, for example, to any molecule 1) capable of being specifically recognized, either in its entirety or fragments thereof, and bound by the “idotypic” portion (antigen-binding region) of a mAb or its derviative; 2) containing peptide sequences which can be bound by MHC and then, in the context of MHC presentation, can specifically engage its cognate T cell antigen receptor. Furthermore, antigens can be derived 36 DM2\19774561.1PATENT Docket No. H3131-00401 from recombinant or genomic DNA. A skilled artisan will understand that any DNA that comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen does not necessarily need to be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen does not necessarily need to be encoded by a “gene” at all. It is readily apparent that an antigen can be synthesized or can be derived from a biological sample, or might be a macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to, e.g., a tissue sample, a cell or a fluid with other biological components.

[0126] Fragments and derivatives of antibodies of this invention (which are encompassed by the term“antibody” as used in this application, unless otherwise stated or clearly contradicted by context), can be produced by techniques that are known in the art.

[0127] “Fragments” comprise a portion of the intact antibody, generally the antigen binding site or variable region. Examples of antibody fragments include Fab, Fab’, Fab’-SH, F(ab’)2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a“single-chain antibody fragment” or“single chain polypeptide”), including without limitation (1) single-chain Fv molecules, (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific antibodies formed from antibody fragments. Fragments of the present antibodies can be obtained using standard methods. The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well- known schemes, including those described by Rabat et al. (1991),“Sequences of Proteins of 37 DM2\19774561.1PATENT Docket No. H3131-00401 Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Rabat” numbering scheme), Al-Lazikani et al, JMB 273:927-948 (1997) (“Chothia” numbering scheme), or a combination thereof.

[0128] The term“graft-versus-host disease” or“GVHD” as used herein refers to a medical complication following the receipt of transplanted tissue from a genetically different person. Immune cells in the donated tissue (the graft) recognize the recipient (the host) as foreign. The transplanted immune cells then attack the host’s body cells. GVHD is commonly associated with stem cell transplant; however, the term includes GVHD arising from other forms of tissue graft. GVHD may also occur after a blood transfusion.

[0129] The “antigen-presenting cell” or “APC” as the term is used herein, encompasses any natural or artificial cell line or cell that express a certain kind of HLA molecule of interest on the cell surface. In an embodiment, it refers to an immune system cell such as an accessory cell (e.g., a B cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHCs) on its surface. T cells may recognize these complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells. In an embodiment.

[0130] The term “knock down” or “” as referred to herein means to reduce expression of a gene, for example, by causing degradation of mRNA or by blocking RNA expression to decrease the protein expression of target genes. The term “metabolic gene” as referred to herein includes but is not limited to PD1, VHL and PPP2R2D.

[0131] The term “Treg cell” refers to a cell capable of suppressing, inhibiting or preventing excessive or unwanted inflammatory responses, such as, for example, autoimmunity or allergic reactions. In some embodiments, the Treg cell population of the invention is capable of suppressive activity. In some embodiments, said suppressive activity is contact independent. In some embodiments, said suppressive activity is contact dependent. In some embodiments, the Treg cell population of the invention presents a suppressive action on effector T cells; in certain embodiments, said suppressive action is dependent on TCR expression and / or activation. 38 DM2\19774561.1PATENT Docket No. H3131-00401

[0132] The term “proliferation” as used herein, means to grow or multiply by producing new cells.

[0133] The terms “purify” or “pure” refer to a molecule isolated from other reaction or cellular components. The term “substantially pure” or “substantially purified” refers to a molecule which is 85, 86, 87, 88, 89, 90, 91, 9, 93, 94, 95, 96, 97, 98, 99, or 100% pure.

[0134] The term “self-cleaving peptide” includes but is not limited to the P2A, T2A, F2A and E2A sequences (SEQ ID NO: 6, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147) that is located between two proteins and can be self-cleaved to separate the two proteins. Ryan, M. D., King, A. M. & Thomas, G. P. Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. J. general virology 72 (Pt 11), 2727-2732, doi:10.1099 / 0022-1317-72-11-2727 (1991).

[0135] As used herein, the term “stimulation”, refers to a primary response induced by ligation of a cell surface moiety. For example, in the context of receptors, such stimulation entails the ligation of a receptor and a subsequent signal transduction event. With respect to stimulation of a T-cell, such stimulation refers to the ligation of a T-cell surface moiety that in one embodiment subsequently induces a signal transduction event, which can include or exclude binding the TCR / CD3 complex. Further, the stimulation event may activate a cell and upregulate or downregulate expression or secretion of a molecule, which can include or exclude downregulation of TGF-β. Thus, ligation of cell surface moieties, even in the absence of a direct signal transduction event, may result in the reorganization of cytoskeletal structures, or in the coalescing of cell surface moieties, each of which could serve to enhance, modify, or alter subsequent cell responses.

[0136] As used herein, the term “vector” includes but is not limited to pMSGV, pMSCV, pFU3W, or any other vector which functions as a carrier of the inserted DNA into live cells.

[0137] In yet another embodiment of the present invention, vectors for the insertion of cDNA encoding TCR alpha chain and / or TCR beta chain are provided. In some embodiments the translational product of the vector includes at least one alpha chain variable region combined with a least one alpha constant region and / or at least one beta chain variable region combined with at least one beta constant region, which are linked by a self-cleaving peptide. 39 DM2\19774561.1PATENT Docket No. H3131-00401 The vectors serve for the delivery of the insertion delivery into naïve T cells by viral transduction.

[0138] The term “viral transduction” as used herein, encompasses a procedure that produces recombinant viruses including but not limiting to retroviruses, lentiviruses, adeno- associated viruses, or other suitable viruses in host cells and uses these recombinant viruses comprising the gene encoding the TCR in their genome to infect, transfect, or transduce target cells. The gene encoding the TCR is integrated to the genome of target cells and is stably expressed and replicated within proliferated cells. The term “target cell” includes but is not limited to CD4+ T cells, CD8+ T cells, tumor cells etc.

[0139] In one embodiment, the invention also provides host cells transfected or transduced with a vector comprising DNA encoding the TCR regions or chains, according to any preceding aspect or any aspect or embodiment disclosed herein, for example, the TCR alpha chain variable region combined with an alpha constant region and a beta chain variable region combined with a beta constant region, which are linked by P2A sequence (SEQ ID NO: 6) for viral production and TCR delivery into naïve T cells.

[0140] The term “host cell” includes but is not limited to cells from the PG-13 cell line, Phoenix-Eco cell line, Phoenix -Ampho cell line, 293GP cell line, or other suitable cell line which can intracellularly assemble the viral genome, package viruses with the capsule proteins, and secret mature viruses extracellularly.

[0141] Chimeric Antigen Receptors (CARs)

[0142] In some embodiments a chimeric antigen receptor (CAR) is an artificially constructed hybrid protein or polypeptide comprising the antigen binding domains of an antibody (scFv) linked to T-cell signaling domains. Characteristics of CARs can include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC- restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition gives T cells expressing CARs the ability to recognize antigens independent of antigen processing, thus bypassing a major mechanism of tumor evasion. Moreover, when expressed in T-cells, CARs advantageously do not dimerize with endogenous T cell receptor (TCR) alpha and beta chains. T cells expressing a CAR are referred 40 DM2\19774561.1PATENT Docket No. H3131-00401 to herein as CAR T cells, CAR-T cells or CAR modified T cells, and these terms are used interchangeably herein. The cell can be genetically modified to stably express an antibody binding domain on its surface, conferring novel antigen specificity that is MHC independent.

[0143] In certain embodiments, the CARs featured herein comprise: an extracellular binding domain comprising an antigen recognition moiety, a hinge domain, a transmembrane domain or a fragment or variant thereof, and an intracellular domain. In certain aspects, the CAR may comprise a T-cell activation moiety.

[0144] In some embodiments, the hinge / TM domain of the CARs featured herein is derived from a transmembrane domain of CD4, CD8, CD28, ZAP70, TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ, LCK, LAT, IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL- 13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα, or the common cytokine receptor γ chain, or any combination thereof. In one embodiment, this disclosure features CARs comprising hinge and transmembrane domains from human or non-human hinge and transmembrane domains, e.g., murine mTCRα, mTCRβ, mTCRγ, mTCRδ, mCD3γ, mCD3δ, mCD3ε, mCD3ζ. In some aspects, the hinge and transmembrane domains from human or non-human hinge and transmembrane domains are selected from, e.g., SEQ ID NOs: 7-48, 90-98, 105-113 and 120- 124.

[0145] In some embodiments, this disclosure features CARs with different lengths of hinge / TM domains of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ, LCK, LAT, IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL- 21Rα, the common cytokine receptor γ chain, 4-1BB-ZAP327, and murine mTCRα, mTCRβ, mTCRγ, mTCRδ, mCD3γ, mCD3δ, mCD3ε, mCD3ζ, including but not limited to the lengths of 23, 37, 65, 95, and 105 amino acids of the hinge / TM domains. In one embodiment, the hinge / TM domain of the featured CARs comprise a polypeptide selected from about 20 to about 110 amino acids of the hinge / TM domains of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ, LCK, LAT, IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα, the common cytokine receptor γ chain, 4-1BB-ZAP327, and murine mTCRα, mTCRβ, mTCRγ, mTCRδ, mCD3γ, mCD3δ, mCD3ε, mCD3ζ or any combination thereof. In one embodiment, this disclosure features a composition comprising a 41 DM2\19774561.1PATENT Docket No. H3131-00401 chimeric antigen receptor (CAR), or cells expressing a CAR, wherein the CAR comprises an antigen recognition moiety, a hinge and transmembrane (hinge / TM) domain, and an intracellular T-cell activation moiety, wherein the hinge / TM domain are a length selected from 23 to 105 amino acids, 37-105, 65-105, 95-105, 37-65, 37-95, 65-95, or 95-105 amino acids of the hinge / TM domains of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ, LCK, LAT, IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL- 21Rα, the common cytokine receptor γ chain, 4-1BB-ZAP327, and murine mTCRα, mTCRβ, mTCRγ, mTCRδ, mCD3γ, mCD3δ, mCD3ε, mCD3ζ or any combination thereof. In one embodiment, this disclosure features a composition comprising a chimeric antigen receptor (CAR), or cells expressing a CAR, wherein the CAR comprises an antigen recognition moiety, a hinge and transmembrane (hinge / TM) domain, and an intracellular T-cell activation moiety, wherein the hinge / TM domain are a length selected from: 23, 37, 65, 95, and 105 amino acids of the hinge / TM domains of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ, LCK, LAT, IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα, the common cytokine receptor γ chain, and 4-1BB-ZAP327, or any combination thereof. In one embodiment, the hinge / TM domain are a length selected from: 23, 37, 65, 95, and 105 amino acids of the hinge / TM domains of CD3γ, IL-7R, IL9-R, IL-21R, and 4-1BB- ZAP327, or any combination thereof. In some embodiments, the hinge and transmembrane domains from human or non-human hinge and transmembrane domains are selected from, e.g., SEQ ID NOs: 7-48, 90-98, 105-113 and 120-124.

[0146] In one embodiment, this disclosure features CARs comprising transmembrane domains derived from cytokine receptors, including but not limited to IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα (such as SEQ ID NO: 49-90, 114-119 and 125-130), and the common cytokine receptor γ chain. In some aspects, this disclosure features CARs comprising an IL-7R intracellular domain with or without co- stimulatory and T cell activation signaling domains. In some embodiments, this disclosure features CARs comprising IL-15R with or without co-stimulatory and T cell activation signaling domains. 42 DM2\19774561.1PATENT Docket No. H3131-00401

[0147] In certain embodiments, the CAR may comprise a T-cell activation moiety. In some embodiments of this disclosure, the intracellular T-cell activation moiety of the CARs featured herein comprises a costimulatory signaling domain. In some embodiments, the CARs or TCRs include or exclude, for example, intracellular T-cell activation moiety comprising a costimulatory signaling domain that include or exclude, or are selected from, e.g., CD28, 4- 1BB (CD137), ICOS (CD278), CD27, OX40 (CD134), MyD88, EphB6, TSLP-R, HLA-DR, CO2, CD4, CD5, CD7, CDS, CD8alpha, CD8beta, CD11a, CD11b, CD11e, CD11d, CD18, CD19, CD19a, CD29, CD30, CD30L, CD40, CD40L (CD154), CD48, CD49a, CD49D, CD49f, CD58, CD53, ICAM-1 (CD54), CD69, CD70, CD80 (B7-l), CD82, CD83, CD84, CD86 (B7-2), CD90, CD96, CD100, CD103, CD122, CD132, CD150 (SLAMFl), CD160 (BY55), CD162 (DNAMl), CD223 (LAG3), CD226, CD229, CD244, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-Ll), CD278, LAT, lymphocyte function-associated antigen-I (LFA-1), LIGHT, NKG2C, NKG2D, NK.p30, NKp44, NKp46, NKp80 (KLRFl), DAPl0, DAP12, LAG-3, 2B4, CARDI, CTLA-4 (CD152), TRIM, ZAP70, FcERIγ, 4-lBBL, BAFF, GADS, GITR, GITR-L, BAFF-R, HVEM, CD27L, OX40L, TACl, BLAME, CRACC, CD2F- 10, NTB-A, integrin α4, integrin α4β1, integrin α4β7, IA4, ICAM-1, IL-2Ralpha, IL-2Rbeta, IL-2Rgamma, IL-4Ralpha, IL-7Ralpha, IL-9Ralpha, IL-12R, IL-21Ralpha, B7-H2, B7-H3, CD83 ligand, PD-1, SLP-76, Toll-like receptors (TLRs, such as TLR2), ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGBl, ITGB2, ITGB7, KIRDS2, LTBR, PAG / Cbp, PSGLI, SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLAl, VLA-6, BTLA, ikaros, LAG-3, LMIR, CEACAMl, CRTAM, TCLIA, DAP12, TIM-1, Dectin-1, PDCD6, PD-1, TIM-4, TSLP, or any combination thereof.

[0148] In some embodiments, the intracellular T-cell activation moiety of the CARs featured herein comprises a costimulatory signaling domain fused with a signaling domain, where the costimulatory signaling domain is selected from a CD28, 4-1BB (CD137), ICOS (CD278), CD27, OX40 (CD134), MyD88, EphB6, TSLP-R, HLA-DR, CO2, CD4, CD5, CD7, CDS, CD8alpha, CD8beta, CD11a, CD11b, CD11e, CD11d, CD18, CD19, CD19a, CD29, CD30, CD30L, CD40, CD40L (CD154), CD48, CD49a, CD49D, CD49f, CD58, CD53, ICAM-1 (CD54), CD69, CD70, CD80 (B7-l), CD82, CD83, CD84, CD86 (B7-2), CD90, 43 DM2\19774561.1PATENT Docket No. H3131-00401 CD96, CD100, CD103, CD122, CD132, CD150 (SLAMFl), CD160 (BY55), CD162 (DNAMl), CD223 (LAG3), CD226, CD229, CD244, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-Ll), CD278, LAT, lymphocyte function-associated antigen-I (LFA-1), LIGHT, NKG2C, NKG2D, NK.p30, NKp44, NKp46, NKp80 (KLRFl), DAPl0, DAP12, LAG-3, 2B4, CARDI, CTLA-4 (CD152), TRIM, ZAP70, FcERIγ, 4-lBBL, BAFF, GADS, GITR, GITR-L, BAFF-R, HVEM, CD27L, OX40L, TACl, BLAME, CRACC, CD2F-10, NTB-A, integrin α4, integrin α4β1, integrin α4β7, IA4, ICAM-1, IL-2Ralpha, IL-2Rbeta, IL-2Rgamma, IL- 4Ralpha, IL-7Ralpha, IL-9Ralpha, IL-12R, IL-21Ralpha, B7-H2, B7-H3, CD83 ligand, PD-1, SLP-76, Toll-like receptors (TLRs, such as TLR2), ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGBl, ITGB2, ITGB7, KIRDS2, LTBR, PAG / Cbp, PSGLI, SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLAl, VLA-6, BTLA, ikaros, LAG-3, LMIR, CEACAMl, CRTAM, TCLIA, DAP12, TIM-1, Dectin-1, PDCD6, PD- 1, TIM-4, TSLP, or any combination thereof. In some embodiments, the costimulatory signaling domain comprises e.g., the replacement of CD3ζ with a ZAP70 kinase domain or a mutant or a variant thereof. In some embodiments, the intracellular T-cell activation moiety comprises or consists of CD28 or 4-1BB costimulatory signaling domain fused with a signaling domain, such as, a ZAP70 kinase domain or a mutant or a variant thereof. In some asepcts, the ZAP70 kinase domain or a mutant or a variant thereof comprises or consists of, for example, a ZAP300 (SEQ ID NO: 1) or ZAP327 (SEQ ID NO: 2) or ZAP338 (SEQ ID NO: 3) or ZAP255 (SEQ ID NO: 4) or ZAP280 (SEQ ID NO: 5) signaling domain or other signaling domain derived from ZAP70.

[0149] In one embodiment, the present invention provides a CAR comprising: at least one extracellular binding domain comprising at least one antigen recognition moiety such as SEQ ID NO: 132 and SEQ id NO: 133; at least one hinge domain and at least one transmembrane domain or a fragment or variant thereof such as SEQ ID: 7-29, 91-98; and at least one intracellular signaling domain (such as ZAP300 (SEQ ID NO: 1), ZAP327 (SEQ ID NO: 2), ZAP338 (SEQ ID NO: 3), ZAP255 (SEQ ID NO: 4) or ZAP280 (SEQ ID NO: 5). 44 DM2\19774561.1PATENT Docket No. H3131-00401

[0150] In some embodiments the CAR may comprise any hinge / TM domain and costimulatory signaling domain fused with a signaling domain featured herein. As featured herein, engineering the hinge and transmembrane domains in CAR constructs improves the therapeutic potential and safety of the CARs.

[0151] In certain embodiments, the extracellular binding domain of a CAR described herein is an antibody or an antigen-binding fragment thereof or receptor or ligand binding fragment thereof. In particular embodiments, the extracellular binding domain is a single chain variable fragment (scFv) such as SEQ ID NO: 133-144. In certain embodiments, the extracellular binding domain may specifically bind, for example, an autoantigen or a B cell antigen, or a receptor ligand.

[0152] In one embodiment, this disclosure relates to methods for generating and functionally validating CARs with hinge and transmembrane domains derived from the T cell receptors and related signaling molecules, e.g., cytokines, for increased T cell signaling, activation, function and persistence, but with low amounts of cytokines produced by CAR-T cells. The strategies to improve or prolong T cell persistence as featured herein are fundamentally important to T-cell immunotherapy, because therapeutic efficacy and long-term cancer cure of CAR-T cell therapy is closely associated with T cell persistence. T cell persistence relies on several key factors or mechanisms, including cell population size of stem- like memory and effector memory T cells, T cell proliferation, and resistance to T cell apoptosis and exhaustion. In one embodiment, the CARs of this disclosure feature hinge and transmembrane domains derived from TCRs, signaling molecules and cytokine receptors are directly involved in T cell signaling, activation and cytokine production. For example, in some aspects, this disclosure relates to generating and functionally validating hinge and transmembrane (hinge / TM) domains derived from T cell receptors and their subunits (TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ), as well as membrane-anchored TCR signaling molecules (such as LCK, LAT), and the second panel of the hinge and transmembrane domains derived from cytokine receptors, including but not limited to IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα, and the common cytokine receptor γ chain. 45 DM2\19774561.1PATENT Docket No. H3131-00401

[0153] In some embodiments the antigen-binding domain of any CARs embodied herein is followed by the hinge and transmembrane domains, and can include or exclude a linker, followed by a costimulatory signaling domain. In some aspects, the CARs embodied herein include or exclude a costimulatory signaling domain derived from a CD28, 4-1BB, CD3ζ, signaling domain, or a ZAP70 kinase domain. In one aspect, any of the CARs embodied herein comprise a costimulatory signaling domain wherein CD3 is replaced by a ZAP70 kinase domain or a mutant or a variant thereof that may include or exclude, e.g., ZAP300 (SEQ ID NO:1), ZAP327 (SEQ ID NO: 2), ZAP338 (SEQ ID NO:3), ZAP255 (SEQ ID NO: 4), or ZAP280 (SEQ ID NO: 5). In another aspect, any of the CARs embodied herein comprise a costimulatory signaling domain wherein the costimulatory signaling domain is derived from a 4-1BB signaling domain and a ZAP70 kinase domain or a mutant or a variant thereof, e.g. ZAP300 (SEQ ID NO:1), ZAP327 (SEQ ID NO: 2), ZAP338 (SEQ ID NO:3), ZAP255 (SEQ ID NO: 4), or ZAP280 (SEQ ID NO: 5).

[0154] In one embodiment, this disclosure features CARs comprising transmembrane domains derived from cytokine receptors, including but not limited to IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα (such as SEQ ID NO: 49-90, 114-119 and 125-130), and the common cytokine receptor γ chain. In some aspects, this disclosure features CARs comprising IL-7R intracellular domain with or without co- stimulatory and T cell activation signaling domains. In some embodiments, this disclosure features CARs comprising IL-15R with or without co-stimulatory and T cell activation signaling domains. In some embodiments, this disclosure features CARs with different lengths of hinge / TM domains of TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ, LCK, LAT, IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL- 21Rα, the common cytokine receptor γ chain, and 4-1BB-ZAP327, including but not limited to the lengths of 23, 37, 65, 95, and 105 amino acids of the hinge / TM domains.

[0155] In one embodiment, this disclosure includes CARs comprising a variable region fused to a modified human constant region, or to a non-human constant region which can be modified or unmodified. In some embodiments the CAR comprises a cancer antigen specific variable region fused to a non-human, e.g., murine TCR constant region. In some embodiments 46 DM2\19774561.1PATENT Docket No. H3131-00401 the TCR variable region comprises an alpha and a beta chain variable region fused to an alpha and a beta TCR constant region, respectively and can include any alpha and / or beta chain of any other aspect of this disclsoure. In some embodiments, fusion of the TCR variable region to a modified or non-human constant region reduces mispairing between the chimeric TCR and an endogenous TCR.

[0156] In one embodiment, the antigen-binding domain of any CAR embodied herein is followed by the hinge and transmembrane domains, with or without a linker. In some embodiments, the antigen-binding domain of any CARs embodied herein is followed by a linker that can be 15-25 amino acids in length. In one aspect the linker is, for example, (X1X2X3X4S)3-5 wherein any of X1-X4 is G or A. In one embodiment the linker is, for example, (GGGGS)3-5 (SEQ ID NO: 148), such as SEQ ID NO: 131). In one aspect, for example, a CD19 ScFv and hinge and transmembrane domain with the linker (GGGGS)3 (SEQ ID NO: 131) followed by a 4-1BB costimulatory signaling domain and ZAP327 T cell activation domain (CD19TMαBBZAP327 CAR).

[0157] In one embodiment, this disclosure relates to the functional analysis of CARs comprising TCR hinge and transmembrane domains. In one embodiment, this disclosure relates to CARs expressing a single TCR transmembrane domain directing the CAR to form a complex with endogenous TCRs.

[0158] In one embodiment, this disclosure features dual antigen CARs using TCR TMα- TMβ and TMγ-TMδ pairing. In some embodiments, this disclosure features pairs of CARs, wherein a first CAR comprising a first ScFv that binds to a first target, TCR hinge / TMα, and T cell signaling domain; and a second CAR comprising a second ScFv that binds to a second target different from the first target, TCR hinge / TMβ, and no signaling domain, such that when paired together, the CARs pair then function to target each ScFv target individually or both antigen-expressing tumor cells. Similarly, in another embodiment, this disclosure features pairs of CARs, wherein a first CAR comprising a first ScFv that binds to a first target, TCR hinge / TMγ, and T cell signaling domain; and a second CAR is comprising a second ScFv that binds to a second target different from the first target, TCR hinge / TMδ, and no signaling domain, such that when paired together, the CARs pair then function to target each ScFv target 47 DM2\19774561.1PATENT Docket No. H3131-00401 individually or both antigen-expressing tumor cells. In some embodiments, the dual antigen CARs use non-human, e.g. murine, TCR α / β and TCR γ / δ pairing.

[0159] In one embodiment, dual antigen CARs comprise leucine zippers to mediate dimerization. In one embodiment, use of leucine zipper-mediated dimerization promotes pairing of a TMα-containing CAR to pair with a TMβ-containing CAR. In some embodiments, CARs comprise a leucine zipper sequence inserted in the intracellular portion of a CAR construct, followed by any one or more co-stimulatory signaling domain featured herein, and any one or more T cell activation domain featured herein. In some embodiments the co- stimulatory signaling domain is from CD28 or 4-1BB, and the T cell activation domain is from CD3ζ or ZAP-70 kinase domain. Leucine zippers derived from human B-ZIP proteins and other aspects of the leucine zippers are reported, for example, in Vinson et al., Classification of Human B-ZIP Proteins Based on Dimerization Properties, MOLECULAR AND CELLULAR BIOLOGY, Sept. 2002, p. 6321–6335; Moll et al., Designed heterodimerizing leucine zippers with a range of pIs and stabilities up to 10−15M, Protein Science (2001), 10:649–655, which are hereby incorporated by reference as if set forth fully herein.

[0160] In some embodiments, dual antigen CARs are associated via coiled-coil peptides, e.g., SYNZIP coiled coils, which are peptide structures in which α helices intertwine to form superhelical bundles. Coiled-coil peptides have a repeating seven-residue pattern, termed a heptad repeat, that is denoted (abcdefg)n. Many of the a and d positions are occupied by hydrophobic residues, forming a well packed interface, while e and g positions on opposing helices tend to be occupied by amino acids that are electrostatically complementary. In some embodiments, coiled coils, PDZ, SH3, and GBD domains, are used to oligomerize dual antigen CARs or any multivalent-multispecific binding protein (e.g., bispecific and trispecific binding proteins) featurered herein. In some embodiments SYNZIPs that interact with leucine zipper peptides are used to oligomerize dual antigen CARs or multivalent-multispecific binding proteins. In other embodiments, SYNZIPs capable of heteroassociating interactions are used to oligomerize dual antigen CARs or multivalent-multispecific binding proteins. Thompson et al., SYNZIP Protein Interaction Toolbox: in Vitro and in Vivo, ACS Synth. Biol. 2012, 1, 118-129, which is hereby incorporated as if set forth fully herein. 48 DM2\19774561.1PATENT Docket No. H3131-00401

[0161] Specifications of Heterospecific Coiled-Coil Interaction Domains

[0162] In one embodiment, this disclosure relates to the development of dual antigen CARs using leucine-zipper dimerization. In some embodiments, the leucine zipper is designed downstream of the TCR hinge / TM domain: the pairs of CARs include a first CAR comprising a first ScFv that binds to a first target, TCR hinge / TMα, a leucine zipper, and T cell signaling domain and a second CAR comprising a second ScFv that binds to a second target different from the first target, TCR hinge / TMβ, a leucine zipper, and no signaling domain, such that when paired together, the CARs pair then function to target each ScFv target individually or both antigen-expressing tumor cells; or the pairs of CARs include a first CAR comprising a first ScFv that binds to a first target, TCR hinge / TMγ, a leucine zipper, and T cell signaling domain and a second CAR comprising a second ScFv that binds to a second target different from the first target, TCR hinge / TMδ, a leucine zipper, and no signaling domain, such that when paired together, the CARs pair then function to target each ScFv target individually or both antigen-expressing tumor cells. In some embodiments, the leucine zipper is designed upstream of the TCR hinge / TM domain. Dual antigens CARs containing TCR TMα-TMβ and TMγ-TMδ pairing through LZ-EE and LZ-RR interaction, fused to CD28-CDζ, 4-BB- CDζ, CD28-ZAP327 or 4-1BB-ZAP327 signaling domains. LZ-EE, LZ-RR and TM are listed as SEQ. ID NOs: 99-104.

[0163] In some embodiments T cells expressing CARs comprising a leucine zipper RR or EE sequence in the downstream of TM domain (intracellular side) can recognize and kill target cells, e.g., tumor cells or cells involved in other diseases or conditions effectively. The amino acid sequences of TM-LZ-RR and TM-LZ-EE are listed as SEQ. ID NOs: 103-104.

[0164] In some embodiments CARs comprise a leucine zipper RR or EE inserted between the ScFv and hinge-TM domain of the CAR construct (extracellular side), followed by co- stimulatory signaling domains (CD28 or 4-1BB), and T cell activation domains (CD3ζ or ZAP- 70 kinase domain). In one aspect the co-stimulatory signaling domain is from CD28 or 4- 1BB), and the T cell activation domain is from CD3ζ or ZAP-70 kinase domain. In some aspects such CARS, are capable of recognizing antigen-expressing cells with decreased cell killing, but trigger cytokine release. 49 DM2\19774561.1PATENT Docket No. H3131-00401

[0165] Extracellular Binding Domains

[0166] In some embodiments, the extracellular domain comprises an antigen-binding domain, e.g.. an antibody or antigen-binding fragment thereof. The portion of the chimeric receptor of the invention comprising an antibody or antigen-binding fragment thereof may exist in a variety of forms where the ligand binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody or a bispecific antibody (Harlow et al, 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al, Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Bird et al, Science 242:423-426 (1988)). In some aspects, the antigen-binding domain of a chimeric receptor described herein comprises an antibody fragment. In some aspects, the chimeric receptor comprises an antibody fragment that comprises an scFv.

[0167] In some embodiments, the CARs or TCRs or cells expressing them include or exclude, or are selected from, for example, antigen recognition moieties, e.g., ScFvs and / or antibody or antibody fragments and / or receptor or receptor fragments that specifically bind to one or more binding parters that include or exclude, or are selected from, e.g., Alpha (α)- fetoprotein (AFP), interferon-inducible protein absent in melanoma 2 (AIM2), adenocarcinoma antigen recognized by T cells 4 (ART-4), B7-H3, a member of the B7 ligand family, BCMA, B antigen (BAGE), CTL-recognized antigen on melanoma (CAMEL), carcinoembryonic antigen peptide-1 (CAP-1), Caspase 8 (CASP8), Cell division cycle 27 (CDC27), Cyclin- dependent kinase 4 (CDK4), CDK12, Carcinoembryonic antigen (CEA), Calcium-activated chloride channel 2 CLCA2), CFTR, CMV, cancer-testis antigen 83 (CT83), desmin, DLK1, DLL3, EBV, EGFRvIII (epidermal growth factor variant III), EGFR and isovariants thereof, EGFR E746-A750del, EGFRVIII, epithelial-specific antigen (ESA), epithelial cell adhesion molecule (EpCAM), Ephrin type-A receptor 2, 3 (EphA2,3), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial membrane protein (EMA), epithelial tumor antigen (ETA), Fibronectin (FN), FGF-5, FGF-6, G antigen 1 (GAGE-1), GAGE-2, GAGaE- 3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, N-Acetylglucosaminyltransferase V (GnT-V), 50 DM2\19774561.1PATENT Docket No. H3131-00401 glycoprotein 100 (GP100), Helicase antigen (HAGE), H3.3K27M, oncofetal antigen (h5T4), IP3KB, influenza hemagglutinin (HA), HA-1, HA-1H, HA-2, Human epidermal receptor 2 / neurological (HER2) / neu), HBV, HERV-E, HIV-1 gag, HMI.24, HMB-45 antigen, HPV E6, HPV E7, HPV-16 E6, HPV-16 E7, Human telomerase reverse transcriptase (hTERT), V-Ki- ras2 Kirsten rat sarcoma viral oncogene (KRAS), KRAS G12D, KRAS G12V, L antigen 1b (LAGE1b), LMP2, LILRB2, LGR5, Ly49, Ly108, LI cell adhesion molecule (LI-CAM), melanoma-associated antigen (MAGE), Melanoma antigen A1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MAGE-C2, c-Met, MICA / B, muscle-specific actin (MSA), protein melan-A (melanoma antigen recognized by T lymphocytes MART-1), Mesothelin (MSLN), Mucin 1 (MUC1), MUC2, Mucin 16 (Muc-16), myo-D1, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2-PK), Necl-2, neurofilament, NKCSI, NKG2D, neuron-specific enolase (NSE), NY-ESO, New York esophageous 1 (NY-ESO-1), Preferentially expressed antigen of melanoma (PRAME), Prostate-specific antigen (PSA), Prostate-specific membrane antigen (PSMA), Renal antigen (RAGE), Ral-B, an abnormal ras protein, ROR1, SLAMF7 / CS1, sperm protein 17 (Sp17), sarcoma antigen (SAGE), squamous antigen rejecting tumor 1, 2, 3 (SART-1,-2,-3), SOX10, synovial sarcoma, X breakpoint 2 (SSX-2), Survivin, OVA1, HE4, DR-70, Total PSA, alpha- Methylacyl-CoA Racemase / AMACR, CA125 / MUC16, ER alpha / NR3A1, ER beta / NR3A2, Thymidine Kinase 1, AG-2, BRCA1, BRCA2, CA15-3 / MUC-1, Caveolin-1, CD117 / c-kit, CEACAM-5 / CD66e, Cytokeratin 14, HIN-1 / SCGB3A1, Ki-67 / MKI67, MKP-3, Nestin, NGF R / TNFRSF16, NM23-H1, PARP, PP4, Serpin E1 / PAI-1, 14-3-3 beta, 14-3-3 sigma, 14-3-3 zeta, 15-PGDH / HPGD, 5T4, TIM-3, TROP-2, Nectin-4, PD1, PD-L1, CTLA-4, PDGFRalpha, VEGF, TRAG-3, T cell receptor gamma alternate reading frame protein (TARP), TGFbII, Thyroglobulin, an abnormal p53 protein, TP53 (p53), TRAIL, Tyrosinase-related protein 1 or gp75 (TRP1), TRP2, TYRP1, Tyrosinase, tumor-associated glycoprotein 72 (TAG-72), TALLA-1, TLR4, TRBCl, TRBC2, Trp-p8, thyroglobulin, thyroid transcription factor-1, Vα24, Wilms’ tumor gene (WT1), CD1a, CD1b, CD1c, CD2, CD3, CD4, CDS, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12, CD13, CD14, CD15 (SSEA-1), CD16, (FcyRIII), CD17, CD18, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, 51 DM2\19774561.1PATENT Docket No. H3131-00401 CD29, CD30, CD31, CD32 (FcyRII), CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD43, CD44, CD44V6, CD45, CD45R / B220, CD45RO, CD49b, CD49d, CD49f, CD52, CD53, CD54, CD56 (NCAM), CD57, CD61 (integrin β3), CD62L, CD63, CD64 (FcγRl), CD66b, CD68, CD69, CD70, CD73, CD74, CD79a (Igα), CD79b (Igβ), CD80, CD83, CD85k (ILT3), CD86, CD88, CD93 (C1Rqp), CD94, CD95, CD99, CD103, CD105 (Endoglin), CD107a, CD107b, CD114 (G-CSFR), CD115, CD117, CD122, CD123, CD129, CD133, CD134, CD138 (Syndecan-1), CD141 (BDCA3), CD146, CD152 (CTLA-4), CD158 (Kir), CD161 (NK-1.1), CD163, CD183, CD191, CD193 (CCR3), CD194 (CCR4), CD195 (CCRS), CD197 (CCR7), CD203c, CD205 (DEC-205), CD207 (Langerin), CD209 (DC- SIGN), CD223, CD235, CD235a, CD244 (2B4), CD252 (OX40L), CD267, CD268 (BAFF- R), CD273 (B7-DC, PD-L2), CD276 (B7-H3), CD279 (PDI), CD282 (TLR2), CD284 (TLR4), CD294, CD304 (Neuropilin-1), CD305, CD314 (NKG2D), CD319 (CRACC), CD326, CD328 (Siglec-7), CD335 (NKp46), HLA-DR, Kappa light chain, Lambda light chain, Pax-5, BCL-2, Ki-67, MPO, TdT, FMC-7, Pro2PSA, ROMA (HE4+CA-125), OVA1 (multiple proteins), HE4, Fibrin / fibrinogen degradation product (DR-70), AFP-L3, Circulating Tumor Cells (EpCAM, CD45, cytokeratins 8, 18+, 19+), Prostate stem cell antigen (PSCA), α2β1, PAP (prostatic acid phosphatase), PAMA, P-cadherin, placental alkaline phosphatase, PRAIVIE, C3AR, carbonic anhydrase IX (CAIX), chromogranin, CLEC12A, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), CS-I, CSPG4, cytokeratin, AC133 antigen, p63 protein, c-Kit, Lewis A (CA19.9), Lewis Y (LeY), Estrogen receptor (ER), Progesterone receptor (PR), Pro2PSA, cancer antigen-125 (CA-125), CA15-3, CA27.29, Free PSA, Thyroglobulin, Nuclear Mitotic Apparatus protein (NuMA / NMP22), A33, ABCB5, ABCB6, ABCG2, ACE / CD143, ACLP, ACP6, Afadin / AF-6, Afamin, AG-2, AG-3, Akt, Aldo-keto Reductase 1C3 / AKR1C3, alpha 1B-Glycoprotein, alpha 1-Microglobulin, AlphaB Crystallin / CRYAB, alpha-Methylacyl-CoA Racemase / AMACR, AMFR / gp78, Annexin A3, Annexin A8 / ANXA8, APC, Apolipoprotein A-I / ApoA1, Apolipoprotein A-II / ApoA2, Apolipoprotein E / ApoE, APRIL / TNFSF13, ASCL1 / Mash1, ATBF1 / ZFHX3, Attractin, Aurora A, BAP1, Bcl-2, Bcl-6, beta 2-Microglobulin, beta-1,3-Glucuronyltransferase 1 / B3GAT1, beta-Catenin, beta-III Tubulin, Bikunin, BMI-1, B-Raf, BRCA1, BRCA2, Brk, 52 DM2\19774561.1PATENT Docket No. H3131-00401 C4.4A / LYPD3, CA15-3 / MUC-1, c-Abl, Cadherin-13, Caldesmon / CALD1, Calponin 1, Calretinin, Carbonic Anhydrase IX / CA9, Catalase, Cathepsin D, Caveolin-1, Caveolin-2, CBFB, CCR1, CCR4, CCR7, CCR9, CEACAM-19, CEACAM-20, CEACAM-4, CHD1L, Chitinase 3-like 1, Cholecystokinin-B R / CCKBR, Chorionic Gonadotropin alpha Chain (alpha HCG), Chorionic Gonadotropin alpha / beta (HCG), CKAP4 / p63, Claudin-18, Clusterin, c-Maf, c-Myc, Coactosin-like Protein 1 / CotL1, COMMD1, Cornulin, Cortactin, COX-2, CRISP-3, CTCF, CTL1 / SLC44A1, CXCL17 / VCC-1, CXCL8 / IL-8, CXCL9 / MIG, CXCR4, Cyclin A1, Cyclin A2, Cyclin D2, Cyclin D3, CYLD, Cyr61 / CCN1, Cytokeratin 14, Cytokeratin 18, Cytokeratin 19, fetal acetylcholine receptor (AChR), ADGRE2, ATM, ALK, ALPK2, DAB2, DCBLD2 / ESDN, DC-LAMP, Dkk-1, DLL3, DMBT1, DNMT1, DPPA2, DPPA4, E6, E- Cadherin, ECM-1, EGF, ELF3, ELTD1, EMMPRIN / CD147, EMP2, Endoglin / CD105, Endosialin / CD248, Enolase 2 / Neuron-specific Enolase, EpCAM / TROP1, Eps15, ER alpha / NR3A1, ER beta / NR3A2, ERBB, EGFR / ErbB1, ERBB2, ErbB3 / Her3, ErbB4 / Her4, ERCC1, ERK1, ERK5 / BMK1, Ets-1, Exostosin 1, EZH2, Ezrin, FABP5 / E-FABP, Fascin, FATP3, FCRLA, Fetuin A / AHSG, FGF acidic, FGF basic, FGF R3, FGF R4, Fibrinogen, folate-binding protein (FBP), Fibroblast Activation Protein alpha / FAP, Follistatin-like 1 / FSTL1, FOLR1, FOLR2, FOLR3, FOLR4, FosB / G0S3, FoxM1, FoxO3, FRAT2, FXYD5 / Dysadherin, FcεRIα, FITC, FLT3, GABA-A R alpha 1, GADD153, GADD45 alpha, Galectin-3, Galectin-3BP / MAC-2BP, galactin, gangiosides, gross cystic disease fluid protein (GCDFP-15), GD2 (ganglioside G2), GD3, GM2, GM3, gamma- Glutamylcyclotransferase / CRF21, Gas1, Gastrin-releasing Peptide R / GRPR, Gastrokine 1, Gelsolin / GSN, glial fibrillary acidic protein (GFAP), GLI-2, Glutathione Peroxidase 3 / GPX3, gpA33, glycopeptides, Glypican 2 (GPC2), Glypican 3, Golgi Glycoprotein 1 / GLG1, gp96 / HSP90B1, GPR10, GPR110, GPR18, GPR31, GPR87, GPRC5A, GPRC6A, GRP78 / HSPA5, HE4 / WFDC2, Heparanase / HPSE, Hepsin, HGF R / c-MET, HIF-2 alpha / EPAS1, HIN-1 / SCGB3A1, HLA-DR, HOXB13, HOXB7, HSP70 / HSPA1A, HSP90, Hyaluronidase 1 / HYAL1, ID1, IgE, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, IGF-I, IGF-I R, IGF-II, IGFL-3, IGFLR1, IL-1 beta / IL-1F2, IL-17E / IL-25, IL-2, IL-6, ICAM-1, IgG, IgD, IgE, IgM, Interleukin 13 receptor a2 chain (IL-13Ra), Interleukin-13 receptor subunit alpha-2 (IL- 53 DM2\19774561.1PATENT Docket No. H3131-00401 13Ra2), integrins, Integrin B7, IMP Dehydrogenase 1 / IMPDH1, Importin alpha 2 / KPNA2, ING1, Integrin beta 1 / CD29, Integrin beta 3 / CD61, IQGAP1, Isocitrate Dehydrogenase 1 / IDH1, ITIH4, ITM2C, Jagged 1, JNK, JunB, JunD, OGR1, Olig2, Osteopontin / OPN, Ovastacin, OXGR1 / GPR80 / P2Y15, p130Cas, p15INK4b / CDKN2B, p16INK4a / CDKN2A, p18INK4c / CDKN2C, p21 / CIP1 / CDKN1A, p27 / Kip1, P2X5 / P2RX5, PARP, PAUF / ZG16B, PBEF / Visfatin, PDCD4, PDCD5, PDGF R alpha, PDGF R beta, PDZD2, PEA-15, Pepsinogen A5 / PGA5, Peptidase Inhibitor 16 / PI16, Peroxiredoxin 2, PGCP, PI 3-Kinase p85 alpha, PIWIL2, PKM2, PLK1, PLRP1, PP4, P-Rex1, PRMT1, Profilin 1, Progesterone R B / NR3C3, Progesterone R / NR3C3, Progranulin / PGRN, Prolactin, Prostaglandin E Synthase 2 / PTGES2, PSAP, PSCA, PSMA / FOLH1 / NAALADase I, PSMA1, PSMA2, PSMB7, PSP94 / MSMB, PTEN, PTEN, PTH1R / PTHR1, PTK7 / CCK4, PTP beta / zeta / PTPRZ, Rab25, RARRES1, RARRES3, Ras, Reg4, Ret, RNF2, RNF43, S100A1, S100A10, S100A16, S100A2, S100A4, S100A6, S100A7, S100A9, S100B, S100P, SART1, SCUBE3, Secretin R, Serpin A9 / Centerin, Serpin E1 / PAI-1, Serum Amyloid A1, Serum Amyloid A4, SEZ6L, SEZ6L2 / BSRP-A, Skp2, SLC16A3, SLC45A3 / Prostein, SLC5A5, SLC5A8 / SMCT1, SLC7A7, Smad4, SMAGP, SOCS-1, SOCS-2, SOCS-6, SOD2 / Mn-SOD, Soggy-1 / DkkL1, SOX11, SOX17, SOX2, SPARC, SPARC-like 1 / SPARCL1, SPINK1, Src, six-transmembrane epithelial antigen of the prostate 1 (STEAP1), STEAP2, STEAP3 / TSAP6, STRO-1, STYK1, Survivin, Synaptotagmin- 1, Syndecan-1 / CD138, Syntaxin 4, Synuclein-gamma, synaptophysin, Kallikrein 2, Kallikrein 6 / Neurosin, KCC2 / SLC12A5, Ki-67 / MKI67, KiSS1R / GPR54, KLF10, KLF17, L1CAM, Lactate Dehydrogenase A / LDHA, Lamin B1, LEF1, Leptin / OB, LIN-28A, LIN-28B, Lipocalin-2 / NGAL, LKB1 / STK11, LPAR3 / LPA3 / EDG-7, LRMP, LRP-1B, LRRC3B, LRRC4, LRRN1 / NLRR-1, LRRN3 / NLRR-3, Ly6K, LYPD1, LYPD8, MAP2, Matriptase / ST14, MCAM / CD146, M-CSF, MDM2 / HDM2, Melanocortin-1 R / MC1R, Melanotransferrin / CD228, Melatonin, Mer, Mesothelin, Metadherin, Metastin / KiSS1, Methionine Aminopeptidase, Methionine Aminopeptidase 2 / METAP2, MFAP3L, MGMT, MIA, MIF, MINA, Mind Bomb 2 / MIB2, Mindin, MITF, MKK4, MKP-1, MKP-3, MMP-1, MMP-10, MMP-13, MMP-2, MMP-3, MMP-8, MMP-9, MRP1, MRP4 / ABCC4, MS4A12, MSH2, MSP R / Ron, MSX2, MUC-4, Musashi-1, NAC1, Napsin A, NCAM-1 / CD56, NCOA3, 54 DM2\19774561.1PATENT Docket No. H3131-00401 NDRG1, NEK2, NELL1, NELL2, Nesfatin-1 / Nucleobindin-2, Nestin, NFkB2, NF-L, NG2 / MCSP, NGF R / TNFRSF16, Nicotinamide N-Methyltransferase / NNMT, NKX2.2, NKX3.1, NM23-H1, NM23-H2, Notch-3, NPDC-1, NTS1 / NTSR1, NTS2 / NTSR2, Tankyrase 1, Tau, TCF-3 / E2A, TCL1A, TCL1B, TEM7 / PLXDC1, TEM8 / ANTXR1, Tenascin C, TFF1, TGF-beta 1, TGF-beta 1, 2, 3, TGF-beta 1 / 1.2, TGF-beta 2 / 1.2, TGF-beta RI / ALK-5, THRSP, Thymidine Kinase 1, Thymosin beta 10, Thymosin beta 4, Thyroglobulin, TIMP Assay Kits, TIMP-1, TIMP-2, TIMP-3, TIMP-4, TLE1, TLE2, TM4SF1 / L6, TMEFF2 / Tomoregulin-2, TMEM219, TMEM87A, TNF-alpha, TOP2A, TopBP1, t-Plasminogen Activator / tPA, TRA-1- 60(R), TRA-1-85 / CD147, TRAF-4, Transgelin / TAGLN, Trypsin 2 / PRSS2, Tryptase alpha / TPS1, TSPAN1, UBE2S, uPAR, u-Plasminogen Activator / Urokinase, Urotensin-II R, VAP-1 / AOC3, VCAM-1 / CD106, VEGFR1 / Flt-1, VEGFR2 / KDR / Flk-1, VEGF / PlGF Heterodimer, VSIG1, VSIG3, YAP1, ZAG, ZAP70, ZMIZ1 / Zimp10, SGK, CNKSR1 / CNK / KSR or any combination thereof.

[0168] In certain embodiments, the chimeric antigen receptor CAR-T or TCR-T cells of the present invention may target a wide range of tumor-associated antigens. In certain embodiments, the antigens comprise one or more of ROR1, CD56, EGFR, CD123, c-met, GD2, HER2, CD20, MUC-1, CD23, and CD30. In certain embodiments, by targeting these antigens, the CAR T cells can be used to treat various solid tumors and hematological malignancies, such as leukemias, lymphomas, breast cancer, lung cancer, colorectal cancer, prostate cancer, ovarian cancer, neuroblastoma, and melanoma.

[0169] Two cancer-testis (CT) antigens, NY-ESO-1, encoded by CTAG1B gene, and CT83 (also known as KK-LC-1), encoded by CT83 gene, are widely expressed in a variety of tumors including lung cancer and breast cancer. Both CD8+ T cells and antibodies have been shown to recognize NY-ESO-1, and a clinical response of 50-80% using NY-ESO-1-specific TCR has been demonstrated in several solid cancers, including melanoma, sarcoma and myeloma. Despite the importance of CD4+ T cells (HLA-DP4 is the most frequent HLA II molecule expressing in general human population, accounting for 70% positive), HLA-DP4-restricted NY-ESO-1 specific TCRs have not been tested in a clinical setting. The inventors previously identified HLA-DR4- and HLA-DP4-restricted NY-ESO-1 epitopes (Zeng, G. et al. 55 DM2\19774561.1PATENT Docket No. H3131-00401 Identification of CD4+ T cell epitopes from NY-ESO-1 presented by HLA-DR molecules. J. Immunol. 165, 1153-1159 (2000); Zeng, G., Wang, X., Robbins, P. F., Rosenberg, S. A. & Wang, R.-F. CD4+ T cell recognition of MHC class II-restricted epitopes from NY-ESO-1 presented by a prevalent HLA-DP4 allele: association with NY-ESO-1 antibody production. Proc. Natl. Acad. Sci. U.S.A.98, 3964-3969 (2001)), and showed that an HLA-DP4-NY-ESO- 1 peptide overlapped with an HLA-A2-restricted NY-ESO-1 peptide. Zeng, G. et al. Generation of NY-ESO-1-specific CD4+ and CD8+ T cells by a single peptide with dual MHC class I and class II specificities: a new strategy for vaccine design. Cancer Res.62, 3630-3635. (2002).

[0170] In addition to the CT antigen NY-ESO-1, CT83 is highly expressed in 60-70% of breast cancer, particularly in TNBC, which is in consistent with previous reports. Fukuyama, T. et al. Identification of a new cancer / germline gene, KK-LC-1, encoding an antigen recognized by autologous CTL induced on human lung adenocarcinoma. Cancer research 66, 4922-4928, doi:10.1158 / 0008-5472.CAN-05-3840 (2006); Paret, C. et al. CXorf61 is a target for T cell-based immunotherapy of triple-negative breast cancer. Oncotarget 6, 25356-25367, doi:10.18632 / oncotarget.4516 (2015). However, relatively little was known about its immunogenicity, T cell epitopes and the cognate TCRs for tumor recognition by T cells. As disclosed herein antigen-specific CD4+ and CD8+ T cells were generated, and used to identify HLA-A2-restricted CT83-specific TCR (A2-CT83 TCR) to determine if CT83 could serve as an attractive target for TCR-T cell immunotherapy.

[0171] In some embodiments, said antibody is an antibody molecule selected from the group consisting of a whole antibody, a humanized antibody, a single chain antibody, a dimeric single chain antibody, a Fv, an scFv, a Fab, a F(ab)’2, a defucosylated antibody, a bi- specific antibody, a diabody, a triabody, and a tetrabody. In some embodiments, said antibody is an antibody fragment selected from the group consisting of a unibody, a single domain antibody, and a nanobody.

[0172] In some embodiments, the antigen-binding domain of a CAR of the invention comprises or consists of an antibody fragment, such as, for example, an scFv. In particular embodiments, the antigen-binding domain is an scFv. 56 DM2\19774561.1PATENT Docket No. H3131-00401

[0173] In some embodiments, the antigen-binding domain of a CAR of the invention recognizes a specific antigen or fragment thereof (e.g., associated with a target cell). Thus, the antigen-binding domain of a CAR may recognize target cells such as, for example, infected cells, damaged cells, or dysfunctional cells. Examples of such target cells may include cells involved in dysfunctional immune reactions (e.g., cells involved in autoimmune diseases or allergy), dysfunctionally activated inflammatory cells (e.g., inflammatory endothelial cells), cancer cells, and infected (e.g., virally, bacterially, or parasitically infected) cells.

[0174] In some embodiments, the antigen is an autoantigen. Examples of autoantigens include, without limitation, an antigen associated with an inflammatory nervous system condition (e.g., a multiple sclerosis-associated antigen), a joint-associated antigen, an eye- associated antigen, a human HSP antigen, a skin-associated antigen or an antigen involved in graft rejection or GVHD.

[0175] In some embodiments, the antigen is an antigen involved in graft rejection or GVHD. Examples of such antigens include, but are not limited to, the MHC specific to the transplanted tissue or to the host, 2-microglobulin, antigens from the ABO system, antigens from the rhesus system (e.g., antigens C, c, E, e and D) and isohaemagglutinins. Other examples of antigens that may be involved in graft rejection or GVHD include, but are not limited to, HLA-DR (in particular during the first six months following grafting), HLA-B (in particular during the first two years following grafting), HLA-A, minor histocompatibility antigens (miHA, e.g., HLA-E, HLA-F and HLA-G), HLAs corresponding to MHC class I (A, B, and C), HLAs corresponding to MHC class II (DP, DM, DOA, DOB, DQ, and DR) and HLAs corresponding to MHC class III (e.g., components of the complement system)

[0176] In some embodiments, the antigen is an inflammatory nervous system condition- associated antigen, e.g., a multiple sclerosis-associated antigen. Examples of inflammatory nervous system condition-associated antigens (e.g., multiple sclerosis-associated antigens) include, but are not limited to, myelin basic protein (MBP), myelin associated glycoprotein (MAG), myelin oligodendrocyte protein (MOG), proteolipid protein (PLP), oligodendrocyte myelin oligoprotein (OMGP), myelin associated oligodendrocyte basic protein (MOBP), oligodendrocyte specific protein (OSP / Claudin-l 1), heat shock proteins, oligodendrocyte 57 DM2\19774561.1PATENT Docket No. H3131-00401 specific proteins (OSP), NOGO A, glycoprotein Po, peripheral myelin protein 22 (PMP22), 2’3’-cyclic nucleotide 3’-phosphodiesterase (CNPase), and fragments, variants and mixtures thereof.

[0177] Knockdown, Knockout and Inactivting Mutations of Negative Signaling Molecules

[0178] It has been demonstrated that CAR-T and TCR-T cell persistence is closely correlated with patient survival. Thus, modulation of TCR-T and CAR-T-cell signaling may enhance T cell persistence and reduce T cell exhaustion through direct regulation of CAR or TCR signaling and knockdown or knockout of negative signaling molecules. In some embodiments, the present disclosure provides an engineered immune (TCR-T or CAR-T) cell comprising an knock down, knockout, inactivating mutation in one or more endogenous genes selected from a group of negative regulators that can include or exclude, or are selected from, e.g., ANKRDJ 1, ARID1A, BACH2, BCL2Ll 1, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3Hl (ROQUIN- 1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2DJA, SMAD2,SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6 and ZC3H12A.

[0179] In some embodiments, the present disclosure provides an engineered immune (TCR-T or CAR-T) cell comprising knockdown, knockout, inactivating mutations in two or more target genes selected from a group of negative regulators that can include or exclude, or are selected from, e.g., ANKRDJ 1, ARID1A, BACH2, BCL2Ll 1, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3Hl (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2DJA, SMAD2,SOCS1, 58 DM2\19774561.1PATENT Docket No. H3131-00401 SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6 and ZC3H12A.

[0180] In some embodiments, knockdown of target gene expression is achieved through an siRNA or an shRNA nucleic acid molecule by targeting one or more genes, where the one or more genes coding for negative regulators can include or exclude, or are selected from, e.g., ANKRDJ 1, ARID1A, BACH2, BCL2Ll 1, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3Hl (ROQUIN- 1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2DJA, SMAD2,SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6 and ZC3H12A, and wherein the siRNA or shRNA molecule comprises about 20-30 nucleotides that bind to an RNA sequence encoded by a DNA sequence of the selected genes.

[0181] In one embodiment of the present invention, the sequences of shRNAs or antisense RNAs or DNAs that specifically knock down, knock out, or alter the expression of metabolic genes to enhance the antitumor activity of TCR or CAR-based therapy in vivo are provided in the form of a stem of 21 base pairs. The TCR may be engineered together with the shRNAs by knocking down target genes to improve the T cell trafficking and persistence in vivo and enhance the antitumor activity.

[0182] In some embodiments, knockout or inactivation of target gene expression is achieved through a CRISPR / Cas9 system comprising a single-strand guide RNA (sgRNA) molecule and Cas endonuclease and is capable of reducing or knocking out gene expression of one or more target genes that can include or exclude, or are selected from, e.g., ANKRDJ 1, ARID1A, BACH2, BCL2Ll 1, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3Hl (ROQUIN-1), 59 DM2\19774561.1PATENT Docket No. H3131-00401 SEMA7A, SERPINA3, SETD5, SH2B3, SH2DJA, SMAD2,SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6 and ZC3H12A, and wherein the sgRNA molecule comprises a targeting RNA sequence that binds to a target DNA sequence selected from a group of negative regulators ANKRDJ 1, ARID1A, BACH2, BCL2Ll 1, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3Hl (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2DJA, SMAD2,SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6 and ZC3H12A.

[0183] The present invention encompasses CD4+ or CD8+ T lymphocytes immunologically recognizing a tumor antigen with the restriction of one HLA class II or class I molecules. The present invention further encompasses at least one T cell receptor derived from the CD4+ T lymphocytes or CD8+ T lymphocytes mentioned above. The T cell receptors are capable of being delivered into naïve CD4+ or CD8+ T lymphocytes that have no immune response to the tumor antigens mentioned above, and changing the function of those CD4+ or CD8+ T lymphocytes to recognize and react with the tumor antigen mentioned above specifically. This reaction between the tumor antigens and the transduced T cell receptor causes the T lymphocytes to respond against, and assist in preventing, eliminating or reducing the human cancers with the tumor antigens mentioned above.

[0184] The CAR T cells of the present invention are particularly advantageous for treating minimal residual disease, which refers to the small number of cancer cells that may remain after primary treatment. Due to their high sensitivity and specificity, CAR T cells can effectively eliminate these residual cancer cells, reducing the risk of relapse. Moreover, the CAR T cells can be employed to treat relapsed or refractory cancers that have become resistant to conventional therapies such as chemotherapy and radiation. In one embodiment, the CAR- T or TCR-T cells of this disclosure are administered to patients following detection of MRD 60 DM2\19774561.1PATENT Docket No. H3131-00401 using molecular testing, e.g., clonoSEQ, Signatera, Plasma Detect, Galleri, Guardant Reveal MRD testing.

[0185] To enhance the persistence and efficacy of the CAR T cells, the cells may be further engineered to co-express a membrane-bound proliferative cytokine, for example, such as interleukin-15 (IL-15). In one embodiment, the CAR T cells express a fusion protein comprising IL-15 and IL-15 receptor alpha (IL-15Ra). This fusion protein may comprise an amino acid sequence at least 85%, 90%, 95%, or 99% identical to SEQ ID NO: 6. The membrane-bound IL-15 provides a sustained proliferative signal to the CAR T cells, enabling them to persist and expand in vivo even in the absence of abundant antigen stimulation.

[0186] B. Compositions

[0187] Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular CAR is disclosed and discussed and a number of modifications that can be made to a number of molecules including the CAR are discussed, specifically contemplated is each and every combination and permutation of CAR and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps 61 DM2\19774561.1PATENT Docket No. H3131-00401 that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0188] In one embodiment, the methods disclosed herein detect or identify CARs that can be used in compositions for the treatment of cancer (either as a therapeutic treatment or prophylactic treatment) as well as in preparing CAR T cells that can be used to treat cancer. Thus, in one embodiment, also disclosed are CAR T cells engineered to express a receptor (which can include or exclude, for example, a T cell receptor) that can recognize antigens disclosed herein.

[0189] In one embodiment, this disclosure also feature methods to enhance CAR-T cell persistence by expression of chemokine receptor and shRNA KO in CAR constructs. In one embodiment, the CAR T cell specific for one antigen (for example, NY-ESO-1, CT83, pp65 and / or IE-1) disclosed herein can be further engineered to knockout or knockdown a negative signaling molecule, for example without limitation, ANKRDJ 1, ARID1A, BACH2, BCL2Ll 1, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3Hl (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2DJA, SMAD2,SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6 and ZC3H12A, to enhance their function which can include or exclude cytotoxic activity and persistence or survival in vivo after adoptive transfer to a cancer patient.

[0190] In some embodiments the negative signaling molecules are, for example, indoleamine (2,3)-dioxygenase (IDO) (including isoforms IDO1 and IDO2), OX40, CTLA-4 (programmed cytotoxic T-lymphocyte antigen 4), PD-1 (programmed death 1), PD-L1 (programmed death ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), and B7 homolog 3 (B7-H3). In certain aspects, the negative signaling molecules are, for example, PD-1, VHL, PPP2R2D and epigenetic factors which can include or exclude JMJD3 and LSD1. 62 DM2\19774561.1PATENT Docket No. H3131-00401

[0191] In one embodiment, this disclosure also features methods to enhance T cell trafficking into tumors and / or the location of tumor cells in vivo by forced expression of chemokine receptors. In some embodiments, expression of chemokine receptors is forced by fusing a CAR construct withi a chemokine. In some embodiments, the chemokine receptor is CCR5, CCR2 of CXCR3. In some embodiments, the chemokine receptor is CCR5.

[0192] In any of the CAR embodiments featured herein, in some embodiments the CAR / chimeric CAR comprises an intracellular T-cell activation moiety, where the intracellular T-cell activation moiety comprises a signaling domain, further wherein the signaling domain comprises the replacement of CD3ζ with a ZAP70 kinase domain or a mutant or a variant thereof comprising a functional ZAP70 kinase. In some embodiments, any of the CARs featured herein comprise a ZAP70 kinase domain derivatized from a functional wild type ZAP70 or a mutant or a variant thereof, wherein the ZAP70 kinase domain derivatized from a functional wild type ZAP70 or a mutant or a variant thereof further comprises a functional ZAP70 kinase domain. In some embodiments, the mutant or variant of ZAP70 which is a functional ZAP70 kinase domain is a truncated biologically active fragment of ZAP70 kinase. In some embodiments, the functional ZAP70 kinase domain comprises an amino acid sequence having at least 55% identity to the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In any of the TCRs disclosed herein, the ZAP70 portion can be any of the fragments ZAP300 (SEQ ID NO: 1, amino acid sequence from 300-619 a.a.), ZAP327 (SEQ ID NO: 2, amino acid sequence from 327-619 a.a.), ZAP338 (SEQ ID NO: 3, amino acid sequence from 338-619 a.a.), ZAP255 (SEQ ID NO: 4, amino acid sequence from 255-619 a.a.), ZAP280 (SEQ ID NO: 5, amino acid sequence from 280-619 a.a.) or a mutant or a variant thereof. In some embodiments the TCR can comprise a ZAP70 kinase domain where the N-terminal amino acid is any amino acid between or including 250 to 338, between or including 256 to 338, between or including 281 to 338, between or including 309 to 338, between or including 300 to 338, between or including 250 to 327, between or including 281 to 327, between or including 309 to 327 or between or including 300 to 327, and the C-terminal amino acid is between or including amino acid 610 to 619. In some of the foregoing embodiments the C-terminal amino acid is 619. In some embodiments the terminal 63 DM2\19774561.1PATENT Docket No. H3131-00401 amino acid is any of 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279. 280, 281, 282, 283, 284, 285, 286. 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338), and the C-terminal amino acid is 610, 611, 612, 613, 614, 615, 616, 617, 618 or 619 a.a.). In some embodiments the ZAP70 kinase domain includes or excludes ZAP255 (255-619 a.a.), ZAP280 (280-619 a.a.), or ZAP308 (308-619 a.a.).

[0193] In any of the CARs featured herein, (e.g., in the CARS comprising chimeric antigen receptors or T cells expressing a CAR / chimeric CAR), in some embodiments the CAR comprises an antigen recognition moiety, a transmembrane domain, and an intracellular T-cell activation moiety, wherein the intracellular T-cell activation moiety further comprises a signaling domain comprising a ZAP70 kinase domain or a variant thereof. In some embodiments, the CARs are specific for a cancer antigen selected from a group consisting of Alpha (α)-fetoprotein (AFP), interferon-inducible protein absent in melanoma 2 (AIM2), adenocarcinoma antigen recognized by T cells 4 (ART-4), BCMA, B antigen (BAGE), CTL- recognized antigen on melanoma (CAMEL), carcinoembryonic antigen peptide-1 (CAP-1), Caspase 8 (CASP8), Cell division cycle 27 (CDC27), Cyclin-dependent kinase 4 (CDK4), CDK12, Carcinoembryonic antigen (CEA), Calcium-activated chloride channel 2 CLCA2), CFTR, CMV, cancer-testis antigen 83 (CT83), desmin, DLK1, DLL3, EBV, EGFRvIII (epidermal growth factor variant III), EGFR and isovariants thereof, EGFR E746-A750del, EGFRVIII, epithelial-specific antigen (ESA), epithelial cell adhesion molecule (EpCAM), Ephrin type-A receptor 2, 3 (EphA2,3), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial membrane protein (EMA), epithelial tumor antigen (ETA), Fibronectin (FN), FGF-5, FGF-6, G antigen 1 (GAGE-1), GAGE-2, GAGaE-3, GAGE- 4, GAGE-5, GAGE-6, GAGE-7, N-Acetylglucosaminyltransferase V (GnT-V), glycoprotein 100 (GP100), HCMV pp65, HCMV IE-1, Helicase antigen (HAGE), H3.3K27M, oncofetal antigen (h5T4), IP3KB, influenza hemagglutinin (HA), HA-1, HA-1H, HA-2, Human epidermal receptor 2 / neurological (HER2) / neu), HBV, HERV-E, HIV-1 gag, HMI.24, HMB- 64 DM2\19774561.1PATENT Docket No. H3131-00401 45 antigen, HPV E6, HPV E7, HPV-16 E6, HPV-16 E7, Human telomerase reverse transcriptase (hTERT), V-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS), KRAS G12D, KRAS G12V, L antigen 1b (LAGE1b), LMP2, LILRB2, LGR5, Ly49, Ly108, LI cell adhesion molecule (LI-CAM), melanoma-associated antigen (MAGE), Melanoma antigen A1 (MAGE- A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MAGE-C2, c-Met, MICA / B, muscle-specific actin (MSA), protein melan-A (melanoma antigen recognized by T lymphocytes MART-1), Mesothelin (MSLN), Mucin 1 (MUC1), MUC2, Mucin 16 (Muc- 16), myo-D1, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2-PK), Necl-2, neurofilament, NKCSI, NKG2D, neuron-specific enolase (NSE), NY-ESO, New York esophageous 1 (NY-ESO-1), Preferentially expressed antigen of melanoma (PRAME), Prostate-specific antigen (PSA), Prostate-specific membrane antigen (PSMA), Renal antigen (RAGE), Ral-B, an abnormal ras protein, ROR1, SLAMF7 / CS1, sperm protein 17 (Sp17), sarcoma antigen (SAGE), squamous antigen rejecting tumor 1, 2, 3 (SART-1,-2,-3), SOX10, synovial sarcoma, X breakpoint 2 (SSX-2), Survivin, OVA1, HE4, DR-70, Total PSA, alpha- Methylacyl-CoA Racemase / AMACR, CA125 / MUC16, ER alpha / NR3A1, ER beta / NR3A2, Thymidine Kinase 1, AG-2, BRCA1, BRCA2, CA15-3 / MUC-1, Caveolin-1, CD117 / c-kit, CEACAM-5 / CD66e, Cytokeratin 14, HIN-1 / SCGB3A1, Ki-67 / MKI67, MKP-3, Nestin, NGF R / TNFRSF16, NM23-H1, PARP, PP4, Serpin E1 / PAI-1, 14-3-3 beta, 14-3-3 sigma, 14-3-3 zeta, 15-PGDH / HPGD, 5T4, TIM-3, TROP-2, Nectin-4, PD1, PD-L1, CTLA-4, PDGFRalpha, VEGF, TRAG-3, T cell receptor gamma alternate reading frame protein (TARP), TGFbII, Thyroglobulin, an abnormal p53 protein, TP53 (p53), TRAIL, Tyrosinase-related protein 1 or gp75 (TRP1), TRP2, TYRP1, Tyrosinase, tumor-associated glycoprotein 72 (TAG-72), TALLA-1, TLR4, TRBCl, TRBC2, Trp-p8, thyroglobulin, thyroid transcription factor-1, Vα24, Wilms’ tumor gene (WT1), CD1a, CD1b, CD1c, CD2, CD3, CD4, CDS, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12, CD13, CD14, CD15 (SSEA-1), CD16, (FcyRIII), CD17, CD18, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32 (FcyRII), CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD43, CD44, CD44V6, CD45, CD45R / B220, CD45RO, CD49b, CD49d, CD49f, CD52, CD53, CD54, CD56 (NCAM), CD57, CD61 (integrin β3), CD62L, CD63, CD64 65 DM2\19774561.1PATENT Docket No. H3131-00401 (FcγRl), CD66b, CD68, CD69, CD70, CD73, CD74, CD79a (Igα), CD79b (Igβ), CD80, CD83, CD85k (ILT3), CD86, CD88, CD93 (C1Rqp), CD94, CD95, CD99, CD103, CD105 (Endoglin), CD107a, CD107b, CD114 (G-CSFR), CD115, CD117, CD122, CD123, CD129, CD133, CD134, CD138 (Syndecan-1), CD141 (BDCA3), CD146, CD152 (CTLA-4), CD158 (Kir), CD161 (NK-1.1), CD163, CD183, CD191, CD193 (CCR3), CD194 (CCR4), CD195 (CCRS), CD197 (CCR7), CD203c, CD205 (DEC-205), CD207 (Langerin), CD209 (DC- SIGN), CD223, CD235, CD235a, CD244 (2B4), CD252 (OX40L), CD267, CD268 (BAFF- R), CD273 (B7-DC, PD-L2), CD276 (B7-H3), CD279 (PDI), CD282 (TLR2), CD284 (TLR4), CD294, CD304 (Neuropilin-1), CD305, CD314 (NKG2D), CD319 (CRACC), CD326, CD328 (Siglec-7), CD335 (NKp46), HLA-DR, Kappa light chain, Lambda light chain, Pax-5, BCL-2, Ki-67, MPO, TdT, FMC-7, Pro2PSA, ROMA (HE4+CA-125), OVA1 (multiple proteins), HE4, Fibrin / fibrinogen degradation product (DR-70), AFP-L3, Circulating Tumor Cells (EpCAM, CD45, cytokeratins 8, 18+, 19+), Prostate stem cell antigen (PSCA), α2β1, PAP (prostatic acid phosphatase), PAMA, P-cadherin, placental alkaline phosphatase, PRAIVIE, C3AR, carbonic anhydrase IX (CAIX), chromogranin, CLEC12A, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), CS-I, CSPG4, cytokeratin, AC133 antigen, p63 protein, c-Kit, Lewis A (CA19.9), Lewis Y (LeY), Estrogen receptor (ER), Progesterone receptor (PR), Pro2PSA, cancer antigen-125 (CA-125), CA15-3, CA27.29, Free PSA, Thyroglobulin, Nuclear Mitotic Apparatus protein (NuMA / NMP22), A33, ABCB5, ABCB6, ABCG2, ACE / CD143, ACLP, ACP6, Afadin / AF-6, Afamin, AG-2, AG-3, Akt, Aldo-keto Reductase 1C3 / AKR1C3, alpha 1B-Glycoprotein, alpha 1-Microglobulin, AlphaB Crystallin / CRYAB, alpha-Methylacyl-CoA Racemase / AMACR, AMFR / gp78, Annexin A3, Annexin A8 / ANXA8, APC, Apolipoprotein A-I / ApoA1, Apolipoprotein A-II / ApoA2, Apolipoprotein E / ApoE, APRIL / TNFSF13, ASCL1 / Mash1, ATBF1 / ZFHX3, Attractin, Aurora A, BAP1, Bcl-2, Bcl-6, beta 2-Microglobulin, beta-1,3-Glucuronyltransferase 1 / B3GAT1, beta-Catenin, beta-III Tubulin, Bikunin, BMI-1, B-Raf, BRCA1, BRCA2, Brk, C4.4A / LYPD3, CA15-3 / MUC-1, c-Abl, Cadherin-13, Caldesmon / CALD1, Calponin 1, Calretinin, Carbonic Anhydrase IX / CA9, Catalase, Cathepsin D, Caveolin-1, Caveolin-2, CBFB, CCR1, CCR4, CCR7, CCR9, CEACAM-19, CEACAM-20, CEACAM-4, CHD1L, 66 DM2\19774561.1PATENT Docket No. H3131-00401 Chitinase 3-like 1, Cholecystokinin-B R / CCKBR, Chorionic Gonadotropin alpha Chain (alpha HCG), Chorionic Gonadotropin alpha / beta (HCG), CKAP4 / p63, Claudin-18, Clusterin, c-Maf, c-Myc, Coactosin-like Protein 1 / CotL1, COMMD1, Cornulin, Cortactin, COX-2, CRISP-3, CTCF, CTL1 / SLC44A1, CXCL17 / VCC-1, CXCL8 / IL-8, CXCL9 / MIG, CXCR4, Cyclin A1, Cyclin A2, Cyclin D2, Cyclin D3, CYLD, Cyr61 / CCN1, Cytokeratin 14, Cytokeratin 18, Cytokeratin 19, fetal acetylcholine receptor (AChR), ADGRE2, ATM, ALK, ALPK2, DAB2, DCBLD2 / ESDN, DC-LAMP, Dkk-1, DLL3, DMBT1, DNMT1, DPPA2, DPPA4, E6, E- Cadherin, ECM-1, EGF, ELF3, ELTD1, EMMPRIN / CD147, EMP2, Endoglin / CD105, Endosialin / CD248, Enolase 2 / Neuron-specific Enolase, EpCAM / TROP1, Eps15, ER alpha / NR3A1, ER beta / NR3A2, ERBB, EGFR / ErbB1, ERBB2, ErbB3 / Her3, ErbB4 / Her4, ERCC1, ERK1, ERK5 / BMK1, Ets-1, Exostosin 1, EZH2, Ezrin, FABP5 / E-FABP, Fascin, FATP3, FCRLA, Fetuin A / AHSG, FGF acidic, FGF basic, FGF R3, FGF R4, Fibrinogen, folate-binding protein (FBP), Fibroblast Activation Protein alpha / FAP, Follistatin-like 1 / FSTL1, FOLR1, FOLR2, FOLR3, FOLR4, FosB / G0S3, FoxM1, FoxO3, FRAT2, FXYD5 / Dysadherin, FcεRIα, FITC, FLT3, GABA-A R alpha 1, GADD153, GADD45 alpha, Galectin-3, Galectin-3BP / MAC-2BP, galactin, gangiosides, gross cystic disease fluid protein (GCDFP-15), GD2 (ganglioside G2), GD3, GM2, GM3, gamma- Glutamylcyclotransferase / CRF21, Gas1, Gastrin-releasing Peptide R / GRPR, Gastrokine 1, Gelsolin / GSN, glial fibrillary acidic protein (GFAP), GLI-2, Glutathione Peroxidase 3 / GPX3, gpA33, glycopeptides, Glypican 2 (GPC2), Glypican 3, Golgi Glycoprotein 1 / GLG1, gp96 / HSP90B1, GPR10, GPR110, GPR18, GPR31, GPR87, GPRC5A, GPRC6A, GRP78 / HSPA5, HE4 / WFDC2, Heparanase / HPSE, Hepsin, HGF R / c-MET, HIF-2 alpha / EPAS1, HIN-1 / SCGB3A1, HLA-DR, HOXB13, HOXB7, HSP70 / HSPA1A, HSP90, Hyaluronidase 1 / HYAL1, ID1, IgE, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, IGF-I, IGF-I R, IGF-II, IGFL-3, IGFLR1, IL-1 beta / IL-1F2, IL-17E / IL-25, IL-2, IL-6, ICAM-1, IgG, IgD, IgE, IgM, Interleukin 13 receptor a2 chain (IL-13Ra), Interleukin-13 receptor subunit alpha-2 (IL- 13Ra2), integrins, Integrin B7, IMP Dehydrogenase 1 / IMPDH1, Importin alpha 2 / KPNA2, ING1, Integrin beta 1 / CD29, Integrin beta 3 / CD61, IQGAP1, Isocitrate Dehydrogenase 1 / IDH1, ITIH4, ITM2C, Jagged 1, JNK, JunB, JunD, OGR1, Olig2, Osteopontin / OPN, 67 DM2\19774561.1PATENT Docket No. H3131-00401 Ovastacin, OXGR1 / GPR80 / P2Y15, p130Cas, p15INK4b / CDKN2B, p16INK4a / CDKN2A, p18INK4c / CDKN2C, p21 / CIP1 / CDKN1A, p27 / Kip1, P2X5 / P2RX5, PARP, PAUF / ZG16B, PBEF / Visfatin, PDCD4, PDCD5, PDGF R alpha, PDGF R beta, PDZD2, PEA-15, Pepsinogen A5 / PGA5, Peptidase Inhibitor 16 / PI16, Peroxiredoxin 2, PGCP, PI 3-Kinase p85 alpha, PIWIL2, PKM2, PLK1, PLRP1, PP4, P-Rex1, PRMT1, Profilin 1, Progesterone R B / NR3C3, Progesterone R / NR3C3, Progranulin / PGRN, Prolactin, Prostaglandin E Synthase 2 / PTGES2, PSAP, PSCA, PSMA / FOLH1 / NAALADase I, PSMA1, PSMA2, PSMB7, PSP94 / MSMB, PTEN, PTEN, PTH1R / PTHR1, PTK7 / CCK4, PTP beta / zeta / PTPRZ, Rab25, RARRES1, RARRES3, Ras, Reg4, Ret, RNF2, RNF43, S100A1, S100A10, S100A16, S100A2, S100A4, S100A6, S100A7, S100A9, S100B, S100P, SART1, SCUBE3, Secretin R, Serpin A9 / Centerin, Serpin E1 / PAI-1, Serum Amyloid A1, Serum Amyloid A4, SEZ6L, SEZ6L2 / BSRP-A, Skp2, SLC16A3, SLC45A3 / Prostein, SLC5A5, SLC5A8 / SMCT1, SLC7A7, Smad4, SMAGP, SOCS-1, SOCS-2, SOCS-6, SOD2 / Mn-SOD, Soggy-1 / DkkL1, SOX11, SOX17, SOX2, SPARC, SPARC-like 1 / SPARCL1, SPINK1, Src, six-transmembrane epithelial antigen of the prostate 1 (STEAP1), STEAP2, STEAP3 / TSAP6, STRO-1, STYK1, Survivin, Synaptotagmin- 1, Syndecan-1 / CD138, Syntaxin 4, Synuclein-gamma, synaptophysin, Kallikrein 2, Kallikrein 6 / Neurosin, KCC2 / SLC12A5, Ki-67 / MKI67, KiSS1R / GPR54, KLF10, KLF17, L1CAM, Lactate Dehydrogenase A / LDHA, Lamin B1, LEF1, Leptin / OB, LIN-28A, LIN-28B, Lipocalin-2 / NGAL, LKB1 / STK11, LPAR3 / LPA3 / EDG-7, LRMP, LRP-1B, LRRC3B, LRRC4, LRRN1 / NLRR-1, LRRN3 / NLRR-3, Ly6K, LYPD1, LYPD8, MAP2, Matriptase / ST14, MCAM / CD146, M-CSF, MDM2 / HDM2, Melanocortin-1 R / MC1R, Melanotransferrin / CD228, Melatonin, Mer, Mesothelin, Metadherin, Metastin / KiSS1, Methionine Aminopeptidase, Methionine Aminopeptidase 2 / METAP2, MFAP3L, MGMT, MIA, MIF, MINA, Mind Bomb 2 / MIB2, Mindin, MITF, MKK4, MKP-1, MKP-3, MMP-1, MMP-10, MMP-13, MMP-2, MMP-3, MMP-8, MMP-9, MRP1, MRP4 / ABCC4, MS4A12, MSH2, MSP R / Ron, MSX2, MUC-4, Musashi-1, NAC1, Napsin A, NCAM-1 / CD56, NCOA3, NDRG1, NEK2, NELL1, NELL2, Nesfatin-1 / Nucleobindin-2, Nestin, NFkB2, NF-L, NG2 / MCSP, NGF R / TNFRSF16, Nicotinamide N-Methyltransferase / NNMT, NKX2.2, NKX3.1, NM23-H1, NM23-H2, Notch-3, NPDC-1, NTS1 / NTSR1, NTS2 / NTSR2, Tankyrase 68 DM2\19774561.1PATENT Docket No. H3131-00401 1, Tau, TCF-3 / E2A, TCL1A, TCL1B, TEM7 / PLXDC1, TEM8 / ANTXR1, Tenascin C, TFF1, TGF-beta 1, TGF-beta 1, 2, 3, TGF-beta 1 / 1.2, TGF-beta 2 / 1.2, TGF-beta RI / ALK-5, THRSP, Thymidine Kinase 1, Thymosin beta 10, Thymosin beta 4, Thyroglobulin, TIMP Assay Kits, TIMP-1, TIMP-2, TIMP-3, TIMP-4, TLE1, TLE2, TM4SF1 / L6, TMEFF2 / Tomoregulin-2, TMEM219, TMEM87A, TNF-alpha, TOP2A, TopBP1, t-Plasminogen Activator / tPA, TRA-1- 60(R), TRA-1-85 / CD147, TRAF-4, Transgelin / TAGLN, Trypsin 2 / PRSS2, Tryptase alpha / TPS1, TSPAN1, UBE2S, uPAR, u-Plasminogen Activator / Urokinase, Urotensin-II R, VAP-1 / AOC3, VCAM-1 / CD106, VEGFR1 / Flt-1, VEGFR2 / KDR / Flk-1, VEGF / PlGF Heterodimer, VSIG1, VSIG3, YAP1, ZAG, ZAP70, ZMIZ1 / Zimp10, SGK, CNKSR1 / CNK / KSR or any combination thereof. In some embodiments the signaling domain of the TCRS or CARs comprises the ZAP70 kinase domain or a mutant or a variant or derivative thereof; the intracellular T-cell activation moiety comprises a signaling domain, further wherein the signaling domain comprises the replacement of CD3ζ with a ZAP70 kinase domain or a mutant or a variant thereof; and the TCR or CAR is fused with a chemokine receptor, wherein the chemokine receptor is optionally selected from CCR5, CCR2, and CXCR3 or chemokine receptors to enhance T cell trafficking. In some embodiments, the signaling domain comprising the ZAP70 kinase domain or a mutant or a variant thereof, further comprises an amino acid sequence having at least 55% identity to the amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0194] In any of the TCRs or CARs featured herein, (including T-cells expressing any of the TCRs or CARs featured herein) in some embodiments the TCR or CAR comprises an antigen recognition moiety, a transmembrane domain, and an intracellular T-cell activation moiety, where the intracellular T-cell activation moiety comprising a costimulatory signaling domain fused with a signaling domain. In some embodiments the costimulatory domain comprises a costimulatory stimulatory domain selected from In some embodiments the costimulatory signaling domain may include or exclude, or is selected from, e.g.CD28, 4-1BB (CD137), ICOS (CD278), CD27, OX40 (CD134), MyD88, EphB6, TSLP-R, HLA-DR, CO2, CD4, CD5, CD7, CDS, CD8alpha, CD8beta, CD11a, CD11b, CD11e, CD11d, CD18, CD19, CD19a, CD29, CD30, CD30L, CD40, CD40L (CD154), CD48, CD49a, CD49D, CD49f, 69 DM2\19774561.1PATENT Docket No. H3131-00401 CD58, CD53, ICAM-1 (CD54), CD69, CD70, CD80 (B7-l), CD82, CD83, CD84, CD86 (B7- 2), CD90, CD96, CD100, CD103, CD122, CD132, CD150 (SLAMFl), CD160 (BY55), CD162 (DNAMl), CD223 (LAG3), CD226, CD229, CD244, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-Ll), CD278, LAT, lymphocyte function-associated antigen-I (LFA-1), LIGHT, NKG2C, NKG2D, NK.p30, NKp44, NKp46, NKp80 (KLRFl), DAPl0, DAP12, LAG-3, 2B4, CARDI, CTLA-4 (CD152), TRIM, ZAP70, FcERIγ, 4-lBBL, BAFF, GADS, GITR, GITR-L, BAFF-R, HVEM, CD27L, OX40L, TACl, BLAME, CRACC, CD2F-10, NTB-A, integrin α4, integrin α4β1, integrin α4β7, IA4, ICAM-1, IL-2Ralpha, IL-2Rbeta, IL-2Rgamma, IL- 4Ralpha, IL-7Ralpha, IL-9Ralpha, IL-12R, IL-21Ralpha, B7-H2, B7-H3, CD83 ligand, PD-1, SLP-76, Toll-like receptors (TLRs, such as TLR2), ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGBl, ITGB2, ITGB7, KIRDS2, LTBR, PAG / Cbp, PSGLI, SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLAl, VLA-6, BTLA, ikaros, LAG-3, LMIR, CEACAMl, CRTAM, TCLIA, DAP12, TIM-1, Dectin-1, PDCD6, PD- 1, TIM-4, TSLP, or any combination thereof, further wherein the signaling domain comprises the ZAP70 kinase domain or a mutant or a variant thereof.

[0195] In any of the CARs featured herein, (e.g., in the CARS comprising chimeric antigen receptors or T cells expressing a CAR / chimeric CAR), in some embodiments the CAR comprises an antigen recognition moiety, a transmembrane domain, and an intracellular T-cell activation moiety, wherein the intracellular T-cell activation moiety comprising a costimulatory signaling domain that may include or exclude, or is selected from, e.g., CD28, 4-1BB (CD137), ICOS (CD278), CD27, OX40 (CD134), MyD88, EphB6, TSLP-R, HLA-DR, CO2, CD4, CD5, CD7, CDS, CD8alpha, CD8beta, CD11a, CD11b, CD11e, CD11d, CD18, CD19, CD19a, CD29, CD30, CD30L, CD40, CD40L (CD154), CD48, CD49a, CD49D, CD49f, CD58, CD53, ICAM-1 (CD54), CD69, CD70, CD80 (B7-l), CD82, CD83, CD84, CD86 (B7-2), CD90, CD96, CD100, CD103, CD122, CD132, CD150 (SLAMFl), CD160 (BY55), CD162 (DNAMl), CD223 (LAG3), CD226, CD229, CD244, CD270 (HVEM), CD273 (PD-L2), CD274 (PD-Ll), CD278, LAT, lymphocyte function-associated antigen-I (LFA-1), LIGHT, NKG2C, NKG2D, NK.p30, NKp44, NKp46, NKp80 (KLRFl), DAPl0, DAP12, LAG-3, 2B4, CARDI, CTLA-4 (CD152), TRIM, ZAP70, FcERIγ, 4-lBBL, BAFF, 70 DM2\19774561.1PATENT Docket No. H3131-00401 GADS, GITR, GITR-L, BAFF-R, HVEM, CD27L, OX40L, TACl, BLAME, CRACC, CD2F- 10, NTB-A, integrin α4, integrin α4β1, integrin α4β7, IA4, ICAM-1, IL-2Ralpha, IL-2Rbeta, IL-2Rgamma, IL-4Ralpha, IL-7Ralpha, IL-9Ralpha, IL-12R, IL-21Ralpha, B7-H2, B7-H3, CD83 ligand, PD-1, SLP-76, Toll-like receptors (TLRs, such as TLR2), ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGBl, ITGB2, ITGB7, KIRDS2, LTBR, PAG / Cbp, PSGLI, SLAMF6 (NTB-A, Lyl08), SLAMF7, SLP-76, TNFR2, TRANCE / RANKL, VLAl, VLA-6, BTLA, ikaros, LAG-3, LMIR, CEACAMl, CRTAM, TCLIA, DAP12, TIM-1, Dectin-1, PDCD6, PD-1, TIM-4, TSLP, or any combination thereof.

[0196] In any of the CARs featured herein, the antigen recognition moiety comprises an antigen-specific antibody, an antigen-binding fragment, an antibody mimetic, a protein receptor or a ligand for a specific receptor, or a TCR-like antibody. In some embodiments, the antibody, antigen-binding fragment, antibody mimetic, protein receptor or a ligand for a specific receptor, or TCR-like antibody comprises ScFv fragments, Fv fragments, Fd fragments, Fab fragments, Fab' fragments, F(ab)'2 fragments, VH domains, VL domains, monoclonal antibodies, polyclonal antibodies, nanobodies, dual or tri-antibody fusion proteins, or any combination thereof.

[0197] In some embodiments, this disclosure features methods to prolong T cell persistence or reducing T cell exhaustion through modulating the CAR or cells signaling and function or through direct manipulation of CAR signaling domains, whereby the CAR signaling domains are regulated by negative signaling molecules, wherein one, two or more negative signaling molecules are knocked down or knocked out, wherein the negative signaling molecules may include or exclude, or is selected from, e.g. ANKRDJ 1, ARID1A, BACH2, BCL2Ll 1, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3Hl (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2DJA, SMAD2,SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6 and ZC3H12A, any combination thereof. 71 DM2\19774561.1PATENT Docket No. H3131-00401

[0198] It is understood and herein contemplated that once the sequence of a CAR is identified, the skilled artisan would have full knowledge of the nucleic acids that would encode said amino acid CAR and it would be well within the skill set of the skilled artisan to make said nucleic acid constructs. Thus, in one aspect, also disclosed herein are nucleic acids encoding a polypeptide for any CAR disclosed herein. Multi-valent, Multispecific Binding Proteins

[0199] In one embodiment, this disclosure features bispecific antibodies or binding proteins, trispecific antibodies or binding proteins and multivalent, multispecific binding proteins. In some embodiments the bispecific antibodies or binding protiens, trispecific antibodies or binding proteins and multivalent, multispecific binding proteins feature a CAR or TCR of this disclosure or binding fragment thereof fused to a binding protein (antibody of antigen binding fragment thereof, or receptor or ligand binding fragment thereof). In some embodiments the components of the bispecific antibodies or binding protiens, trispecific antibodies or binding proteins and multivalent, multispecific binding proteins may be fused using any of the hinge domains featured herein or any fusion structure known in the art. Examples of multispecific antibodies comprising three or more different antigen-binding regions include but are not limited to (i) bispecific antibodies coupled with an additional single chain variable Fragment (scFv) at their Fc part (Weidle et al., Cancer Genomics Proteomics. 2013 January-February; 10(1):1-18), (ii) fusion proteins consisting of three or more scFv (triabody, tetrabody; Chames et al., FEMS Microbiol Lett. 2000 Aug. 1; 189(1):1-8) and (iii) fusion proteins connected to scFv (Kermer et. al. Mol Cancer Ther. 2014 January; 13(1):112- 21). The bispecific antibodies or binding proteins, trispecific antibodies or binding proteins and multivalent, multispecific binding proteins may bind to two, three, or two or more, respectively, binding partners disclosed herein.

[0200] In one embodiment, the present invention provides a multispecific antibody, wherein said multispecific antibody is a trispecific antibody. The trispecific antibody is engineered to simultaneously engage multiple tumor-associated antigens and immune cell receptors, such as CD3 on T cells or CD16 on natural killer (NK) cells. This engagement enhances tumor cell targeting and immune cell activation, thereby potentiating the anti-tumor 72 DM2\19774561.1PATENT Docket No. H3131-00401 immune response. In a specific embodiment, the trispecific antibody is a T cell-engaging antibody that targets CD19, CD33, and CD3, and is suitable for the treatment of mixed lineage leukemia. In another specific embodiment, the trispecific antibody is a killer cell-engaging antibody that binds to tumor antigens and CD16 on NK cells, thus promoting antibody- dependent cell-mediated cytotoxicity (ADCC) against tumor cells.

[0201] In another embodiment, the multispecific antibody is a tetraspecific antibody, which is designed to target four distinct tumor-associated antigens concurrently. The simultaneous targeting of multiple tumor antigens by the tetraspecific antibody provides enhanced tumor specificity and inhibition compared to monospecific or bispecific antibodies. In one example, a tetraspecific antibody targeting epidermal growth factor receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R), hepatocyte growth factor receptor (cMet), and human epidermal growth factor receptor 3 (HER3) has been developed to inhibit solid tumor growth. In another example, navy-type tetraspecific antibodies have been engineered to bind four different tumor targets, further expanding the range of tumor antigens that can be targeted simultaneously.

[0202] In a further embodiment, the multispecific antibody is a bispecific antibody that is coupled with additional binding domains. These additional binding domains can be single- chain variable fragments (scFv) that are fused to the light chain, heavy chain, or Fc region of the bispecific antibody. In one example, a bispecific antibody targeting CD3 and a tumor antigen is coupled with an scFv directed against another tumor antigen, thereby enhancing the tumor specificity of the antibody. In some embodiments, other binding domains, such as nanobodies or peptides, can be fused to the bispecific antibody to further improve its targeting capabilities. In one embodiment, the bispecific, trispecific or multivalent multispecific antibody and / or binding moiety specifically binds to epitopes on CD3 and a second antigen selected from CD19, CD33, and CD16.

[0203] In yet another embodiment, the multispecific fusion protein comprises three or more scFv, forming a triabody or tetrabody. The scFv components of the triabody or tetrabody can be directed against different tumor antigens or a combination of tumor antigens and immune cell receptors. For example, a triabody targeting CD19, CD33, and CD16 has been developed to engage NK cells and enhance ADCC against mixed lineage leukemia cells, demonstrating 73 DM2\19774561.1PATENT Docket No. H3131-00401 the potential of multispecific fusion proteins in harnessing the anti-tumor activity of immune cells.

[0204] In a further embodiment, the multispecific fusion protein is composed of scFv fused to effector proteins or peptides. These effector molecules can be cytokines, such as interleukin- 2 (IL-2), IL-12, IL-7, IL-15, IL-21, or tumor necrosis factor (TNF), which are fused to tumor- targeting scFv to promote immune cell activation and tumor cell killing. The fusion of cytokines to tumor-targeting scFv has been shown to enhance the anti-tumor immune response and improve the therapeutic efficacy of the multispecific fusion protein. In alternative embodiments, other effector proteins or peptides can be fused to scFv to achieve desired therapeutic effects, such as enhanced tumor cell apoptosis or increased immune cell infiltration into the tumor microenvironment.

[0205] In a specific embodiment, a T cell receptor (TCR)-like antibody such as A2 / ESO scFv antibody (SEQ ID NO: 134-144). The TCR-like scFv antibody can be used to generate CAR-T cells for immunotherapy.

[0206] In a specific embodiment, the multispecific antibody is a TCR-like scFv-anti-CD3 bispecific antibody. This bispecific antibody uses the TCR as a tumor-targeting binding moiety, while the anti-CD3 moiety activates T cells. The TCR component of the bispecific antibody is specific for tumor-associated peptide-major histocompatibility complex (MHC) complexes, allowing for targeted T cell activation and tumor cell killing. In one aspect, this disclosure features an mRNA encoding the TCR-anti-CD3 bispecific antibody.

[0207] In another embodiment, the multispecific antibody is a tandem single-chain variable fragment (scFv) fusion protein. This type of multispecific antibody is composed of two or more scFv molecules that are connected by flexible linkers. Each scFv molecule can be specific for a different tumor antigen or immune cell receptor, allowing for the simultaneous targeting of multiple targets. In one example, a tandem scFv fusion protein targeting CD19 and CD3 has been developed for the treatment of B-cell malignancies. The anti-CD19 scFv directs the fusion protein to CD19-expressing tumor cells, while the anti-CD3 scFv recruits and activates T cells, leading to enhanced tumor cell killing. 74 DM2\19774561.1PATENT Docket No. H3131-00401

[0208] In a further embodiment, the multispecific antibody is a dual-affinity re-targeting (DART) protein. DART proteins are composed of two antigen-binding domains that are connected by a linker. The antigen-binding domains can be scFv or Fab fragments, and they can be specific for different tumor antigens or immune cell receptors. DART proteins have been shown to exhibit improved stability and pharmacokinetic properties compared to other multispecific antibody formats. In one example, a DART protein targeting CD19 and CD3 has been developed for the treatment of B-cell malignancies, demonstrating potent anti-tumor activity in preclinical models.

[0209] In yet another embodiment, the multispecific antibody is a bispecific T-cell engager (BiTE). BiTE antibodies are composed of two scFv molecules that are connected by a short linker. One scFv is specific for a tumor antigen, while the other scFv is specific for CD3 on T cells. By simultaneously engaging tumor cells and T cells, BiTE antibodies promote T cell activation and tumor cell lysis. Blinatumomab, a BiTE antibody targeting CD19 and CD3, has been approved for the treatment of acute lymphoblastic leukemia, highlighting the clinical potential of this multispecific antibody format.

[0210] In a further embodiment, the multispecific antibody is an IgG-scFv fusion protein. This type of multispecific antibody is composed of an intact IgG molecule that is fused to one or more scFv molecules. The IgG component provides effector functions, such as antibody- dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), while the scFv component allows for the targeting of additional tumor antigens or immune cell receptors. In one example, an IgG-scFv fusion protein targeting HER2 and CD3 has been developed for the treatment of HER2-positive solid tumors, demonstrating potent anti-tumor activity in preclinical models.

[0211] In another embodiment, the multispecific antibody is a knobs-into-holes (KiH) bispecific antibody. KiH bispecific antibodies are generated by introducing complementary mutations into the CH3 domains of two different antibody heavy chains. These mutations promote the heterodimerization of the heavy chains, allowing for the production of bispecific antibodies with defined heavy chain pairings. KiH bispecific antibodies have been shown to exhibit improved stability and manufacturability compared to other bispecific antibody 75 DM2\19774561.1PATENT Docket No. H3131-00401 formats. In one example, a KiH bispecific antibody targeting EGFR and cMet has been developed for the treatment of solid tumors, demonstrating potent anti-tumor activity in preclinical models.

[0212] In a specific embodiment, the multispecific antibody is a triple-action antibody. Triple-action antibodies are composed of an intact IgG molecule that is fused to two different scFv molecules. The IgG component provides effector functions, while the two scFv components allow for the targeting of three different tumor antigens or immune cell receptors. In one example, a triple-action antibody targeting HER2, CD3, and PD-1 has been developed for the treatment of HER2-positive solid tumors. The anti-HER2 component directs the antibody to HER2-expressing tumor cells, the anti-CD3 component recruits and activates T cells, and the anti-PD-1 component blocks the immunosuppressive PD-1 / PD-L1 pathway, leading to enhanced anti-tumor immune responses. Identity / Homology

[0213] It is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein is through defining the variants and derivatives in terms of identity or homology and / or identify to specific known sequences. For example, SEQ ID NO: 3 sets forth a particular sequence of a CAR signaling domain comprising ZAP338. Specifically disclosed are variants of these and other genes and proteins herein disclosed which have at least, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 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 percent homology or identity to the stated sequence and any range between any two of the recited percentage homologies or identities. Those of skill in the art readily understand how to determine the homology or identity of two proteins or nucleic acids, which can include or exclude genes. For example, the homology or identity can be calculated after aligning the two sequences so that the homology is at its highest level.

[0214] Another way of calculating homology or identity can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math.2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search 76 DM2\19774561.1PATENT Docket No. H3131-00401 for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

[0215] The same types of homology or identity can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989 which are herein incorporated by reference for at least material related to nucleic acid alignment. Nucleic acids

[0216] There are a variety of molecules disclosed herein that are nucleic acid based, including for example the nucleic acids that encode, for example SEQ ID NO: 1, or any of the nucleic acids disclosed herein or fragments thereof, as well as various functional nucleic acids. In some embodiments, the nucleic acids included herein can include or exclude cDNA encoding TCR alpha chain and / or TCR beta chain. As used herein the term “cDNA” refers to a nucleic acid which joins exon-exon, or exon-only coding sequences. The disclosed nucleic acids are made up of for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that for example, when a vector is expressed in a cell, that the expressed mRNA will typically be made up of A, C, G, and U. Likewise, it is understood that if, for example, an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantageous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment. In some embodiments the nucleotide analogs comprise one or more modifications to one or more base, sugar, or phosphate moieties in the nucleic acid as disclosed further herein.

[0217] The present invention also provides a nucleic acid sequence encoding a CAR or TCR as described herein, as well as a vector comprising the nucleic acid sequence and a host cell comprising the nucleic acid sequence or the vector. 77 DM2\19774561.1PATENT Docket No. H3131-00401

[0218] The invention also provides a vector comprising a CAR-encoding nucleic acid sequence as described herein. Examples of vectors that may be used in the present invention include, but are not limited to, a DNA vector, a RNA vector, a plasmid, a phagemid, a phage derivative, a virus and a cosmid.

[0219] Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.

[0220] In order to assess the expression of a CAR polypeptide or portions thereof, the expression vector to be introduced into a T cell can also contain either a selectable marker gene such as CD34, CD271 or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neomycin and the like, e.g., NeoR, BsdR, HygR, PuroR, and BleoR genes, which confer resistance to the selective antibiotics neomycin / G418 / geneticin, blasticidin, hygromycin B, puromycin, and zeocin, respectively. Nucleotides and related molecules

[0221] A nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage. The base moiety of a nucleotide can be adenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T). The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate moiety of a nucleotide is pentavalent phosphate. A non-limiting example of a nucleotide would be 3'-AMP (3'- adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate). There are many varieties of these types of molecules available in the art and available herein. 78 DM2\19774561.1PATENT Docket No. H3131-00401

[0222] A nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to nucleotides are well known in the art and would include for example, 5-methylcytosine (5-me-C), 2-methylcytosine (2-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications at the sugar or phosphate moieties. There are many varieties of these types of molecules available in the art and available herein.

[0223] Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, which can include or exclude peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson-Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid. There are many varieties of these types of molecules available in the art and available herein.

[0224] It is also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. Conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties which can include or exclude a cholesterol moiety. (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556). There are many varieties of these types of molecules available in the art and available herein.

[0225] A Watson-Crick interaction is at least one interaction with the Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute. The Watson-Crick face of a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, N1, and C6 positions of a purine based nucleotide, nucleotide analog, or nucleotide substitute and the C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or nucleotide substitute.

[0226] A Hoogsteen interaction is the interaction that takes place on the Hoogsteen face of a nucleotide or nucleotide analog, which is exposed in the major groove of duplex DNA. The Hoogsteen face includes the N7 position and reactive groups (NH2 or O) at the C6 position of purine nucleotides. 79 DM2\19774561.1PATENT Docket No. H3131-00401 Primers and probes

[0227] Disclosed are compositions including primers and probes, which are capable of interacting with the disclosed nucleic acids, which can include or exclude the tumor antigens, epitopes and CARs disclosed herein. In certain embodiments the primers are used to support DNA amplification reactions. Typically, the primers will be capable of being extended in a sequence specific manner. Extension of a primer in a sequence specific manner includes any methods wherein the sequence and / or composition of the nucleic acid molecule to which the primer is hybridized or otherwise associated directs or influences the composition or sequence of the product produced by the extension of the primer. Extension of the primer in a sequence specific manner therefore includes, but is not limited to, PCR, DNA sequencing, DNA extension, DNA polymerization, RNA transcription, or reverse transcription. Techniques and conditions that amplify the primer in a sequence specific manner are preferred. In certain embodiments the primers are used for the DNA amplification reactions, which can include or exclude PCR or direct sequencing. It is understood that in certain embodiments the primers can also be extended using non-enzymatic techniques, where for example, the nucleotides or oligonucleotides used to extend the primer are modified such that they will chemically react to extend the primer in a sequence specific manner. Typically, the disclosed primers hybridize with the disclosed nucleic acids or region of the nucleic acids or they hybridize with the complement of the nucleic acids or complement of a region of the nucleic acids. Peptides Protein variants

[0228] As discussed herein there are numerous variants of the CARs that are known and herein contemplated. Protein variants and derivatives are well understood to those of skill in the art and in can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and / or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Immunogenic fusion protein derivatives, which can include or 80 DM2\19774561.1PATENT Docket No. H3131-00401 exclude those described in the examples, are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any one site within the protein molecule. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example M13 primer mutagenesis and PCR mutagenesis. Amino acid substitutions are typically of single residues, but can occur at a number of different locations at once; insertions usually will be on the order of about from 1 to 10 amino acid residues; and deletions will range about from 1 to 30 residues. Substitutions may include or exclude substitutions in one or more of the six TCR CDR regions. Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 1 and 2 and are referred to as conservative substitutions. TABLE 1: Amino Acid Abbreviations Amino Acid Abbreviations Alanine Ala A allosoleucine AIle Arginine Arg R asparagine Asn N aspartic acid Asp D Cysteine Cys C glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isolelucine Ile I 81 DM2\19774561.1PATENT Docket No. H3131-00401 Leucine Leu L Lysine Lys K phenylalanine Phe F proline Pro P pyroglutamic acid pGlu Serine Ser S Threonine Thr T Tyrosine Tyr Y Tryptophan Trp W Valine Val V TABLE 2: Amino Acid Substitutions Original Residue Exemplary Conservative Substitutions, others are known in the art. Ala Ser Arg Lys; Gln Asn Gln; His Asp Glu Cys Ser Gln Asn, Lys Glu Asp Gly Pro His Asn;Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

[0229] Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 2, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the protein 82 DM2\19774561.1PATENT Docket No. H3131-00401 properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine, in this case, (e) by increasing the number of sites for sulfation and / or glycosylation.

[0230] For example, the replacement of one amino acid residue with another that is biologically and / or chemically similar is known to those skilled in the art as a conservative substitution. For example, a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another. The substitutions include combinations which can include or exclude, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.

[0231] Substitutional or deletional mutagenesis can be employed to insert sites for N- glycosylation (Asn-X-Thr / Ser) or O-glycosylation (Ser or Thr). Deletions of cysteine or other labile residues also may be desirable. Deletions or substitutions of potential proteolysis sites, e.g. Arg, is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.

[0232] Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post- translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86

[1983] ), acetylation of the N- terminal amine and, in some instances, amidation of the C-terminal carboxyl. 83 DM2\19774561.1PATENT Docket No. H3131-00401

[0233] It is understood that one way to define the variants and derivatives of the disclosed proteins herein is through defining the variants and derivatives in terms of homology / identity to specific known sequences. Specifically disclosed are variants of these and other proteins herein disclosed which have at least, 55%, 60% or 65%, 70% or 75% or 80% or 85% or 90% or 95% homology / identity to the stated sequence. Those of skill in the art readily understand how to determine the homology / identity of two proteins. For example, the homology / identity can be calculated after aligning the two sequences so that the homology / identiy is at its highest level.

[0234] Another way of calculating homolog / identity can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math.2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by inspection.

[0235] The same types of homology / identity can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M. Science 244:48-52, 1989, Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger et al. Methods Enzymol. 183:281-306, 1989.

[0236] The description of conservative mutations and homology / identity can be combined together in any combination, which can include or exclude embodiments that have at least 55% homology / identity to a particular sequence wherein the variants are conservative mutations.

[0237] As this specification discusses various proteins and protein sequences it is understood that the nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences. Thus, while each particular nucleic acid sequence may 84 DM2\19774561.1PATENT Docket No. H3131-00401 not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence.

[0238] It is understood that there are numerous amino acid and peptide analogs which can be incorporated into the disclosed compositions. For example, there are numerous D amino acids or amino acids which have a different functional substituent then the amino acids shown in Table 1 and Table 2. The opposite stereo isomers of naturally occurring peptides are disclosed, as well as the stereo isomers of peptide analogs. These amino acids can readily be incorporated into polypeptide chains by charging tRNA molecules with the amino acid of choice and engineering genetic constructs that utilize, for example, amber codons, to insert the analog amino acid into a peptide chain in a site-specific way.

[0239] Molecules can be produced that resemble peptides, but which are not connected via a natural peptide linkage. For example, linkages for amino acids or amino acid analogs can include CH2NH--, --CH2S--, --CH2--CH2 --, --CH=CH-- (cis and trans), --COCH2 --, -- CH(OH)CH2--, and --CHH2SO—(These and others can be found in Spatola, A. F. in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, Issue 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci (1980) pp. 463-468; Hudson, D. et al., Int J Pept Prot Res 14:177-185 (1979) (--CH2NH--, CH2CH2--); Spatola et al. Life Sci 38:1243-1249 (1986) (--CH H2--S); Hann J. Chem. Soc Perkin Trans. I 307-314 (1982) (--CH--CH--, cis and trans); Almquist et al. J. Med. Chem. 23:1392-1398 (1980) (-- COCH2--); Jennings-White et al. Tetrahedron Lett 23:2533 (1982) (--COCH2--); Szelke et al. European Appln, EP 45665 CA (1982): 97:39405 (1982) (--CH(OH)CH2--); Holladay et al. Tetrahedron. Lett 24:4401-4404 (1983) (--C(OH)CH2--); and Hruby Life Sci 31:189-199 (1982) (--CH2--S--); each of which is incorporated herein by reference. A particularly preferrednon-peptide linkage is --CH2NH--. It is understood that peptide analogs can have more thanone atom between the bond atoms, which can include or exclude b-alanine, g-aminobutyric acid, and the like.

[0240] Amino acid analogs and analogs and peptide analogs often have enhanced or desirable properties, which can include or exclude, more economical production, greater 85 DM2\19774561.1PATENT Docket No. H3131-00401 chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.

[0241] D-amino acids can be used to generate more stable peptides, because D amino acids are not recognized by peptidases and such. Systematic substitution of one or more amino acids of a consensus sequence with a D-amino acid of the same type (e.g., D-lysine in place of L- lysine) can be used to generate more stable peptides. Cysteine residues can be used to cyclize or attach two or more peptides together. This can be beneficial to constrain peptides into particular conformations. Pharmaceutical carriers / Delivery of pharmaceutical products

[0242] In one aspect, disclosed herein are compositions comprising a therapeutically effective amount of one or more CAR T-cells; wherein CAR T cell has been engineered to express a receptor for one tumor antigen disclosed herein. In one aspect, the CAR T cell specific for one tumor antigen disclosed herein can be further engineered to knockout or knockdown programmed cell death protein (PD1), von Hippel-Lindau tumor suppressor (VHL), and / or protein phosphatase 2 regulatory subunit B delta (PPP2R2D) to enhance their function which can include or exclude cytotoxic activity and persistence or survival in vivo after adoptive transfer to a cancer patient.

[0243] As described above, the compositions can also be administered in vivo in a pharmaceutically acceptable carrier. By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

[0244] The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, including topical intranasal administration or administration by inhalant. As used 86 DM2\19774561.1PATENT Docket No. H3131-00401 herein, “topical intranasal administration” means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation. The exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

[0245] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., a mammalian, bacterial, yeast, or insect cell, by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0246] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, of the invention, a polynucleotide is introduced into a host cell using calcium phosphate transfection.

[0247] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0248] Parenteral administration of the composition, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or 87 DM2\19774561.1PATENT Docket No. H3131-00401 suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.

[0249] The materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-2065, (1991)). Vehicles which can include or exclude “stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo. The following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general, receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes. The internalization pathways serve a variety of functions, which can include or exclude nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor- level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)). 88 DM2\19774561.1PATENT Docket No. H3131-00401 Pharmaceutically Acceptable Carriers

[0250] The compositions, including antibodies, can be used therapeutically in combination with a pharmaceutically acceptable carrier.

[0251] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations which can include or exclude semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

[0252] Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions which can include or exclude sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

[0253] Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients which can include or exclude antimicrobial agents, antiinflammatory agents, anesthetics, and the like.

[0254] The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection. The disclosed antibodies can be administered 89 DM2\19774561.1PATENT Docket No. H3131-00401 intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

[0255] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils which can include or exclude olive oil, and injectable organic esters which can include or exclude ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (which can include or exclude those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present which can include or exclude, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

[0256] Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0257] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.

[0258] Some of the compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids which can include or exclude hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids which can include or exclude formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base which can include or exclude sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases which can include or exclude mono-, di-, trialkyl and aryl amines and substituted ethanolamines. 90 DM2\19774561.1PATENT Docket No. H3131-00401 Therapeutic Uses

[0259] Effective dosages and schedules for administering the compositions may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms of the disorder are effected. The dosage should not be so large as to cause adverse side effects, which can include or exclude unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. For example, guidance in selecting appropriate doses for antibodies can be found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. A typical daily dosage of the antibody used alone might range from about 1 µg / kg to up to 100 mg / kg of body weight or more per day, depending on the factors mentioned above. It can generally be stated that a pharmaceutical compositioncomprising the subject engineered T cells, may be administered at a dosage of 104 to 107engineered T cells / kg body weight, preferably 105to 106engineered T cells / kg body weight, including all integer values within those ranges. Engineered T cells compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly. Methods of using the compositions

[0260] Method of treating diseases and disorders 91 DM2\19774561.1PATENT Docket No. H3131-00401

[0261] The present invention further relates to a method for treating a disease or disorder in a subject in need thereof, comprising administering to a subject at least one cell population or composition according to the present invention. In some embodiments, the method is a cell therapy method. In some embodiments, said diseases or disorders include inflammatory diseases, autoimmune diseases, allergic diseases, organ transplantation conditions, cancers and infectious diseases. In some embodiments, the cell population is a regulatory immune cell population or a Treg cell population, and said disease or disorder is an inflammatory disease, an autoimmune disease, an allergic disease, or an organ transplantation condition (e.g., graft rejection or graft-versus-host disease). In some embodiments, the immune cell population is a T effector cell population, a NK cell population or a γδ cell population and said disease or disorder is a cancer or an infectious disease.

[0262] The disclosed compositions can be used to treat any disease where uncontrolled cellular proliferation occurs which can include or exclude cancers. Accordingly, in one aspect, disclosed herein are methods of stimulating an immunological response against a cancer or treating, inhibiting, and / or preventing a cancer comprising administering to a subject a composition comprising a therapeutically effective amount of T cells engineered with the CARs disclosed herein.

[0263] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination. The precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient.

[0264] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment 92 DM2\19774561.1PATENT Docket No. H3131-00401 designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.

[0265] A non-limiting list of different types of cancers that can be treated by the disclosed methods is as follows: lymphomas (Hodgkins and non-Hodgkins), leukemias, carcinomas, carcinomas of solid tissues, squamous cell carcinomas, adenocarcinomas, sarcomas, gliomas, high grade gliomas, blastomas, neuroblastomas, plasmacytomas, histiocytomas, melanomas, adenomas, hypoxic tumors, myelomas, AIDS-related lymphomas or sarcomas, metastatic cancers, or cancers in general.

[0266] A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers which can include or exclude small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical cancer, cervical carcinoma, breast cancer, and epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon cancer, and / or rectal cancers.

[0267] Furthermore, the CAR T cells of the present invention may be combined with other immunomodulatory strategies to enhance their therapeutic efficacy. For example, the CAR T cells may be administered in combination with immune checkpoint inhibitors, such as antibodies targeting PD-1, PD-L1, or CTLA-4. By blocking inhibitory pathways, these checkpoint inhibitors can unleash the full cytotoxic potential of the CAR T cells and promote a more robust anti-tumor immune response. Additionally, the CAR T cells may be combined 93 DM2\19774561.1PATENT Docket No. H3131-00401 with chemotherapy, radiation therapy, or targeted small molecule inhibitors to achieve synergistic anti-tumor effects. C. Methods

[0268] In one aspect, disclosed herein are methods of identifying T cell receptors disclosed herein, wherein the T cell activity (which can include or exclude, for example, release of cytokines including, but not limited to IFN-γ, TGF-β, lymphotoxin-α, IL-2, IL-4, IL-10, IL-17, or IL-25) is measured by any immunodetection disclosed herein, for example, without limitation, by ELISA, ELISpot, Intracellular cytokine staining, or Chromium Release.

[0269] Immunoassays are binding assays involving antibodies and antigens. There are many types, including chemiluminescent assays, ELISAs, RIAs, Western blots, flow cytometry, protein arrays, and multiplexed bead arrays. They involve contacting a sample with an antibody under conditions to allow immunocomplex formation, washing to remove unbound antibody, and detecting the bound complexes.

[0270] Detection commonly uses labels like enzymes, fluorescent dyes (fluorochromes), radioactive tags, or epitope tags. Direct labeling has the detecting antibody include the label, while indirect labeling uses an additional labeled molecule that binds the immunocomplex.

[0271] ELISAs detect antigen or antibody by enzyme-linked immunosorbent assay in different formats like sandwich assays and competitive binding. ELISpot is a variation that produces localized colored spots.

[0272] Protein arrays immobilize proteins on surfaces like glass slides, membranes or beads for parallel, miniaturized ligand-binding assays. Various immobilization chemistries and fabrication methods are used. Detection is often by fluorescence using instrumentation similar to DNA microarrays. Novel label-free methods are also employed.

[0273] Antibody arrays use immobilized antibodies, antibody fragments, engineered protein scaffolds, or aptamers to capture and detect target proteins. Large-scale proteome chips can analyze biochemical activities and interactions. Multiplexed bead assays use spectrally distinct particles to capture and quantitate multiple analytes simultaneously by flow cytometry.

[0274] It is understood and herein contemplated that the disclosed T cell receptors (for example, without limitation, T cell receptors that bind cancer antigens that can include or 94 DM2\19774561.1PATENT Docket No. H3131-00401 exclude any of DP4-ESO-1 TCR, A2-CT83 TCR, DR13-CT83 TCR, A2-pp65-TCR and / or A2-IE-1-TCR) can be used to treat any cancer that express certain types of MHC molecules and antigens, including, but not limited to B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers, small cell lung cancer, non-small cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancers, melanoma, basal cell carcinoma, squamous cell carcinoma, liver cancer, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon and rectal cancers, prostatic cancer, AIDS- related lymphomas, or AIDS-related sarcomas. Therefore, in one aspect, also disclosed herein are methods of identifying TCR disclosed herein, wherein the cancer is selected from the group consisting of B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, lung cancers, small cell lung cancer, non-small cell lung cancer, neuroblastoma, glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancers, melanoma, basal cell carcinoma, squamous cell carcinoma, liver cancer, squamous cell carcinomas of the mouth, throat, larynx, and lung, cervical cancer, cervical carcinoma, breast cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon and rectal cancers, prostatic cancer, AIDS-related lymphomas, or AIDS-related sarcomas.

[0275] The present invention employs standard PCR techniques for amplification of DNA sequences. PCR is a widely used method for amplifying specific DNA sequences, which involves repeated cycles of DNA denaturation, primer annealing, and extension by a thermostable DNA polymerase. Overlap PCR is used to join DNA fragments, enabling the construction of chimeric sequences. The use of PCR allows for efficient generation and modification of CAR constructs. 95 DM2\19774561.1PATENT Docket No. H3131-00401

[0276] The CAR constructs of the present invention are cloned into retroviral vectors, such as pMSGV1, for stable gene delivery into T cells. Retroviral packaging is performed by transfecting HEK293T cells with the CAR-encoding retroviral vector, along with envelope (e.g., RD114) and packaging (e.g., Gag-pol) plasmids. The resulting retroviral particles are used to transduce activated human T cells in the presence of RetroNectin, which enhances transduction efficiency. This retroviral gene delivery system allows for the efficient generation of CAR-expressing T cells.

[0277] Flow cytometry is used to analyze the expression of CARs on the surface of transduced T cells and to characterize T cell phenotypes. Cells are stained with specific antibodies or reagents (e.g., protein L for CD19 CAR) conjugated to fluorescent dyes, allowing for the quantification of CAR expression and the identification of T cell subsets (e.g., memory T cells) and activation / exhaustion markers. Flow cytometry enables the rapid and sensitive analysis of CAR-T cell properties at the single-cell level.

[0278] The present invention employs animal models, such as immunodeficient NOD / SCID / γ-chain- / - (NSG) mice, to assess the in vivo function and antitumor activity of CAR-T cells. Mice are intravenously injected with luciferase-expressing tumor cells, followed by treatment with CAR-T cells. Tumor burden is monitored using bioluminescence imaging, and survival is tracked over time. These animal studies provide crucial insights into the efficacy and safety of CAR-T cell therapies in a living system.

[0279] The present invention employs enzyme-linked immunosorbent assays (ELISAs) to quantify the release of cytokines, such as interferon-gamma (IFN-γ), by CAR-T cells upon co- culture with target tumor cells. Supernatants from co-cultures are added to plates coated with capture antibodies specific for the cytokine of interest, followed by detection using biotinylated secondary antibodies and avidin-HRP. The use of ELISAs allows for the sensitive and specific measurement of CAR-T cell functional responses.

[0280] The present invention employs lactate dehydrogenase (LDH) release assays to assess the cytolytic activity of CAR-T cells against tumor cell targets. LDH is a stable cytosolic enzyme released upon cell lysis, serving as a marker for cell death. Supernatants from CAR-T cell and tumor cell co-cultures are analyzed for LDH activity using a colorimetric assay, with 96 DM2\19774561.1PATENT Docket No. H3131-00401 the percentage of specific lysis calculated based on the relative LDH release compared to maximum and spontaneous release controls. LDH assays provide a quantitative measure of CAR-T cell-mediated tumor cell killing.

[0281] Sequences SEQ ID NO: 1 ZAP300 (Truncated human Zap70, starting from aa300) TSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGV YRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALML VMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNV LLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVW SYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIY KWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA SEQ ID NO: 2 ZAP327 (Truncated human Zap70, starting from aa327) DKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEE MMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNV AELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYY TARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEV MAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKV EGPPGSTQKAEAACA SEQ ID NO: 3 ZAP338 (Truncated human Zap70, starting from aa338) LIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQL DNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGM KYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLK WYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRME CPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAE AACA SEQ ID NO: 4 ZAP255 (Truncated human Zap70, starting from aa255) PNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPMPMDTS VYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKV LKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKF LVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFG LSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYG QKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQR MRACYYSLASKVEGPPGSTQKAEAACA 97 DM2\19774561.1PATENT Docket No. H3131-00401 SEQ ID NO: 5 ZAP280 (Truncated human Zap70, starting from aa280) PQRRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNL LIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQL DNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGM KYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLK WYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRME CPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAE AACA SEQ ID NO: 6 P2A (Synthetic peptide): ATNFSLLKQAGDVEENPGP T2A (Synthetic peptide): EGRGSLLTCGDVEENPGP (SEQ ID NO: 145) F2A (Synthetic peptide): VKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 146) E2A (Synthetic peptide):QCTNYALLKLAGDVESNPGP (SEQ ID NO: 147) SEQ ID NO: 7 Human TCR hinge-transmembrane α-1 (H / TMα-1) DFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAG FNLLMTLRLW SEQ ID NO: 8 Human TCR H / TMα-2 IPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLW SEQ ID NO: 9 Human TCR H / TMβ-1 ENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAV LVSALVLMAMV SEQ ID NO: 10 Human TCR H / TMβ-2 VTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMV SEQ ID NO: 11 Human TCR H / TMβ-3 ADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMV SEQ ID NO: 12 Human TCR H / TMδ-1 STDFEVKTDSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFA 98 DM2\19774561.1PATENT Docket No. H3131-00401 KTVAVNFLLTAKLFF SEQ ID NO: 13 Human TCR H / TMδ-2 VKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKL FF SEQ ID NO: 14 Human TCR H / TMδ-3 KSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFLLTAKLFF SEQ ID NO: 15 Human TCR H / TMγ-1 PIKTDVTTVDPKYNYSKDANDVITMDPKDNWSKDANDTLLLQLTNTSAYYTYLLLL LKSVVYFAIITCCLL SEQ ID NO: 16 Human TCR H / TMγ-2 NYSKDANDVITMDPKDNWSKDANDTLLLQLTNTSAYYTYLLLLLKSVVYFAIITCCL L SEQ ID NO: 17 Human TCR H / TMγ-3 PKDNWSKDANDTLLLQLTNTSAYYTYLLLLLKSVVYFAIITCCLL SEQ ID NO: 18 Mouse TCR H / TMα-1 TCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNL LMTL SEQ ID NO: 19 Mouse TCR H / TMα-2 ATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTL SEQ ID NO: 20 Mouse TCR H / TMα-3 DATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTL SEQ ID NO: 21 Mouse TCR H / TMβ-1 GSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVL MAMV SEQ ID NO: 22 99 DM2\19774561.1PATENT Docket No. H3131-00401 Mouse TCR H / TMβ-2 SAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMV SEQ ID NO: 23 Mouse TCR H / TMβ-3 GITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMV SEQ ID NO: 24 Mouse TCR H / TMδ-1 STDFEPYANSFNNEKLPEPENDTQISEPCYGPRVTVHTEKVNMMSLTVLGLRLLFAK TIAINFLLTVKLFF SEQ ID NO: 25 Mouse TCR H / TMδ-2 KLPEPENDTQISEPCYGPRVTVHTEKVNMMSLTVLGLRLLFAKTIAINFLLTVKLFF SEQ ID NO: 26 Mouse TCR H / TMδ-3 PCYGPRVTVHTEKVNMMSLTVLGLRLLFAKTIAINFLLTVKLFF SEQ ID NO: 27 Mouse TCR H / TMγ-1 VKHENNKGGADQEIFFPSIKKVAVSTKPTTCWQDKNDVLQLQFTITSAYYTYLLLLL KSVIYLAIISFSLL SEQ ID NO: 28 Mouse TCR H / TMγ-2 FFPSIKKVAVSTKPTTCWQDKNDVLQLQFTITSAYYTYLLLLLKSVIYLAIISFSLL SEQ ID NO: 29 Mouse TCR H / TMγ-3 PTTCWQDKNDVLQLQFTITSAYYTYLLLLLKSVIYLAIISFSLL SEQ ID NO: 30 Human CD3G-1 KKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIV SIFVLAVGVYFIA SEQ ID NO: 31 Human CD3G-2 GTYQCQGAKETSNPLQVYYRMCENCIELNIGTISGFLFAEIVSIFVLAVGVYFIA SEQ ID NO: 32 Human CD3G-3 100 DM2\19774561.1PATENT Docket No. H3131-00401 PLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIA SEQ ID NO: 33 Human CD3D-1 LDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIAT LLLALGVFCFA SEQ ID NO: 34 Human CD3D-2 RCNGTDIYKDKESTVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFA SEQ ID NO: 35 Human CD3D-3 TVQVHYRMCQSCVELDPATVAGIIVTDVIATLLLALGVFCFA SEQ ID NO: 36 Human CD3E-1 EDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVI VDICITGGLLLLVYYWS SEQ ID NO: 37 Human CD3E-2 SGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVY YWS SEQ ID NO: 38 Human CD3E-3 EDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWS SEQ ID NO: 39 Mouse CD3G-1 KNTWNLGNNAKDPRGTYQCQGAKETSNPLQVYYRMCENCIELNIGTISGFIFAEVISI FFLALGVYLIA SEQ ID NO: 40 Mouse CD3G-2 GMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIA SEQ ID NO: 41 Mouse CD3G-3 PLQVYYRMCENCIELNIGTISGFIFAEVISIFFLALGVYLIA SEQ ID NO: 42 101 DM2\19774561.1PATENT Docket No. H3131-00401 Human TCR TMα-3 SCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLW SEQ ID NO: 43 Mouse CD3D-1 LNLGKGVLDPRGIYLCNGTEQLAKVVSSVQVHYRMCQNCVELDSGTMAGVIFIDLIA TLLLALGVYCFA SEQ ID NO: 44 Mouse CD3D-2 LCNGTEQLAKVVSSVQVHYRMCQNCVELDSGTMAGVIFIDLIATLLLALGVYCFA SEQ ID NO: 45 Mouse CD3D-3 SVQVHYRMCQNCVELDSGTMAGVIFIDLIATLLLALGVYCFA SEQ ID NO: 46 Mouse CD3E-1 QKHDKHLVLQDFSEVEDSGYYVCYTPASNKNTYLYLKARVCEYCVEVDLTAVAIIII VDICITLGLLMVIYYWS SEQ ID NO: 47 Mouse CD3E-2 VEDSGYYVCYTPASNKNTYLYLKARVCEYCVEVDLTAVAIIIIVDICITLGLLMVIYY WS SEQ ID NO: 48 Mouse CD3E-3 SNKNTYLYLKARVCEYCVEVDLTAVAIIIIVDICITLGLLMVIYYWS SEQ ID NO: 49 Human IL2Rα-1 FPGEEKPQASPEGRPESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLI SVLLLSGL SEQ ID NO: 50 Human IL2Rα-2 PESETSCLVTTTDFQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGL SEQ ID NO: 51 Human IL2Rα-3 FQIQTEMAATMETSIFTTEYQVAVAGCVFLLISVLLLSGL SEQ ID NO: 52 102 DM2\19774561.1PATENT Docket No. H3131-00401 Human IL2Rβ-1 ICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWLGHLLV GLSGAFGFIILVYLLI SEQ ID NO: 53 Human IL2Rβ-2 QVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTIPWLGHLLVGLSGAFGFIILVYLL I SEQ ID NO: 54 Human IL2Rβ-3 WSPWSQPLAFRTKPAALGKDTIPWLGHLLVGLSGAFGFIILVYLLI SEQ ID NO: 55 Human IL4Rα-1 RIAASTLKSGISYRARVRAWAQCYNTTWSEWSPSTKWHNSYREPFEQHLLLGVSVS CIVILAVCLLCYVSIT SEQ ID NO: 56 Human IL4Rα-2 ARVRAWAQCYNTTWSEWSPSTKWHNSYREPFEQHLLLGVSVSCIVILAVCLLCYVSI T SEQ ID NO: 57 Human IL4Rα-3 WSEWSPSTKWHNSYREPFEQHLLLGVSVSCIVILAVCLLCYVSIT SEQ ID NO: 58 Human IL7Rα-1 QRKLQPAAMYEIKVRSIPDHYFKGFWSEWSPSYYFRTPEINNSSGEMDPILLTISILSFF SVALLVILACVLW SEQ ID NO: 59 Human IL7Rα-2 RSIPDHYFKGFWSEWSPSYYFRTPEINNSSGEMDPILLTISILSFFSVALLVILACVLW SEQ ID NO: 60 Human IL7Rα-3 EWSPSYYFRTPEINNSSGEMDPILLTISILSFFSVALLVILACVLW SEQ ID NO: 61 Human IL9R-1 RLRVQMATLEDDVVEEERYTGQWSEWSQPVCFQAPQRQGPLIPPWGWPGNTLVAV SIFLLLTGPTYLLF 103 DM2\19774561.1PATENT Docket No. H3131-00401 SEQ ID NO: 62 Human IL9R-2 EEERYTGQWSEWSQPVCFQAPQRQGPLIPPWGWPGNTLVAVSIFLLLTGPTYLLF SEQ ID NO: 63 Human IL9R-3 QPVCFQAPQRQGPLIPPWGWPGNTLVAVSIFLLLTGPTYLLF SEQ ID NO: 64 Human IL15Rα-1 PSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLC GLSAVSLLACYL SEQ ID NO: 65 Human IL15Rα-2 HGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYL SEQ ID NO: 66 Human IL15Rα-3 LTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYL SEQ ID NO: 67 Human IL21R-1 LLPLEFRKDSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKEGWNPHLLLL LLLVIVFIPAFW SEQ ID NO: 68 Human IL21R-2 QVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKEGWNPHLLLLLLLVIVFIPAFW SEQ ID NO: 69 Human IL21R-3 GTWSEWSDPVIFQTQSEELKEGWNPHLLLLLLLVIVFIPAFW SEQ ID NO: 70 Mouse IL2Rα-1 RFLASEESQGSRNSSPESETSCPITTTDFPQPTETTAMTETFVLTMEYKVAVASCLFLLI SILLLSGLTW SEQ ID NO: 71 Mouse IL2Rα-2 PESETSCPITTTDFPQPTETTAMTETFVLTMEYKVAVASCLFLLISILLLSGLTW 104 DM2\19774561.1PATENT Docket No. H3131-00401 SEQ ID NO: 72 Mouse IL2Rα-3 FPQPTETTAMTETFVLTMEYKVAVASCLFLLISILLLSGLTW SEQ ID NO: 73 Mouse IL2Rβ-1 QWLFLEMLIPSTSYEVQVRVKAQRNNTGTWSPWSQPLTFRTRPADPMKEILPMSWL RYLLLVLGCFSGFFSCVYILV SEQ ID NO: 74 Mouse IL2Rβ-2 VQVRVKAQRNNTGTWSPWSQPLTFRTRPADPMKEILPMSWLRYLLLVLGCFSGFFS CVYILV SEQ ID NO: 75 Mouse IL2Rβ-3 TWSPWSQPLTFRTRPADPMKEILPMSWLRYLLLVLGCFSGFFSCVYILV SEQ ID NO: 76 Mouse IL4Rα-1 RLSFPINILMSGVYYTARVRVRSQILTGTWSEWSPSITWYNHFQLPLIQRLPLGVTISC LCIPLFCLFCYFSIT SEQ ID NO: 77 Mouse IL4Rα-2 ARVRVRSQILTGTWSEWSPSITWYNHFQLPLIQRLPLGVTISCLCIPLFCLFCYFSIT SEQ ID NO: 78 Mouse IL4Rα-3 WSEWSPSITWYNHFQLPLIQRLPLGVTISCLCIPLFCLFCYFSIT SEQ ID NO: 79 Mouse IL7Rα-1 IPQRKLRPKAMYEIKVRSIPHNDYFKGFWSEWSPSSTFETPEPKNQGGWDPVLPSVTI LSLFSVFLLVILAHVLW SEQ ID NO: 80 Mouse IL7Rα-2 RSIPHNDYFKGFWSEWSPSSTFETPEPKNQGGWDPVLPSVTILSLFSVFLLVILAHVL W SEQ ID NO: 81 Mouse IL7Rα-3 SEWSPSSTFETPEPKNQGGWDPVLPSVTILSLFSVFLLVILAHVLW 105 DM2\19774561.1PATENT Docket No. H3131-00401 SEQ ID NO: 82 Mouse IL9R-1 RLRVQMTLESYEDKTEGEYYKSHWSEWSQPVSFPSPQRRQGLLVPRWQWSASILVV VPIFLLLTGFVHLLF SEQ ID NO: 83 Mouse IL9R-2 GEYYKSHWSEWSQPVSFPSPQRRQGLLVPRWQWSASILVVVPIFLLLTGFVHLLF SEQ ID NO: 84 Mouse IL-9R-3 PVSFPSPQRRQGLLVPRWQWSASILVVVPIFLLLTGFVHLLF SEQ ID NO: 85 Mouse IL15Rα-1 SQTTSAGTTGTGSHKSSRAPSLAATMTLEPTASTSLRITEISPHSSKMTKVAISTSVLLV GAGVVMAFLAW SEQ ID NO: 86 Mouse IL15Rα-2 SRAPSLAATMTLEPTASTSLRITEISPHSSKMTKVAISTSVLLVGAGVVMAFLAW SEQ ID NO: 87 Mouse IL15Rα-3 PTASTSLRITEISPHSSKMTKVAISTSVLLVGAGVVMAFLAW SEQ ID NO: 88 Mouse IL21R-1 EEFHKDSSYQLQVRAAPQPGTSFRGTWSEWSDPVIFQTQAGEPEAGWDPHMLLLLA VLIIVLVFMGLKIHL SEQ ID NO: 89 Mouse IL21R-2 PQPGTSFRGTWSEWSDPVIFQTQAGEPEAGWDPHMLLLLAVLIIVLVFMGLKIHL SEQ ID NO: 90 Mouse IL21R-3 WSDPVIFQTQAGEPEAGWDPHMLLLLAVLIIVLVFMGLKIHL SEQ ID NO: 91 Mouse TCR H / TMα-4 (19TMA-BBZAP327 (1)) DVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS 106 DM2\19774561.1PATENT Docket No. H3131-00401 SEQ ID NO: 92 Mouse TCR H / TMβ-4 (19TMB-BBZAP327 (1)) RADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS SEQ ID NO: 93 Mouse TCR H / TMα-5 (19TMA-BBZAP327 (2)) TCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNL LMTLRLWSS SEQ ID NO: 94 Mouse TCR TMβ-5 (19TMB-BBZAP327 (2)) GSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVL MAMVKKKNS SEQ ID NO: 95 Mouse TCR H / TMα-6 (19TMA-BBZAP327 (3)) DVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 96 Mouse TCR H / TMβ-6 (19TMB-BBZAP327 (3)) RADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS SEQ ID NO: 97 Mouse TCR H / TMα-7 (19TMA-BBZAP327(4)) TCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNL LMTLRLWSS SEQ ID NO: 98 Mouse TCR H / TMβ-7 (19TMB-BBZAP327 (4)) GSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVL MAMVKKKNS SEQ ID NO: 99 RR- H / TMα (1) (Synthetic peptide) LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGKDVPCDATLTE KSFETDMNLNFQNLLVMVLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 100 EE- H / TMβ (1) (Synthetic peptide) LEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGKRADCGITSAS YHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS SEQ ID NO: 101 107 DM2\19774561.1PATENT Docket No. H3131-00401 RR- H / TMα (2) (Synthetic peptide) LEIRAAFLRQRNTALRTEVAELEQEVQRLENEVSQYETRYGPLGGGKSFETDMNLNF QNLLVMVLRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 102 EE- H / TMβ (2) (Synthetic peptide) LEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGKHQGVLSATIL YEILLGKATLYAVLVSGLVLMAMVKKKNS SEQ ID NO: 103 H / TMα-RR (Synthetic peptide) TCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVMVLRILLLKVAGFNL LMTLRLWSSLEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK SEQ ID NO: 104 H / TMβ-EE (Synthetic peptide) GSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVL MAMVKKKNSLEIEAAFLERENTALETRVAELRQRVQRLRNRVSQYRTRYGPLGGGK SEQ ID NO: 105 Human CD3G-23 QSIKGNHLVKVYDYQEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSN AKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVY FIA SEQ ID NO: 106 Human CD3G-37 QEDGSVLLTCDAEAKNITWFKDGKMIGFLTEDKKKWNLGSNAKDPRGMYQCKGSQ NKSKPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIA SEQ ID NO: 107 Human CD3G-65 LTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQVYYRMCQNCIELNAATISGFLF AEIVSIFVLAVGVYFIA SEQ ID NO: 108 Human CD3G-95 KPLQVYYRMCQNCIELNAATISGFLFAEIVSIFVLAVGVYFIA SEQ ID NO: 109 Human CD3G-105 QNCIELNAATISGFLFAEIVSIFVLAVGVYFIA SEQ ID NO: 110 108 DM2\19774561.1PATENT Docket No. H3131-00401 Human TCR H / TMα-4 (19-hTCR-TMA) : DVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS SEQ ID NO: 111 Human TCR H / TMβ-4 (19-hTCR-TMB): DRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVL MAMVKRKDSRG SEQ ID NO: 112 Human TCR H / TMγ-4 (19-hTCR-TMG): DVITMDPKDNCSKDANDTLLLQLTNTSAYYMYLLLLLKSVVYFAIITCCLLRRTAFC CNGEKS SEQ ID NO: 113 Human TCR H / TMδ-4 (19-hTCR-TMD): DSTDHVKPKETENTKQPSKSCHKPKAIVHTEKVNMMSLTVLGLRMLFAKTVAVNFL LTAKLFFL SEQ ID NO: 114 Human IL7R-4 (197RBBZ-C1): KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVE SEQ ID NO: 115 Human IL7R-5 (197RBBZ-C2): KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEG FLQDTFPQQLEESEKQRLGGDVQSPNCPSEDV SEQ ID NO: 116 Human IL9R-4 (1999RBBZ-C1): KLSPRVKRIFYQNVPSPAMFFQPLYSVHNGNFQTWMGAHGAGVLLSQDCAGTPQGA LEPCVQEATALLTCGP SEQ ID NO: 117 Human IL9R-5 (199RBBZ-C2): KLSPRVKRIFYQNVPSPAMFFQPLYSVHNGNFQTWMGAHGAGVLLSQDCAGTPQGA LEPCVQEATALLTCGPARPWKSVALEEEQEGPGTRLPGNLSSEDVLPAGCTEWRVQT LAYLPQEDWAPTSLTRPAPPDSEGSR SEQ ID NO: 118 Human IL21R-4 (1921RBBZ-C1): SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPE VPSTLEVYSCHP SEQ ID NO: 119 109 DM2\19774561.1PATENT Docket No. H3131-00401 Human IL21R-5 (1921RBBZ-C2): SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPE VPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWP TLEVYSCHP SEQ ID NO: 120 Mouse CD3G-23 QTNKAKNLVQVDGSRGDGSVLLTCGLTDKTIKWLKDGSIISPLNATKNTWNLGNNA KDPRGTYQCQGAKETSNPLQVYYRMCENCIELNIGTISGFIFAEVISIFFLALGVYLIA SEQ ID NO: 121 Mouse CD3G-37 RGDGSVLLTCGLTDKTIKWLKDGSIISPLNATKNTWNLGNNAKDPRGTYQCQGAKE TSNPLQVYYRMCENCIELNIGTISGFIFAEVISIFFLALGVYLIA SEQ ID NO: 122 Mouse CD3G-65 LNATKNTWNLGNNAKDPRGTYQCQGAKETSNPLQVYYRMCENCIELNIGTISGFIFA EVISIFFLALGVYLIA SEQ ID NO: 123 Mouse CD3G-95 NPLQVYYRMCENCIELNIGTISGFIFAEVISIFFLALGVYLIA SEQ ID NO: 124 Mouse CD3G-105 ENCIELNIGTISGFIFAEVISIFFLALGVYLIA SEQ ID NO: 125 Mouse IL7R-4 KKRIKPVVWPSLPDHKKTLEQLCKKPKTSLNVSFNPESFLDCQIHEVKGVEARDEVE SEQ ID NO: 126 Mouse IL7R-5 KKRIKPVVWPSLPDHKKTLEQLCKKPKTSLNVSFNPESFLDCQIHEVKGVEARDEVE SFLPNDLPAQPEELETQGHRAAVHSANRSPETS SEQ ID NO: 127 Mouse IL9R-4 KLSPRLKRIFYQNIPSPEAFFHPLYSVYHGDFQSWTGARRAGPQARQNGVSTSSAGSE SSIWEAVATLTYSPACP SEQ ID NO: 128 Mouse IL9R-5 110 DM2\19774561.1PATENT Docket No. H3131-00401 KLSPRLKRIFYQNIPSPEAFFHPLYSVYHGDFQSWTGARRAGPQARQNGVSTSSAGSE SSIWEAVATLTYSPACPVQFACLKWEATAPGFPGLPGSEHVLPAGCLELEGQPSAYLP QEDWAPLGSARPPPPDSDSGSSDYC SEQ ID NO: 129 Mouse IL21R-4 PWRLWKKIWAPVPTPESFFQPLYREHSGNFKKWVNTPFTASSIELVPQSSTTTSALHL SLYPAKEKKFP SEQ ID NO: 130 Mouse IL21R-5 PWRLWKKIWAPVPTPESFFQPLYREHSGNFKKWVNTPFTASSIELVPQSSTTTSALHL SLYPAKEKKFPGLPGLEEQLECDGMSEPGHWCIIPLAAGQAV SEQ ID NO: 131 Flexible linker (Synthetic peptide): GGGGSGGGGSGGGGS SEQ ID NO: 132 CD19 scFv (Synthetic protein): MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSL PDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQ TDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS SEQ ID NO: 133 B7H3 scFv (Synthetic protein): MEFGLSWLFLVAILKGVQCDIVMTQSHKFMSTSIGARVSITCKASQDVRTAVAWYQ QKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYGTP PWTFGGGTKLEIKGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLKLSCEASRFTF SSYAMSWVRQTPEKRLEWVAAISGGGRYTYYPDSMKGRFTISRDNAKNFLYLQMSS LRSEDTAMYYCARHYDGYLDYWGQGTTLTVSS SEQ ID NO: 134 A2 / ESO scFv-1 (Synthetic protein): EVQLVESGGGLVQPGRSLRLSCRGSGFTFGDKAIHWFRQAPGKGLQWMGFISNKAY GATTKYAASVNGRFIISRDDSRNVAYLQMNGLRTEDTAVYYCTRNYGAGCSAGSCY GAFDIWGQGTMVIVSSGGGGSGGGGSGGGGSEIVMTQSPATLSLSPGERATLSCRAS QSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAV YYCQQRSNWPPGLTFGGGTRGYQS 111 DM2\19774561.1PATENT Docket No. H3131-00401 SEQ ID NO: 135 A2 / ESO scFv-2 (Synthetic protein): VQLQESGPGLVKPSETLSLTCTVSGGSISNYYWSWIRQTPGKGMEWIGYIYYTGTTN YNPSLKSRVTISVDTSKNQFSLRLSSVTAADTAVYYCARVLTNCGGDCSLDYYYYY GMDVWGQGTMVTVSSGGGGSGGGGSGGGGSNIQMTQSPSSLSASVGDRVTITCRVS QGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFA TYYCLQDYNYPPWTFGQGTKVEI SEQ ID NO: 136 A2 / ESO scFv-3 (Synthetic protein): VQLVQSGTEVKKPGASVKVSCKASGYTFTDYYIHWVRQAPGQGLEWMGWINPNRG ATKYAQKFQGWATMTRDTSISTVYMELSRLNSDDTAVYYCATARAGGDPGDNSPL DYWGQGTMVTVSSGGGGSGGGGSGGGGSSYVLTQPPSASGSPGQTVTISCSGSRSNL GNNYACWYQQVPGTAPKLLIYRNNERLSGVPERFSASKSGASASLAISGLRSDDEAD YYCSSWEDSLGGSWVFGGGTKVTVL SEQ ID NO: 137 A2 / ESO scFv-4 (Synthetic protein): GTLQQSGPGLVKPSETLSLTCTVSNGSVNDYYWGYIRQPPGKGLEWIGYVYYSGSSY YNPSLKSRVSISINTSKNQFSLKLTSVTAADTAVYYCARGYFDWLSLPGGFDYWGQG TLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSFSASTGDRVTITCRASQGISSYLAWY QQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISCLQSEDFATYYCQQYYSY PRLTFGGGTKLDIK SEQ ID NO: 138 A2 / ESO scFv-5 (Synthetic protein): GQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARDWKGYNWFDPWGQGTLVT VSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTITCRASQSISSHLNWYQQKP GKAPNLLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYFCQQSYTTPLTFGQ GRRLEIK SEQ ID NO: 139 A2 / ESO scFv-6 (Synthetic protein): QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCARVTWERFGEDASD YYYYGMDVWGQGTMVTVSSGGGGSGGGGSGGGGSDIQLTQSPSSLSASVGDRVTIT CRASQNFSRYLNWYQQKPGKAPKLLIYAVSSLQSGVPSRFSGSGSGSDFTLTISSLQP EDFATYYCQQSYSTPLTFGGGTTVDIK SEQ ID NO: 140 A2 / ESO scFv-7 (Synthetic protein): QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYN GNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDAGNYDYVWGS 112 DM2\19774561.1PATENT Docket No. H3131-00401 YSVVPDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSVLTQPASVSGSPGQSITIS CTGTSSDIGAYNYVSWYQQHPGKAPKLMIYDVRNRPSGVPDRFSGSKSGNTASLTIS GLQAEDEANYYCSSYTRSGKGVFGTGTQLTVL SEQ ID NO: 141 A2 / ESO scFv-8 (Synthetic protein): VQLQESGPGLVKPSETLSLTCTVSGGSISSSNYYWGWIRQPPGKGLEWIGSIYYSGST YYNPSLKSRVTISVDPSKSQFSLKLSSVTAADTAVYYCARSYGDYALFDYWGQGTL VTVSSGGGGSGGGGSGGGGSESVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLSIRRLEPEDSAVYYCQQYGSSQ FTFGPGTTVDIK SEQ ID NO: 142 A2 / ESO scFv-9 (Synthetic protein): QVELQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGST NYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGLSGDRNLRYFDLWGR GTLVTVSSGGGGSGGGGSGGGGSNFMLTQPPSASGTPGQRVTISCSGSNSNIGSNSVN WYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAW DDNLNGKIFGGGTKLTVL SEQ ID NO: 143 A2 / ESO scFv-10 (Synthetic protein): VQLQESGPGLVKPSETLSLTCTVSGGSISSSNYYWGWIRQPPGKGLEWIGSIYYSGST YYNPSLKSRVTISVDPSKSQFSLKLSSVTAADTAVYYCARSYGDYALFDYWGQGTL VTVSSGGGGSGGGGSGGGGSESVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY QQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLSIRRLEPEDSAVYYCQQYGSSQ FTFGPGTTVDIK SEQ ID NO: 144 A2 / ESO scFv-11 (Synthetic protein): AQLQESGPGLVTPSETLSLTCNVSGESMNNYYWTWIRQPPGKGLEWIGYVYYSGTT KYNPSFKSRVTILVDTSKRHLSLRLNSVTAADTGVYYCARVREKATKYHAAFDYWG QGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPGALSLSPGERATLSCRASQSVSSNY LAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVFYCQQ YGDSPWTFGQGTKVEIK Experimental Methods and Constructions

[0282] CAR constructs: The 19BBz CAR-encoding gene was generated by linking the sequences encoding the FMC63-derived ScFv to those of the CD8α extracellular, transmembrane and cytoplasmic domain of 4-1BB, and CD3ζ signaling domain. 1928z used 113 DM2\19774561.1PATENT Docket No. H3131-00401 CD28 hinge, transmembrane domain and intracellular domain. ZAP70 kinase domain- containing fragments were generated by overlap PCR, and replaced CD3ζ signaling domain in the CAR constructs. All constructs were cloned into the pMSGV1 vector with sequencing verification.

[0283] The development of CAR-T cell therapy with long-term remission and little or no toxicity is urgently needed for cancer patients. It was recently shown that CD3ζ signaling domain could be replaced with ZAP-70 kinase signaling domain (from a.a. 300-619, and 327- 619, or ZAP300 and ZAP327, respectively for short) (WO / 2021 / 263211).

[0284] 1928ZAP327 CAR-T cells prepared from multiple healthy donors could recognize Raji tumor target cells but secreted significantly lower amounts of cytokines, compared with 1928z CAR-T cells. Importantly, 1928ZAP327 and 1928z CAR-T cells showed similar cytolytic activity against Raji cancer cells. Similar results were obtained with 19BBZAP327 versus 19BBz CAR-T cells. 19BBZAP327 CAR-T cells secreted significantly lower amounts of cytokines than 19BBz CAR-T cells, but maintained similar or even better cytolytic activity.

[0285] It was further demonstrated that 1928ZAP327 CAR-T cells outperformed 1928z CAR-T cells in vivo experiments, and dramatically prolonged overall survival in a Raji lymphoma NSG mouse model. Similar results were obtained with 19BBZAP327 versus 19BBz CAR-T cells. These results suggest that the kinase domain (ZAP300 and ZAP327) of ZAP-70 is critically important to boost the therapeutic immunity of CAR-T cells (WO / 2021 / 263211).

[0286] T cell survival at day 35 after T cell transfer into NSG mice was tested to understand why 1928ZAP327 CAR-T cells produce more potent antitumor immunity, and it was found that higher percentages of human CD3+ 1928ZAP327 CAR-T cells could be detected in the bone marrows, lung and spleens, compared with mice treated with 1928z CAR-T cells. Furthermore, the percentages of Tcm and Tscm populations in the total 1928ZAP327 CAR-T cells were much higher than 1928z CAR-T cells. 1928ZAP327 CAR-T cells expressed lower amounts of PD-1, TIM3 and LAG3 molecules (an exhaustion markers) than 1928z CAR-T cells, suggesting that the ZAP327 signaling domain promotes T cell memory function and persistence in vivo. The ZAP327 signaling domain promotes stem-like memory T cells and 114 DM2\19774561.1PATENT Docket No. H3131-00401 Applicants therefore call this Synthetic TCR signaling for Enhancing Memory T cell technology as STEM for short (WO / 2021 / 263211).

[0287] Hinge and transmembrane domain sequences from CD8, CD28, IgG1 or IgG4 protein have been used in the CAR constructs. Among them, the hinge and transmembrane domain sequences from CD8 and CD28 are commonly used in all CAR constructs. It has been shown that the hinge and transmembrane domain could regulate CAR-T cell function, cytokine production and T cell persistence in preclinical and clinical studies (Alabanza, L. et al. Function of Novel Anti-CD19 Chimeric Antigen Receptors with Human Variable Regions Is Affected by Hinge and Transmembrane Domains. Mol Ther 25, 2452-2465 (2017). Ying, Z. et al. A safe and potent anti-CD19 CAR T cell therapy. Nat Med 25, 947-953 (2019). Hudecek, M. et al. The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol Res 3, 125-135 (2015).).

[0288] Generation of retroviral constructs and transduction: Primers and gBlocks were obtained from Integrated DNA Technologies, Inc (Skokie, Illinois, USA). The pMSGV1- 19bbz 19bbZAP327 vector served as the template, with NotI and PmeI restriction sites introduced after the ScFv and in front of BBZ or BBZAP327, respectively. Cloning was performed using PCR products or gBlocks containing various lengths of hinge and transmembrane regions. To construct 19TMAs and 19TMBs, TCR alpha and beta chain were utilized as templates and designed lengths were obtained using primers. TMA-RR, TMB-EE, RR-TMAs, and EE-TMBs were synthesized and then cloned into the NotI and PmeI cut vector via homozygous recombination (NEB, E2621X). SalI cut 19TMA plasmid was hemizygously combined with GFP fragment obtained from PCR to generate 19TMA-GFP, while 2A-GFP was used to construct 19TMA-2A-GFP. For CD3 gamma chain equipped CAR constructs, different lengths of CD3 gamma truncates were PCR amplified using primers. The intracellular domain of IL7R was PCR amplified from an entry clone (Thermo Fisher Scientific), and fragments were overlapped by PCR and assembled with the vector. Similarly, constructs with common gamma chain receptor incorporated CARs were cloned using the same methods. All constructs were sequenced to confirm their accuracy. 115 DM2\19774561.1PATENT Docket No. H3131-00401

[0289] Flow cytometry: Flow cytometry was performed using an Attune™ Acoustic Focusing Cytometer. T cells were stained with protein L for CD19 CAR-T cells, followed by PE-conjugated streptavidin. Data were analyzed using FlowJo V.10.0.7 software (TreeStar).

[0290] Confocal imaging: T cells were fixed with 4% PFA and mounted on a glass slide. Imaging was acquired using a Nikon A1 confocal microscope.

[0291] Cell lines: HEK293T and MDA-MB-231-CD19 cell lines were cultured in DMEM supplemented with 10% inactivated FBS and 100 units / mL penicillin and 100 µg / mL streptomycin. Raji cells were cultured in RPMI-1640 medium supplemented with 10% inactivated FBS and 100 units / mL penicillin and 100 µg / mL streptomycin. All cells were tested regularly to confirm they were mycoplasma negative.

[0292] Human PBMC and transduction: Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples of healthy donors using Ficoll reagent according to the manufacturer's instructions. PBMCs were seeded into an anti-human CD3 antibody (OKT3)- coated plate for activation, and retrovirus was packaged in HEK293T cells with envelope plasmid RD114 and packaging plasmid Gag-pol. Virus was harvested at 48 and 72 hours post- transfection and filtered with a 0.45 µm filter. Activated PBMCs were transduced twice with retrovirus in the presence of RetroNectin according to the manufacturer's instructions. T cells were cultured for 3-7 days before use.

[0293] ELISA and cytokine release assays: Tumor cells (Raji and MDA-MB-231-CD19) were seeded in a 96-well round-bottom plate with triplicates (104 cells / well). T cells (0.1 million / well) were cocultured with tumor cells overnight. On the following day, supernatants (50 µL) were added to a human IFN-γ (1:1000, Thermo Fisher, M700A) precoated and 1% BSA blocked plate and incubated for 1 hour at room temperature with gentle shaking. The plate was washed twice and incubated with a biotin-conjugated IFN-γ antibody (1:1000, Thermo Fisher, M700B) for 1 hour, followed by another two washes and incubation with avidin-HRP (1:5000) for 30 min in the dark. After washing, 100 µL TMB was added for the reaction, which was stopped by adding 50 µL of 2.5 N sulfuric acid after 15 min. The absorbance was read at 450 nm on a spectrophotometer (Bio-Tek). 116 DM2\19774561.1PATENT Docket No. H3131-00401

[0294] LDH cytotoxicity release assay: Transduced T cells were co-cultured with tumor cells in 96-well plates at different effector to target (E:T) ratios for 4 hours. The supernatants containing LDH were transferred into a new enzymatic plate for cytotoxicity assay as per the manufacturer’s instructions. The absorbance was read on a spectrophotometer at 490 nm. The percentage of specific lysis was calculated using the formula: % cytotoxicity = (experimental - effector spontaneous - target spontaneous) / (target maximum - target spontaneous) * 100. All values were first subtracted by the medium control.

[0295] Statistical analysis: Statistically significant differences between two groups were assessed using a two-tailed paired or unpaired t-test. Comparisons between more than two groups were carried out by an ANOVA with Tukey’s multiple-comparisons test. Differences were considered statistically significant at a P value <0.05. In the mouse experiments, the overall survival of the mice that were treated with the T cells were depicted by a Kaplan–Meier curve, and the survival difference between the groups were compared using the log-rank test. All statistical analyses were performed using GraphPad Prism 8 software. Examples Example 1: New CAR constructs with the hinge and transmembrane derived from TCR and related signaling molecules

[0296] In this invention, new CAR constructs were generated by replacing CD28 hinge / TM in CD28-containing CAR constructs by the hinge / TM derived from TCR and their signaling molecules such as hTCR-hinge / TMα, hTCR-hinge / TMβ, hTCR-hinge / TMγ, hTCR- hinge / TMδ, hCD3γ-hinge / TM, hCD3δ-hinge / TM, hCD3ε-hinge / TM, hCD3ζ-hinge / TM, hLCK-hinge / TM, hLAT-hinge / TM, mTCR-TMα, mTCR-TMβ, mTCR-TMγ, mTCR-TMδ, as well as transmembrane domains from mCD3γ, mCD3δ, mCD3ε, and mCD3ζ. The representative examples of new CD19TM28Z CAR constructs containing the hinge / TM from human and murine TCR-hinge / TMα, TCR-hinge / TMβ, TCR-hinge / TMγ, and TCR-hinge / TMδ are illustrated in FIG.1A. For example, CD19 ScFv was linked to the hinge / TMα from TCRα chain with or without a linker [such as (GGGGS)3-5 (SEQ ID NO: 148)], followed by a costimulatory signaling domain derived from CD28 and CD3ζ TCR signaling domain. 117 DM2\19774561.1PATENT Docket No. H3131-00401

[0297] Similarly, new CAR constructs were also generated by replacing CD8 hinge / TM in 4-1BB-containing CAR constructs by the hinge / TM derived from TCR and their signaling molecules such as hTCR-hinge / TMα, hTCR-hinge / TMβ, hTCR-hinge / TMγ, hTCR- hinge / TMδ, hCD3γ-hinge / TM, hCD3δ-hinge / TM, hCD3ε-hinge / TM, hCD3ζ-hinge / TM, hLCK-hinge / TM, hLAT-hinge / TM, mTCR-TMα, mTCR-TMβ, mTCR-TMγ, mTCR-TMδ, as well as transmembrane domains from mCD3γ, mCD3δ, mCD3ε, and mCD3ζ. The representative examples of new CD19TMBBZ CAR constructs containing the hinge / TM from from human and murine TCR-hinge / TMα, TCR-hinge / TMβ, TCR-hinge / TMγ, and TCR- hinge / TMδ are illustrated in FIG. 1B. For example, CD19 ScFv was linked to the hinge / TMα from TCRα chain with or without a linker [such as (GGGGS)3-5 (SEQ ID NO: 148)], followed by a costimulatory signaling domain derived from 4-1BB and CD3ζ TCR signaling domain.

[0298] ZAP327-containing CAR-T cells released significantly lower amounts of cytokines compared with CD3ζ-containing CAR-T cells, but maintained similar specific lysis against target cancer cells. Importantly, ZAP327-containing CAR-T cells outperformed CD3ζ- containing CAR-T cells in vivo antitumor immunity, and remarkedly prolonged mouse survival. Therefore, ZAP327-containing CAR constructs were generated with different hinge / TM domains derived from human and murine TCR and their signaling molecules. The representative examples of new CD19TM28ZAP327 CAR constructs containing the hinge / TM from human and murine TCR-hinge / TMα, TCR-hinge / TMβ, TCR-hinge / TMγ, and TCR- hinge / TMδ are illustrated in FIG. 1C. For example, CD19 ScFv was linked to the hinge / TMα from TCRα chain with or without a linker [such as (GGGGS)3-5 (SEQ ID NO: 148)], followed by a costimulatory signaling domain derived from CD28, and ZAP70 kinase domain (such as ZAP327) as a TCR signaling domain.

[0299] Furthermore, new CAR constructs were also generated by replacing CD8 hinge / TM in 4-BB-containing CAR constructs by the hinge / TM derived from TCR and their signaling molecules. The representative examples of new CD19TMBBZAP327 CAR constructs containing the hinge / TM from human and murine TCR-hinge / TMα, TCR-hinge / TMβ, TCR- hinge / TMγ, and TCR-hinge / TMδ are illustrated in FIG. 1D. For example, CD19 ScFv was linked to the hinge / TMα from TCRα chain with or without a linker [such as (GGGGS)3-5 (SEQ 118 DM2\19774561.1PATENT Docket No. H3131-00401 ID NO: 148)], followed by a costimulatory signaling domain derived from 4-1BB, and ZAP70 kinase domain (such as ZAP327) as a TCR signaling domain. The amino acid sequences of both human and mouse TCR hinge and transmembranes with different lengths are listed in SEQ ID NO: 7-48 and SEQ ID NO: 110-113. Example 2: Functional analysis of novel CARs containing TCR hinge / TM domains

[0300] To test whether CARs containing a TCR hinge / TM domain could express and function in T cells in response to target antigen-expressing tumor cells, CD19TMαBBZAP327 CAR containing different lengths of hinge-TM with a linker (GGGGS)3 (SEQ ID NO: 131) between CD19 ScFv and hinge-TM (45 or 61 amino acids) were constructed, followed by a 4- 1BB costimulatory signaling domain and ZAP327 T cell activation domain (BBZAP327) (FIG. 2A). Similarly, CD19TMβBBZAP327 CAR containing the same set of hinge and TM with different lengths were constructed, followed by a 4-1BB costimulatory signaling domain and ZAP327 T cell activation domain (FIG. 2A). T cells were transduced with CARs containing different length combinations of hinge and TM, and then co-cultured with CD19- expressing tumor cells. The conventional CD19BBZ was used as a positive control. It was found that CD19TMαBBZAP327 and CD19TMβBBZAP327 CAR-T cells strong recognized tumor cells and released IFN-γ from T cells (FIG. 2B). The bars in FIG 2B are grouped as sets for T cell only and +Raji, in the order 19TMA(1), 19TMA(2), 19TMA(3), 19TMA(4), 19BBZ, 19TMB(1), 19TMB(2), 19TMB(3), 19TMB(4), and Ctrl. For the T cell only group, only 19TMB(4) appears to show IFN-γ production. Further, these CAR-T cells also strongly lysed tumor cells (FIG. 2C). These results suggest that CAR constructs with a single TCR TM domain can function well in respond to tumor cells for specific recognition and lysis, regardless of the length of hinge region. The amino acid sequences of mouse TCR-h / TMα and TCR- h / TMβ are listed in the SEQ. ID s: 91-98. The amino acid sequences of the (G4S)3 linker and anti-CD19 scFv are listed in the SEQ. ID NOs: 131-132. Example 3: A single TCR transmembrane domain directs CARs to form a complex with endogenous TCRs

[0301] Unexpectedly, the surface expression of the TMα- or TMβ-containing CAR could not be detected by using L-protein staining for CD19CAR, which generally strongly detects 119 DM2\19774561.1PATENT Docket No. H3131-00401 conventional CAR expression in T cells through FACS analysis (FIG.3A). The TMα- or TMβ- containing CAR could interact with endogenous TCR complexes through its TM domain to form complexes with endogenous TCRs. To test this possibility, a CAR construct with GFP fusion or with T2A-GFP (CAR and GFP being separated after protein translation) was generated (FIG. 3B). The TMα- containing CARs were found to be expressed in the TCR complexes on the cell surface (FIG. 3C). By contrast, GFP in the CAR-T2A-GFP construct was expressed in the whole cell (FIG. 3C). Both CAR-GFP fusion and CAR-T2A-GFP could be detected by FACS analysis (FIG. 3D). These results suggest that the L-protein could not stain and detect the TMα- or TMβ-containing CAR expression once CAR forms a complex with endogenous TCR. Although the direct fusions of antibody (anti-CD19) ScFv to TCR CD3 subunits (Helsen, C.W. et al. The chimeric TAC receptor co-opts the T cell receptor yielding robust anti-tumor activity without toxicity. Nat Commun 9, 3049 (2018). Baeuerle, P.A. et al. Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor response. Nat Commun 10, 2087 (2019).) or TCRα and β chain constant regions (Liu, Y. et al. Chimeric STAR receptors using TCR machinery mediate robust responses against solid tumors. Sci Transl Med 13 (2021). Mansilla-Soto, J. et al. HLA-independent T cell receptors for targeting tumors with low antigen density. Nat Med 28, 345-352 (2022).) have been reported, this is the first demonstration that a single TCRα and β TM domain is sufficient to drive the CAR fusion protein to form a complex with endogenous TCR subunits for its biological function, antigen recognition and tumor killing (Fig. 3E). Thus, TCR-TM-based CAR likely form a complex with endogenous TCR subunits for potent biological function and immunity. Example 4: CARs containing human TCR hinge-transmembrane domain are functional in target recognition and killing

[0302] To test the hinge-TM domains derived from human TCR-TMα, -TMβ, TMγ and TMδ can direct CAR T cell functional recognition and killing of target cells, CD19 CARs containing anti-CD19 ScFv, hinge / TMα (or -hinge / TMβ, hinge / TMγ, hinge / TMδ), a costimulatory domain from 4-1BB, and T cell signaling domain (ZAP327). The final products are 19-hTCR-TMA, 19-hTCR-TMB, 19-hTCR-TMG and 19-hTCR-TMD (FIG. 4A, 4B). 120 DM2\19774561.1PATENT Docket No. H3131-00401 These hTCR-TM-containing CAR-T cells were capable of recognizing MDA-MB-231-CD19 cells and specifically killed these tumor cells (FIG.4C, 4D). These results suggest that human TCR hinge-TM domains derived from human TCR-TMα, -TMβ, TMγ and TMδ are functional. Based on these results, additional human TCR-TM CARs containing P2A and MyD88 components were designed to activate NF-kB signaling pathway in T cells. MyD88 can be replaced with Toll-like receptors, TRAF, TAK1, IKKalpha, IKKbeta, NEMO (IKKgamma) or MALT-1 gene to enhance CAR-T cell function and persistence (Fig. 4E). Example 5: Development of dual antigen CARs using TCR TMα-TMβ and TMγ-TMδ pairing

[0303] TCR transmembrane domains play a critical role in the correct TCR pairing, complex assembling and surface expression (Haga-Friedman, A., Horovitz-Fried, M. & Cohen, C.J. Incorporation of transmembrane hydrophobic mutations in the TCR enhance its surface expression and T cell functional avidity. J Immunol 188, 5538-5546 (2012). Brazin, K.N. et al. The T Cell Antigen Receptor alpha Transmembrane Domain Coordinates Triggering through Regulation of Bilayer Immersion and CD3 Subunit Associations. Immunity 49, 829-841 e826 (2018).). More importantly, these results suggest that a single TCR-TM domain alone can direct CARs to form the CAR / TCR complexes through endogenous TCR subunits. Thus, whether one CAR containing TMα could pair with another CAR containing TMβ domain was contemplated. To test this possibility, a first CD19 CAR containing anti-CD19 ScFv, hinge / TMα, a costimulatory domain from either CD28 or 4-1BB, and T cell signaling domain (CD3ζ or ZAP327), and a second CD22 CAR containing a ScFv, hinge / TMβ, but no signaling domain were constructed (FIG. 5A, 5B, 5C and 5D). The second CD22 CAR cannot function itself until it pairs with the first CD19 CAR due to its lack of a signaling domain. When paired together, the dual antigens CARs can recognize CD19 alone, CD22 alone or both antigen- expressing tumor cells (FIG. 5). Similarly, dual antigens CARs using TMγ-TMδ containing CARs were constructed, as shown in FIG. 5. The functionality of these dual antigen CARs is tested experimentally. To increase the specificity and correct pairing, a modified murine TCR TMα and TMβ was used for the proposed CAR constructs, since the modified murine TCR 121 DM2\19774561.1PATENT Docket No. H3131-00401 TMα and TMβ preferentially pair each other, rather than pairing with human endogenous TCRs. Example 6: Enhancing the stability and specificity of dual antigens CARs using leucine- zipper dimerization

[0304] Despite use of modified murine TCR TMα and TMβ it is still likely that the TM- containing CARs can pair with endogenous TCRs. Thus, dual antigens CARs may not work well. For this reason, use of leucine zipper-mediated dimerization was contemplated, in order to force TMα-containing CAR to pair with TMβ-containing CARs. To test this possibility, CARs with a leucine zipper sequence inserted in the intracellular portion of a CAR construct were constructed, followed by co-stimulatory signaling domains (CD28 or 4-1BB), and T cell activation domains (CD3ζ or ZAP-70 kinase domain) (FIG. 6A, 6B, 6C and 6D). CARs containing TM-leucine zipper (LZ)-4-1BB-ZAP327 signaling domains. The amino acid sequences of LZ-TM and TM-LZ are listed as SEQ. ID NOs: 99-104.

[0305] As an example shown in FIG. 7A, CD19BBZAP327 containing leucine zipper RR or EE right between TM and 4-1BBZAP327 was constructed. It was found that leucine zipper RR or EE containing CAR-T cells were capable of recognizing tumor antigen position and efficiently kill Raji tumor cells (FIG. 7B and 7C). In FIG 7B, the bars are grouped as sets for T cell only and +Raji, in the order 19TMA-RR, 19TMB-EE, 19BBZ, and Ctrl. For the T cell only group, only 19BBZ appears to show IFN-γ production. These results suggest that T cells expressing CARs containing a leucine zipper RR or EE sequence in the downstream of TM domain (intracellular side) can recognize and kill tumor cells effectively. The amino acid sequences of TM-LZ-RR and TM-LZ-EE are listed as SEQ. ID NOs: 103-104.

[0306] Alternatively, leucine zipper RR or EE could be inserted between the ScFv and hinge-TM domain of CAR construct (extracellular side), followed by co-stimulatory signaling domains (CD28 or 4-1BB), and T cell activation domains (CD3ζ or ZAP-70 kinase domain) (FIG. 8A, 8B, 8C and 8D). To test this possibility, CD19RRTMA (1), CD19EETMB (1), CD19RRTMA (2) and CD19EETMB (2) were constructed (FIG. 9A) It was found that LZ RR-containing CD19RRTMA (1), CD19RRTMA (2) and CD19EETMB (2) CAR-T cells were capable of recognizing CD19-expressing tumor cells and release cytokine (IFN-γ), but LZ EE- 122 DM2\19774561.1PATENT Docket No. H3131-00401 containing CD19EETMB (1) CAR-T cells failed to do so (FIG. 9B). The bars in FIG 8B are grouped as sets for T cell only and +Raji, in the order 19RR-TMA(1), 19EE-TMB(1), 19RR- TMA(2), 19RR-TMB(2), 19BBZ, and Ctrl. For the T cell only group, only 19BBZ appears to show IFN-γ production. Furthermore, LZ RR-containing CAR-T cells generally showed the reduced ability to lyse tumor cells, while LZ EE-containing CD19EETMB (1) CAR-T cells completely lost killing ability (FIG. 9C). These results suggest that the CAR constructs containing LZ sequence between ScFv and TM (extracellular side) remain active for cytokine release, but markedly reduce their ability to kill tumor cells. Therefore, dual antigen CARs with ScFv designed to target CD19 and CD22 are constructed, followed by the paired TMα-TMβ with LZ pairs (RR-EE), or TMγ-TMδ with LZ pairs (RR-EE), as shown in FIG.10A, 10B and 10C. The BBZAP327 signaling domain is only fused to TCR TMα or TMγ. The antigen recognition and functional lysis of CAR-T cells are further analyzed.

[0307] To further demonstrate the function and specificity of dual antigens CARs using TCR-TMα and TMβ, we generated three dual antigens CARs: (1) 19-B7TMAB CAR contained the first anti-CD19 scFv, TCR-TMα, P2A, the second anti-B7H3 scFv and TCR- TMβ; (2) 19Β7ΤΜΑΒΖ ^ ^ CAR contained the first anti-CD19 scFv, TCR-TMα, 4-1BB-ZAP, P2A, the second anti-B7H3 scFv and TCR-TMβ; (3) 19-B7TMABLZ contained CAR contained the first anti-CD19 scFv, TCR-TMα, LZ-RR, P2A, the second anti-B7H3 scFv, TCR-TMβ and LZ-EE (FIG.11A). These dual antigens CARs can form the TMα-TMβ paired complex or form a TCR complex with endogenous TCR subunits (a CAR-TCR complex) (FIG 11B). However, the TMα-TMβ paired complex of 19-B7TMAB and 19-B7TMABLZ CAR-T cells lack signaling domains, while the TMα-TMβ paired complex of 19-B7TMABZAP CAR- T cells have 4-1BB-ZAP327 signaling domain (FIG.11B). To test whether these dual antigens CAR-T cells can recognize target cells and specifically kill tumor cells, we used Raji cells (CD19+ B7H3-) and U937 (CD19- B7H3+) cells to determine their antigen specificities. We found that all three CAR-T cells (19-B7TMAB, 19-B7TMABZAP and 19-B7TMABLZ) recognized and killed Raji and U937 cells, suggesting that CD19 and B7H3 dual antigens CAR- T cells are functional and specific (FIG.11C). Importantly, 19-B7TMAB and 19-B7TMABLZ CAR-T cells (without signaling domains) are functional, suggesting that these CAR may form 123 DM2\19774561.1PATENT Docket No. H3131-00401 a CAR-TCR complex for signaling actionation and function, as shown in FIG.11B. The amino acid sequences of anti-CD19 scFv and anti-B7H3 scFv are listed in the SEQ ID NO 132-133. Example 6: Development of new CARs containing TM and signaling domains from cytokine receptors

[0308] A panel of CARs containing TM domains derived from transmembrane domains derived from cytokine receptors, including but not limited to IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα, and the common cytokine receptor γ chain was generated. The representative examples of these new CARs are shown in FIG. 12. Anti-CD19 ScFv was directly fused to the hinge / TM and signaling domains of cytokine receptors, followed by costimulatory and T cell activation signaling domains (FIG. 12A, 12B and 12C). The amino acid sequences of human and mouse cytokine receptors are listed in SEQ ID NO 114-119 and 125-130.

[0309] Furthermore, whether IL7R or IL-15R intracellular signaling domains could be inserted into the CAR constructs was tested, as shown in FIG. 13A. It was found that CAR containing IL-7R intracellular domain but without co-stimulatory and T cell activation signaling domains (197) could recognize tumor cells, but failed to lyse tumor cells (FIG. 13B and 13C). By contrast, CAR containing IL-15R intracellular domain without co-stimulatory and T cell activation signaling domains (1915) could not function for tumor recognition and lysis. CAR containing IL-7R or IL-15R plus co-stimulatory and T cell activation signaling domains (197BBZ and 1915BBZ) could recognize and kill tumor cells (FIG. 13B and 13C). These results suggest that IL-7R intracellular domain can function for tumor recognition to release cytokine, but co-stimulatory and T cell activation signaling domains are needed for tumor lysis.

[0310] To further test whether TM domain and IL-7R intracellular domain can work together in CAR constructs, CARs (3G-23, 3G-37, 3G-65, 3G-95 and 3G-105) containing different lengths of hinge and TM of CD3γ, plus IL-7R and 4-1BB-ZAP327 were constructed (FIG. 14A). It was found that CARs containing different lengths of hinge and TM of CD3γ could reduce the cytokine release, but remained or even increased their lyses of tumor cells compared with conventional 19BBz (FIG. 14B and 14C). Particularly, 3G-105 CAR 124 DM2\19774561.1PATENT Docket No. H3131-00401 containing a short of hinge and TM of CD3γ function well for tumor lysis, with reduced cytokine release (FIG. 14B and 14C). These results suggest that the length of CD3γ hinge regulates T cells for cytokine release, but remained similar or even better cytolytic activity. The cytokine release from T cells is not correlated with their cytolytic activity. The amino acid sequences of TCR subunits (CD3E, CD3D, CD3G) are listed in the SEQ ID NO 30-48, 105- 109 and 120-124.

[0311] Based on these novel findings, a series of CARs with different signaling domains derived from cytokine receptors such as IL-7R, IL-9R and IL-21R are constructed (FIG. 15 and FIG. 16) to identify the optimal signaling domains or combinations to improve the therapeutic potential CAR-T cell function. Importantly, these signaling domains or combinations may apply to TCR engineering to increase therapeutic immunity against cancer. The amino acid sequences of human and mouse cytokine receptors (such as IL-7R, IL-9R and IL-21R) are listed in SEQ ID NO 49-90, 114-119 and 125-130.

[0312] To demonstrate the function of CARs with different signaling domains derived from IL-7R, IL-9R and IL-21R, we generated 197RBBZ-C1, 197RBBZ-C2, 199RBBZ-C1, 1999RBBZ-C2, 1921RBBZ-C1 and 1921RBBZ-C2 (FIG. 17A) and found that these CAR-T cells could recognized and killed CD19+ Raji tumor cells (FIG. 17B, 17C). These results indicate that CARs with different TM and signaling domains derived from IL-7R, IL-9R and IL-21R are functional.

[0313] Having thus described in detail preferred embodiments of the present invention, it is to be understood that the invention defined by the above paragraphs is not to be limited to particular details set forth in the above description as many apparent variations thereof are possible without departing from the spirit or scope of the present invention. Example 7: Development of TCR-like CARs containing TCR-TM and 4-1BB-ZAP327

[0314] Because conventional CAR-T cells recognize surface targets, but not intracellular molecules, it limits the power and scope of CAR-T technology’s application. To address this issue, we developed and identified scFv antibodies that were capable of recognizing HLA- A2 / NY-ESO-1 peptide complex from a phage-display library screening (FIG. 18A, 18B). These A2 / ESO scFv antibodies are listed as A2 / ESO scFV-1 to scFV-11 (SEQ NO 134-144). 125 DM2\19774561.1PATENT Docket No. H3131-00401

[0315] Using one of the selected A2 / ESO scFv, we constructed an A2 / ESO CAR containing A2 / ESO scFv, TCR-TMβ, and 4-1BB-ZAP327 signaling domain (FIG. 19A). A2 / ESO CAR-T cells recognized EO771 / A2-ESO cells for cytokine release (FIG. 19B), and specifically killed EO771 / A2-ESO cells (FIG. 19C). These results suggest that TCR-like CARs can recognize and kill target tumor cells, thus expanding the scope and power of CAR- T technology. Example 8: Bi-specific antibody therapeutics using TCR-like scFv

[0316] Bi-specific antibodies have been extensively developed and approved for cancer treatment (Nature Reviews Drug Discovery, 23, 301-319, 2024). A2 / ESO scFv antibody can be engineered to fuse with CD3ζ as part of the TCR on T cells, NKG2D or CD16 on NK cells, PD-1, CTLA4 on T cells, PD-L1 on tumor cells, and other molecules on immune cells (FIG. 20). 126 DM2\19774561.1

Claims

PATENT Docket No. H3131-00401 CLAIMS What is claimed is:

1. A composition comprising a chimeric antigen receptor (CAR), or cells expressing a CAR, wherein the CAR comprises at least one antigen recognition moiety, at least one hinge and transmembrane (hinge / TM) domain, and at least one intracellular T-cell activation moiety, wherein: a. the at least one intracellular T-cell activation moiety comprises at least one costimulatory signaling domain fused with a signaling domain, wherein the costimulatory signaling domain selected from a CD28, 4-1BB, CD3ζ, IL-2Rβ, IL-4Rα, IL-7R, IL9-R, IL- 12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21R, or a combination thereof; and b. the at least one hinge / TM domain with different length of hinge region is derived from a transmembrane domain of CD4, CD8, CD28, ZAP70, TCRα, TCRβ, TCRγ, TCRδ, CD3γ, CD3δ, CD3ε, CD3ζ, LCK, LAT, IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, IL-21Rα, or the common cytokine receptor γ chain, or any combination thereof.

2. The composition of claim 1, wherein each of the at least one intracellular T-cell activation moiety comprises a costimulatory signaling domain, further wherein the at least one costimulatory signaling domain comprises the replacement of CD3ζ with a ZAP70 kinase domain or a mutant or a variant thereof.

3. The composition of claim 1, wherein the each of the at least one intracellular T-cell activation moiety comprises at least one costimulatory signaling domain, further wherein the at least one costimulatory signaling domain is derived from a 4-1BB signaling domain and a ZAP70 kinase domain or a mutant or a variant thereof.

4. The composition of claim 2 or 3, wherein the ZAP70 kinase domain or a mutant or a variant thereof is ZAP255 (SEQ ID NO: 4), ZAP280 (SEQ ID NO: 5), ZAP300 (SEQ ID NO: 127 DM2\19774561.1PATENT Docket No. H3131-00401 1), ZAP327 (SEQ ID NO: 2), or ZAP338 (SEQ ID NO: 3), or a mutant or a variant thereof, wherein the N-terminal amino acid is any of 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279. 280, 281, 282, 283, 284, 285, 286. 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338), and the C-terminal amino acid is 610, 611, 612, 613, 614, 615, 616, 617, 618 or 619 a.a.) of ZAP70 kinase.

5. The composition of claim 3, wherein the ZAP70 kinase domain or a mutant or a variant thereof is a polypeptide comprising the amino acid sequence that has at least 70% identity to the sequence of SEQ ID NO: 1 (ZAP300) or SEQ ID NO: 2 (ZAP327), SEQ ID NO: 3 (ZAP338), SEQ ID NO: 4 (ZAP255), and SEQ ID NO: 5 (ZAP280), or a mutant or a variant thereof, wherein the N-terminal amino acid is any of 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279. 280, 281, 282, 283, 284, 285, 286. 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338), and the C-terminal amino acid is 610, 611, 612, 613, 614, 615, 616, 617, 618 or 619 a.a.) of ZAP70 kinase.

6. The composition of claim 1, wherein the CAR comprises an IL-7R intracellular domain with or without the costimulatory and T-cell activation signaling domains.

7. The composition of claim 1, wherein the CAR comprises an IL-15R intracellular domain with or without the costimulatory and T-cell activation signaling domains.

8. The composition of claim 1, wherein the hinge / TM domain is derived from CD3γ, IL- 7R, IL9-R, IL-21R, or 4-1BB-ZAP327 and is 23-105 amino acids in length. 128 DM2\19774561.1PATENT Docket No. H3131-00401 9. The composition of claim 8, wherein the hinge / TM domain is derived from CD3γ, IL- 7R, IL9-R, IL-21R, or 4-1BB-ZAP327, further wherein the hinge / TM domain is a length selected from 23, 37, 65, 95, and 105 amino acids in length.

10. The composition of claim 8, wherein the hinge / TM domain derived from CD3γ and is 105 amino acids in length.

11. The composition of claim 1, wherein the antigen recognition moiety is followed by the hinge / TM domain with or without a linker followed by the costimulatory signaling domain of the intracellular T-cell activation moiety.

12. The composition of claim 11, wherein the linker is 15-25 amino acids in length.

13. The composition of claim 12, wherein the linker is (X1X2X3X4S)3-5, wherein any of X1-X4 is G or A.

14. The composition of claim 1, wherein then CAR comprises hinge and transmembrane domains selected from SEQ ID NO; 7-48, 90-98, 105-113 and 120-124.

15. The composition of claim 1 wherein antigen recognition moiety comprises SEQ ID NO: 132 or SEQ ID NO: 133; further wherein the at least one transmembrane domain comprises a polypeptide having a sequence selected from SEQ ID NOs: 7-29, and SEQ ID NOs 91-98; and wherein the at least one intracellular signaling domain comprises a polypeptide domain having a sequence selected from ZAP300 (SEQ ID NO: 1), ZAP327 (SEQ ID NO: 2), ZAP338 (SEQ ID NO: 3), ZAP255 (SEQ ID NO: 4) and ZAP280 (SEQ ID NO: 5).

16. A composition comprising a chimeric antigen receptor (CAR), or cells expressing a CAR, wherein the CAR comprises an antigen recognition moiety, a hinge and transmembrane 129 DM2\19774561.1PATENT Docket No. H3131-00401 (hinge / TM) domain, and an intracellular T-cell activation moiety, wherein the hinge / TM domain are a length selected from: 23, 37, 65, 95, and 105 amino acids of the hinge / TM domains of CD3γ, IL-7R, IL9-R, IL-21R, and 4-1BB-ZAP327, or any combination thereof.

17. A composition comprising a chimeric antigen receptor polypeptide comprising a hinge / TM domain selected from a human or a non-human hinge and transmembrane domain, wherein the non-human hinge / TM domain is selected from the list of: mTCRα, mTCRβ, mTCRγ, mTCRδ, mCD3γ, mCD3δ, mCD3ε, and mCD3ζ, or any combination thereof.

18. A composition comprising pairs of chimeric antigen receptors or cells expressing a first CAR and a second CAR, wherein the CARs target dual antigens using TMα-TMβ or TMγ- TMδ TCR transmembrane pairing, the CARs comprising: a. a first CAR comprising a first ScFv that binds to a first target, TCR hinge / TMα, and T cell signaling domain; and a second CAR comprising a second ScFv that binds to a second target different from the first target, TCR hinge / TMβ, and no signaling domain; or b. a first CAR comprising a first ScFv that binds to a first target, TCR hinge / TMγ, and T cell signaling domain; and a second CAR comprising a second ScFv that binds to a second target different from the first target, TCR hinge / TMδ, and no signaling domain.

19. A composition comprising pairs of chimeric antigen receptors or cells expressing pairs of CARs, wherein the CARs are targeting dual antigens using leucine zipper dimerization and TMα-TMβ or TMγ-TMδ TCR transmembrane pairing, the CARs comprising: a. a first CAR comprising a first ScFv that binds to a first target, TCR hinge / TMα, a leucine zipper, and T cell signaling domain; and a second CAR comprising a second ScFv that binds to a second target different from the first target, TCR hinge / TMβ, a leucine zipper, and no signaling domain; or b. a first CAR comprising a first ScFv that binds to a first target, TCR hinge / TMγ, a leucine zipper, and T cell signaling domain; and a second CAR comprising a second ScFv that binds to a second target different from the first target, TCR hinge / TMδ, a leucine zipper, and no 130 DM2\19774561.1PATENT Docket No. H3131-00401 signaling domain; wherein the leucine zipper RR or EE sequence is downstream of the hinge / TM domain, the leucine zipper sequences include, but not limited to RR, EE and other any leucine zipper sequences.

20. The composition of claim 19, wherein the leucine zipper is designed upstream of the hinge / TM domains.

21. The composition of claim 18 or 19, wherein the first CAR comprises a T cell signaling domain, further wherein the T cell signaling domain comprises the ZAP70 kinase domain or a mutant or a variant thereof.

22. The composition of claim 20, wherein the ZAP70 kinase domain or a mutant or a variant thereof is ZAP255, ZAP280, ZAP300, ZAP327, or ZAP338.

23. The chimeric antigen receptor (CAR), or cells expressing a CAR of claim 1 or 16, wherein the antigen recognition moiety of the CAR specifically binds to an antigen from a protein selected from Alpha (α)-fetoprotein (AFP), interferon-inducible protein absent in melanoma 2 (AIM2), adenocarcinoma antigen recognized by T cells 4 (ART-4), BCMA, B antigen (BAGE), CTL-recognized antigen on melanoma (CAMEL), carcinoembryonic antigen peptide-1 (CAP-1), Caspase 8 (CASP8), Cell division cycle 27 (CDC27), Cyclin- dependent kinase 4 (CDK4), CDK12, Carcinoembryonic antigen (CEA), Calcium-activated chloride channel 2 CLCA2), CFTR, CMV, cancer-testis antigen 83 (CT83), desmin, DLK1, DLL3, EBV, EGFRvIII (epidermal growth factor variant III), EGFR and isovariants thereof, EGFR E746-A750del, EGFRVIII, epithelial-specific antigen (ESA), epithelial cell adhesion molecule (EpCAM), Ephrin type-A receptor 2, 3 (EphA2,3), epithelial glycoprotein 2 (EGP2), epithelial glycoprotein-40 (EGP-40), epithelial membrane protein (EMA), epithelial tumor antigen (ETA), Fibronectin (FN), FGF-5, FGF-6, G antigen 1 (GAGE-1), GAGE-2, GAGaE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, N-Acetylglucosaminyltransferase V 131 DM2\19774561.1PATENT Docket No. H3131-00401 (GnT-V), glycoprotein 100 (GP100), Helicase antigen (HAGE), H3.3K27M, oncofetal antigen (h5T4), IP3KB, influenza hemagglutinin (HA), HA-1, HA-1H, HA-2, Human epidermal receptor 2 / neurological (HER2) / neu), HBV, HERV-E, HIV-1 gag, HMI.24, HMB- 45 antigen, HPV E6, HPV E7, HPV-16 E6, HPV-16 E7, Human telomerase reverse transcriptase (hTERT), V-Ki-ras2 Kirsten rat sarcoma viral oncogene (KRAS), KRAS G12D, KRAS G12V, L antigen 1b (LAGE1b), LMP2, LILRB2, LGR5, Ly49, Ly108, LI cell adhesion molecule (LI-CAM), melanoma-associated antigen (MAGE), Melanoma antigen A1 (MAGE-A1), MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MAGE-C2, c-Met, MICA / B, muscle-specific actin (MSA), protein melan-A (melanoma antigen recognized by T lymphocytes MART-1), Mesothelin (MSLN), Mucin 1 (MUC1), MUC2, Mucin 16 (Muc-16), myo-D1, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2-PK), Necl-2, neurofilament, NKCSI, NKG2D, neuron-specific enolase (NSE), NY-ESO, New York esophageous 1 (NY-ESO-1), Preferentially expressed antigen of melanoma (PRAME), Prostate-specific antigen (PSA), Prostate-specific membrane antigen (PSMA), Renal antigen (RAGE), Ral-B, an abnormal ras protein, ROR1, SLAMF7 / CS1, sperm protein 17 (Sp17), sarcoma antigen (SAGE), squamous antigen rejecting tumor 1, 2, 3 (SART-1,-2,-3), SOX10, synovial sarcoma, X breakpoint 2 (SSX-2), Survivin, OVA1, HE4, DR-70, Total PSA, alpha-Methylacyl-CoA Racemase / AMACR, CA125 / MUC16, ER alpha / NR3A1, ER beta / NR3A2, Thymidine Kinase 1, AG-2, BRCA1, BRCA2, CA15- 3 / MUC-1, Caveolin-1, CD117 / c-kit, CEACAM-5 / CD66e, Cytokeratin 14, HIN-1 / SCGB3A1, Ki-67 / MKI67, MKP-3, Nestin, NGF R / TNFRSF16, NM23-H1, PARP, PP4, Serpin E1 / PAI- 1, 14-3-3 beta, 14-3-3 sigma, 14-3-3 zeta, 15-PGDH / HPGD, 5T4, TIM-3, TROP-2, Nectin-4, PD1, PD-L1, CTLA-4, PDGFRalpha, VEGF, TRAG-3, T cell receptor gamma alternate reading frame protein (TARP), TGFbII, Thyroglobulin, an abnormal p53 protein, TP53 (p53), TRAIL, Tyrosinase-related protein 1 or gp75 (TRP1), TRP2, TYRP1, Tyrosinase, tumor- associated glycoprotein 72 (TAG-72), TALLA-1, TLR4, TRBCl, TRBC2, Trp-p8, thyroglobulin, thyroid transcription factor-1, Vα24, Wilms' tumor gene (WT1), CD1a, CD1b, CD1c, CD2, CD3, CD4, CDS, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CD12, CD13, CD14, CD15 (SSEA-1), CD16, (FcyRIII), CD17, CD18, CD20, CD21, CD22, CD23, 132 DM2\19774561.1PATENT Docket No. H3131-00401 CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32 (FcyRII), CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD43, CD44, CD44V6, CD45, CD45R / B220, CD45RO, CD49b, CD49d, CD49f, CD52, CD53, CD54, CD56 (NCAM), CD57, CD61 (integrin β3), CD62L, CD63, CD64 (FcγRl), CD66b, CD68, CD69, CD70, CD73, CD74, CD79a (Igα), CD79b (Igβ), CD80, CD83, CD85k (ILT3), CD86, CD88, CD93 (C1Rqp), CD94, CD95, CD99, CD103, CD105 (Endoglin), CD107a, CD107b, CD114 (G- CSFR), CD115, CD117, CD122, CD123, CD129, CD133, CD134, CD138 (Syndecan-1), CD141 (BDCA3), CD146, CD152 (CTLA-4), CD158 (Kir), CD161 (NK-1.1), CD163, CD183, CD191, CD193 (CCR3), CD194 (CCR4), CD195 (CCRS), CD197 (CCR7), CD203c, CD205 (DEC-205), CD207 (Langerin), CD209 (DC-SIGN), CD223, CD235, CD235a, CD244 (2B4), CD252 (OX40L), CD267, CD268 (BAFF-R), CD273 (B7-DC, PD- L2), CD276 (B7-H3), CD279 (PDI), CD282 (TLR2), CD284 (TLR4), CD294, CD304 (Neuropilin-1), CD305, CD314 (NKG2D), CD319 (CRACC), CD326, CD328 (Siglec-7), CD335 (NKp46), HLA-DR, Kappa light chain, Lambda light chain, Pax-5, BCL-2, Ki-67, MPO, TdT, FMC-7, Pro2PSA, ROMA (HE4+CA-125), OVA1 (multiple proteins), HE4, Fibrin / fibrinogen degradation product (DR-70), AFP-L3, Circulating Tumor Cells (EpCAM, CD45, cytokeratins 8, 18+, 19+), Prostate stem cell antigen (PSCA), α2β1, PAP (prostatic acid phosphatase), PAMA, P-cadherin, placental alkaline phosphatase, PRAIVIE, C3AR, carbonic anhydrase IX (CAIX), chromogranin, CLEC12A, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), CS-I, CSPG4, cytokeratin, AC133 antigen, p63 protein, c-Kit, Lewis A (CA19.9), Lewis Y (LeY), Estrogen receptor (ER), Progesterone receptor (PR), Pro2PSA, cancer antigen-125 (CA-125), CA15-3, CA27.29, Free PSA, Thyroglobulin, Nuclear Mitotic Apparatus protein (NuMA / NMP22), A33, ABCB5, ABCB6, ABCG2, ACE / CD143, ACLP, ACP6, Afadin / AF-6, Afamin, AG-2, AG-3, Akt, Aldo-keto Reductase 1C3 / AKR1C3, alpha 1B-Glycoprotein, alpha 1-Microglobulin, AlphaB Crystallin / CRYAB, alpha-Methylacyl-CoA Racemase / AMACR, AMFR / gp78, Annexin A3, Annexin A8 / ANXA8, APC, Apolipoprotein A-I / ApoA1, Apolipoprotein A-II / ApoA2, Apolipoprotein E / ApoE, APRIL / TNFSF13, ASCL1 / Mash1, ATBF1 / ZFHX3, Attractin, Aurora A, BAP1, Bcl-2, Bcl-6, beta 2-Microglobulin, beta-1,3-Glucuronyltransferase 133 DM2\19774561.1PATENT Docket No. H3131-00401 1 / B3GAT1, beta-Catenin, beta-III Tubulin, Bikunin, BMI-1, B-Raf, BRCA1, BRCA2, Brk, C4.4A / LYPD3, CA15-3 / MUC-1, c-Abl, Cadherin-13, Caldesmon / CALD1, Calponin 1, Calretinin, Carbonic Anhydrase IX / CA9, Catalase, Cathepsin D, Caveolin-1, Caveolin-2, CBFB, CCR1, CCR4, CCR7, CCR9, CEACAM-19, CEACAM-20, CEACAM-4, CHD1L, Chitinase 3-like 1, Cholecystokinin-B R / CCKBR, Chorionic Gonadotropin alpha Chain (alpha HCG), Chorionic Gonadotropin alpha / beta (HCG), CKAP4 / p63, Claudin-18, Clusterin, c-Maf, c-Myc, Coactosin-like Protein 1 / CotL1, COMMD1, Cornulin, Cortactin, COX-2, CRISP-3, CTCF, CTL1 / SLC44A1, CXCL17 / VCC-1, CXCL8 / IL-8, CXCL9 / MIG, CXCR4, Cyclin A1, Cyclin A2, Cyclin D2, Cyclin D3, CYLD, Cyr61 / CCN1, Cytokeratin 14, Cytokeratin 18, Cytokeratin 19, fetal acetylcholine receptor (AChR), ADGRE2, ATM, ALK, ALPK2, DAB2, DCBLD2 / ESDN, DC-LAMP, Dkk-1, DLL3, DMBT1, DNMT1, DPPA2, DPPA4, E6, E-Cadherin, ECM-1, EGF, ELF3, ELTD1, EMMPRIN / CD147, EMP2, Endoglin / CD105, Endosialin / CD248, Enolase 2 / Neuron-specific Enolase, EpCAM / TROP1, Eps15, ER alpha / NR3A1, ER beta / NR3A2, ERBB, EGFR / ErbB1, ERBB2, ErbB3 / Her3, ErbB4 / Her4, ERCC1, ERK1, ERK5 / BMK1, Ets-1, Exostosin 1, EZH2, Ezrin, FABP5 / E- FABP, Fascin, FATP3, FCRLA, Fetuin A / AHSG, FGF acidic, FGF basic, FGF R3, FGF R4, Fibrinogen, folate-binding protein (FBP), Fibroblast Activation Protein alpha / FAP, Follistatin-like 1 / FSTL1, FOLR1, FOLR2, FOLR3, FOLR4, FosB / G0S3, FoxM1, FoxO3, FRAT2, FXYD5 / Dysadherin, FcεRIα, FITC, FLT3, GABA-A R alpha 1, GADD153, GADD45 alpha, Galectin-3, Galectin-3BP / MAC-2BP, galactin, gangiosides, gross cystic disease fluid protein (GCDFP-15), GD2 (ganglioside G2), GD3, GM2, GM3, gamma- Glutamylcyclotransferase / CRF21, Gas1, Gastrin-releasing Peptide R / GRPR, Gastrokine 1, Gelsolin / GSN, glial fibrillary acidic protein (GFAP), GLI-2, Glutathione Peroxidase 3 / GPX3, gpA33, glycopeptides, Glypican 2 (GPC2), Glypican 3, Golgi Glycoprotein 1 / GLG1, gp96 / HSP90B1, GPR10, GPR110, GPR18, GPR31, GPR87, GPRC5A, GPRC6A, GRP78 / HSPA5, HE4 / WFDC2, Heparanase / HPSE, Hepsin, HGF R / c-MET, HIF-2 alpha / EPAS1, HIN-1 / SCGB3A1, HLA-DR, HOXB13, HOXB7, HSP70 / HSPA1A, HSP90, Hyaluronidase 1 / HYAL1, ID1, IgE, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, IGF-I, IGF-I R, IGF-II, IGFL-3, IGFLR1, IL-1 beta / IL-1F2, IL-17E / IL-25, IL-2, IL-6, ICAM-1, IgG, IgD, 134 DM2\19774561.1PATENT Docket No. H3131-00401 IgE, IgM, Interleukin 13 receptor a2 chain (IL-13Ra), Interleukin-13 receptor subunit alpha-2 (IL-13Ra2), integrins, Integrin B7, IMP Dehydrogenase 1 / IMPDH1, Importin alpha 2 / KPNA2, ING1, Integrin beta 1 / CD29, Integrin beta 3 / CD61, IQGAP1, Isocitrate Dehydrogenase 1 / IDH1, ITIH4, ITM2C, Jagged 1, JNK, JunB, JunD, OGR1, Olig2, Osteopontin / OPN, Ovastacin, OXGR1 / GPR80 / P2Y15, p130Cas, p15INK4b / CDKN2B, p16INK4a / CDKN2A, p18INK4c / CDKN2C, p21 / CIP1 / CDKN1A, p27 / Kip1, P2X5 / P2RX5, PARP, PAUF / ZG16B, PBEF / Visfatin, PDCD4, PDCD5, PDGF R alpha, PDGF R beta, PDZD2, PEA-15, Pepsinogen A5 / PGA5, Peptidase Inhibitor 16 / PI16, Peroxiredoxin 2, PGCP, PI 3-Kinase p85 alpha, PIWIL2, PKM2, PLK1, PLRP1, PP4, P-Rex1, PRMT1, Profilin 1, Progesterone R B / NR3C3, Progesterone R / NR3C3, Progranulin / PGRN, Prolactin, Prostaglandin E Synthase 2 / PTGES2, PSAP, PSCA, PSMA / FOLH1 / NAALADase I, PSMA1, PSMA2, PSMB7, PSP94 / MSMB, PTEN, PTEN, PTH1R / PTHR1, PTK7 / CCK4, PTP beta / zeta / PTPRZ, Rab25, RARRES1, RARRES3, Ras, Reg4, Ret, RNF2, RNF43, S100A1, S100A10, S100A16, S100A2, S100A4, S100A6, S100A7, S100A9, S100B, S100P, SART1, SCUBE3, Secretin R, Serpin A9 / Centerin, Serpin E1 / PAI-1, Serum Amyloid A1, Serum Amyloid A4, SEZ6L, SEZ6L2 / BSRP-A, Skp2, SLC16A3, SLC45A3 / Prostein, SLC5A5, SLC5A8 / SMCT1, SLC7A7, Smad4, SMAGP, SOCS-1, SOCS-2, SOCS-6, SOD2 / Mn-SOD, Soggy-1 / DkkL1, SOX11, SOX17, SOX2, SPARC, SPARC-like 1 / SPARCL1, SPINK1, Src, six-transmembrane epithelial antigen of the prostate 1 (STEAP1), STEAP2, STEAP3 / TSAP6, STRO-1, STYK1, Survivin, Synaptotagmin-1, Syndecan- 1 / CD138, Syntaxin 4, Synuclein-gamma, synaptophysin, Kallikrein 2, Kallikrein 6 / Neurosin, KCC2 / SLC12A5, Ki-67 / MKI67, KiSS1R / GPR54, KLF10, KLF17, L1CAM, Lactate Dehydrogenase A / LDHA, Lamin B1, LEF1, Leptin / OB, LIN-28A, LIN-28B, Lipocalin- 2 / NGAL, LKB1 / STK11, LPAR3 / LPA3 / EDG-7, LRMP, LRP-1B, LRRC3B, LRRC4, LRRN1 / NLRR-1, LRRN3 / NLRR-3, Ly6K, LYPD1, LYPD8, MAP2, Matriptase / ST14, MCAM / CD146, M-CSF, MDM2 / HDM2, Melanocortin-1 R / MC1R, Melanotransferrin / CD228, Melatonin, Mer, Mesothelin, Metadherin, Metastin / KiSS1, Methionine Aminopeptidase, Methionine Aminopeptidase 2 / METAP2, MFAP3L, MGMT, MIA, MIF, MINA, Mind Bomb 2 / MIB2, Mindin, MITF, MKK4, MKP-1, MKP-3, MMP-1, 135 DM2\19774561.1PATENT Docket No. H3131-00401 MMP-10, MMP-13, MMP-2, MMP-3, MMP-8, MMP-9, MRP1, MRP4 / ABCC4, MS4A12, MSH2, MSP R / Ron, MSX2, MUC-4, Musashi-1, NAC1, Napsin A, NCAM-1 / CD56, NCOA3, NDRG1, NEK2, NELL1, NELL2, Nesfatin-1 / Nucleobindin-2, Nestin, NFkB2, NF- L, NG2 / MCSP, NGF R / TNFRSF16, Nicotinamide N-Methyltransferase / NNMT, NKX2.2, NKX3.1, NM23-H1, NM23-H2, Notch-3, NPDC-1, NTS1 / NTSR1, NTS2 / NTSR2, Tankyrase 1, Tau, TCF-3 / E2A, TCL1A, TCL1B, TEM7 / PLXDC1, TEM8 / ANTXR1, Tenascin C, TFF1, TGF-beta 1, TGF-beta 1, 2, 3, TGF-beta 1 / 1.2, TGF-beta 2 / 1.2, TGF-beta RI / ALK-5, THRSP, Thymidine Kinase 1, Thymosin beta 10, Thymosin beta 4, Thyroglobulin, TIMP Assay Kits, TIMP-1, TIMP-2, TIMP-3, TIMP-4, TLE1, TLE2, TM4SF1 / L6, TMEFF2 / Tomoregulin-2, TMEM219, TMEM87A, TNF-alpha, TOP2A, TopBP1, t-Plasminogen Activator / tPA, TRA-1-60(R), TRA-1-85 / CD147, TRAF-4, Transgelin / TAGLN, Trypsin 2 / PRSS2, Tryptase alpha / TPS1, TSPAN1, UBE2S, uPAR, u- Plasminogen Activator / Urokinase, Urotensin-II R, VAP-1 / AOC3, VCAM-1 / CD106, VEGFR1 / Flt-1, VEGFR2 / KDR / Flk-1, VEGF / PlGF Heterodimer, VSIG1, VSIG3, YAP1, ZAG, ZAP70, ZMIZ1 / Zimp10, SGK, CNKSR1 / CNK / KSR or any combination thereof.

24. The cells expressing CARs of claim 1 or 16, wherein the cells are CAR-T, CAR-NK, CAR-NKT or CAR-M cells.

25. A bispecific antibody comprising a TCR that specifically binds to CD19 fused to an antibody or fragment thereof that specifically binds to CD3.

26. A cell selected from a CAR-T, CAR-NK, CAR-NKT and CAR-M cell expressing the bispecific antibody of claim 25.

27. The composition of claim 1, wherein the hinge and transmembrane domains are selected from SEQ ID NO: 7-48, 90-98, 105-113 and 120-124.

28. The composition of claim 1, wherein the at least one costimulatory signaling domain is 136 DM2\19774561.1PATENT Docket No. H3131-00401 derived from a 4-1BB signaling domain and the signaling domain is selected from ZAP300 (SEQ ID NO:1), ZAP327 (SEQ ID NO: 2), ZAP338 (SEQ ID NO:3), ZAP255 (SEQ ID NO: 4), or ZAP280 (SEQ ID NO: 5), or a mutant or a variant thereof, wherein the N-terminal amino acid is any of 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279. 280, 281, 282, 283, 284, 285, 286. 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338), and the C-terminal amino acid is 610, 611, 612, 613, 614, 615, 616, 617, 618 or 619 a.a.) of ZAP70 kinase.

29. The composition of claim 1, wherein the at least one hinge / TM domain is derived from a transmembrane domain selected from the group consisting of IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, and IL-21Rα.

30. The composition of claim 1, wherein the antigen recognition moiety is followed by the hinge / TM domain with a linker comprising the sequence (GGGGS)3-5 (SEQ ID NO: 148).

31. The composition of claim 1, wherein the CAR comprises: a. an antigen recognition moiety comprising SEQ ID NO: 132 or SEQ ID NO: 133; b. at least one transmembrane domain comprising a polypeptide selected from SEQ ID NOs: 7-29 and SEQ ID NOs 91-98; and c. at least one intracellular signaling domain comprising a polypeptide domain selected from ZAP300 (SEQ ID NO: 1), ZAP327 (SEQ ID NO: 2), ZAP338 (SEQ ID NO: 3), ZAP255 (SEQ ID NO: 4) and ZAP280 (SEQ ID NO: 5), or a mutant or a variant thereof, wherein the N-terminal amino acid is any of 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279. 280, 281, 282, 283, 284, 285, 286. 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 137 DM2\19774561.1PATENT Docket No. H3131-00401 332, 333, 334, 335, 336, 337, 338), and the C-terminal amino acid is 610, 611, 612, 613, 614, 615, 616, 617, 618 or 619 a.a.) of ZAP70 kinase. .

32. A method of prolonging CAR-T or TCR-T cell persistence by knocking down, knocking out or introducing an inactivating mutation in one or more negative signaling regulators selected from ANKRDJ 1, ARID1A, BACH2, BCL2Ll 1, BCL3, BCOR, BATF, CALM2, CBLB, CHIC2, CTLA4, DHODH, DHX37, DNMT3A, E2F8, EGR2, FLII, FOXP3, GATA3, GNAS, HAVCR2, IKZF1, IKZF2, IKZF3, JMJD1C, JMJD3 (KDM6B), LAG3, LSD1, MAP4K, MED12, NFKBIA, NR4A1, NR4A2, NR4A3, NRPJ, PBRMJ, PCBPJ, PDCDJ, PELII, PIK3CD, PPP2R2D, PTPN1, PTPN6, PTPN2, PTPN22, RASA2, RBM39, RC3Hl (ROQUIN-1), SEMA7A, SERPINA3, SETD5, SH2B3, SH2DJA, SMAD2,SOCS1, SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, ZC3H12A, indoleamine (2,3)-dioxygenase (IDO) (including isoforms IDO1 and IDO2), OX40, CTLA-4 (programmed cytotoxic T-lymphocyte antigen 4), PD-1 (programmed death 1), PD-L1 (programmed death ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), B7 homolog 3 (B7-H3), VHL, PPP2R2D and epigenetic factors which can include or exclude JMJD3 and LSD1. SUV39H1, TANK, TET2, TGFBR1, TGFBR2, TIGIT, TNFAIP3, TOX1, TOX2, TRAF6, UMPS, VHL, WDR6, ZC3H12A, , indoleamine (2,3)- dioxygenase (IDO) (including isoforms IDO1 and IDO2), OX40, CTLA-4 (programmed cytotoxic T-lymphocyte antigen 4), PD-1 (programmed death 1), PD-L1 (programmed death ligand 1), PD-L2, lymphocyte activation gene 3 (LAG3), and B7 homolog 3 (B7-H3).

33. The method of claim 32, wherein the knocking down, knocking out or introducing an inactivating mutation is achieved through siRNA, shRNA or a CRISPR / Cas9 system comprising a single-strand guide RNA (sgRNA) molecule and Cas endonuclease. 138 DM2\19774561.1PATENT Docket No. H3131-00401 34. A method of treating a subject having an inflammatory disease, an autoimmune disease, an allergic disease, an organ transplantation condition, a cancer, an infectious disease, or aging, the method comprising administering the composition of any one of claims 1-29.

35. A composition comprising a chimeric antigen receptor (CAR), or cells expressing a CAR, wherein the CAR comprises an antigen recognition moiety, a hinge and transmembrane (hinge / TM) domain, and an intracellular T-cell activation moiety, wherein the hinge / TM domain are a length selected from: 23, 37, 65, 95, and 105 amino acids of the hinge / TM domains of CD3γ, IL-7R, IL9-R, IL-21R, and 4-1BB-ZAP327, or any combination thereof.

36. The composition of claim 1 for the manufacture of a medicament for the treatment of a disease or disorder in a human subject in need thereof, wherein the disease or disorder that is treated is selected from the group consisting of an inflammatory disease, an autoimmune disease, an allergic disease, an organ transplantation condition, a cancer, and an infectious disease.

37. The composition of claim 1, wherein the hinge and transmembrane domains are selected from murine mTCRα, mTCRβ, mTCRγ, mTCRδ, mCD3γ, mCD3δ, mCD3ε, and mCD3ζ.

38. The composition of claim 1, wherein the signaling domain is selected from ZAP300 (SEQ ID NO:1), ZAP327 (SEQ ID NO: 2), ZAP338 (SEQ ID NO:3), ZAP255 (SEQ ID NO: 4), or ZAP280 (SEQ ID NO: 5), or a mutant or a variant thereof, wherein the N-terminal amino acid is any of 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264,265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279. 280, 281, 282, 283, 284, 285, 286. 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338), and the C-terminal amino acid is 610, 611, 612, 613, 614, 615, 616, 617, 618 or 619 a.a.) of ZAP70 kinase. 139 DM2\19774561.1PATENT Docket No. H3131-00401 .

39. The composition of claim 1, wherein the at least one hinge / TM domain is derived from a transmembrane domain selected from the group consisting of IL-1R, IL-2Rβ, IL-4Rα, IL-7α, IL-9Rα, IL-12R, IL-13Rα, IL-15Rα, IL-17Rα, IL-17RC, and IL-21Rα.

40. The composition of claim 1, wherein the linker between the antigen recognition moiety and the hinge / TM domain comprises the sequence (X1X2X3X4S)3-5, wherein any of X1-X4 is G or A.

41. The composition of claim 1, wherein the linker between the antigen recognition moiety and the hinge / TM domain comprises the sequence (X1X2X3X4S)3-5, wherein any of X1-X4 is G or A.

42. The composition of claim 18 or 19, wherein: a. the first CAR comprises an anti-CD19 scFv, a TCRα hinge / TM domain, a P2A domain, a 4- 1BB costimulatory domain, and a ZAP327 signaling domain; and b. the second CAR comprises an anti-B7H3 scFv, a TCRβ hinge / TM domain, and no signaling domain.

43. The composition of claim 19, wherein: a. the first CAR comprises an anti-CD19 scFv, a TCRα hinge / TM domain, a leucine zipper RR domain, and a signaling domain; and b. the second CAR comprises an anti-B7H3 scFv, a TCRβ hinge / TM domain, and a leucine zipper EE domain. 140 DM2\19774561.1

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